Global Protein Expression Profiling Underlines Reciprocal Regulation of Caveolin 1 and Endothelial Nitric Oxide Synthase Expression in Ovariectomized Sheep Uterine Artery by Estrogen/Progesterone Replacement Therapy1

Abstract

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.

Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

“…In the uterine circulation, numerous studies have shown that NO produced locally by uterine artery endothelial cells (UAEC) is the most important, though not the only, factor to regulate uterine vasodilatation during normal pregnancy and the follicular phase of the estrous cycle as well as estrogen replacement therapy (Chen et al, 2006;Magness et al, 1997;Weiner et al, 1994). Uterine vasodilatation as shown by dramatic rises in uterine blood flow during pregnancy is required for delivering nutrients and oxygen to the developing fetus and thus is detrimental to fetal development (Lang et al, 2003).…”

“…However, many studies have also demonstrated that the expression level of NOS3 gene can be up-regulated by many physiological and pathophysiological factors, including estrogen (Chen et al, 2006;Weiner et al, 1994;Zheng et al, 2005), growth factors (Inoue et al, 1995;Zheng et al, 1999), shear stress (Li et al, 2003), and protein kinase C activators (NavarroAntolin et al, 2000), whereas the inflammatory cytokines tumor necrosis factor-α (Yoshizumi et al, 1993) and bacterial toxin lipopolysaccharide (Schwartz et al, 1997) down-regulates the expression of this vasodilatory enzyme. The regulation of NOS3 expression in endothelial cells can take place at the levels of transcription (i.e., NOS3 promoter activity) and post-transcription (i.e., mRNA stability) (Fleming and Busse, 2003).…”

“…The regulation of NOS3 expression in endothelial cells can take place at the levels of transcription (i.e., NOS3 promoter activity) and post-transcription (i.e., mRNA stability) (Fleming and Busse, 2003). Despite a large body of evidence accumulated showing that increased uterine artery endothelial NOS3 protein coincides with the pregnancy-associated and estrogen-induced uterine vasodilatation (Chen et al, 2006;Magness et al, 1997;Weiner et al, 1994), the mechanisms by which NOS3 expression is controlled in the UAEC are generally unknown. Uterine artery endothelial NOS3 mRNA is increased in ovariectomized sheep receiving estrogen replacement therapy (Rosenfeld et al, 2003), suggesting that uterine artery endothelial NOS3 expression might be regulated at the level of gene transcription.…”

Le paradoxe cardiovasculaire de la grossesse

“…Nous estimons que la progestérone et l'oestradiol, ainsi que l'aldostérone, via le récepteur minéralocorticoïde, sont des joueurs clés (flèches pointillées, Figure 4). L'administration d'oestrogènes provoque une vasodilatation généralisée, une augmentation du débit cardiaque, une faible diminution de la pression artérielle et une réduction de la résistance systémique [25]. Cette situation ressemble étrangement à celle qui prévaut durant la gestation [9,26].…”

“…Cette situation ressemble étrangement à celle qui prévaut durant la gestation [9,26]. De plus, l'augmentation d'oestrogènes contribuerait à une augmentation de l'expression de la synthase endothéliale du monoxyde d'azote qui affecterait l'ensemble de la circulation sanguine [25].…”