Advertisement

Gli1 activation and protection against hepatic encephalopathy is suppressed by circulating transforming growth factor β1 in mice

      Background & Aims

      Hepatic encephalopathy (HE) is a neurologic disorder that develops during liver failure. Few studies exist investigating systemic-central signalling during HE outside of inflammatory signalling. The transcription factor Gli1, which can be modulated by hedgehog signalling or transforming growth factor β1 (TGFβ1) signalling, has been shown to be protective in various neuropathies. We measured Gli1 expression in brain tissues from mice and evaluated how circulating TGFβ1 and canonical hedgehog signalling regulate its activation.

      Methods

      Mice were injected with azoxymethane (AOM) to induce liver failure and HE in the presence of Gli1 vivo-morpholinos, the hedgehog inhibitor cyclopamine, Smoothened vivo-morpholinos, a Smoothened agonist, or TGFβ-neutralizing antibodies. Molecular analyses were used to assess Gli1, hedgehog signalling, and TGFβ1 signalling in the liver and brain of AOM mice and HE patients.

      Results

      Gli1 expression was increased in brains of AOM mice and in HE patients. Intra-cortical infusion of Gli1 vivo-morpholinos exacerbated the neurologic deficits of AOM mice. Measures to modulate hedgehog signalling had no effect on HE neurological decline. Levels of TGFβ1 increased in the liver and serum of mice following AOM administration. TGFβ neutralizing antibodies slowed neurologic decline following AOM administration without significantly affecting liver damage. TGFβ1 inhibited Gli1 expression via a SMAD3-dependent mechanism. Conversely, inhibiting TGFβ1 increased Gli1 expression.

      Conclusions

      Cortical activation of Gli1 protects mice from induction of HE. TGFβ1 suppresses Gli1 in neurons via SMAD3 and promotes the neurologic decline. Strategies to activate Gli1 or inhibit TGFβ1 signalling might be developed to treat patients with HE.

      Graphical abstract

      Abbreviations:

      HE (hepatic encephalopathy), AOM (azoxymethane), TGFβ1 (transforming growth factor beta 1), Gli1 (glioma-associated oncogene homolog 1), TGFβR2 (TGFβ receptor 2), Shh (sonic hedgehog), Ihh (indian hedgehog), HBC ((2-hydropropyl)-β-cyclodextrin), VM (vivo-morpholino), SAG (smoothened agonist), RT-PCR (reverse transcriptase polymerase chain reaction), H&E (hematoxylin and eosin), SIS3 (specific inhibitor of SMAD3), CNS (central nervous system), DAPI (4′,6-diamidino-2-phenylindole), ELISA (enzyme-linked immunosorbent assay)

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic and Personal
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Cash W.J.
        • McConville P.
        • McDermott E.
        • McCormick P.A.
        • Callender M.E.
        • McDougall N.I.
        Current concepts in the assessment and treatment of hepatic encephalopathy.
        QJM. 2010; 103: 9-16
        • Bernal W.
        • Auzinger G.
        • Dhawan A.
        • Wendon J.
        Acute liver failure.
        Lancet. 2010; 376: 190-201
        • Hazell A.S.
        • Butterworth R.F.
        Hepatic encephalopathy: an update of pathophysiologic mechanisms.
        Proc Soc Exp Biol Med. 1999; 222: 99-112
        • Cauli O.
        • Rodrigo R.
        • Llansola M.
        • Montoliu C.
        • Monfort P.
        • Piedrafita B.
        • et al.
        Glutamatergic and gabaergic neurotransmission and neuronal circuits in hepatic encephalopathy.
        Metab Brain Dis. 2009; 24: 69-80
        • Suwelack D.
        • Hurtado-Lorenzo A.
        • Millan E.
        • Gonzalez-Nicolini V.
        • Wawrowsky K.
        • Lowenstein P.R.
        • et al.
        Neuronal expression of the transcription factor Gli1 using the Talpha1 alpha-tubulin promoter is neuroprotective in an experimental model of Parkinson’s disease.
        Gene Ther. 2004; 11: 1742-1752
        • Ji H.
        • Miao J.
        • Zhang X.
        • Du Y.
        • Liu H.
        • Li S.
        • et al.
        Inhibition of sonic hedgehog signaling aggravates brain damage associated with the down-regulation of Gli1, Ptch1, and SOD1 expression in acute ischemic stroke.
        Neurosci Lett. 2012; 506: 1-6
        • Ji H.
        • Zhang X.
        • Du Y.
        • Liu H.
        • Li S.
        • Li L.
        Polydatin modulates inflammation by decreasing NF-kappaB activation and oxidative stress by increasing Gli1, Ptch1, SOD1 expression and ameliorates blood-brain barrier permeability for its neuroprotective effect in pMCAO rat brain.
        Brain Res Bull. 2012; 87: 50-59
        • Matise M.P.
        • Joyner A.L.
        Gli genes in development and cancer.
        Oncogene. 1999; 18: 7852-7859
        • Katoh Y.
        • Katoh M.
        Integrative genomic analyses on GLI1: positive regulation of GLI1 by Hedgehog-GLI, TGFbeta-Smads, and RTK-PI3K-AKT signals, and negative regulation of GLI1 by Notch-CSL-HES/HEY, and GPCR-Gs-PKA signals.
        Int J Oncol. 2009; 35: 187-192
        • Dennler S.
        • Andre J.
        • Alexaki I.
        • Li A.
        • Magnaldo T.
        • ten Dijke P.
        • et al.
        Induction of sonic hedgehog mediators by transforming growth factor-beta: Smad3-dependent activation of Gli2 and Gli1 expression in vitro and in vivo.
        Cancer Res. 2007; 67: 6981-6986
        • Derynck R.
        • Zhang Y.E.
        Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
        Nature. 2003; 425: 577-584
        • Omenetti A.
        • Choi S.
        • Michelotti G.
        • Diehl A.M.
        Hedgehog signaling in the liver.
        J Hepatol. 2011; 54: 366-373
        • Pereira Tde A.
        • Witek R.P.
        • Syn W.K.
        • Choi S.S.
        • Bradrick S.
        • Karaca G.F.
        • et al.
        Viral factors induce Hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma.
        Lab Invest. 2010; 90: 1690-1703
        • Witek R.P.
        • Yang L.
        • Liu R.
        • Jung Y.
        • Omenetti A.
        • Syn W.K.
        • et al.
        Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.
        Gastroenterology. 2009; 136: e322
        • Gressner A.M.
        • Weiskirchen R.
        • Breitkopf K.
        • Dooley S.
        Roles of TGF-beta in hepatic fibrosis.
        Front Biosci. 2002; 7: d793-d807
        • Roth S.
        • Michel K.
        • Gressner A.M.
        (Latent) transforming growth factor beta in liver parenchymal cells, its injury-dependent release, and paracrine effects on rat hepatic stellate cells.
        Hepatology. 1998; 27: 1003-1012
        • Eguchi S.
        • Kamlot A.
        • Ljubimova J.
        • Hewitt W.R.
        • Lebow L.T.
        • Demetriou A.A.
        • et al.
        Fulminant hepatic failure in rats: survival and effect on blood chemistry and liver regeneration.
        Hepatology. 1996; 24: 1452-1459
        • Frampton G.
        • Invernizzi P.
        • Bernuzzi F.
        • Pae H.Y.
        • Quinn M.
        • Horvat D.
        • et al.
        Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism.
        Gut. 2012; 61: 268-277
        • Quinn M.
        • Ueno Y.
        • Pae H.Y.
        • Huang L.
        • Frampton G.
        • Galindo C.
        • et al.
        Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat.
        Am J Physiol Gastrointest Liver Physiol. 2012; 302: G182-G193
        • Lyck L.
        • Kroigard T.
        • Finsen B.
        Unbiased cell quantification reveals a continued increase in the number of neocortical neurones during early post-natal development in mice.
        Eur J Neurosci. 2007; 26: 1749-1764
        • Amankulor N.M.
        • Hambardzumyan D.
        • Pyonteck S.M.
        • Becher O.J.
        • Joyce J.A.
        • Holland E.C.
        Sonic hedgehog pathway activation is induced by acute brain injury and regulated by injury-related inflammation.
        J Neurosci. 2009; 29: 10299-10308
        • Omenetti A.
        • Diehl A.M.
        The adventures of sonic hedgehog in development and repair. II. Sonic hedgehog and liver development, inflammation, and cancer.
        Am J Physiol Gastrointest Liver Physiol. 2008; 294: G595-G598
        • Omenetti A.
        • Yang L.
        • Li Y.X.
        • McCall S.J.
        • Jung Y.
        • Sicklick J.K.
        • et al.
        Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation.
        Lab Invest. 2007; 87: 499-514
        • Yang L.
        • Wang Y.
        • Mao H.
        • Fleig S.
        • Omenetti A.
        • Brown K.D.
        • et al.
        Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells.
        J Hepatol. 2008; 48: 98-106
        • Ochoa B.
        • Syn W.K.
        • Delgado I.
        • Karaca G.F.
        • Jung Y.
        • Wang J.
        • et al.
        Hedgehog signaling is critical for normal liver regeneration after partial hepatectomy in mice.
        Hepatology. 2010; 51: 1712-1723
        • Pratap A.
        • Panakanti R.
        • Yang N.
        • Lakshmi R.
        • Modanlou K.A.
        • Eason J.D.
        • et al.
        Cyclopamine attenuates acute warm ischemia reperfusion injury in cholestatic rat liver: hope for marginal livers.
        Mol Pharm. 2011; 8: 958-968
        • Seth D.
        • Haber P.S.
        • Syn W.K.
        • Diehl A.M.
        • Day C.P.
        Pathogenesis of alcohol-induced liver disease: classical concepts and recent advances.
        J Gastroenterol Hepatol. 2011; 26: 1089-1105
        • Sicklick J.K.
        • Li Y.X.
        • Choi S.S.
        • Qi Y.
        • Chen W.
        • Bustamante M.
        • et al.
        Role for hedgehog signaling in hepatic stellate cell activation and viability.
        Lab Invest. 2005; 85: 1368-1380
        • Lipinski R.J.
        • Hutson P.R.
        • Hannam P.W.
        • Nydza R.J.
        • Washington I.M.
        • Moore R.W.
        • et al.
        Dose- and route-dependent teratogenicity, toxicity, and pharmacokinetic profiles of the hedgehog signaling antagonist cyclopamine in the mouse.
        Toxicol Sci. 2008; 104: 189-197
        • Lanz T.V.
        • Ding Z.
        • Ho P.P.
        • Luo J.
        • Agrawal A.N.
        • Srinagesh H.
        • et al.
        Angiotensin II sustains brain inflammation in mice via TGF-beta.
        J Clin Invest. 2010; 120: 2782-2794
        • Luo J.
        • Ho P.P.
        • Buckwalter M.S.
        • Hsu T.
        • Lee L.Y.
        • Zhang H.
        • et al.
        Glia-dependent TGF-beta signaling, acting independently of the TH17 pathway, is critical for initiation of murine autoimmune encephalomyelitis.
        J Clin Invest. 2007; 117: 3306-3315
        • Zemtsova I.
        • Gorg B.
        • Keitel V.
        • Bidmon H.J.
        • Schror K.
        • Haussinger D.
        Microglia activation in hepatic encephalopathy in rats and humans.
        Hepatology. 2011; 54: 204-215
        • Jiang W.
        • Desjardins P.
        • Butterworth R.F.
        Direct evidence for central proinflammatory mechanisms in rats with experimental acute liver failure: protective effect of hypothermia.
        J Cereb Blood Flow Metab. 2009; 29: 944-952
        • Bemeur C.
        • Qu H.
        • Desjardins P.
        • Butterworth R.F.
        IL-1 or TNF receptor gene deletion delays onset of encephalopathy and attenuates brain edema in experimental acute liver failure.
        Neurochem Int. 2010; 56: 213-215
        • Shahi M.H.
        • Afzal M.
        • Sinha S.
        • Eberhart C.G.
        • Rey J.A.
        • Fan X.
        • et al.
        Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6, and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma.
        BMC Cancer. 2010; 10: 614
        • Doyle K.P.
        • Cekanaviciute E.
        • Mamer L.E.
        • Buckwalter M.S.
        TGFbeta signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke.
        J Neuroinflammation. 2010; 7: 62