Abstract
We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.
Highlights
We have recently demonstrated that fibroblast-con- tide that stimulates theproliferation of cultured hepatocytes ditioned medium induces Madin-Darby canine kidney (Nakamura et al, 1989; Miyazawa et al, 1989) and other cell (MDCK) epithelialcellstoformbranchingtubules types (Rubin et al, 1991; Nakamura, 1991; Matsumoto and when grown in three-dimensional collagen or fibrin Nakamura, 1992; Michalopoulos and Zarnegar, 1992)
We have foundthat in collagen or fibrin gels in the presence of fibroblastsor urokinase-type P A (u-PA) activity and mRNA are in- fibroblast-conditionedmedium (Montesanoet al., 1991a), and creased 4.9-fold by conditioned medium (CM) fromhuman MRC-5 fibro- that the fibroblast-derived factor responsible forepithelial blasts, which has tubulogenic activity, but not byCM tubulogenesis is Hepatocyte growth factor (HGF) (Montesanoet al., 1991b)
We have found that tubule formation cells, an effect which was inhibited by 71%by prein- by MDCK cells infibrin gels is prevented by addition of cubating theCM with antibodies to recombinant humanHGF (rhHGF), and whichexogenous serineproteaseinhibitorstotheculture system was mimicked by exogenously added rhHGF(31.%fold (Montesano et al, 1991a)
Summary
We have recently demonstrated that fibroblast-con- tide that stimulates theproliferation of cultured hepatocytes ditioned medium induces Madin-Darby canine kidney (Nakamura et al, 1989; Miyazawa et al, 1989) and other cell (MDCK) epithelialcellstoformbranchingtubules types (Rubin et al, 1991; Nakamura, 1991; Matsumoto and when grown in three-dimensional collagen or fibrin Nakamura, 1992; Michalopoulos and Zarnegar, 1992). Added rhHGF increased u-PA activity andmRNA 5.9-fold in MDCK cells, with anoptimal effect at approximately 10 ng/ml. MRC-5 CM increased u-PA receptor mRNA 34.9-fold in MDCK
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