Requirement for receptor‐bound urokinase in plasmin‐dependent cellular conversion of latent TGF‐β to TGF‐β

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Abstract
The role of receptor‐bound urokinase‐type plasminogen activator (uPA) in cellular activation of latent transforming growth factor‐beta (LTGF‐β) was investigated in a model system of mouse LB6 cells transfected with either a human uPA receptor cDNA (LhuPAR+). a human prouPA cDNA (LhuPA), or a control neomycinresistance cDNA (Lneo). When LhuPAR+ cells were co‐cultured with LhuPA cells, the plasmin‐dependent fibrinolytic activity generated was more than that observed in either homotypic cultures with fivefold greater number of LhuPA cells or co‐cultures containing LhuPA and Lneo cells instead of the LhuPAR+ cells. The preferential activation of TGF‐β by co‐cultures with the greatest plasmin‐generation potential, LhuPAR+ and LhuPA cells, was confirmed by three independent bioassays. In the first assay, a 48% decrease in PA activity, a measure of active TGF‐β production, was observed with BAE cells treated with conditioned medium (CM) from co‐cultures of LhuPA and LhuPAR+ cells. Inclusion of neutralizing antibodies to TGF‐β abrogated the inhibitory effect of CM on PA activity demonstrating that the inhibitory molecule was TGF‐β. Addition of the amino terminal fragment of uPA (ATF) or omission of plasminogen from co‐cultures blocked both the fibrinolytic activity and the generation of TGF‐β activity in the CM. In the second assay, CM from co‐cultures of LhuPA and LhuPAR+ cells inhibited the migration of BAE cells in a wound assay. Controls with anti‐TGF‐β IgG indicated that the inhibition was due to TGF‐β. In the third assay, proliferation of mink lung epithelial cells was inhibited by CM generated by co‐cultures of LhuPA and LhuPAR+ cells as compared to CM from the same cells cultured in the absence of plasminogen or to CM from a co‐culture of LhuPA with LhuPAR− cells. Excess mannose‐6‐phosphate (M6P) blocked the generation of TGF‐β as assayed by both the BAE migration and PA assays, presumably because it interfered with cellsurface localization of LTGF‐β. Additionally, small numbers of LhuPA and LhuPAR+ cells co‐cultured with BAE cells inhibited the BAE cell PA activity via the paracrine action of TGF‐β. These results support the conclusion that plasmindependent activation LTGF‐β by LB6 cells is promoted by the surface localization of uPA by its receptor.