Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: role of nitric oxide and cyclic guanosine monophosphate signaling pathways.
Recent studies from our laboratory have shown that insulin induces relaxation of vascular smooth muscle cells (VSMCs) via stimulation of myosin phosphatase and inhibition of Rho kinase activity. In this study, we examined the mechanism whereby insulin inhibits Rho signaling and its impact on actin cytoskeleton organization. Incubation of confluent serum-starved VSMCs with thrombin or phenylephrine (PE) caused a rapid increase in glutathione S-transferase-Rhotekin-Rho binding domain-associated RhoA, Rho kinase activation, and actin cytoskeleton organization, which was blocked by preincubation with insulin. Preexposure to N(G)-monomethyl L-arginine acetate (L-NMMA), a nitric oxide synthase inhibitor, and Rp-8 CPT-cyclic guanosine monophosphate (RpcGMP), a cyclic guanosine monophosphate (cGMP) antagonist, attenuated the inhibitory effect of insulin on RhoA activation and restored thrombin-induced Rho kinase activation, and site-specific phosphorylation of the myosin-bound regulatory subunit (MBS(Thr695)) of myosin-bound phosphatase (MBP), and caused actin fiber reorganization. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked the inhibitory effects of insulin and abolished thrombin-mediated Rho activation. Insulin inactivation of RhoA was accompanied by inhibition of isoprenylation via reductions in geranylgeranyl transferase-1 activity as well as increased RhoA phosphorylation, which was reversed by pretreatment with RpcGMP and L-NMMA. We conclude that insulin may inhibit Rho signaling by affecting posttranslational modification of RhoA via nitric oxide/cGMP signaling pathway to cause MBP activation, actin cytoskeletal disorganization, and vasodilation.
Begum
Najma
N
Diabetes Research Laboratory, Winthrop University Hospital, 259 First Street, Mineola, NY 11501, USA. nbegum@winthrop.org
Sandu
Oana A
OA
Duddy
Noreen
N
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000199:Actins / Q000378:metabolism*; D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000502:physiology*; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D000199:Actins; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D011993:Recombinant Fusion Proteins; D010656:Phenylephrine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D019323:omega-N-Methylarginine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D006152:Cyclic GMP