Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells.

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor (TFPI) gene expression through the androgen receptor in endothelial cells. This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-alpha. TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. To study the cellular mechanism of testosterone's action, nuclear factor-kappa B (NF-kappaB) translocation was confirmed by electrophoretic mobility shift assays. We found that after NF-kappaB was activated by TNF-alpha, TFPI protein levels declined significantly by 37.3% compared with controls (P < 0.001), and the mRNA levels of TFPI also decreased greatly (P < 0.001). A concentration of 30 nmol L(-1) testosterone increased the secretion of TFPI compared with the TNF-alpha-treated group. NF-kappaB DNA-binding activity was significantly suppressed by testosterone (P < 0.05). This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-kappaB activity.

[Effects of testosterone on extracellular signal-regulated kinase 1/2 phosphorylation in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effects of testosterone on extracellular signal-regulated kinase l/2 ( ERK1/2) phosphorylation in human umbilical vein endothelial cells (HUVEC). METHODS: Activations of ERK1/2 stimulated by physiological testosterone were detected by Western blotting in cultured HUVEC. RESULTS: A rapid phosphorylation expression of ERK1/2 was observed by treatment of the HUVECs with 3 x 10(-8) mol/L testosterone, especially at 30 minutes. This phosphorylation was greatly inhibited by incubation with androgen receptor antagonist flutamide for 3 hours previously. CONCLUSION: Testosterone at physiological concentrations induces the mitogen-activated protein kinase (MAPK, ERK1/2 and MEK1/2) phosphorylation within a short time, and flutamide could impair the process.

Mitogen-activated protein kinases pathway is involved in physiological testosterone-induced tissue factor pathway inhibitor expression in endothelial cells.

The mechanism of testosterone inducing the tissue factor pathway inhibitor (TFPI) in protecting against thrombosis is unknown. We aimed to elucidate the mechanisms involved in the induction by observing, in human umbilical vein endothelial cells (HUVECs), the phosphorylation of mitogen-activated protein kinases (MAPKs), a major cell signaling system. The level of testosterone regulating several signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 MAPK, was measured by western blot in HUVECs. ELISA and quantitative real-time reverse transcriptase-PCR were used to analyze TFPI expression after blocking ERK1/2 (with PD98059) or JNK (with SP600125) pathway in HUVECs. Testosterone-induced a rapid phosphorylation of ERK1/2, JNK and p38 MAPK in HUVECs, which could not be inhibited by androgen receptor antagonist flutamide. Blocking ERK1/2 or JNK pathway could significantly impair testosterone-induced TFPI at both translational and transcriptional levels in HUVECs. Testosterone at a physiological concentration may help to prevent thrombosis development by stimulating TFPI expression in HUVECs, partly through the ERK1/2 and JNK MAPK pathway.