Lack of EGFR catalytic activity in hepatocytes improves liver regeneration following DDC-induced cholestatic injury by promoting a pro-restorative inflammatory response

Despite the well-known hepatoprotective role of the epidermal growth factor receptor (EGFR) pathway upon acute damage, its specific actions during chronic liver disease, particularly cholestatic injury, remain ambiguous and unresolved. Here, we analyzed the consequences of inactivating EGFR signaling in the liver on the regenerative response following cholestatic injury. For that, transgenic mice overexpressing a dominant negative mutant human EGFR lacking tyrosine kinase activity (ΔEGFR) in albumin-positive cells were submitted to liver damage induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), an experimental model resembling human primary sclerosing cholangitis. Our results show an early activation of EGFR after 1–2 days of a DDC-supplemented diet, followed by a signaling switch-off. Furthermore, ΔEGFR mice showed less liver damage and a more efficient regeneration following DDC injury. Analysis of the mechanisms driving this effect revealed an enhanced activation of mitogenic/survival signals, AKT and ERK1/2-MAPKs, and changes in cell turnover consistent with a quicker resolution of damage in response to DDC. These changes were concomitant with profound differences in the profile of intrahepatic immune cells, consisting of a shift in the M1/M2 balance towards M2 polarity, and the Cd4/Cd8 ratio in favor of Cd4 lymphocytes, overall supporting an immune cell switch into a pro-restorative phenotype. Interestingly, ΔEGFR livers also displayed an amplified ductular reaction, with increased expression of EPCAM and an increased number of CK19-positive ductular structures in portal areas, demonstrating an overexpansion of ductular progenitor cells. In summary, our work supports the notion that hepatocyte-specific EGFR activity acts as a key player in the crosstalk between parenchymal and non-parenchymal hepatic cells, promoting the pro-inflammatory response activated during cholestatic injury and therefore contributing to the pathogenesis of cholestatic liver disease. © 2022 The Pathological Society of Great Britain and Ireland.This work was supported by the Ministry of Science,Innovation and Universities (MICIU) and AgenciaEstatal de Investigacion (AEI), Spain (co-funded byFEDER funds/Development Fund–a Way to BuildEurope): RTI2018-099098-B-100 to AS/BH andRTI2018-094052-B-100 to AMV; and the RamonAreces Foundation: 20th National Competition forScientific and Technical Research in Life and MatterScience (2020) to IF. NL and JGS were recipients ofresearch assistant contracts linked to grant SAF2015-69145-R and RTI2018-099098-B-100, respectively. CMR was the recipient of a researchcontract (PEJD-2019-POST/BMD-16090) from the Education, Universities, Research and Spokesperson Counseling of the Community of Madrid

Papel de señalización del TGF-β, HGF y EGF en la regulación de la proliferación, diferenciación y muerte de las células progenitoras adultas hepáticas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Bioquímica y Biología Molecular II, leída el 09-07-2012Sección Deptal. de Bioquímica y Biología Molecular (Farmacia)Fac. de FarmaciaTRUEunpu

Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-Induced oxidative stress and apoptosis.

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Metflx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Metflx/flx and Met−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met-/- oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Metflx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Metflx/flx oval cells, whereas no effect was observed in Met−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis

Increased sensitivity to TGF-β-induced apoptosis in oval cells lacking a functional Met receptor. A.

<p>Mouse Met^flx/flx and Met^−/− oval cell lines were serum-starved and incubated in the absence (C) or presence of 1 ng/ml TGF-β (Tβ) for different periods of time. Cells were lysed and caspase-3 activity was measured. Data are mean±SEM of eight independent experiments. <b>B.</b> Cells were treated as in A, detached by tripsinization and incubated with annexin V and PI. Subsequently, fluorescence intensity was measured in a FACScan flow cytometer and the percentage of annexin V positive/PI negative cells was calculated. Data are expressed as fold induction over untreated cells and are mean±SEM of four independent experiments. <b>C.</b> Cells were infected with Ad-CMVCre or Ad-CMVNull for 24 hours, then serum-starved for 4 hours and treated with 1 ng/ml TGF-β for 24 hours. Apoptotic nuclei were visualized and counted after PI staining under a fluorescence microscope. A minimum of 1000 nuclei was counted per condition. <b>D.</b> PCR genotyping of genomic DNA to confirm <i>met</i> deletion in infected cells. <b>Black bars</b>, Met^flx/flx cells. <b>White bars</b>, Met^−/− cells. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 (treated versus untreated); #<i>P</i><0.05 (treated Met^−/− versus treated Met^flx/flx).</p

Comparison of the TGF-β-induced canonical and non-canonical signaling in oval cell expressing a functionally active or inactive Met receptor. A.

<p>Mouse Met^flx/flx and Met^−/− oval cell lines were serum-starved and treated with 1 ng/ml TGF-β for different periods of time as indicated. Untreated cells were included as control. Whole cell lysates were collected and used for immunoblotting with anti-phosphoSmad2 antibody. β-actin was analyzed as loading control. A representative experiment of two is shown. <b>B.</b> Mouse Met^flx/flx and Met^−/− oval cell lines were transiently transfected with SBE containing construct linked to a luciferase reporter (Cignal Smad reporter). Cells were serum-starved and incubated in the absence (C) or presence of 1 ng/ml TGF-β (Tβ) for 8 hours. Normalized luciferase activity is shown as fold induction relative to untreated cells. Data are mean±SEM of three independent experiments run in triplicates. <b>C.</b> Cells were treated as in A. Whole cell lysates were collected and used for immunoblotting with indicated antibodies. A representative experiment of two is shown. <b>Black bars</b>, Met^flx/flx cells. <b>White bars</b>, Met^−/− cells. ***<i>P</i><0.001 (treated versus untreated).</p

Effect of PI3K inhibition on TGF-β-induced apoptotic and HGF-induced anti-apoptotic activities in oval cells. A.

<p>Mouse Met^flx/flx oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± HGF (40 ng/ml) and/or LY294002 (7.5 µM) for 24 hours. Apoptotic index was calculated by counting apoptotic nuclei after PI staining under a fluorescence microscope. A minimum of 1000 nuclei was counted per condition. Data are mean±S.D. of a representative experiment performed with triplicate dishes. <b>B.</b> Mouse Met^flx/flx and Met^−/− oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± LY294002 (7.5 µM) for different periods of time. Fluorescence intensity was measured in a FACScan flow cytometer and the percentage of annexin V positive/PI negative cells was calculated. Data are expressed as fold induction over untreated cells and are mean±SEM of four independent experiments. <b>C, D and E.</b> Mouse Met^flx/flx oval cells were serum-starved and incubated in the absence or presence of TGF-β (1 ng/ml) ± LY294002 (7.5 µM). <b>C.</b> After 8 hours of treatment, cells were collected for spectrophotometric determination of intracellular glutathione. Results are expressed as fold change over untreated cells and are mean±SEM of two independent experiments run in duplicate. <b>D and E.</b> After 12 hours of treatment RNA was isolated and <i>Nox4</i> and <i>Bmf</i> mRNA levels were analyzed by qRT-PCR and normalized to the housekeeping gene <i>Gusb</i>. Data represent fold change relative to untreated samples and are mean±S.E.M of three independent experiments. <b>Black bars</b>, Met^flx/flx cells. <b>White bars</b>, Met^−/− cells. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 (treated versus untreated); #<i>P</i><0.05 (T+LY treated versus T treated).</p

c‐Met signaling is essential for mouse adult liver progenitor cells expansion after TGF‐β‐induced EMT and regulates cell phenotypic switch

16 p.-6 fig.-1 graph. abst.Adult hepatic progenitor cells (HPCs)/oval cells are bipotential progenitors that participate in liver repair responses upon chronic injury. Recent findings highlight HPCs plasticity and importance of the HPCs niche signals to determine their fate during the regenerative process, favoring either fibrogenesis or damage resolution. Transforming Growth Factor-β (TGF-β) and Hepatocyte Growth Factor (HGF) are among the key signals involved in liver regeneration and as component of HPCs niche regulate HPCs biology. Here, we characterize the TGF-β-triggered epithelial-mesenchymal transition (EMT) response in oval cells, its effects on cell fate in vivo, and the regulatory effect of the HGF/c-Met signaling. Our data show that chronic treatment with TGF-β triggers a partial EMT in oval cells, based on co-expression of epithelial and mesenchymal markers. The phenotypic and functional profiling indicate that TGF-β-induced EMT is not associated with stemness but rather represents a step forward along hepatic lineage. This phenotypic transition confers advantageous traits to HPCs including survival, migratory/invasive and metabolic benefit, overall enhancing the regenerative potential of oval cells upon transplantation into a carbon tetrachloride-damaged liver. We further uncover a key contribution of the HGF/c-Met pathway to modulate the TGF-β-mediated EMT response. It allows oval cells expansion after EMT by controlling oxidative stress and apoptosis, likely via Twist regulation, and it counterbalances EMT by maintaining epithelial properties. Our work provides evidence that a coordinated and balanced action of TGF-β and HGF are critical for achievement of the optimal regenerative potential of HPCs, opening new therapeutic perspectives. SIGNIFICANCE STATEMENT: The findings from this study support that a balanced action of TGF-ß and HGF could determine liver progenitors fate and the outcome of liver regeneration, and open possibilities for targeted therapies oriented at improving the regenerative capacity of these cells in chronic liver diseases.This work was supported by a Marie Curie Action FP7-2012(Grant #PITN-GA-2012-316549; IT-LIVER); Ministry of Science,Innovation and Universities, Spain (Grant #SAF2015-69145-R) and General Direction of Universities and Research of theAutonomous Community of Madrid, Spain (Grant #S2010/BMD-2402, MITOLAB consortium). LA was recipient of a pre-doctoral contract from UCM. MG-A, AMP and NL were recipients from research-training contracts from Grant #S2010/BMD-2402 and SAF2006-12025 (Ministry of Education and Sci-ence) and SAF2015-69145-R, respectively.Peer reviewe

Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-Induced oxidative stress and apoptosis.

We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Metflx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Metflx/flx and Met−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met-/- oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Metflx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Metflx/flx oval cells, whereas no effect was observed in Met−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis