The specific objective of this project is on the cell and molecular biological mechanisms regulating skeletal development and mesenchymal cell differentiation, specifically the action of growth factors.? ? A summary of our research progress s presented below:? 1. Cellular signaling mechanism of TGF-beta in osteoblasts? TGF-beta1 action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF-beta1 and BMP-2 treatment of primary human osteoblasts (HOB) enhances cell-substrate adhesion. In this report, we demonstrate that TGF-beta1 elicits a rapid and transient rise in the intracellular calcium concentration, Ca2+i, that is necessary for enhancement of cell adhesion in HOB. This rise in Ca2+i in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L-type calcium channel blocker, nifedipine, but is stimulated upon treatment with the L-type calcium channel agonist, Bay K 8644, or under high K+ conditions. The rise in Ca2+i is severely attenuated after treatment of the HOB with thapsigargin, a selective endoplasmic reticulum calcium pump inhibitor. TGF-beta1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular calcium signaling is an important second messenger of the TGF-beta1 signal transduction pathway in osteoblast function.? ? 2. Spatiotemporal protein distribution of TGF-betas, their receptors, and extracellular matrix molecules during embryonic tendon development? Tendon is one of the least understood tissues of the musculoskeletal system in terms of development and morphogenesis. Collagen fibrillogenesis has been the most studied aspect of tendon development, focusing largely on the role of matrix molecules such as collagen type III and decorin. While involvement of matrix molecules in collagen fibrillogenesis during chick tendon development is well understood, the role of growth factors has yet to be elucidated. This work examines the expression patterns of TGF-beta1, -2, and -3, and their receptors with respect to expression patterns of collagen type III, decorin, and fibronectin. We focus on the intermediate stages of tendon development in the chick embryo, a period during which the tendon micro- and macro-architecture are being established. Our findings demonstrate for the first time that TGF-beta1, -2, and -3? have distinct spatiotemporal developmental protein localization patterns in the developing tendon and strongly suggest that these isoforms have independent roles in tendon development.? ? 3. Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan? Cartilage oligomeric matrix protein (COMP), also known as thrombospondin 5 (TSP5), is a pentameric extracellular matrix protein. Based on its sequence COMP/TSP5 belongs to the TSP family (1,2). It is a major component of the extracellular matrix (ECM) of the musculoskeletal system including cartilage, tendon, and ligament. The physiological function of COMP/TSP5 remains largely unclear, but its importance is underscored by its association with several joint diseases. To study the functions of COMP/TSP5 in cartilage ECM, we have examined its interaction with glycosaminoglycans (GAGs) and aggrecan, a major and critical components of cartilage extracellular matrix. We also tested MUT3, a COMP/TSP5 mutant found to account for 30% of human pseudoachondroplasia cases, to determine if the mutation affects the function of the protein. Using affinity co-electrophoresis (ACE), we show that COMP/TSP5 bound to heparin, chondroitin sulfates, and heparan sulfate in its native calcium-replete conformation. EDTA treatment of COMP/TSP5 reduces heparin binding and inhibits chondroitin sulfate binding, implying a conformation-dependent GAG-binding site in the molecule. MUT3 showed weaker binding to the GAGs than COMP/TSP5 in the presence of Ca, and EDTA treatment of MUT3 further decreased binding. Using a solid binding assay, we showed that COMP/TSP5 can bind aggrecan, which is decreased upon EDTA chelation, in agreement with the ACE data. MUT3 showed weak interaction with aggrecan. Also, soluble GAGs inhibited aggrecan binding to COMP/TSP5, suggesting that the interaction of aggrecan with COMP/TSP5 was through binding to its GAG side chains. Using recombinant COMP/TSP5 fragments covering various COMP/TSP5 domains were tested, the C-globe region alone was found to bind to aggrecan in solution, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. These data show that COMP/TSP5 is a proteoglycan-binding protein, and it binds aggrecan, possibly through the GAg side chains on aggrecan and the C-globe regain of COMP/TSP5. This interaction is regulated by calcium-binding sensitiv conformation of the molecule. Our results suggest that COMP/TSP5 may function to support matrix interaction in the extracellular matrix of cartilage. ? ? 4. Role of fibronectin splice isoform and splicing factor SRp40 in mesenchymal chondrogenesis and chondrocyte maturation.? Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that include the type II repeat extra domain B (EDB) but exclude extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for progression of chondrogenesis. To test this, we examined the effect of the chondrogenic growth factor, TGF-beta1 on mesenchymal cells, on the modulation of EDA and EDB exon expression as well as markers of late chondrocyte maturation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. Overexpression of the long-form of SRp40 yielded a FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.? A variant spliced form of SRp40, SRp40LF, is expressed preferentially in chondrocytes and in chondrifying mesenchymal cells. Forced overexpression of SRp40 or SRp75, but not SRp55, enhanced chondrogenic differentiation of chick limb mesenchymal cells in a high-density micromass assay. SRp40LF overexpression, which produces a truncated form of SRp40, also was strongly pro-chondrogenic. In a HeLa cell-based assay, SRp40LF fails to substitute for SRp40 in mediating an increase in exon EIIIA inclusion, suggesting that the latter event is not essential for the pro-chondrogenic effect. These results demonstrate the ability of these highly conserved splicing factors to modulate chondrogenesis, consistent with earlier results implicating exon EIIIA-contaiining isoforms of fibronectin in formation of chondrogenic condensations.? ? 5. Nitric oxide and embryonic skeletal dysmorphogenesis? Based on previous results on the teratogenic effects of CO and NO on chick embryos, our recent study showed that NO regulation is essential for normal axial development, that sites of altered NO expression correlate to those of altered apoptosis and dymorphogenesis, and that CO coadministration rectified normal NO expression. Collectively, these results suggest that alteration in endogenous NO/CO signaling is responsible, in part, for the observed NO-induced teratogenesis.
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