CaMKII knockdown attenuates H2O2-induced phosphorylation of ERK1/2, PKB/Akt, and IGF-1R in vascular smooth muscle cells.
We have shown earlier a requirement for Ca(2+) and calmodulin (CaM) in the H(2)O(2)-induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key mediators of growth-promoting, proliferative, and hypertrophic responses in vascular smooth muscle cells (VSMC). Because the effect of CaM is mediated through CaM-dependent protein kinase II (CaMKII), we have investigated here the potential role of CaMKII in H(2)O(2)-induced ERK1/2 and PKB phosphorylation by using pharmacological inhibitors of CaM and CaMKII, a CaMKII inhibitor peptide, and siRNA knockdown strategies for CaMKII alpha. Calmidazolium and W-7, antagonists of CaM, as well as KN-93, a specific inhibitor of CaMKII, attenuated H(2)O(2)-induced responses of ERK1/2 and PKB phosphorylation in a dose-dependent fashion. Similar to H(2)O(2), calmidazolium and KN-93 also exhibited an inhibitory effect on glucose/glucose oxidase-induced phosphorylation of ERK1/2 and PKB in these cells. Transfection of VSMC with CaMKII autoinhibitory peptide corresponding to the autoinhibitory domain (aa 281-309) of CaMKII and with siRNA of CaMKII alpha attenuated the H(2)O(2)-induced phosphorylation of ERK1/2 and PKB. In addition, calmidazolium and KN-93 blocked H(2)O(2)-induced Pyk2 and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation. Moreover, treatment of VSMC with CaMKII alpha siRNA abolished the H(2)O(2)-induced IGF-1R phosphorylation. H(2)O(2) treatment also induced Thr(286) phosphorylation of CaMKII, which was inhibited by both calmidazolium and KN-93. These results demonstrate that CaMKII plays a critical upstream role in mediating the effects of H(2)O(2) on ERK1/2, PKB, and IGF-1R phosphorylation.
Bouallegue
Ali
A
Laboratory of Cell Signaling, Montreal Diabetes Research Centre, Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Technopole Angus Campus, Montreal, Canada.
Pandey
Nihar R
NR
Srivastava
Ashok K
AK
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D001013:Aorta, Thoracic / Q000473:pathology; D001596:Benzylamines / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
D064413:3-Phosphoinositide-Dependent Protein Kinases; D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D004791:Enzyme Inhibitors; D051418:Focal Adhesion Kinase 2; D006861:Hydrogen Peroxide; D007093:Imidazoles; D048052:Mitogen-Activated Protein Kinase 3; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D017526:Receptor, IGF Type 1; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides; C031938:calmidazolium; C017967:W 7