Disruption of Estrogen Receptor Alpha in Rats Results in Faster Initiation of Compensatory Regeneration Despite Higher Liver Injury After Carbon Tetrachloride Treatment

Abstract

Estrogen receptor alpha (ESR1) is 1 of the 2 intracellular receptors for estrogen and is expressed by hepatocytes in the liver. The role of ESR1 in the regulation of toxicant-induced liver injury and compensatory regeneration is not completely clear. We investigated the role of ESR1 in liver regeneration after carbon tetrachloride (CCl₄)-induced liver injury using wild type (WT) and ESR1 knockout (ESR1-KO) rats. Adult female WT and ESR1-KO rats were treated with 1 mL/kg CCl₄ and euthanized over a time course of 0 to 48 hours. Liver injury measured by serum alanine amino transaminase, and histopathological analysis showed significantly higher liver injury in ESR1-KO as compared to WT rats. Hematoxylin and eosin staining revealed 2-fold higher necrosis and significant inflammatory cell infiltration in ESR1-KO rats. Chloracetate esterase staining revealed higher neutrophil infiltration in ESR1-KO rat livers. Interestingly, proliferating cell nuclear antigen immunohistochemistry showed that in spite of 2-fold higher liver injury, the ESR1-KO rats had equal liver regeneration as compared to WT rats. Western blot analysis of cyclin D1 and phosphorylated Rb, proteins involved in the initiation of the cell cycle, was significantly higher at all time points in ESR1-KO rats. Further analysis revealed faster activation of canonical Wnt/β-catenin and NF-κB signaling in ESR1-KO rats characterized by higher activated β-catenin and phosphorylated p65 at 12 hours after CCl₄ treatment. Taken together, these data indicate that ESR1-mediated signaling inhibits liver regeneration by downregulation of Wnt signaling resulting in lower cyclin D1 activation after chemical-induced liver injury.

Keywords: PCNA; estrogen; necrosis; proliferation; β-catenin.

Figure 1

CCl₄-induced liver injury in WT and ESR1-KO rats. (A) Representative photomicrographs (400x) of H&E stained liver sections of WT and ESR1-KO rat liver at 12, 24 and 48 hr after CCl₄ treatment. Black arrowheads point to centrilobular necroinflammatory foci. Bar graphs showing serum ALT (B) and (C) percent necrosis in WT and ESR1-KO rats after CCl₄ treatment. * denotes significant difference at P≤0.05 between KO and WT, # denoted significant difference at P≤0.05 at that time point from the 0 hr of the respective genotype.

Figure 2

Increased neutrophil infiltration in ESR1-KO rat livers after CCl₄ treatment. (A) Representative photomicrographs (400x) of chloracetate esterase (CAE) stained liver sections of WT and ESR1-KO rat liver at 12 and 24 hr after CCl₄ treatment. Yellow arrowheads point to neutrophils. (B) Line graph showing neutrophil count of CAE stained liver section of WT and ESR1 rat livers at various time points. * denotes significant difference at P≤0.05 between KO and WT, # denoted significant difference at P≤0.05 at that time point from the 0 hr of the respective genotype.

Figure 3

Accelerated compensatory cell proliferation in ESR1-KO rats after CCl₄ treatment. (A) Western blot analysis of cyclin D1, Cyclin E, CDK4, and phosphorylated Rb proteins at 12, 24 and 48 hr after CCl₄ treatment performed using total liver cell extracts of WT and ESR1-KO rat livers. (B–C) Densitometric analysis of Cyclin D1 and pRb blots. (D) Representative photomicrographs of PCNA immunohistochemistry (400x) performed on WT and ESR1-KO rat livers at 24 and 48 hr after CCl₄ treatment. Black arrowheads point to cells in S-phase of cell cycle (E) bar graph showing number of PCNA positive cells. * denotes significant difference at P≤0.05 between WT and KO.

Figure 4

Increased β-catenin activation in ESR1-KO livers after CCl4 treatment. (A) Western blot analysis of total and activated β-catenin, phosphorylated nuclear p65; and total and phosphorylated GSK3β proteins at 12, 24 and 48 hr after CCl₄ treatment performed using total liver cell extracts of WT and ESR1-KO rat livers. (B) Bar graphs showing densitometric analysis of the active β-catenin western blot. (C) Bar graphs showing densitometric analysis of the phosphorylated GSK3β western blot. * denotes significant difference at P≤0.05 between WT and KO.