Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage

Annexins II, V, and VI are major components of matrix vesicles (MV), i.e. particles that have the critical role of initiating the mineralization process in skeletal tissues. Furthermore, types II and X collagen are associated with MV, and these interactions mediated by annexin V stimulate Ca(2+) uptake and mineralization of MV. However, the exact roles of annexin II, V, and VI and the interaction between annexin V and types II and X collagen in MV function and initiation of mineralization are not well understood. In this study, we demonstrate that annexin II, V, or VI mediate Ca(2+) influx into phosphatidylserine (PS)-enriched liposomes, liposomes containing lipids extracted from authentic MV, and intact authentic MV. The annexin Ca(2+) channel blocker, K-201, not only inhibited Ca(2+) influx into fura-2-loaded PS-enriched liposomes mediated by annexin II, V, or VI, but also inhibited Ca(2+) uptake by authentic MV. Types II and X collagen only bound to liposomes in the presence of annexin V but not in the presence of annexin II or VI. Binding of these collagens to annexin V stimulated its Ca(2+) channel activities, leading to an increased Ca(2+) influx into the liposomes. These findings indicate that the formation of annexin II, V, and VI Ca(2+) channels in MV together with stimulation of annexin V channel activity by collagen (types II and X) binding can explain how MV are able to rapidly take up Ca(2+) and initiate the formation of the first crystal phase.     

L-type calcium channels in growth plate chondrocytes participate in endochondral ossification

Longitudinal bone growth occurs by a process called endochondral ossification that includes chondrocyte proliferation, differentiation, and apoptosis. Recent studies have suggested a regulatory role for intracellular Ca(2+) (Ca(i) (2+)) in this process. Indirect studies, using Ca(2+) channel blockers and measurement of Ca(i) (2+), have provided evidence for the existence of Ca(2+) channels in growth plate chondrocytes. Furthermore, voltage-gated Ca(2+) channels (VGCC), and specifically L- and T-type VGCCs, have been recently described in murine embryonic growth plates. Our aim was to assess the effect of L-type Ca(2+) channel blockers on endochondral ossification in an organ culture. We used cultures of fetal rat metatarsal rudiments at 20 days post gestational age, with the addition of the L-type Ca(2+) channel blockers verapamil (10-100 microM) or diltiazem (10-200 microM) to the culture medium. Longitudinal bone growth, chondrocyte differentiation (number of hypertrophic chondrocytes), and cell proliferation (incorporation of tritiated thymidine) were measured. Verapamil dose-dependently decreased growth, the number of hypertrophic chondrocytes, and cell proliferation, at concentrations of 10-100 microM. Growth and the number of hypertrophic chondrocytes decreased significantly with diltiazem at 50-100 microM, and proliferation decreased significantly at concentrations of 10-200 microM. Additionally, there was no increase in apoptosis over physiological levels with either drug. We confirmed the presence of L-type VGCCs in rat rudiments using immunohistochemistry, and showed that the antagonists did not alter the pattern of VGCC expression. In conclusion, our data suggest that L-type Ca(2+) channel activity in growth plate chondrocytes is necessary for normal longitudinal growth, participating in chondrocyte proliferation and differentiation.     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Tissue transglutaminase expression in quail epiphyseal chondrocytes.

Tissue transglutaminase (tTGase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification via epsilon(gamma-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation.tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation.Our findings lend support to the idea that tTGase might play a role in non-dividing cultured chondrocytes.     

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.     

Annexin V counteracts apoptosis while inducing Ca(2+) influx in human lymphocytic T cells

We have previously shown that when annexin V is present during the execution of a cell death program, apoptosis is delayed. This is reflected by the inhibition of DNA cleavage and of the release of apoptotic membrane particles, and by reduction of the proteolytic processing of caspase-3. Here, we have studied the mechanism(s) through which annexin V counteracts apoptosis in the human CEM T cell line. The degree of apoptosis inhibition was associated with an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). Reduction of the extracellular Ca(2+) concentration by EGTA abolished the anti-apoptotic effect, suggesting that annexin V favors Ca(2+) influx and that Ca(2+) acts as an inhibitor rather than an activator of apoptosis in CEM T cells. The effects on apoptosis and [Ca(2+)](i) of several modified annexins with different electrophysiological properties indicate that the N-terminal domain of annexin V is necessary for the Ca(2+)-dependent anti-apoptotic action of annexin V. These results suggest that annexin V regulates membrane Ca(2+) permeability and is protective against apoptosis by increasing [Ca(2+)](i) in CEM T cells.     

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues, but the ways in which they exert this key function remain poorly understood. This issue is made even more intriguing by the fact that matrix vesicles are also present in nonmineralizing tissues. Thus, we tested the novel hypothesis that matrix vesicles produced and released by mineralizing cells are structurally and functionally different from those released by nonmineralizing cells. To test this hypothesis, we made use of cultures of chick embryonic hypertrophic chondrocytes in which mineralization was triggered by treatment with vitamin C and phosphate. Ultrastructural analysis revealed that both control nonmineralizing and vitamin C/phosphatetreated mineralizing chondrocytes produced and released matrix vesicles that exhibited similar round shape, smooth contour, and average size. However, unlike control vesicles, those produced by mineralizing chondrocytes had very strong alkaline phosphatase activity and contained annexin V, a membrane-associated protein known to mediate Ca²⁺ influx into matrix vesicles. Strikingly, these vesicles also formed numerous apatite-like crystals upon incubation with synthetic cartilage lymph, while control vesicles failed to do so. Northern blot and immunohistochemical analyses showed that the production and release of annexin V-rich matrix vesicles by mineralizing chondrocytes were accompanied by a marked increase in annexin V expression and, interestingly, were followed by increased expression of type I collagen. Studies on embryonic cartilages demonstrated a similar sequence of phenotypic changes during the mineralization process in vivo. Thus, chondrocytes located in the hypertrophic zone of chick embryo tibial growth plate were characterized by strong annexin V expression, and those located at the chondro–osseous mineralizing border exhibited expression of both annexin V and type I collagen. These findings reveal that hypertrophic chondrocytes can qualitatively modulate their production of matrix vesicles and only when induced to initiate mineralization, will release mineralization-competent matrix vesicles rich in annexin V and alkaline phosphatase. The occurrence of type I collagen in concert with cartilage matrix calcification suggests that the protein may facilitate crystal growth after rupture of the matrix vesicle membrane; it may also offer a smooth transition from mineralized type II/type X collagen-rich cartilage matrix to type I collagen-rich bone matrix.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.     

Parathyroid hormone-related peptide (PTHrP)-dependent and -independent effects of transforming growth factor beta (TGF-beta) on endochondral bone formation.

Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in skeletal tissue resulted in increased hypertrophic differentiation in growth plate and articular chondrocytes, suggesting a role for TGF-beta in limiting terminal differentiation in vivo. Parathyroid hormone-related peptide (PTHrP) has also been demonstrated to regulate chondrocyte differentiation in vivo. Mice with targeted deletion of the PTHrP gene demonstrate increased endochondral bone formation, and misexpression of PTHrP in cartilage results in delayed bone formation due to slowed conversion of proliferative chondrocytes into hypertrophic chondrocytes. Since the development of skeletal elements requires the coordination of signals from several sources, this report tests the hypothesis that TGF-beta and PTHrP act in a common signal cascade to regulate endochondral bone formation. Mouse embryonic metatarsal bone rudiments grown in organ culture were used to demonstrate that TGF-beta inhibits several stages of endochondral bone formation, including chondrocyte proliferation, hypertrophic differentiation, and matrix mineralization. Treatment with TGF-beta1 also stimulated the expression of PTHrP mRNA. PTHrP added to cultures inhibited hypertrophic differentiation and matrix mineralization but did not affect cell proliferation. Furthermore, terminal differentiation was not inhibited by TGF-beta in metatarsal rudiments from PTHrP-null embryos; however, growth and matrix mineralization were still inhibited. The data support the model that TGF-beta acts upstream of PTHrP to regulate the rate of hypertrophic differentiation and suggest that TGF-beta has both PTHrP-dependent and PTHrP-independent effects on endochondral bone formation.     

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

The growth arrest and DNA damage-inducible 45beta (GADD45beta) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45beta in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45beta as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45beta mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45beta protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45beta(-/-) mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45beta in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45beta stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45beta plays an essential role during chondrocyte terminal differentiation.     

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.     

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453–462, 1998. © 1998 Wiley-Liss, Inc.     

Altered expression and localization of N-myristoyltransferase in experimentally induced rat model of ischemia-reperfusion

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation.     

Growth plate chondrocyte maturation is regulated by basal intracellular calcium

Among the cellular events that are associated with the process of endochondral ossification is an incremental increase in chondrocyte basal intracellular free Ca(2+) concentration ([Ca(2+)](i)) from 50 to 100 nM. To determine if this rise in [Ca(2+)](i) functionally participates in the maturational process of growth plate chondrocytes (GPCs), we examined its effect on several markers of hypertrophy, including annexin V, bone morphogenetic protein-6, type X collagen, and indian hedgehog. Expression of these genes was determined under conditions either where the Ca(2+) chelator EGTA was used to deplete extracellular Ca(2+) and lower [Ca(2+)](i) to < 50 nM or where the extracellular addition of 5 mM CaCl(2) was used to elevate [Ca(2+)](i) to > 100 nM. Although no effect on the expression of these genes was observed following treatment with 5 mM CaCl(2), 4 mM EGTA significantly inhibited their expression. This effect was recapitulated in sternal chondrocytes and was reversed following withdrawal of EGTA. Based on these findings, we hypothesized that the EGTA-induced suppression of these genes was mediated by a factor whose expression is responsive to changes in basal [Ca(2+)](i). Since EGTA mimicked the effect of parathyroid hormone-related peptide (PTHrP) on GPC maturation, we examined the effect of low [Ca(2+)](i) on PTHrP expression. Suggesting that low [Ca(2+)](i) suppression of hypertrophy was PTHrP-dependent in GPCs, (a) treatment with 4 mM EGTA increased PTHrP expression, (b) the EGTA effect was rescued by blocking PTHrP binding to its receptor with the competitive antagonist TIP(7-39), and (c) EGTA could mimic the PTHrP stimulation of AP-1 binding to DNA. Additionally, PTHrP promoter analysis identified a domain (-1498 to -862, relative to the start codon) involved with conferring Ca(2+) sensitivity to the PTHrP gene. These findings underscore the importance of cellular Ca(2+) in GPC function and suggest that PTHrP action in the growth plate is at least partially regulated by changes in basal [Ca(2+)](i).