Development of Sonic hedgehog (SHH) transgenic mouse model to investigate its role in hepatic fibrosis and cancer

Abstract

Liver fibrosis is a major health problem worldwide. Nevertheless, pathogenesis and underlying mechanisms of liver fibrosis are not fully understood. Sonic hedgehog (SHH) signaling regulates critical cell fate decisions and modulates wound healing responses in adult tissue, including the liver. The hedgehog signaling pathway is found frequently activated in liver injury.To investigate the role of persistent activation of hedgehog signaling in the pathogenesis of liver fibrosis and cirrhosis, we developed the SHH transgenic mouse models for liver fibrosis using a hydrodynamic injection method and the Sleeping Beauty Transposon System.Transposon vectors encoding SHH were constructed. Plasmids encoding the Sleeping Beauty transposase and SHH-encoding transposons were mixed and injected into the lateral tail veins of 6-week-old C57BL/6 mice.The mice were observed at 2 months and 6 months post hydrodynamic injection. The development of liver fibrosis was observed histologically.Liver fibrosis was induced by the stable expression of SHH. Development of liver fibrosis was determined via Masson’s trichrome and picro-sirius red staining. The histological examination showed that the fibrotic area in the control group was less than 2% of the total hepatic area examined, whereas the degree of hepatic fibrosis in the SHH group increased more than 6-folds (12.5%). Immunohistochemical analysis revealed nuclear localization of Gli1 and Gli2, the downstream transcription factors of SHH signaling. Expression levels of fibrosis-related genes significantly increased compared to the control group, such as type I collagen α1, α-smooth muscle actin, MMP9, TGF-β1, desmin, and platelet-derived growth factor (PDGF). Western blotting for collagenIαI, α-SMA, desmin, TIMP1, MMP2, MMP9, TGF-β1 and phospho-Smad2 showed similar results. The levels of serum AST, ALP, and ALT in SHH group were

significantly higher than those in the control, suggesting SHH-induced liver injury.Persistent expression of SHH in the liver failed to induce HCC until 11 months after hydrodynamic injection. To accelerate carcinogenesis, we employed a transgenic mouse model expressing an activated RAS and a gain-of-function P53 mutant, called 2PLEASE mouse. The 2PLEASE mice were hydrodynamically transfected with transposons encoding SHH and GFP (a control) and then analyzed for tumor initiation and progression. In 2PLEASE+SHH group, tumor incidence rate (42.9%) was not significantly different from that in 2PLEASE+EGFP group. However, histological examination of tumors from the 2PLEASE+SHH group revealed more malignant phenotypes compared to those from 2PLEASE+EGFP group; Tumors of 2PLEASE+SHH were diagnosed as HCC, while 2PLEASE+EGFP tumors were hepatic adenoma, suggesting the role of SHH in tumor progression.Our data indicated that over-expression of SHH in the mouse liver promoted liver fibrogenesis, suggesting its role in the development of liver fibrosis and genetic interaction with other pro-fibrogenic signaling such as TGF-β1 and PDGF signaling.Models expressing other fibrogenic genes can be generated using a similar approach which will contribute to a better understanding of the molecular pathogenesis of human liver fibrosis.