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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

Palladin Expression is Induced by TGF-β.(A) Human VSMCs were incubated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and the level of palladin was analyzed using a specific antibody. β-actin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs con and ***p<0.001 vs con. Con = control cells untreated with TGF-β. (B) Human VSMCs were preincubated with 100 μM cyclohexamide (CHX) for 30 min. Cells were then treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of palladin were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ***p<0.001 vs con, ****p<0.0001 vs con, and #### p<0.0001 vs TGF-β.
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pone.0153199.g005: Palladin Expression is Induced by TGF-β.(A) Human VSMCs were incubated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and the level of palladin was analyzed using a specific antibody. β-actin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs con and ***p<0.001 vs con. Con = control cells untreated with TGF-β. (B) Human VSMCs were preincubated with 100 μM cyclohexamide (CHX) for 30 min. Cells were then treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of palladin were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ***p<0.001 vs con, ****p<0.0001 vs con, and #### p<0.0001 vs TGF-β.

Mentions: To explore a role for other potential Nox4 effectors in TGF-β-induced SMA and CNN expression, we focused on palladin because, like knockdown of Nox4, deletion of palladin results in a loss of actin fibers [23] and, like activation of Nox4, overexpression of palladin activates Rho and induces stress fibers [24]. We first determined whether TGF-β alters palladin expression in VSMCs. Cells were treated with TGF-β for 24 hr and analyzed by western blot. As shown in Fig 5A, TGF-β increased expression of the 90-kDa palladin isoform and caused neo-expression of the 140-kDa isoform of palladin. To determine if the effect of TGF-β on palladin is due to its increased expression or due to its decreased degradation, we treated cells with the protein synthesis inhibitor cyclohexamide (Fig 5B). Cyclohexamide treatment blocked TGF-β-induced palladin expression, suggesting that TGF-β increases palladin protein expression rather than decreasing its degradation. Treatment with NAC significantly reduced TGF-β-stimulated palladin expression in VSMCs (Fig 6A), as did knockdown of Nox4 using siRNA (Fig 6B), an effect that was accompanied by loss of SMA expression (Fig 3B). This effect on palladin appears to be independent of ROCK, because Y-27632 treatment had no effect on palladin expression (palladin 140 kDa; TGF-β 0.52 ± 0.06 vs TGF-β + inhibitor 0.58 ± 0.02, palladin 90 kDa; TGF-β 1.39 ± 0.04 vs TGF-β + inhibitor 1.27 ± 0.07).


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)

Palladin Expression is Induced by TGF-β.(A) Human VSMCs were incubated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and the level of palladin was analyzed using a specific antibody. β-actin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs con and ***p<0.001 vs con. Con = control cells untreated with TGF-β. (B) Human VSMCs were preincubated with 100 μM cyclohexamide (CHX) for 30 min. Cells were then treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of palladin were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ***p<0.001 vs con, ****p<0.0001 vs con, and #### p<0.0001 vs TGF-β.
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pone.0153199.g005: Palladin Expression is Induced by TGF-β.(A) Human VSMCs were incubated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and the level of palladin was analyzed using a specific antibody. β-actin was used as a loading control. Bars are means ± SE of 3 independent experiments. *p<0.05 vs con and ***p<0.001 vs con. Con = control cells untreated with TGF-β. (B) Human VSMCs were preincubated with 100 μM cyclohexamide (CHX) for 30 min. Cells were then treated with TGF-β (2 ng/ml) for 24 hr. Total protein was extracted and levels of palladin were analyzed using a specific antibody. β-tubulin was used as a loading control. Bars are means ± SE of 4 independent experiments. ***p<0.001 vs con, ****p<0.0001 vs con, and #### p<0.0001 vs TGF-β.
Mentions: To explore a role for other potential Nox4 effectors in TGF-β-induced SMA and CNN expression, we focused on palladin because, like knockdown of Nox4, deletion of palladin results in a loss of actin fibers [23] and, like activation of Nox4, overexpression of palladin activates Rho and induces stress fibers [24]. We first determined whether TGF-β alters palladin expression in VSMCs. Cells were treated with TGF-β for 24 hr and analyzed by western blot. As shown in Fig 5A, TGF-β increased expression of the 90-kDa palladin isoform and caused neo-expression of the 140-kDa isoform of palladin. To determine if the effect of TGF-β on palladin is due to its increased expression or due to its decreased degradation, we treated cells with the protein synthesis inhibitor cyclohexamide (Fig 5B). Cyclohexamide treatment blocked TGF-β-induced palladin expression, suggesting that TGF-β increases palladin protein expression rather than decreasing its degradation. Treatment with NAC significantly reduced TGF-β-stimulated palladin expression in VSMCs (Fig 6A), as did knockdown of Nox4 using siRNA (Fig 6B), an effect that was accompanied by loss of SMA expression (Fig 3B). This effect on palladin appears to be independent of ROCK, because Y-27632 treatment had no effect on palladin expression (palladin 140 kDa; TGF-β 0.52 ± 0.06 vs TGF-β + inhibitor 0.58 ± 0.02, palladin 90 kDa; TGF-β 1.39 ± 0.04 vs TGF-β + inhibitor 1.27 ± 0.07).

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