CXCL6-EGFR-induced Kupffer cells secrete TGF-β1 promoting hepatic stellate cell activation via the SMAD2/BRD4/C-MYC/EZH2 pathway in liver fibrosis

Liver fibrosis is the excessive accumulation of extracellular matrix proteins in response to the inflammatory response that accompanies tissue injury, which at an advanced stage can lead to cirrhosis and even liver failure. This study investigated the role of the CXC chemokine CXCL6 (GCP-2) in liver fibrosis. The expression of CXCL6 was found to be elevated in the serum and liver tissue of high stage liver fibrosis patients. Furthermore, treatment with CXCL6 (100 ng/mL) stimulated the phosphorylation of EGFR and the expression of TGF-beta in cultured Kupffer cells (KCs). Although treatment with CXCL6 directly did not activate the hepatic stellate cell (HSC) line, HSC-T6, HSCs cultured with media taken from KCs treated with CXCL6 or TGF-beta showed increased expression of alpha-SMA, a marker of HSC activation. CXCL6 was shown to function via the SMAD2/BRD4/C-MYC/EZH2 pathway by enhancing the SMAD3-BRD4 interaction and promoting direct binding of BRD4 to the C-MYC promoter and CMY-C to the EZH2 promoter, thereby inducing profibrogenic gene expression in HSCs, leading to activation and transdifferentiation into fibrogenic myofibroblasts. These findings were confirmed in a mouse model of CCl4-induced chronic liver injury and fibrosis in which the levels of CXCL6 and TGF-beta in serum and the expression of alpha-SMA, SMAD3, BRD4, C-MYC, and EZH2 in liver tissue were increased. Taken together, our results reveal that CXCL6 plays an important role in liver fibrosis through stimulating the release of TGF-beta by KCs and thereby activating HSCs.