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. 2011 Dec 8;287(4):2666–2677. doi: 10.1074/jbc.M111.246173

FIGURE 2.

FIGURE 2.

A, Western blotting results show phosphorylation of STAT3 (Tyr-705/Ser-727) upon treatment of cultured cardiac fibroblasts with IL-6 for 20 min (second lane, IL-6+DMSO; third lane, IL-6+NSsiRNA). p-STAT3-Tyr-705 level was reduced significantly with STAT3-specific siRNA (fourth lane, IL-6+STAT3 siRNA) as well as STAT3 inhibitor S31-201 (fifth lane, IL-6+S31-201) treatment together with IL-6. The p-STAT3-Ser-727 level was also reduced by 1.3 ± 0.14-fold (p < 0.05) upon siRNA treatment. Although, S31-201 treatment did not change the total STAT3 level, siRNA treatment resulted in a 1.52 ± 0.2-fold (p < 0.01) decrease in total STAT3 level. α-Actin was used as the internal loading control. B, Western blotting results show phosphorylation (second lane) of p38 MAPK, ERK1/2, and STAT1 (Tyr-701 and Ser-727) in cultured cardiac fibroblasts upon IL-6 treatment (20 min) that were significantly reduced (third lane) by treatment with specific inhibitors for STAT1 (fludarabine, 50 μm), p38 MAPK (SB203580, 10 μm), and ERK1/2 (U0126, 10 μm). No significant change in p-STAT-Ser-727 level was observed upon fludarabine treatment. Total STAT1, p38 MAPK, and ERK1/2 proteins were unchanged during the treatment. C, down-regulation of collagen gene expression with STAT3 and p38 MAPK inhibitors is shown. Significant down-regulation of collagen 1 (1.47-fold, p < 0.001) and collagen 3 transcripts (1.4-fold, p < 0.001) was observed in IL-6-induced (24 h) fibroblasts and treated with STAT3 inhibitor S31-201 as shown by real time PCR. Both collagen 1 and 3 gene expressions were also decreased significantly (1.5- and 1.39-fold, respectively; p < 0.01) with STAT3 siRNA treatment in IL-6-induced cardiac fibroblasts. Fludarabine (STAT1 inhibitor) and U0126 (ERK1/2 inhibitor) had no effect on collagen gene expression. SB203580 treatment resulted in down-regulation of both collagen 1 (1.14-fold, p < 0.05) and collagen 3 transcript (1.1-fold, p < 0.05) levels in IL-6-treated fibroblasts. Results are expressed as ±S.E. of three independent experiments. ***, p < 0.001 with respect to control; †††, p < 0.001 and †, p < 0.05 with respect to IL-6+DMSO treatment; ‡‡, p < 0.01 with respect to IL-6+NSsiRNA treatment). D, hydroxyproline assay shows a 43.68 and 44.9% decrease in IL-6-induced (24 h) collagen synthesis in cultured fibroblasts using S31-201 and STAT3 siRNA, respectively. STAT1and ERK1/2 inhibitors had no effect on IL-6-induced collagen synthesis. p38 MAPK inhibition also resulted in significant regression of total secreted collagen (19.71% regression) in IL-6-induced fibroblast supernatants. Results are expressed as ±S.E. of three independent experiments. **, p < 0.01 with respect to control; ††, p < 0.01 and †, p < 0.05 with respect to IL-6+DMSO treatment; ‡‡, p < 0.01 with respect to IL-6+NSsiRNA treatment.