Article |
Address correspondence to Thorsten Kirsch, Dept. of Orthopaedics and Rehabilitation, H089, Penn State College of Medicine, Hershey Medical Center, 500 University Dr., Hershey, PA 17033. Tel.: (717) 531-7788. Fax: (717) 531-1607. E-mail: tkirsch{at}psu.edu
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Abstract |
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Key Words: annexin; calcium; matrix vesicles; mineralization; retinoic acid
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Introduction |
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Annexins II, V, and VI are highly expressed in hypertrophic and mineralizing growth plate cartilage and in bone. These molecules belong to the annexin protein family, which have in common that they bind to acidic phospholipids in the presence of calcium (Crompton et al., 1988; Genge et al., 1989; Kirsch et al., 2000c, 2001). Annexin II and V each contain four 7080 amino acid repeats with an annexin consensus sequence, whereas annexin VI contains eight such repeats. These four or eight repeats form the conserved core region, which is responsible for the Ca2+-dependent binding of the proteins to phospholipids. In contrast, the NH2-terminal regions of the annexins are highly variable and may contribute to the specific functions of the various annexins (Geisow et al., 1988). Previous studies have shown that annexins II, V, and VI form Ca2+ channels when inserted into artificial phospholipid bilayers or mediate Ca2+ influx into liposomes (Berendes et al., 1993; Chen et al., 1993; Benz et al., 1996; Burger et al., 1996; Matsuda et al., 1997; Kirsch et al., 2000b). Recently, we have demonstrated that annexins II, V, and VI form Ca2+ channels in matrix vesicles (Kirsch et al., 2000b). Matrix vesicles are particles that are released from the plasma membrane of mineralizing cells. These vesicles have the critical role of initiating the mineralization process (Anderson, 1995; Kirsch et al., 1997b). The first mineral phase forms inside the vesicles in a protected environment. Once these intralumenal crystals have reached a certain size they rupture the vesicle membrane and grow out into the extracellular matrix (Anderson, 1995). For the formation of the first intralumenal crystals, phase channel or transporter systems are required to mediate influx of mineral ions into the vesicles. Annexins II, V, and VI enable the influx of Ca2+ into matrix vesicles and the formation of the first intralumenal mineral phase. The inhibition of annexin Ca2+ channel activities led to a loss of the ability of these vesicles to mineralize (Kirsch et al., 2000b). Other studies have shown that matrix vesicles contain a Na+-coupled Pi symport system that mediates the influx of inorganic phosphate into the vesicles (Montessuit et al., 1991, 1995). The total amounts of Ca2+ and Pi in matrix vesicles are significantly higher than those in adjacent cells or in the extracellular fluid. The vast majority of Ca2+ and about half of Pi are in insoluble, protein/lipid-bound form in the vesicles, which allows permanent annexin-mediated Ca2+ influx into the vesicles.
Ultrastructural studies have shown a high concentration of matrix vesicles in the reserve zone of growth plate cartilage and another peak concentration in the hypertrophic zone just before the onset of matrix mineralization (Reinholt et al., 1982; Buckwalter et al., 1987). Furthermore, we have described a qualitative difference between vesicles released by the various growth plate chondrocytes, and that only mineralization-competent growth plate chondrocytes release specialized matrix vesicles that are capable of initiating the mineralization process, whereas nonmineralizing growth plate chondrocytes release vesicles that do not mineralize (Kirsch et al., 1997b, 2000a). Thus, it is reasonable to hypothesize that chondrocytes regulate the mineralization process by controlling the release of specialized matrix vesicles and that only mineralization-competent chondrocytes release matrix vesicles that initiate the mineralization process. However, very little is known about the mechanisms involved in regulating the release of these mineralization-competent matrix vesicles from the plasma membrane of growth plate chondrocytes.
Calcium is recognized as an important regulatory element for many cellular processes. Increase in the cytosolic calcium concentration, [Ca2+]i, which occurs in many cell types after stimulation by hormones, controls a diverse range of cell functions including adhesion, motility, gene expression, and proliferation. Several studies have provided evidences that growth plate chondrocytes accumulate large amounts of cytosolic calcium just before the initiation of mineralization (Gunter et al., 1990; Kirsch et al., 1992; Iannotti et al., 1994). Nevertheless, little is known about the mechanisms involved in alterations of Ca2+ homeostasis in growth plate chondrocytes and the role of altered Ca2+ homeostasis in chondrocyte differentiation and mineralization. Interestingly, vesiculation or ectocytosis of the plasma membrane has been shown to be dependent on an influx of calcium in the cytoplasm of many cell types. For example, Ca2+ ionophores can cause microvesiculation and membrane shedding in a variety of cell types including chondrocytes, fibroblasts, neutrophils, and platelets (Wiedmer et al., 1990; Iannotti et al., 1994; Bucki et al., 1998). Thus, it is possible that annexins II, V, and VI form Ca2+ channels in hypertrophic growth plate chondrocytes leading to influx of Ca2+ and alterations of Ca2+ homeostasis. The altered Ca2+ homeostasis leads to the release of annexin-containing, mineralization-competent matrix vesicles.
Previously we and others have shown that retinoic acid (RA)* affects the release of annexin-containing matrix vesicles and stimulates mineralization of hypertrophic growth plate chondrocytes (Iwamoto et al., 1993; Kirsch et al., 2000a). To test our hypothesis that annexin-mediated alterations in Ca2+ homeostasis of growth plate chondrocytes might regulate the mineralization process of growth plate chondrocytes, we cultured hypertrophic growth plate chondrocytes in the presence of RA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable Ca2+ chelator, K-201, a specific annexin Ca2+ channel blocker (Kaneko, 1994; Kaneko et al., 1997; Kirsch et al., 2000b), or antibodies specific for annexin II, V, or VI. We measured changes in [Ca2+]i, and characterized matrix vesicles isolated from these cultures and the degree of mineralization in these cultures.
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Results |
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Discussion |
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Our results reveal that under certain physiological conditions, annexins are able to form Ca2+ channels. These findings present a new concept to regulate Ca2+ homeostasis by annexin Ca2+ channels. Annexins are cytosolic proteins, which in the presence of Ca2+, can bind to acidic phospholipids. However, channel formation not only requires membrane binding, but also membrane insertion. Previous studies have provided evidence that annexins V, VI, and XII can insert into the membrane and form Ca2+ channels at low pH in a Ca2+-independent manner (Isas et al., 2000; Golczak et al., 2001a, b). For example, annexin VI undergoes major conformational changes under low pH that leads to an increase of hydrophobicity (Golczak et al., 2001a,b). Furthermore, a biophysical study has demonstrated that the secondary structure of annexin V is affected by the combined interactions with Ca2+ and membrane components (Wu et al., 1999). Interestingly, a recent study has shown that annexin V mediates peroxide-induced Ca2+ influx into B cells under physiological pH (Kubista et al., 1999). These and our findings indicate that the cell not only can regulate gene expression of annexins, but also their channel formation, allowing a precise regulation of the biological consequences of annexin channel formation. It is not clear which factors regulate physiological channel formation by annexins. It is possible that a certain Ca2+ concentration is required for membrane binding and channel formation. Other possibilities are that intracellular acidification or, as we have shown previously, the lipid composition of the membrane, may play regulatory roles in annexin channel formation (Kirsch et al., 1997a). Furthermore, a previous study has shown that vitamin D caused an increase in fluidity of the plasma membrane (Swain et al., 1993). Thus, it is possible that RA has a similar effect on the plasma membrane that could favor annexin channel formation.
Our results and the findings showing that annexin V mediated peroxide-induced Ca2+ influx into B cells (Kubista et al., 1999) establish that annexin Ca2+ channels have important physiological functions in altering cell behavior and differentiation. In case of growth plate chondrocytes, annexin channel formation regulates Ca2+ homeostasis in these cells, and thereby the mineralization process including the release of mineralization-competent matrix vesicles. These vesicles contain annexins II, V, and VI as major components (Genge et al., 1989). We have demonstrated that these annexins also form Ca2+ channels in matrix vesicles, allowing Ca2+ influx into these particles and the formation of the first mineral phase inside the vesicles (Kirsch et al., 2000b). Thus, these annexins not only regulate Ca2+ homeostasis in growth plate chondrocytes, but they also play a direct role in the initiation of mineralization. Interestingly, our data indicate that annexin-mediated Ca2+ influx into growth plate chondrocytes up-regulates annexin gene expression. Thus, these annexins regulate their own gene expression, thereby possibly enabling sufficient amounts of annexins II, V, and VI to be available for the release of mineralization-competent matrix vesicles.
[Ca2+]i in hypertrophic chondrocytes is higher than the concentration in immature chondrocytes (Iannotti and Brighton, 1989; Gunter et al., 1990). As indicated by previous studies, this increase is most likely mediated by other Ca2+ channels than the annexins (Zimmermann et al., 1994; Zuscik et al., 1997). Initial increased [Ca2+]i might lead to annexin Ca2+ channel formation and subsequent further increase in [Ca2+]i. K-201 treatment, which inhibits Ca2+ channel activities of annexin II, V, and VI (Kirsch et al., 2000b), did not completely reduce RA-mediated increase in [Ca2+]i to levels in untreated cultures. However, K-201 reduced the increase by 87%, indicating that the majority of Ca2+ influx into growth plate chondrocytes is mediated by annexins. There are several reasons why K-201 did not completely inhibit RA-mediated increase in [Ca2+]i. First, we have shown that the concentration of K-201 required to completely block annexin II, V, and VImediated Ca2+ influx into artificial liposomes or matrix vesicles is 100 µM (Kirsch et al., 2000b). However, 100 µM K-201 is toxic to chondrocytes (unpublished data). Thus, it is possible that 20 µM K-201, which was not toxic to the cells, might not completely block annexin channels. Another possibility is that growth plate chondrocytes contain other Ca2+ channels that might contribute to the Ca2+ influx into RA-treated chondrocytes. Previous characterizations of Ca2+ channels in growth plate chondrocytes are controversial. One study provided evidence that growth plate chondrocytes contain N-type Ca2+ channels, whereas another study suggested that growth plate chondrocytes contain L-type Ca2+ channels (Zimmermann et al., 1994; Zuscik et al., 1997). Each antibody specific for annexin II, V, or VI decreased RA-mediated increase in [Ca2+]i by between 57 and 75%, revealing that all three annexins contribute to Ca2+ influx into growth plate chondrocytes and blocking the channel activities of one annexin only partially reduces RA-mediated increase in [Ca2+]i. Thus, our findings indicate that the annexins contribute the majority to the alteration of Ca2+ homeostasis in RA-treated growth plate chondrocytes and thereby regulate the mineralization process.
Annexin-mediated Ca2+ influx into growth plate chondrocytes may be directly involved in the release of mineralization-competent matrix vesicles. Vesiculation or ectocytosis of the plasma membrane has been shown to be dependent on an influx of Ca2+ into the cytoplasm of many cell types. Ca2+ ionophores can cause microvesiculation and membrane shedding in a variety of cell types, including chondrocytes, fibroblasts, neutrophils, and platelets (Wiedmer et al., 1990; Iannotti et al., 1994; Bucki et al., 1998). In contrast, when platelets were suspended in a solution containing EDTA, microparticle formation was inhibited (Wiedmer et al., 1990). Furthermore, we and others have provided evidence that matrix vesicles contain Ca2+-Pi-phospholipid or nucleational core complex. This core complex is required for the formation of the first mineral phase inside the vesicles (Kirsch et al., 1994; Wu et al., 1997). There is some evidence to suggest that this complex might already be assembled at the plasma membrane of the cell before matrix vesicles are released (Wuthier, 1982). Thus, it is possible that high [Ca2+]i is required for the assembly of this complex.
Our study indicates that RA action is required for completion of the chondrocyte maturation process, and it provides the first important insight into the underlying mechanisms. Interestingly, a previous study demonstrated that the perichondral tissue surrounding the cartilage contained large amounts of retinoids and that implantation of beads containing RA antagonist near the humeral anlagen severely affected humerus development (Koyama et al., 1999). Thus, it is likely that RA plays an important role in regulating mineralization and terminal differentiation of growth plate chondrocytes in vivo.
In conclusion, our findings provide evidence that RA treatment of growth plate chondrocytes leads to Ca2+ channel formation by annexins II, V, and VI and influx of Ca2+ into these cells. The annexin-mediated alteration in Ca2+ homeostasis regulates a whole sequence of events eventually leading to matrix mineralization, including up-regulation of annexin II, V, and VI gene expression, relocation of these annexins to the plasma membrane, and release of APase- and annexin-containing matrix vesicles that initiate the mineralization process.
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Materials and methods |
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Measurement of [Ca2+]i
Chondrocytes were trypsinized first, and then 2 x 106 cells were incubated with 4 µM of fura-2AM (Molecular Probes, Inc.) at 37°C for 15 min in complete medium. [Ca2+]i was measured as described previously (Kirsch et al., 1992). Briefly, labeled cells were resuspended in measuring buffer ([mM] 140 NaCl, 5 KCl, 1 CaCl2, 20 Hepes, 1 NaH2PO4, 5.5 glucose, pH 7.4), transferred to a cuvette (magnetically stirred), and fluorescence was measured in a fluorimeter (Photon Technology Instruments) using the excitation wavelength of 340 nm (Ca2+-bound form of fura-2AM) and the emission wavelength of 510 nm. The fluorescence maximum (Fmax) was determined by addition of 2 pM ionomycin (Calbiochem-Novabiochem">Calbiochem-Novabiochem), and the fluorescence minimum (Fmin) was determined in the presence of 1 mM EDTA/10 mM Tris. [Ca2+]i was calculated according to the following equation: ([Ca2+]i = Kd x [(F - Fmin)/(Fmax - F)] with Kd = 224 nM (Grynkiewicz et al., 1985).
Isolation of total RNA and real-time PCR
Total RNA was isolated from chondrocyte cultures after 1-, 2-, 3-, 4-, 5-, and 6-d treatments using RNeasy Mini Kit (QIAGEN). 1 µg RNA was reverse transcribed using Ominiscript RT Kit (QIAGEN). Then a 1:100 dilution of the resulting cDNA was used as the template to quantitate the relative content of mRNA by real-time PCR (ABI PRISM 7700 sequence detection system) using respective primers and SYBR Green. The following primers for real-time PCR were designed using Primers Express software: annexin II, forward primer 5'-CATGCCTATCTGCTCTTCGTT-3', reverse primer 5'-AGCCACCACACCGTCCATAA-3'; annexin V, forward primer 5'-AGAGACATCAGGCCATTTTCAGA-3', reverse primer 5'-CTGCCATCAGGATCTCTATTTGC-3'; annexin VI, forward primer 5'-GCGGCTGATTGTAAGCTTGAT-3', reverse primer 5'-GTCGGTGGTCCAGCACTTA-3'; and type X collagen, forward primer 5'-AGTGCTGTCATTGATCTCATGGA-3', reverse primer 5'-TCAGAGGAATAGAGACCATTGGATT-3'. PCR reactions were performed with TaqMan PCR master mix kit (Applied Biosystems). The 18 S RNA was amplified at the same time and used as an internal control. The cycle threshold values for 18 S RNA and that of the samples were measured and calculated by computer software. Relative transcript levels were calculated as x = 2-Ct, in which
Ct =
E -
C, and
E = Ctexp - Ct18S;
C = Ctctl - Ct18S.
Isolation of matrix vesicles
Matrix vesicles were isolated from chondrocyte cultures by enzymatic digestion as described previously (Kirsch et al., 1997b). Briefly, adherent chondrocyte layers were washed with PBS and incubated with crude collagenase (500 U/ml; type IA; Sigma Chemical Co.) in HBSS at 37°C for 3 h. Matrix vesicles were harvested by different ultracentrifugation.
Ca2+ uptake by matrix vesicles
Ca2+ uptake by matrix vesicles was assayed by incubating matrix vesicle aliquots (100 µg of protein) in 400 µl of synthetic cartilage lymph solution at 37°C for 24 h. After incubation, matrix vesicles were pelleted, washed twice in TNE buffer (TES pH 7.4, NaCl 1.5 mM and EDTA 2 µM), resuspended in TNE buffer containing 1 µM fura-2, transferred to a cuvette, and fluorescence was measured in fluorimeter using the excitation wavelength of 340 nm and the emission wavelength of 510 nm. Matrix vesicles were lysed with 1% Triton X-100 to release intravesicular calcium.
Recombinant annexin proteins and antibodies
Recombinant annexins II, V, and VI were prepared using the pGEX expression vector (Amersham Pharmacia Biotech) as described previously (Kirsch et al., 1997a). The preparation of antibodies specific for annexins II, V, and VI were also described previously (Kirsch et al., 2000c, 2001).
SDS-PAGE and immunoblotting
Samples were dissolved in 3% SDS sample buffer with DTT, denatured at 100°C for 3 min, and analyzed by electrophoresis in 10 or 12% (wt/vol) SDSpolyacrylamide gels. Samples were electroblotted onto nitrocellulose filters after electrophoresis. After blocking with a solution of low-fat milk protein, blotted proteins were immunostained with primary antibodies followed by peroxidase-conjugated secondary antibody, and the signal was detected by enhanced chemiluminescene (Pierce Chemical Co.).
Measurement of APase activity and protein content
The measurements of APase activity and protein content were described previously (Kirsch et al., 1997b). Cells or vesicles were washed in PBS and suspended in 10 mM Tris-HCl, pH 7.5, 0.1% Triton X-100, and 0.5 mM MgCl2. Protein content was analyzed by BCA assay (Pierce Chemical Co.). APase activity was determined using p-nitrophenyl phosphate as a substrate (Tietz et al., 1983).
Alizarin red S staining
To determine the degree of mineralization in chondrocyte cultures, cultures were stained with alizarin red S. After washing, chondrocyte cultures were fixed with 70% ethanol for 10 min, and then stained with 0.5% alizarin red S in H2O, pH 4.0, for 5 min at room temperature. After staining, cultures were washed three times with H2O followed by 70% ethanol. To quantify matrix mineralization, the alizarin red Sstained cultures were incubated with 100 mM cetylpyridinium chloride for 1 h to solubilize and release calcium-bound alizarin red into solution (Johnson et al., 2001). The absorbance of the released alizarin red S was measured at 570 nm using a spectrophotometer. Data are expressed as units of alizarin red S released per milligram of protein in each culture.
Statistical analysis
Numerical data are presented as mean + SD (n > 4) and statistical significance between groups was identified using the two-tailed t test (P values are reported in the figure legends).
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Footnotes |
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Acknowledgments |
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Submitted: 4 March 2002
Revised: 17 April 2002
Accepted: 19 April 2002
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References |
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