Regulation of hepatocyte proliferation by ligand-activated aryl hydrocarbon receptor

Abstract

Ligands of the aryl hydrocarbon receptor (AhR), a basic helix-loop-helix transcription factor, induce hepatocellular carcinomas and alter hepatocyte proliferation in rodents. In the studies described in this thesis, primary hepatocytes and the rat hepatoma 5L cell line were used as models to characterize the actions of TCDD on hepatocyte proliferation. TCDD inhibited DNA synthesis in primary hepatocytes and 5L cells, as measured by (\sp3H) thymidine incorporation into DNA. 5L cell proliferation, as determined by total cell number, was also inhibited. Inhibition of DNA synthesis and cell proliferation were dose-dependent and stereospecific responses, supporting the involvement of AhR. Further studies were carried out to elucidate the mechanisms for this response. TCDD suppressed DNA synthesis induced by the hepatocyte mitogens epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor $\alpha$ (TGF$\alpha).$ Characterization of the EGF mitogenic pathway revealed that TCDD did not alter the specific binding of EGF, or change the levels of EGF receptor protein. TCDD-dependent inhibition of DNA synthesis occurred independently of transforming growth factor-\beta\sb1 (TGF-\beta\sb1). Cell cycle analysis of 5L cells indicated that TCDD arrested cells in G\sb1 of the cell cycle (91% TCDD-treated versus 49% control cells in G\sb1 after 24 h treatment). This arrest is associated with the maintenance of the G\sb1 regulatory protein, Rb, in a hypophosphorylated state. Accumulation of Rb cleavage products was also observed. Rb hypophosphorylation is not due to changes in the levels of cdk4 or of its catalytic partners, cyclin D1 or D3; however, TCDD increased the levels of the cdk inhibitor p27\rm\sp{Kip1}. In whole cell extracts, AhR immunoprecipitates with cdk4 in the absence of ligand, whereas in the presence of TCDD this interaction is not detected or is attenuated. This interaction is detected in nuclear extracts, but the ligand-dependency is inconclusive. AhR co-immunoprecipitates with Rb with an apparent ligand-dependent increase in the AhR/Rb interaction. These studies demonstrate that TCDD, the prototype AhR ligand, induces G\sb1 arrest and Rb hypophosphorylation. TCDD-dependent regulation of G\sb1 progression may result from elevated levels of p27\rm\sp{Kip1}, from ligand-dependent changes in the interactions of AhR with cdk4 and Rb, or both