Estrogen Increases Expression of the Human Prostacyclin Receptor within the Vasculature through an ERα-Dependent Mechanism

Abstract

Prostacyclin and the prostacyclin receptor (IP) are implicated in mediating many of the atheroprotective effects of estrogen in both humans and in animal models but through unknown mechanisms. Hence, herein the influence of estrogen on IP gene expression in endothelial EA.hy926, human erythroleukemia 92.1.7 and primary human aortic smooth muscle cells was investigated. Estrogen increased hIP mRNA levels, promoter (PrmIP)-directed reporter gene expression and cicaprost-dependent cAMP generation in all cell types, effects that were abrogated by actinomycin D and the general estrogen receptor (ER)-α/ERβ antagonist ICI 182,780. Furthermore, the ERα-selective agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), but not the ERβ-agonist 2,3-bis(4-hydroxyphenyl)-propionitrile, significantly increased hIP mRNA and PrmIP-directed gene expression. Deletional and mutational analysis of PrmIP uncovered an evolutionary conserved estrogen-response element, while electrophoretic mobility shift, antibody-supershift and chromatin immunoprecipitations assays confirmed the direct binding of ERα, but not ERβ, to PrmIP both in vitro and in vivo. Moreover, immunofluorescence microscopy corroborated that estrogen and PPT increased hIP expression in primary human aortic smooth muscle cells. In conclusion, the hIP gene is directly regulated by estrogen that largely occurs through an ERα-dependent transcriptional mechanism and thereby provides critical insights into the role of prostacyclin/hIP in mediating the atheroprotective effects of estrogen within the human vasculature.

Introduction

The prostanoid prostacyclin plays a central role in haemostasis, acting as a potent inhibitor of platelet aggregation and as an endothelium-derived vasodilator.1, 2 The prostacyclin receptor (IP) is abundantly expressed throughout the vasculature, including in platelets/megakaryocytes, macrophages, vascular endothelial and smooth muscle cells, various other tissues including the heart, kidney, lung, thymus and spleen and in sensory neurons of the dorsal root ganglion.2, 3 The IP is primarily coupled to Gs/adenylyl cyclase activation, mediating prostacyclin inhibition of platelet aggregation and vascular tone.2, 3

The cardioprotective effects of prostacyclin within the myocardium and vasculature are well documented.4, 5 Alterations in the levels of prostacyclin, its synthase or receptor, the IP, are associated with a range of vascular dysfunctions including stroke and myocardial infarction.6, 7 Multiple and frequent single-nucleotide polymorphisms occur within the coding sequence of the human IP that correlate with predisposition to cardiovascular (CV) disease including enhanced intimal hyperplasia and platelet activation in deep vein thrombosis.⁸ As a major product of cyclooxygenase (COX)-2, prostacyclin acts as a potent pro-inflammatory mediator and is abundantly produced during myocardial ischemia and hypoxia, offering cardioprotection.9, 10 Recognition of the importance of prostacyclin for haemostasis and CV integrity has been critically highlighted by various clinical trials that established that certain COXIBs, the subclass of non-steroidal anti-inflammatory drugs that selectively inhibit COX2, depress prostacyclin generation predisposing patients to increased risk of thrombotic stroke and myocardial infarction.11, 12 While IP^−/− null mice display normal vascular function, they exhibit enhanced thrombotic tendency in response to vascular injury in addition to reduced acute inflammatory responses.³

The protective role of estrogens in the heart and vasculature has also been established, and gender-specific differences in the incidence of CV disease occur both clinically and in animal studies.⁵ For example, hormone/estrogen replacement therapy can prevent the primary onset of coronary artery disease in post-menopausal women,⁵ although such effects are not without controversy.13, 14 The effects of estrogen are largely mediated through its binding to one of two estrogen receptor (ER) α and β subtypes, members of the nuclear receptor superfamily.15, 16 ERα and ERβ display distinct patterns of expression and biological function, largely acting as transcription factors to modulate expression of target genes by either direct binding to the estrogen-responsive element (ERE) with the consensus 5′-GGTCAnnnTGACC-3′ palindromic sequence¹⁷ or by indirect interaction with other transcription factors, such as Sp1 and Ap1.¹⁵ Ligand-activated ERs may, in turn, recruit coactivators such as CBP/p300, SRC-1, TIF2 or co-repressors including N-CoR and SMRT.18, 19

In addition to such classic genomic regulation by estrogen and analogues, more rapid non-genomic effects also occur, and it is thought that some of these CV protective actions may be mediated by direct effects on the vessel wall.5, 16, 20 Consistent with this, there is accumulating evidence that many of the cardioprotective effects of estrogen are mediated due to its increased synthesis and release of the endothelial-derived vasodilators nitric oxide and prostacyclin.²¹ For example, estrogen induces the synthesis and expression of COX1, COX2 and prostacyclin synthase, resulting in up to sixfold increases in systemic prostacyclin levels. Moreover, in the female low-density lipoprotein receptor null mice (LDLR^−/−), estrogen stimulated both COX2 expression and prostacyclin formation resulting in a substantial atheroprotection.²² In the same study, further disruption of the IP gene abrogated the atheroprotective effects of estrogen and accelerated atherogenesis in the double LDLR^−/−/IP^−/− null mouse.²² However, despite this, the actual molecular basis of the role of the IP in mediating such estrogen-induced atheroprotection remains to be established. Moreover, it is currently unknown whether estrogen may directly, or indeed indirectly, affect IP expression levels possibly accounting for such effects²² and hence, critically, remains to be investigated.

The overall aim and rationale of the current study is to address this deficit by characterizing the hIP gene, focusing primarily on delineating the mechanism determining its aforementioned role in mediating the response to estrogen within the vasculature. Herein, we have uncovered a consensus cis-acting ERE in the hIP promoter critical for the transcriptional regulation of hIP expression by estrogen in a host of cells of vascular origin, including in the human endothelial EA.hy926 and megakaryocytic human erythroleukemia (HEL) 92.1.7 cell lines²³ and cultured primary human aortic smooth muscle cells (1° hAoSMCs). The data outlined provide compelling evidence that the hIP gene is a direct target of estrogen that occurs through an ERα-dependent mechanism and, accordingly, not only provides a molecular genetic basis for understanding the modes of regulation of hIP expression in health and disease but also for the combined protective roles of estrogen and prostacyclin within the CV system.