Faculty Bibliography

Chondrocyte proliferation is important for skeletal development and growth, but the mechanisms regulating it are not completely clear. Previously, we showed that syndecan-3, a cell surface heparan sulfate proteoglycan, is expressed by proliferating chondrocytes in vivo and that proliferation of cultured chondrocytes in vitro is sensitive to heparitinase treatment. To further establish the link between syndecan-3 and chondrocyte proliferation, additional studies were carried out in vivo and in vitro. We found that the topographical location of proliferating chondrocytes in developing chick long bones changes with increasing embryonic age and that syndecan-3 gene expression changes in a comparable manner. For in vitro analysis, mitotically quiescent chondrocytes were exposed to increasing amounts of fibroblast growth factor-2 (FGF-2). Proliferation was stimulated by as much as 8-10-fold within 24 h; strikingly, this stimulation was significantly prevented when the cells were treated with both fibroblast growth factor-2 (FGF-2) and antibodies against syndecan-3 core protein. This neutralizing effect was dose-dependent and elicited a maximum of 50-60% inhibition. To establish specificity of neutralizing effect, cultured chondrocytes were exposed to FGF-2, insulin-like growth factor-1, or parathyroid hormone, all known mitogens for chondrocytes. The syndecan-3 antibodies interfered only with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone. Protein cross-linking experiments indicated that syndecan-3 is present in monomeric, dimeric, and oligomeric forms on the chondrocyte surface. In addition, molecular modeling indicated that contiguous syndecan-3 molecules might form stable complexes by parallel pairing of beta-sheet segments within the ectodomain of the core protein. In conclusion, the results suggest that syndecan-3 is a direct and selective regulator of the mitotic behavior of chondrocytes and its role may involve formation of dimeric/oligomeric structures on their cell surface

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

Biomineralization is a cell-regulated process. Matrix vesicles, which are released from mineralization-competent cells, initiate the mineralization process. These particles contain channel-forming annexins II, V and VI, and an Na⁺/Pi symport system, which enable Ca²⁺ and inorganic phosphate (Pi) influx into the vesicles. Rapid Ca²⁺ and Pi influxes are required for the formation of the first mineral phase inside the vesicles. Furthermore, matrix vesicles contain plasma cell membrane glycoprotein 1 (PC-1), an enzyme which generates pyrophosphate (PPi). Extracellular PPi is also regulated by ank, a transmembrane protein that transports intracellular PPi to the extracellular milieu. PPi is an inhibitor of mineralization; however, matrix vesicle-associated alkaline phosphatase (TNAP) may degrade PPi and thus may not only provide Pi but more importantly remove an inhibitor of mineralization. The release of mineralization-competent matrix vesicles is regulated by annexin-mediated alteration of Ca²⁺ homeostasis, suggesting that targeting annexin functions might provide a novel therapeutic strategy to prevent pathological mineralization. Furthermore, the possible inhibitory function of other factors, such as matrix gla protein (MGP), in mineralization and cell differentiation is discussed

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

Type IIA procollagen is an alternatively spliced product of the type II collagen gene and uniquely contains the cysteine (cys)-rich globular domain in its amino (N)-propeptide. To understand the function of type IIA procollagen in cartilage development under normal and pathologic conditions, the detailed expression pattern of type IIA procollagen was determined in progressive stages of development in embryonic chicken limb cartilages (days 5-19) and in human adult articular cartilage. Utilizing the antibodies specific for the cys-rich domain of the type IIA procollagen N-propeptide, we localized type IIA procollagen in the pericellular and interterritorial matrix of condensing pre-chondrogenic mesenchyme (day 5) and early cartilage (days 7-9). The intensity of immunostaining was gradually lost with cartilage development, and staining became restricted to the inner layer of perichondrium and the articular cap (day 12). Later in development, type IIA procollagen was re-expressed at the onset of cartilage hypertrophy (day 19). Different from type X collagen, which is expressed throughout hypertrophic cartilage, type IIA procollagen expression was transient and restricted to the zone of early hypertrophy. Immunoelectron microscopic and immunoblot analyses showed that a significant amount of the type IIA procollagen N-propeptide, but not the carboxyl (C)-propeptide, was retained in matrix collagen fibrils of embryonic limb cartilage. This suggests that the type IIA procollagen N-propeptide plays previously unrecognized roles in fibrillogenesis and chondrogenesis. We did not detect type IIA procollagen in healthy human adult articular cartilage. Expression of type IIA procollagen, together with that of type X collagen, was activated by articular chondrocytes in the upper zone of moderately and severely affected human osteoarthritic cartilage, suggesting that articular chondrocytes, which normally maintain a stable phenotype, undergo hypertrophic changes in osteoarthritic cartilage. Based on our data, we propose that type IIA procollagen plays a significant role in chondrocyte differentiation and hypertrophy during normal cartilage development as well as in the pathogenesis of osteoarthritis

OBJECTIVE: To determine the levels of vascular endothelial growth factor (VEGF) mRNA and protein expression in normal and osteoarthritic (OA) human articular cartilage, and whether VEGF expression alters during the progression of OA. METHODS: Sections from normal and OA human knee cartilage were immunotained with a polyclonal antibody recognising VEGF. In addition, total RNA was isolated from normal and osteoarthritic human knee cartilage and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) for VEGF mRNA expression. RESULTS: VEGF was found to be present in normal and OA human knee cartilage in all cartilage layers. A significant increase of VEGF immunopositive chondrocytes to up to approximately 82% was detected in severe OA cartilage compared with normal articular cartilage (approximately 56% of immunopositive chondrocytes). RT-PCR analysis showed the expression of VEGF also on the mRNA level. CONCLUSIONS: VEGF is expressed by articular chondrocytes in normal and OA human knee cartilage. The percentage of VEGF immunopositive chondrocytes significantly increases in late stages of the disease. The VEGF transcript levels encoding all four isoforms shows a big variability in samples from different donors, suggesting a distinct regulation of the expression of the four VEGF isoforms in normal and OA cartilage

Although osteoarthritis is characterized by a progressive loss of the extracellular cartilage matrix, very little is known about the fate of articular chondrocytes during the progression of the disease. In this study we examined the expression of syndecan-3, a marker of early chondrocyte differentiation, and annexin VI, a marker of late chondrocyte differentiation, in mammalian embryonic growth plate cartilage and normal and osteoarthritic human articular cartilage. Whereas syndecan-3 was expressed in the proliferative and hypertrophic zones of growth platecartilage, immunostaining for annexin VI waspredominately found in the hypertrophic and mineralizing zones of fetal bovine growth plate cartilage. Approximately 20% of chondrocytes were immunopositive for syndecan-3 in normal human articular cartilage, the number of syndecan-3-expressing chondrocytes significantly increased during the progression of osteoarthritis with more than 80% syndecan-3-positive cells in the upper zone of severely affected osteoarthritic cartilage. Similarly, the number of annexin VI-expressing cells significantly increased in the upper cartilage zones during the progression of osteoarthritis. Furthermore, immunostaining for proliferating cell nuclear antigen, a marker for cell proliferation, was detected in chondrocytes in the upper zone of osteoarthritic cartilage. Double-labeling experiments with antibodies against syndecan-3 and annexin VI revealed chondrocytes that expressed only syndecan-3, and cells that expressed both syndecan-3 and annexin VI. These results suggest that the expression of early (proliferating cell nuclear antigen, syndecan-3) and late differentiation markers (annexin VI, alkaline phosphatase) is activated in chondrocytes of osteoarthritic cartilage

Background: Bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) belong to the large transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. Signaling of the BMPs requires the binding of the BMP to the BMP cell surface receptors BMPR-IA, BMPR-IB, and BMPR-II. Similar to other cytokines, members of the TGF-beta superfamily exhibit stringent specificity in their ligand-receptor interactions, which may be a reason for the qualitative and quantitative differences in cellular responses. To understand how BMPs and GDFs activate their receptors, it is important to determine structure and binding mechanisms of ligand-receptor complexes. We have used BMP-2 as a key representative of the BMPs to identify the epitopes for type I and type II receptor binding by mutational interaction analyses and have solved the crystal structure of a BMP-2:BMPR-IA receptor ectodomain complex. Methods: To identify amino acid side chains involved in receptor binding, a collection of in vitro mutagenized human BMP-2 variants was prepared and subjected to interaction analyses with use of the receptor ectodomains of BMPR-IA, BMPR-II, and ActR-Il immobilized on a biosensor system. The biological activity of the BMP-2 variants was measured by BMP-2 dependent expression of alkaline phosphatase (ALP) in C2C12 cells. For crystallization, a complex of BMP-2 and the ectodomain of BMPR-IA was formed in solution, purified, and crystallized as described(12). Results: The ligand-receptor interaction analysis of the BMP-2 variants identified distinct epitopes for type I and type II receptor binding. Because the structure of TGF-beta -like proteins has been compared with that of an open hand, the binding epitope for the type I receptor was-on the basis of its location-termed 'wrist' epitope. The crystal structure of the BMP-2:BMPR-IA ectodomain complex revealed a key feature of the ligand-receptor interaction: a large hydrophobic residue (Phe85) within a hydrophobic patch of BMPR-IA fit into a hydrophobic pocket composed of residues of both BMP-2 monomers. A second epitope identified by alanine mutagenesis scanning was termed the 'knuckle' epitope on the basis of its location on the outer side of the 'finger' segments of BMP-2. Mutations in either the wrist epitope or the knuckle epitope produced variants with altered biological activities. Variants with antagonistic properties were exclusively generated by mutations in the knuckle epitope of BMP-2. Conclusions and Clinical Relevance: The identification and characterization of the two receptor binding epitopes in BMP-2 provide new insight into the primary steps of BMP-receptor activation. Because of the structural similarities between members of the TGF-beta superfamily, it can be assumed that the data presented in this work are transferable to other TGF-beta receptor systems. Because of the association with various diseases, the generation of antagonists of other TGF-beta superfamily members might generate potent tools for basic research and therapeutic approaches. $$:

Mineralization and ossification of human thyroid cartilage first starts after the end of adolescence when the previously cartilaginous human skeleton has become ossified and the epiphyseal discs are in the process of closing. However, the mechanisms involved in mineralization and ossification of human thyroid cartilage are not well understood. Ultrastructural analysis of human thyroid cartilage revealed that mineralization started close to cartilage canals in a matrix containing gigantic collagen fibers (asbestoid fibers). Matrix vesicles were detected in mineralized areas and were often associated with needle-like crystals. For the first time we were able to isolate matrix vesicles from human thyroid cartilage by mild enzymatic digestions and ultracentrifugation. These particles were oval and varied in size; some were heavily calcified. They were enriched in alkaline phosphatase, calcium, and inorganic phosphate, suggesting that the particles contain Ca2+-Pi complexes. Immunoblot analysis of these vesicles revealed the presence of annexins II, V, and VI, membrane-associated, channel-forming proteins, which allow influx of Ca2+ into the vesicles and intralumenal crystal growth. In addition, the vesicles were associated with types II and X collagen, suggesting that this association not only anchors the vesicles to the extracellular matrix, but, as shown previously, also stimulates Ca2+ influx into these particles. In conclusion, matrix vesicles isolated from human thyroid cartilage contain all the components, enabling them to initiate and mediate the mineralization process in human thyroid cartilage.

Zinc (Zn2+) has long been known to play important roles in mineralization and ossification of skeletal tissues, but the mechanisms of Zn2+ action are not well understood. In this study we investigated the effects of Zn2+ on mineralization in a cell culture system in which terminal differentiation and mineralization of hypertrophic growth plate chondrocytes was induced by retinoic acid (RA) treatment. Addition of Zn2+ to RA-treated cultures decreased mineralization in a dose-dependent manner without affecting alkaline phosphatase (APase) activity. Characterization of matrix vesicles (MVs), particles that initiate the mineralization process, revealed that vesicles isolated from RA-treated and RA/Zn2+-treated cultures showed similar APase activity, but vesicles from RA/Zn2+-treated cultures contained significantly less Ca2+ and Pi. MVs isolated from RA-treated cultures were able to take up Ca2+ and mineralize in vitro, whereas vesicles isolated from RA/Zn2+-treated cultures were not able to do so. Detergent treatment, which ruptures the MV membrane and exposes preformed intravesicular Ca2+-Pi-phospholipid complexes, did not restore the Ca2+ uptake abilities of MVs isolated from RA/Zn2+-treated cultures, suggesting that vesicles from RA/Zn2+-treated cultures did not contain functional Ca2+-Pi-phospholipid complexes. Zn2+ treatment did not affect the content of annexins II, V, and VI in MVs or the Ca2+-dependent, EDTA-reversible binding of these molecules to the membrane surface. However, Zn2+ treatment did affect the EDTA-nonreversible binding of these molecules to the MV membrane, suggesting that Zn2+ interferes with the assembly of annexins in the MV membrane. In addition, Zn2+ inhibited annexin II-, V-, and VI-mediated Ca2+ influx into liposomes. In conclusion, Zn2+ inhibits the mineralizing competence of intravesicular Ca2+-Pi-phospholipid complexes and function of annexin channels, thereby controlling Ca2+ influx into MVs, the formation of the first crystal phase inside the vesicles and initiation of mineralization.