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Angiotensin II induced reactive oxygen species modulate smooth muscle α‐actin gene expression via increased SRF binding to CArG cis elements in SHR renal microvascular smooth muscle cells
Author(s) -
Luo Zaiming,
Chen Shiyou,
Gill Pritmohinder,
Kawada Noritaka,
Andresen Bradley T,
Welch William J.,
Jose Pedro A.,
Wilcox Chrostopher S.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1148-a
Subject(s) - chromatin immunoprecipitation , serum response factor , microbiology and biotechnology , angiotensin ii , histone , gene expression , actin , chemistry , reactive oxygen species , activator (genetics) , biology , promoter , gene , biochemistry , receptor
We tested the hypothesis that Superoxide (O 2 ·− ) mediates Ang II‐dependent increase in expression of smooth muscle α‐actin (SMα‐actin) gene in cultured renal preglomerular microvascular smooth muscle cells (PGSMCs) from SHR. O 2 ·− quantified by lucigenin method was significantly increased by 85% in intact PGSMCs and by 47% in cell membranes at 12 hr after incubation with Ang II (1uM). Expression of SMα‐actin mRNA determined by real time RT‐PCR was enhanced by 81% by Ang II and inhibited by 50% in Ang II‐treated cells by the thiol antioxidant, N‐acetyl‐L‐cysteine (NAC, 20mM) which also reduced Ang II‐induced O 2 ·− generation to 20%. Serum response factor (SRF) and its co‐activator, myocardin (Myo) mediate CArG‐dependent transcription of SMα‐actin gene. Although Ang II treatment did not modify mRNA for SRF, it increased Myo expression by 22%. Promoter repoter assays indicated that Ang II increased SMα‐actin promoter activity by 2.3 fold and this was inhibited by 43% by NAC. Site‐directed mutations demonstrated that CArG elements are required for SMα‐actin promoter activity. Quantitative chromatin immunoprecipitation assay revealed that Ang II increased SRF enrichment of CArG regions in SMα‐actin promoter in intact chromatin by 57%, whereas NAC reduced it by 43% without affecting histone 3 (H3) acetylation or histone 3 lysine‐9 (H3K9) dimethylation. In conclution; Ang II induces O 2 ·− in SHR PGSMCs that increases SMα‐actin gene expression via up‐regulation of Myo and enhanced SRF bound to CArG elements without modifications of H3 acetylation and H3K9 methylation in the SMα‐actin promoter regions.

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