Figure 7.

TGFβ induces proteasome-dependent degradation of SMAD7 that is inhibited by NR4A1 ligand CDIM8 (DIM-C-pPhOH). A549 cells were treated with TGFβ (A) and MKK4(WT) (alone, transfected) (B) alone or in combination with MG132 and various agents. Whole cell lysates were analyzed for SMAD7 expression by western blots. (C) A549 cells were transfected with MKK7(CA) alone or in combination with MG132 and various agents and transfected with MKK7(DN) ± TGFβ, and whole cell lysates were analyzed for SMAD7 expression by western blots. A549 cells were treated with DMSO, TGFβ, transfected with MKK7(CA) and MKK4(WT) alone or in combination with MG132 and effects on cell migration (D) and SMAD7 expression (E) were determined in Boyden chamber and western blot assays, respectively. (F) Cells were treated with TGFβ alone or transfected with pCMV6 (empty vector), pCMV6-SMAD7 alone or in combination with TGFβ, and effects on A549 cell migration were determined; cell lysates from these treatment groups were also analyzed for SMAD7 expression by western blots. Results (D and F) are means ± SE for 3 separate determinations, and significantly (p<0.05) enhanced migration (*) and inhibition of this response (**) are indicated. (G) Summary of TGFβ-PKA-MKK4/7-JNK phosphorylation and nuclear export of NR4A1 and inhibition by CDIM8/NR4A1 antagonist. The NR4A1 band intensities relative to β-actin in the western blots (A-C, E) were determined.