Solange M. F. Ribeiro

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

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.

Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-beta, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-beta. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-beta by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-beta activity observed when vitronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent TGF-beta with vitronectin in a cell-free system does not result in increased TGF-beta activity. Assays of total TGF-beta levels in heat-treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-beta in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with antibodies specific for latent TGF-beta. These data suggest that vitronectin regulates expression and/or secretion of TGF-beta by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by alpha v beta 3 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)