HMGB2: A Novel Regulator and Potential Therapeutic Target for Liver Fibrosis

Abstract

Background and AimHigh mobility group box 2 (HMGB2) is a member of the non‐histone chromosomal high mobility group protein family. Liver fibrosis is characterized by the excessive deposition of the extracellular matrix proteins largely produced by activated hepatic stellate cells (HSCs). The current study aimed at elucidating a novel functional role of HMGB2 in hepatic fibrogenesis.MethodsLiver fibrosis was induced by CCl4 in C57BL/6J mice. Overexpression of Hmgb2 in liver was produced by AAV8‐Hmgb2, whereas loss of function studies were performed in HMGB2−/− mice. HMGB2 inhibitor Inflachromene (ICM) and liposome‐shHMGB2 (lipo‐shHMGB2) were used to reverse liver fibrosis. Other methods: liver histology, immunohistochemistry (IHC), qPCR, Western blots, Co‐immunoprecipitation (Co‐IP), and primary stellate cell (HSC) culture.ResultsHepatic HMGB2 expression was high during the embryonic stage but its level drastically decreased in adult liver, as revealed by RNA‐seq. However, the HMGB2 gene was reactivated in CCl4‐induced fibrotic liver in mice and in human fibrotic and cirrhotic liver. Overexpression of HMGB2 initiated fibrogenesis and augmented the effect of CCl4 as determined by Masson Trichrome staining. In contrast, liver fibrosis was prevented in HMGB2−/− mice. Plasma ALT levels were elevated in AAV‐HMGB2 mice and by CCl4 and were diminished in HMGB2−/− mice. Major fibrosis markers were markedly activated by HMGB2. HMGB2 protein expressed by AAV8‐HMGB2 was mainly observed in hepatocytes and HSCs, whereas the majority of endogenous HMGB2 protein induced by CCl4 was found in HSCs. Primary HSCs were culture‐activated as monitored for morphologic changes that are characteristic of transdifferentiation as well as induction of GFAP. As noted, HMGB2−/− HSCs showed impaired transdifferentiation and diminished activation of α‐SMA, AKT and ERK signaling. We introduced lipo‐shHMGB2 to knockdown the endogenous HMGB2 induced by CCl4 and observed a marked reduction in liver fibrosis. Intriguingly, mice treated with a small molecule inhibitor of HMGB2 ICM showed improved liver gross morphology and were prevented from developing liver fibrosis. This was accompanied by the decreased plasma ALT levels and diminished HMGB2 protein. Treatment of ICM in LX2 cells induced HMGB2 protein cytoplasmic localization and degradation, which was associated with its decreasing CCl4‐induced HMGB2 phosphorylation status.ConclusionHMGB2 is a novel regulator of liver fibrosis. Diminishing HMGB2 expression by ICM or lipo‐shHMGB2 provides a promising therapeutic strategy to prevent or reverse liver fibrosis.All authors have nothing to disclose.Support or Funding InformationGrant support: L.W. is supported by NIH R01DK104656, R01DK080440, R01ES025909, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634, and P30 DK34989 (Yale Liver Center, Michael Nathanson).