Estrogen Induced Uterine Artery Endothelial Nitrosyl-Proteome: Potential Roles of Protein Nitrosylation in Uterine Blood Flow Regulation.

Abstract

Estrogen is one of the major driving forces for dilating uterine arteries to facilitate the dramatic rises in uterine blood flow that parallels the exponential growth phase of fetal weight during late gestation. Estrogen-induced and pregnancy-associated uterine vasodilatations occur via enhanced endothelial nitric oxide (NO) synthase (eNOS) expression/activation thereby increasing NO production locally at the level of the uterine artery. However, it is unknown whether increased NO production by estrogen stimulation directly affects proteins and their functions in uterine arteries. Formation of S-nitrothiols via addition of NO-derived nitrosyl groups to cysteines regulates the function of a plethora of proteins. This post-translational protein modification has been named as S-nitrosylation with biological significance analogous to phosphorylation. To determine the effects of exogenous estrogen and NO treatments on protein nitrosylation and to profile the estrogen-induced nitrosyl-proteome in uterine artery endothelial cells. Primary cultured sheep uterine artery endothelial cells were treated with or without etradiol-17β (E2β, 10 nM) or a NO donor S-nitrosoglutathione (GSNO, 1 mM) for 30 min. Protein nitrosylation was measured by a modified 3-step biotin switch technique including blocking free thiols in the nitrosyl-proteomes of total protein samples by methylmethanethiosulfonate, followed by ascorbate reduction and labeling with the CyDye fluorescence tags. E2β or GSNO treated samples (Cy5 labeled, red) were mixed with equal amounts of control sample (Cy3 labeled, green) and then analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). High resolution digitalized fluorescence images of the 2D-DIGE were captured and changes in densities of nitrosyl-proteins by E2α or GSNO were quantified off-line. Protein spots of interest (based on increases or decreases in intensity) on the 2D-DIGE were picked up and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF) mass spectrometry. There were >1000 nitrosylated proteins identified on 2D-DIGE. Among those, at least 25 proteins were significantly stimulated by E2β or GSNO. Interestingly, many proteins were also unexpectedly denitrosylated by estrogen or GSNO. With MALDI-TOF mass spectrometry we sequenced 100 nitrosyl-proteins of interest. We found that E2β and GSNO stimulated significantly different nitrosyl-proteomes. Some proteins were nitrosylated or denitrosylated by both E2β and GSNO, but others were only affected by one or the other. Pathway analysis of the identified nitrosyl-proteins suggested that protein nitrosylation is clearly involved in estrogen regulation of protein translation and degradation, mitochondrial redox balance, cell structure and metabolism, and ion homeostasis, etc. Our data showing estrogen stimulation of protein nitrosylation in uterine artery endothelial cells signifies a significant next step for the understanding of the biological targets of enhanced NO production by estrogen in uterine arteries that are associated with the regulation of uterine blood flow. The physiological consequences of nitrosylated/denitrosylated proteins by estrogen in uterine artery endothelial cells warrants for further investigation (NIH HL70562, HL74947 HL49210, HD38843, HL87144 and HL86939). (platform)