Bone morphogenesis

Abstract: DURING gastrulation in vertebrates, ectodermal cells choose between two fates, neural and epidermal. The nervous system forms in response to signals from the Spemann organizer1,2; ectoderm that does not receive these signals becomes epidermis. Unexpectedly, however, in Xenopus, neural tissue also forms when cell-cell communication within the ectoderm is disrupted by cell dissociation3,4 or by antagonists of the growth factor activin5-7. These observations suggest that epidermal specification depends on local signalling, by activin or a close relative, and that neural tissue forms when this communication is blocked6. Here we report that bone morphogenesis protein 4 (Bmp-4), a relative of activin that is expressed in the embryo at the time of ectodermal fate determination8,9, is a potent epidermal inducer and neural inhibitor, the first reported in any vertebrate. Activin can inhibit neuralization by inducing mesoderm, but does not induce epidermis. Moreover, the dominant-negative activin receptor, which stimulates neuralization when expressed in the embryo5,6, blocks Bmp-4 in our assay. Our findings demonstrate that epidermal fate can be induced, and thus provide further evidence that neural specification is under inhibitory control in vertebrates.

Abstract: TNF and TNFR family proteins play important roles in the control of cell death, proliferation, autoimmunity, the function of immune cells, or the organogenesis of lymphoid organs. Recently, novel members of this large family have been identified that have critical functions in immunity and that couple lymphoid cells with other organ systems such as bone morphogenesis and mammary gland formation in pregnancy. The TNF-family molecule RANK-L (RANK-L, TRANCE, ODF) and its receptor RANK are key regulators of bone remodeling, and they are essential for the development and activation of osteoclasts. Intriguingly, RANK-L/RANK interactions also regulate T cell/dendritic cell communications, dendritic cell survival, and lymph node formation; T cell-derived RANK-L can mediate bone loss in arthritis and periodontal disease. Moreover, RANK-L and RANK are expressed in mammary gland epithelial cells, and they control the development of a lactating mammary gland during pregnancy and the propagation of mammalian species. Modulation of these systems provides us with a unique opportunity to design novel therapeutics to inhibit bone loss in arthritis, periodontal disease, and osteoporosis.

Abstract: A bone morphogenetic protein (BMP) obtained in solution by digestion of demineralized rabbit cortical bone matrix with bacterial collagenase retains its biologically active conformation in a neutral salt/ethylene glycol mixture. BMP may be insolubilized by coprecipitation with calcium phosphate and resolubilized by chemical extraction with a neutral salt in the same solvent mixture. Upon concanavalin A-Sepharose chromatography, BMP is bound by hydrophobic interaction and carbohydrate recognition and is recovered by elution with either alpha-methyl mannoside or ethylene glycol solvent mixture. Implants of both eluates and the extracts of the coprecipitate in double-walled diffusion chambers induce transmembrane bone morphogenesis. BMP is not species specific; rabbit BMP induces new bone formation in the rat. The present observations indicate that BMP is a glycoprotein.

Abstract: We previously showed that anti-fibronectin antibodies or soluble fibronectin fragments containing the central cell-binding domain inhibit formation of mineralized nodules by fetal calvarial osteoblasts in vitro. These findings suggest a critical role for fibronectin in osteoblast differentiation and morphogenesis. In this study we tested the hypothesis that fibronectin's effects on osteogenesis are mediated via direct interactions with integrin receptors for fibronectin on osteoblasts. Immunocytochemical analysis identified the integrin fibronectin receptor alpha5ss1 in fetal rat calvarial tissue and in cultured osteoblasts at all stages of differentiation. Three other integrins, alpha3ss1, alpha8ss1 and alphavss3, which can bind fibronectin, as well as other matrix components, were also identified in tissue and at all stages of cell culture. Immunoprecipitation data showed that alpha5ss1 levels are constant throughout osteoblast differentiation whereas levels of alpha3ss1 and alpha8ss1 decline in mature mineralized cultures. To determine whether integrin fibronectin receptors are required for osteoblast formation of mineralized nodules, we examined the extent of nodule formation in the presence and absence of function-perturbing anti-integrin antibodies. The antibodies were present continuously in cultures beginning at confluence (day 3), and nodule formation was measured at days 10 and 20. An anti-alpha5 integrin subunit antibody reduced nodule formation to less than 5% of control values at both time points. Inhibition of nodule formation was reversible and did not affect cell attachment and viability. Function-perturbing antibodies against alpha3ss1 and alpha8ss1 also reduced nodule formation, to less than 20% of control values. In contrast, function-perturbing antibodies to alphavss3 and alphavss5 did not affect nodule formation, indicating that the inhibitions noted were indeed specific. To determine the effect of antibody treatment on gene expression, steady-state mRNA expression was examined and found to be suppressed for osteoblast markers alkaline phosphatase and osteocalcin. Together, these results indicate that direct osteoblast interactions with the extracellular matrix are mediated by a select group of integrin receptors that includes alpha5ss1, alpha3ss1 and alpha8ss1. We further conclude that the specific alpha5ss1 fibronectin receptor mediates critical interactions between osteoblasts and fibronectin required for both bone morphogenesis and osteoblast differentiation.