Estrogen upregulates cyclooxygenase gene expression in fetal pulmonary artery endothelial cells

Prostacyclin (PGI₂) is a key mediator of pulmonary vasomotor tone and lung development in late fetal life. Its production in the pulmonary vasculature rises dramatically during late gestation as a result of increasing expression of the rate-limiting enzyme cyclooxygenase (COX). Estrogen may mediate this increase in COX since fetal estrogen levels rise in late gestation and estrogen enhances PGI₂ synthesis in certain non-pulmonary vascular cells. We therefore studied the effect of estrogen on COX gene expression in fetal pulmonary artery endothelial cells (PAEC). Ovine fetal PAEC from third generation intrapulmonary arteries were treated with 10^-10 to 10^-6 M estradiol-17β (E₂β) for 48 or 96 hours. To examine COX mRNA expression, a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed using specific primers designed for the constitutive isoform of COX. There was a linear relationship between the quantity of RNA subjected to RT-PCR and the amount of COX PCR product generated (r=0.98). To control for mRNA stability and the RT step, RT-PCR was also done for the housekeeping gene malate dehydrogenase (MDH). Exposure to E₂β for 48 hours caused a dose-related increase in COX mRNA expression which was maximal at 10^-8 M (4.4 fold increase). MDH mRNA expression was unaffected. Findings were similar after 96 hours of E₂β. Since estrogen-mediated regulation of gene transcription involves activation of the estrogen receptor (ER), we also determined whether fetal PAEC express ER by performing RT-PCR using primers designed from the estrogen binding domain of the human ER. In the presence of RT, the correct size PCR product was generated and its identity was confirmed by Southern analysis and by sequencing. Thus, estrogen upregulates COX gene expression in fetal PAEC, and this may be mediated by activation of PAEC ER. This process may play a role in optimizing the capacity for PGI₂-mediated pulmonary vasodilatation during cardiopulmonary transition at birth.