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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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Cell type specific activation of Erk.(A) AKR-2B and NIH 3T3 fibroblast were treated with TGF-β (2 ng/ml) for times ranging from 0 to 4 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Typical results are shown representing four independent time course experiments. (B) Graph of densitometric analysis of western blots from time courses from 0–3 h (n = 4) of phospho-Erk. Shown is the mean fold increase of Phospho-Erk relative to total Erk for each time point with the 0 time set as 1. Statistically significant change from 0 time is noted as, (*) P<0.05 and (**) P<0.01. (C) Mv1Lu and NMuMG epithelial cell lines were treated with TGF-β (2 ng/ml) for times ranging from 0 to 3 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Triplicate blots were performed on three independent time course experiments with typical results shown.
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pone-0042513-g001: Cell type specific activation of Erk.(A) AKR-2B and NIH 3T3 fibroblast were treated with TGF-β (2 ng/ml) for times ranging from 0 to 4 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Typical results are shown representing four independent time course experiments. (B) Graph of densitometric analysis of western blots from time courses from 0–3 h (n = 4) of phospho-Erk. Shown is the mean fold increase of Phospho-Erk relative to total Erk for each time point with the 0 time set as 1. Statistically significant change from 0 time is noted as, (*) P<0.05 and (**) P<0.01. (C) Mv1Lu and NMuMG epithelial cell lines were treated with TGF-β (2 ng/ml) for times ranging from 0 to 3 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Triplicate blots were performed on three independent time course experiments with typical results shown.

Mentions: Since TGF-β has been known to stimulate fibroblast replication and had been shown to activate PI3K [10], [12] we wished to further define activation of the Erk pathway in normal (untransformed) cell lines. Temporal changes in Erk phosphorylation as the result of fibroblast cell lines treated with TGF-β over the course of 4 h were determined (Figure 1). Taking into account protein loading (total Erk) between lanes, we found Erk phosphorylation increased significantly above background between 60–90 minutes after TGF-β addition (Figure 1B). Previous reports have shown Erk activation at earlier time points [7], [13]. We likewise saw an increase in phospho-Erk earlier, but only when cells were allowed to cool when taken from the incubator during addition of TGF-β. Cells maintained near 37°C, demonstrated no significant activation of Erk prior to the 60–90 minute window. Earlier activation was consistent with Erk signaling being indicative of an induced stress response [32], [33]. Since TGF-β activation of PI3K has been shown to be cell type dependent [10], [12] the previous experiment was repeated using Mv1Lu and NMuMG epithelial cells. No increase in Erk phosphorylation was identified at any time point (Figure 1C) following TGF-β treatment. Together these results confirm that activation of Erk upon TGF-β treatment occurs in phenotypically normal cells of mesenchymal origin, but not epithelial cells.


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

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

Cell type specific activation of Erk.(A) AKR-2B and NIH 3T3 fibroblast were treated with TGF-β (2 ng/ml) for times ranging from 0 to 4 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Typical results are shown representing four independent time course experiments. (B) Graph of densitometric analysis of western blots from time courses from 0–3 h (n = 4) of phospho-Erk. Shown is the mean fold increase of Phospho-Erk relative to total Erk for each time point with the 0 time set as 1. Statistically significant change from 0 time is noted as, (*) P<0.05 and (**) P<0.01. (C) Mv1Lu and NMuMG epithelial cell lines were treated with TGF-β (2 ng/ml) for times ranging from 0 to 3 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Triplicate blots were performed on three independent time course experiments with typical results shown.
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Related In: Results  -  Collection

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pone-0042513-g001: Cell type specific activation of Erk.(A) AKR-2B and NIH 3T3 fibroblast were treated with TGF-β (2 ng/ml) for times ranging from 0 to 4 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Typical results are shown representing four independent time course experiments. (B) Graph of densitometric analysis of western blots from time courses from 0–3 h (n = 4) of phospho-Erk. Shown is the mean fold increase of Phospho-Erk relative to total Erk for each time point with the 0 time set as 1. Statistically significant change from 0 time is noted as, (*) P<0.05 and (**) P<0.01. (C) Mv1Lu and NMuMG epithelial cell lines were treated with TGF-β (2 ng/ml) for times ranging from 0 to 3 h. Cell lysates were probed with antibodies specific to phospho-Erk (P-Erk). Blots were then stripped and reprobed for total Erk as a loading control. Triplicate blots were performed on three independent time course experiments with typical results shown.
Mentions: Since TGF-β has been known to stimulate fibroblast replication and had been shown to activate PI3K [10], [12] we wished to further define activation of the Erk pathway in normal (untransformed) cell lines. Temporal changes in Erk phosphorylation as the result of fibroblast cell lines treated with TGF-β over the course of 4 h were determined (Figure 1). Taking into account protein loading (total Erk) between lanes, we found Erk phosphorylation increased significantly above background between 60–90 minutes after TGF-β addition (Figure 1B). Previous reports have shown Erk activation at earlier time points [7], [13]. We likewise saw an increase in phospho-Erk earlier, but only when cells were allowed to cool when taken from the incubator during addition of TGF-β. Cells maintained near 37°C, demonstrated no significant activation of Erk prior to the 60–90 minute window. Earlier activation was consistent with Erk signaling being indicative of an induced stress response [32], [33]. Since TGF-β activation of PI3K has been shown to be cell type dependent [10], [12] the previous experiment was repeated using Mv1Lu and NMuMG epithelial cells. No increase in Erk phosphorylation was identified at any time point (Figure 1C) following TGF-β treatment. Together these results confirm that activation of Erk upon TGF-β treatment occurs in phenotypically normal cells of mesenchymal origin, but not epithelial cells.

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