Abstract. Transforming growth factor-13 (TGFI3) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGFI3 were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGFI3 was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGF[3 was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGFI3 as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved by pH 1.5. In an effort to define more physiological means of TGFI3 activation, the effects of some proteases were tested. Plasmin and cathepsin D were found to generate 25-kD bands corresponding to the active form of TGFI3 as shown by immunoprecipitation analysis of radiolabeled cell-conditioned medium. Plasmin treatment of the medium resulted in activity that was quantitatively similar to that of mild acid treatment as measured by radioreceptor and soft agar assays. In addition, the plasmin-generated activity was inhibited by anti-TGFI3 antibodies. Sequential treatments of AKR-MCA cell-conditioned medium with mild acid followed by plasmin or plasmin followed by mild acid gave activation comparable to either treatment alone. The data suggest that conditioned medium may contain at least two different pools of latent TGFI3. One pool is resistant to mild acid and/or plasmin and requires strong acid or alkali treatment for activation. A second pool is activated by mild pH change and/or plasmin. Activation of this form of latent TGFI3 may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGFl3-binding protein complex.

Abstract. Medium conditioned by Chinese hamster ovary (CHO) cells transfected with the simian pre-pro-TGF/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.

No abstract

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.