A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17β (E₂) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E ₂ increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E₂ but not with E ₂-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERα and ERβ. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERα mRNA (100-200%) but decreased ERβ mRNA (30-40%) expression as measured by real time PCR. ERα protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERβ protein expression declines (25-55%). Using ovalbumin bound to E₂ (Ov-E₂) linked to Eu (Eu-Ov-E₂), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of ³[H] E₂, that nearly entirely represents intracellular E₂ binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERα/ERβ as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.