Role of SMAD 3 in TGF-β1-induced Itgβ6 mRNA expression in MMNK-1 cells. A, MMNK-1 cells were stimulated with 5 ng/ml TGF-β1 or vehicle (0.1% BSA in PBS) for 4 h. Itgβ6 mRNA levels were determined by qPCR. B, MMNK-1 cells were treated with 5 ng/ml TGF-β1 or vehicle for various times. Levels of C-terminally phosphorylated SMAD2 and SMAD3, total SMAD2, and total SMAD3 levels were determined in nuclear extracts by Western blotting. A representative Western blot is shown. C, MMNK-1 cells transiently transfected with a SMAD-responsive luciferase reporter construct (SBE4) were stimulated with 5 ng/ml TGF-β1 or vehicle for 4 h and lysed, and luciferase activity was determined. Data are expressed as fold change ± S.E.M. compared with vehicle-treated MMNK-1 cells. D and E, MMNK-1 cells transiently transfected with SMAD3ΔC (D) or SMAD2ΔC (E) mutants were treated with 5 ng/ml TGF-β1 or vehicle for 4 h, and Itgβ6 mRNA levels were determined by qPCR. Data are expressed as mean ± S.E.M. relative to vehicle-treated cells transfected with an empty control plasmid (pRK7) from at least three independent experiments. *, significantly different from 0.1% BSA vehicle-treated group, P < 0.05. #, significantly different from cells transfected with control plasmid, P < 0.05.

Role of AP-1 in TGF-β1-dependent Itgβ6 mRNA induction in MMNK-1 cells. A, MMNK-1 cells transfected with an AP-1 luciferase reporter construct (pAP-1-Luc) were treated with 5 ng/ml TGF-β1 or vehicle for 4 h and lysed, and luciferase activity was determined. B, MMNK-1 cells were transfected with a plasmid containing a dominant-negative AP-1 (TAM67) mutant for 24 h and then treated with 5 ng/ml TGF-β1 or vehicle for 4 h. Itgβ6 mRNA levels were determined by qPCR. *, significantly different from 0.1% BSA vehicle-treated cells, P < 0.05. #, significantly different from cells transfected with a control plasmid (pCMV4), P < 0.05. Data are expressed as mean ± S.E.M. relative to vehicle-treated cells from three independent experiments.

TGF-β1-dependent Itgβ6 mRNA induction requires protein synthesis in MMNK-1 cells. MMNK-1 cells were treated with 5 ng/ml TGF-β1 or vehicle for various times. A and B, JunB mRNA (A) and nuclear protein (B) levels were determined. C and D, MMNK-1 cells were pretreated with 10 μM SB203580 or vehicle (0.1% DMSO) for 30 min before treatment with 5 ng/ml TGF-β1 for 30 min (C) or 2 h (D), and mRNA (C) and nuclear protein (D) levels were determined. E, MMNK-1 cells were pretreated with 10 μg/ml cycloheximide (CHX) or vehicle (0.1% DMSO) for 30 min before treatment with 5 ng/ml TGF-β1 or vehicle for 4 h, and Itgβ6 mRNA levels were determined by qPCR. Data are expressed as mean ± S.E.M. from three independent experiments. *, significantly different from 0.1% BSA vehicle-treated cells, P < 0.05. #, significantly different from cells pretreated with DMSO, P < 0.05.

Role of p38 MAPK in TGF-β1-induced Itgβ6 mRNA expression in MMNK-1 cells. MMNK-1 cells were treated with 5 ng/ml TGF-β1 or vehicle for 10, 30, or 60 min. A, representative Western blot showing phosphorylated p38 and total p38 levels. B, densitometry was performed on Western blots from three independent experiments. Data are expressed as a ratio of phosphorylated p38 to total p38 relative to vehicle-treated cells at 10 min. C to E, MMNK-1 cells were pretreated with 10 μM SB203580 (C), 10 μM SB202190 (D), 10 μM SB202474 (E), or DMSO vehicle (0.1% final concentration) (C–E) for 30 min and then treated with 5 ng/ml TGF-β1 or vehicle for 4 h. Itgβ6 mRNA levels were determined by qPCR. *, significantly different from 0.1% BSA vehicle-treated cells, P < 0.05. #, significantly different from cells pretreated with DMSO, P < 0.05. Data are expressed as mean ± S.E.M. from at least three independent experiments.

Role of ATF-2 in TGF-β1-dependent Itgβ6 mRNA induction in MMNK-1 cells. A, MMNK-1 cells were treated with 5 ng/ml TGF-β1 or vehicle (0.1% BSA in PBS) for 10 or 20 min. A representative Western blot indicating levels of phosphorylated ATF-2 and total ATF-2 is shown. B, densitometry was performed on Western blots from three independent experiments. Data are expressed as a ratio of phosphorylated ATF-2 to total ATF-2 relative to vehicle-treated cells. *, significantly different from 0.1% BSA vehicle-treated cells, P < 0.05. C, MMNK-1 cells were treated with 5 ng/ml TGF-β1 or vehicle for 30 min and nuclear proteins were collected. SMAD3 immunoprecipitation was performed and levels of coimmunoprecipitated ATF-2 were determined by Western blot. A representative Western blot is shown. D, MMNK-1 cells transfected with a SMAD-dependent reporter construct were pretreated with 10 μM SB203580 or vehicle (0.1% DMSO vehicle) for 30 min before treatment with 5 ng/ml TGF-β1 for 4 h and lysed, and luciferase activity was determined. Data are expressed as fold change ± S.E.M. compared with DMSO-treated cells. *, significantly different from DMSO-treated cells, P < 0.05. E to G, MMNK-1 cells were transfected with an ATF-2 siRNA or control siRNA for 48 h before treatment with 5 ng/ml TGF-β1 or vehicle for 4 h. ATF-2 mRNA (E), ATF-2 protein (F), and Itgβ6 mRNA (G) levels were determined. Representative Western blots are shown. Data are expressed as mean ± S.E.M. relative to vehicle-treated cells from three independent experiments. E, *, significantly different from the same group treated with control siRNA, P < 0.05. G, *, significantly different from the same group treated with 0.1% BSA vehicle, P < 0.05.