Hepatocyte Rho-Associated Kinase Signaling is Required to Survive Liver Injury

Rho-associated kinases 1 and 2 (ROCK1 and ROCK2) are downstream effectors of RhoA and regulate various critical cell functions such as actomyosin contractility, apoptosis, proliferation, and cell migration. Some studies utilizing inhibitors suggested that ROCK inhibition could serve as a treatment for liver fibrosis. However, more investigation is needed to understand the role of ROCK signaling in hepatocytes with injury in vivo. Rock1fl/fl, Rock2fl/fl, and Rock1fl/fl;Rock2fl/fl mice were injected with adeno-associated virus serotype 8 (AAV8)-thyroid hormone-binding globulin (TBG)-Cre for targeted gene deletion in hepatocytes, or injected with AAV8-TBG-Null to generate littermate controls (WT). Mice were then given a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce cholestatic liver injury. Serum liver function tests, liver histology, and RNAseq analyses were performed. Mice deficient in both ROCK1 and ROCK2 (DKO) showed early mortality, decreased hepatic synthetic function, and increased hepatocellular injury compared to WT. Mice deficient in ROCK1 only (R1KO) or ROCK2 only (R2KO) demonstrated minimal differences compared to WT, indicating that the two ROCK isoforms had redundant roles in this disease model. Compared to WT, histologic analysis showed that DKO mice had significantly greater loss of liver parenchyma. RNAseq analysis indicated upregulation of apoptotic and inflammatory genes in DKO compared to WT liver. Immunostaining showed an increase in p21 and cleaved caspase 3 in DKO mice compared to WT, indicating that deletion of ROCK potentially leads to p21 overexpression and cell death. These results demonstrate that hepatocyte ROCK signaling is essential in promoting cell survival in the setting of liver cholestatic injury.