Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Abstract

Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. We detected an estrogen receptor alpha (ERalpha) transcript in human endothelial cells that encodes a truncated 46-kDa ERalpha (Delta1a-hERalpha-46). A corresponding 46-kDa ERalpha protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ERalpha (ERalpha-66) and Delta1a-hERalpha-46 resulted in appropriately sized recombinant proteins identified by anti-ERalpha antibodies. Confocal microscopy revealed that a proportion of both ERalpha-66 and hERalpha-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ERalpha isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by Delta1a-hERalpha-46 was much less than with ERalpha-66. Furthermore, Delta1a-hERalpha-46 inhibited classical hERalpha-66-mediated transcriptional activation in a dominant-negative fashion. These findings suggest that expression of an alternatively spliced, truncated ERalpha isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.