Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype.
OBJECTIVE
The mechanisms responsible for maintaining the differentiated phenotype of adult vascular smooth muscle cells (VSMCs) are incompletely understood. Reactive oxygen species (ROS) have been implicated in VSMC differentiation, but the responsible sources are unknown. In this study, we investigated the role of Nox1 and Nox4-derived ROS in this process.


RESULTS
Primary VSMCs were used to study the relationship between Nox homologues and differentiation markers such as smooth muscle alpha-actin (SM alpha-actin), smooth muscle myosin heavy chain (SM-MHC), heavy caldesmon, and calponin. We found that Nox4 and differentiation marker genes were downregulated from passage 1 to passage 6 to 12, whereas Nox1 was gradually upregulated. Nox4 co-localized with SM alpha-actin-based stress fibers in differentiated VSMC, and moved into focal adhesions in de-differentiated cells. siRNA against nox4 reduced NADPH-driven superoxide production in serum-deprived VSMCs and downregulated SM-alpha actin, SM-MHC, and calponin, as well as SM-alpha actin stress fibers. Nox1 depletion did not decrease these parameters.


CONCLUSIONS
Nox4-derived ROS are critical to the maintenance of the differentiated phenotype of VSMCs. These findings highlight the importance of identifying the specific source of ROS involved in particular cellular functions when designing therapeutic interventions.
Clempus
Roza E
RE
Emory University, Division of Cardiology, 1639 Pierce Dr, Atlanta, GA 30322, USA.
Sorescu
Dan
D
Dikalova
Anna E
AE
Pounkova
Lily
L
Jo
Patricia
P
Sorescu
George P
GP
Schmidt
Harald H H
HH
Lassègue
Bernard
B
Griendling
Kathy K
KK
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D026362:Serum Response Factor / Q000378:metabolism; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D008840:Microfilament Proteins; D018995:Myosin Heavy Chains; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; D017382:Reactive Oxygen Species; D026362:Serum Response Factor; D000096985:Calponins; C498862:Acta2 protein, rat; C000614712:NOX1 protein, rat; C498911:Nox4 protein, rat