Growth Hormone & IGF Research 13 (2003) 203–222 www.elsevier.com/locate/ghir
Abstracts S1 REGIONAL EXPRESSION OF THE IGF SYSTEM DURING DEVELOPMENT: EVIDENCE FOR AUTOCRINE/PARACRINE ACTIONS OF IGFBPs Victor Han, Carole Watson, Anthony Carter. Department of Paediatrics, CIHR Group in Fetal and Neonatal Health and Development, Child Health Research Institute, University of Western Ontario. London, Ont., Canada The IGF system is the major regulator of embryonic and placental growth and diﬀerentiation. In contrast to IGF receptors, which are ubiquitously and uniformly expressed in almost every tissue during development, the IGFs and IGFBPs are expressed in spatial and temporally speciﬁc manner in fetal and placental tissues. Since IGFBPs modulate IGF–IGF receptor interactions, either negatively or positively, we hypothesized that the sites of expression of IGFBPs indicate the sites of IGF actions in during development. We have cloned cDNAs encoding IGFBPs 1–6 of several species (human, ovine, guinea pig, rat, and mouse) and have utilized these probes for expression studies, using Northern analysis and in situ hybridization on diﬀerent fetal and placental tissues of various gestational ages. Comparative histochemical studies showed similarities in expression patterns for most IGFs and IGFBPs among diﬀerent species, as well as signiﬁcant diﬀerences. These ﬁndings suggest that diﬀerent IGFBPs are utilized similarly in modulating IGF actions in cellular growth and diﬀerentiation, but there are also species-speciﬁc actions of IGFBPs. In particular, the diﬀerences in expression pattern of IGFs and IGFBPs are observed in the placentae of all the species studied, indicating that the uniqueness of placenta of each species is reﬂected in the expression pattern of these peptides. Delineation of the expression patterns of mRNAs encoding these peptides in mice provided crucial guidance in developing transgenic and gene-targeted models to further dissect out the biological actions of IGFs and IGFBPs during fetal development in vivo. Since circulating IGFBP-1 concentrations in the fetus are elevated in growth restriction of many diﬀerent species, including humans, and IGFBP-1 mRNA is expressed only in the fetal hepatocytes, we addressed the question of whether hepatic overexpression of IGFBP-1 with resultant increase in circulating IGFBP-1 is suﬃcient in causing a growth restrictive phenotype. We generated transgenic mice using a-fetoprotein promoter-hIGFBP-1 construct. The resulting newborns were growth restricted by 20% compared to wild type. The growth restriction was attributed mainly to a reduction in carcass tissues. These ﬁndings indicate that elevated IGFBP-1 in fetal circulation in growth restriction has a causal relationship with the phenotype. Our studies to date have indicated both the paracrine as well as endocrine role for diﬀerent IGFBPs during development. Supported by the Canada Research Chair Award and CIHR grants.
S2 WHAT ARE FREE IGFs? Jan Frystyk. Medical Research Laboratories and Medical Department M, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: [email protected]
The vast majority of circulating IGF-I is bound to speciﬁc high-aﬃnity IGFBPs. Since the ligand aﬃnity of the IGFBPs is generally higher 1096-6374/03/$ - see front matter 2003 Published by Elsevier Science Ltd. doi:10.1016/S1096-6374(03)00036-4
than that of the IGF-I receptor (IGF-IR), free unbound IGF-I has been hypothesized to constitute the primary bioactive fraction. Throughout literature, several diﬀerent approaches to determine immunoreactive serum levels of free IGF-I have been described. At present, most researchers use direct non-competitive immunoassays based on antibodies speciﬁc for unbound IGF-I, or ultraﬁltration by centrifugation followed by immunoassay of the ultraﬁltrate. The ﬁrst part of this presentation will discuss the current methodologies used for determination of immunoreactive levels of free IGF-I, focusing on assay diﬀerences and pitfalls. It has been argued that bioactive IGF-I is composed of the sum of free IGF-I plus readily dissociable IGF-I, i.e., IGF-I being liberated from the IGFBPs at the level of the IGF-IR. In order to study this, we have developed a highly speciﬁc cell based IGF-I kinase receptor activation assay (KIRA). This bioassay has enabled us to determine the concentration of IGF-I being able to activate (i.e., phosphorylate) the IGF-IR. The second part of the presentation will present comparative data on serum levels of free and bioactive IGF-I. In order to clarify which IGF-I measurement that best reﬂects the true IGF-I bioactivity in vivo, one needs to compare the different IGF-I measurements with a biological endpoint. In this context GH secretion may be useful, since it is feedback regulated by IGF-I. The last part of the presentation will focus on the relationship between serum GH and the diﬀerent measurements of IGF-I (total IGF-I, free/dissociable IGF-I, ultraﬁltered free IGF-I and KIRA IGF-I) in various physiological and pathophysiological conditions.
S3 WHAT IS THE ROLE OF CIRCULATING IGF-I? Derek LeRoith, Shoshana Yakar. Diabetes Branch, NIH, Bethseda, MD, USA To study the role of circulating (‘‘endocrine’’) IGF-I compared with tissue (‘‘paracrine/autocrine’’) IGF-I in normal growth and development we created the liver-speciﬁc IGF-I gene-deleted mouse model (LID). Using this model we have demonstrated that the 70% reduction in circulating IGF-I leads to the following: 1. Elevated circulating GH levels that leads to insulin resistance (Diabetes 50:1110, 2001). 2. Accumulation of brain amyloid Ab protein (associated with Alzheimers) Nature Medicine 8:1390, 2002. To extend these ﬁndings we have crossed the LID mice with a mouse line carrying a null mutation of the acid-labile subunit (ALS) gene. These mice demonstrate a further reduction in circulating IGF-I levels and post-natal growth retardation, suggesting an important role for circulating IGF-I. Interestingly, the levels of free IGF-I are elevated compared to controls; however periosteal bone circumference is markedly reduced, out of proportion to the reduction in bone length. These ﬁndings have led us to conclude that biologically active IGF-I should be complexed to its major binding protein (IGFBP-3) for maximum eﬀectiveness. In addition, it is likely that circulating IGF-I plays an important role on bone formation whereas circulating and locally produced IGF-I are important for bone length during the growth phase. We are presently studying mice that are null for the liver
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
IGF-I gene (LID) as well as for the IGFBP-3 and ALS genes to conﬁrm and extend these ﬁndings.
S4 PERTURBATIONS IN GLUCOSE METABOLISM IN IGFBP TRANSGENIC MICE Josef V. Silha, Liam J. Murphy. Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, Canada Insulin-like growth factor-I (IGF-I) exerts hypoglycemic activity and can potentiate insulin action. IGF binding proteins (IGFBPs) can both enhance and attenuate eﬀects of IGF-I in vitro and in vivo. To investigate the eﬀects of elevated levels of IGFBP-3 on glucose homeostasis, we examined male transgenic (Tg) mice that constitutively overexpressed the human IGFBP-3 cDNA driven by either the cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. The IGFBP-3 Tg mice of both lineages demonstrated ubiquitous expression of hIGFBP-3 mRNA, serum levels of hIGFBP-3 5 lg/ml, and 1.5 fold increase in total serum IGF-I compared to wild-type (Wt) mice. Fasting blood glucose levels were signiﬁcantly elevated in 8-week-old CMVBP-3 and PGKBP-3. Plasma insulin was signiﬁcantly elevated only in CMVBP-3 compared to Wt mice; 6.45 ± 0.71 vs. 4.08 ± 0.64 lIU/ml. Growth hormone levels were signiﬁcantly elevated only in PGKBP-3 mice. Unlike PGKBP-3 Tg mice, CMV BP-3 Tg mice had increased visceral adiposity which was accompanied by with elevated plasma leptin and adiponectin levels. The glycemic responses to an intraperitoneal glucose challenge was signiﬁcantly increased in both Tg strains: AUCglucose = 1824 ± 65, 1910 ± 115 versus 1590 ± 67 mmol Æ min/l for CMVBP-3, PGKBP-3 and Wt mice, respectively. The hypoglycemic eﬀects of insulin and IGF-I were signiﬁcantly attenuated in IGFBP-3 Tg mice compared to Wt mice. When measured by IVGTT followed by minimal model analysis, insulin sensitivity was signiﬁcantly reduced in IGFBP-3 Tg mice. IGFBP-3 has been described to interact with retinoid X receptor-a (RXRa) in vitro. There were no signiﬁcant diﬀerences in adipose tissue resistin, RXRa or peroxisome proliferator-activated receptor-c (PPARc) mRNA levels, measured by ribonuclease protection assay, and RXRa and PPARc protein abundance between any group of mice. The phenotype of IGFBP-1 Tg mice overexpressing rat IGFBP-1 under control of PGK promoter led to elevated fasting glycemia, increased fasting insulin, and impaired glucose tolerance. These data from independent Tg mouse lineages demonstrate that constitutive overexpression of IGFBP-1 and IGFBP-3 at high levels results in elevated fasting glycemia and impaired glucose tolerance.
S5 MULTIFUNCTIONAL ROLE OF IGFBPs IN BONE Subburaman Mohan, Donna D. Strong, Yousef Amaar, David J. Baylink. Musculoskeletal Diseases Center, JLP VA Medical Center, Loma Linda, CA 92357, USA There is now convincing experimental evidence that the IGF system plays a critical role in the development and maintenance of the skeleton. The anabolic eﬀects of IGFs on bone are mediated via both endocrine and local autocrine/paracrine mechanisms. The hormonal and local growth factor actions of IGFs depend not only on the amount of IGFs themselves but also on the types (inhibitory or stimulatory) and amounts of various IGFBPs present in serum (endocrine) and/or extracellular body ﬂuids. Interestingly, in addition to regulating IGF actions, IGFBPs also exhibit unique growth factor actions on osteoblasts in an IGF-independent manner. The growth factor actions of IGFBPs can be either stimulatory (e.g., IGFBP-5) or inhibitory (e.g., IGFBP-6). To identify the intracellular pathways by which IGFBP-5 and IGFBP-6 exert diﬀering intrinsic biological activity in osteoblasts, we undertook studies to identify proteins that
interacted with these IGFBPs using either IGFBP-5 or IGFBP-6 as bait in yeast two hybrid screens of a U2 human osteosarcoma cell cDNA library. In these studies, we have identiﬁed FHL2 and RASSF1C as interacting partners for IGFBP-5 and Lim Mineralizing Protein-1 (LMP-1) as an interacting partner for IGFBP-6. FHL2, a four and half LIM domain protein, has recently been shown to stimulate transcription of Fos and Jun (Morlon and Sassone-Corsi, 2003), key genes involved in osteoblast regulation. RASSF1C, the C isoform of the Ras association family 1 gene, is expressed in various osteoblast cell types and blockage of RASSF1C expression by means of a siRNA duplex caused an inhibition of osteoblast cell proliferation. RASSF1C belongs to a family of proteins known to be involved in mediating Ras-dependent activation of p38MAP kinase and ERK1/2 pathways. In contrast to IGFBP-5, IGFBP-6 (a strong inhibitor of osteoblast diﬀerentiation) speciﬁcally interacted with LMP-1, a positive regulator of osteoblast diﬀerentiation. Based on these and other ﬁndings, we conclude that (1) IGFBPs exert multifunctional roles in bone; and (2) IGFBPs recruit key transcription factors and signaling proteins that are either novel or not previously known to be involved in regulating osteoblast cell proliferation/diﬀerentiation to mediate their IGF-independent eﬀects on osteoblasts.
S6 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN PROTEOLYSIS: 2003 AND BEYOND John L. Fowlkes, Kathryn M. Thrailkill, R. Clay Bunn. Division of Pediatric Endocrinology and Diabetes, UMAS/ACH/ACHRI, Little Rock, AR, USA High-aﬃnity interactions between insulin-like growth factors (IGF-I and IGF-II) and insulin-like growth factor binding proteins (IGFBP-1, -2, -3, -4, -5 and -6) antagonize the binding of IGF to the type-1 IGF receptor. Proteases found in a variety of biological ﬂuids can degrade IGFBP 1–6 into fragments that have a greatly reduced aﬃnity for IGF-I and IGF-II, increasing the concentration of free IGFs at the cell surface and allowing IGFs to bind to and activate the IGF receptor. Therefore, IGFBP proteolysis can directly impact on the initial step in IGF receptor signaling, thereby eﬀecting cell survival, mitogenesis and diﬀerentiation. Our understanding of IGFBP proteolysis has grown exponentially over the past ﬁve years, with the identiﬁcation of several new IGFBP proteases, a growing appreciation of the potential for IGF-independent actions of IGFBP fragments and the realization that perturbations of IGFBP proteolysis are observed in and might contribute to, several pathological conditions. Proteolytic cleavage has now been demonstrated for all six IGFBPs and has gained wide acceptance as the predominant mechanism for IGF release from IGFBPs. For this overview, we will focus on newly described IGFBPdegrading proteinases, IGF-independent eﬀects of proteolytic fragments derived from IGFBPs, regulation of IGFBP proteolysis and IGFBP proteolysis in disease states.
S7 IGFBP SIGNALING PATHWAYS ACTIVATED INDEPENDENTLY OF IGF-BINDING Jean-Marc Ricorta,b, Alain Lombetc, Claudine Lassarrea, Michel Binouxa. aINSERM U515, Hoˆpital Saint-Antoine, Paris, France; b CNRS UMR 8113, Ecole Normale Supe´rieure de Cachan, Cachan, France; cINSERM U339, Hoˆpital Saint-Antoine, Paris, France Apart from their well-established ability to modulate IGF action via binding to their ligands, IGFBPs are increasingly becoming recognized as capable of intrinsic eﬀects. There is mounting evidence that certain IGFBPs modulate cell proliferation and apoptosis independently of their binding to IGFs. Nevertheless, although the IGFBPs have recently been reported to inﬂuence a variety of individual cellular events, little is known of the signaling pathways or molecular mechanisms that they activate.
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 The aim of our work has therefore been to identify and characterize these signaling pathways, using wild-type and mutated recombinant IGFBPs obtained in both Escherichia coli and baculovirus systems. We intentionally investigated two very diﬀerent cell models, human MCF-7 breast carcinoma cells and murine C2 myoblast cells. Our results indicate that the IGFBPs are capable of inducing speciﬁc cellular signals and that the response to the IGFBP-activated signal is cell type-dependent. This would mean expression of speciﬁc cell-surface receptors. Our results also indicate that for a given IGFBP, the mechanisms of intracellular communication may diﬀer from one cell type to another, which would account for the speciﬁcity of intracellular response and signaling pathway elicited by each IGFBP. These ﬁndings open new perspectives for research on the signaling pathways activated by IGFBPs.
S8 IGFBP-1 AND THE ADAPTIVE RESPONSE TO LIVER INJURY Rebecca Taub. Bristol-Myers Squibb Pharmaceuticals, Wallingford, CT, USA After a two-thirds hepatectomy, normally quiescent liver cells are stimulated to reenter the cell cycle and proliferate to restore the original liver mass. One of the most rapidly and highly induced genes and proteins in regenerating liver is insulin-like growth factor binding protein 1 (IGFBP-1), a secreted protein that may modulate the activities of insulin-like growth factors (IGFs) or signal via IGF-independent mechanisms. To assess the functional role of IGFBP-1 in liver regeneration, mice with a targeted disruption of the IGFBP-1 gene were generated. Although IGFBP-1-/- mice demonstrated normal development, they had abnormal liver regeneration after partial hepatectomy, characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis. The abnormal regenerative response was associated with speciﬁc signal transduction defects that could be corrected by treatment of the mice with IGFBP-1. These ﬁndings are the ﬁrst demonstration of the involvement of IGFBP-1 in the regulation of in vivo mitogenic signaling pathways. IGFBP-1 deﬁcient livers were also associated with massive liver injury following Fas agonist treatment or toxic damage, which could be corrected by pretreatment with IGFBP-1. IGFBP-1-deﬁcient livers had enhanced signaling via the integrin receptor at early times after Fas agonist treatment accompanied by elevated activated matrix metalloproteinase-9, a known target of ﬁbronectin signaling and activator of TGF-b. Within 3 hours of Fas agonist treatment, elevated expression of active TGF-b1, a hepatocyte apoptogen, was observed in IGFBP-1-deﬁcient livers that correlated with the appearance of the apoptotic process. Both MMP-9 and TGF-b1 expression were suppressed by IGFBP-1 treatment, supporting their role in the apoptotic process. Taken together, these ﬁndings indicate that IGFBP-1 functions as a critical hepatic survival factor in the liver by reducing the level of proapoptotic signals and stimulating hepatic regeneration.
S9 IGFBPs AND IGF-I RECEPTOR SIGNALING Laura A. Maile, Anna Moralez, Jane Badley Clarke, Walker H. Busby, David R. Clemmons. Division of Endocrinology and Metabolism, University of North Carolina School of Medicine, Chapel Hill, NC, USA IGF-I is a potent stimulator of smooth muscle cell (SMC) migration and proliferation. The responsiveness of SMCs to IGF-I is however also regulated by ligand occupancy of at least three other transmembrane proteins in addition to the IGF-IR, namely aVb3 integrin, integrin-associated protein (IAP) and SHPS-1. Thrombospondin-1 (TS-1), a ligand for both aVb3 and IAP enhances IGF-I stimulated eﬀects. Since blocking TS-1 binding to IAP using an anti IAP antibody inhibits the enhancement eﬀect of TS-1 on IGF-I signaling and a
synthetic peptide homologous to the IAP binding domain of TS-1 mimics the eﬀect of TS-1 it seems likely that the eﬀect of TS-1 is mediated via its association with IAP. Since IGFBP-5 also binds to TS1 we investigated the eﬀect of IGFBP-5 on the ability of TS-1 to regulate IGF-I signaling. The addition of IGFBP-5 to cells treated with IGF-I alone had no eﬀect however; the addition of IGFBP-5 to cells that were exposed to both TS-1 and IGF-I inhibited the enhancing eﬀect of TS-1. To determine whether the eﬀect of IGFBP-5 was mediated via its ability to bind TS-1 or IGF-I two IGFBP-5 mutants were used. An IGFBP-5 mutant that did not bind IGF-I was equally effective as wild type IGFBP-5 in inhibiting the eﬀect of TS-1 however, a mutant IGFBP-5 that bound poorly to TS-1 had little eﬀect of the ability of TS-1 to enhance IGF-I stimulated signaling. Since TS-1 mediates its eﬀects via binding to IAP we investigated whether the eﬀect of IGFBP-5 was due to its ability to inhibit TS-1 binding to IAP. The addition of IGFBP-5 blocked TS-1 association with IAP suggesting that the eﬀect of IGFBP-5 on TS-1 enhancement of IGF-I signaling is mediated via its ability to block TS-1 association with IAP. The results from our studies demonstrate a role for IGFBP-5 in regulating IGF signaling that is independent of its ability to bind IGF-I. Our results also demonstrate that the responsiveness of a cell to IGF-I is determined by the interplay between several transmembrane and extracellular proteins in addition to IGF-I itself.
S10 GENE REGULATION OF PROGRAMMED CELL DEATH Boris Zhivotovsky. Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden Programmed cell death (PCD), or apoptosis, is a genetically controlled form of death that is of importance for normal embryonic development and for the maintenance of tissue homeostasis in the adult organism. Apoptosis is triggered by extrinsic or intrinsic (mitochondrially mediated) signalling pathways that induce death-associated proteolytic and/or nucleolytic activities. Apoptosis occurs in a wellorganized sequence of morphological events. The cell ﬁrst undergoes nuclear and cytoplasmic condensation with blebbing of the plasma membrane. It subsequently breaks up into membrane-enclosed apoptotic bodies, which are rapidly recognized and engulfed by neighbouring cells or macrophages. Our understanding of the regulation of apoptosis is based on studies of PCD in Caenorhabditis elegans. PCD involves a cell-death protease (Ced-3), which is associated with activating (Ced-4) and inhibitory (Ced-9) proteins. Homologous proteins have been found in organisms throughout the animal kingdom. The homologues of Ced-3 are termed caspases (cysteine-dependent aspartate-speciﬁc proteases), and their overexpression results in death with apoptotic morphology. The proteolytic activity of caspases provides a biochemical basis for the apoptotic phenotype. Structural components are cleaved by caspases, which precedes nuclear condensation and membrane blebbing. Exposure of phosphatidylserine on the cell surface, which is a signal for phagocytosis of apoptotic cells, is blocked by caspase inhibitors. Furthermore, caspases cleave negative regulators of apoptosis and either inactivate them or produce fragments that promote death. Among proteins that regulate cell death is the Bcl-2 family. At present, more than 20 proteins of this family have been identiﬁed. Based on their role in apoptosis they can be divided into two groups: anti- and pro-apoptotic members. Many of these proteins act at the level of the mitochondria, and translocation of pro-apoptotic members from the cytosol to mitochondria is a key initiating event in apoptosis. IGFs are acting as cell survival factors by inhibiting apoptosis and stimulating cell proliferation. Consistently, IGFBPs can modulate antiapoptotic eﬀect of IGFs by regulation of IGF-IGFR interaction. Proteolysis of IGFBPs also modulates their activity and might play a role in regulation of their antiproliferative eﬀects. IGFBP-3 might fulﬁl
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
pro-apoptotic properties via activation of IGFRI-independent pathway and also potentiate the apoptotic eﬀects of diﬀerent DNAdamaging stimuli. It is likely, that IGFBPs-mediated apoptosis implies the intrinsic signalling pathway, which might be inhibited by caspase inhibitors.
S11 USE OF IGFBPs IN CANCER THERAPY Jeﬀ Holly, Claire Perks. Bristol Royal Inﬁrmary, Bristol, UK The important role of the IGF-system in the development and progression of epithelial cancers has led to many diﬀerent strategies aimed at targeting this system. The activity of IGFs is tightly regulated by the IGFBPs, which are expressed in a tissue speciﬁc manner in a way that could confer speciﬁcity on the pluripotent IGFs. The IGFBPs are however not only key determinants of the mitogenic and survival actions of the IGFs but they also have well-established intrinsic actions on cell growth and apoptosis. IGFBP-3 expression by epithelial cells is increased by a number of negative growth regulators including retinoids, TGF-b and p53. We have shown that IGFBP-3 alone does not aﬀect epithelial cell survival but when the cells are stressed it can accentuate apoptosis induced by radiation and chemotherapeutic agents. This can be the result of blocking the survival actions of IGFs, but can also be due to intrinsic actions which we have demonstrated on breast, prostate, colorectal and oesophageal cancer cells. In contrast IGFBP-3 acted as a potent survival factor on non-transformed breast epithelial cells. Furthermore, the enhancing eﬀect of IGFBP-3 on ceramide induced apoptosis of the Hs578T breast cancer cells was reversed when these cells were plated on ﬁbronectin, which induces a more polarized, anchorage-dependent phenotype. We have also demonstrated that the actions of IGFBP-5 on epithelial cells are similarly the opposite on anchorage-dependent compared to anchorage-independent cells and these could again be switched by manipulations of the matrix-integrin interactions. In the case of IGFBP-3, a number of short synthetic peptides have been developed which retain their intrinsic actions on epithelial cells, but which do not interact with IGFs or ALS and may simplify potential problems of in vivo distribution and clearance. The intact molecule may however have additional beneﬁt due to its ability to negate the actions of the IGFs. A number of diﬀerent IGFBP-based strategies are therefore being developed with the exciting potential that some of these may confer speciﬁcity and enhance the kill of cancer cells, but at the same time protect normal anchorage-dependent cells.
S12 THERAPEUTIC INDICATIONS FOR IGF-I/ IGFBP-3 COMPLEX Cecilia Camacho-Hu¨bner. St Bartholomew’s and Royal London School of Medicine and Dentistry, UK
OR1 IGF-I STIMULATES CELL PROLIFERATION AND INDUCES IGFBP-3 AND IGFBP-5 GENE EXPRESSION IN CULTURED GROWTH PLATE CHONDROCYTES BY USE OF DISTINCT SIGNALING PATHWAYS D. Kiepea, S. Ciarmatoria, A. Ho¨ﬂichb, E. Wolfb, O. Mehlsa, B. To¨nshoﬀa. aDivision of Pediatric Nephrology, Univ.-Children’s Hospital, INF 150, 69120 Heidelberg, Germany; bInst. Mol. Animal Breeding and Genetics, Munich, Germany The activity of IGF-I in the growth cartilage may be modulated by the synthesis of stimulatory and/or inhibitory IGFBPs. We therefore studied the eﬀect of IGF-I on (i) cell proliferation and IGFBP production in rat growth plate chondrocytes in primary culture and (ii) the possible involvement of the mitogen-activated protein kinase (MAPK) and the phophatidylinositol 3-kinase (PI 3-kinase) signaling pathways for these diﬀerential eﬀects of IGF-I by use of speciﬁc inhibitors. IGFBP gene expression was analyzed by multiplex RT-PCR and solution hybridization RNase protection assay, IGFBP protein concentration in conditioned medium by [125I]-IGF-II-ligand blot and immunoprecipitation. Under baseline conditions, growth plate chondrocytes expressed mRNA species for IGFBP-2 to -6. In conditioned media, the predominant IGFBP was IGFBP-3 and IGFBP-5, and in lower abundance IGFBP-4 and IGFBP-2. IGF-I-stimulated IGFBP-3 gene expression was maximal after 12–18 h of incubation and increased in a dose-dependent manner (20–100 ng/ml) up to 2-fold. IGFBP-3 protein concentration increased by 200%. IGF-I (60 ng/ml) also stimulated IGFBP-4 mRNA and protein concentration 2-fold. IGF-I-stimulated IGFBP-5 expression was maximal after 6–12 h of incubation. Both IGFBP-5 gene expression and protein concentration increased in a dosedependent manner in response to IGF-I (20–100 ng/ml) up to 2-fold. Coincubation of IGF-I and U126 (2.5 lM), a speciﬁc inhibitor of the MAPK pathway, inhibited IGF-I-stimulated ERK1 and ERK2 phosphorylation (downstream of MAPK kinase) by 80%. U126 completely abolished IGF-I-stimulated cell proliferation and the synthesis of IGFBP-3 on the level of gene expression and protein concentration. In contrast, LY 294002 (25 lM), a speciﬁc inhibitor of the PI 3-kinase signaling pathway, abolished the IGF-I-stimulated cell proliferation and the synthesis of IGFBP-3 and IGFBP-5 both on the level of gene expression and protein concentration. Conclusions: IGF-I modulates its activity in growth plate chondrocytes by the synthesis of both inhibitory (IGFBP-3, IGFBP-4) and stimulatory (IGFBP-5) binding proteins in a feed-back manner. The action of IGF-I on cell proliferation and IGFBP3 gene expression in these cells is mediated both by the MAPK and PI 3kinase signaling pathways, whereas the PI 3-kinase pathway plays a primary role for IGF-I-mediated IGFBP-5 production.
OR2 OXYGEN-DEPENDENT MODULATION OF BIOSYNTHESIS OF IGF BINDING PROTEINS IN PRIMARY CULTURES OF RAT HEPATOCYTES
Raymond L. Hintz. Stanford University, Stanford, CA 94305, USA
Jens-Gerd Scharfa, Thomas Kietzmannb. aMedizinische Klinik und Poliklinik, Abteilung Gastroenterologie und Endokrinologie, Germany; bInstitut fu¨r Biochemie und Molekulare Zellbiologie, Georg-August-Universita¨t, Go¨ttingen, Germany
In the fourteen years since the ﬁrst IGF Binding Protein Workshop in 1989, research in the IGFBP ﬁeld has exploded with exciting ideas, data, and workers. The three IGF Binding Protein Workshops since the ﬁrst one – in Opio, Tu¨bingen and Terrigal – have all served to stimulate and advance the ﬁeld. This 5th International Workshop on IGF Binding Proteins demonstrates that this ﬁeld of research will continue to provide important insights into the biology of growth and growth factors as new people and new ideas continue to enter into the ﬁeld, and are welcomed and nurtured by veteran investigators.
In rat liver, higher levels of IGFBP-1 mRNA are expressed in the perivenous compared with the periportal zone of the liver. Since gradients in oxygen tension (pO2) may contribute to the zonated gene expression, the inﬂuence of arterial and venous pO2 on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA levels (100%) were observed under venous pO2 whereas less than 30% of maximal mRNA levels were observed under arterial pO2. Accordingly, secretion of IGFBP-1 protein was maximal under venous pO2 and reduced under arterial pO2. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D in-
S13 WHERE HAVE WE BEEN AND WHERE ARE WE GOING?
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 dicated a transcriptional regulation. Desferrioxamine with the ability to chelate iron also increased steady state levels of IGFBP-1 mRNA under both venous and arterial pO2. Furthermore, the response of hypoxia appeared to involve radical oxygen species since in hepatocytes treated with H2O2, a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO2, was observed, whereas IGFBP-1 mRNA expression under arterial pO2 was not aﬀected. The response to desferrioxamine and radical oxygen species indicated a possible participation of the hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated an involvement of HIF2a and HIF3a and to a lesser extent of HIF1a for the induction of IGFBP-1 mRNA under venous conditions. These results support the concept that iron, radical oxygen species and the HIF-2 and -3 pathway are crucial for the modulation of IGFBP-1 expression by hypoxia in rat hepatocytes.
OR3 THE ROLE OF IGFBP-5 TERNARY COMPLEXES IN IGF-I BIOAVAILABILITY Sue M. Firth, Fiona K. McDougall, Patric J.D. Delhanty, Robert C. Baxter. Kolling Institute of Medical Research, University of Sydney, RNSH, St Leonards, NSW 2065, Australia IGF-I circulates in three forms: unbound peptide, binary complex with one of the six IGFBPs, and ternary complex with either IGFBP-3 or -5 and ALS. It is thought that the ternary complexes regulate IGF-I bioavailability in circulation. This study examined the role of IGFBP-5 ternary complexes in the regulation of IGF-I bioavailability. Circulating levels of IGFBP-5 in male Sprague-Dawley rats increased from birth to adulthood (10 weeks), returning to pubertal levels by 8 months. Examined by size exclusion chromatography, 58% of IGFBP-5 was ternary complexed in adult rats, compared to 43% in juveniles, suggesting that more IGFBP-5 might be available in juveniles to transport IGFs to the tissues. To examine the role of IGFBP-5 ternary complexes in regulating the accessibility of IGF-I, male Wistar rats were administered with an intravenous bolus injection of recombinant human (rh)IGF-I, rhIGFBP-5, or rhIGF-I + rhIGFBP-5 (binary complex) via a jugular vein cannula, and blood samples were taken at various time points post injection. Coadministration of IGF-I + IGFBP-5 prevented IGF-I induced hypoglycaemia seen with IGF-I alone. The clearance rate of coadministered IGF-I + IGFBP-5 from the circulation was signiﬁcantly slower than when IGF-I (p < 0.05) or IGFBP-5 (p < 0.0001) was administered alone. This was due to the rapid sequestration of IGF-I and IGFBP-5 into ternary complexes, more of which was retained in the circulation compared to the binary form. This suggests that the clearance rate of the ternary complex will have an impact on IGF-I bioavailability. To determine the contribution of ALS to the clearance rate of the ternary complex, we have conducted similar infusion studies of IGFBP-5 binary or ternary complexes or rhALS alone in male GH-deﬁcient spontaneous dwarf rats (SDRs) that have undetectable levels of circulating ALS. ALS was cleared signiﬁcantly faster when administered alone than when administered as IGFBP-5 ternary complexes (p < 0.0001). Surprisingly, there was no signiﬁcant diﬀerence in the clearance proﬁles of IGFBP-5 when administered as binary or ternary complexes, both of which were cleared more slowly than IGFBP-5 alone (p < 0.01). These results suggest that IGF-I is the determining factor in the clearance of both binary and ternary IGFBP-5 complexes.
OR4 THREE-DIMENSIONAL STRUCTURE OF THE C-DOMAIN OF IGFBP-6 IN SOLUTION USING NUCLEAR MAGNETIC RESONANCE (NMR) Leon A. Bacha, Stephen J. Headeya,b, Shenggen Yaob, David W. Keizerb, Nigel J. Parkera, Phillip Kantharidisa, Raymond S. Nortonb. aUniversity of Melbourne, Department of Medicine, Austin and Repatriation Medical Centre, Heidelberg, Vic. 3084, Australia; bWalter and Eliza Hall Institute of Medical Research, Parkville, Vic. 3050, Australia
IGFBPs 1–6 share a three-domain structure. The N- and C-domains are highly conserved and contain internal disulﬁde linkages, and they are joined by a non-conserved linker domain. Both the N- and C-domains contribute to high aﬃnity IGF binding. In addition, binding to glycosaminoglycans and nuclear localization of IGFBPs -3 and -5 are dependent on C-domain residues. To date, the only three-dimensional structure of any IGFBP is conﬁned to a 50-residue IGF-binding region in the N-domain of IGFBP-5. We therefore expressed the C-domain (residues 161–240 based on proIGFBP-6) of IGFBP-6 as a His6-tagged protein in Escherichia coli and puriﬁed it by Ni–NTA chromatography followed by SP-FF Sepharose cation-exchange chromatography (pH 4.5). Its three-dimensional structure was determined using NMR on protein uniformly labelled with 15N and 13C. Assignment of NOEs and structure calculations were performed using the CANDID module of CYANA. The structure reveals the presence of an a-helix-b-strand at the beginning of the C-domain followed by a two-stranded b-sheet linked by less well-deﬁned loops. This fold is similar to that of the type 1 thyroglobulin domain. The C-terminal region (residues 224–240) remains relatively mobile. The structural core of the C-domain consists of Tyr186, Pro188, Tyr196, Gln200, Trp203 and Val205. A putative IGF-binding surface was identiﬁed and is currently being evaluated by coincubation of labelled C-domain with unlabelled IGF-II. Residues homologous with those involved in nuclear localization of IGFBP-3 and -5 form a distinct surface adjacent to and overlapping that involved in glycosaminoglycan binding. Availability of the three-dimensional structure of the C-domain of an IGFBP for the ﬁrst time will accelerate our understanding of the structure-function relationships of IGFBPs.
OR5 DISULFIDE STRUCTURE OF THE PROFORM OF EOSINOPHIL MAJOR BASIC PROTEIN; MECHANISTIC IMPLICATIONS FOR THE PAPP-A/ProMBP COMPLEX FORMATION Simon G. Pedersen, Michael T. Overgaard, Claus Oxvig. Department of Molecular Biology, University of Aarhus, Aarhus, Denmark The proform of eosinophil major basic protein (proMBP) has recently been shown to be a physiological inhibitor of the IGF-dependent IGFBP-4 proteolysis by PAPP-A. Inhibition only occurs when proMBP is covalently complexed to PAPP-A. ProMBP contains 12 cysteine residues, three of which are in the prodomain. The status of the cysteine residues in the mature form of major basic protein (MBP) and in the PAPP-A/proMBP complex has previously been accounted for by our group. In order to elucidate the mechanism of PAPP-A/proMBP complex formation and the inhibitory mechanism of proMBP, we have cloned and expressed recombinant human proMBP in HEK 293 cells. Recombinant proMBP has been puriﬁed and the disulﬁde structure has been partially determined by mass spectrometry and sequence analysis of isolated Cys-containing peptides generated by thermolytic digestion. The initial results show that the two bridges previously found in mature MBP and in proMBP in the PAPP-A/proMBP complex are conserved in free proMBP. In addition, several of the cysteine residues known from MBP to have free sulfhydryl groups, were found to participate in intrachain disulﬁde bridges connecting the mature MBP domain with the prodomain. Knowledge of the status of cysteine residues in proMBP, will determine whether the inhibitory mechanism could be based on ligation of a free sulfhydryl group to the Zn-ion of the active site in PAPP-A. This would be identical to the ‘Cysteine-switch’ mechanism known from the inactive zymogens of several other Metzincin metalloproteinases. In addition, the determination of the disulﬁde pattern of proMBP will have important implications for the understanding of the mechanism underlying the PAPP-A/proMBP complex formation.
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
OR6 CHARACTERIZATION OF AN IGF-II-BINDING PROTEIN COMPLEX WITH PROTEOLYTIC ACTIVITY ISOLATED FROM HUMAN PLASMA Sandra Oesterreichera, Werner F. Blumb, Thomas Braulkea, Bernd Ku¨blera. aChildren’s Hospital-Biochemistry, University of Hamburg, Hamburg, Germany; bEli Lilly and Company, Bad Homburg, and University Children’s Hospital, Leipzig, Germany To identify new proteins interacting in vivo with IGF-II, Cohn fraction IV of human plasma was subjected to IGF-II aﬃnity chromatography. Bound proteins were eluted at acidic pH followed by further puriﬁcation by C18 reversed phase-HPLC. In addition to a fraction containing mainly IGFBP-3, several other protein-containing fractions were eluted with increasing acetonitrile concentration. The majority of IGFBP-3 and a 30-kDa IGF-binding protein was eluted in fraction 46. Two other polypeptides with apparent molecular masses of 73- and 100-kDa were eluted in fraction 50–57. Fingerprint analysis revealed that the 73-kDa protein is transferrin which was conﬁrmed by Western blotting. The 100-kDa protein could not be identiﬁed yet and failed to bind IGF-II. However, in vitro IGFBP-protease assays showed that fractions containing the 100-kDa protein are able to cleave [125I]IGFBP-2, -3, and -4. 125I-substrate zymographies showed proteolytic active bands at 100-kDa. Protease inhibitor studies indicate that the proteolytic activity against [125I]IGFBP-2, -3 and -4 can be completely inhibited by aprotinin whereas EDTA, 1,10-phenantroline and TAPI (TNFa-activating protein inhibitor) had no eﬀect on the degradation of the IGFBPs suggesting that the 100-kDa IGFBP-protease belongs to the family of serine proteases. Furthermore, the cleaving pattern of [125I]IGFBP-4 catalyzed by the 100-kDa protease diﬀers compared to that in pregnancy serum. These data indicate that in serum: (i) there are IGFBP-serine proteases in addition to matrixins and disintegrin-metalloproteases and (ii) that serum proteins can form heteromultimer complexes interacting with IGF binding proteins and/or IGF-II.
OR7 NOVEL IGF:IGFBP COMPLEXES FOR APPLICATIONS IN TISSUE REPAIR AND REGENERATION Z. Upton, D. Harkin, C. Hyde, C. Towne, B. Hollier, P. Gillies, A. Noble, J. Kricker, C. Wilson, S. Richards, R. Dawson, D. Leavesley, A. Herington. Tissue BioRegeneration and Integration Program, Science Research Centre, Queensland University of Technology, GPO Box 2434, Brisbane, Q4001, Australia. Fax: +61 7 3864 1534, E-mail: [email protected]
We recently discovered novel links between IGFs, IGFBPs and vitronectin (VN) and have reported that IGF-II can bind directly to VN, while IGF-I binds indirectly via IGFBP-2, -3, -4 and -5. Moreover, our functional studies have revealed that these complexes signiﬁcantly enhance cell migration and proliferation in a range of cells including Chinese Hamster Ovary and human osteoblast-like cells, as well as in skin and corneal epithelial cell lines. Importantly, studies in cultures of keratinocytes derived from adult skin and cornea have validated these ﬁndings. Furthermore, assays using IGF analogues with reduced afﬁnity for the IGF-IR (but which retain binding to IGFBPs and VN) and a function-blocking antibody against the VN-binding av integrins, indicate that these complexes enhance proliferation and migration through coordinate activation of both the IGF-IR and VN-binding integrins. Recognising the key importance of this, and realising that this phenomena could be exploited as a biological system to deliver IGFs and IGFBPs in situations where cell proliferation and migration
are wanted, led us to commence studies examining the potential of the complexes in a number of situations including wound healing and ex vivo expansion of progenitor cells for tissue regeneration. Indeed we have recently examined the potential of the complexes to replace serum and feeder cells used in current best clinical practice for ex vivo expansion of keratinocytes for split thickness autografting. These studies revealed that cells seeded at low density on complex-coated dishes were not only able to survive, but also, to expand more rapidly than those grown using current clinical protocols (i.e., seeded at the same density and grown in the presence of serum and growth-arrested feeder cells). The use of this technology for ex vivo keratinocyte expansion is currently being further developed with the Australian Red Cross to provide a safe, animal-product free, autologous cell-based therapy for burn patients. Moreover we anticipate that a similar approach will also be useful to developing safe cell-based therapies for diabetic and venous ulcers, or other cell-based applications that use similar culture technologies, including embryonic stem cells. Indeed our recent preliminary studies with these cells strongly suggest that this will be the case.
OR8 INTERPLAY OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 AND TGFb IN MESENCHYMAL CHONDROPROGENITOR STEM CELLS Anna Spagnolia, Lynda O’Reara, Monica Torellob, Lara Longobardia, Harold L. Mosesc. aDepartment of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, USA; bResearch Center, Shriners Hospital for Children, Portland, OR, USA; cDepartment of Cancer Biology and Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Introduction Cartilage is essential for the skeletal growth and the functional integrity of the skeleton. Mesenchymal chondroprogenitor stem cells (MCC) condensation, proliferation and diﬀerentiation are critical sequential steps required for the cartilage formation. There is compelling evidence that MCC can be used for cellular and gene therapy in cartilage repair. However, little is known about the molecular mechanisms by which growth factors regulate the MCC fate. We have previously reported that: IGFBP-3 has IGF-independent antiproliferative and apoptotic eﬀects in MCC; IGFBP-3 apoptotic eﬀect in MCC requires STAT-1 expression and activation; IGFBP-3 modulates the chondrocytic diﬀerentiation rate by regulating MCC growth. TGFb is a key chondroinductive growth factor. In several cell lines, IGFBP-3 has been demonstrated to mediate the TGFb eﬀects or to enhance TGFb signaling. Aim The aim of the study is to deﬁne the IGFBP-3 and TGFb interface in MCC. Results We found that in MCC,TGFb antagonized the IGF-independent antiproliferative eﬀect of IGFBP-3; the EC50 for the antiproliferative eﬀect of IGFBP-3 increase from 2.2 to 3.8 nM in the presence of TGFb (p < 0.05). IGFBP-3 induced a dramatic increase of the CDK inhibitor p21 (more than 300% of control) protein expression. This eﬀect was clearly IGF-independent, in fact the IGFBP-3 analog with abolished aﬃnity for IGFs, GGG-IGFBP-3, had similar eﬀects. The IGFBP-3 eﬀect on p21 was totally abolished by TGFb. Furthermore, TGFb inhibited STAT-1 phosphorylation induced by IGFBP-3 and GGG-IGFBP-3. Similarly to TGFb, STAT-1 morpholino oligo-antisense inhibited the STAT-1 phoshorylation and the antiproliferative eﬀect induced by IGFBP-3 and GGG-IGFBP-3. We were also able to demonstrate that IGFBP-3 had IGF-independent antagonistic eﬀect on TGFb transcriptional activity and signaling. Using dual-luciferase reporter
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 assays, we determined that IGFBP-3 and GGG-IGFBP-3 inhibited SMAD-mediated TGF-induced transcriptional activity, respectively, by 44 ± 11% of TGFb activity (p < 0.01) and by 54 ± 10% (p < 0.01). Furthermore, IGFBP-3 and GGG-IGFBP-3 determined a decrease of SMAD2 phosphorylation induced by TGFb, respectively, by 30% and 50%. Conclusions We have determined the IGFBP-3 and TGFb interface in MCC. Diﬀerently from other cancer cell systems, IGFBP-3 has antagonistic eﬀects on TGFb signaling and transcriptional activity. Furthermore in MCC, TGFb antagonizes the STAT-1-p21 mediated antiproliferative eﬀects of IGFBP-3.
OR9 INTERACTION OF IGFBP-3 WITH AUTOCRINE MOTILITY FACTOR ON THE CELL SURFACE OF T-47D BREAST CANCER CELLS S. Mishraa, A. Razb, L.J. Murphya. aDepartments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, Man., Canada; b Karmanos Cancer Institute, Wayne State University, School of Medicine Detroit, MI, USA Cross-linking of biotinylated IGFBP-3 to intact T-47D cells identiﬁed complexes of 100 and 75 kDa. The IGFBP-3 binding partners in solubilized T-47D cell membranes were puriﬁed by gel ﬁltration and ion-exchange chromatography followed by aﬃnity chromatography. An IGFBP-3–Sepharose 4B column retained a 50 kDa protein which, when analyzed by tandem mass spectroscopy was identiﬁed as autocrine motility factor (AMF). Identiﬁcation was conﬁrmed by immunoblot using AMF antiserum. AMF is a multifunctional membrane-associated cytokine which is also known as phosphoglucoisomerase (PGI), maturation factor (MF), and neuroleukin (NLK). Increased expression of AMF/PGI and its receptor has been found in wide spectrum of malignancies, and is associated with cancer progression and metastasis. To verify the interaction between AMF/PGI and IGFBP-3, biotinylated IGFBP-3 was cross-linked to breast cancer cells and precipitated with streptavidin–agarose conjugate and analyzed by SDS–PAGE. The same 75 kDa complex was identiﬁed when the nitrocellulose membranes were probed either with streptavidin-HRP conjugate or AMF antiserum conﬁrming the cross-linking of IGFBP-3 to AMF/PGI. Interaction between IGFBP-3 and AMF were further conﬁrmed by ligand blotting using biotinylated IGFBP-3. Phosphorylation of AMF have been reported to be associated with its translocation to the membrane and secretion, therefore we examined the eﬀect of IGFBP-3 on AMF/PGI phosphorylation in cell lysate and in conditioned media using [32P]orthophosphate. IGFBP-3 signiﬁcantly inhibited AMF phosphorylation in cell lysate and subsequently its secretion from cells. In summary we have identiﬁed AMF/PGI as a membrane associated binding partner for IGFBP-3 in breast cancer cells. Further studies are underway to determine the role of AMF/ PGI in the IGF-independent eﬀects of IGFBP-3
OR10 IGFBP-3-INDUCED APOPTOSIS IN PC-3 HUMAN PROSTATE CANCER CELLS IS INITIATED BY FADD-DEPENDENT ACTIVATION OF CASPASE 8 Jiang Hong, Matthew M. Rechler. National Institutes of Health, Bethesda, MD 20892, USA We recently reported that a hIGFBP-3 mutant that does not bind IGFI or IGF-II, 6m-hIGFBP-3, induces apoptosis by IGF-independent mechanisms in the PC-3 human prostate cancer cell line as eﬀectively as wild-type IGFBP-3 (J. Biol. Chem. 277 (2002) 10489). The present
study examines the pathway by which apoptosis is induced by the IGFBP-3 mutant. The characteristic changes of apoptosis result from the proteolysis of key substrates by eﬀector caspases such as caspase 3 which are activated from inactive precursors by one of two initiator caspase pathways: caspase 8 (typically activated by death receptors via an adapter protein, FADD) or caspase 9 (activated following the release of cytochrome c from mitochondria). Induction of PC-3 cell death (measured by trypan blue uptake) by puriﬁed 6m-hIGFBP-3 was abolished by coincubation with speciﬁc inhibitors of either caspase 8 (z-IETD-fmk) or caspase 9 (z-LEHD-fmk), suggesting that both initiator caspase pathways are involved. The same conclusion was reached when 6m-hIGFBP-3-induced apoptosis was examined in stably transfected PC-3 cells that overexpressed dominant negative FADD (FADD-DN) to selectively inhibit caspase 8, or Bcl-2, an antiapoptotic protein that stabilizes mitochondria and prevents the release of cytochrome c and activation of caspase 9. Apoptosis induced by 6mhIGFBP-3 was decreased signiﬁcantly in both PC-3-FADD-DN and PC-3-Bcl-2 cells. Participation of both initiator caspase pathways can be explained by cross-talk between the two pathways: caspase 8 can activate caspase 9 (by proteolysis of BID to generate a protein fragment that translocates to mitochondria and induces cytochrome c release), and caspase 9 (directly or indirectly) can activate caspase 8. To determine whether 6m-hIGFBP-3-induced apoptosis was triggered by activation of caspase 8, we measured caspase 8 activity in a colorimetric assay using a speciﬁc chromogenic tetrapeptide substrate. Peak activity was seen 1 h after addition of 6m-hIGFBP-3. Caspase 8 induction was abolished in PC-3 cells overexpressing FADD-DN, but not in PC-3 cells overexpressing Bcl-2. We conclude that 6m-hIGFBP3 induces apoptosis by triggering FADD-dependent activation of caspase 8. Ampliﬁcation of the caspase 8 signal by activation of the mitochondrial-caspase 9 pathway, however, is required to activate eﬀector caspases and induce apoptosis.
OR11 THE ALZHEIMER’S SURVIVAL PEPTIDE HUMANIN BINDS IGFBP-3 AND ANTAGONIZES ITS PRO-APOPTOTIC FUNCTIONS Maaria Ikonena, Bingrong Liua, Yuichi Hashimotob, Liqun Maa, Kuk-Wha Leea, Ikuo Nishimotob, Pinchas Cohena. aDepartment of Pediatrics, Mattel Children’s Hospital, Division of Endocrinology, David Geﬀen School of Medicine, University of California, Los Angeles, CA, USA; bDepartment of Pharmacology and Neurosciences, KEIO University School of Medicine, Tokyo, Japan Increasing evidence points to multi-mechanistic role of IGFBP-3 in the regulation of cellular survival. To further decipher IGFBP-3 actions, we used yeast two-hybrid screening to identify IGFBP-3interacting proteins. As a result, we cloned Humanin, HN, a novel anti-apoptotic peptide, as an IGFBP-3 binding partner. HN is a 24 amino acid peptide that has been shown to speciﬁcally inhibit neuronal cell death induced by familial Alzheimer’s disease (FAD) mutant genes and amyloid-b in vitro. Multiple colonies expressing the HN cDNA showed potent GAL4 activation together with IGFBP-3. We also showed that (His)-6-tagged HN could pull-down IGFBP-3 in a HN-displaceable manner. Using ligand blot experiments, we found that HN bound IGFBP-3 with high aﬃnity and speciﬁcity. The binding of IGFBP-3 to HN did not inhibit the binding of IGF-I to IGFBP-3, suggesting the possibility of ternary complex formation of IGFBP-3, HN and IGF-I. This also indicates that it is likely that the interaction between HN and IGFBP-3 involves the mid-region of the IGFBP-3 molecule. Furthermore, HN potently, at low nanomolar concentrations, inhibited IGFBP-3-induced apoptosis in human glioblastoma (A172) cells. However, in SH-SY5Y neuroblastoma cells HN had only a minimal eﬀect on IGFBP-3-induced apoptosis. In all cell lines used, HN had no caspase-inhibiting eﬀects on its own, unlike IGF-I and TPA that independently promoted cell survival in
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
the absence of added IGFBP-3. Of note is that the A172 and SHSY5Y cell lines exhibit a diﬀerent time course in the responses to IGFBP-3 induced caspase activation. Whereas A172 cells respond to IGFBP-3 early, SH-SY5Y cells have a slower response, indicating diﬀerences in IGFBP-3 signaling mechanisms in these models, with the early response to IGFBP-3 being HN-inhibited, while the late response being HN-independent. Interestingly, although HN protects from IGFBP-3-induced apoptosis, it does not inhibit transport of IGFBP-3 into the nucleus. In conclusion, we identiﬁed a novel, highaﬃnity interaction between the survival peptide, Humanin, and the pro-apoptotic IGFBP-3, which may have important implications to disease processes and could provide an important target for drug development.
OR12 ENHANCEMENT OF TUMOUR NECROSIS FACTOR-MEDIATED GROWTH INHIBITION BY IGFBP-5 BUT NOT IGFBP-3 IN HUMAN BREAST CANCER CELLS Alison J. Butt, Kristie A. Dickson, Robert C. Baxter. Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia IGFBP-3 and IGFBP-5 modulate the mitogenic and survival eﬀects of IGFs by regulating their interactions with the IGF type I receptor. However, both IGFBPs have IGF-independent antiproliferative and proapoptotic eﬀects on cancer cell growth, with some evidence that these eﬀects are mediated intracellularly. Two distinct apoptotic pathways have been elucidated thus far – the ‘extrinsic’ pathway involving the extracellular binding of death ligands such as tumour necrosis factor (TNF) to the TNF receptor leading to the activation of caspase-8, and the ‘intrinsic’ pathway modulated by Bcl-2 proteins and involving the release of apoptotic factors from the mitochondria. We have previously demonstrated the involvement of Bcl-2 proteins in IGFBP-3 and -5mediated apoptosis. This study aims to further elucidate the apoptotic pathways utilised by these binding proteins in human breast cancer cells. MDA-MB-231 human breast cancer cells are resistant to the inhibitory eﬀects of TNF. We examined the eﬀects of stable IGFBP expression on the response of these cells to TNF. Cells stably expressing human IGFBP-3, IGFBP-5 cDNA or vector controls, were grown in the presence or absence of 10 ng/ml TNF, and counted on days 3, 5 and 7 posttreatment. While IGFBP-3 and IGFBP-5 expression was inhibitory to the growth of MDA cells, IGFBP-5 further sensitised MDA cells to the inhibitory eﬀects of TNF (p < 0.003 by repeated measures ANOVA), whereas IGFBP-3 expression had no such eﬀect. TNF (10 ng/ml) also signiﬁcantly inhibited DNA synthesis in MDA/BP-5 cells but not vector controls (p < 0.0001), an eﬀect that was not seen in IGFBP-3-expressing cells. Preliminary data have demonstrated that apoptosis induced following adenoviral-mediated expression of IGFBP-5 but not IGFBP-3, results in a rapid activation of caspase-8 and is inhibited by the caspase8-speciﬁc inhibitor, z-IETD-fmk (80 lM). These results suggest that IGFBP-5 may elicit an ‘extrinsic’ apoptotic pathway in human breast cancer cells, that may be further ampliﬁed at the level of the mitochondria. Currently, we are examining potential direct interactions between IGFBPs and the intracellular apoptotic machinery.
OR13 DISRUPTION OF CHOLESTEROL STABILISED MEMBRANE COMPLEXES MODULATES THE ACTIONS OF IGFBP-3 ON APOPTOSIS IN HS578T HUMAN BREAST CANCER CELLS C.M. Perks, C. Burrows, C. McCaig, J.M.P. Holly. Division of Surgery, Level 7, Bristol Royal Inﬁrmary, Bristol BS2 8HW, UK
Introduction In response to radiotherapy or chemotherapeutic agents, we have shown that IGFBP-3 directly enhances apoptosis of Hs578T cells, but when these cells are plated onto ﬁbronectin IGFBP-3 then acts in an opposite manner and acts as a potent survival factor. The mechanism by which IGFBP-3 elicits these matrix-modulated IGF-independent actions is still largely unknown. Aims Since integrin receptor signalling involves lipid rafts, we determined if raft disruption using the cholesterol chelating agents ﬁlipin or nystatin, aﬀected these intrinsic actions of IGFBP-3. Having demonstrated previously that the b1 integrin sub-unit is most abundant in the Hs578T cells, we further investigated if the accentuating actions of IGFBP-3 were also modulated by a b1 integrin receptor blocking antibody (anti-b1). Methods Cells were treated with IGFBP-3 (100 ng/ml) ± C2-ceramide (10 lM), ±ﬁlipin (5 lg/ml), or ±nystatin (50 lg/ml), or ±anti-b1 (200 ng/ml). Cell death was measured by trypan blue cell counting and ﬂow cytometry. Results The induction of apoptosis by ceramide was not aﬀected by either of the cholesterol chelators and IGFBP-3 alone had no eﬀect under any conditions. Ceramide increased cell death from 11.9% to 26.2% and this was accentuated to 37.1% by IGFBP-3. The accentuation of cell death by IGFBP-3 was completely blocked by ﬁlipin and nystatin, but reversed in the presence of anti-b1, such that IGFBP-3 then conferred signiﬁcant (p < 0.05) cell survival. Conclusion These data suggest that modulation of the b1 integrin switches the ability of IGFBP-3 from accentuating apoptosis to acting as a potent survival factor. These results further demonstrate that intact cholesterol-stabilised membrane complexes, which optimise signalling for a number of diﬀerent receptors, including integrin receptors, are critical for IGFBP-3 to exert its IGF-independent accentuating actions on apoptosis. Acknowledgements Funding from AICR and The Needham Cooper Trust.
OR14 IGFBP-3 LEVELS ARE INCREASED IN EARLY-STAGE MYELODYSPLASTIC SYNDROME AND MAY CONTRIBUTE TO DISEASE PATHOPHYSIOLOGY BY ALTERING APOPTOTIC AND PROLIFERATIVE RESPONSES IN HEMOPOIETIC CELLS H.-M.P. Wilson, V. Lesnikov, J. Ward, H.J. Deeg. Department of Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA The pathophysiology of myelodysplastic syndrome (MDS) is incompletely understood. Tumor necrosis factor (TNF) a, TNF-related apoptosis-inducing ligand (TRAIL), and Fas-L are dysregulated in patients with MDS. These cytokines and their receptors inﬂuence expression of various components of the insulin-like growth factor (IGF) system, including IGF-binding protein-3 (IGFBP-3), which is required for TNFa-induced apoptosis. IGFBP-3 has dual functions that are tissue/cell speciﬁc and are inﬂuenced by exposure to cytokines. IGFBP-3 enhances proliferation and protects cells from apoptotic stimuli. However, IGFBP-3 also exerts potent antiproliferative eﬀects against the potentially transforming properties of growth hormone and IGF-I. We hypothesize that altered IGFBP-3 levels contribute to the dysregulation of hemopoiesis by aﬀecting proliferation and apoptosis. Addition of IGFBP-3 reduces the number of apoptotic cells by 20% (p = 0.05 for all comparisons, Wilcoxon test) in myeloid leukemic and primary marrow cells treated with
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 TNFa-, TRAIL-, or agonistic anti-Fas antibody (aFas); TNFa alters IGFBP-3 protein secretion in a cell-speciﬁc manner (upregulates in ML-1 myeloid leukemic cells, downregulates in HS-5 stroma cells); and IGFBP-3 modulates hemopoiesis in vitro: while glycosylated IGFBP-3 increases, non-glycosylated IGFBP-3 decreases burst forming units-erythroid and granulocytic/monocytic colony formation. We show that IGFBP-3 protein levels are signiﬁcantly higher in marrow plasma from MDS patients with refractory anemia (RA), who also show higher levels of TNFa, compared to normal donors (p = 0.04). Furthermore, Westerns analysis, using marrow plasma from normal and MDS patients to detect diﬀerences in IGFBP-3 isoform expression, suggest that marrow plasma from patients with advanced MDS contained less glycosylated IGFBP-3 protein. Therefore, we hypothesize that TNFa-induced changes in IGFBP-3 expression contribute to dysregulation of cell proliferation and death associated with MDS. Understanding the action of IGFBP-3 in hemopoiesis may allow for development of novel therapeutic strategies for treating patients with MDS.
OR15 UPREGULATION OF INSULIN LIKE GROWTH FACTOR BINDING PROTEINS-1 AND -6 IN OLIGODENDROCYTES IN CHRONIC MULTIPLE SCLEROSIS LESIONS Nadine Wilczak, Nicole Ku¨hl, Daniel Chesik, Jacques de Keyser. Department of Neurology, Academisch Ziekenhuis Groningen (AZG), The Netherlands Multiple sclerosis (MS) is a disease of the central nervous system in which myelin; oligodendrocytes and axons are destroyed. As the disease progresses most lesions fail to remyelinate. Insulin-like growth factor-I (IGF-I) plays a pivotal role in oligodendrocyte development, survival, and myelin synthesis. We investigated IGF-I receptors and IGF binding proteins (IGFBPs) in oligodendrocytes at the edges of chronic lesions of MS. Sections of cerebral white matter containing chronic plaques from 11 MS cases and cerebral white matter from 12 controls without neurologic disease were studied by immunohistochemistry and confocal microscopy. Oligodendrocytes in normal white matter stained for IGFBPs-1, -2, -3, -4 and -5. Oligodendrocytes at the margins of the MS plaques displayed enhanced IGFBP-1 immunoreactivity and were also IGFBP-6 positive, while immunoreactivity for IGFBPs-2, -3, -4, and -5 was similar to that detected in oligodendrocytes of normal white matter. Experiments in cultures of rat oligodendrocyte progenitor cells showed that both IGFBP-1 and IGFBP-6 reduced IGF-I mediated cell survival and formation of the myelin protein CNPase. Consistent with the observation that these IGFBPs reduce the activity of IGFI, we found that IGF-I receptors on oligodendrocytes at the margins of MS plaques were upregulated. Our results suggest that an upregulation of IGFBPs-1 and -6 in oligodendrocytes may contribute to the loss of oligodendrocytes and failure of remyelination in MS lesions.
OR16 IGFBP-3 LOCALISES TO AND MODULATES PROLIFERATIVE EPIDERMAL KERATINOCYTES IN VIVO Stephanie Edmondsona, Susan Thumigera, Brian Loha, Rachel Koelmeyerb, Amy Lib, Josef Silhac, Liam Murphyc, George Werthera, Christopher Wraighta, Pritinder Kaurb. aCutaneous Biology Group, Dermal Therapeutics, Centre for Hormone Research, Murdoch Childrens Research Institute, Parkville 3052, Vic., Australia; bPeterMacCallum Cancer Institute, East Melbourne 3002, Vic., Australia; cDepartment of Medicine and Physiology, University of Manitoba, Winnipeg, Man., Canada R3E 3P4
The epidermis undergoes a continuous choreographed process of keratinocyte growth and terminal diﬀerentiation (death). Beginning in the basal layer, keratinocyte stem cells (KSC) become transit amplifying (TA) and then post-mitotic diﬀerentiating (PMD) cells that metamorphose through the upper epidermal layers. IGF-IR activation is essential for epidermal homeostasis. IGFBP-3 expression is limited to selected basal keratinocytes, grossly altered in hyperplastic epidermis and reduced on diﬀerentiation. We aimed to: (i) clarify the basal keratinocyte sub-type expression of IGFBP-3 mRNA and (ii) clarify the functional role of epidermal IGFBP-3. Human basal keratinocytes were FACS fractionated on the basis of a6 integrin (a6) and transferrin receptor (CD71) abundance (KSC: a6bright/CD71dim; TA: a6bright /CD71bright; PMD: a6dim). Real timePCR revealed that IGFBP-3 mRNA abundance was signiﬁcantly higher in TA cells when compared with KSCs (6 ± 2.1 SE) and PMD (1.7 ± 1.2 SE). In contrast, IGF-IR mRNA levels were similar in all keratinocyte basal sub-types. To further elucidate the role of IGFBP-3 in keratinocyte proliferation, we examined the eﬀect of IGFBP-3 over-expression in a transgenic mouse model. When compared with wild-type, back, belly and tail skin of transgenic mice (CMV-humanIGFBP-3) exhibited: (i) overexpression of human IGFBP-3 cDNA and protein in epidermal keratinocytes without grossly altered morphology and (ii) reduced proliferative (Ki67 positive) keratinocytes (mean normal: transgenic; 8.2 ± 1.2 SE : 1.5 ± 0.3 SE). In conclusion, these data conﬁrm that in skin: (i) IGFBP-3 mRNA is primarily expressed by proliferative keratinocytes (TA) and (ii) IGFBP-3 over-expression results in the inhibition of epidermal keratinocyte proliferation. These data provide the ﬁrst functional evidence for epidermal IGFBP-3 and strongly support a role for IGFBP-3 in modulating keratinocyte proliferation in vivo.
P1 IGFBP-1 RESPONSE TO NUTRITIONAL DEPRIVATION IS IMPAIRED IN TYPE 2 DIABETES MELLITUS Kerstin Brismar, Kerstin Hall, Moira S. Lewitt Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Background and aim IGFBP-1 is transcriptionally inhibited by insulin. When hepatic insulin sensitivity is normal, there is a close inverse relationship between circulating levels of IGFBP-1 and insulin, and a characteristic diurnal variation in relation to food intake. In type 2 diabetes, however, the relationship between IGFBP-1 and insulin may be lost, with a relative increase in IGFBP-1 concentrations The aim of this study was to study the diurnal pattern of IGFBP-1 in diabetes mellitus and normal subjects and the eﬀect of caloric restriction. IGF and IGFBP-3 responses in this study have previously been reported (Bang et al., JCEM 78 960). Materials and methods After a baseline assessment period, food intake was reduced to 150 kcal/day, consumed as fruit or vegetable juice at mealtimes, for 72 h, and increased again to baseline (refeeding). Subjects with type 2 diabetes (n = 6) were compared with two control groups, both with normal fasting glucose and insulin concentrations. One control group had a normal BMI (n = 6), while the second (n = 6) was matched for obesity with the type 2 diabetic individuals. Results During a 24-h baseline assessment, mean IGFBP-1 concentrations were similar in all three groups, however the obese controls had reduced diurnal variation, and lower 6 am values. The IGFBP-1 concentrations were relatively increased, in relation to insulin levels, in the diabetic group. During nutritional deprivation, insulin concentrations fell by 50% in all 3 groups and the diurnal variation and the 6 am IGFBP-1 concentrations increased in the lean and obese control groups, but were unchanged in the diabetic subjects. With refeeding after 72-h caloric restriction, 6 am IGFBP-1 concentrations and its
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
diurnal variation were suppressed in patients with type 2 diabetes, to a similar extent to the control groups. Conclusions Nutritional deprivation is normally associated with enhanced ﬂuctuations in IGFBP-1 in response to fasting and the consumption of small quantities of carbohydrate. Patients with type 2 diabetes mellitus have altered regulation of IGFBP-1 that may relate to impaired hepatic insulin sensitivity, which is improved by caloric restriction.
P2 FURTHER EVIDENCE FOR INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-1 (IGFBP-1) AS A BIOMARKER OF HEPATIC INSULIN SENSITIVITY A.H. Healda, J.K. Cruickshankb, A. Vyasb, J. Patelb, K. Siddalsa, A. Rudenskia, S. Andersonb, J.M. Gibsona. aDepartment of Diabetes and Endocrinology, University of Manchester, Salford Royal Hospitals University Trust, Salford M6 8HD, UK; bClinical Epidemiology Group, University of Manchester Medical School M13 9PT, UK Objective Impaired hepatic insulin sensitivity is increasingly being recognized as integral to the pathophysiology of insulin resistance and type 2 diabetes. We have previously proposed insulin-like growth factor binding protein-1 (IGFBP-1) is a marker for hepatic insulin sensitivity. In this study we examined whether lifestyle change that accompanies population migration from North-West India to the UK, with increase in cardiovascular risk and diabetes prevalence, is mirrored by changes in insulin sensitivity and IGFBP-1 levels. Design In a population-based sample, we compared a speciﬁc Gujarati community in Sandwell UK (n = 161) with people still resident in the same villages of origin in India (n = 228). Mean age of both groups was 49 years. Fasting plasma glucose, insulin, IGF-I, IGFBP-1 and IGFBP-3 were measured. HOMA-S (insulin sensitivity) was calculated from fasting insulin and glucose concentrations. This was done using an iterative computer program which found the values for HOMA-S and HOMA-B at which the non-linear model for glucose and insulin homeostasis was at equilibrium in steady state for the observed fasting insulin and glucose values. Results Body mass index and waist–hip ratio were elevated in both men and women in UK Gujaratis compared with Indian Gujaratis. Systolic and diastolic blood pressure were higher in both men and women from the UK group. HOMA-S as a measure of insulin sensitivity was lower in UK Gujaratis (men 0.46 95% CI 0.38–0.54; women 0.51 (0.43–0.60)) than Indian Gujaratis (men 0.73 (0.65–0.81); women 0.63 (0.55–0.71) p < 0.001. The decrease in hepatically synthesised IGFBP-1 for a given increase in its principal negative regulator insulin, was signiﬁcantly less in UK Gujaratis (ln IGFBP-1 = )0.40 · ln insulin + 4.1) than Indian Gujaratis (ln IGFBP-1 = )0.54 · ln insulin + 4.9) suggesting greater hepatic insulin resistance in the UK group. Conclusion The diﬀerences seen in the relationship between insulin and IGFBP-1 levels associated with migration are in keeping with lower hepatic insulin sensitivity in the UK Gujarati group, who also have lower circulating IGFBP-1 levels. IGFBP-1 thus appears to be a valid marker for hepatic insulin sensitivity.
P3 A RANDOMISED CONTROLLED INTERVENTION STUDY TO EXAMINE DIRECT MODULATION OF THE IGF-SYSTEM BY DIET A.H. Healda, C. Goldingb, R. Sharmaa, K. Siddalsa, S. Andersona, J.E. Cadeb, J.M. Gibsona. aDepartment of Diabetes and Endocrinology,
University of Manchester, Salford Royal Hospitals University Trust, Salford M6 8HD, UK; bUniversity of Leeds Nutrition Epidemiology Group, Nuﬃeld Institute for Health, 71-75 Clarendon Road, Leeds LS2 9PL, UK Introduction There has recently been great interest in links between the insulin-like growth factor (IGF)-system and cancer. Increased cancer risk correlates with raised concentrations of IGF-I and lower concentrations of IGF binding proteins (IGFBPs), such as IGFBP-3, that are thought to modulate the bioavailability of IGFs. The IGF-system has also been implicated in the pathogenesis of diabetes and cardiovascular disease. Cross-sectional studies have shown associations between total energy and macronutrient and circulating IGF/IGFBP levels. In this study we have examined the eﬀect of direct dietary intervention on the IGFsystem. Method 80 volunteers were recruited from the University of Leeds campus. Potential subjects (high fat consumers) were identiﬁed using the DINE questionnaire by telephone. None of the subjects were taking any drugs known to aﬀect appetite or had a history of eating disorders. Each subject was randomly allocated into one of the following groups for 3 months. Substitution: Substitution of high fat foods was made with reduced fat products; Omission: Omission of high fat foods; Combination of substitution and omission strategies; Control group, where no advice was given. The eﬀects of the diﬀerent interventions were compared after 3 months. Fasting blood samples were taken at baseline and follow-up and analysed for glucose, insulin, IGF-I, IGFBP-1 and IGFBP-3. Insulin sensitivity was calculated using the HOMA model. Results Substitution of dietary saturated fat with reduced fat products resulted in weight loss (mean ± standard error of mean )1.4 ± 0.6%, p = 0.06) whereas there was no signiﬁcant weight change with other interventions (omission )0.5 ± 0.5%; combination )0.2 ± 0.8%, control )0.2 ± 0.3%). Circulating IGF-I increased with both the substitution protocol (baseline 184 ± 14 ng/ml; follow-up 215 ± 20 ng/ml) and omission protocol (baseline 167 ± 18ng/ml; follow-up 186 ± 16 ng/ ml), p = 0.02 but there was no diﬀerence in IGF-I for the combination protocol (baseline 167 ±16 ng/ml; follow-up 166 ±16 ng/ml). There was no change in circulating IGFBP-1, insulin concentrations or insulin sensitivity (HOMA-S) with any intervention. Fasting cholesterol and triglycerides concentration fell with the substitution of saturated fat but not with other interventions. The diﬀerence in cholesterol (p = 0.05) and triglycerides (p = 0.01) between baseline and follow-up was signiﬁcantly greater for substitution compared with other interventions. Conclusion The increase in circulating IGF-I with both the substitution and omission protocols is further evidence for a direct role of dietary fat intake in modulating IGF-I synthesis. The increased level of circulating IGF-I may have beneﬁcial eﬀects on glucose and fatty acid utilisation in peripheral tissues.
P4 FOREARM SKIN MICROVASCULAR REACTIVITY, IGF-I, AND IGFBP-1 IN HEALTHY SUBJECTS WITH AND WITHOUT HEREDITY FOR TYPE 2 DIABETES ¨ stenssonb, G. Jo¨rneskoga, J. Kuhlb, M. Kalanic, S. Efendicb, C.-G. O b a K. Brismar . Internal Medicine, Danderyds Hospital, Stockholm, Sweden; bEndocrinology and Diabetology, Karolinska Hospital, Stockholm, Sweden; cCardiology, Karolinska Hospital, Stockholm, Sweden
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 Background and aims An impaired skin microvascular reactivity has been demonstrated in healthy subjects with heredity for type 2 diabetes, which might be an early marker for vascular disease. Low levels of insulin-like growth factor binding protein-1 (IGFBP-1) have been related to decreased insulin sensitivity and cardiovascular disease. The aim of the present study was to investigate associations between skin microvascular reactivity, IGF-1, and IGFBP-1 in healthy subjects with and without heredity for type 2 diabetes. Materials and methods Thirty-seven male subjects, free of medication, non-smokers, and with normal oral glucose tolerance tests were investigated. Twenty-one of the subjects had heredity (Group A), and 16 had no heredity (Group B) for type 2 diabetes. The two groups were matched for age and body mass index. Forearm skin microvascular reactivity was investigated by laser Doppler ﬂuxmetry (AU) before and after iontophoresis of endothelial(acetylcholine; Ach) and non-endothelial- (sodium nitroprusside; SNP) dependent substances. Plasma levels of IGF-1 and IGFBP-1 were investigated by RIA. Results Group A showed impaired (p < 0.03) skin microvascular responses (Ach: 2.5 ± 1.2 AU; SNP: 3.0 ± 1.6 AU), higher levels of IGF-1 (184 ± 38 lg/L p < 0.03), while IGFBP-1 was not signiﬁcantly diﬀerent (20 ± 14 lg/L; p = 0.22), as compared to Group B (Ach: 4.0 ± 1.9 AU; SNP: 4.4 ± 1.6 AU; IGF-1: 157 ± 20 lg/L; IGFBP-1: 23 ± 11 lg/L). So far the analyses of IGF-1 and IGFBP-1 have been performed in only 24 of the subjects, i.e., 13 in Group A and 11 in Group B. In Group B, signiﬁcant correlations were observed between the maximal microvascular responses to Ach (r = 0.75; p < 0.01), SNP (r = 0.64; p = 0.02), and IGFBP-1, respectively. In Group A, a non-signiﬁcant correlation was seen between the maximal microvascular response to Ach and IGFBP-1 (r = 0.57; p = 0.07), while no correlation was seen between the maximal response to SNP and IGFBP-1. No signiﬁcant diﬀerences were seen between the two groups regarding systolic and diastolic arm blood pressures, pulse rate and blood lipids. Conclusion The preliminary results of the present study indicate that healthy subjects with heredity for type 2 diabetes have higher levels of IGF-1 than healthy subjects without heredity. Furthermore, only the group without diabetes heredity demonstrated signiﬁcant correlations between skin microvascular reactivity and IGFBP-1 levels. Hence, the present data suggest that IGF-1 and IGFBP-1 may inﬂuence skin microvascular reactivity, e.g., by changes in smooth muscle cell function of the vessel walls.
P5 RELATIONSHIP BETWEEN IGFBP-2 AND RISK FACTORS FOR MACROVASCULAR DISEASE IN TYPE 2 DIABETES K. Kaushal, S. Anderson, A. Rudenski, A.H. Heald, J.M. Gibson. Department of Diabetes and Endocrinology, Hope Hospital, Stott Lane, Salford M6 8HD, UK Introduction Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are known to have an important contributory role in cardiovascular disease and diabetes. We have previously shown that, in patients with type 2 diabetes mellitus, lower IGFBP-1 levels are found in the presence of established macrovascular disease and are associated with factors known to increase cardiovascular risk. Little is known about the role of the other lower molecular-weight IGFBPs, such as IGFBP-2, in relation to cardiovascular disease. Method We investigated the association of circulating IGFBP-2 levels with known cardiovascular risk factors and diabetes treatment (diet only n = 20; sulphonylurea (SU) n = 70; insulin n = 31; insulin + SU n = 41)
in a cohort of 162 well characterised patients with type 2 diabetes. IGFBP-2 was measured by ELISA using the (DSL, Webster, Texas, USA). Insulin sensitivity was calculated using the HOMA-model. Results Higher circulating IGFBP-2 levels were signiﬁcantly and negatively associated with raised fasting plasma glucose (Spearman’s q = –0.23, p = 0.003), fasting triglycerides (q = )0.19, p = 0.01), LDL-cholesterol (q = )0.20, p = 0.01), IGF-I (q = )0.16, p = 0.04) and IGF-II (q = )0.27, p = 0.001). There was a trend for negative association between IGFBP-2 and fasting total cholesterol (q = )0.15, p = 0.06). There was a strong positive correlation between IGFBP-2 and IGFBP1 (q = 0.37, p < 0.001). In patients receiving diet and/or sulphonyurea therapy, IGFBP-2 correlated positively with insulin sensitivity (HOMA-S) (q = 0.261, p = 0.01). Patients treated with insulin alone had signiﬁcantly higher IGFBP-2 levels (482ng/ml 95% CI 365–600ng/ ml) than the other treatment groups (diet alone 333(276–390) ng/ml; SU 312(276–349) ng/ml; insulin+SU 345(292–398) ng/ml); F = 4.1, p = 0.008 for lnIGFBP-2. Conclusion In type 2 diabetes a low circulating concentration of IGFBP-2 is associated with hyperglycaemia, hyperlipidaemia and insulin resistance. This is further evidence in support of the role of the lower molecular weight IGFBPs in modulating the metabolic eﬀects of the IGFs. IGFBP-2 displays the same variation with treatment group as IGFBP-1 and low circulating levels are similar associated with markers of cardiovascular risk.
P6 MARKED HETEROGENEITY IN GROWTH HORMONE DOSES REQUIRED TO NORMALIZE IGF-I IN IGF DEFICIENT CHILDREN WITH OR WITHOUT GH DEFICIENCY: POTENTIAL PREDICTIVE ROLE OF BASELINE IGFBP-3 George M Brighta, Pinchas Cohenb, Bob Ana, Ron Rosenfeldc on behalf of the American Norditropin Clinical Studies Group (ANCSG). aNovo Nordisk Pharmaceuticals, Princeton; bUniversity of California at Los Angeles; cOregon Health Sciences University Individualized dosing of human growth hormone (hGH) based on serum IGF-I responses increasingly replaces body weight-based dosing. However, no algorithms are available to calculate eﬀective doses of hGH, hence most IGF-based dosing is by trial and error. The ANCSG is conducting a two year proof of concept trial for IGF-based dosing in IGF deﬁcient, short-stature children with or without documented GH deﬁciency. Of 172 randomized prepubertal subjects, 80% are randomized to adjust rhGH doses to an IGF-I z-score of zero or plus two; 20% are dosed at 40 mcg rhGH/kg*day. IGF-I levels (DSL IRMA Assay #5600) are drawn at 30 days and every 90 days thereafter. A 20% change in total rhGH dose is used for each z-score unit removed from the IGF target (0 or 2). The randomization code was not broken for this analysis. The IGF-I z-score at baseline was –1.81 ± 0.37 and at 6 months was + 0.11 ± 1.1. At 6 months, there was an extraordinary range of doses needed for adjustment to IGF-I targets. The average dose was 63 ± 33 lg/kg *day with a 17-fold range (12–202 lg/kg).
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
Forward, stepwise multiple regression analysis was used to discover pretreatment variables predictive of the eventual rhGH dose (dependent variable). Age, gender, bone age, maximal stimulated GH response, baseline IGF-I and BP3 concentrations and the change in IGF-I concentrations and z-scores were tested as independent variables. IGFBP3 was negatively correlated (p = 0.002) and age positively correlated (p = 0.04) with the dose (per kg) at 6 months (r = 0.33). We conclude 1) a surprisingly large range of rhGH doses are needed normalize IGF-I levels in GH and/or IGF deﬁcient children and 2) basal IGFBP-3 may become useful in IGF-I based dosing algorithms. IGFBP-3 may be a predictor of IGF-based dosing because a) IGFBP-3 concentration is a direct indicator of an individual subject’s exposure and sensitivity to GH signaling and b) IGFBP-3 positively inﬂuences the half-life of circulating IGF-I.
P7 APPROPRIATE INCREASE IN IGF-I AND IGFBP3 DURING GH TREATMENT IN HIV POSITIVE SUBJECTS WITH WASTING AND LIPOHYPERTROPHY Joseph M. Gertnera, Daena Bocka, Norma Muurahainena, Fanny O’Briena, Elisabeth Svanbergb. aSerono Inc, Rockland MA, USA; b Serono SA, Geneva CH Assessment of spontaneous and stimulated growth hormone (GH) secretion and the response of the GH-IGF axis to exogenous GH in HIVAIDS have given conﬂicting results. Mynarcik et al (JAIDS 1999; 22:49– 55) suggested that exposure to exogenous GH might induce a normal IGF-I response in HIV-AIDS, including patients with wasting, but that GH might not increase IGFBP3 signiﬁcantly in AIDS wasting. This could imply an increased potential risk of neoplasia due to a disproportionate increase in unbound IGF-I during GH treatment of HIV+ individuals. We measured IGF-I and IGFBP3 in two recent placebocontrolled trials using pharmacological doses of GH in HIV+ subjects. In the ﬁrst, patients with AIDS wasting (weight loss > 10%) were treated for 12 weeks with placebo (n = 204), GH 6 mg alternate days (n = 193), or 6 mg daily (n = 181). At the end of treatment respective mean±SD IGF-I levels in the three groups were 167 ± 101, 320 ± 151, and 475 ± 240 ng/ ml, with corresponding IGFBP3 3053 ± 1077, 3958 ± 1109, and 4579 ± 1500 ng/ml, respectively, (p < 0.0001 for 6 mg daily vs placebo for both substances). In the 6 mg daily group age and sex corrected standard deviation (z) scores for IGF-I and IGFBP3 rose from –0.6 ± 1.2 and –0.2 ± 1.2 at baseline to 2.4 ± 1.7 and 1.3 ± 1.4 at week 12, respectively, (p < 0.05 for both). In the second study, 83 patients with HIV-associated lipohypertrophy, transferred from a 24 week trial of treatment with up to 4 mg GH daily, and completed a maintenance extension during which they received GH 2 mg alternate days (n = 60) or daily (n = 23) over a 24 week period. At the end of that period mean IGFI levels were 525 ± 216 and 645 ± 342 ng/ml, respectively, in the alternate day and daily dosage groups while corresponding IGFBP3 levels were 5026 ± 1020 and 4981 ± 1314 ng/ml. Z scores for IGF-I were 3.9 ± 2.3 (alt day) 4.8 ± 3.4 (daily) and for IGFBP3 3.6 ± 2.1 and 3.8 ± 3, respectively. As expected, IGF-I elevation was more robust in lipohypertrophy than in wasting. In wasting IGFBP3 rose signiﬁcantly and on-treatment levels were elevated concomitantly with IGF-I. These data demonstrate that GH treatment of HIV+ persons, including those with wasting, induces an appropriate increase in IGFBP3 levels.
P8 EFFECTS OF GROWTH HORMONE ON SERUM LEVELS AND HEPATIC EXPRESSION OF IGFBP-6 IN HUMANS Henrik von Horn, Tomas J. Ekstro¨m, Kerstin Hall, Michael Tally. Department of Molecular Medicine, Karolinska Institute, Stockholm, Sweden. E-mail: [email protected]
Background We have previously determined the hepatic expression and serum protein levels of IGF-I, IGF-II, ALS and IGFBPs 1-3 in humans receiving growth hormone (GH) (JCE&M 84:553–560, 1999). We now expand the study to include IGFBPs 4, 5, and 6. IGFBP-4 and IGFBP-5 showed no diﬀerences after GH administration (unpublished observations). Here we present the eﬀect of GH on IGFBP-6 expression. Materials and methods Thirtyone patients undergoing liver biopsy before cholecystectomy were divided into three groups. 11 received 12IU of GH for ﬁve consecutive days, 10 received 12IU of GH 4-5 hours before surgery and there were 10 untreated controls. Serum samples were drawn in all subjects before the liver biopsy was taken and also before GH administration. The mRNA expression was measured with RNase protection assay and serum protein was determined by a commercially available RIA. Results The geometrical mean of IGFBP-6 serum levels at the time of biopsy was signiﬁcantly lower in both GH treated groups (32 and 33 lg/l) as compared with controls (73 lg/l). In those treated with GH for ﬁve days there was a signiﬁcant decline in IGFBP-6 (46%, p = 0.020) concomitantly with the decrease of IGFBP-1 and-2 levels previously reported. No change of IGFBP-6 was observed 4–5 h after a single GH dose. There was no diﬀerence in the hepatic mRNA expression between the GH treated and untreated subjects. However, there was a signiﬁcant diﬀerence between the two GH treated groups and this diﬀerence seemed to be associated to BMI. No correlation was found between the hepatic expression and the serum levels of IGBP-6. Conclusion High GH doses during ﬁve days decreased the serum levels of IGFBP6, but no diﬀerence of hepatic mRNA levels was observed between the two GH treated groups and the controls. We therefore conclude that in contrast to IGFBP-1 and -2 the liver is not likely to be the main source of IGFBP-6 in the circulation.
P9 CHANGES IN SEMINAL PLASMA IGF-I, IGF-II, AND IGFBP-3 WITH AGEING Li Fua, Aye Nyein Tinta, Andrew Hoﬀmanb, Arief T. Bongsoc, K.-O. Leea. aDivision of Endocrinology, Dept of Medicine, National University of Singapore, Singapore 119074; bDepartment of Medicine, Stanford University School of Medicine, Palo Alto, California 94304; cDepartment of Obstetrics & Gynaecology, National University of Singapore, Singapore 119074 We have shown previously that IGFBP-3 is present in seminal plasma (SP) and that there is no qualitative nor signiﬁcant quantitative diﬀerence in the IGFBPs between normal and vasectomized men, indicating the predominant contribution of the prostate gland. We have continued our investigations on the hypothesis that prostatic IGFs and IGFBPs might be related to the increase in incidence of BPH and prostate cancer with aging by measuring SP concentrations of IGF-I, IGF-II, and IGFBP-3 in 73 normal healthy virile men (Age range: 22–58 yrs). Semen samples, after assessment of standard sperm parameters (sperm count, percentage normality and percentage motility), had IGF-I, IGF-II and PSA measured by standard immunoassay, and a special radioimmunoassay previously validated for the measurement of IGFBP-3 fragments in seminal plasma (DSL, Webster, TX, USA). Results Seminal plasma IGF-II concentrations increased signiﬁcantly with increasing age (r = 0.332, p < 0.005) but SP IGF-I (r = )0.019, p = 0.87) did not show any statistically signiﬁcant correlation with age. In addition, SP IGFBP-3 (r = 0.087, p = 0.47) and sperm parameters
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 did not show statistically signiﬁcant correlation with increasing age. Seminal plasma PSA concentration, as expected, had a signiﬁcant positive correlation with age (r = 0.301, p < 0.01), but this correlation was weaker than that of IGF-II. Conclusion Prostatic IGF-II may be an important factor in the pathogenesis of benign prostatic hypertrophy (BPH) and prostate cancer in ageing men.
P10 EXPRESSION OF IGF-I, IGF-I RECEPTOR, AND IGF-BINDING PROTEINS IN HUMAN SYNOVIAL CELLS OF PATIENTS WITH RHEUMATOID ARTHRITIS Tomoko Matsumoto, Toshiyuki Tsurumoto, Hiroyuki Shindo. Department of Orthopedic Surgery, Nagasaki University, Nagasaki, Japan Objective Rheumatoid arthritis (RA) is characterized by the proliferation of an inﬂammatory synovia that destroys the articular cartilage and bone. Although elevated levels of insulin-like growth factor (IGF)-I and IGFbinding proteins (IGFBPs) were reported in synovial ﬂuids of patients with RA, little is known about the IGF-axis in synovial cells. The object of this study is to clarify the expression of IGF-I, IGF-1 receptor, and IGF-binding proteins in cultured synovial cells from patients with RA. Methods Synoviocytes isolated from synovial tissues of RA were cultured in D-MEM containing 10% fetal calf serum for 7 days and subsequently subjected to 4 passages. After treatment with or without reagents under serum-free medium, total RNA was extracted from cell layer. The expression of mRNA was identiﬁed by reverse transcriptase-polymerase chain reaction (RT-PCR). We also characterized IGFBPs in conditioned medium by Western-ligand blotting (WLB) with [125I]IGF-I and [125I]IGF-II. Each band of IGFBP was identiﬁed by immunoblotting. Results The expression of mRNA for IGF-I, IGF-I receptor, IGFBP- 2, -3, -4, and -5 was found in synovial cells of RA patients. WLB of conditioned medium also showed mainly 40-kDa, 29-kDa, and 24-kDa IGFBPs. These bands are identiﬁed as IGFBP-3, IGFBP-2, IGFBP-4, respectively, by immunoblotting. Comparing to the high expression of IGFBP-5 mRNA in RT-PCR, the band for IGFBP-5 was hardly seen in WLB, suggesting the proteolysis of IGFBP-5. In addition, the expression of IGF-I and IGFBPs was stimulated by interleukin (IL)-1, and the eﬀect of IL-I was in part inhibited by indomethacin, or dexamethasone. Conclusion RA synovial cells express IGF-I, IGF-I receptor, and IGFBPs, which was modulated by inﬂammatory cytokines. These ﬁndings suggest that the IGF-I and IGFBPs produced by synovial membrane might play a important role in pathogenesis of inﬂammatory arthritis.
P11 INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-4 AND RECEPTORS FOR IGF-I AND INSULIN ARE EXPRESSED IN HUMAN ENDOTHELIUM Simona I. Chisalita, Marloes Dekker-Nitert, Hans Arnqvist. Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden Background Micro- and macroangiopathy are major causes of morbidity and mortality in diabetes mellitus. Endothelial cells are considered to play an important role in the development of vascular disease. Little is known about insulin-like growth factor binding proteins-4 (IGFBP-4), insulin-like growth factor -I receptors (IGF-IR) and insulin receptors (IR) in human micro- and macrovascular endothelial cells.
Aim To characterize IGFPB-4, IGF-I and IR and in human micro- and macrovascular endothelial cells. Methods Cultured human dermal microvascular endothelial cells (HMVEC) and aortic endothelial cells (HAEC) were used. The cells were serum starved for 24 h before experiments and then cultured for 12 h in diﬀerent condition: insulin (10–8 M), IGF-I (10–8 M), angiotensin (10–6 M) and growth medium. Gene expression was measured by quantitative real-time RT-PCR analysis. Phosphorylation of IGF-IR protein was analyzed by immunoprecipitation and Western blot. Results In both human micro- and macrovascular endothelial cells IGFBP4 was more express than IGF-IR and IR in this order IGFBP4>IGF-IR>IR. The relative gene expression of IGFBP-4 standardize to GAPDH in HMVEC was about 70 times higher than in HAEC, (p < 0.001). Both IGF-IR and IR were approximately 3–4 more times more express in HMVEC than HAEC. The diﬀerent condition mentioned above did not result in any regulation of these peptides mRNA. IGF-IR was phosphorylated by IGF-I 10–8 M, but not by insulin 10–8 M. Conclusion Our study shows that, in both cultured human micro- and macrovascular endothelial cells, IGFBP-4 is more expressed than IGF-IR and IR. Furthermore IGF-IR is more expressed than IR. The IGF-IR was activated by IGF-I (10–8 M), but not by insulin (10–8 M). Our results provide experimental evidence for a signiﬁcant role of IGFBP-4, IGF-IR and IR in vascular physiology.
P12 IGFBP-4 IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA Heike Wex, Do¨rte Ahrens, Bianka Hohmann, Uwe Mittler, Peter Vorwerk. Otto-von-Guericke-University, Dept. Paediatric Haematology and Oncology, Magdeburg D-39112, Germany The IGF system plays a central role in the normal proliferation and diﬀerentiation as well as in the malignant transformation of diﬀerent cell systems. The availability of the IGFs for biological action at their receptor is regulated by six high aﬃnity IGFBPs. From these well characterised binding proteins IGFBP-4 seems to be the only one that act exclusively in an inhibitory way by sequestering IGF from its receptor and therefore prevent IGF induced DNA synthesis. Furthermore IGFBP-4 is a known inducer of apoptosis. Aim of the present study was the investigation of IGFBP-4 expression in malignant lymphoblasts and the determination of IGFBP-4 serum availability in children with acute lymphoblastic leukaemia. The IGFBP-4 plasma level was measured in bone marrow from 110 ALL patients (ALL-BFM 90/95 trial) at the time of diagnosis, 27 patients at day 33 (remission) and 57 peripheral blood samples of healthy age matched controls. The mean values were 631, 391 and 761 ng/ml IGFBP-4, respectively, detected by a currently available IGFBP-4 ELISA (DSL, Sinsheim, Germany). The control IGFBP-4 level is signiﬁcantly higher compared to ALL patient at diagnosis as well as at day 33. There was no diﬀerence regarding the age, gender, immunology or risk groups of the ALL patients and no correlation of IGFBP-4 to IGF-I, IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 plasma levels. The majority of ALL patients did not express IGFBP-4 mRNA (53 out of 92), investigated by standard RT-PCR, and there were no diﬀerence between B- or Tcell immunology. Looking at mean IGFBP-4 values in dependence of IGFBP-4 mRNA expression we found a signiﬁcant higher level of IGFBP-4 in samples with positive mRNA expression at day 33, but not at diagnosis. Interestingly, patients with high IGFBP-4
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
plasma level (above 700 ng/ml) had a better chance of event free survival than patients with IGFBP-4 below 700 ng/ml. The diﬀerence comes even more clearly for patients with positive mRNA expression. Taking together, we found a reduced plasma level of IGFBP-4 of ALL patients at diagnosis and a further reduction at day 33 compared to controls. If low IGFBP-4 level as an IGF inhibitory binding protein is involved in development or progress of leukaemia needs to be further investigated.
P13 POPULATION MIGRATION PROFOUNDLY INFLUENCES THE IGF-SYSTEM IN SUBJECTS ORIGINALLY FROM GUJARAT, INDIA A.H. Healda, J.K. Cruickshankb, A. Vyasb, J. Patelb, K. Siddalsa, S. Andersonb, J.M. Gibsona. aDepartment of Diabetes & Endocrinology, University of Manchester, Salford Royal Hospitals University Trust, Salford, M6 8HD, UK; bClinical Epidemiology Group, University of Manchester Medical School M13 9PT Objective We have previously established epidemiological links between the IGF-system and the development of impaired glucose tolerance, obesity and macrovascular disease. We therefore examined whether lifestyle change that accompanies population migration with increase in cardiovascular risk and diabetes, is mirrored by changes in the IGFsystem. Design In a population-based sample, we compared a speciﬁc Gujarati community in Sandwell UK (n = 161) with people still resident in the same villages of origin in India (n = 228). Mean age of both groups was 49 years. We measured circulating fasting plasma glucose, insulin, IGF-I, IGFBP-1 and IGFBP-3 concentrations. Results Body mass index (BMI) was signiﬁcantly higher in the UK Gujaratis (men = 25.9 (95% Conﬁdence interval, 25.1–26.7); women = 26.6 (25.7–27.3)) than Indian Gujaratis (men = 21.0 (20.3–21.7); women = 20.8 (20.3–21.6)). IGFBP-1 was signiﬁcantly lower in UK migrants (29.5 (25.9–33.0) mcg/l) vs (56.5 (50.6–62.5) mcg/l); F = 48.4, p < 0.001. Conversely fasting insulin was higher in UK Gujaratis (81.5 (74.9–88.1 pmol/l) F = 12.9, p = 0.004) as was IGF-I (145.9 (138.1– 153.6 ng/ml); F = 76.6, p < 0.001), than Indian Gujaratis (insulin 64.7 (58.2–71.1) pmol/l; IGF-I 100.9 (94.6–107.3 ng/ml)). Circulating IGFBP-3 was signiﬁcantly higher in both UK migrant Gujarati men (3.9 (3.7–4.1) mg/l) and women (3.9 (3.7–4.1) mg/l) than Indian Gujarati men (3.5 (3.3–3.7) mg/l) and women (3.5 (3.3–3.7) mg/l F = 6.4, p < 0.001). Conclusion The diﬀerences in circulating IGFBP-1, IGFBP-3 and IGF-I appear to reﬂect changes in cardiovascular risk, BMI and dietary change related to migration in these two samples. Our results provide important evidence for the eﬀect of environmental factors in modulating genetic inﬂuences on the concentration and biological eﬀects of the IGFs.
P14 MARKEDLY DIFFERENT EFFECTS OF HORMONE REPLACEMENT REGIMENS ON THE IGF-SYSTEM ACCORDING TO PROGESTIN FORMULATION A.H. Healda, K. Kaushala, S. Andersona, A.P. Yatesb, P. Durringtonb, P. Selbyb, J.M. Gibsona. aDepartment of Diabetes & Endocrinology, University of Manchester, Salford Royal Hospitals University Trust,
Salford, M6 8HD, UK; bBone Disease Research Centre, University of Manchester, Manchester M13 9PT, UK Introduction There remains much controversy about the risks vs beneﬁts of hormone replacement in post-menopausal women. The insulin-like growth factor/IGF binding protein (IGF/IGFBP) system has been implicated in the pathophysiological mechanisms underlying cardiovascular disease, cancer and osteoporosis. We here report the results of a comprehensive examination of the eﬀects of diﬀering estrogen/progestin combinations on the IGF-system. Method Oral conjugated equine estrogen (CEE) alone (0.625 mg Premarin) or in combination with the increasingly androgenic progestins, Medroxyprogesterone acetate (MPA) 10 mg, Desogestrel (DG) 75 mcg or Norethisterone (NE) 1.05 mg were given in a randomised triple crossover fashion to 35 healthy postmenopausal women. Serum concentrations of IGFs and their principal circulating binding proteins were measured. Results Baseline circulating IGF-I levels were signiﬁcantly reduced by CEE (134 ± 12 vs 83 ± 11ng/ml, p = 0.001). This eﬀect was reversed by progestins according to their androgenicity (MPA 96 ± 9 ng/ml, DG 109 ± 9 ng/ml, NE 131 ± 10 ng/ml) (F = 6.2, p = 0.001). IGF-II levels were unaﬀected by CEE or progestins. In contrast plasma IGFBP-1 concentration increased signiﬁcantly from baseline to CEE alone (29 ± 3 vs 67 ± 6 lg/l, p < 0.001). This rise was opposed by progestins of increasing androgenicity (MPA 60±5lg/l, DG 57 ± 5 lg/l, NE 45 ± 5 lg/l) (F = 4.1, p = 0.02). IGFBP-2 levels fell with CEE and their was no further change with the addition of progestins p = 0.03 (baseline 757 ± 125 ng/ml, CEE 493 ± 80 ng/ml). Changes in IGFBP-3 were similar to those in IGF-I. IGFBP-4 levels were signiﬁcantly higher in subjects receiving estrogen alone (738 ± 55 ng/ml) than at baseline (no HRT) (573 ± 33 ng/ml), p = 0.001. IGFBP-4 levels remained higher than baseline with addition of progestins (MPA 654 ± 32 ng/ml, DG 672 ± 44 ng/ml, NE 680 ± 31 ng/ml) (p = 0.002). There was no diﬀerence in IGFBP-4 between progestins of diﬀering androgenicity. With CEE there was an increase in the proportional contribution of the lowest molecular weight IGFBPs, particularly IGFBP-1 and IGFBP-4 to total IGFBP binding with a corresponding decrease in the contribution of IGFBP-3. This eﬀect was partially reversed by the addition of progestins. Conclusion There are marked diﬀerences in the response of circulating IGFs and their principal binding proteins to diﬀering HRT combinations with corresponding changes in molar ratios of the IGFBPs in relation to the progestin component. Given the established links of the IGF-system with cancer and cardiovascular disease, the eﬀect of progestins on IGF bioavailability could be an important determinant of the longer term risks of speciﬁc HRT preparations.
P15 EXPRESSION AND REGULATION OF THE INSULIN-LIKE GROWTH FACTOR AXIS COMPONENTS IN RAT LIVER MYOFIBROBLASTS Ruslan Novosyadlyy, Kyrylo Tron, Jozsef Dudas, Giuliano Ramadori, Jens-Gerd Scharf. Department of Internal Medicine, Division of Gastroenterology and Endocrinology, Georg-AugustUniversita¨t, Go¨ttingen, Germany Apart from hepatic stellate cells liver myoﬁbroblasts represent a second mesenchymal cell population involved in hepatic ﬁbrogenesis. The
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 IGF system including the insulin-like growth factors I and II (IGF-I, -II), their receptors (IGF-I receptor, IGF-IR; IGF-II/mannose 6-phosphate receptor, IGF-II/M6-PR) and six high aﬃnity IGF binding proteins (IGFBPs) participate in the regulation of growth and diﬀerentiation of cells of the ﬁbroblast lineage, possibly contributing to the ﬁbrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF (MF). Methods Cultures of MF from passages 1 to 4 were studied. IGFBP secretion was analyzed by [125I]-IGF-I ligand and immunoblotting. IGF-I, IGFIR, IGF-II/M6-PR and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5-bromo-20 -deoxyuridine incorporation assay. Results MF from passages 1 to 4 constitutively expressed mRNA transcripts speciﬁc for IGF-I, IGF-IR and IGF-II/M6-PR. In MF, biosynthesis of IGFBP-3 and -2 was observed that was stimulated by IGF-I, insulin and transforming growth factor b (TGF-b), whereas platelet-derived growth factor (PDGF-BB) revealed inhibitory eﬀects. IGF-I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP-2 or -3 with IGF-I diminished the mitogenic eﬀects of IGFI on MF whereas preincubation of MF with IGFBP-2 or -3 further potentiated the IGF-I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of ﬁbrogenesis during acute and chronic liver injury.
P16 IGFBP-3 FRAGMENTS IN PLASMA OF A CHILD WITH AN AUTOIMMUNE DISORDER Victoria Schebek-Fu¨rstenberga, Dirk E. Mu¨ller-Wiefela, Werner F. Blumb, Thomas Braulkea, Bernd Ku¨blera. aChildrens Hospital, University of Hamburg, Hamburg, Germany; bEli Lilly and Company, Bad Homburg, and University Childrens Hospital, Leipzig, Germany In sera from patients with catabolic states or severe diseases increased serum IGFBP-3 protease activity has been reported, suggesting that proteolysis of IGFBP-3 is involved in the regulation of the bioavailability of the IGF mitogens. In the present study, IGFBPs and IGFBP fragments were analyzed in the plasma of a 14-year old girl who underwent repeated plasmapheresis due to an autoimmune disorder (systemic lupus erythematosus (SLE) with severe glomerulonephritis). The plasma IGF-I and IGFBP-3 levels were drastically reduced but reached normal values one year after the 5th plasmapheresis. Three liters of plasma were subjected to a 50 kDa size exclusion ultraﬁltration and fractionated by cation exchange chromatography and reverse phase -HPLC. Ligand blotting of the fractions with monobiotinylated IGF-II revealed two strong bands of 29 and 17 kDa, and weak signals of IGFII-binding polypeptides with apparent molecular masses of 45/43, 22, and 12 kDa. The 45/43, 29, 22 and 17 kDa polypeptides were immunoreactive with an anti IGFBP-3 antibody. N-terminal sequence analysis of the 17 kDa IGFBP-3 peptide yielded the sequence KVDHESQ_TDT, indicating the isolation of a C-terminal IGFBP-3 fragment starting with the residue 160. When the IGF-II binding protein pattern in the hemoﬁltrate from patients with chronic renal failure was compared with the pattern of the patient with acute renal failure, almost all IGF binding protein forms were detected in both preparations. The data suggests that in acute and chronic renal failure similar or identical protease (s) are involved in cleavage of circulating IGFBP-3 after amino acid residues 98, 138, 157 and 159.
P17 QUANTIFICATION OF SERUM IGFBP-3 PROTEOLYSIS IN PATIENTS WITH COLORECTAL CANCER A.G. Renehana, K. Khosravib, A. Diamandib, S.T. O’Dwyera, S.M. Shaleta. aDepartments of Surgery and Endocrinology, Christie Hospital NHS Trust, Manchester, UK; bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Background Semi-quantitative studies have shown that serum IGFBP-3 proteolysis is common in patients with cancer. IGFBP-3 fragmentation may increase IGF-I bio-availability and decrease IGFBP-3 bio-activity, favouring tumourogeneis. To examine this further, we quantiﬁed the extent of IGFBP-3 proteolysis in colorectal cancer patients using a panel of previously validated immunoassays for intact (mAb2), fragmented (mAb5), and total IGFBP-3 (pAb) (Diamandi et al. J Clin Endo Metab 85:2327–2333, 2000). Methods Determinants were measured in three patient groups: (i) normal colonoscopy as controls (N = 41: age 56–77); (ii) benign adenomas (N = 29: age 56–65); (iii) colorectal cancer (N = 57: age 48–80) categorised in early (Dukes’ A and B: N = 26) and advanced (Dukes’ C and D: N = 31). Among the cancer patients, thirteen had repeat measurements at 6–8 weeks post-surgery. Serum IGF-I, IGF-II and common capture IGFBP-3 (mAb3) were also measured. Clinico-pathological data included age, sex, nutritional status, and tumour characteristics. Results were expressed as means with standard deviations (SD), and the fragmented to intact IGFBP-3 ratio (F/I) calculated. Parametric tests were used. Results Among the controls, total IGFBP-3 correlated positively with IGFI (r = 0.37, p = 0.02), IGF-II (r = 0.59, p IGFBP-3 (r = 0.78, p < 0.001). Means for intact, fragmented and total IGFBP-3 were similar for controls versus adenomas, and controls versus cancers. However, when considered by cancer stage, there were signiﬁcant reductions in mean intact IGFBP-3 and total IGFBP-3 levels in patients with advanced cancers (Table). Furthermore, mean levels for intact and fragmented IGFBP-3 normalised after surgery.
Means (SD) Intact BP3 Fragmented BP3 Total BP3 Ratio (F/I)
Controls 1.17 (0.27) 0.67 (0.33)
Early Ca. 1.28 (0.42) 0.81 (0.35)
Advanced Ca. P* P** 0.95 (0.48) 0.02 0.008 0.77 (0.38) 0.23 0.70
1.40 (0.34) 0.58 (0.23)
1.39 (0.35) 0.68 (0.33)
1.19 (0.36) 1.65 (3.22)
0.01 0.04 0.04 0.13
*t test: advanced versus controls. **t test: advanced versus early.
Conclusions Serum IGFBP-3 proteolysis, as quantiﬁed by immunoassays, is signiﬁcant increased in patients with advanced colorectal cancer. Further study is merited to determine whether these changes inﬂuence cancer progression and prognosis.
P18 GENDER SPECIFIC PATTERN OF IGFBP-3 PROTEASE ACTIVITY IN MOUSE THYROID Jing Wang, Inga-Lena Wivall-Helleryd, Jacob Gru¨nler, Moira Lewitt. Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
Background The IGF system is believed to play a role in the growth and function of the thyroid gland. Proteolysis is an important post-transcriptional process that modulates the IGF-binding aﬃnity of IGFBPs, thus regulating IGF activity. IGFBP-3 protease activity has been demonstrated in a thyroid cell line (Wang et al, J Endocrinol 152: 265, 1997). PSA and human kallikrein 2, which are both known to cleave IGFBP3, are expressed in human thyroid tissue (Magklara et al, Clin Chim Acta 300:171, 2000). Aim The aim of this study is to determine whether there is IGFBP-3 protease activity in thyroid tissue in vivo, and to characterise this activity. Methods Male and female Balb/c mice, aged 16 week, were fasted for 16 h and divided into 3 groups; fasted, 1-h refed and 4-h refed (n = 5 in each). Blood was collected by cardiac puncture and thyroid tissues were dissected and homogenised. IGFBP-3 protease activity was determined by incubating samples, adjusted for protein content, with [125I]-human nonglycosylated IGFBP-3 at 37 C, pH 7 for 2 h. After electrophoresis through 14% SDS–PAGE under non-reducing conditions, the gels were ﬁxed, dried, and autoradiographed. Results IGFBP-3 protease activity was present in thyroid tissue, and was substantially greater in tissue from male mice compared to that from female animals. Two predominant IGFBP-3 fragments (approx. 25 and 15 kDa) were found and this pattern of fragmentation was similar to that induced by human PSA. There was no eﬀect of nutritional status, however serum from fasted mice had a greater inhibitory eﬀect on the activity than refed serum. IGFBP-3 protease activity was maximal at neutral pH and was inhibited by the serine protease inhibitors, PMSF and aprotinin, and by a kallikrein inhibitor (cyclohexylacetyl-Phe-Arg-Ser–Val–Gln amide). Conclusions Mouse thyroid contains IGFBP-3 protease(s). The gender dimorphism, along with the patterns of fragmentation and inhibition by protease inhibitors, suggests it is PSA-like. Inhibitors of tissue IGFBP3 protease(s) are present in the circulation and their activity is regulated by fasting and feeding.
P19 UV-INDUCED APOPTOSIS OF HUMAN KERATINOCYTES IS ASSOCIATED WITH IGFBP-3 PROTEOLYSIS. Susan Thumigera, Tim Adamsb, George Werthera, Christopher Wraighta, Stephanie Edmondsona. aCutaneous Biology Group, Dermal Therapeutics, Centre for Hormone Research, Murdoch Childrens Research Institute, Parkville, Vic. 3052, Australia; bCSIRO, Division of Health Sciences and Nutrition, Parkville, Vic. 3052, Australia Epidermal keratinocytes respond to UV insult via cell cycle arrest, DNA repair and apoptosis, primarily via the tumour suppressor p53. IGF-IR and IGFBP-3, p53 transcriptional targets, regulate cell survival and are co-localised to the basal layer of the epidermis. IGF-IR activation is also critical for normal epidermal growth. We have utilised normal human keratinocytes (NHK; p53+) and a human keratinocyte cell line, (HaCaT; p53)) to assess the integrity, modulation and requirement of components of the IGF system following UV irradiation. Keratinocytes (90% conﬂuence) were exposed to 0, 240 or 960 mJ/ cm2 UVB and then grown in keratinocyte growth or basal medium ±50 ng/ml IGF-I (24 h). IGF-IR and IGFBP-3 mRNA and protein abundance (conditioned medium/cell lysate) and/or integrity were determined by either real time PCR, Western ligand blot and/or immunoblot. Apoptosis was assessed by cell Death Detection ELISA and histology.
Results Compared with untreated cells (1) Exposure to 960 mJ/cm2, not 240 mJ/cm2, induced NHK and HaCaT apoptosis. (2) UV-induced apoptosis in HaCaTs was greater than NHKs. (3) HaCaT IGFBP-3 mRNA and protein was reduced after 960 mJ/cm2 but not altered in NHKs. (4) IGFBP-3 was proteolysed, to at least two fragments between 15 and 11 kDa, following exposure of HaCaTs and NHK to 960 mJ/cm2. (5) HaCaT IGF-IR protein, not mRNA, abundance was reduced following exposure to 960 mJ/cm2 but not consistently altered in NHKs. (6) IGF-I reduced NHK, and not HaCaT, apotosis. In conclusion, these data indicate that following UV insult, irrespective of p53 status: (i) keratinocytes undergo apoptosis in response to high doses of UV and (ii) IGFBP-3 is proteolysed. Thus these data suggest a pro-apoptotic role for proteolysed IGFBP-3. Additionally, these data suggest that HaCaTs exhibit greater apoptotic susceptibility to high UV doses, possibly related to their inability to maintain intact IGFBP-3 abundance and the inability of IGF-I to promote cell survival.
P20 SITE DIRECTED MUTAGENESIS TO ALTER hIGFBP-2 CELL SURFACE ASSOCIATION AND PROTEOLYTIC PROCESSING D.G. Cossensa, S.I. Ymera, E. Andaloroa, B. Schutta, A. Hoeﬂichb, D.D. Mikolc, E. Feldmanc, L.A. Bachd, G.A. Werthera, V.-C. Russoa. a Centre for Hormone Research, Murdoch Childrens Research Institute, Dept. of Paediatrics University of Melbourne, Parkville 3052, Vic., Australia; bLudwig-Maximilians University, D-81377 Munich, Germany; cDepartment of Neurology, University of Michigan, Ann Arbor, MI, USA; dDepartment of Medicine, University of Melbourne, Austin and Repatriation Medical Centre, Heidelberg 3084, Vic., Australia. Modulation of IGF action by the IGFBPs occurs in the pericellular space involving interactions with matrix proteins including proteoglycans. IGFBP/IGF interaction might be further modulated by speciﬁc proteases, which cleave IGFBPs to fragments with reduced aﬃnity for IGF-I. We have demonstrated IGFBP-2 binding to brain proteoglycans (PGs) and in neuronal cells we have shown that FGF-2 induced proteolysis of soluble and cell surface associated IGFBP-2. Of interest was that one of the IGFBP-2 fragments retained the ability to complex with IGF-I on the cell surface. However, the role of IGFBP-2 cell surface association and the biological consequences of IGFBP-2 proteolytic processing in IGF action remain unclear. We therefore aimed to alter hIGFBP-2 cell surface association and proteolytic processing by mutating the putative HBD and proteolytic cleavage sites of hIGFBP2 by mutagenesis. HBD-IGFBP-2 mutant The putative HBD of hIGFBP2, (218PKKLRP223-) was mutated to (218PNNLAP223-). Mutations were introduced in hIGFBP-2 by overlap extension PCR. Both cDNA HBD-IGFBP-2 mutant and WT-IGFBP-2 control were transiently expressed in SK-N-SHEP neuroblastoma cells (IGFBP-2 negative) and as His6-tagged HBDor WT- IGFBP-2 in Escherichia coli. The expressed HBD/WTIGFBP2 were puriﬁed from conditioned medium by IGF-I aﬃnity chromatography and the purity was veriﬁed by SDS–PAGE followed by silver staining and immunoblotting. In both mammalian and bacterial systems HBD- and WT-hIGFBP-2 maintained the ability to bind 125I-IGF-I as determined by WLB and crosslinking analysis. Scatchard analysis revealed that the binding aﬃnity of the HBD-mutant for IGF1 was not signiﬁcantly diﬀerent from WT- IGFBP-2 or commercial IGFBP-2. However, the HBD-IGFBP-2 mutant showed reduced binding to heparin, thus
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 suggesting that the predicted HBD at 218PKKLRP223 is involved in this interaction. Protease resistant IGFBP-2 The published sequence of IGFBP2 fragments has suggested the presence of two potential proteolytic cleavage sites (207KGGK210-) and (219KKLR222-). We have mutated both of these sites and made various mutants using the methodology described above for HBD-mutants. The identiﬁcation of protease resistant IGFBP-2 and characterisation of these mutants is in progress. The generation of IGFBP-2 with altered functional domains is central to understanding the role of IGFBP2 in modulation of IGF action.
P21 ANALYSIS OF THE CLEAVAGE OF IGFBP-4 AND -5 CHIMERAS BY PAPP-A; DIFFERENTIAL EFFECT OF IGF Michael T. Overgaard, Vivien Rodacker, Lisbeth S. Laursen, Claus Oxvig. Department of Molecular Biology, University of Aarhus, Aarhus, Denmark Pregnancy-associated plasma protein-A (PAPP-A) has been identiﬁed as the IGFBP-4 proteinase in a number of diﬀerent biological systems. We have previously shown that PAPP-A, in addition to IGFBP-4, cleaves IGFBP-5 and IGFBP-2, and demonstrated an opposite inﬂuence of IGF on the cleavage of these diﬀerent IGFBP’s by PAPP-A. The presence of IGF is required or dramatically increases the cleavage of IGFBP-4 and IGFBP-2, whereas IGF inhibits cleavage of IGFBP-5. We have also previously demonstrated that the inﬂuence of IGF is due to formation of the IGF/IGFBP complex, and that there is no direct interaction between IGF and the proteinase. To investigate the role of individual binding protein domains responsible for this diﬀerential IGF eﬀect, we have generated IGFBP-4/-5 chimeras by swapping either the N- or C-terminal domains in the two IGFBP’s. The chimeras and the wild-type IGFBP’s where expressed in HEK-293 cells, puriﬁed and labeled. The rate of proteolysis by PAPP-A and the ability of IGF-binding were analyzed for all chimeras and wild-type binding proteins. The results provide insight into the underlying mechanism of the diﬀerential eﬀect of IGF on IGFBP cleavage, and have implications for the overall structure of IGFBP’s with or without IGF bound.
P22 THE DIVERGENT EFFECT OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP)-1 ON IGF-INDUCED STEROIDOGENESIS IN BOVINE ADRENOCORTICAL CELLS IS NOT DUE TO ITS PHOSPHORYLATION STATUS Christian Fottnera, Gerald Spo¨ttlb, Dieter Engelhardtb, Matthias M. Webera. aSchwerpunkt Endokrinologie und Stoﬀwechselerkrankungen, I. Medizinische Klinik und Poliklinik der Johannes Gutenberg Universita¨t Mainz, Germany; bMedizinische Klinik II, Klinikum Grosshadern, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany In previous studies we have shown that insulin-like growth factor (IGF)-II stimulates basal as well as ACTH-induced cortisol secretion from bovine adrenocortical cells more potently than IGF-I. The steroidogenic eﬀect of both, IGF-I and IGF-II, is mediated through the IGF-I receptor and modiﬁed by locally produced IGF
binding proteins. Further studies demonstrate that bovine adrenocortical cells do synthesize IGFBP-1 to -4 and that their secretion is regulated diﬀerentially by IGFs and ACTH. Since IGFBP-1 selectively is induced 4-fold by ACTH, the aim of the following studies was to evaluate the eﬀect of IGFBP-1 on IGF-induced cortisol secretion from bovine adrenocortical cells in primary culture. Coincubation of bovine adrenocortical cells with puriﬁed IGFBP-1 (30 nM) induced a time- and dose-dependent potentiating eﬀect (38 ± 2%) on IGF-I-stimulated (6.5 nM) cortisol-secretion, wheras the IGF-II induced steroidogenic eﬀect was inhibited (40 ± 3%) by IGFBP-1. In order to evaluate the inﬂuence of different phosphorylation states of IGFBP-1 on the steroidogenic effect of IGF-I and IGF-II and on the aﬃnity to IGFs, a highly phosphorylated (pIGFBP-1) and a dephosphorylated isoform (dIGFBP-1) of IGFBP-1 have been separated by anion exchange chromatography for further incubation experiments and binding studies. No diﬀerent eﬀects on IGF-I or IGF-II-induced steroidogenesis were observed when a highly phosphorylated IGFBP-1 isoform was used, compared to the dephosphorylated IGFBP-1 isoforms. In binding studies IGFBP-1 did show high aﬃnity binding for IGF-I with a calculated association constant (Ka) of 2.17 · 109 M)1. Similar association constants were calculated for dIGFBP-1 and pIGFBP-1 (1.93 · 109 and 2.67 · 109 M)1 respectively) and no diﬀerence in binding capacity was observed when IGF-II was used as ligand. In conclusion these results demonstrate that in bovine adrenocortical cells IGFBP-1 time- and dose-dependently inhibits the steroidogenic eﬀect of IGF-II and stimulates the eﬀect of IGF-I. The observed modulatory eﬀect of IGFBP-1 is independent of the mode of incubation and its phosphorylation status. The mechanisms by which IGFBP-1 divergently regulates the steroidogenic eﬀect of IGF-I and IGF-II remain unclear at present and further investigation is necessary to elucidate the mechanisms by which IGFBP-1 modulates IGF action in the adult adrenal gland.
P23 KIDNEY GROWTH IN NORMAL AND DIABETIC MICE IS NOT AFFECTED BY hIGFBP-1 ADMINISTRATION Vesna Cingel-Ristic´**a, Bieke F. Schrijversb, Arle`ne K. van Vlieta, Ruth Raschc, Stenvert L.S. Dropa, Allan Flyvbjergb. aLaboratory of Pediatrics, Subdivision of Molecular Endocrinology, Erasmus b MC, Rotterdam, The Netherlands; Medical Research Laboratories, Institute of Experimental Clinical Research, Aarhus University Hospital, Aarhus, Denmark; cDepartment of Cellular Biology, Department of Anatomy, Aarhus University, Aarhus, Denmark Insulin-like growth factor I (IGF-I) accumulates in the kidney following the onset of diabetes, initiating diabetic renal hypertrophy. Increased renal IGF-I protein content, not reﬂected in mRNA levels, suggests that renal IGF-I accumulation is due to sequestration of circulating IGF-I rather than local synthesis. It has been suggested that IGF-I is trapped in the kidney by IGF binding protein 1 (IGFBP-1). We administered puriﬁed human (h)IGFBP-1 to non-diabetic and diabetic mice as 3 daily s.c. injections for 14 days, starting 6 days after induction of streptozotocin-diabetes when the animals were overtly diabetic. Accumulation of renal IGF-I despite decreased mRNA levels in 20 days diabetic mice coincided with markers of early diabetic renal changes, i.e. increased kidney weight, glomerular volume and albuminuria. Increased IGF-I levels in kidney of normal mice receiving hIGFBP-1, were not reﬂected on
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222
kidney parameters. Renal IGFBP-1, -3, -4 and -5 mRNA levels were decreased in diabetic mice. Administered hIGFBP-1 did not have any signiﬁcant eﬀect on IGFBPs mRNA expression pattern in diabetic kidney. However, it increased renal IGFBP-1, -2 and -4 protein content in diabetic mice. In conclusion, hIGFBP-1 administration had no eﬀect on increased kidney weight and albuminuria in early diabetes, although it abolished renal IGF-I accumulation and glomerular hypertrophy in diabetic mice.
P24 ROLE OF ENDOGENOUS IGFBP-1 IN THE SURVIVAL OF INS-1 PANCREATIC BETA CELLS Moira S. Lewitt, Jing Wang, A. Majid. Department of Molecular Medicine, Karolinska Institutet, Stockholm, Sweden Background and aim It has been demonstrated that IGFBP-1 has an endocrine role, regulating IGF actions. IGF-independent activity has also been documented in vitro, including an eﬀect on apoptosis. Most of the circulating IGFBP-1 is derived from liver, however it is expressed in other tissues, including pancreas. We have recently shown that secretion of IGFBP-1 by the glucose-sensitive rat pancreatic beta cell line, INS-1, is stimulated by dexamethasone and inhibited by glucose. INS-1 cells have also been shown to express IGF-II. The aim of this study was to determine the eﬀect of IGFBP-1 on INS-1 cell survival. Materials and methods Studies were performed in monolayer cultures of INS-1 cells. After a 6-h serum free period, cells were exposed to eﬀectors in the absence of serum for a further 18 h. IGFBP-1 expression was determined by a quantitative RT-PCR method using a europium-labeled probe. IGFBP-1 secretion was determined by sensitive immunoassay and cell viability assessed by MTT assay. Results In the presence of dexamethasone (100 ng/ml), IGFBP-1 expression and secretion was stimulated approx 5-fold while cell viability decreased by 25%. The eﬀect of dexamethasone on cell viability was partially reversed by IGF-II (25 ng/ml). The addition to INS-1 cells of exogenous human IGFBP-1, puriﬁed from amniotic ﬂuid, decreased cell numbers by 20% while exposure to a polyclonal rabbit anti-rat IGFBP-1 antibody (B1) increased cell numbers by 30%, compared to non-immune rabbit serum. Conclusions IGFBP-1 secreted by INS-1 pancreatic beta cells reduces cell survival. While some of this eﬀect is due inhibition of endogenous IGF activity, we speculate that IGF-independent mechanisms are also involved.
P25 IGF:IGFBP:VITRONECTIN (VN) COMPLEXES STIMULATE CELL MIGRATION AND PROTEIN SYNTHESIS IN HaCAT HUMAN KERATINOCYTES Carolyn E. Hyde, Damien Harkin, Zee Upton. Tissue BioRegeneration and Integration Program, Science Research Centre, Queensland University of Technology, GPO Box 2434, Brisbane, Q4001, Australia The insulin-like growth factor (IGF) system plays an important role in a number of disease states, such as cancer and has also been implicated in wound and burn healing processes. Two IGF receptors, the type-1 IGF and type-2 IGF receptors, as well as six insulin-like growth factor binding proteins (IGFBP-1 to 6),
have well established roles in mediating IGF activity. Earlier studies in this laboratory demonstrated that IGF-II binds to the ECM protein vitronectin (VN), and although IGF-I does not bind directly to VN it can bind indirectly via speciﬁc IGFBPs. Therefore the aim of this study was determine whether binary and ternary complexes of IGF-I/II, IGFBPs and VN aﬀect human keratinocyte cell function. The strategy of pre-binding these complexes to the culture dishes was adopted in this study in an attempt to more accurately reﬂect the extracellular environment in vivo. These studies demonstrated that the binary complex of IGF-II and VN and the ternary complexes comprised of IGF-I, IGFBP-2, or 3, or 4, or 5 and VN signiﬁcantly stimulated HaCAT cell protein synthesis, as measured by de novo protein synthesis. Interestingly these latter experiments demonstrated that although large increases in protein synthesis were observed using the ternary complexes, IGF-I/IGFBP complexes alone were responsible for the signiﬁcant increases in protein synthesis. In addition, both the binary and ternary complexes signiﬁcantly enhanced cell migration through 12 lm TranswellsTM. Unlike the protein synthesis assays, VN was critically important in these migratory responses and highlighted the important role that intergrins play in cell migration. Cell attachment assays on the other hand demonstrated that the interactions of IGFs with IGFBPs and VN did not eﬀect cell attachment. This is the ﬁrst study to provide a functional role for the interaction between IGFs, IGFBPs and VN in human keratinocytes. These results suggest that IGF/IGFBP/VN complexes maybe useful in situations where enhanced keratinocyte cell migration and proliferation is required, such as in wound healing and skin engineering applications.
P26 MANIPULATION OF LIPID RAFTS HAS DIFFERENTIAL EFFECTS ON IGF-I AND IGFBP-3-INDUCED PROLIFERATION IN THE RELATIVELY NORMAL MCF10A BREAST EPITHELIAL CELLS Martin A Clark, Zoe E. Winters, Jeﬀ M.P. Holly, Claire M. Perks. University Department of Surgery, Level 7, Bristol Royal Inﬁrmary, Bristol, BS2 8HW. UK Introduction IGF-I is a potent mitogen and survival factor for many cell types. IGFBP-3 is the main IGF-carrier protein in human serum and in addition to controlling IGF activity also has direct actions on cell growth. In addition to the proliferative eﬀects of IGF-I in MCF10A cells, we have shown previously that IGFBP3 can also promote cell growth in an IGF-independent manner. The mechanism by which IGFBP-3 elicits these actions is still largely unknown. Cell matrix interactions ensure normal growth constraints and intact lipid rafts are critical to co-ordinate such signalling. Therefore we investigated if manipulation of lipid rafts modulated the ability of either IGFBP-3 or IGF-I to aﬀect cell growth. Methods MCF10A cells were dosed with either IGF-I (100 ng/ml) or IGFBP-3 (100 ng/ml) following a period of serum starvation. The cholesterol depleting agent, b-methyl cyclodextrin (b-MCD; 30 min pre-incubation of 4 mM), and a lipid raft-disrupting agent, ﬁlipin (5 lg/ml) were used to control and alter the cholesterol status of the MCF10A cells. Cell growth was assessed by cell counting using trypan blue dye exclusion and tritiated thymidine incorporation. Results In the presence of b-MCD or ﬁlipin alone there was a decrease in growth rates from 100% to 81% and 98%, respectively. IGFBP-3 (100 ng/ml) increased cell growth (from 100% to 144%) after 24 h exposure.
Abstracts / Growth Hormone & IGF Research 13 (2003) 203–222 This response was blocked by both b-MCD (from 144% to 83.6%) and ﬁlipin (from 144% to 97.8%). IGF-I (100 ng/ml) increased growth (from 100% to 160%) after 48 h exposure. In contrast to the block in IGFBP-3-induced cell proliferation observed in the presence of bMCD, stimulation of cell growth by IGF-I was actually enhanced (from 160% to 180%). Conclusion These data suggest that intact lipid rafts are critical for IGFBP-3 to exert its intrinsic eﬀects on cell growth. However, in contrast manipulation of cholesterol, and hence lipid rafts, does not block but actually enhances IGF-I-induced cell proliferation. Acknowledgements We would like to acknowledge the Needham cooper Trust and the Association for International Cancer Research for their continued support.
P27 IGFBP-3 MEDIATES ENDOTHELIAL CELL SURVIVAL AND APOPTOSIS, LIKELY VIA THE CERAMIDE SIGNALING PATHWAY Riccarda Granataa, Marzia De Petrinia, Daniela Atragenea, Marina Talianoa, Fabio Broglioa, Giulia Somenzib, Riccardo Ghidonib, Ezio Ghigoa. aDivision of Endocrinology and Metabolism, Department of Internal Medicine; University of Turin, Italy; bLaboratory of Genetics and Biochemistry, San Paolo University Hospital; University of Milan, Italy IGFBP-3 modulates IGFs action and exerts direct IGF-independent eﬀects inducing apoptosis or promoting cell growth in diﬀerent cell types. IGFBP-3 expression has been shown in both cardiomyocytes and endothelial cells. Doxorubicin exerts its apoptotic eﬀect through the ceramide death pathway and we have found that IGFBP-3 mediates its apoptotic action in cardiomyocytes. In this study we aimed to investigate the role of IGFBP-3 in endothelial cell apoptosis induced by doxorubicin. We evaluated (1) the expression of IGFBP-3 and IGF-I in human endothelial cells (HUVEC) exposed to doxorubicin (0.5 lM) by Western blot and radio-immunoassay; (2) the eﬀect of IGFBP-3 (1000 ng/ml) on cell survival and apoptosis of HUVEC exposed to doxorubicin and fumonisin B1 (FB1), a ceramide synthase inhibitor, by MTT, and FACS analysis; (3) IGF-I secretion in cells exposed to IGFBP-3 with or without doxorubicin and/or FB1; (4) the eﬀect of IGFBP-3 antisense oligonucleotides on cell survival and apoptosis (by HOECHST staining) in all experimental conditions; (5) cell motility and cytoskeletal changes of cells treated with IGFBP-3, doxorubicin and/or FB1. We found that IGFBP-3 was up-regulated upon doxorubicin-induced cell death, with no change in IGF-I secretion. Exogenous IGFBP-3 increased apoptosis of doxorubicin treated cells. The protective eﬀect of FB1 over doxorubicin-induced cell death and apoptosis was unexpectedly enhanced by IGFBP-3 while IGF-I secretion was dramatically increased under these conditions. IGFBP-3 antisense oligonucleotides counteracted apoptosis induced by doxorubicin but also reduced the protective eﬀect of both FB1 and FB1 + IGFBP-3 over doxorubicin-induced cell death. Finally, IGFBP-3 increased cell motility of HUVEC exposed to doxorubicin and FB1; under these conditions, cytoskeleton alterations appeared reduced by IGFBP-3. In conclusion, IGFBP-3 mediates endothelial cell apoptosis induced by doxorubicin but it is noteworthy that under blockade of ceramide synthesis, IGFBP-3 potentiates the protective eﬀect of fumonisin B1. Thus, it is likely that IGFBP-3 exerts complex modulation of the ceramide signaling pathway. IGFBP-3 directly or indirectly regulates even IGF-I release that, in turn, would per se exert antiapoptotic action. Study supported by University of Turin, MURST and SMEM Foundation
P28 DIFFERENTIAL EFFECTS OF IGFBP-3 ON APOPTOSIS OF BREAST EPITHELIAL CELLS ACCORDING TO APOPTOTIC TRIGGER C. Burrowsa, L.J. Shiryb, J.M.P. Hollya, C.M. Perksa. aDivision of Surgery, Bristol Royal Inﬁrmary, Marlborough Street, Bristol, BS2 8HW; bInsmed Incorporated, P.O. Box 2400, 4851 Lake Brook Drive, Glen Allen, VA 23058, USA Compelling evidence indicates that the IGF-system plays an important role in the development and progression of a number of epithelial cancers. The activity of IGFs within the body are tightly regulated by speciﬁc high aﬃnity binding proteins of which IGFBP-3 is the most prevalent. Epithelial cell expression of IGFBP-3 is increased by a number of negative growth regulators and cell stress mediators, including p53. We have previously demonstrated that IGFBP-3 can potentiate epithelial cell apoptosis induced by radiation and chemotherapeutic agents. This can be eﬀected by reducing the anti-apoptotic action of IGF-I, but also due to direct intrinsic actions of IGFBP-3. In view of further developing IGFBP-3 as a potential therapeutic, this study aimed to investigate the interactions of recombinant human (rhIGFBP-3) with various apoptotic triggers in a human breast cancer cell line Hs578T and a non-transformed breast epithelial line MCF10A. With the transformed Hs578T cells, rhIGFBP-3 potently enhanced apoptosis induced by ceramide, antimycin A and the UV-mimetic 4NQO with just 100 ng/ml enhancing cell death by between 50 and 200%. rhIGFBP-3 was less potent at enhancing paclitaxel-induced apoptosis with 500 ng/ml enhancing cell death by 20–25%. In contrast with the non-transformed MCF-10A cells, rhIGFBP-3 had a diﬀerential eﬀect depending upon the apoptotic trigger: enhancing paclitaxel-induced death (by 20–60%), but reducing ceramide-induced death (by 50%). The enhancing eﬀect of rhIGFBP-3 on ceramide induced apoptosis of the Hs578T cells was reversed when the cells were plated on ﬁbronectin, which induced a more polarized, anchorage-dependent phenotype; whereas the accentuating eﬀects on antimycin induced death were unchanged. These results conﬁrm that rhIGFBP-3 can potentiate apoptosis induced by a variety of chemotherapeutic agents. It further indicates that with selected agents, rhIGFBP-3 not only enhances eﬃciency of cell-death induction, but also adds speciﬁcity accentuating the death of transformed cells while protecting nontransformed cells. Acknowledgements Funding from AICR and The Needham Cooper Trust.
P29 IMPACT OF c-myb ON THE EXPRESSION OF INSULIN-LIKE GROWTH FACTORS (IGFs) AND IGF-BINDING PROTEINS IN HUMAN CHRONIC MYELOGENOUS LEUKEMIA CELLS Dae-Yeol Lee, Ho Keun Yi, Sun Yong Kim, Pyoung Han Hwang. Department of Pediatrics, Chonbuk National University Medical School, Jeonju, Jeonbuk, Korea The c-myb plays an important role in regulation of cell growth and diﬀerentiation. The c-myb gene is expressed at high levels in immature hematopoietic cells, and c-myb gene functions in normal and malignant hematopoiesis by regulating the G1/S transition in cycling hematopoietic cells. The present study examined the eﬀect of overexpression of c-myb on proliferation, and expression of IGF system in leukemia cells. K562 cells, human chronic myelogenous leukemia cells, and recombinant adenovirus containing human c-myb cDNA constructed by using standard technique were used in present study. To block c-myb function, we employed dominant negative (DN) gene construct strategy. Western immunoblot analysis of cell lysate
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showed that the level of c-myb protein was increased in c-myb virus infected K562 cells compared with empty virus infected control cells. K562 cells infected with c-myb virus showed signiﬁcant increase of growth at a MOI 100 with maximum 2 fold more growth rate compared with control cells. However, DN-myb infected leukemia cells is showed signiﬁcant reduction of cell growth compared with c-myb infected cells. The c-myb overexpression in K562 cells induced increase of IGF-I, -II and IGF-IR expressions with maximum 2 fold more expression. Of the six IGFBPs, the expressions of IGFBP-1, 3 and -6
were decreased in c-myb infected cells compared with control cells. In contrast, IGFBP-2, -4 and -5 expressions were markedly increased up to 2.7-fold by infection of c-myb in K562 cells. In addition, IGFBP3 protein was signiﬁcantly reduced in c-myb infected cell. The alteration in expression of IGF system induced by c-myb overexpression was completely blocked by DN-myb overexpression in K562 cells. These ﬁndings suggest that the role of c-myb in cell growth of leukemia cells is, at least in part, mediated through modulation of IGF–IGFBP axis.