Proteolytic activation of latent transforming growth factor-β from fibroblast-conditioned medium

Abstract

Transforming growth factor-β (TGFβ) is produced by most cultured cells in an inactive form. Potential activation mechanisms of latent TGFβ were studied using fibroblastic (NRK-49F and AKR-MCA) cell-conditioned medium as a model. Active TGFβ was monitored by radioreceptor and soft agar assays as well as by antibody inhibition and immunoprecipitation. Little or no TGFβ was detected in untreated conditioned medium. Treatment of the medium with extremes of pH (1.5 or 12) resulted in significant activation of TGFβ as shown by radioreceptor assays, while mild acid treatment (pH 4.5) yielded only 20-30% of the competition achieved pH 1.5. In an effort to define more physiological means of TGFβ 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 TGFβ 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 sof agar assays. In addition, the plasmid-generated activity was inhibited by anti-TGFβ 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 TGFβ. 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 TGFβ may take place by dissociation or proteolytic digestion from a precursor molecule or hypothetical TGFβ-binding protein complex.