[Study on the molecular expression and regulation of toll pathway in HT-29 cells].
OBJECTIVE
To investigate the changes of TLR4, MD2 and HBD2 in transcription and protein expression levels in HT-29 colon cancer cell line after inducing by cytokines TNF-alpha, IL-1beta, IFN-gamma and LPS, and its relationship with NF-kappaB activation.


METHODS
HT-29 cells were divided into 8 groups, by adding RPMI 1640, TNF-alpha (20 ng/mL), IL-1beta (20 ng/mL), IFN-gamma (20 ng/mL), LPS (50 ng/mL), TNF-alpha (20 ng/mL) + LPS (50 ng/ mL), IL-1beta (20 ng/mL) + LPS (50 ng/mL), IFN-gamma (20 ng/mL) + LPS(50 ng/mL) respectively for intervention. ELISA was applied to detect the IL-8 in the supernatants of each group. The level of TLR4, MD2 and HBD2 mRNA were assayed by RT-PCR. The expressions of TLR4 and NF-kappaB protein of each group were determined by western blot.


RESULTS
IL-8 expressions in supernatant of cytokines and cytokine plus LPS group were higher than that of control (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further augmented IL-8 secretion (P < 0.01). Increased levels of TLR4 and MD2 mRNA in HT-29 cells with Cytokines and cytokine plus LPS stimulating were observed (P < 0.01). The level of HBD2 mRNA were elevated in IL-1beta and LPS plus (TNF-alpha, IL-1beta, IFN-gamma) groups (P < 0.05). Elevated expressions of TLR4 and NF-kappaB protein in HT-29 cells with cytokine and cytokine plus LPS were also observed (P < 0.01). Pre-incubation with cytokines, following by LPS stimulation, HT-29 cells further up-regulated NF-kappaB protein expression (P < 0.05).


CONCLUSIONS
Cytokines (TNF-alpha, IL-1beta, IFN-gamma) can enhance TLR4 and MD-2 expressions and promote the reaction with LPS in intestinal epithelial cells (IEC), which leads to over activity of IEC with commensal bacteria and initiation or aggravation of intestinal inflammation. Inflammatory stimuli (IL-1beta, LPS + TNF-alpha, LPS + IFN-gamma) may induce the transcription and expression of HBD2 gene in IEC, which leads to enhancing intestinal adaptive immune responses and affects the progress of intestinal inflammation.
Chen
Xi
X
Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu 610041, China.
Ouyang
Qin
Q
D016207:Cytokines / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D023083:beta-Defensins / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; C106641:DEFB4A protein, human; D053583:Interleukin-1beta; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D012333:RNA, Messenger; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D023083:beta-Defensins; D007371:Interferon-gamma