Transforming growth factor-β (TGF-β) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-β receptors. We studied TGF-β–induced rearrangements of the actin filament system and found that TGF-β1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-β1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38MAPK; treatment of the cells with the p38MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38MAPK, abrogated the TGF-β–induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-β1–induced formation of stress fibers. Collectively, these data indicate that TGF-β–induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.

The inhibitory Smad7, a direct target gene for transforming growth factor-β (TGF-β), mediates TGF-β1–induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-β1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-β–activated kinase 1 (TAK1)- and mitogen-activated protein kinase kinase 3 (MKK3)-dependent manner. Expression of dominant negative p38, dominant negative MKK3, or incubation with the p38 selective inhibitor [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], prevented TGF-β1–induced apoptosis. The expression of Smad7 was required for TGF-β–induced activation of MKK3 and p38 kinases, and endogenous Smad7 was found to interact with phosphorylated p38 in a ligand-dependent manner. Ectopic expression of wild-type TAK1 promoted TGF-β1–induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-β1–induced phosphorylation of p38 and apoptosis. Endogenous Smad7 was found to interact with TAK1, and TAK1, MKK3, and p38 were coimmunoprecipitated with Smad7 in transiently transfected COS1 cells. Moreover, ectopically expressed Smad7 enhanced the coimmunoprecipitation of HA-MKK3 and Flag-p38, supporting the notion that Smad7 may act as a scaffolding protein and facilitate TAK1- and MKK3-mediated activation of p38.

Members of the transforming growth factor ␤ (TGF-␤) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with ␤-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-␤-dependent manner. Smad7 was found to be required for TGF-␤1-induced accumulation of ␤-catenin and LEF1 in human prostate cancer (PC-3U) cells as well as in human keratinocytes (HaCaT cells). Moreover, when the endogenous Smad7 was repressed by specific small interfering RNA, TGF-␤-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3␤ phosphorylated on Ser9 was prevented, as well as the TGF-␤-induced association between ␤-catenin and LEF1. Notably, the observed physical association of Smad7 and ␤-catenin was found to be important for TGF-␤-induced apoptosis, since suppression of ␤-catenin expression by small interfering RNA decreased the apoptotic response to TGF-␤.

Transforming growth factor β (TGF-β) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-β receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-β/Smad signaling cascade. In a previous study, we found that TGF-β induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-β signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-β-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-β-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-β-dependent activation of Rho GTPases.

A novel function for Smad7 is demonstrated in the establishment of cell polarity during cell migration in certain cell types. TGFβ type I receptors, Smad7, active p38, and APC are localized in the leading edge of polarized, migrating cells.