1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.     

Vitamin D3 metabolites regulate LTBP1 and latent TGF-beta1 expression and latent TGF-beta1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.     

Transforming growth factor-beta and Wnt signals regulate chondrocyte differentiation through Twist1 in a stage-specific manner

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-beta and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, whereas growth plate-extracted cells displayed a hypertrophic phenotype. TGF-beta significantly induced beta-catenin in immature chondrocytes, whereas it repressed it in mature chondrocytes. TGF-beta further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it inhibited Topflash activity in a time-dependent manner in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-beta induced Sma- and Mad-related protein 3 interaction with T-cell factor 4 in immature chondrocytes, whereas it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin immunoprecipitation showing that TGF-beta differentially shifts T-cell factor 4 occupancy on the Runx2 promoter in lower sternal chondrocytes vs. growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-beta and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, whereas TGF-beta induced Twist1 in mature chondrocytes, it inhibited Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment, which predominates over TGF-beta effects on these cells. Finally, overexpression of chick Twist1 in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression toward terminal maturation in response to TGF-beta and canonical Wnt signaling.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid

1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of lysophospholipid.     

Regulation of fracture repair by growth factors.

Fractured bones heal by a cascade of cellular events in which mesenchymal cells respond to unknown regulators by proliferating, differentiating, and synthesizing extracellular matrix. Current concepts suggest that growth factors may regulate different steps in this cascade (10). Recent studies suggest regulatory roles for PDGF, aFGF, bFGF, and TGF-beta in the initiation and the development of the fracture callus. Fracture healing begins immediately following injury, when growth factors, including TGF-beta 1 and PDGF, are released into the fracture hematoma by platelets and inflammatory cells. TGF-beta 1 and FGF are synthesized by osteoblasts and chondrocytes throughout the healing process. TGF-beta 1 and PDGF appear to have an influence on the initiation of fracture repair and the formation of cartilage and intramembranous bone in the initiation of callus formation. Acidic FGF is synthesized by chondrocytes, chondrocyte precursors, and macrophages. It appears to stimulate the proliferation of immature chondrocytes or precursors, and indirectly regulates chondrocyte maturation and the expression of the cartilage matrix. Presumably, growth factors in the callus at later times regulate additional steps in repair of the bone after fracture. These studies suggest that growth factors are central regulators of cellular proliferation, differentiation, and extracellular matrix synthesis during fracture repair. Abnormal growth factor expression has been implicated as causing impaired or abnormal healing in other tissues, suggesting that altered growth factor expression also may be responsible for abnormal or delayed fracture repair. As a complete understanding of fracture-healing regulation evolves, we expect new insights into the etiology of abnormal or delayed fracture healing, and possibly new therapies for these difficult clinical problems.     

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse- chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.     

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.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Growth plate chondrocytes store latent transforming growth factor (TGF)-β1 in their matrix through latent TGF-β1 binding protein-1

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-β) as well as a 290 kDa form containing latent TGF-β binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-β in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-β. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-β1 were found in these digests by TGF-β1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-β, in addition to LTBP1 that is associated with the 100 kDa latent TGF-β complex. These studies show for the first time that latent TGF-β is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-β, even at sites distant from the cell, in a relatively avascular environment. J. Cell. Physiol. 177:343–354, 1998. © 1998 Wiley-Liss, Inc.     

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.     

1,25-(OH)2D3 modulates growth plate chondrocytes via membrane receptor-mediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2.

1,25-(OH)2D3 (1,25) exerts its effects on growth plate chondrocytes through classical vitamin D (VDR) receptor-dependent mechanisms, resulting in mineralization of the extracellular matrix. Recent studies have shown that membrane-mediated mechanisms are involved as well. 1,25 targets cells in the prehypertrophic and upper hypertrophic zones of the costochondral cartilage growth plate (GC cells), resulting in increased specific activity of alkaline phosphatase (ALP), phospholipase A2 (PLA2), and matrix metalloproteinases (MMPs). At the cellular level, 1,25 action results in rapid changes in arachidonic acid (AA) release and re-incorporation, alterations in membrane fluidity and Ca ion flux, and increased prostaglandin E1 and E2 (PGE2) production. Protein kinase C (PKC) is activated in a phospholipase C (PLC) dependent-mechanism, due in part to the increased production of diacylglycerol (DAG). In addition, AA acts directly on the cell to increase PKC specific activity. AA also provides a substrate for cyclooxygenase (COX), resulting in PGE2 production. 1,25 mediates its effects through COX-1, the constitutive enzyme, but not COX-2, the inducible enzyme. Time course studies using specific inhibitors of COX-1 show that AA stimulates PKC activity and PKC then stimulates PGE2 production. PGE2 acts as a mediator of 1,25 action on the cells, also stimulating PKC activity. The rapid effects of 1,25 on PKC are nongenomic, occurring within 3 min and reaching maximal activation by 9 min. It promotes translocation of PKC to the plasma membrane. When 1,25 is incubated directly with isolated plasma membranes, PKCalpha is stimulated although PKCzeta is also present. In contrast, when isolated matrix vesicles (MVs) are incubated with 1,25, PKCzeta is inhibited and PKCalpha is unaffected. These membrane-mediated effects are due to the presence of a specific membrane vitamin D receptor (mVDR) that is distinct from the classical cytosolic VDR. Studies using 1,25 analogs with reduced binding affinity for the classical VDR, confirm that rapid activation of PKC by 1,25 is not VDR dependent. The membrane-mediated effects of 1,25 are critical to the regulation of events in the extracellular matrix produced by the chondrocytes. MVs are extracellular organelles associated with maturation of the matrix, preparing it for mineralization. MV composition is under genomic control, involving VDR-mechanisms. In the matrix, no new gene expression or protein synthesis can occur, however. Differential distribution of PKC isoforms and their nongenomic regulation by 1,25 is one way for the chondrocyte to control events at sites distant from the cell. GC cells contain 1a-hydroxylase and produce 1,25; this production is regulated by 1,25, 24,25, and dexamethasone. 1,25 stimulates MMPs in the MVs, resulting in increased proteoglycan degradation in mineralization gels, and increased activation of latent transforming growth factor-beta 1 (TGF-beta1).     

Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.     

Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3

Vitamin D metabolites 1alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) regulate endochondral ossification in a cell maturation-dependent manner via membrane-mediated mechanisms. 24R,25(OH)(2)D(3) stimulates PKC activity in chondrocytes from the growth plate resting zone, whereas 1alpha,25(OH)(2)D(3) stimulates PKC in growth zone chondrocytes. We used the rat costochondral growth plate cartilage cell model to study how these responses are differentially regulated. 1alpha,25(OH)(2)D(3) acts on PKC, MAP kinase, and downstream physiological responses via phosphatidylinositol-specific PLC-beta; 24R,25(OH)(2)D(3) acts via PLD. In both cases, diacylglycerol (DAG) is increased, activating PKC. Both cell types possess membrane and nuclear receptors for 1alpha,25(OH)(2)D(3), but the mechanisms that render the 1alpha,25(OH)(2)D(3) pathway silent in resting zone cells or the 24R,25(OH)(2)D(3) pathway silent in growth zone cells are unclear. PLA(2) is pivotal in this process. 1alpha,25(OH)(2)D(3) stimulates PLA(2) activity in growth zone cells and 24R,25(OH)(2)D(3) inhibits PLA(2) activity in resting zone cells. Both processes result in PKC activation. To understand how negative regulation of PLA(2) results in increased PKC activity in resting zone cells, we used PLA(2) activating peptide to stimulate PLA(2) activity and examined cell response. PLAP is not expressed in resting zone cells in vivo, supporting the hypothesis that PLA(2) activation is inhibitory to 24R,25(OH)(2)D(3) action in these cells.     

Uncoupling of chondrocyte death and vascular invasion in mouse galectin 3 null mutant bones

Galectin 3 is a beta-galactoside binding protein which localizes to the cytoplasm of proliferative, mature, and hypertrophic chondrocytes in the growth plate cartilage of developing long bones. To elucidate the function of galectin 3 during bone development, we examined the epiphyseal femurs and tibias of fetal mice carrying a null mutation for the galectin 3 gene. Detailed histological and ultrastructural studies identified abnormalities in the cells of the proliferative, mature, and hypertrophic zones and in the extracellular matrix of the hypertrophic zone, as well as a reduction in the total number of hypertrophic chondrocytes. The expression patterns of several chondrocyte and bone cell markers were analyzed and revealed a subtle modification of Ihh expression in the galectin 3 mutant growth plate. A striking difference was observed at the chondrovascular junction where many empty lacunae are present. In addition, large numbers of condensed chondrocytes exhibiting characteristic signs of cell death were found in the late hypertrophic zone, indicating that the rate of chondrocyte death is increased in the mutants. These results suggest a role for galectin 3 as a regulator of chondrocyte survival. In addition, this unique phenotype shows that the elimination of chondrocytes and vascular invasion can be uncoupled and indicates that galectin 3 may play a role in the coordination between chondrocyte death and metaphyseal vascularization.     

Smad4 is required for the normal organization of the cartilage growth plate

Smad4 is the central intracellular mediator of transforming growth factor-beta (TGF-beta) signals. To study the role of Smad4 in skeletal development, we introduced a conditional mutation of the gene in chondrocytes using Cre--loxP system. We showed that Smad4 was expressed strongly in prehypertrophic and hypertrophic chondrocytes. The abrogation of Smad4 in chondrocytes resulted in dwarfism with a severely disorganized growth plate characterized by expanded resting zone of chondrocytes, reduced chondrocyte proliferation, accelerated hypertrophic differentiation, increased apoptosis and ectopic bone collars in perichondrium. Meanwhile, Smad4 mutant mice exhibited decreased expression of molecules in Indian hedgehog/parathyroid hormone-related protein (Ihh/PTHrP) signaling. The cultured mutant metatarsal bones failed to response to TGF-beta1, while the hypertrophic differentiation was largely inhibited by Sonic hedgehog (Shh). This indicated that Ihh/PTHrP inhibited the hypertrophic differentiation of chondrocytes independent of the Smad4-mediated TGF-beta signals. All these data provided the first genetic evidence demonstrating that Smad4-mediated TGF-beta signals inhibit the chondrocyte hypertrophic differentiation, and are required for maintaining the normal organization of chondrocytes in the growth plate.     

Authentic matrix vesicles contain active metalloproteases (MMP). a role for matrix vesicle-associated MMP-13 in activation of transforming growth factor-beta

Matrix vesicles (MV) play a key role in the initiation of cartilage mineralization. Although many components in these microstructures have been identified, the specific function of each component is still poorly understood. In this study, we show that metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV isolated from growth plate cartilage. In addition, we provide evidence that MV contain transforming growth factor-beta (TGF-beta) and that MV-associated MMP-13 is capable of activating latent TGF-beta. To determine whether MMPs are associated directly with MV, vesicles isolated from growth plate cartilage were sequentially treated with hyaluronidase, NaCl, and bacterial collagenase to remove matrix proteins and other components attached to their outer surface. Finally, the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV fraction was subjected to substrate zymography, immunoblotting, and substrate activity assay. Whereas active MMP-13 was lost after combined treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were still retained in the pellet fraction even after detergent treatment, suggesting that the gelatinases, MMP-2 and -9, are integral components of MV. In addition, MV contain TGF-beta in the small latent complex, and MMP-13 associated with the MV surface was responsible for activation of TGF-beta. Since the amount of TGF-beta activated by hypertrophic chondrocytes increased with mineral appearance in serum-free chondrocyte cultures, a role for active MV-associated MMPs is suggested in activation of TGF-beta seen during late chondrocyte hypertrophy and mineralization of growth plate cartilage.     

Stanniocalcin 1 acts as a paracrine regulator of growth plate chondrogenesis

During embryogenesis, the expression of mammalian stanniocalcin (STC1) in the appendicular skeleton suggests its involvement in the regulation of longitudinal bone growth. Such a role is further supported by the presence of dwarfism in mice overexpressing STC1. Yet, the STC 1 inhibitory effect on growth may be related to both postnatal metabolic abnormalities and prenatal defective bone formation. In our study, we used an organ culture system to evaluate the effects of STC on growth plate chondrogenesis, which is the primary determinant of longitudinal bone growth. Fetal rat metatarsal bones were cultured in the presence of recombinant human STC (rhSTC). After 3 days, rhSTC suppressed metatarsal growth, growth plate chondrocyte proliferation and hypertrophy/differentiation, and extracellular matrix synthesis. In addition, rhSTC increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, rhSTC increased phosphate uptake, reduced chondrocyte proliferation and matrix synthesis, and induced apoptosis. All these effects were reversed by culturing chondrocytes with rhSTC and phosphonoformic acid, an inhibitor of phosphate transport. The rhSTC-mediated inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was abolished by culturing metatarsals with rhSTC and phosphonoformic acid. Taken together, our findings indicate that STC1 inhibits longitudinal bone growth directly at the growth plate. Such growth inhibition, likely mediated by an increased chondrocyte phosphate uptake, results from suppressed chondrocyte proliferation, hypertrophy/differentiation, and matrix synthesis and by increased apoptosis. Last, the expression of both STC1 and its binding site in the growth plate would support an autocrine/paracrine role for this growth factor in the regulation of growth plate chondrogenesis.