Faculty Bibliography

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. $$:

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

OBJECTIVE: To test the hypothesis that terminal differentiation of chondrocytes in human osteoarthritic cartilage might lead to the failure of repair mechanisms and might cause progressive loss of structure and function of articular cartilage. DESIGN: Markers for terminally differentiated chondrocytes, such as alkaline phosphatase, annexin II, annexin V and type X collagen, were detected by immunohistochemical analysis of human normal and osteoarthritic knee cartilage from medial and lateral femoral condyles. Apoptosis in these specimens was detected using the TUNEL labeling. Mineralization and matrix vesicles were detected by alizarin red S staining and electron microscopic analysis. RESULTS: Alkaline phosphatase, annexin II, annexin V and type X collagen were expressed by chondrocytes in the upper zone of early stage and late stage human osteoarthritic cartilage. However, these proteins, which are typically expressed in hypertrophic and calcifying growth plate cartilage, were not detectable in the upper, middle and deep zones of healthy human articular cartilage. TUNEL labeling of normal and osteoarthritic human cartilage sections provided evidence that chondrocytes in the upper zone of late stage osteoarthritic cartilage undergo apoptotic changes. In addition, mineral deposits were detected in the upper zone of late stage osteoarthritic cartilage. Needle-like mineral crystals were often associated with matrix vesicles in these areas, as seen in calcifying growth plate cartilage. CONCLUSION: Human osteoarthritic chondrocytes adjacent to the joint space undergo terminal differentiation, release alkaline phosphatase-, annexin II- and annexin V-containing matrix vesicles, which initiate mineral formation, and eventually die by apoptosis. Thus, these cells resume phenotypic changes similar to terminal differentiation of chondrocytes in growth plate cartilage culminating in the destruction of articular cartilage in osteoarthritis

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.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments

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.

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues