Testosterone induced downregulation of migration and proliferation in human Umbilical Vein Endothelial Cells by Androgen Receptor dependent and independent mechanisms.

Recent research has emphasized the potential unfavorable effects of declining testosterone (T) levels in men and the putative beneficial effect of androgen therapy in select women. Some controversy surrounding the mechanism of action and the effects of T on endothelium remains. In this study, we evaluated the mechanism of T action on pooled primary Human Umbilical Vein Endothelial Cells (HUVEC) of mixed gender by focusing on two important processes, proliferation and migration. In our in vitro model system, we found that only the supra-physiological dose of T affected these two processes irrespective of the ratio of male to female cells in the pools. At a concentration of 1 µM, T downregulated the proliferation of HUVEC by inducing arrest in the G1 cell cycle phase in an Androgen Receptor (AR)-independent manner. We show that treatment with 1 µM T also induced downregulation of HUVEC migration. This process was AR-dependent and was associated with persistent phosphorylation of ezrin, radixin and moesin. Regardless of the mechanism of action, the treatment of HUVEC with both supra- and physiological doses of T was associated with posttranscriptional stabilization of the AR upon ligand binding.

Testosterone dependent androgen receptor stabilization and activation of cell proliferation in primary human myometrial microvascular endothelial cells.

OBJECTIVE: To clarify, whether uterine endothelial proliferation could be regulated via an autocrine estrogen producing mechanism or direct actions of testosterone. DESIGN: In vitro study. SETTING: Tertiary care facility. PATIENT(S): Human myometrial tissue obtained from 40 women undergoing hysterectomy without further intrauterine pathology. INTERVENTION(S): Cell culture, proliferation assay and CYP19 activity assay on human myometrial endothelial cells treated with testosterone, estradiol, letrozole, flutamide, PD98059, MG-132 alone or in combination. MAIN OUTCOME MEASURE(S): We analyzed whether aromatase is expressed in human myometrial microvascular endothelial cells (HMMECs) and whether it affects proliferation and converts androgens to estrogens. In addition, we aimed to define whether or not T could have a direct capability to affect HMMEC proliferation. RESULT(S): Using quantitative real-time PCR and Western analysis, primary passage four HMMECs were shown to express low levels of aromatase mRNA and protein, respectively. However, HMMECs were unable to convert radioactively labeled 3∗H-1ß-androstenedione to estrogen. Pharmacologic doses of T (10(-6) and 10(-4) M) increased HMMEC proliferation, assessed through a bromodeoxyuridine ELISA. This effect of T on proliferation could not be blocked after pretreatment of cells with the aromatase inhibitor letrozole. In addition, HMMECs were found to express androgen receptors (ARs), and the AR antagonist flutamide abolished T-dependent proliferation. T was shown to increase AR protein levels, which was due to T-dependent receptor stabilization and not activation of gene transcription. CONCLUSION(S): We conclude that myometrial endothelial proliferation is not regulated through myometrial endothelial estrogen production. However, pharmacologic doses of T increase myometrial endothelial proliferation through a receptor-dependent and -stabilizing mechanism.

Differential regulation of proteasome-dependent estrogen receptor alpha and beta turnover in cultured human uterine artery endothelial cells.

Estrogen-induced loss of estrogen receptor (ER) alpha expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER alpha and/or ER beta levels is unclear. Using RT-PCR assays, we did not find any regulation of ER alpha or ER beta mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17 beta-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17 beta-estradiol was capable of down-regulating both ER alpha and ER beta protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER alpha, but not ER beta, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER alpha as well as ER beta through different proteasomal uptake mechanisms.