Prostacyclin, or PGI/2, is a critical mediator of vasodilation in the developing lung produced in endothelium by cyclooxygenase (COX). We have shown that there is a 30-fold maturational rise in PGI/2 in ovine fetal and newborn intrapulmonary arteries due to increasing expression of the COX-1 isoform. The increase in COX-1 in the fetus may be induced by rising plasma estrogen (E/2) levels because we have shown that 48h exposure to E/2 up-regulates COX-1 in ovine fetal pulmonary artery endothelial cells (PAEC). Preliminary studies indicated that E/2 also causes rapid PGI/2 activation (15 min), and that an estrogen receptor (ER) subpopulation is associated with the plasma membrane. The OBJECTIVE of this proposal is to determine the molecular mechanisms by which prolonged E/2 exposure up- regulates COX-1 expression and by which E/2 also acutely activates PGI/2 in cultured ovine fetal PAEC.
Aim 1 is to determine the mechanisms underlying E/2-induced COX-1 up-regulation in transient transfection studies focused on the roles of the ER subtypes, ERalpha and ERbeta, and tyrosine kinase (TK)/MAP kinase (MAPK) signaling. E/2-induced transcriptional activation will also be studied using COX-1 promoter- reporter gene constructs.
Aim 2 is to determine the basis for acute PGI/2 activation by E/2, elucidating the step in the PGI/2 synthetic cascade hat is involved. The roles of ERalpha and ERbeta and TK/MAPK signalling in the acute response will also be discerned by pharmacologic approaches and cell transfection.
Aim 3 is to identify the ER subtypes expressed in PAEC and to further delineate their localization in studies of plasma membrane caveolae and other cell fractions using immunodetection and radio-ligand binding. Results will be confirmed by immunofluorescence and immunoelectron microscopy in intact cells. Cell surface ER function will also be evaluated in studies of PGI/2 stimulation by immobilized E/2 ligand.
Aim 4 is to determine the mechanisms regulating PAEC ER expression, following up on the observation that ERalpha abundance is potently up-regulated by E/2. The molecular basis for this process will be determined, including studies of transcriptional events using ERalpha promoter activity assays. The effects of E/2 on ERbeta expression will also be examined. These studies will provide fundamental new information about the regulation of pulmonary PGI/2 synthesis in the perinatal period, and about the roles of E/2 and ERs in vascular biology.
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