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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 Molecular Weight Shift is Due To Phosphorylation.Human VSMCs were treated with TGF-β (2 ng/ml) for 24 hr. (A) Lysates were incubated with or without alkaline phosphatase at 37°C for 30 min. Western blot was performed using MRTF-A antibody. Bars represent mean ± SE of 5 independent experiments. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and immunoprecipitated with rabbit anti- MRTF-A antibody. Membranes were immunoblotted with phospho-Ser/Thr and goat anti-MRTF-A antibodies. This figure is representative of 3 independent experiments.
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pone.0153199.g001: TGF-β-induced MRTF-A Molecular Weight Shift is Due To Phosphorylation.Human VSMCs were treated with TGF-β (2 ng/ml) for 24 hr. (A) Lysates were incubated with or without alkaline phosphatase at 37°C for 30 min. Western blot was performed using MRTF-A antibody. Bars represent mean ± SE of 5 independent experiments. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and immunoprecipitated with rabbit anti- MRTF-A antibody. Membranes were immunoblotted with phospho-Ser/Thr and goat anti-MRTF-A antibodies. This figure is representative of 3 independent experiments.

Mentions: We first investigated the effect of TGF-β on MRTF-A phosphorylation in human aortic VSMCs. Although MRTF-A is known to be phosphorylated on multiple sites [9], there is little specific information and no available reagents to probe individual sites; therefore, we evaluated phosphorylation by measuring the upward shift in molecular weight, as has been done by others [9, 12]. As shown in Fig 1A, a 24-h treatment with TGF-β (2 ng/ml) induced a significant shift in molecular weight. Moreover, incubation of the lysate with alkaline phosphatase largely abolished the upward shift of MRTF-A in response to TGF-β, indicating that the molecular weight shift is due to phosphorylation of MRTF-A (Fig 1A). To confirm that the upward shift of MRTF-A is due to phosphorylation, we immunoprecipitated MRTF-A and blotted with phospho-Ser/Thr antibody (Fig 1B). The results show a predominant phospho-Ser/Thr band near 150 kDa that overlaps the MRTF-A band, supporting our conclusion that the molecular weight shift of MRTF-A is due to phosphorylation.


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 Molecular Weight Shift is Due To Phosphorylation.Human VSMCs were treated with TGF-β (2 ng/ml) for 24 hr. (A) Lysates were incubated with or without alkaline phosphatase at 37°C for 30 min. Western blot was performed using MRTF-A antibody. Bars represent mean ± SE of 5 independent experiments. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and immunoprecipitated with rabbit anti- MRTF-A antibody. Membranes were immunoblotted with phospho-Ser/Thr and goat anti-MRTF-A antibodies. This figure is representative of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153199.g001: TGF-β-induced MRTF-A Molecular Weight Shift is Due To Phosphorylation.Human VSMCs were treated with TGF-β (2 ng/ml) for 24 hr. (A) Lysates were incubated with or without alkaline phosphatase at 37°C for 30 min. Western blot was performed using MRTF-A antibody. Bars represent mean ± SE of 5 independent experiments. *p<0.05 vs con and # p<0.05 vs TGF-β. (B) Total protein was extracted and immunoprecipitated with rabbit anti- MRTF-A antibody. Membranes were immunoblotted with phospho-Ser/Thr and goat anti-MRTF-A antibodies. This figure is representative of 3 independent experiments.
Mentions: We first investigated the effect of TGF-β on MRTF-A phosphorylation in human aortic VSMCs. Although MRTF-A is known to be phosphorylated on multiple sites [9], there is little specific information and no available reagents to probe individual sites; therefore, we evaluated phosphorylation by measuring the upward shift in molecular weight, as has been done by others [9, 12]. As shown in Fig 1A, a 24-h treatment with TGF-β (2 ng/ml) induced a significant shift in molecular weight. Moreover, incubation of the lysate with alkaline phosphatase largely abolished the upward shift of MRTF-A in response to TGF-β, indicating that the molecular weight shift is due to phosphorylation of MRTF-A (Fig 1A). To confirm that the upward shift of MRTF-A is due to phosphorylation, we immunoprecipitated MRTF-A and blotted with phospho-Ser/Thr antibody (Fig 1B). The results show a predominant phospho-Ser/Thr band near 150 kDa that overlaps the MRTF-A band, supporting our conclusion that the molecular weight shift of MRTF-A is due to phosphorylation.

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