Lipopolysaccharide initiates a bypass feedback loop of epidermal growth factor receptor signaling by HPS70-induced COX-2 in H22 hepatocarcinoma cells

Abstract

Abstract. LPS can induce TACE upregulation via signaling from TLR4-derived EGFR activation in tumor cells. The regulation and activity of TACE have been investigated with the observation that gene expression is upregulated in response to LPS followed by EGFR activation, however, the process remains poorly understood. In this study, we examined the effects of LPS on H22 hepatocarcinoma cells that displayed constitutively active TLR4 expression. Upon TLR4 shRNA transfection into H22 cells, HSP70 expression significantly increased. However, LPS induced early phosphorylation of EGFR in H22 cells, which reached maximum levels within 30 min. Inhibition of TLR4 in H22 cells resulted in a significant rise in both EGFR phosphorylation and TACE upregulation 24 h after exposure to LPS. Exogenous HSP70 also induced rapid phosphorylation of EGFR, upregulated the expression of COX-2 via a signaling pathway that involved TACE-dependent TGF-α release. Furthermore, inhibition of EGFR activation and reduction of COX-2 expression by COX-2 inhibitor prevented HSP70-induced cell invasion in vitro. These findings demonstrate that the biological importance of HSP70/ COX-2 is crucial to the second, but not the first, phase of EGFR phosphorylation in tumor cells. The growth of tumor cells by inserting shRNA plasmid TLR4 combination with COX-2 inhibitor could be effectively reduced in LPS stimulation. We concluded that LPS triggered a bypass feedback loop of EGFR activation and involved HSP70/COX-2 in H22 cells by inhibition of TLR4 and that EGFR phosphorylation is implicated in tumor growth by LPS stimulation. IntroductionChronic inflammation has long been associated with increased incidence of malignancy, and similarities in regulatory mechanisms have been suggested for more than a century. Additionally, supporting tumor cells evade antitumor immunity and the infiltration of innate immune cells, such as macrophages and neutrophils, into tumors promotes tumor development by various mechanisms including increased matrix metalloprotease activities, angiogenesis and vasculature density. Although chronic inflammation exhibits a spectrum of malignant potential behavior similar to most tumors, the molecular mechanisms of tumor progression, growth and invasion have not been fully elucidated. Studies on these mechanisms should provide opportunities to design new molecular therapeutic targets.LPS is a major integral component of the outer membrane of gram-negative bacteria and is one of the most potent stimuli of inflammation. Chronic inflammation promotes neoplastic cell growth, may contribute to neovascularization (1), and is proposed to be an initiating factor in many tumors (2). LPS induces a ligand for TLR4-dependent EGFR phosphorylation, which triggers the MAPK signaling to the nucleus that subsequently causes TACE production. The proposed surfacesignaling pathway responsible for the initial activation of EGFR is based on its demonstration in tumor cells (3).Though initially Hsp70 was thought to be a typical intracellular protein, recent s...