Lipoxin A4 inhibits TNF-α-induced production of interleukins and proliferation of rat mesangial cells

Abstract

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). 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. 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. 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.