Abstract
Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E2 (PGE2) generation mediated by S1P receptors/PKCα/MAPKs cascade-dependent activation of NF-κB. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to Gq- and Gi-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE2 system induced by S1P, in turn leading to apoptosis in HCFs.
Highlights
Cardiac fibroblasts, one kind of the main cell types in the cardiac tissue, play key roles in normal myocardial function and myocardial remodeling, including myofibroblast differentiation, proliferation, migration, secretion of cytokines and growth factors, matrix protein synthesis, and inflammation [1]
We further determined whether Sphingosine 1-phosphate (S1P) receptor subtypes and G proteins were involved in the enhanced MMP9 activity; Human cardiac fibroblasts (HCFs) were pretreated with S1PR1 antagonist (W123), S1PR3 antagonist (CAY10444), Gq protein antagonist (GPA2A), Gi antagonist (pertussis toxin (PTX)), or the inhibitor of HB-epidermal growth factor (EGF) (CRM197) and challenged with S1P for 3 min
The findings showed that pretreatment with W123, CAY10444, GPA2A, or PTX reduced MMP9 activity induced by S1P, but not with CRM197 in HCFs (Figure 1(e))
Summary
One kind of the main cell types in the cardiac tissue, play key roles in normal myocardial function and myocardial remodeling, including myofibroblast differentiation, proliferation, migration, secretion of cytokines and growth factors, matrix protein synthesis, and inflammation [1]. Cyclooxygenase- (COX-) 2, one of two kinds of COXs, could be inducible during various inflammatory conditions by several proinflammatory factors leading to prostaglandin (PG) synthesis in various types of cells. Some studies showed that S1P could induce PGE2 synthesis related to the upregulation of COX-2 in numerous types of cells and organs [10–14]. We recently reported that S1P induces the expression of COX-2/PGE2 through NF-κB activity enhanced by PKCα-mediated mitogen-activated protein kinases (MAPKs) in HCFs [15]. S1P could exert a key role in cardiovascular inflammatory disorders
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