Altered AP-1/Ref-1 redox pathway and reduced proliferative response in iNOS-deficient vascular smooth muscle cells.
We previously reported that injury-induced medial vascular smooth muscle cell (VSMC) proliferation and neointima formation in carotid arteries of inducible nitric oxide synthase knockout (iNOS KO) mice were significantly reduced compared with wild type (WT). However, the molecular pathway underlying such differences is not known. In this in vitro study, we discovered that the AP-1/Ref-1/thioredoxin signaling pathway is altered in aortic VSMC from iNOS KO mice, which leads to reduced growth response when compared with aortic VSMC from WT mice. After equal initial seeding, the cell number after 7 days in serum medium was less in iNOS KO cells compared with WT VSMC (1.2 +/- 0.6 x 10(5) vs 3.2 +/- 1.1 x 10(5); p < 0.05). Significantly more iNOS KO cells remained in the G0/G1 phase compared with WT cells after 24-h serum treatment (82.6 +/- 13.7% vs 62.3 +/- 14.6%; p < 0.05) by cell-cycle analysis. Nuclear PCNA expression was also less in the iNOS KO cells, which was not affected by exogenous NO or superoxide. Superoxide generation after 24-h serum stimulation was less in the iNOS KO cells compared with WT cells. After 30-min serum stimulation, AP-1 DNA binding was reduced and a lack of increase in nuclear c-Jun protein was observed in iNOS KO VSMC. RT-PCR analysis confirmed a lack of inducible c-Jun mRNA after serum stimulation in the KO cells. In addition, KO cells had less nuclear reducing factor-1 (Ref-1) and serum-inducible thioredoxin protein expression. Reduced proliferative response of iNOS KO VSMC to serum treatment is associated with altered AP-1 /Ref-1 /thioredoxin pathway activation.
Chyu
Kuang-Yuh
KY
Atherosclerosis Research Center, Burns and Allen Research Institute, Division of Cardiology, Cedars-Siffai Medical Center/David Geffen School of Medicine, UCLA, Los Angeles, CA 90048, USA. Chyuk@cshs.org
Dimayuga
Paul C
PC
Zhao
Xiaoning
X
Nilsson
Jan
J
Shah
Prediman K
PK
Cercek
Bojan
B
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D020030:Nitric Oxide Donors / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000172:deficiency*; D010084:Oxidation-Reduction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D020030:Nitric Oxide Donors; D018809:Proliferating Cell Nuclear Antigen; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D013481:Superoxides; D013879:Thioredoxins; D052247:Nitric Oxide Synthase Type II; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase