Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle.
We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.
Lee
Chang-Kwon
CK
Department of Physiology, Konkuk University, Danwol-dong, Chungju City, Chungbuk, Korea.
Park
Hyo-Jun
HJ
So
Hyeon Ha
HH
Kim
Hyo Jin
HJ
Lee
Keun Sang
KS
Choi
Wahn Soo
WS
Lee
Hwan Myung
HM
Won
Kyung-Jong
KJ
Yoon
Taek Joon
TJ
Park
Tae-Kyu
TK
Kim
Bokyung
B
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D051340:Cofilin 2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D040081:Protein Array Analysis / Q000379:methods*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014638:Vanadates / Q000494:pharmacology
D051340:Cofilin 2; D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D014638:Vanadates; D006861:Hydrogen Peroxide; D017027:Protein Tyrosine Phosphatases