title: AP-1 regulates sphingosine kinase 1 expression in a positive feedback manner in glomerular mesangial cells exposed to high glucose.
abstract: Our previous studies have confirmed that the sphingosine kinase 1 (SphK1)-sphingosine 1-phosphate (S1P) signaling pathway in the kidney under diabetic conditions is closely correlated with the pathogenesis of diabetic nephropathy (DN). The activation of SphK1-S1P pathway by high glucose (HG) can increase the expression of fibronectin (FN), an important fibrotic component, in glomerular mesangial cells (GMCs) by promoting the DNA-binding activity of transcription factor AP-1. However, the mechanism responsible for the sustained activation of SphK1-S1P pathway remains unclear. Given the binding motifs for AP-1 within the first intron of the SphK1 gene, we speculated that the activated AP-1 in the kidney under HG condition possibly regulates SphK1 expression in a positive feedback manner, thereby promoting the sustained activation of SphK1-S1P pathway and mediating the pathological progression of DN. Here, we observed the effect of AP-1 on SphK1 expression in GMCs and explored the molecular mechanism involved in the sustained activation of SphK1-S1P pathway. We found two consensus binding motifs for AP-1 in the promoter sequences and non-coding region downstream of the transcriptional initiation of the rat SphK1 gene by chromatin immunoprecipitation assay. The treatment of GMCs with both HG and S1P significantly increased the protein expression of c-Jun and c-Fos, and obviously enhanced the phosphorylation of c-Jun at Ser63 and Ser73, and c-Fos at Ser32. Knockdown of c-Jun and c-Fos with siRNAs substantially inhibited the expression of SphK1 and FN, whereas overexpression of c-Jun and c-Fos significantly increased the expression of SphK1 and FN. Curcumin treatment greatly decreased the levels of c-Jun, c-Fos, SphK1, and FN in the kidney tissues of diabetic rats. SiRNAs targeting SphK1 and S1P2 receptor respectively inhibited the phosphorylation of c-Jun (ser63 and ser73) and c-Fos (ser32), as well as FN expression under both normal and HG conditions. Our data demonstrated that the activated SphK1-S1P signaling pathway in GMCs under diabetic conditions is closely associated with AP-1 to form a positive feedback loop. This positive feedback loop functions as an important molecular basis for the sustained activation of SphK1-S1P pathway and increased FN expression that lead to the initiation and progression of DN.
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Kaipeng
authors.initials: K
authors.lastname: Huang
authors.affiliation: Yunnan Baiyao Group Co. Ltd, Kunming 650500, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Huang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Chen
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Hao
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Shaogui
authors.initials: S
authors.lastname: Wang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Junying
authors.initials: J
authors.lastname: Huang
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Liu
authors.affiliation: Laboratory of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China. Electronic address: huangheq@mail.sysu.edu.cn.
authors.forename: Heqing
authors.initials: H
authors.lastname: Huang
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D003474:Curcumin / Q000494:pharmacology; D004268:DNA-Binding Proteins; D003920:Diabetes Mellitus / Q000473:pathology; D003928:Diabetic Nephropathies / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005353:Fibronectins / Q000096:biosynthesis; D005947:Glucose / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D008246:Lysophospholipids / Q000096:biosynthesis / Q000494:pharmacology; D008297:Male; D050527:Mesangial Cells / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D049349:Receptors, Lysosphingolipid / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000494:pharmacology; D013549:Sweetening Agents / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D000099157:Sphingosine Kinase
chemical_list: D003474:Curcumin; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005353:Fibronectins; D005947:Glucose; D048031:JNK Mitogen-Activated Protein Kinases; D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D016760:Proto-Oncogene Proteins c-fos; D034741:RNA, Small Interfering; D049349:Receptors, Lysosphingolipid; D013110:Sphingosine; D013549:Sweetening Agents; D018808:Transcription Factor AP-1; D000099157:Sphingosine Kinase; C060506:sphingosine 1-phosphate
keywords: AP-1; ChIP; DN; Diabetic nephropathy; ECM; FN; GMCs; HG; NG; S1P; S1P2 receptor; STZ; SphK; Sphingosine 1-phosphate; Sphingosine kinase 1; TGF-β1; activator protein-1; c-Fos; c-Jun; chromatin immunoprecipitation; diabetic nephropathy; extracellular matrix; fibronectin; glomerular mesangial cells; high glucose; normal glucose; sphingosine 1-phosphate; sphingosine kinase; streptozocin; transforming growth factor-β1; type 2 receptor specific for S1P