Gene Expression in Endocrine Tissues
(W459) JUN/AP-1 ADENOVIRAL OVEREXPRESSION RECIPROCALLY REGULATES UTERINE ARTERY ENDOTHELIAL CELL eNOS AND CAVEOLIN-1 EXPRESSION.
Chen, Dongbao1, 1 University of California San Diego, La Jolla, CA
ABSTRACT- Uterine vasodilatation requires increased eNOS and decreased caveolin-1 expression in uterine artery endothelium (UAE). However, the mechanism of the reciprocal regulation of UAE eNOS and caveolin-1 expresison is unknown. We have recently isolated sheep eNOS and caveolin-1 gene promoter sequences in which both contain a consensus AP-1 site. This led us to hypothesize that AP-1 is important in regulating UAE eNOS and caveolin-1 expression. The coding regions of rat c-jun and junB cDNAs were subcloned into the E1 region-deleted pACCMVpLpA recombinant adenoviral shuttle vector in sense and antisense orientation to yield the pSR-sense-c-jun (S-c-jun) or -junB (S-junB) and pSR-antisense-c-jun (A-c-jun) or -junB (A-junB) constructs, and propagated in HEK293 cells to yield adenoviral stocks (1010 pfu/ml). Ovine UAEC (60%) were infected with adenovirus at 25 pfu/cell in DMEM-1% FBS for 6h, allowed to recover for 16h in DMEM-20% FCS, and cultured in M-199-25mM Hepes-1%CS-FCS for up to 72h. Total RNA was extracted for determining c-Jun, eNOS and caveolin-1 mRNA levels by RT-PCR. Total protein extracts were prepared for determining c-Jun, JunB, eNOS and caveolin-1 protein expression by Western blotting. Basal c-Jun, JunB, eNOS and caveolin-1 mRNAs were detected in non- and sham-infected controls. A 5-fold induction of c-Jun and JunB mRNAs was observed in antisense and sense c-Jun and junB infectants. eNOS mRNA was slightly but not statistically significant increased whereas caveolin-1 mRNA was decreased by 45% (P<0.05) in sense, but not antisense, c-jun and junB infectants at 24h. Baseline c-Jun and JunB proteins were detected in controls. c-Jun and JunB proteins were increased by 10-fold and 7-fold in their respective infectants, but unchanged in antisense Jun infectants. eNOS and caveolin-1 proteins are constitutively expressed in UAEC, and both were unchanged in control cells up to 72h. Both S-c-jun and S-junB, but not A-c-Jun and A-JunB, increased eNOS protein significantly (P<0.05) at 48h, while decreased caveolin-1 protein by 70% (P<0.05) at 48 and 72h. It is concluded that Jun/AP-1 reciprocally regulates uterine artery endothelial cell eNOS and caveolin-1. (Supported by RO1 HL70562 & HL74947).
KEY WORDS: Jun/AP-1 adenoviral delivery, eNOS and caveolin-1 expression, Uterine artery endothelial cells