ABSTRACT
Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3ß (GSK-3ß). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-ß (TGFß) and is known to inhibit various TGFß-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFß stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3ß kinases to facilitate local TGFß/p38-dependent inactivation of GSK-3ß, accumulation of ß-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFß type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFß.
Subject(s)
Adenomatous Polyposis Coli Protein/metabolism , Cell Movement , Microtubules/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptors, Transforming Growth Factor beta/metabolism , Smad7 Protein/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Polarity/drug effects , Enzyme Activation/drug effects , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Male , Mice , Microtubules/drug effects , Models, Biological , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , Protein Binding/drug effects , Pseudopodia/drug effects , Pseudopodia/metabolism , Receptor, Transforming Growth Factor-beta Type I , Transforming Growth Factor beta/pharmacology , beta Catenin/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Members of the transforming growth factor beta (TGF-beta) and Wnt/wingless superfamilies regulate cell fate during development and tissue maintenance. Here we report that Smad7 interacts with beta-catenin and lymphoid enhancer binding factor 1/T-cell-specific factor (LEF1/TCF), transcriptional regulators in Wnt signaling, in a TGF-beta-dependent manner. Smad7 was found to be required for TGF-beta1-induced accumulation of beta-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-beta-induced increase of activated p38, Akt phosphorylated on Ser473, glycogen synthase kinase 3beta phosphorylated on Ser9 was prevented, as well as the TGF-beta-induced association between beta-catenin and LEF1. Notably, the observed physical association of Smad7 and beta-catenin was found to be important for TGF-beta-induced apoptosis, since suppression of beta-catenin expression by small interfering RNA decreased the apoptotic response to TGF-beta.
Subject(s)
Apoptosis/physiology , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/pharmacology , Animals , Apoptosis/drug effects , COS Cells , Cell Fractionation , Chlorocebus aethiops , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Lymphoid Enhancer-Binding Factor 1 , Male , Mice , Phosphorylation/drug effects , Prostatic Neoplasms/metabolism , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , RNA, Small Interfering/metabolism , Serine/metabolism , Smad7 Protein , Tumor Cells, Cultured , beta Catenin , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Transforming growth factor beta (TGF-beta) 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-beta receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-beta/Smad signaling cascade. In a previous study, we found that TGF-beta 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-beta signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-beta-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-beta-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-beta-dependent activation of Rho GTPases.
Subject(s)
DNA-Binding Proteins/metabolism , Monomeric GTP-Binding Proteins/metabolism , Trans-Activators/metabolism , Transforming Growth Factor beta/pharmacology , cdc42 GTP-Binding Protein/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/metabolism , Cadmium Chloride/pharmacology , Cell Line, Tumor , Cell Surface Extensions/drug effects , Chromones/pharmacology , DNA-Binding Proteins/genetics , Enzyme Activation/drug effects , Guanosine Triphosphate/metabolism , Humans , Imidazoles/pharmacology , MAP Kinase Signaling System/drug effects , Morpholines/pharmacology , Oligodeoxyribonucleotides, Antisense , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Pyridines/pharmacology , Smad7 Protein , Stress Fibers/drug effects , Stress Fibers/metabolism , Trans-Activators/genetics , Transforming Growth Factor beta/agonists , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The inhibitory Smad7, a direct target gene for transforming growth factor-beta (TGF-beta), mediates TGF-beta1-induced apoptosis in several cell types. Herein, we report that apoptosis of human prostate cancer PC-3U cells induced by TGF-beta1 or Smad7 overexpression is caused by a specific activation of the p38 mitogen-activated protein kinase pathway in a TGF-beta-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-beta1-induced apoptosis. The expression of Smad7 was required for TGF-beta-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-beta1-induced phosphorylation of p38 and apoptosis, whereas dominant negative TAK1 reduced TGF-beta1-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.
Subject(s)
Apoptosis , DNA-Binding Proteins/metabolism , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Prostatic Neoplasms/pathology , Protein-Tyrosine Kinases/metabolism , Trans-Activators/metabolism , Transforming Growth Factor beta/physiology , Animals , Blotting, Western , COS Cells , DNA Fragmentation , Enzyme Activation , Enzyme Inhibitors/pharmacology , Genes, Dominant , Humans , In Situ Nick-End Labeling , MAP Kinase Kinase 3 , Male , Microscopy, Fluorescence , Phosphorylation , Precipitin Tests , Protein Binding , Smad7 Protein , Time Factors , Transfection , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1 , Tumor Cells, Cultured , p38 Mitogen-Activated Protein KinasesABSTRACT
Transforming growth factor-beta (TGF-beta) 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-beta receptors. We studied TGF-beta-induced rearrangements of the actin filament system and found that TGF-beta 1 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-beta 1 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 p38(MAPK); treatment of the cells with the p38(MAPK) inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38(MAPK), abrogated the TGF-beta-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-beta 1-induced formation of stress fibers. Collectively, these data indicate that TGF-beta-induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.
