TY - JOUR
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
AU - Poole, Leslie B.
AU - Lassègue, Bernard
AU - Zhang, Guogang
AU - Griendling, Kathy K.
N1 - Funding Information:
We thank Dr Vsevolod Belousov for the pC1-HyPer-3 plasmid. The electron microscopy study was supported in part by the Robert P. Apkarian Integrated Electron Microscopy Core (RPAIEMC), which is subsidized by the Emory College of Arts and Sciences and the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. An adenoviral shuttle vector for Poldip2 was prepared by the Emory Integrated Genomics Core, which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. Additional support was provided by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR000454. We also acknowledge the help of Hong Yi and Jeannette Taylor in the RPAIEMC.
Funding Information:
This work was supported by the National Institutes of Health grants HL38206 and HL095070, as well as the IMAT (Innovative Molecular Analysis Technologies) program at NCI (R33 CA177461).
Publisher Copyright:
© 2018 American Heart Association, Inc.
PY - 2018
Y1 - 2018
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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U2 - 10.1161/ATVBAHA.118.311668
DO - 10.1161/ATVBAHA.118.311668
M3 - Article
C2 - 30354218
AN - SCOPUS:85055606405
VL - 38
SP - 2423
EP - 2434
JO - Arteriosclerosis, Thrombosis, and Vascular Biology
JF - Arteriosclerosis, Thrombosis, and Vascular Biology
SN - 1079-5642
IS - 10
ER -