The roles of TM4SF5 in the promotion of fibrotic and tumorigenic potentials of hepatocytes

Abstract

Chronic injury and inflammation causes liver fibrosis, through a process involving epithelial–mesenchymal transition (EMT), which is characterized by excessive accumulation of extracellular matrix proteins such as collagen [1-3]. Fibrosis can eventually lead to cirrhosis, liver failure, and the development of hepatocellular carcinoma (HCC). TM4SF5 (transmembrane 4 L6 family member 5) is expressed at much higher levels in liver tumours than in normal hepatic tissues [4]. TM4SF5 is a transmembrane glycoprotein that has four transmembrane domains

its N- and C-terminal tails are located in the cytosol [5]. TM4SF5 expression causes morphological changes (with aberrant actin reorganization) and EMT [4], mediates aberrant growth in multilayers, accelerates G1-to-S phase progression [4,6] and enhances cellular migration and invasion [7]. In addition, TM4SF5 can form massive tetraspanin web structures (Teraspanin-enriched microdomain, TREM) by forming complexes with other tetraspanins or cell adhesion molecules, such as integrins, and can play a role in regulation of matastasis. However, it is not known TM4SF5 roles during liver fibrosis/cirrhosis and how TM4SF expression is regulated. Also, any hierarchy among teraspanins has not been reported. In this study, I explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal features, using human and mouse normal hepatocytes and animal model. And then, I examined the correlations between TM4SF5 and other teraspanins ( CD151 and CD63 ) using TM4SF5- expressing and -none-expressing cells. First, Using a CCl4-mediated mouse liver in vivo model, I examined whether TM4SF5 is expressed during liver fibrosis mediated by CCl4 administration and whether treatment with anti-TM4SF5 reagent blocks the fibrotic liver features. In the CCl4-mediated mouse liver model, TM4SF5 was expressed during the liver fibrosis mediated by CCl4 administration and correlated with α-smooth muscle actin expression and collagen I deposition in fibrotic septa regions. Interestingly, treatment with anti-TM4SF5 reagent blocked the TM4SF5-mediated liver fibrotic features. These results suggest that TM4SF5 expression is induced by fibrotic processes during chronic liver injuries. TM4SF5 is thus a candidate target for prevention of liver fibrosis following chronic liver injury. I also explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. I found that TGFβ1-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR pathway. Inhibition of EGFR activity following TGFβ1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFβ1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFβ1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features. Next, I investigated the relationship between TM4SF5-positive TERMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumorsuppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface. Altogether, this study reveals that TM4SF5 expression is induced by fibrotic processes during chronic liver injuries and TGFβ1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features. Thus, TM4SF5 induction may be involved in the development of liver pathologies. And TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins (CD151 and CD63) on the cell surface. Taken together, TM4SF5 could provide a promising therapeutic target for prevention and treatment of liver malignancies.

-----------------------------------------------------

Keywords : tetraspanin, TM4SF5, liver fibrosis, EMT, TGFβ1, anti- TM4SF5, cytokine, EGFR, tetraspanin web, CD151, CD63