Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan 33302, Taiwan.
School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan 33302, Taiwan.
Oxid Med Cell Longev. 2022 Feb 22;2022:7664290. doi: 10.1155/2022/7664290. eCollection 2022.
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 E (PGE) generation mediated by S1P receptors/PKC/MAPKs cascade-dependent activation of NF-B. 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/PGE 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/PGE 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/PGE 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 G- and G-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE 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/PGE system induced by S1P, in turn leading to apoptosis in HCFs.
人心脏成纤维细胞（HCFs）在心脏的正常生理功能和病理过程中发挥关键作用。我们最近的研究发现，在 HCFs 中，1-磷酸鞘氨醇（S1P）可以上调环氧化酶-2（COX-2）的表达，导致 S1P 受体/蛋白激酶 C/丝裂原活化蛋白激酶（MAPKs）级联依赖性 NF-B 的激活导致前列腺素 E（PGE）的产生。或者，已经证明 G 蛋白偶联受体（GPCR）介导的受体酪氨酸激酶（RTKs）的转激活诱导炎症反应。然而，S1P 在 HCFs 中诱导的 COX-2/PGE 系统中，GPCR 介导的 RTKs 的转激活是否参与其中尚不清楚。我们假设 GPCR 介导的 RTKs 依赖性信号级联的转激活参与了 S1P 诱导的反应。本研究旨在探讨 S1P 促进 COX-2/PGE 表达和诱导 HCFs 凋亡的综合机制。在这里，我们使用药理学抑制剂和 siRNA 转染来评估基质金属蛋白酶（MMP）2/9、肝素结合-（HB-）表皮生长因子（EGF）、表皮生长因子受体（EGFR）、PI3K/Akt、MAPKs 和转录因子 AP-1 是否参与了通过 Western blot、实时聚合酶链反应（RT-PCR）、染色质免疫沉淀（ChIP）和启动子报告基因测定在 HCFs 中确定的 S1P 诱导的 COX-2/PGE 系统。我们的结果表明，S1P 通过 S1PR1/3 激活与 G-和 G-偶联的 MMP9 活性，刺激 EGFR/PI3K/Akt/MAPKs/AP-1 依赖性转录活性，上调 COX-2 并伴有 PGE 产生，从而刺激半胱天冬酶-3 活性和凋亡。此外，S1P 增强的 c-Jun 结合到 COX-2 启动子上的相应结合位点，通过 ChIP 测定，该结合位点被这些蛋白激酶抑制剂减弱。这些结果表明，MMP9/EGFR 介导的 PI3K/Akt/MAPKs 依赖性 AP-1 活性的转激活参与了 S1P 诱导的 COX-2/PGE 系统的上调，进而导致 HCFs 凋亡。
