NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion

Sasa Vukelic, Qian Xu, Bonnie Seidel-Rogol, Elizabeth A. Faidley, Anna E. Dikalova, Lula L. Hilenski, Ulrich P. Jorde, Leslie B. Poole, Bernard Lassègue, Guogang Zhang, Kathy K. Griendling

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

Original languageEnglish (US)
Pages (from-to)2423-2434
Number of pages12
JournalArteriosclerosis, Thrombosis, and Vascular Biology
Volume38
Issue number10
DOIs
StatePublished - Oct 1 2018

Fingerprint

Vinculin
NADPH Oxidase
Cell Adhesion
Integrins
Actins
Focal Adhesions
Proteins
Reactive Oxygen Species
Actin Cytoskeleton
Oxidation-Reduction
Cell Movement
Vascular Smooth Muscle
Point Mutation
Catalase
Hydrogen Peroxide
Small Interfering RNA
Smooth Muscle Myocytes
Transfection

Keywords

  • actins
  • cell adhesion
  • NADPH oxidase
  • reactive oxygen species
  • vinculin

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion. / Vukelic, Sasa; Xu, Qian; Seidel-Rogol, Bonnie; Faidley, Elizabeth A.; Dikalova, Anna E.; Hilenski, Lula L.; Jorde, Ulrich P.; Poole, Leslie B.; Lassègue, Bernard; Zhang, Guogang; Griendling, Kathy K.

In: Arteriosclerosis, Thrombosis, and Vascular Biology, Vol. 38, No. 10, 01.10.2018, p. 2423-2434.

Research output: Contribution to journalArticle

Vukelic, Sasa ; Xu, Qian ; Seidel-Rogol, Bonnie ; Faidley, Elizabeth A. ; Dikalova, Anna E. ; Hilenski, Lula L. ; Jorde, Ulrich P. ; Poole, Leslie B. ; Lassègue, Bernard ; Zhang, Guogang ; Griendling, Kathy K. / NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion. In: Arteriosclerosis, Thrombosis, and Vascular Biology. 2018 ; Vol. 38, No. 10. pp. 2423-2434.
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abstract = "Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26{\%}, 99±1{\%}, and 98±1{\%}, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.",
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T1 - NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion

AU - Vukelic, Sasa

AU - Xu, Qian

AU - Seidel-Rogol, Bonnie

AU - Faidley, Elizabeth A.

AU - Dikalova, Anna E.

AU - Hilenski, Lula L.

AU - Jorde, Ulrich P.

AU - Poole, Leslie B.

AU - Lassègue, Bernard

AU - Zhang, Guogang

AU - Griendling, Kathy K.

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N2 - Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

AB - Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

KW - actins

KW - cell adhesion

KW - NADPH oxidase

KW - reactive oxygen species

KW - vinculin

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