Endothelial regulation of eNOS, PAI-1 and t-PA by testosterone and dihydrotestosterone in vitro and in vivo.

The aim of this study is the identification of direct endothelial regulation by the androgens testosterone (T) and dihydrotestosterone (DHT). We tested the effects of T and DHT on nitric oxide (NO) synthesis and on tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) expression in human endothelial cells and in ovariectomized (OVX) rats. The results showed that at physiological concentrations T and DHT increase endothelial synthesis of NO. This depends on a rapid recruitment of the extracellular-related kinase (ERK) 1/2 and of the phosphatidylinositol 3-OH kinase (PI3K)/Akt cascades, resulting in endothelial nitric oxide synthase (eNOS) Ser(1177)-phosphorylation. In addition, a later increase of eNOS expression is found. With supra-physiological amounts of T or DHT the induction of NO synthesis is lost. A concentration-related increase of t-PA expression starting from physiological concentrations of T or DHT is found, whereas PAI-1 is augmented only with higher doses. Although DHT exerts these actions through androgen receptors (AR), T acts in part through aromatase-dependent conversion to 17ß-estradiol. Ovariectomy is associated with significant changes in eNOS, t-PA and PAI-1 expression in the aorta of Wistar rats and T and DHT result in modifications on eNOS, PAI-1 and t-PA that are in line with the in vitro experiments. In conclusion, T and DHT act on endothelial cells through AR or via conversion to estradiol. Physiological, but not higher amounts are associated with enhanced NO synthesis and an increased t-PA/PAI-1 ratio. These findings are useful to understand the impact of androgens in ageing individuals.

Differential actions of estrogen and SERMs in regulation of the actin cytoskeleton of endometrial cells.

Estrogen and selective estrogen receptor modulators (SERMs) differentially impact endometrial cell function, however, the biological basis of these differences is not established. Deregulated cell adhesion to the extracellular matrix, cell movement and invasion are related to endometrial disorders, such as endometriosis or endometrial cancer. Remodeling of the actin cytoskeleton is required to achieve cell adhesion and movement. Estrogen receptor (ER) regulates actin and cell membrane remodeling through extra-nuclear signaling cascades. In this article, we show that administration of 17beta-estradiol (E2) and tamoxifen (TAM) to immortalized Ishikawa endometrial cells or to human endometrial stromal cells (ESC) results in remodeling of actin fibers and cell membrane. This is linked to rapid phosphorylation on Thr(558) of the actin-binding protein moesin and enhanced migration and invasion of normal and Ishikawa cells. Raloxifene (RAL) does not result in moesin activation or actin remodeling. When endometrial cells are exposed to E2 in the presence of TAM or RAL, both SERMs interfere with the recruitment of moesin, with the remodeling of the cytoskeleton, and with cell movement and migration induced by E2. The differential actions of E2, TAM and RAL are linked to a distinct modulation of the extra-nuclear signaling of ER to G proteins and to the Rho-associated kinase. These findings increase our understanding of the actions of estrogen and SERMs in endometrial cells and highlight potential molecular targets to interfere with the estrogen-related altered cell adhesion encountered in endometrial disorders.