title: Lipoxin A4 inhibits TNF-alpha-induced production of interleukins and proliferation of rat mesangial cells.
abstract: BACKGROUND
  Studies have shown that lipoxin A(4) (LXA(4)) and its analogues inhibited proliferation of glomerular mesangial cells induced by leukotriene D(4) (LTD(4)) or platelet-derived growth factor (PDGF), reduced the production of proinflammatory cytokines such as interleukin (IL)-1beta and IL-6 in renal tissue of ischemic injury. In the present studies, we examine whether LXA(4) have inhibitory effects on tumor necrosis factor-alpha (TNF-alpha)-induced productions of IL-1beta and IL-6 and proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway of LXA(4).
  
  
  METHODS
  Cultured glomerular mesangial cells were treated with TNF-alpha (10 ng/mL), with or without preincubation with LXA(4) at the different concentrations. Cell proliferation was assessed by [(3)H]-thymidine incorporation. Proteins of IL-1beta and IL-6 in supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA). Expressions of mRNA of IL-1beta and IL-6 were determined by real-time polymerase chain reaction (PCR) and cyclin E by reverse transcription (RT)-PCR. Proteins of cyclin E, threonine phosphorylated Akt(1) at 308 site (Thr(308)) and p27(kip1) were analyzed by Western blotting studies. Activities of signal transducers and activators of transcription-3 (STAT(3)), nuclear factor-kappaB (NF-kappaB) were determined by electrophroretic mobility shift assay (EMSA). Expression of Src homology (SH) 2-containing protein-tyrosine phosphatase (SHP-2) was assessed by immunoprecipitation and immunoblotting.
  
  
  RESULTS
  TNF-alpha-stimulated proliferation, release of proteins and expressions of mRNA of IL-1beta and IL-6 in mesangial cells were inhibited by LXA(4) in a dose-dependent manner. The marked increments in mRNA expression and protein synthesis of cyclin E induced by TNF-alpha in parallel with proliferation of mesangial cells were down-regulated by LXA(4). LXA(4) antagonized the phosphorylation of SHP-2 and activity of NF-kappaB induced by TNF-alpha. Pretreatment of the cells with NF-kappaB inhibitor pyrrolidine dithio-carbamate (PDTC) blocked the productions of IL-1beta, IL-6, and activation of NF-kappaB induced by TNF-alpha. Stimulation of mesangial cells with TNF-alpha resulted in enhanced DNA-binding activity of STAT(3). This increment was inhibited by LXA(4) in a dose-dependent manner. Threonine phosphorylated Akt(1) protein at 308 site stimulated by TNF-alpha was reduced by LXA(4.) TNF-alpha-induced decrement in expression of p27(kip1) protein was ameliorated by LXA(4) in a dose-dependent manner.
  
  
  CONCLUSIONS
  TNF-alpha-induced proliferation and increment of cyclin E of rat mesangial cells can be inhibited by LXA(4), and these inhibitory effects might be through the mechanisms of STAT(3) and Akt(1)/p27(kip1) pathway-dependent signal transduction. LXA(4) also antagonized TNF-alpha-stimulated IL-1beta and IL-6 synthesis, and these antagonisms were related to SHP-2 and NF-kappaB pathway-dependent signal transduction.
authors.affiliation: Department of Pediatrics, Central Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China. kad-yc@163.com
authors.forename: Sheng-Hua
authors.initials: SH
authors.lastname: Wu
authors.forename: Chao
authors.initials: C
authors.lastname: Lu
authors.forename: Ling
authors.initials: L
authors.lastname: Dong
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhou
authors.forename: Zha-Guang
authors.initials: ZG
authors.lastname: He
authors.forename: Zi-Qing
authors.initials: ZQ
authors.lastname: Chen
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019927:Cyclin E / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D005920:Glomerular Mesangium / Q000166:cytology* / Q000187:drug effects* / Q000276:immunology; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D044045:Lipoxins / Q000494:pharmacology*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D015534:Trans-Activators / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D044045:Lipoxins; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C494088:Stat3 protein, rat; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D025521:Tumor Suppressor Proteins; C040527:lipoxin A4; D050760:Cyclin-Dependent Kinase Inhibitor p27; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516727:Ptpn11 protein, rat