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.

Interleukin-10 Superfamily and Cancer

“…In addition, Chang et al (2012) revealed that the production of IL-10 in human monocyte-derived DCs from patients with gastric cancer is dependent on p38 mitogen-activated protein kinase (MAPK) signaling and on NF-κB activation. Consistent with previous reports, Ying et al (2013) demonstrated that the activation of MAPK, c-Jun, N-terminal kinase (JNK -Janus kinase), and signal transducer and activator of transcription 3 (STAT3) was associated with an increased IL-10 production in bladder epithelial cells. Finally, a recent study by Sanda et al (2013) revealed that T cell acute lymphoblastic leukemia cell lines and tissue samples showed the activation of a pathway involving TYK2, STAT1, and bcl-2, an antiapoptotic protein associated with numerous cancers (Barille-Nion et al, 2012).…”

“…The activation of IL-10 expression is known to be mediated by tolllike receptor (TLR)2, TLR3, and TLR4 in the presence of an infectious agent or pathogen-associated molecular patterns (PAMPs) (Bogunovic et al, 2011;Chang et al, 2012;Ying et al, 2013). It was found that IL-10 promotes tumor aggressiveness through the upregulation of CIP2A, an oncoprotein correlated with an accelerated cell growth and cancer progression .…”

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

“…Further supporting the potential role of S100A8/A9 proteins in metatases, high expression of their receptor, RAGE, was present not only in metastatic Capan1, but also in the primary MiaPaCa2 cells, which cause a liver metastases rate overlapping that of Capan1 [47]. The other S100A8/A9 receptor, TLR4 [29], is reported to activate STAT3 [48]. The finding that this signal transducer was constitutively activated in all PDAC cell lines, supports the hypothesis that it plays role in this cancer type [49]; however the fact that it was not induced by the S100 studied molecules, suggests that they act mainly through RAGE engagement.…”

The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism

“…Because STAT3 can be activated by cytokines, including IL-6 in the tumor microenvironment, potential paracrine effects are likely to occur in vivo. Other factors are known to contribute to BCL-XL expression (41), and STAT3 can also be regulated by multiple signaling pathways, including G proteincoupled receptor and Toll-like receptor 4 (42,43). Of note, unphosphorylated STAT3 represents the bulk of total STAT3 and has been described to be oncogenic, in part, by modulating p-STAT3 gene targets with pro-oncogenic or pro-survival effects (c-Myc, c-fos, and BCL-XL as well as genes such as RANTES, cdc2, cyclin B1, IL-6, IL-8, Met, and M-RAS) through its cooperative binding with NF-B to specific B elements and/or other novel mechanisms (44).…”