Limits...
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

Nox4 is Necessary for TGF-β-induced MRTF-A Phosphorylation and SMC Differentiation Marker Expression.Human VSMCs were transfected with siRNA (siNeg) or siRNA against Nox4 (siNox4). Two different sequences for siNox4 were used. After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Nox4 mRNA was analyzed by real-time RT-PCR. Bars are means ± SE of 4 independent experiments. *p<0.05 vs siNeg, # p<0.05 vs siNeg + TGF-β. ND = not detectable. (B) Total protein was extracted and levels of MRTF-A and SMA were analyzed using specific antibodies. β-tubulin was used as a loading control. (C, D) Bars are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, and ### p<0.001 vs TGF-β. E. siNo4 #2 was used for this experiment. Total protein was extracted and the level of CNN was analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 5 independent experiments. ***p<0.001 vs siNeg, ### p<0.001 vs siNeg + TGF-β.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4835087&req=5

pone.0153199.g003: Nox4 is Necessary for TGF-β-induced MRTF-A Phosphorylation and SMC Differentiation Marker Expression.Human VSMCs were transfected with siRNA (siNeg) or siRNA against Nox4 (siNox4). Two different sequences for siNox4 were used. After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Nox4 mRNA was analyzed by real-time RT-PCR. Bars are means ± SE of 4 independent experiments. *p<0.05 vs siNeg, # p<0.05 vs siNeg + TGF-β. ND = not detectable. (B) Total protein was extracted and levels of MRTF-A and SMA were analyzed using specific antibodies. β-tubulin was used as a loading control. (C, D) Bars are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, and ### p<0.001 vs TGF-β. E. siNo4 #2 was used for this experiment. Total protein was extracted and the level of CNN was analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 5 independent experiments. ***p<0.001 vs siNeg, ### p<0.001 vs siNeg + TGF-β.

Mentions: To determine if TGF-β -induced MRTF-A phosphorylation requires ROS, VSMCs were treated with the antioxidant N-acetylcysteine (NAC) prior to TGF-β addition and then MRTF-A phosphorylation was evaluated. TGF-β- stimulated MRTF-A phosphorylation was abolished by NAC treatment (Fig 2A). Importantly, impaired MRTF-A phosphorylation correlated with inhibition of the expression of two of its target genes, SMA and CNN (Fig 2A and 2B). We have previously shown that 20 mM NAC completely scavenges NADPH oxidase-derived ROS in VSMCs [5, 13]. We therefore investigated if Nox4, the NADPH oxidase activated by TGF-β in these cells [5], affects MRTF-A phosphorylation. Using two different but effective siRNAs against Nox4 (Fig 3A), we found that knockdown of Nox4 reduced TGF-β-mediated MRTF-A phosphorylation and, in confirmation of our previous results, SMA and CNN expression as well (Fig 3B–3E).


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)

Nox4 is Necessary for TGF-β-induced MRTF-A Phosphorylation and SMC Differentiation Marker Expression.Human VSMCs were transfected with siRNA (siNeg) or siRNA against Nox4 (siNox4). Two different sequences for siNox4 were used. After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Nox4 mRNA was analyzed by real-time RT-PCR. Bars are means ± SE of 4 independent experiments. *p<0.05 vs siNeg, # p<0.05 vs siNeg + TGF-β. ND = not detectable. (B) Total protein was extracted and levels of MRTF-A and SMA were analyzed using specific antibodies. β-tubulin was used as a loading control. (C, D) Bars are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, and ### p<0.001 vs TGF-β. E. siNo4 #2 was used for this experiment. Total protein was extracted and the level of CNN was analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 5 independent experiments. ***p<0.001 vs siNeg, ### p<0.001 vs siNeg + TGF-β.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153199.g003: Nox4 is Necessary for TGF-β-induced MRTF-A Phosphorylation and SMC Differentiation Marker Expression.Human VSMCs were transfected with siRNA (siNeg) or siRNA against Nox4 (siNox4). Two different sequences for siNox4 were used. After 48 hr, the cells were treated with TGF-β (2 ng/ml) for 24 hr. (A) Nox4 mRNA was analyzed by real-time RT-PCR. Bars are means ± SE of 4 independent experiments. *p<0.05 vs siNeg, # p<0.05 vs siNeg + TGF-β. ND = not detectable. (B) Total protein was extracted and levels of MRTF-A and SMA were analyzed using specific antibodies. β-tubulin was used as a loading control. (C, D) Bars are means ± SE of 3 independent experiments. ****p<0.0001 vs siNeg, and ### p<0.001 vs TGF-β. E. siNo4 #2 was used for this experiment. Total protein was extracted and the level of CNN was analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 5 independent experiments. ***p<0.001 vs siNeg, ### p<0.001 vs siNeg + TGF-β.
Mentions: To determine if TGF-β -induced MRTF-A phosphorylation requires ROS, VSMCs were treated with the antioxidant N-acetylcysteine (NAC) prior to TGF-β addition and then MRTF-A phosphorylation was evaluated. TGF-β- stimulated MRTF-A phosphorylation was abolished by NAC treatment (Fig 2A). Importantly, impaired MRTF-A phosphorylation correlated with inhibition of the expression of two of its target genes, SMA and CNN (Fig 2A and 2B). We have previously shown that 20 mM NAC completely scavenges NADPH oxidase-derived ROS in VSMCs [5, 13]. We therefore investigated if Nox4, the NADPH oxidase activated by TGF-β in these cells [5], affects MRTF-A phosphorylation. Using two different but effective siRNAs against Nox4 (Fig 3A), we found that knockdown of Nox4 reduced TGF-β-mediated MRTF-A phosphorylation and, in confirmation of our previous results, SMA and CNN expression as well (Fig 3B–3E).

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