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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 and SMA Expression Require Palladin.(A) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of MRTF-A were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg and # p<0.05 vs siNeg + TGF-β. (B,C) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against MRTF-A (siMRTF-A) and treated as in A. Total protein was extracted and levels of palladin, SMA, and CNN were analyzed using specific antibodies. β-tubulin was used as a loading control. Bars (B) are means ± SE of 4 independent experiments. Bars (C) are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, # p<0.05 vs siNeg + TGF-β, and ### p<0.001 vs siNeg + TGF-β. (D) Human VSMCs were treated as in A. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg, **p<0.01 vs siNeg, ## p<0.01 vs siNeg + TGF-β, #### p<0.0001 vs siNeg + TGF-β, and &&&& p<0.0001 vs siNeg. (E) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 48 hr. Total protein was extracted and levels of CNN were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ****p<0.0001 vs siNeg, ## p<0.01 vs siNeg + TGF-β.
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pone.0153199.g007: TGF-β-induced MRTF-A Phosphorylation and SMA Expression Require Palladin.(A) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of MRTF-A were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg and # p<0.05 vs siNeg + TGF-β. (B,C) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against MRTF-A (siMRTF-A) and treated as in A. Total protein was extracted and levels of palladin, SMA, and CNN were analyzed using specific antibodies. β-tubulin was used as a loading control. Bars (B) are means ± SE of 4 independent experiments. Bars (C) are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, # p<0.05 vs siNeg + TGF-β, and ### p<0.001 vs siNeg + TGF-β. (D) Human VSMCs were treated as in A. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg, **p<0.01 vs siNeg, ## p<0.01 vs siNeg + TGF-β, #### p<0.0001 vs siNeg + TGF-β, and &&&& p<0.0001 vs siNeg. (E) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 48 hr. Total protein was extracted and levels of CNN were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ****p<0.0001 vs siNeg, ## p<0.01 vs siNeg + TGF-β.

Mentions: Jin et al. [14] showed that palladin binds to MRTF-A, but the function of this interaction remains unclear. Based on this finding and considering that palladin expression is controlled by TGF-β/Nox4 in VSMC, we tested if knockdown of palladin affects TGF-β-induced MRTF-A phosphorylation. Indeed, knockdown of palladin prevented TGF-β-stimulated MRTF-A phosphorylation (Fig 7A).


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 and SMA Expression Require Palladin.(A) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of MRTF-A were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg and # p<0.05 vs siNeg + TGF-β. (B,C) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against MRTF-A (siMRTF-A) and treated as in A. Total protein was extracted and levels of palladin, SMA, and CNN were analyzed using specific antibodies. β-tubulin was used as a loading control. Bars (B) are means ± SE of 4 independent experiments. Bars (C) are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, # p<0.05 vs siNeg + TGF-β, and ### p<0.001 vs siNeg + TGF-β. (D) Human VSMCs were treated as in A. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg, **p<0.01 vs siNeg, ## p<0.01 vs siNeg + TGF-β, #### p<0.0001 vs siNeg + TGF-β, and &&&& p<0.0001 vs siNeg. (E) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 48 hr. Total protein was extracted and levels of CNN were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ****p<0.0001 vs siNeg, ## p<0.01 vs siNeg + TGF-β.
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pone.0153199.g007: TGF-β-induced MRTF-A Phosphorylation and SMA Expression Require Palladin.(A) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of MRTF-A were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg and # p<0.05 vs siNeg + TGF-β. (B,C) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against MRTF-A (siMRTF-A) and treated as in A. Total protein was extracted and levels of palladin, SMA, and CNN were analyzed using specific antibodies. β-tubulin was used as a loading control. Bars (B) are means ± SE of 4 independent experiments. Bars (C) are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, # p<0.05 vs siNeg + TGF-β, and ### p<0.001 vs siNeg + TGF-β. (D) Human VSMCs were treated as in A. Bars are means ± SE of 3 independent experiments. *p<0.05 vs siNeg, **p<0.01 vs siNeg, ## p<0.01 vs siNeg + TGF-β, #### p<0.0001 vs siNeg + TGF-β, and &&&& p<0.0001 vs siNeg. (E) Human VSMCs were transfected with control siRNA (siNeg) or siRNA against palladin (siPall). After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 48 hr. Total protein was extracted and levels of CNN were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ****p<0.0001 vs siNeg, ## p<0.01 vs siNeg + TGF-β.
Mentions: Jin et al. [14] showed that palladin binds to MRTF-A, but the function of this interaction remains unclear. Based on this finding and considering that palladin expression is controlled by TGF-β/Nox4 in VSMC, we tested if knockdown of palladin affects TGF-β-induced MRTF-A phosphorylation. Indeed, knockdown of palladin prevented TGF-β-stimulated MRTF-A phosphorylation (Fig 7A).

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