Mechanism of Activation of Latent Recombinant Transforming Growth Factor by Plasmin

Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-proTGF/31 cDNA contains high levels of latent TGF/31. The amino-terminal region of the TGF/~I precursor is secreted and can be detected in the conditioned medium by immunoblotting using peptide antibodies specific for amino-terminal peptides. Chemical crosslinking of CHO-conditioned medium using bis-(sulfosuccinimidyl)-suberate (BS 3) followed by immunoblot analyses indicates that latent recombinant TGF/31 contains both the cleaved amino-terminal glycopeptide and mature TGF/31 polypeptide in a noncovalent association and that this association confers latency. The data presented here do not support the involvement of a unique TGF/3 binding protein(s) in latent recombinant TGF/31. Plasmin treatment of CHO-conditioned medium resulted in the appearance of TGF/3 competing activity. In addition, immunoblot analysis of plasmintreated CHO-conditioned medium indicates that the amino-terminal glycopeptide is partially degraded and that mature TGF/31 is released. Thus, activation of latent TGFB1 may occur by proteolytic nicking within the amino-terminal glycopeptide thereby causing a disruption of tertiary structure and noncovalent bonds, which results in the release of active, mature TGF/~I. Acid activation of latent TGF/~, in comparison, appears to be due to dissociation of the amino-terminal glycopeptide from the mature polypeptide. T RANSFORMING growth factor/3 (TGFB) ~ is an important regulator of cell growth and differentiation. TGF/3 has been shown to be a potent inhibitor of proliferation of many cell types, both normal and transformed (Tucker et al., 1984b; Moses et al., 1985). In addition, TGF/3 may stimulate growth under specific conditions in certain cell types. The majority of cultured cells studied to date have been shown to secrete TGF/~I in an inactive form (Lawrence et al., 1985; Pircher et al., 1986) that neither interacts with specific TGF/~ cell surface receptors nor elicits any of the known TGF/3-induced biological responses. The presence of specific TGF/3 receptors on most cell types (Tucker et al., 1984a) and the ubiquity of the TGF/3 molecule itself(Roberts et al., 1983; Moses et al., 1985) suggest that activation of latent TGF/~ must be an integral component in the sequence of events leading to growth regulation by TGFB. The active 25-kD TGF/3 molecule consists of two identical disulfide-linked 12.5-kD polypeptide chains (Assoian et al., 1983). The 12.5-kD monomer contains 112 amino acids and is synthesized as a larger pre-pro-TGFffl molecule of 390 amino acid residues as determined by cDNA sequencing (Derynck et al., 1985). The carboxy-terminal 12.5-kD mature peptide is proteolyticaUy cleaved from the aminoterminal glycopeptide at arginine 278 (Derynck et al., 1985; I. Abbreviations used in this paper: BS 3, bis(sulfosuccinimidyl)suberate; TGF/3, transforming growth factor/3. Gentry et al., 1987). In addition, a signal sequence of 29 amino acids is cleaved by a signal peptidase during transit of the precursor through the rough endoplasmic reticulum (Gentry et al., 1988). Other posttranslational modifications of TGF/~ that take place before secretion include glycosylation, mannose-6-phosphorylation of the glycoprotein and disulfide isomerization (Gentry et al., 1988; Purchi0 et al., 1988; Brunner et al., 1988). These events have been designated "processing" steps, and, while their order has not been well characterized, they are distinct from "activation" events that are required for the generation of biologically active TGF/$ from the latent molecule. Activation may be achieved by extremes of pH or by treatment with selected chaotrophic agents, including SDS and urea (Lawrence et al., 1985; Pireher et al., 1986). More recently, we have reported protease, specifically plasmin, activation of latent TGF/3 secreted by fibroblasts (Lyons et al., 1988). Recent findings of Sato and Rifkin (1989) indicate that activation of latent TGF/~ occurring during co-culture of bovine endothelial cells and pericytes is due to plasmin. This suggests that plasmin may be a physiological activator of latent TGF/3. Chinese hamster ovary (CHO) ceils transfected with simian TGF/~I eDNA secrete 200-1,000-fold more latent TGF/~I than nontransfected cells providing an excellent source of material for use in activation studies (Gentry et al., 1987). The overexpression of latent recombinant TGF/$1 enabled the detection of the amino-terminal glycopeptide and mature © The Rockefeller University Press, 0021-95251901041136117 $2.00 The Journal of Cell Biology, Volume 110, April 199