Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts

Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts

Osteoarthritis and Cartilage (1998) 6, 324–329 7 1998 Osteoarthritis Research Society 1063–4584/98/050324 + 06 $12.00/0 Intracellular calcium respon...

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Osteoarthritis and Cartilage (1998) 6, 324–329 7 1998 Osteoarthritis Research Society

1063–4584/98/050324 + 06 $12.00/0

Intracellular calcium responses to basic calcium phosphate crystals in fibroblasts By Paul B. Halverson*, Andrew Greene† and Herman S. Cheung‡ *Division of Rheumatology and †Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226; (Department of Medicine [Arthritis], University of Miami School of Medicine and Geriatric Research, Education and Clinical Center, VA Medical Center, Miami, Florida 33135, U.S.A. Summary Objective: To examine the intracellular calcium response to basic calcium phosphate (BCP) crystals in fibroblasts. Design: In this study, intracellular calcium [Ca2 + ]i levels in fibroblasts were determined using the photoactive dye, fura-2. Interruption of these responses was accomplished by either removal of Ca2 + from the extracellular medium or addition of ammonium chloride that inhibits intracellular dissolution of BCP crystals by alkalinizing phagolysosomes. The effects of such interruptions on BCP induction expression of proto-oncogenes were demonstrated by the Northern blot analysis. Results: Addition of media containing BCP crystals yielded an immediate 10-fold rise of [Ca2 + ]i over the baseline level in human fibroblasts. This peak was derived mostly from extracellular calcium and was not seen when BCP crystals in calcium-free media were added to fibroblasts. The [Ca2 + ]i concentration returned to the baseline level within 8 min. A second rise of [Ca2 + ]i started at 60 min and continued to increase up to at least 3 h. This peak was derived from intracellular dissolution of phagocytosed crystals and almost completely inhibited by 10 mm ammonium chloride. Conclusion: The initial transient [Ca2 + ]i increase probably serves as a second messenger leading to activation of early cellular responses such as c-fos expression which is important in BCP crystal-induced mitogenesis. The second, slower and more sustained rise of [Ca2 + ]i probably initiates other cellular processes needed for fibroblast mitogenesis. Key words: Calcium, Fura-2, Basic calcium phosphate crystals, Fibroblasts.


by a process similar to that of platelet-derived growth factor (PDGF) [3]. Moreover, BCP crystals and PDGF exert other similar biologic effects on cultured cells, such as stimulation of prostaglandin E2 (PGE2) production via the phospholipase A2/cyclo-oxygenase pathway [4], activation of phospholipase C and inositol phospholipid hydrolysis [5], induction of collagenase and neutral protease synthesis [6, 7], and induction of protooncogenes (c-fos and c-myc) [8, 9]. Based on these data, we proposed a mechanism for BCP crystal-induced mitogenesis in which at least two distinct events were required [9]. The first is a fast membrane associated event in which phospholipase C activation leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate [PIP2] to inositol trisphosphate (IP3) and diacylglycerol (DAG) [5, 9]. Both IP3 and DAG are intracellular messengers [10]. IP3 releases calcium from endoplasmic reticulum, modulating the activities of

The arthropathies associated with calcium-containing crystals, which include calcium pyrophosphate dihydrate (CPPD) [1] and basic calcium phosphate (BCP) [2] are a group of clinically heterogeneous arthritides representing a significant source of morbidity in the elderly. The etiology of these diseases is unknown. Concentrations of calcium-containing crystals found in human joint fluid stimulate fibroblast, synovial cell, and chondrocyte mitogenesis in vitro Received 6 January 1998; accepted 7 May 1998. Address correspondence to: Herman S. Cheung, Ph.D., Dept. of Medicine [Arthritis], University of Miami, School of Medicine and Geriatric Research, Education, and Clinical Center, VA Medical Center, Miami, Florida 33135. Tel: 001 (305) 243-5735, Fax: 001 (305) 243-5655; E-mail: hcheung This work is supported in part by USPHS grant AR-38421-10 and a VA Merit Review grant.


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calcium-dependent and calcium/calmodulin-dependent enzymes such as protein kinases and proteases [11]. DAG is the other hydrolysis product of PIP2. It is a potent activator of the serine/ threonine protein kinase C, which can phosphorylate intracellular proteins such as growth factor receptors, ribosomal subunit, and cytoskeletal components [12–14]. DAG also may activate membrane Na + /H + antiporter, causing a rise in cellular pH, a correlate of stimulated DNA synthesis [15]. The second event occurs at least an hour later and involves endocytosis and intracellular dissolution of BCP crystals. BCP crystal dissolution raises the intracellular calcium level and is required for induction of mitogenesis by calciumcontaining crystals [9]. In the present studies, we have tested this hypothesis by examining the [Ca2 + ]i in human fibroblasts in response to BCP crystal stimulation. Further, interruption of these responses was accomplished by removal of Ca2 + from the extracellular medium or addition of ammonium chloride [NH4Cl] which inhibits intracellular dissolution of BCP crystals by alkalinizing phagolysosomes [16, 17]. The effects of such interruptions on BCP induction expression of proto-oncogenes were demonstrated by the Northern blot analysis. Materials and Methods Tritiated thymidine (50 Ci/mmole) was from Amersham (Arlington Heights, IL, U.S.A.). Alpha labeled 32P dATP (3000 Ci/mmole) was obtained from ICN (Irvine, CA, U.S.A.). Fetal bovine serum [FBS], Hank’s buffered saline solution [HBSS], penicillin–streptomycin–fungizone, and Dulbecco’s modified Eagle’s medium [DMEM] were supplied by GIBCO Laboratories (Grand Island, NY, U.S.A.). Human foreskin fibroblasts were grown to confluence on a glass coverslip (or plastic culture dishes where applicable) in DMEM with 10% FBS and 1% penicillin–streptomycin–fungizone. Cultures were ‘downshifted’ to 0.5% FBS 24 h before experiments in order to synchronize cells in a Go phase. Intracellular calcium Cells were loaded with 3 mm Fura-2 AM and 0.3% Pluronic acid (Molecular Probes, Eugene, OR) for 45 min. After incubation, cells were superfused with HBSS to remove any nonhydrolyzed dye. Coverslips were placed in a specially designed,


thermostatically controlled perfusion chamber affixed to the stage of an inverted microscope (Diaphot, Nikon Inc., Japan) equipped for epifluorescence. Video images were obtained with an intensified CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). The excitation wavelengths were selected by narrow band filters (10 nm) centered at 340 and 380 nm. Quartz neutral-density filters were used to equalize the delivered intensities for the two wavelengths. Illumination frequencies were switched rapidly ( Q 10 ms) by a filter wheel (Sutter Instruments, Raleigh, NC, U.S.A.). Fluorescence emission was filtered through a longpass filter and a 500 nm narrow band filter. Images were digitized using a 512 × 512 A/D board (Matrox, Dorval, Quebec) on a 486 personal computer (Gateway, North Sioux City, SD, U.S.A.), and analyzed using the Image-1 software (Universal Imaging, Westchester, PA, U.S.A.) by the ratiometric method described below. Data analysis to obtain [Ca2 + ]i was computed by the ratio method as follows. Individual cells or groups of cells in each field were identified in the images and manually traced on the computer screen to obtain the intracellular fluorescent intensity at each wavelength and time point. The following formula was used the for subsequent computations of [Ca2 + ]i: [Ca2 + ]i = Kd (Sf2/Sb2) [(R − Rmin)/(Rmax − R)] where Sf2 and Sb2 are the fluorescence of the free and bound form of the dye respectively, R is the measure of the 340/380 fluorescence, Rmax is the maximum ratio determined by adding 50:M of the calcium ionophore BR-A23187 (Molecular Probes) to approximate [Ca2 + ]i equal to [Ca2 + ]o. Rmin is the minimum ratio obtained by adding 10 mm EGTA to the bath to chelate Ca2 + . Kd was determined as described below. The determination of the intracellular dissociation constant (Kd) of the fura-2–Ca2 + complex was carried out in a separate group of cells using methods which have been described by others [18]. Cells were labeled with fura-2, treated as described above and studied on the same imaging microscope. Calcium Standard solutions (Molecular Probes) buffered with ethylene glycolbis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and containing 30 mm 4-bromo-A23187 (Molecular Probes) were introduced into the cell chamber. At least 10 cells were imaged at each of the 10 calcium concentrations (1 mm–1 mm). Kd was determined as the x intercept of the plot of log[8(R − Rmin)/(Rmax − R)] on the y axis versus − log[Ca2 + ] on the x axis assuming a 1:1 complex of fura-2 and Ca2 + [19].


Halverson et al.: Intracellular calcium to calcium crystals

In situ calibration in our system yielded an estimate of Kd of 367 2 16 nm. This value was used in all subsequent determinations of [Ca2 + ]i. BCP crystals were synthesized by the method of Bett et al. [20] and sized to Q 10 mm. ‘Large’ BCP crystals, 46 mm, were provided by CeraMed (Lakewood, CO, U.S.A.). The early time course of [Ca2 + ]i was determined by rapid sampling after the addition of 25 mg/cm2 BCP crystals with observations made up to 14 min. Intracellular calcium levels were also determined in separate groups of cells at fixed time points up to 3 h after addition of crystals. In these experiments, BCP crystals were added earlier to culture dishes and the dishes replaced in the incubator. Fura-2 was added 45 min before testing. The maximum length of time any slide was on the microscope was 20 min. Monosodium urate monohydrate [MSU] crystals, sized 10–30 mm, were prepared from twicerecrystallized uric acid and sodium hydroxide (Sigma, St. Louis, MO, U.S.A.) as previous described [21]. Control cell stimulation was accomplished with 5 ng/ml epidermal growth factor (EGF) (Upstate Biotechnology, Inc. NY, U.S.A.) or 200 mg/ml of MSU crystals. Inhibition of intracellular crystal dissolution was achieved by addition 10 mm NH4Cl to the culture media at the time BCP crystals were added [22]. cdna probes The c-fos probe was a 1.3 kb Pst1 v-fos fragment from the pfos-1/plasmid supplied by I Verma (Salk Institute, San Diego, CA, U.S.A.) [23]. Probes were randomly primed with kits from Boehringer Mannheim (Indianapolis, IN, U.S.A.). northern blot analysis Northern blot analysis of total cellular RNA was used to examine the effect of Ca2 + -free media on the expression of c-fos after stimulation by either BCP crystals. Control cultures were incubated in HBSS with or without Ca2 + . At times specified after stimulation, cells were scraped from 100 mm plates, washed in physiological-buffered saline and disrupted in guanidinium isothiocyanate. The RNA was then precipitated in 4 m lithium chloride as described by Cathala et al. [24]. RNA (5 mg/lane) was electrophoresed on formaldehyde gels and tranferred to nitrocellulose filters as described previously [25]. The filters were hybridized with inserts purified from plasmid carrying sequences of the desired gene. The inserts were labeled to a specific activity greater than 108 cpm/mg using the random primer method [26].

Fig. 1. Time course of intracellular calcium in fibroblasts following addition BCP crystals in calcium-containing HBSS (open circles) or in Ca2 + and Mg2 + free HBSS. Later time points represent different experiments.

Results [Ca2 + ]i The [Ca2 + ]i concentration in response to BCP crystal stimulation is summarized in Fig. 1. The baseline [Ca2 + ]i concentration in fibroblasts was 268 2 35 nm (mean 2 s.e.m.) in HBSS and 255 2 16 nm in calcium free HBSS. Following the addition of BCP crystals, calcium level rose within 1 min to 2288 2 169 nm. Calcium concentrations continued to rise during the first 6 min reaching approximately 3400 nm and then declined to near baseline by 8 min. A second slow rising calcium peak appeared at about 60 min and gradually increased to 3000 nm at 180 min. In the absence of crystals neither peak was observed. When the cells were stimulated with BCP crystals in calcium free HBSS, the initial calcium peak was marked reduced but the second peak was unaffected. This suggested that Ca2 + influx from extracellular media was primarily responsible for the initial rise of [Ca2 + ]i (Fig. 1). To examine whether the second rise in [Ca2 + ] is due to endocytosis and intracellular dissolution of BCP crystal, cells were stimulated with BCP crystals in the presence or absence of 10 mm NH4Cl. While NH4Cl did not altered the initial rise of [Ca2 + ]i, it did abolish the second calcium peak

Fig. 2. Time course of intracellular calcium in fibroblasts following addition of non-phagocytosable 46 mm BCP crystals (open circles) and the effect of 10 mm NH4Cl on the dissolution of Q 20 mm BCP crystals (closed circles).

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BCP crystals, in contrast to MSU and CPPD crystals, do not produce severe gout-like inflammatory attacks or accumulation of PMNs within a joint. Nevertheless, in osteoarthritis, the presence of BCP crystals is associated with larger joint effusions [28] and greater joint damage [29] suggesting a possible role for BCP crystals in joint destruction. In vitro, fibroblasts which phagocytose BCP crystals undergo multifaceted responses including prostaglandin release, protease production, and mitogenesis. Prostaglandin release and metalloproteinase production are not specific to BCP crystals, having been observed with other crystals [30] and noncrystalline particles [31].

Fig. 3. Time course of intracellular calcium in fibroblasts following addition of monosodium urate monohydrate crystals in calcium-containing HBSS (circles) or in Ca2 + and Mg2 + free HBSS (squares).

(Fig. 2). Large crystals (46 mm) which were not phagocytosed as observed by light microscopy (data not shown) yielded an early peak of approximately 2000 nm but only a small elevation in [Ca2 + ]i level after 2 h (Fig. 2). Stimulation by control agents (Fig. 3) such as EGF and MSU (non-calcium containing crystals) yielded only the initial calcium rise, e.g., EGF stimulated an initial calcium peak of 2629 2 462 nm but no secondary calcium rise.



significance of [Ca2 + ]i BCP crystal induction of c-fos expression was significantly reduced in the Ca2 + free HBSS media as compared with control [Fig. 4]. Since removal of calcium from the extracellular media abolished the initial transient rise of [Ca2 + ]I, but did not block the expression of C-fos completely, it suggested that other signals are also required for c-fos responses and mitogenesis [8, 9].

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Discussion MSU and CPPD crystals form in joints and may cause attacks of gout or pseudogout, respectively. These attacks of arthritis are associated with influxes of polymorphonuclear leukocytes (PMNs), and crystals are subsequently phagocytosed by PMNs and macrophages. Experimentally, addition of MSU or CPPD crystals to PMN causes a dramatic rise in intracellular calcium which peaks at 60–80 s and then declines to baseline [27].




Fig. 4. Northern blot analysis demonstrating c-fos accumulation in fibroblasts as follows: (a) HBSS alone, (b) BCP crystals + Ca2 + and Mg2 + free HBSS, (c) BCP crystals + HBSS.


Halverson et al.: Intracellular calcium to calcium crystals

Mitogenesis, though, requires calcium containing crystals. Thus, we have hypothesized that calcium containing crystals are metabolized by cells, releasing calcium which drives the cells to divide [32]. In these experiments, we have observed a biphasic calcium response to BCP crystals. The initial transient calcium peak lasted only 8 min before baseline [Ca2 + ]i concentration was re-established. The source of the transient calcium peak appeared to be primarily from extracellular calcium. The small transient rise of calcium observed when cells were stimulated with BCP crystals in the Ca2 + -free HBSS may be due to IP3 mediated calcium release from intracellular storage as suggested from our previous study [5] [Fig. 1]. Since both the large [46 mm] nonphagocytosable and the smaller [10 mm] BCP crystals induced this initial calcium response, it would suggest that crystal endocytosis is not required. The physical contact between crystal and cell was sufficient to initiate the Ca2 + influx. The mechanism of Ca2 + transposition induced by BCP crystals is unknown at present but could result from the opening of calcium channels or, alternatively, creation of membrane ‘pores’ within the cell membrane as a result of the crystal–membrane interaction [33]. This transient [Ca2 + ]i increase probably serves as a second messenger leading to activation of early cellular responses such as c-fos expression [Fig. 5]. C-fos expression is one of earliest cellular responses in BCP crystal and peptide growth factor stimulation and appeared to be important in both BCP crystal or peptide growth factor-induced mitogenesis [9, 23, 34]. A second rise in Ca2 + was detected at 60 min and continued to increase for at least 180 min up to 3000 nm (Fig. 1). Some error is encountered in the measurement of intracellular calcium at levels greater than 2000 nm, but we believe the error is small ( Q 15%) up to 4000 nm. The source of this calcium appeared to be derived from intracellular BCP crystal dissolution since it was observed even in the absence of extracellular sources of calcium. This calcium rise was largely inhibitable by NH4Cl which had been shown to block lysosomal dissolution of BCP crystals [22]. Also, the second calcium rise was not observed with large, nonphagocytosable BCP crystals [Fig. 2] nor with control agents such as EGF and non-calcium containing crystals [MSU crystals], even though the primary, membrane-related transient calcium peak was still present [Fig. 3]. These data taken together confirming that the requirement of calcium-containing crystal endocytosis and

intracellular dissolution for the second calcium peak. In summary, the present studies substantiate our original hypothesis that BCP crystals induce a biphasic [Ca2 + ]i response in fibroblasts. However, the data suggest a modification of the model in that most of the initial calcium rise appears to come from the extracellular environment rather than from IP3 mediated intracellular calcium release as suggested by the hypothesis [9, 35]. This transient [Ca2 + ]i increase probably serves as a second messenger leading to activation of early cellular responses such as c-fos expression [Fig. 4] which appeared to be important in BCP crystal and peptide growth factor-induced mitogenesis [9, 23, 34]. Following the initial calcium rise, BCP crystals produce a persisting state of intracellular ‘hypercalcemia’ which appears to result from lysosomal dissolution of BCP crystals. The second, slower sustained rise of [Ca2 + ]i probably initiates other cellular processes needed for fibroblast mitogenesis.

Acknowledgments The authors gratefully acknowledge comments of Dr L. M. Ryan.



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