Limits...
Proteomic analysis identifies an NADPH oxidase 1 (Nox1)-mediated role for actin-related protein 2/3 complex subunit 2 (ARPC2) in promoting smooth muscle cell migration.

Al Ghouleh I, Rodríguez A, Pagano PJ, Csányi G - Int J Mol Sci (2013)

Bottom Line: Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC.Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1.These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.

View Article: PubMed Central - PubMed

Affiliation: Vascular Medicine Institute, 12th Floor BST, 200 Lothrop Street, University of Pittsburgh, PA 15261, USA. pagano@pitt.edu.

ABSTRACT
A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 µM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.

Show MeSH

Related in: MedlinePlus

Representative 2D gel images. Vascular smooth muscle cells (VSMC) incubated with vehicle (control) or transfected with Scrmb or Nox1 siRNA and treated with H2O2 (50 μM, 3 h) were lysed and fluorescently labeled with Cy3, Cy2, or Cy5, respectively and run on a 2D gel (n = 1). Images show the comparisons of two fluorescent channels at a time (assigned green and red pseudocolors) as presented in Table 1. Vertical scale indicates the molecular weight ladder; lower horizontal scale denotes pH gradient. The 96 gel spots selected by DeCyder are annotated by white numbers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3821612&req=5

f1-ijms-14-20220: Representative 2D gel images. Vascular smooth muscle cells (VSMC) incubated with vehicle (control) or transfected with Scrmb or Nox1 siRNA and treated with H2O2 (50 μM, 3 h) were lysed and fluorescently labeled with Cy3, Cy2, or Cy5, respectively and run on a 2D gel (n = 1). Images show the comparisons of two fluorescent channels at a time (assigned green and red pseudocolors) as presented in Table 1. Vertical scale indicates the molecular weight ladder; lower horizontal scale denotes pH gradient. The 96 gel spots selected by DeCyder are annotated by white numbers.

Mentions: To identify proteins upregulated or downregulated by feed-forward ROS-induced Nox1 activation, rat aortic VSMC transfected with scrambled (Scrmb) or Nox1 short interfering RNA (siRNA) were incubated with H2O2 (50 μM, 3 h) and compared to each other and to vehicle-treated non-transfected VSMC (control). Utilization of fluorescent dyes that complex with all proteins in a sample allows for use of 2D gel technology to compare multiple samples simultaneously. Taking advantage of this technique, each of the three samples were labeled with one particular CyDye (Cy3 for control, Cy2 for Scrmb siRNA + H2O2, and Cy5 for Nox1 siRNA + H2O2) and loaded simultaneously onto a 2D gel. For the analysis, two conditions were compared at a time using the DeCyder 2D Differential Analysis Software (version 6.5) as detailed in Table 1. Ninety six gel spots were selected for further analysis based on spot intensity changes and in-gel resolution (Figure 1).


Proteomic analysis identifies an NADPH oxidase 1 (Nox1)-mediated role for actin-related protein 2/3 complex subunit 2 (ARPC2) in promoting smooth muscle cell migration.

Al Ghouleh I, Rodríguez A, Pagano PJ, Csányi G - Int J Mol Sci (2013)

Representative 2D gel images. Vascular smooth muscle cells (VSMC) incubated with vehicle (control) or transfected with Scrmb or Nox1 siRNA and treated with H2O2 (50 μM, 3 h) were lysed and fluorescently labeled with Cy3, Cy2, or Cy5, respectively and run on a 2D gel (n = 1). Images show the comparisons of two fluorescent channels at a time (assigned green and red pseudocolors) as presented in Table 1. Vertical scale indicates the molecular weight ladder; lower horizontal scale denotes pH gradient. The 96 gel spots selected by DeCyder are annotated by white numbers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821612&req=5

f1-ijms-14-20220: Representative 2D gel images. Vascular smooth muscle cells (VSMC) incubated with vehicle (control) or transfected with Scrmb or Nox1 siRNA and treated with H2O2 (50 μM, 3 h) were lysed and fluorescently labeled with Cy3, Cy2, or Cy5, respectively and run on a 2D gel (n = 1). Images show the comparisons of two fluorescent channels at a time (assigned green and red pseudocolors) as presented in Table 1. Vertical scale indicates the molecular weight ladder; lower horizontal scale denotes pH gradient. The 96 gel spots selected by DeCyder are annotated by white numbers.
Mentions: To identify proteins upregulated or downregulated by feed-forward ROS-induced Nox1 activation, rat aortic VSMC transfected with scrambled (Scrmb) or Nox1 short interfering RNA (siRNA) were incubated with H2O2 (50 μM, 3 h) and compared to each other and to vehicle-treated non-transfected VSMC (control). Utilization of fluorescent dyes that complex with all proteins in a sample allows for use of 2D gel technology to compare multiple samples simultaneously. Taking advantage of this technique, each of the three samples were labeled with one particular CyDye (Cy3 for control, Cy2 for Scrmb siRNA + H2O2, and Cy5 for Nox1 siRNA + H2O2) and loaded simultaneously onto a 2D gel. For the analysis, two conditions were compared at a time using the DeCyder 2D Differential Analysis Software (version 6.5) as detailed in Table 1. Ninety six gel spots were selected for further analysis based on spot intensity changes and in-gel resolution (Figure 1).

Bottom Line: Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC.Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1.These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.

View Article: PubMed Central - PubMed

Affiliation: Vascular Medicine Institute, 12th Floor BST, 200 Lothrop Street, University of Pittsburgh, PA 15261, USA. pagano@pitt.edu.

ABSTRACT
A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 µM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing "scratch" assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.

Show MeSH
Related in: MedlinePlus