17-beta estradiol attenuation of hypoxia-induced erythropoietin expression

Previous studies from our laboratory found that ovariectomy (OVX) exacerbates the polycythemic response to hypoxia while treatment with 17-β estradiol (E2-β) inhibits this effect. We hypothesized that E2-β decreases EPO gene expression during hypoxia. This hypothesis was tested in OVX catheterized rats treated with E2-β (20 μg/24 hrs, 7 days) or vehicle (polypropylene glycol) and exposed to hypoxia (12% O₂) or normoxia (room air) for 8 or 12 hrs. We found that E2-β attenuated hypoxic-induction of EPO gene expression.; E2-β attenuates hypoxic-induction of endothelin-1 (ET-1) gene expression by decreasing HIF-1 activity. We speculated the same mechanism may cause E2-β attenuation of EPO expression. Immunofluorescence studies in Hep3B cells found that E2-β (100 pM) attenuates hypoxic increases in EPO and HIF-1α staining. This decrease was prevented by non-selective estrogen receptor (ER) antagonist, ICI 182,780 (25 μM). E2-β similarly inhibited hypoxic increases in luciferase activity in construct containing the hypoxia response element (HRE) from the human EPO enhancer region. These studies suggest that E2-β attenuates HRE-mediated EPO expression by interfering with hypoxic-induction of HIF-1α in an ER-dependent manner.; E2-β induces NO synthesis in a number of organs and NO can attenuate HIF-1 activity during hypoxia. We hypothesized that E2-β increases NO to attenuate EPO synthesis during hypoxia. To test this, OVX catheterized rats were administered a bolus of the nitric oxide synthase (NOS) inhibitor, Nω-nitro-L-arginine (L-NNA, 15 mg/kg) or vehicle (saline) followed by a continuous infusion of L-NNA (15 mg/kg/hr) during the hypoxic exposure (8 hrs). Both hypoxia and E2-β induced NO synthesis in saline-treated animals. This was abolished with L-NNA treatment demonstrating this dose inhibits NO synthesis. Immunoblot studies revealed that renal expression of eNOS and iNOS were not altered by E2-β, L-NNA or hypoxia. We could not detect nNOS in these preparations. This demonstrates increased renal NOS expression doesn't cause the increase in plasma NO with E2-β treatment. To further evaluate the role of NO in this response, plasma EPO levels were determined at 0 and 8 hrs of hypoxia. We found that NOS inhibition did not alter E2-β-dependent decreases in EPO synthesis demonstrating that this attenuation is independent of increased NO availability. We conclude that E2-β attenuates hypox-induction of EPO gene expression by decreasing HIF-1 activity independent of increased NO availability.