Thrombospondin binds and activates the small and large forms of latent transforming growth factor-beta in a chemically defined system.

Abstract

Transforming growth factor-beta (TGF-beta) is a potent growth regulatory protein normally secreted by cells in a latent form. Primary regulation of TGF-beta activity occurs through factors which control the processing of the latent to the biologically active molecule. Thrombospondin (TSP), a platelet alpha-granule and extracellular matrix protein, forms specific complexes with active TGF-beta in platelet releasate and activates endogenous latent TGF-beta secreted by endothelial cells via a cell- and protease-independent mechanism. In order to better understand TSP-mediated activation of cell-secreted latent TGF-beta, we examined the consequences of interactions of the large (platelet-derived) and small (recombinant) forms of latent TGF-beta with TSP in a chemically defined system. Data from these studies show that interactions between TSP and both forms of latent TGF-beta result in the generation of biologically active TGF-beta as assayed by the ability of NRK-49F cells to form colonies in soft agar, by the ability to compete for binding to TGF-beta receptors on endothelial cells, and by an enzyme-linked immunosorbent assay selective for the active form of TGF-beta. Activation of latent TGF-beta by TSP stripped of associated TGF-beta activity (sTSP) is time- and concentration-dependent, but temperature-independent. The mechanism whereby sTSP activates latent TGF-beta appears to involve the direct binding of sTSP to the latent molecule as shown by gel permeation chromatography. In addition, a polyclonal antibody specific for the amino-terminal region of the latency-associated peptide (amino acids 81-94) inhibits sTSP-mediated activation of latent TGF-beta in both the chemically defined system and in endothelial cell conditioned medium. These data and the observation that similar concentrations of sTSP activate latent TGF-beta in both the chemically defined system and in the endothelial cell system indicate that there is a common mechanism by which TSP activates the small, large, and endothelial cell-derived latent TGF-beta complexes. The ability of TSP to convert latent TGF-beta to biologically active TGF-beta suggests that TSP is a major regulatory factor in the control of TGF-beta activity.