MOLECULAR INSIGHTS AND PHYSIOLOGICAL CONSEQUENCES OF ARYL HYDROCARBON RECEPTOR REGULATED PLASMINOGEN ACTIVATOR INHIBITOR-1 EXPRESSION
The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, attenuates liver regeneration in vivo when activated by its prototypical agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) following 70% partial hepatectomy (PH). One reported target of the AhR that may account for suppression of the regenerative response is plasminogen activator inhibitor-1 (PAI-1), which negatively regulates the cleavage and activation of hepatocyte growth factor (HGF) from its latent form in the extracellular matrix. Once activated, HGF signalling through its receptor cMet is a crucial component early in regeneration. Recent studies identified a sequence distinct from the canonical AhR binding site, the ncXRE, which confers TCDD-inducible expression to the PAI-1 promoter. Since the ncXRE shares partial sequence homology with the Kruppel-like factor 6 (KLF6) consensus binding site; I hypothesize that the AhR interacts with KLF6 at the ncXRE, inducing transcription of PAI-1, suppressing HGF processing and its activation of cMet, inhibiting liver regeneration. To test this hypothesis, coimmunoprecipitation on liver nuclear extracts and recombinant proteins confirmed that KLF6 and the AhR interact, likely dependent on the C-terminus transactivating domain of AhR and the DNA binding domain of KLF6. Both proteins bind the ncXRE in vitro and deletion analyses revealed that the N-terminal 27 amino acids of hKLF6 were required for complex formation. Chromatin immunoprecipitation studies demonstrated that the AhR and KLF6 bind to the PAI-1 promoter in vivo. To assess the effects of AhR activation in vivo, C57BL/6 and PAI-1-/- mice were pretreated with TCDD, underwent PH, and liver samples and serum were collected at multiple time points post-PH to monitor PAI-1 expression, HGF processing, and cMet phosphorylation (activation) and DNA synthesis in the liver. I found that PAI-1 transcript and corresponding serum PAI-1 protein levels were markedly increased in TCDD-pretreated C57BL/6 mice, and this rise in PAI-1 levels inversely correlated to HGF processing and cMet phosphorylation. Hepatocytes in the periportal region of PAI-1-/- mice were able to overcome TCDD-mediated suppression of regeneration. The AhR-KLF6 interaction at the PAI-1 promoter, resulting in increased PAI-1 expression and decreased HGF processing and cMet activation, reveals a novel mechanism by which the AhR may contribute to liver homeostasis.