Yang Chien-Chung, Hsiao Li-Der, Su Mei-Hsiu, Yang Chuen-Mao
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Tao-Yuan, Taiwan.
School of Traditional Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.
Front Pharmacol. 2020 Oct 30;11:569802. doi: 10.3389/fphar.2020.569802. eCollection 2020.
In the regions of tissue injuries and inflammatory diseases, sphingosine 1-phosphate (S1P), a proinflammatory mediator, is increased. S1P may induce the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E (PGE) system in various types of cells to exacerbate heart inflammation. However, the detailed molecular mechanisms by which S1P induces COX-2 expression in human cardiac fibroblasts (HCFs) remain unknown. HCFs were incubated with S1P and analyzed by Western blotting, real time-Polymerase chain reaction (RT-PCR), and immunofluorescent staining. Our results indicated that S1P activated S1PR-dependent transcriptional activity to induce COX-2 expression and PGE production. S1P recruited and activated PTX-sensitive G or -insensitive G protein-coupled S1PR and then stimulated PKCα-dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, leading to activating transcription factor NF-κB. Moreover, S1P-activated NF-κB was translocated into the nucleus and bound to its corresponding binding sites on COX-2 promoters determined by chromatin immunoprecipitation (ChIP) and promoter-reporter assays, thereby turning on COX-2 gene transcription associated with PGE production in HCFs. These results concluded that in HCFs, activation of NF-κB by PKCα-mediated MAPK cascades was essential for S1P-induced up-regulation of the COX-2/PGE system. Understanding the mechanisms of COX-2 expression and PGE production regulated by the S1P/S1PRs system on cardiac fibroblasts may provide rationally therapeutic interventions for heart injury or inflammatory diseases.
在组织损伤和炎症性疾病区域,促炎介质1 -磷酸鞘氨醇(S1P)水平会升高。S1P可能会诱导多种细胞中环氧合酶-2(COX-2)/前列腺素E(PGE)系统上调,从而加重心脏炎症。然而,S1P在人心脏成纤维细胞(HCFs)中诱导COX-2表达的详细分子机制仍不清楚。将HCFs与S1P一起孵育,并用蛋白质免疫印迹法、实时聚合酶链反应(RT-PCR)和免疫荧光染色进行分析。我们的结果表明,S1P激活S1PR依赖性转录活性,从而诱导COX-2表达和PGE生成。S1P招募并激活百日咳毒素敏感的G或不敏感的G蛋白偶联S1PR,然后刺激PKCα依赖性的p42/p44 MAPK、p38 MAPK和JNK1/2磷酸化,导致转录因子NF-κB激活。此外,S1P激活的NF-κB易位进入细胞核,并通过染色质免疫沉淀(ChIP)和启动子报告基因分析确定其与COX-2启动子上相应结合位点结合,从而开启HCFs中与PGE生成相关的COX-2基因转录。这些结果得出结论,在HCFs中,PKCα介导的MAPK级联反应激活NF-κB对于S1P诱导的COX-2/PGE系统上调至关重要。了解心脏成纤维细胞上S1P/S1PRs系统调节COX-2表达和PGE生成的机制,可能为心脏损伤或炎症性疾病提供合理的治疗干预措施。