INTERFERON-γ UP-REGULATES TOLL-LIKE RECEPTOR 4 AND COOPERATES WITH LIPOPOLYSACCHARIDE TO PRODUCE MACROPHAGE-DERIVED CHEMOKINE AND INTERFERON-γ INDUCIBLE PROTEIN-10 IN HUMAN BLADDER CANCER CELL LINE RT4

Abstract

Previously we have reported that RT4, a well differentiated human bladder cancer line, increases the expression of macrophage derived chemokine (MDC) and interferon (IFN)-gamma-inducible protein-10 (IP-10) in response to IFN-gamma and tumor necrosis factor (TNF)-alpha. In this study we examined the signal mechanisms for inducting these 2 chemokines in RT4 cells by lipopolysaccharide (LPS), IFN-gamma and TNF-alpha. MDC and IP-10 expression was evaluated by sandwich enzyme-linked immunosorbent assay. Signal molecule activation was examined by Western blotting and electrophoretic mobility shift assay. The expression of toll-like receptor (TLR)-4 was analyzed by reverse transcriptase-polymerase chain reaction and flow cytometry. LPS did not induce RT4 cells to produce IP-10 and MDC. However, LPS plus IFN-gamma synergized the productions of the 2 chemokines. IFN-gamma up-regulated the expression of TLR-4, which is an LPS binding receptor. Although LPS and IFN-gamma alone marginally activated nuclear factor (NF)-kappaB but not AP-1, LPS plus IFN-gamma augmented NF-kappaB and AP-1. Specific inhibition of NF-kappaB and AP-1 pathways decreased the production of MDC and IP-10. Extracellular regulated kinase (ERK)1/2, an upstream signal of AP-1, was also responsive to LPS and/or IFN-gamma. TNF-alpha also activated NF-kappaB, AP-1 and ERK1/2. However, TNF-alpha plus IFN- gamma was associated with the activation of NF-kappaB but not of AP-1/ERK1/2 for the induction of MDC and IP-10. IFN-gamma enhances LPS for the induction of MDC and IP-10 through up-regulation of TLR-4, and the signal pathways of NF-kappaB and AP-1/ERK1/2. This mechanism may help us understand inflammatory responses of the bladder to localized bacterial infection.