Browsing by Subject "liver regeneration"
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Item MOLECULAR INSIGHTS AND PHYSIOLOGICAL CONSEQUENCES OF ARYL HYDROCARBON RECEPTOR REGULATED PLASMINOGEN ACTIVATOR INHIBITOR-1 EXPRESSION(2013-06-03) Wilson, Shelly; Elferink, Cornelis; Barton, Michelle; Cicalese, Luca; Boor, Paul; Papaconstantinou, JohnThe 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.Item PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy(2008-11-25) Lindsey Nicole Jackson; B. Mark Evers, M.D.; Sundararajah Thevananther, Ph.D.; Kathleen O'Connor, Ph.D.; Frank C. Schmalstieg, M.D., Ph.D.; Cornelis Elferink, Ph.D.Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85Ą regulatory and a p110Ą catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In these studies, I used the potent PI3K inhibitor, wortmannin, and small-interfering RNA (siRNA) targeting the p85Ą and p110Ą subunits to determine if total or selective PI3K inhibition would abrogate the proliferative response of the liver following resection. After partial hepatectomy in mice, there is an increase in PI3K activity; total PI3K blockade with wortmannin, and selective inhibition of p85Ą or p110Ą with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest timepoints (ie, 48h and 72h). Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85Ą or p110Ą, as reflected by a paucity of F4/80+ cells present by immunohistochemistry (IHC). Additionally, PI3K inhibition led to an aberrant hepatocyte architecture characterized by vacuolization, lipid deposition, and glycogen accumulation. My data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, perhaps by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.Item PI3K/Akt activation is critical for early hepatic regeneration after partial hepatectomy(2008-11-25) Lindsey Nicole Jackson, M.D.; B. Mark Evers, M.D.; Sundararajah Thevananther, Ph.D.; Kathleen O'Connor, Ph.D.; Frank C. Schmalstieg, M.D., Ph.D.; Cornelis Elferink, Ph.D.Hepatic resection is associated with rapid proliferation and regeneration of the remnant liver. Phosphatidylinositol 3-kinase (PI3K), composed of a p85Ą regulatory and a p110Ą catalytic subunit, participates in multiple cellular processes, including cell growth and survival; however, the role of PI3K in liver regeneration has not been clearly delineated. In these studies, we used the potent PI3K inhibitor, wortmannin, and small-interfering RNA (siRNA) targeting the p85Ą and p110Ą subunits to determine if total or selective PI3K inhibition would abrogate the proliferative response of the liver following resection. After partial hepatectomy in mice, there is an increase in PI3K activity; total PI3K blockade with wortmannin, and selective inhibition of p85Ą or p110Ą with siRNA resulted in a significant decrease in hepatocyte proliferation, especially at the earliest timepoints (ie, 48h and 72h). Fewer macrophages and Kupffer cells were present in the regenerating liver of mice treated with wortmannin or siRNA to p85Ą or p110Ą, as reflected by a paucity of F4/80+ cells present by immunohistochemistry (IHC). Additionally, PI3K inhibition led to an aberrant hepatocyte architecture characterized by vacuolization, lipid deposition, and glycogen accumulation. Our data demonstrate that PI3K/Akt pathway activation plays a critical role in the early regenerative response of the liver after resection; inhibition of this pathway markedly abrogates the normal hepatic regenerative response, perhaps by inhibiting macrophage infiltration and cytokine elaboration and thus hepatocyte priming for replication.