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Testosterone induces activation of the extrinsic apoptotic pathway in VSMC by mechanisms involving ROS generation
Author(s) -
Lopes Rheure,
Neves Karla,
Chignalia Andréia,
Zanotto Camila,
Pestana Cézar,
Curti Carlos,
Tostes Rita
Publication year - 2013
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.27.1_supplement.1092.8
Subject(s) - apoptosis , cytochrome c , reactive oxygen species , annexin , mitochondrion , calcein , microbiology and biotechnology , chemistry , fas ligand , programmed cell death , biology , biochemistry , membrane
The mechanisms by which testosterone (testo) affects the cardiovascular system may involve reactive oxygen species (ROS). ROS activate cell death signaling pathways. We hypothesized that testo induces ROS generation via mitochondria, culminating in caspase 3 activation and apoptosis. Cultured vascular smooth muscle cells (VSMCs‐mesentery of Wistar rats) were pre‐treated with flutamide(flu,10 −5 M), CCCP(10 −6 M), MnTmPyP(3×10 −5 M), ZIETD( 10 −5 M) or vehicle and then stimulated with testo(10 −7 M) (2–12h). ROS was measured by lucigenin and DCF, apoptosis by calcein and annexin V, procaspases/caspases 3/8 expression by immunoblotting. Cytochrome c, Bax and Bcl‐2 by immunoblotting of cytoplasmic and mitochondrial fractions. Testo (2h) induced ROS generation [162.6 ± 16 vs. 100], an effect abolished by CCCP (90.4 ± 15), MnTmPyP (26.2 ± 11.6) and flu (122.7 ± 10.6). Testo (6h) induced caspase 3 activation (166.2 ± 19 vs 100), which was abolished by flu (122.7 ± 10.6), MnTmPyP (61.8 ± 2,5), CCCP (71.6 ± 13,2) and Z‐IETD (125.1 ± 6). Testo (6h) increased annexin V fluorescence [197.6 ± 21,5 vs. 100] and decreased calcein fluorescence [34.4 ± 6.4 (2h) vs. 100]. Bax/Bcl‐2 ratio was reduced and there was no cytochrome c translocation in response to testo. Testo (6h) induced cleavage of procaspase 8 [161.1 ± 13.5 vs 100] and increased FasL [3.6 ± 1.2 vs 0.7 ± 0.5] and TNF [1.7 ± 0.4 vs 0.3 ± 0.1] gene expression, an effect abolished by CCCP, MnTmPyP and flu. In conclusion, testo induces apoptosis in VSMC by mechanisms involving ROS generation via mitochondria, androgen receptor and the extrinsic apoptotic pathway. Financial Support: CNPQ, FAPESP.