Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA.
Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21(Waf1/Cip1) and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA(63L)) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21(Waf1/Cip1) correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO.
Zuckerbraun
Brian S
BS
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Stoyanovsky
Detcho A
DA
Sengupta
Rajib
R
Shapiro
Richard A
RA
Ozanich
Brett A
BA
Rao
Jayashree
J
Barbato
Joel E
JE
Tzeng
Edith
E
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D015538:Nitrosation; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D009569:Nitric Oxide; D048049:Extracellular Signal-Regulated MAP Kinases; D020742:rhoA GTP-Binding Protein; D006152:Cyclic GMP