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Redox-Sensitive Regulation of Myocardin-Related Transcription Factor (MRTF-A) Phosphorylation via Palladin in Vascular Smooth Muscle Cell Differentiation Marker Gene Expression.

Lee M, San Martín A, Valdivia A, Martin-Garrido A, Griendling KK - PLoS ONE (2016)

Bottom Line: We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression.Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway.Knockdown of palladin also decreases MRTF-A phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, United Sates of America.

ABSTRACT
Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-β (TGF-β) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle α-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.

No MeSH data available.


Related in: MedlinePlus

TGF-β-induced MRTF-A Phosphorylation is Partially Mediated by ROCK.Human VSMCs were preincubated with 10 μM ROCK inhibitor (Y-27632) for 1 hr. Then the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Total protein was extracted and levels of MRTF-A was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. Y; Y-27632. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and levels of CNN was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. **p<0.01 vs con and # p<0.05 vs TGF-β.
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pone.0153199.g004: TGF-β-induced MRTF-A Phosphorylation is Partially Mediated by ROCK.Human VSMCs were preincubated with 10 μM ROCK inhibitor (Y-27632) for 1 hr. Then the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Total protein was extracted and levels of MRTF-A was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. Y; Y-27632. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and levels of CNN was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. **p<0.01 vs con and # p<0.05 vs TGF-β.

Mentions: As noted above, RhoA is downstream of Nox4 in VSMCs [13] and in NIH3T3 cells, C3 transferase abolishes MRTF-A phosphorylation in response to fetal calf serum [9]. These observations suggest a role for ROCK in MRTF-A phosphorylation, although this has not been tested in VSMCs or with TGF-β as the stimulus. We therefore used Y-27632, a specific ROCK inhibitor, to determine if ROCK mediates MRTF-A phosphorylation in response to TGF-β in VSMCs. Y-27632 treatment significantly reduced MRTF-A phosphorylation and SMA and CNN expression (Fig 4A and 4B), suggesting that in VSMCs, ROCK participates in MRTF-A phosphorylation after TGF-β treatment.


Redox-Sensitive Regulation of Myocardin-Related Transcription Factor (MRTF-A) Phosphorylation via Palladin in Vascular Smooth Muscle Cell Differentiation Marker Gene Expression.

Lee M, San Martín A, Valdivia A, Martin-Garrido A, Griendling KK - PLoS ONE (2016)

TGF-β-induced MRTF-A Phosphorylation is Partially Mediated by ROCK.Human VSMCs were preincubated with 10 μM ROCK inhibitor (Y-27632) for 1 hr. Then the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Total protein was extracted and levels of MRTF-A was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. Y; Y-27632. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and levels of CNN was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. **p<0.01 vs con and # p<0.05 vs TGF-β.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835087&req=5

pone.0153199.g004: TGF-β-induced MRTF-A Phosphorylation is Partially Mediated by ROCK.Human VSMCs were preincubated with 10 μM ROCK inhibitor (Y-27632) for 1 hr. Then the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Total protein was extracted and levels of MRTF-A was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. Y; Y-27632. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and levels of CNN was analyzed using specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. **p<0.01 vs con and # p<0.05 vs TGF-β.
Mentions: As noted above, RhoA is downstream of Nox4 in VSMCs [13] and in NIH3T3 cells, C3 transferase abolishes MRTF-A phosphorylation in response to fetal calf serum [9]. These observations suggest a role for ROCK in MRTF-A phosphorylation, although this has not been tested in VSMCs or with TGF-β as the stimulus. We therefore used Y-27632, a specific ROCK inhibitor, to determine if ROCK mediates MRTF-A phosphorylation in response to TGF-β in VSMCs. Y-27632 treatment significantly reduced MRTF-A phosphorylation and SMA and CNN expression (Fig 4A and 4B), suggesting that in VSMCs, ROCK participates in MRTF-A phosphorylation after TGF-β treatment.

Bottom Line: We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression.Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway.Knockdown of palladin also decreases MRTF-A phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Division of Cardiology, Emory University, Atlanta, Georgia, United Sates of America.

ABSTRACT
Vascular smooth muscle cells (VSMCs) undergo a phenotypic switch from a differentiated to synthetic phenotype in cardiovascular diseases such as atherosclerosis and restenosis. Our previous studies indicate that transforming growth factor-β (TGF-β) helps to maintain the differentiated phenotype by regulating expression of pro-differentiation genes such as smooth muscle α-actin (SMA) and Calponin (CNN) through reactive oxygen species (ROS) derived from NADPH oxidase 4 (Nox4) in VSMCs. In this study, we investigated the relationship between Nox4 and myocardin-related transcription factor-A (MRTF-A), a transcription factor known to be important in expression of smooth muscle marker genes. Previous work has shown that MRTF-A interacts with the actin-binding protein, palladin, although how this interaction affects MRTF-A function is unclear, as is the role of phosphorylation in MRTF-A activity. We found that Rho kinase (ROCK)-mediated phosphorylation of MRTF-A is a key event in the regulation of SMA and CNN in VSMCs and that this phosphorylation depends upon Nox4-mediated palladin expression. Knockdown of Nox4 using siRNA decreases TGF-β -induced palladin expression and MRTF-A phosphorylation, suggesting redox-sensitive regulation of this signaling pathway. Knockdown of palladin also decreases MRTF-A phosphorylation. These data suggest that Nox4-dependent palladin expression and ROCK regulate phosphorylation of MRTF-A, a critical factor in the regulation of SRF responsive gene expression.

No MeSH data available.


Related in: MedlinePlus