Truncated Estrogen Receptor &agr; 46-kDa Isoform in Human Endothelial Cells: Relationship to Acute Activation of Nitric Oxide Synthase

Background—Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results—We detected an estrogen receptor &agr; (ER&agr;) transcript in human endothelial cells that encodes a truncated 46-kDa ER&agr; (&Dgr;1a-hER&agr;-46). A corresponding 46-kDa ER&agr; protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER&agr; (ER&agr;-66) and &Dgr;1a-hER&agr;-46 resulted in appropriately sized recombinant proteins identified by anti-ER&agr; antibodies. Confocal microscopy revealed that a proportion of both ER&agr;-66 and hER&agr;-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER&agr; isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by &Dgr;1a-hER&agr;-46 was much less than with ER&agr;-66. Furthermore, &Dgr;1a-hER&agr;-46 inhibited classical hER&agr;-66–mediated transcriptional activation in a dominant-negative fashion. Conclusions—These findings suggest that expression of an alternatively spliced, truncated ER&agr; isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

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