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TLR4 Mediates MAPK–STAT3 Axis Activation in Bladder Epithelial Cells

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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.

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Abbreviations

BEC:

Bladder epithelial cell

BSA:

Bovine serum albumin

CG:

Cystitis glandularis

ERK:

Extracellular signal-regulated kinase

ELISA:

Enzyme-linked immunosorbent assay

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IL:

Interleukin

JNK:

c-Jun NH2-terminal kinase

LPS:

Lipopolysaccharide

MAP kinase:

Mitogen-activated protein kinase

NF-κB:

Nuclear factor-κB

PCR:

Polymerase chain reaction

STAT:

Signal transducer and activator of transcription

TLR:

Toll-like receptor

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Acknowledgments

This project was supported by the Natural Science Foundation of China (30900368) and by the Ministry of Education research fund for the doctoral program of China (20092104120011).

Conflict of interest

None.

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Authors and Affiliations

  1. Department of Ultrasound, Shengjing Hospital of China Medical University, 110004, Shenyang, China

    Huang Ying, Xia Yu, Liu Li-xia, Xie Li-mei & Ren Wei-dong

  2. Department of Orthopaedic Surgery, Shengjing Hospital of China Medical University, 110004, Shenyang, China

    Liu Da

  3. Department of Embryology, China Medical University, Liaoning Shenyang, 110000, China

    Shi Yu-xiu

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  1. Huang Ying
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  2. Liu Da
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  3. Shi Yu-xiu
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  4. Xia Yu
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Correspondence to Huang Ying.

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Ying, H., Da, L., Yu-xiu, S. et al. TLR4 Mediates MAPK–STAT3 Axis Activation in Bladder Epithelial Cells. Inflammation 36, 1064–1074 (2013). https://doi.org/10.1007/s10753-013-9638-7

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