Figure 4.
Determination of MMP-1 expression in HT-29 colon carcinoma cells dependent on STAT3 activation. (A) Influence of STAT3 activation by cell stimulation with IL-6 and/or (over)expression of STAT3 variants. Parental HT-29 cells and derivatives stably expressing STAT3 or STAT3 c.a. were grown for 24 hours in the absence or presence of 20 ng/ml IL-6 as indicated. Total RNA was isolated, transcribed into cDNA, and subjected to quantitative RT-PCR with specific primers and probes for MMP-1 as described in Materials and Methods. Three independent experiments, each run in triplicate, were performed, and the results were normalized to 18S rRNA expression. Relative MMP-1 expression was calculated by arbitrarily setting the expressions from samples with STAT3 c.a. as 1 (100%). (B) Influence of STAT3 activation on enzymatic MMP-1 activity. HT-29 cells (over)expressing STAT3, the constitutively active variant STAT3 c.a. or the dominant-negative STAT3 mutant Y705F (STAT3 d.n.) were cultured for 4 days in the absence or presence of 20 ng/ml IL-6 as indicated. Culture supernatants were assayed for active MMP-1 by the turnover of a fluorogenic substrate as described in Materials and Methods. Data represent the means of three independent experiments. (C) Influence of STAT3 expression in HT-29 cells on MMP-1 promoter activity. HT-29 cells or HT-29 cells stably (over)expressing STAT3 or STAT3 c.a., respectively, were transiently transfected with 1 µg of the -4372 hMMP-luci plasmid together with 0.1 µg of Renilla plasmid as an internal control. After 14 hours, cells were harvested and protein lysates were assayed for luciferase and renilla luciferase activities as described in Materials and Methods. (D) Influence of IL-6-induced STAT3 activation on MMP-1 promoter activity in HT-29 cells. The experiment was performed as described in (C), but after 14 hours cells were washed and, before lysis, incubated for 8 hours with fresh medium with or without 20 ng/ml IL-6 as indicated. Results in (C) and (D) represent the means of three independent transfections, each run in triplicate.