Sphingosine kinase 2 plays a critical role in bile acid‐induced cholestatic liver injury via regulating miR‐155 and macrophage activation
Authors: Zhang Yuan , Yang Wilson , Wang Yanyan , Liu Runping , Li Xiaojiaoyang , Zhao Derrick , Wu Jilin , Tai Yunling , Ge Pu , Wang Xuan , Gurley Emily C , Hylemon Phillip B , Zhou Huiping 
Published: April 2020

Primary sclerosing cholangitis (PSC) is a devastating chronic cholestatic liver disease without effective therapy due to a limited understanding of its pathogenesis. The multidrug resistance gene 2‐knockout (Mdr2KO) mice spontaneously develop hepatobiliary injury, fibrosis, and hepatocellular carcinoma (HCC) closely resembling PSC. Previous studies have shown that accumulation of conjugated primary bile acid, such as taurocholic acid (TCA), contributes to the disease progression via activation of sphingosine‐1 phosphate receptor 2 (S1PR2)/sphingosine kinase 2 (SphK2). However, the underlying mechanisms remain to be fully identified. MicroRNAs (miRs) are small non‐coding RNAs that play essential roles in regulating various cellular pathways under both physiological and pathological conditions. The expression level of miR155 is correlated to the activation of inflammation and hepatic fibrosis. The aim of this study is to examine the role of SphK2 in bile acid‐induced cholestatic injury.

Methods

Wild type (WT) and Mdr2KO mice (age and gender‐matched) were treated with a specific Sphk2 inhibitor ABC294640 (50 mg/kg) by daily gavage for 4 weeks. Blood and liver tissues were collected. The serum levels of ALT, AST, and ALP levels were measured using the commercial kits. Liver tissues were processed for H&E and Masson’s trichrome staining, immunohistochemistry (IHC), and isolation of total RNA. The hepatic miR155 expression levels were analyzed using Taqman microRNA assays. The mRNA levels of the key genes involved in hepatic inflammation, fibrotic injury, and bile acid metabolism were measured by real‐time RT PCR. The protein levels of S1PR2 and SphK2 were determined by Western Blot analysis. The effect of TCA on miR155 expression in mouse cholangiocyte was determined.

Results

Compared to the gender/age‐matched WT mice, Mdr2 KO mice developed severe hepatobiliary injury as indicated by the upregulation of ALP, AST, and ALT, activation of the inflammatory response, and increase of liver fibrosis. The expression levels of S1PR2, SphK2, and miR155 were significantly increased in Mdr2KO mice. Treatment of ABC294640 significantly reduced hepatic macrophage infiltration, fibrotic liver injury, and expression of inflammatory mediators, including IL‐1β, MCP‐1, IL‐18, and IL‐6. In cultured mouse cholangiocytes, TCA significantly increased miR155 expression.

Conclusion

Bile acid‐induced activation of S1PR2/SphK2 may play critical roles in cholestatic liver fibrosis by the upreguation of miR155 and inflammatory mediators. Targeting SphK2 represents a potential therapeutic strategy for the treatment of the cholestatic liver injury.

Support or Funding Information

This work was supported by VA Merit Award I01BX004033 and 1I01BX001390, VA Career Scientist Award IK6BX004477 and National Institutes of Health Grant R01 DK104893 and R01DK‐057543