TGF-Beta Induced Erk Phosphorylation of Smad Linker Region Regulates Smad Signaling

Abstract

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Mathematical Modeling and Analysis of Crosstalk between MAPK Pathway and Smad-Dependent TGF-β Signal Transduction

“…It is assumed that PSmadL attenuates R-Smad to inhibit its association with C-Smad which causes the negative regulation of TGF-β/Smad signaling by the MAPK pathway [19]. However, this argument cannot explain the opposite effect of the MAPK pathway on TGF-β/Smad signaling reported in several sources in the literature [1,9,[11][12][13]27]. As previously mentioned, some studies suggest that the levels of Ras activity could dictate the outcome of the cross talk [18,19].…”

“…Experiments by Hough et al [1] and Funaba et al [12] showed that most of PSmadL was found in the nucleus of cell fibroblasts. These data do not preclude the possibility of cytoplasmic phosphorylation of Smad2 in the linker region, followed by a rapid nuclear translocation [13,35].…”

“…Rapid and transient p38 MAPK activation by TGF-β has been described in certain cell types, including human neutrophils, HEK293, and C2C12 cells, whereas the prolonged and sustained p38 MAPK activation was observed in pancreatic carcinoma cells, hepatocytes and osteoblasts [26]. Hough et al reported that Erk phosphorylation in mesenchymal cells became significant between 60 and 90 min and increased for at least 3 h after TGF-β addition [1]. In mesenchymal AKR-2B cells, Erk phosphorylation became detectable between 20 and 40 min of TGF-β treatment and remained elevated for at least 120 min [23].…”

“…A number of studies mention that the linker region phosphorylation of Smad proteins plays an important role on how MAPKs, i.e., Erk, p38, or JNK, affect the TGF-β/Smad pathway [1,2,[9][10][11][12][13][14][15]. Reported mechanisms include activated MAPKs phosphorylating R-Smad proteins, regulating nuclear translocation or DNA binding of Smads [9], all of which either enhance or inhibit the TGF-β induced gene expression.…”

“…Similarly, there are fewer studies investigating the interaction at point III. A few experimental studies [1,2] have concluded that activated MAPKs increased stability of C-terminal phosphorylated R-Smad and the amount of the nuclear Smad complex. However, the underlying interaction kinetics have not been disclosed.…”