E. Chu1, T. Liu1, N. Vanli1, G. F. Hu1, J. Yoo1 1Tufts Medical Center,Colon And Rectal Surgery,Boston, MA, USA
Introduction: The myofibroblast is an important stromal cell of the gastrointestinal (GI) tract that is a target of tumor necrosis factor-alpha (TNF-α ), a potent pro-inflammatory cytokine that has been strongly implicated in the pathophysiology of colitis-associated cancer. Crosstalk mechanisms are known to exist between TNF-α and other pro-inflammatory mediators, including multiple G protein-coupled receptor (GPCR)-mediated agonists, that amplify inflammatory signaling. However, the mechanism has not been previously determined. Angiogenin (ANG) is a 14-kDa member of the ribonuclease superfamily that was the first tumor-derived angiogenesis protein. Like TNF-α , ANG levels are elevated in patients with inflammatory bowel disease (IBD) and colorectal cancer. However, the role of ANG on inflammatory mediator crosstalk in the myofibroblast is unknown.
Methods: The human colonic myofibroblast cell line 18Co was grown to confluence on 35×10 mm cell culture dishes and was used from passages 8-14. 18Co cells were exposed to TNF-α (10 ng/ml) and bradykinin (100nM) for varying times. ANG was quantified from the supernatant of serum-starved 18Co cells by ELISA. The monoclonal antibody 26-2F was used to block the activity of ANG. The expression of cyclo-oxygenase-2 (COX-2) was assessed by Western Blot.
Results:We have previously reported that 18Co cells exposed to both TNF-α and the pro-inflammatory GPCR bradykinin (BK) lead to the synergistic expression of COX-2, evident after 4 h (P<0.05). To determine whether ANG was involved in this process, we first measured ANG levels in the cell culture supernatant of 18Co cells by ELISA. 18Co cells secrete high levels of ANG (265.5 ± 4.7 pg/ml in serum-free media over 24 h). Exposure of 18Co cells to TNF-α (10ng/ml) led to a rapid (4 h, 127.8 ± 9.7 pg/ml, P<0.05) and sustained (24 h, 124.6 ± 25.1 pg/ml, P<0.05) reduction in the concentration of ANG in the supernatant, corresponding to an uptake of ANG by these cells. The anti-ANG monoclonal antibody 26-2F, which neutralizes the activity of ANG, inhibited the synergistic expression of COX-2 induced by TNF-α and BK at 4 h (P<0.05).
Conclusion:TNF-α stimulates ANG uptake by the myofibroblast, and inhibition of ANG blocks synergistic COX-2 expression induced by TNF-α and BK. Crosstalk signaling between TNF-α and BK appears to be mediated by ANG. Angiogenin may play an important role in the regulation of COX-2 expression in the setting of inflammation, and may be a novel therapeutic target for the management of colitis-associated cancer.
