Prostacyclin receptor suppresses cardiac fibrosis: Role of CREB phosphorylation

Abstract

Cardiac fibrosis is a consequence of many cardiovascular diseases and contributes to impaired ventricular function. Activation of the prostacyclin receptor (IP) protects against cardiac fibrosis, but the molecular mechanisms are not totally understood. Using mouse cardiac fibroblasts, we found that IP activation with cicaprost suppressed expression of collagen I and other target genes of transforming growth factor-β. This effect of cicaprost was unlikely to be mediated by inhibition of the Smad2/3 or mitogen-activated protein kinase (MAPK) activities, but was associated with cAMP elevation and phosphorylation of the transcription factor cAMP response element binding protein (CREB). Expression of a non-phosphorylated CREB mutant suppressed the inhibitory effect of cicaprost. It appears that phosphorylated CREB binds to and sequestrates the transcription coactivator CBP/p300 from binding to Smad. Inhibition of the intrinsic histone acetyl-transferase activity of CBP/p300 with garcinol significantly suppressed collagen I expression in fibroblasts. Using apolipoprotein E and IP double knockout mouse, we demonstrated that endogenous prostacyclin/IP signaling had an inhibitory effect on angiotensin II-induced cardiac fibrosis under hypercholesterolemic conditions. Taken together, our results suggest that the prostacyclin/IP pathway suppresses cardiac fibrosis, at least partly, by inducing CREB phosphorylation.

Introduction

Cardiac fibrosis is a consequence of many cardiovascular diseases including hypertension, myocarditis, dilated cardiomyopathy, and myocardial infarctions [1], [2], [3], [4]. Such diseases trigger inflammatory responses in the myocardium, leading to production of inflammatory cytokines, leukocyte infiltration, fibroblast proliferation and transdifferentiation to myofibroblasts. All of these changes may contribute to accumulation of extracellular matrix proteins, in particular collagens, ultimately leading to impaired myocardial contractile and/or diastolic dysfunctions. Many factors have been implicated in the initiation and progression of cardiac fibrosis, including mechanical overload, inflammatory cytokines, angiotensin II, endothelin, growth factors and oxidative stress [3], [4], [5], [6]. Despite many attempts to define the causal roles of these factors in cardiac fibrosis, the precise mechanisms underlying this pathological process remain poorly understood.

Amongst these biological factors, transforming growth factor (TGF)-β has been recognized to have a pivotal role in cardiac fibroblast activation and extracellular matrix production [5], [7]. TGF-β-induced intracellular signaling is mainly mediated by the Smad pathway, as well as activation of MAPK members such as ERK and p38. Ligand binding on TGF receptors induces Smad2/3 phosphorylation, which then recruits Smad4 and the formed Smad complex translocates into the nucleus, where it binds to the TGF-responsive element and modulates gene expression [7]. In addition to the Smad pathway, TGF can also activate p38 MAPK via the upstream kinases TAK1 and MEK3/6 [8], and ERK via activation of the Ras cascade [9]. Experimental evidence suggests that all of the Smad, p38 and ERK pathways may be involved in mediating TGF-induced collagen expression in fibroblasts [10], [11], [12].

Prostacyclin (PGI₂) is an endothelium-derived eicosanoid with multiple cardiovascular protective actions, mainly mediated by activation of its G protein coupled receptor IP and subsequent accumulation of intracellular cAMP [13]. Using IP-deficient animals, it has been shown that cardiac fibrosis induced by overload was enhanced in the absence of IP signaling [14], [15]. However, these studies were carried out in normolipidemic animals. The effect of prostacyclin/IP signaling on cardiac fibrosis under hypercholesterolemic condition, which is a prominent risk factor for cardiovascular disease, has not been explored. Moreover, the molecular mechanisms of the anti-fibrotic action of IP in the heart are poorly understood. In vitro experiments showed that the prostacyclin analogue suppressed collagen expression in cardiac fibroblasts [16]. In skin fibroblasts, IP activation inhibited collagen expression by antagonizing TGF-β-induced ERK activation [17]. However, whether the same mechanism underlies the anti-fibrotic effects of prostacyclin/IP in the heart is unclear. Therefore in this study we investigated (1) whether the prostacyclin/IP system protects against cardiac fibrosis in vivo under hypercholesterolemic conditions; and (2) the effects and mechanisms of IP activation on collagen expression in primary murine cardiac fibroblasts. Moreover, recent studies have suggested that NADPH oxidase mediated-oxidative stress may have an important role in the development of cardiac fibrosis [6]. For example, Looi et al. demonstrated that Nox2 NADPH oxidase deficiency attenuated myocardial fibrosis and expression of connective tissue growth factor and collagen after infarction [18]. Therefore, we also tested whether the protective effect of IP activation could be related to altered expression of Nox NADPH oxidases.