Addition of 25 and 50% MMT cell-derived CM for 24 h stimulated a 4-5 fold increase in the release of HGF from normal human dermal fibroblasts in culture. Treatment with specific antibodies and antagonists showed that this effect was not due to PDGF, FGF2, IL-1 and TGFα, all of which have been shown to be released from carcinoma cells.
MMT CM was concentrated and purified by RP HPLC, and HGF production by fibroblasts was used as the assay for the inducing factor. PGE2 not only stimulated the production of HGF by cultured fibroblasts but also co-eluted with the peak of HGF-inducing activity in MMT CM. Both the HPLC peak and its activity were eliminated in the presence of the specific cyclo-oxygenase inhibitor, indomethacin, in a dose-dependent manner as was the secretion of PGE2 from the MMT cells. No consistent differences were found between the amount of HGF released from normal dermal fibroblasts and those found associated with human breast tumour cells, although both PGE2 and MMT CM could stimulate an increase in HGF release from both cell types.
HGF stimulated both the growth, motility and Matrigel invasion of the MMT cells. In co-culture invasion assays, normal human dermal fibroblasts seeded in the lower compartment of an invasion chamber stimulated the invasion of epithelial cells from the upper compartment through a layer of Matrigel. Similarly, the level of HGF produced by the fibroblasts was increased in the presence of MMT cells. Both activities were inhibited by indomethacin and an antibody to HGF. The simultaneous addition of 10-6M PGE2 reversed the inhibition by indomethacin.
HGF treatment also increased the release of u-PA protease, but not gelatinase activity from MMT cells.