Elsevier

Biochemical Pharmacology

Volume 71, Issue 9, 28 April 2006, Pages 1308-1323
Biochemical Pharmacology

Synthetic peroxisome proliferator-activated receptor γ agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells

https://doi.org/10.1016/j.bcp.2006.01.011Get rights and content

Abstract

The human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPα and TPβ, that are regulated by distinct promoters designated promoter Prm1 and Prm3, respectively. Previous studies established that 15d-Δ12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TPβ expression through a peroxisome proliferator-activated receptor (PPAR)γ mechanism without affecting Prm1 activity or TPα expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARγ ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPβ mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPα mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARγ-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARγ in regulating the human TP gene; they highlight further differences in TPα and TPβ expression/regulation and point to essential differences between natural and synthetic PPARγ agonists in mediating those effects.

Introduction

Peroxisome proliferator-activated receptor (PPAR)γ has been implicated in a broad range of cellular functions including adipocyte differentiation [1], glucose homeostasis and insulin sensitization [2], [3], inflammatory responses, carcinogenesis and apoptosis [4], [5]. In addition, there is a significant association between PPARγ polymorphism and coronary artery disease [6] and PPARγ mRNA expression in human adipose is inversely associated with cardiovascular risk factors [7]. Of the natural PPARγ ligands, the prostaglandin derivative 15-deoxy-Δ12,14 prostaglandin J2 (15d-PGJ2) and 9- or 13-hydroxy-octadecadienoic acid (9-HODE or 13-HODE) mediate potent adipogenic and anti-inflammatory effects [8]. Although 15d-PGJ2 exerts the majority of it's effects through the activation of PPARγ, a number of PPARγ-independent effects have been reported. These PPARγ-independent effects are believed to be due to direct 15d-PGJ2-covalent modification of target proteins [9].

Several synthetic PPARγ agonists such as the thiazolidinedione (TZD) derivatives, rosiglitazone (Avandia®) and pioglitazone (Actos®) are being used in the treatment of type II diabetes mellitus. Both of these drugs are believed to ameliorate type II diabetes in vivo by improving the body's sensitivity to insulin [10]. Specifically, TZD-mediated activation of the peroxisome proliferator-activated receptor (PPAR) family of transcription factors has been shown to augment insulin sensitivity through the activation of insulin-responsive genes involved in the control of glucose production, transport, and utilization [11].

PPARγ is a member of the nuclear hormone receptor superfamily that can positively or negatively regulate gene expression in response to ligand binding. Typically, PPARγ positively regulates gene expression by binding as a heterodimer with the retinoic X receptor (RXR) to PPAR response elements (PPREs) within target genes [12]. When either the PPARγ or RXR components of the heterodimer are bound by agonists, their respective ligand binding domains undergo a conformational change that leads to the recruitment of co-activators and consequent transcription of target genes [13]. Liganded PPARγ can also inhibit the expression of a number of genes, typically those associated with inflammation [14], [15] and thrombosis [16], [17].

Such inhibitory effects of liganded PPARγ are generally mediated by trans-repression, as they do not appear to involve direct binding to the target promoter. This trans-repression reduces the activity of transcription factors key to inflammation and thrombosis, such as NF-κB, SP-1 and AP-1, by acting at multiple levels [18]. These include interference with the signalling pathway leading to AP-1 activation through the direct inhibition of cJun [19], the PPARγ-dependent sequestration of co-activators including CBP [20] and the impairment of SP-1 binding to DNA [16], as exemplified by PPARγ-mediated suppression of the proinflammatory/prothrombotic cyclooxygenase-2, inducible nitric oxide synthase and rat thromboxane A2 receptor genes, respectively. PPARγ is reported to inhibit signalling by NF-κB in human aortic smooth muscle cells by direct protein:protein interaction, PPARγ: NF-κB complex formation and trans-repression of NF-κB action and function [18]. Consistent with this, it has recently been discovered that non-pathogenic commensal microflora (e.g. Bacteroides thetaiotaomicron) attenuate inflammation within the intestinal epithelium through a trans-repression mechanism involving both NF-κB and PPARγ[21]. In this mechanism, it was established that commensal anaerobic gut bacteroides-induce nuclear association between the RelA (p65) subunit of NF-κB and PPARγ. The subsequent export of the newly formed NF-κB:PPARγ complex out of the nucleus attenuates NF-κB-mediated gene transcription, thereby impeding the inflammatory response [21].

Thromboxane (TX)A2 is an unstable arachidonate metabolite that plays a key role in haemostasis but is also widely implicated as a mediator in cardiovascular diseases such as thrombosis, atherosclerosis, myocardial infarction, stroke and bronchial asthma [22], [23], [24], [25], [26]. Binding of TXA2 to its receptor TP, a G-protein coupled receptor, induces vasoconstriction and platelet aggregation as well as mitogenic- and hypertrophic-growth of vascular smooth muscle [27], [28]. In humans, but not in non-primates, TXA2 signals through two TP isoforms termed TPα and TPβ that arise through differential splicing [29], [30]. While the biologic significance of two TP isoforms in humans is unclear, there is increasing evidence that they may be physiologically distinct displaying certain differences in their intracellular signalling [31], [32], in their homologous and heterologous desensitization [33], [34], [35], [36] and in their patterns of expression [37]. Consistent with this, recent studies have established that TPα and TPβ expression is actually under the transcriptional control of distinct promoters within the single human TP gene located on chromosome 19 [38], [39]. While the originally identified promoter (Prm) 1 directs TPα expression, a novel promoter (Prm3) was identified that exclusively directs TPβ expression [38], [40].

In keeping with the critical role of PPARγ within the CV system, as stated, it has been established that both 15d-PGJ2 and troglitazone suppress expression of the rat TP gene via an interaction of PPARγ with SP-1 [16]. However, in order to examine the effect of PPARγ agonists on human TP expression, their effect on two independently regulated TP isoforms through two distinct promoters, namely TPα and TPβ through Prm1 and Prm3, respectively, must be determined. In a recent study, we established that the endogenous PPARγ agonist 15d-PGJ2 specifically suppressed Prm3-directed gene expression and TPβ mRNA expression in the megakaryocytic human erythroleukemic (HEL) 92.1.7 cell line while TPα mRNA expression and Prm1-directed gene expression was unaffected by 15d-PGJ2[41]. Moreover, the effect of 15d-PGJ2 on TPβ expression occurred through a novel mechanism involving direct binding of activated PPARγ-RXR heterodimers to a PPRE located within the −168 to −141 region of Prm3 [41]. It is, however, now increasingly recognized that PPARγ ligands, such as 15d-PGJ2 and the TZDs, can elicit a range of both shared but also entirely distinct biological effects [42]. For example, although PPARγ-mediated repression of the GLUT4 promoter is augmented by 15d-PGJ2, it is completely alleviated by rosiglitazone [42].

Hence, in view of such distinctions in the biologic actions between endogenous and synthetic PPARγ ligands, in the present study, we extended our previous investigations by determining the effect of the synthetic rosiglitazone (Avandia®) and troglitazone (Rezulin®) on Prm1 and Prm3-directed reporter gene expression and on TPα and TPβ mRNA expression. Our data herein data provide further evidence for the role of PPARγ in the regulation of the human TP gene; they highlight further differences in the modes of regulation of TPα and TPβ expression but also point to critical differences between natural versus synthetic PPARγ ligands in mediating those effects.

Section snippets

Materials

pGL3Basic, pGL3Enhancer, pRL-Thymidine Kinase (pRL-TK) and Dual Luciferase® Reporter Assay System were obtained from Promega Corporation, Madison, WI, USA. [γ32P] ATP (6000 Ci/mmol at 10 m Ci/ml) was from Valeant Pharmaceuticals (ICN), Costa Mesa, USA. The endogenous PPARγ ligand 15-deoxy-Δ12,14-PGJ2 was obtained from Calbiochem-Novabiochem, Nottingham, UK. Cicaprost was obtained from Schering AG, Berlin, Germany. The agonist 17 phenyl trinor prostaglandin (PG) E2 was obtained from Cayman,

The effect of rosiglitazone and troglitazone on promoter (Prm) 1, 2 and 3 activity

Previous studies have established that the PPARγ ligands 15d-PGJ2 and troglitazone suppress expression of the TXA2 receptor (TP) gene in rat vascular smooth muscle cells [16]. In humans, the TP gene encodes two TP isoforms, termed TPα and TPβ, and is under the transcriptional regulation of three distinct promoter (Prm) regions termed Prm1, Prm2 and Prm3 [38]. Recent studies have revealed that 15d-PGJ2 selectively inhibits Prm3-directed reporter gene expression and TPβ mRNA expression through a

Discussion

There is growing evidence that along with its classical role in β-oxidation and adipogenesis, PPARγ also plays an important part in the control of inflammatory responses, cell growth and differentiation [46]. Within the vasculature, PPARγ appears to broadly offer a cardioprotective role against inflammatory and thrombotic disorders. Moreover, expression of both the rat TXA2 synthase and rat TXA2 receptor/TP are suppressed through PPARγ dependent mechanisms [16], [17]. Consistent with this, it

Acknowledgements

This work was supported by grants from The Wellcome Trust and The Health Research Board, Ireland. We are grateful to Dr. Martina B. O’Keeffe for assistance with the calcium studies. We are also very grateful to Dr. Stephen Smith, Glaxo-SmithKline, Essex, UK, for providing Rosiglitazone for these studies.

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