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Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

Hough C, Radu M, Doré JJ - PLoS ONE (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: BioMedical Sciences, Memorial University, St. John's, Newfoundland, Canada.

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.

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Erk activity is integral for TGF-β signaling and inducing growth in fibroblasts.(A) Fold change in expression levels of two smad regulated genes, PAI-1 and smad7, relative to untreated controls levels are shown from AKR-2B fibroblasts treated for 10 minutes with or without TGF-β (2 ng/ml), with or without U0126 (10 µM) for 3 h. The mean values (±SEM) of six independent experiments are shown with statistical evaluation indicating differences between groups as (NS) not significant, (*) P<0.05, (***) P<0.001. (B) Thymidine incorporation was determined in serum deprived NIH 3T3 cells treated with TGF-β (5 ng/ml) with or without infection with Ad-dnPAK2 or Ad-EGFP (MOI = 125∶1) or U0126 (10 µM) prior to treatment. The effect of treatment is expressed as a fold change in thymidine incorporation relative to untreated cells (control = 1). The mean (±SEM) of triplicate assays is shown with statistical evaluation indicating differences between groups as previously describe. (C) Schematic representation of TGF-β signaling pathways in fibroblasts, indicating the proposed interactions and their subcellular locations. Arrows represent a functional link between the subsequent proteins, not necessarily a direct interaction.
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pone-0042513-g006: Erk activity is integral for TGF-β signaling and inducing growth in fibroblasts.(A) Fold change in expression levels of two smad regulated genes, PAI-1 and smad7, relative to untreated controls levels are shown from AKR-2B fibroblasts treated for 10 minutes with or without TGF-β (2 ng/ml), with or without U0126 (10 µM) for 3 h. The mean values (±SEM) of six independent experiments are shown with statistical evaluation indicating differences between groups as (NS) not significant, (*) P<0.05, (***) P<0.001. (B) Thymidine incorporation was determined in serum deprived NIH 3T3 cells treated with TGF-β (5 ng/ml) with or without infection with Ad-dnPAK2 or Ad-EGFP (MOI = 125∶1) or U0126 (10 µM) prior to treatment. The effect of treatment is expressed as a fold change in thymidine incorporation relative to untreated cells (control = 1). The mean (±SEM) of triplicate assays is shown with statistical evaluation indicating differences between groups as previously describe. (C) Schematic representation of TGF-β signaling pathways in fibroblasts, indicating the proposed interactions and their subcellular locations. Arrows represent a functional link between the subsequent proteins, not necessarily a direct interaction.

Mentions: Since nuclear localized, C-terminal phospho-smads bind DNA and regulate gene expression [38], this implies that increased stability would likely increase duration of function. By transiently treating fibroblasts with TGF-β (10 min.), we wished to assess the stability of smad mediated transcription. The 3 h time point chosen to assess mRNA stability was based on the differences in western blot phospho-smad densities in figure 5C. As shown in figure 5C/D, smads remained phosphorylated longer when Erk was active. Likewise, smad mediated gene transcription of both PAI-1 and Smad7 remained elevated greater than 2 fold (P>0.001; Figure 6A) in the presence of active Erk, three hours after removal of TGF-β treatment.


Tgf-beta induced Erk phosphorylation of smad linker region regulates smad signaling.

Hough C, Radu M, Doré JJ - PLoS ONE (2012)

Erk activity is integral for TGF-β signaling and inducing growth in fibroblasts.(A) Fold change in expression levels of two smad regulated genes, PAI-1 and smad7, relative to untreated controls levels are shown from AKR-2B fibroblasts treated for 10 minutes with or without TGF-β (2 ng/ml), with or without U0126 (10 µM) for 3 h. The mean values (±SEM) of six independent experiments are shown with statistical evaluation indicating differences between groups as (NS) not significant, (*) P<0.05, (***) P<0.001. (B) Thymidine incorporation was determined in serum deprived NIH 3T3 cells treated with TGF-β (5 ng/ml) with or without infection with Ad-dnPAK2 or Ad-EGFP (MOI = 125∶1) or U0126 (10 µM) prior to treatment. The effect of treatment is expressed as a fold change in thymidine incorporation relative to untreated cells (control = 1). The mean (±SEM) of triplicate assays is shown with statistical evaluation indicating differences between groups as previously describe. (C) Schematic representation of TGF-β signaling pathways in fibroblasts, indicating the proposed interactions and their subcellular locations. Arrows represent a functional link between the subsequent proteins, not necessarily a direct interaction.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412844&req=5

pone-0042513-g006: Erk activity is integral for TGF-β signaling and inducing growth in fibroblasts.(A) Fold change in expression levels of two smad regulated genes, PAI-1 and smad7, relative to untreated controls levels are shown from AKR-2B fibroblasts treated for 10 minutes with or without TGF-β (2 ng/ml), with or without U0126 (10 µM) for 3 h. The mean values (±SEM) of six independent experiments are shown with statistical evaluation indicating differences between groups as (NS) not significant, (*) P<0.05, (***) P<0.001. (B) Thymidine incorporation was determined in serum deprived NIH 3T3 cells treated with TGF-β (5 ng/ml) with or without infection with Ad-dnPAK2 or Ad-EGFP (MOI = 125∶1) or U0126 (10 µM) prior to treatment. The effect of treatment is expressed as a fold change in thymidine incorporation relative to untreated cells (control = 1). The mean (±SEM) of triplicate assays is shown with statistical evaluation indicating differences between groups as previously describe. (C) Schematic representation of TGF-β signaling pathways in fibroblasts, indicating the proposed interactions and their subcellular locations. Arrows represent a functional link between the subsequent proteins, not necessarily a direct interaction.
Mentions: Since nuclear localized, C-terminal phospho-smads bind DNA and regulate gene expression [38], this implies that increased stability would likely increase duration of function. By transiently treating fibroblasts with TGF-β (10 min.), we wished to assess the stability of smad mediated transcription. The 3 h time point chosen to assess mRNA stability was based on the differences in western blot phospho-smad densities in figure 5C. As shown in figure 5C/D, smads remained phosphorylated longer when Erk was active. Likewise, smad mediated gene transcription of both PAI-1 and Smad7 remained elevated greater than 2 fold (P>0.001; Figure 6A) in the presence of active Erk, three hours after removal of TGF-β treatment.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: BioMedical Sciences, Memorial University, St. John's, Newfoundland, Canada.

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.

Show MeSH
Related in: MedlinePlus