A Mutation in the Gene Encoding the Transcription Factor Zinc Fingers and Homeoboxes 2 (Zhx2) is Responsible for Increased Liver Fibrosis in BALB/cJ Mice

Abstract

Liver fibrosis is a consequence of persistent liver injury that can result from many insults, including exposure to natural and man‐made environmental hepatotoxins, viral infection, excessive alcohol consumption, and non‐alcoholic fatty liver disease. Carbon tetrachloride (CCl4) is an environmental toxin that can be metabolized into highly reactive toxic radicals by hepatic enzymes, primarily cytochrome P‐450 2E1 (Cyp2E1), that can damage proteins, lipids, DNA and other macromolecules. Chronic CCl4 exposure to mice injures hepatocytes and is a well‐established experimental model for human liver fibrosis. Our understanding of environmental factors influencing liver fibrosis far exceeds our knowledge of genetic factors. However, human studies provide strong evidence the fibrosis has a robust heritable component, and several genes associated with fibrosis and other liver diseases have been identified. Studies in genetically tractable organisms, including mice, complement human studies and continue to identify genes that contribute to liver disease. Moreover, mice provide experimental systems to elucidate disease mechanisms. Previously, a Quantitative Trait Locus (QTL) analysis identified a region on Chromosome 15 called hFib1 that contributed to the high liver fibrosis phenotype observed in BALB/cJ mice when given CCl4. The region of this QTL contains the gene encoding the transcription factor Zhx2, which we showed previously to be mutated in BALB/cJ mice (called Zhx2Afr1), but not in other BALB/c substrain. Furthermore, our studies indicate that Zhx2 controls the expression of hepatic genes and influences the extent of liver damage in mice maintained on a high‐fat diet. These findings led us to hypothesize that the increased liver fibrosis phenotype seen in BALB/cJ mice treated with CCl4 was due to reduced Zhx2 expression. To test this, BALB/cJ mice were crossed with C57BL/6 mice that are heterozygous for a null Zhx2 allele (Zhx2WT/KO) generated by gene targeting to obtain male and female mice that expressed (Zhx2WT/Afr1) or did not express (Zhx2KO/Afr1) Zhx2. Mice at 8 weeks of age were treated with 0.5mL/kg of 1:10 CCl4 in mineral oil or mineral oil alone twice weekly for six weeks. Forty‐eight hours after the last injection, plasma and liver were harvested for further analysis. Formalin‐fixed liver sections stained for Hematoxylin and Eosin, Masson Trichrome, and Sirius Red showed increased damage and fibrosis in the livers of the CCl4 –treated male and female Zhx2KO/Afr1 mice compared to the CCl4 –treated Zhx2WT/Afr1 mice. RT‐qPCR analysis indicated that fibrosis and inflammatory markers were altered in these mice in comparison to the control. These findings support our hypothesis that Zhx2 is the causative gene for the hFib1 phenotype. Future studies will test whether Zhx2 overexpression can block or possibly reverse liver fibrosis and identify pathways and cell types (hepatocytes, Kupffer cells, stellate cells) that are impacted by Zhx2.Support or Funding InformationR01DK074816P30 GM127211