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

Hough C, Radu M, Dor├ę 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-╬▓) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-╬▓ 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-╬▓ receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-╬▓ induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-╬▓ stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-╬▓ 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-╬▓/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-╬▓-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 is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 ┬ÁM) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-╬▓ (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 ┬ÁM) 30 minutes before TGF-╬▓ was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 ┬ÁM or 20 ┬ÁM, respecitively) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (┬▒SEM) shown.
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pone-0042513-g002: Erk is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 ┬ÁM) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-╬▓ (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 ┬ÁM) 30 minutes before TGF-╬▓ was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 ┬ÁM or 20 ┬ÁM, respecitively) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (┬▒SEM) shown.

Mentions: Having shown Erk activation, via TGF-╬▓, as a cell type specific response, we next wanted to confirm the mechanism through which the TGF-╬▓ signal was propagated. Previous studies have shown that TGF-╬▓ induced activation of Pak2 and/or Ras [10], [12], [13], we sought to identify the pathway resulting in Erk activation. Using LY294002, a specific inhibitor of PI3K, Erk phosphorylation was inhibited (Figure 2). Additionally, the MEK1/2 inhibitor U0126, blocked Erk phosphorylation, demonstrating that not only is PI3K necessary but also the MAPKK, MEK is involved in TGF-╬▓ activation of Erk. There was also a temporal increase in c-Raf phosphorylation (Figure 2B) dependent on TGF-╬▓ stimulation and subsequent to PI3K activation as shown by the phosphorylation of S338, a known Pak activation site [34], [35] and its inhibition by the PI3K inhibitor LY294002. In the canonical Erk pathway, activated Ras initiates a cascade resulting in Erk activation. Using small molecular weight inhibitors, we evaluated the activation of two different PI3K activated pathways, Erk and Akt (Figure 2C). Although both pathways were activated by TGF-╬▓ through PI3K, only Erk phosphorylation was sensitive to U0126 and neither pathway was inhibited by the Ras farnysylation inhibitor, FPT II [36]. TGF-╬▓ induced Erk phosphorylation was significantly increased, above control levels (untreated and FTPII), with no significant difference found between the two TGF-╬▓ treated groups (TGF-╬▓ alone and TGF- ╬▓+FTPII).


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

Hough C, Radu M, Dor├ę JJ - PLoS ONE (2012)

Erk is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 ┬ÁM) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-╬▓ (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 ┬ÁM) 30 minutes before TGF-╬▓ was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 ┬ÁM or 20 ┬ÁM, respecitively) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (┬▒SEM) shown.
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Related In: Results  -  Collection

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pone-0042513-g002: Erk is activated in fibroblasts via the PI3K/c-Raf/MEK pathway.(A) AKR-2B fibroblasts were treated with the PI3K inhibitor LY294002 or MEK inhibitor U0126 (10 ┬ÁM) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with an antibody specific to phospho-Erk, blots were then stripped and reprobed for total Erk as a loading control. (B) AKR-2B fibroblasts were treated with TGF-╬▓ (2 ng/ml) for the indicated times. Cells were also treated with LY294002 (10 ┬ÁM) 30 minutes before TGF-╬▓ was added for 2 h. Blots were probed using an antibody specific to phospho-c-Raf (Ser338), and total Erk as a loading control. The loading control blot was obtained using the lower molecular mass portion of the same blot. (C) AKR-2B fibroblasts were treated with LY294002, U0126 or Ras inhibitor FPTII (10 ┬ÁM or 20 ┬ÁM, respecitively) 30 minutes prior to addition of TGF-╬▓ (2 ng/ml) for 2 h. Cell lysates were probed with antibodies specific to phospho-Erk or phospho-Akt (S473) with the blots stripped and reprobed for the corresponding total protein as a loading control. Comparisons of the relative intensity of bands of Phopho-Erk relative to total Erk loading control was expressed as fold increase relative to untreated control (set at 1). Statistical analysis was used to determine if treatments were not significant (NS), or significant at P<0.05 (*) or P<0.01 (**). Analysis was performed on four independent blots and the mean values (┬▒SEM) shown.
Mentions: Having shown Erk activation, via TGF-╬▓, as a cell type specific response, we next wanted to confirm the mechanism through which the TGF-╬▓ signal was propagated. Previous studies have shown that TGF-╬▓ induced activation of Pak2 and/or Ras [10], [12], [13], we sought to identify the pathway resulting in Erk activation. Using LY294002, a specific inhibitor of PI3K, Erk phosphorylation was inhibited (Figure 2). Additionally, the MEK1/2 inhibitor U0126, blocked Erk phosphorylation, demonstrating that not only is PI3K necessary but also the MAPKK, MEK is involved in TGF-╬▓ activation of Erk. There was also a temporal increase in c-Raf phosphorylation (Figure 2B) dependent on TGF-╬▓ stimulation and subsequent to PI3K activation as shown by the phosphorylation of S338, a known Pak activation site [34], [35] and its inhibition by the PI3K inhibitor LY294002. In the canonical Erk pathway, activated Ras initiates a cascade resulting in Erk activation. Using small molecular weight inhibitors, we evaluated the activation of two different PI3K activated pathways, Erk and Akt (Figure 2C). Although both pathways were activated by TGF-╬▓ through PI3K, only Erk phosphorylation was sensitive to U0126 and neither pathway was inhibited by the Ras farnysylation inhibitor, FPT II [36]. TGF-╬▓ induced Erk phosphorylation was significantly increased, above control levels (untreated and FTPII), with no significant difference found between the two TGF-╬▓ treated groups (TGF-╬▓ alone and TGF- ╬▓+FTPII).

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-╬▓) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-╬▓ 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-╬▓ receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-╬▓ induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-╬▓ stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-╬▓ 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-╬▓/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-╬▓-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