Growth factors are potent cytoregulatory peptides that require strict controls to avoid inappropriate cell growth. Transforming growth factor- Beta (TGF-Beta) is synthesized as a latent precursor and its activity is primarily controlled by processes that regulate conversion of inactive precursor to the active molecule. Physiologic mechanisms of TGF-Beta activation are poorly understood. We found that active TGF-Beta is associated with the extracellular matrix protein, thrombospondin (TSP), in platelet releasates; there is a specific interaction between TSP and TGF-Beta; and TSP stripped of TGF-Beta activity (sTSP) retains its TGF- Beta-dependent endothelial cell growth inhibition. Furthermore, we show that sTSP activates cell-secreted latent TGF-Beta by a novel mechanisms that does not require cell-cell/surface interactions and does not involve plasmin. Activation may occur through direct interactions of sTSP with latent TGF-B, since sTSP binds latent TGF-Beta and stimulates activation. Based on these data, we propose that sTSP is a major regulator of TGF- Beta activity.
The specific aims are 1) to determine which biological function of TGF-Beta are modulated by sTSP; 2) to determine the mechanisms involved in sTSP-mediated stimulation of TGF-Beta activity; and 3) to characterize the TGF-Beta binding site on the trimeric 50kDa fragment of sTSP. We will determine whether sTSP modulates, TGF-Beta growth regulation and if sTSP modulates TGF-Beta regulated gene expression of matrix, protease inhibitor,and cell-cycle related genes by measuring mRNA levels of TGF-Beta inducible proteins. Proposed mechanisms of TGF-Beta regulation by sTSP that will be examined include a) the role of the latency associated peptide in sTSP-TGF-Beta interactions and of potential conformational changes due to TGF-Beta binding to sTSP that could activate latent TGF-Beta; b) cysteine protease activation by sTXP or sTSP acting as a protease; c) modulation of TGF-Beta transcription and synthesis; d) sTSP as a potential carrier of TGF-Beta to prolong TGF-Beta half-life in vitro; and e) sTSP protection of TGF-Beta from inactivation by decorin and alpha2- macroglobulin. To characterize the TGF-Beta binding site on the 50 kDa fragment of sTSP, constructs of this region will be made using a baculovirus expression system and studied for TGF-Beta binding and for the ability to activate latent TGF-Beta. These data should enhance our understanding of the mechanism(s) of sTSP regulation of TGF-Beta activity and of how extracellularly matrix modulates the """"""""context"""""""" in which growth factors act. This will be important for understanding matrix-growth factor control of neovascular responses, such as occur in wound healing and tumor angiogenesis.
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