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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL050061-01A1
Application #
2226170
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1994-07-01
Project End
1999-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Pathology
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Belmadani, Souad; Bernal, Juan; Wei, Chih-Chang et al. (2007) A thrombospondin-1 antagonist of transforming growth factor-beta activation blocks cardiomyopathy in rats with diabetes and elevated angiotensin II. Am J Pathol 171:777-89
Zhou, Yong; Poczatek, Maria H; Berecek, Kathleen H et al. (2006) Thrombospondin 1 mediates angiotensin II induction of TGF-beta activation by cardiac and renal cells under both high and low glucose conditions. Biochem Biophys Res Commun 339:633-41
Young, Geoffrey D; Murphy-Ullrich, Joanne E (2004) The tryptophan-rich motifs of the thrombospondin type 1 repeats bind VLAL motifs in the latent transforming growth factor-beta complex. J Biol Chem 279:47633-42
Daniel, Christoph; Wiede, Julia; Krutzsch, Henry C et al. (2004) Thrombospondin-1 is a major activator of TGF-beta in fibrotic renal disease in the rat in vivo. Kidney Int 65:459-68
Young, Geoffrey D; Murphy-Ullrich, Joanne E (2004) Molecular interactions that confer latency to transforming growth factor-beta. J Biol Chem 279:38032-9
Zhang, Hong; Akman, Hasan O; Smith, Eric L P et al. (2003) Cellular response to hypoxia involves signaling via Smad proteins. Blood 101:2253-60
Wang, Shuxia; Shiva, Sruti; Poczatek, Maria H et al. (2002) Nitric oxide and cGMP-dependent protein kinase regulation of glucose-mediated thrombospondin 1-dependent transforming growth factor-beta activation in mesangial cells. J Biol Chem 277:9880-8
Harpel, J G; Schultz-Cherry, S; Murphy-Ullrich, J E et al. (2001) Tamoxifen and estrogen effects on TGF-beta formation: role of thrombospondin-1, alphavbeta3, and integrin-associated protein. Biochem Biophys Res Commun 284:11-4
Murphy-Ullrich, J E; Poczatek, M (2000) Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology. Cytokine Growth Factor Rev 11:59-69
Patel, R P; Moellering, D; Murphy-Ullrich, J et al. (2000) Cell signaling by reactive nitrogen and oxygen species in atherosclerosis. Free Radic Biol Med 28:1780-94

Showing the most recent 10 out of 17 publications