title: TLR4 mediates MAPK-STAT3 axis activation in bladder epithelial cells.
abstract: The role of Toll-like receptor 4 (TLR4) in immune cells is well characterized, but its biological properties in bladder epithelial cells (BECs), especially reciprocal crosstalk between mitogen-activated protein (MAP) kinase pathway and signal transducer and activator of transcription (STAT)3-mediated signal transduction elicited by TLR4 have not been demonstrated so far. The present studies were to demonstrate the signal transduction and inflammatory cytokine response elicited through activation of TLR4 in BECs with a special focus on the crosstalk between the MAPK-pathway and STAT3-mediated signals and its regulatory relevance for the inflammatory response towards lipopolysaccharide (LPS). We selected human bladder cancer T24 cell line in the present study and examined its expression of TLR4 by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression of p38, extracellular signal regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and STAT3 were performed by RT-PCR, quantitative PCR, and Western blotting. Signal transduction was analyzed by Western blotting. Interleukin-6 (IL-6) and interleukin-10 (IL-10) secretion in culture supernatants were tested by human enzyme-linked immunosorbent assay (ELISA) kit. BECs of rat infection in vivo model and patients with cystitis glandularis (CG) were analyzed as described above. Our study demonstrated that TLR4 was significantly upregulated following LPS treatment, with the maximum mRNA expression occurring at 4 h after stimulation. Activation of TLR4 signaling by LPS resulted in phosphorylation of MAPK and STAT pathways and upregulation of IL-10 in dose- and time-dependent manners in T24 cells. Pretreatment of cells with SB203580 (inhibitor of p38) and SP600125 (inhibitor of JNK) attenuated LPS-induced IL-10 expression, whereas it markedly inhibited the STAT3 expression. IL-10 mRNA expression was increased in inflamed lesions compared to noninflamed tissue in rats and patients with CG disease. Our results demonstrate that activation of TLR4 signaling in BECs induces IL-10 expression via activation of p38 and JNK, and the activation of STAT-3 was upregulated. Our data indicated that the reciprocal crosstalk between the MAPK pathway and STAT3-mediated signal transduction forms a critical axis successively activated by LPS in BECs.
authors.affiliation: Department of Ultrasound, Shengjing Hospital of China Medical University, 110004, Shenyang, China, huangying712@163.com.
authors.forename: Huang
authors.initials: H
authors.lastname: Ying
authors.forename: Liu
authors.initials: L
authors.lastname: Da
authors.forename: Shi
authors.initials: S
authors.lastname: Yu-xiu
authors.forename: Xia
authors.initials: X
authors.lastname: Yu
authors.forename: Liu
authors.initials: L
authors.lastname: Li-xia
authors.forename: Xie
authors.initials: X
authors.lastname: Li-mei
authors.forename: Ren
authors.initials: R
authors.lastname: Wei-dong
mesh_terms: D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D045744:Cell Line, Tumor; D003556:Cystitis / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000096:biosynthesis; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000096:biosynthesis / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D008070:Lipopolysaccharides; D020935:MAP Kinase Signaling System; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor / Q000096:biosynthesis / Q000378:metabolism*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D001743:Urinary Bladder / Q000166:cytology / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D000873:Anthracenes; C508609:IL10 protein, human; C508600:IL6 protein, human; D007093:Imidazoles; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011725:Pyridines; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D016753:Interleukin-10; C432165:pyrazolanthrone; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580