Faculty Bibliography

Syndecans are single-pass integral membrane components that serve as co-receptors for growth factors and cytokines and can elicit signal transduction via their cytoplasmic tails. We review here previous studies from our groups on syndecan-3 biology and function in the growth plates of developing long bones in chick and mouse embryos. Gain- and loss-of-function data indicate that syndecan-3 has important roles in restricting mitotic activity to the proliferative zone of growth plate and may do so in close cooperation and interaction with the signaling molecule Indian hedgehog (IHH). Biochemical and protein-modeling data suggest a dimeric/oligomeric syndecan-3 configuration on the chondrocyte's cell surface. Analyses of embryos misexpressing syndecan-3 or lacking IHH provide further clues on syndecan-3/IHH interdependence and interrelationships. The data and the conclusions reached provide insights into mechanisms fine-tuning chondrocyte proliferation, maturation, and function in the developing and growing skeleton and into how abnormalities in these fundamental mechanisms may subtend human congenital pathologies, including osteochondromas in hereditary multiple exostoses syndrome

BACKGROUND: Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS: A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS: At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS: Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. Clinical Relevance: Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors

Mineralization often occurs in areas of apoptotic changes. Our findings indicate that physiological mineralization is mediated by matrix vesicles. These matrix vesicles use mechanisms to induce mineralization that are different from the mechanisms used by apoptotic bodies released from apoptotic cells. Therefore, different therapeutic approaches must be chosen to inhibit pathological mineralization depending on the mechanism of mineralization (matrix vesicles versus apoptotic bodies). INTRODUCTION: Physiological mineralization in growth plate cartilage is restricted to regions of terminally differentiated and apoptotic chondrocytes. Pathological mineralization of tissues also often occurs in areas of apoptosis. We addressed the question of whether apoptotic changes control mineralization events or whether both events are regulated independently. METHODS: To induce mineralization, we treated growth plate chondrocytes with retinoic acid (RA); apoptosis in these cells was induced by treatment with staurosporine, anti-Fas, or TNFalpha. The degrees of mineralization and apoptosis were determined, and the structure and function of matrix vesicles and apoptotic bodies were compared. RESULTS: Release of matrix vesicles and mineralization in vivo in the growth plate occurs earlier than do apoptotic changes. To determine the functional relationship between apoptotic bodies and matrix vesicles, growth plate chondrocytes were treated with RA to induce matrix vesicle release and with staurosporine to induce release of apoptotic bodies. After 3 days, approximately 90% of staurosporine-treated chondrocytes were apoptotic, whereas only 2-4% of RA-treated cells showed apoptotic changes. RA- and staurosporine-treated chondrocyte cultures were mineralized after 3 days. Matrix vesicles isolated from RA-treated cultures and apoptotic bodies isolated from staurosporine-treated cultures were associated with calcium and phosphate. However, matrix vesicles were bigger than apoptotic bodies. Furthermore, matrix vesicles but not apoptotic bodies contained alkaline phosphatase and Ca2+ channel-forming annexins II, V, and VI. Consequently, matrix vesicles but not apoptotic bodies were able to take up Ca2+ and form the first mineral phase inside their lumen. Mineralization of RA-treated cultures was inhibited by antibodies specific for annexin V but not mineralization of staurosporine-treated cultures. CONCLUSION: Physiological mineralization of growth plate chondrocytes is initiated by specialized matrix vesicles and requires alkaline phosphatase and annexins. In contrast, mineral formation mediated by apoptotic bodies occurs by a default mechanism and does not require alkaline phosphatase and annexins

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes

Purpose of review: Osteoarthritis is the most common form of arthritis, affecting a large population of mostly elderly people. No cure for osteoarthritis currently exists. Ultimate treatment is joint replacement. Understanding the mechanisms causing onset and progression is critical. This review describes recent findings that provide new insights into changes of cellular phenotype in osteoarthritis as a possible reason for tissue failure. Recent findings: Recent findings suggest that articular chondrocytes, when stimulated, can undergo hypertrophic and terminal differentiation events similar to those occurring during endochondral bone formation. Interestingly, collagenase-3 (matrix metalloproteinase-13), a main matrix-degrading enzyme in osteoarthritis, is expressed only in terminally differentiated chondrocytes during normal development. Summary: Although terminal differentiation events are required for endochondral bone formation, they lead to cartilage destruction when occurring in articular chondrocytes. Maintaining the articular chondrocyte phenotype and preventing these cells from undergoing hypertrophic and terminal differentiation might provide novel therapeutic targets to prevent onset or progression of osteoarthritis

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