tPA induces MMP-9 protein secretion and gene expression in renal interstitial fibroblasts. (a and b) Zymographic analysis shows an induction of MMP-9 secretion by tPA. Rat renal interstitial fibroblasts (NRK-49F) were incubated with different concentrations (10^–9 to 10^–7 M) of human tPA protein for 48 hours (a), or with 10^–8 M tPA for various periods of time (b). Samples of the cell culture supernatants were separated on a polyacrylamide gel containing 1 mg/ml gelatin. Proteolytic activity was demonstrated by digestion of gelatin, resulting in the bands of clearing. The locations of bands corresponding to MMP-9 and MMP-2 are indicated. (c and d) RT-PCR analysis demonstrates that tPA induces MMP-9 gene expression in renal interstitial fibroblasts. NRK-49F cells were incubated with different concentrations (10^–9 and 10^–8 M) of human tPA protein for 24 hours (c), or with 10^–8 M tPA for various periods of time (d). Cellular MMP-9 mRNA levels were determined by RT-PCR analysis. Amplified MMP-9 cDNA fragment with the correct size (1.16 kb) is indicated. RT-PCR amplification of housekeeping β-actin was performed in an identical manner to serve as control. (e and f) Southern blot analysis of the RT-PCR product. To confirm the RT-PCR product as MMP-9 cDNA, Southern blot analysis of the RT-PCR product in c and d was performed using an internal oligonucleotide corresponding to MMP-9 cDNA as a probe.