Roles of CYP2C29 and RXR gamma in vascular EET synthesis of female mice.
We aimed to identify which cytochrome P-450 (CYP) family/subfamily, as well as related transcription factor(s), is responsible for the estrogen-dependent synthesis of epoxyeicosatrienoic acids (EETs) to initiate shear stress-induced vasodilation. Microarray analysis indicated a significant upregulation of CYP2C29 and retinoid X receptor gamma (RXRgamma) in isolated mesenteric arteries/arterioles of female endothelial nitric oxide synthase-knockout mice, a result that was validated by real-time RT-PCR. The cannulated vessels were then perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress dose-dependently stimulated the release of EETs into the perfusate, associated with an EET-mediated vasodilation, in which predominantly 14,15-EET and 11,12-EET contributed to the responses ( approximately 87.4% of total EETs). Transfection of vessels with CYP2C29 siRNA eliminated the release of EETs into the perfusate, which was evidenced by an abolished vasodilation, and confirmed by RT-PCR and Western blot analyses. Knockdown of RXRgamma in these vessels significantly inhibited the production of EETs, parallel to a reduced vasodilation. RXRgamma siRNA not only silenced the vascular RXRgamma expression, but synchronously downregulated CYP2C29 expression, leading to a reduced EET synthesis. In conclusion, our data provide the first evidence for a specific signaling cascade, by which estrogen potentially activates the CYP2C29 gene in the absence of nitric oxide, to synthesize EETs in response to shear stress, via an RXRgamma-related regulatory mechanism.
Department of Physiology, New York Medical College, Valhalla, NY 10595, USA.
Dong
D
Sun
Yang-Ming
YM
Yang
Houli
H
Jiang
Hongyan
H
Wu
Caroline
C
Ojaimi
Gabor
G
Kaley
An
A
Huang
D000818:Animals; D001160:Arterioles / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis; D005260:Female; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis / Q000502:physiology*; D008297:Male; D008638:Mesenteric Arteries / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D019001:Nitric Oxide Synthase / Q000172:deficiency; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D034622:RNA Interference; D047491:Retinoid X Receptor gamma / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012727:Sex Characteristics; D014664:Vasodilation
D005231:Fatty Acids, Unsaturated; D006893:Hydroxyeicosatetraenoic Acids; D047491:Retinoid X Receptor gamma; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D019001:Nitric Oxide Synthase; C086292:Cyp2c29 protein, mouse; D000072467:Cytochrome P450 Family 2