McGrath KCY, Li XH, Gaus K, Williams P, Celermajer DS, Handelsman DJ and Heather AK
There exists a striking gender difference in the incidence of atherosclerosis. Androgen exposure may predispose men to earlier onset atherosclerosis. We previously demonstrated that the potent androgen, dihydrotestosterone, enhanced the binding of monocytes to endothelial cells, via androgen receptor/nuclear factor kappa B-dependent expression of the cell adhesion molecules, vascular cell adhesion molecule-1 and intercellular cell adhesion molecule-1. We now show that testosterone and dihydrotestosterone can also induce a novel, non-genomic pathway that leads to the rapid activation of nuclear factor-kappa B via intracellular Ca2+ signalling, initiated at the plasma membrane. Human umbilical vein endothelial cells exposed to 6-60 nM testosterone or dihydrotestosterone showed a rapid increase in intracellular calcium levels. The testosterone or dihydrotestosterone effect on increased intracellular calcium could not be abrogated by pre-incubation with androgen receptor antagonist, hydroxyflutamide, or by U73122, an inhibitor of intracellular calcium release from endoplasmic reticulum stores. However, pre-incubation with both Ni2+or an extracellular Ca2+ chelator blocked the testosterone-induced intracellular Ca2+ surge. Testosterone conjugated to bovine serum albumin was equal to free testosterone in its ability to induce the intracellular Ca2+ surge. Binding studies showed testosterone does bind to the plasma membrane, however, classical androgen receptor was unable to be detected in the plasma membrane of human umbilical vein endothelial cells. Testosterone was found to rapidly increase nuclear factor-kappa B activity, an effect that was blocked when cells were incubated in calcium-free media. This study demonstrates for the first time that testosterone induces a non-genomic membrane-initiated Ca2+ dependent signalling pathway that leads to the rapid activation of nuclear factor-kappa B.