Author ORCID Identifier

https://orcid.org/0000-0001-6299-8062

Date Available

8-3-2021

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Pharmacology and Nutritional Sciences

First Advisor

Dr. Brett T. Spear

Abstract

The liver is the largest internal organ in mammals and responsible for carrying out various processes, including lipid and carbohydrate metabolism, detoxification of chemicals, and production of serum proteins. Liver damage, which can be caused by a variety of agents including viral infection, environmental toxins, alcohol and excessive dietary fats, can cause dysregulation of these critical functions, leading to worsening liver pathophysiology and impacting health. However, the liver has the remarkable ability to regenerate when damaged. Hepatocytes, which comprise a majority of liver cells, are relatively quiescent under healthy conditions. Upon injury, remaining hepatocytes can proliferate to recover from liver damage while performing their metabolic functions. Unfortunately, persistent injury can progressively lead to hepatitis, fibrosis, and ultimately end-stage liver disease, cirrhosis, and liver cancer. Overall, liver disease is the fifth leading cause of death worldwide and a growing healthcare problem in the United States and other western countries. A better understanding of liver regeneration could be used clinically to treat a wide range of liver diseases.

Studies in different mouse strains have provided genetic models to understand aspects of liver gene regulation and liver disease. Two regulators of the liver-derived serum protein alpha-fetoprotein (AFP), alpha-fetoprotein regulator 1 and 2 (Afr1 and Afr2), have been implicated to play a role in liver disease and regeneration. AFP is normally expressed in the fetal liver, silenced at birth, and reactivated in liver regeneration and liver cancer. Based on the unusually high AFP expression in adult BALB/cJ mice, our lab identified Zinc Fingers and Homeoboxes 2 (Zhx2) as the gene responsible for the Afr1 trait. BALB/cJ mice contain a natural mutation in the Zhx2 gene. Additional studies have shown that Zhx2 is involved in several liver diseases, including diet-induced fatty liver disease and hepatocellular carcinoma. In addition to these traits, BALB/cJ mice have been shown to have increased liver fibrosis after chronic treatment with the hepatotoxin carbon tetrachloride (CCl4). The locus responsible for this trait, called Hepatic fibrosis 1 (Hfib1), was mapped to the same region of Chromosome 15 (Chr15) as Zhx2, but the Hfib1 gene has not been identified.

During liver regeneration after acute treatment with CCl4, it was found that liver AFP mRNA levels were much higher in C3H/HeJ mice than in C57BL/6J mice. The locus that controls this strain-specific difference in AFP reactivation was called Afr2; C3H/HeJ and C57BL/6 mice are thought to contain the Afr2a and Afr2b alleles, respectively. The Afr2 locus has been mapped to Chr2, but the Afr2 gene has not been identified.

This dissertation tested the hypothesis that Zhx2 is responsible for the Hfib1 trait in BALB/cJ mice. Using BALB/cJ mice and C57BL/6J mice with a targeted mutation in the Zhx2 gene, my data indicates that Zhx2 is not responsible for the Hfib1 trait. Mice with low Zhx2 expression did not have more significant inflammation, liver damage, or fibrosis than mice with wild-type Zhx2 levels. These data suggest that another gene, presumably within the same region as Zhx2, is responsible for the Hfib1 phenotype in BALB/cJ mice. I also analyzed the Afr2 trait across several different strains of mice. My studies indicate that the strains 129X1/SvJ, C3H/HeJ, and DBA/2J contain the Afr2a allele, whereas mice in the C57 lineage (C57BL/6J, C57BL/6N, C57BL/10) contain the Afr2b allele. I also demonstrate that F1 offspring of 129X1/SvJ mice and C57BL/6J mice display an intermediate AFP reactivation phenotype, suggesting that the Afr2a and Afr2b alleles are co-dominant. These data provide the framework for future studies to identify the Afr2 gene. Taken together, my results indicate that regulators of gene expression within the liver, as defined by differences within mouse strains, can provide insight into liver disease and regeneration.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.352

Funding Information

This study was supported by the National Institutes of Health Pharmacology and Nutritional Sciences: Multidisciplinary Approaches for Metabolic Disease grant (T32 DK007778) from 2017 to 2019.

This study was supported by the National Institutes of Health RORalpha and Hepatic Zonal Regulation grant (5R01DK074816) from 2018 to 2021.

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