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• W2896226433 abstract "Although hedgehog (Hh) signaling pathway is inactive in adult healthy liver, it becomes activated during acute and chronic liver injury and, thus, modulates the reparative process and disease progression. We developed a novel mouse model with liver-specific knockout of Smoothened (Smo LKO), and animals were subjected to Fas-induced liver injury in vivo. Results showed that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Hh signaling in hepatocytes in the setting of Fas-induced injury was indicated by the fact that Jo2 treatment enhanced hepatic expression of Ptch1, Smo, and its downstream target Gli1 in control but not Smo LKO mice. Primary hepatocytes from control mice showed increased Hh signaling activation in response to Jo2 treatment in vitro. On the other hand, the Smo KO hepatocytes were devoid of Hh activation and were more susceptible to Jo2-induced apoptosis. The levels of NF-κB and related signaling molecules, including epidermal growth factor receptor and Akt, were lower in Smo KO livers/hepatocytes than in control livers/hepatocytes. Accordingly, hydrodynamic gene delivery of active NK-κB prevented Jo2-induced liver injury in the Smo LKO mice. Our findings provide important evidence that adult hepatocytes become responsive to Hh signaling through up-regulation of Smo in the setting of Fas-induced liver injury and that such alteration leads to activation of NF-κB/epidermal growth factor receptor/Akt, which counteracts Fas-induced hepatocyte apoptosis. Although hedgehog (Hh) signaling pathway is inactive in adult healthy liver, it becomes activated during acute and chronic liver injury and, thus, modulates the reparative process and disease progression. We developed a novel mouse model with liver-specific knockout of Smoothened (Smo LKO), and animals were subjected to Fas-induced liver injury in vivo. Results showed that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Hh signaling in hepatocytes in the setting of Fas-induced injury was indicated by the fact that Jo2 treatment enhanced hepatic expression of Ptch1, Smo, and its downstream target Gli1 in control but not Smo LKO mice. Primary hepatocytes from control mice showed increased Hh signaling activation in response to Jo2 treatment in vitro. On the other hand, the Smo KO hepatocytes were devoid of Hh activation and were more susceptible to Jo2-induced apoptosis. The levels of NF-κB and related signaling molecules, including epidermal growth factor receptor and Akt, were lower in Smo KO livers/hepatocytes than in control livers/hepatocytes. Accordingly, hydrodynamic gene delivery of active NK-κB prevented Jo2-induced liver injury in the Smo LKO mice. Our findings provide important evidence that adult hepatocytes become responsive to Hh signaling through up-regulation of Smo in the setting of Fas-induced liver injury and that such alteration leads to activation of NF-κB/epidermal growth factor receptor/Akt, which counteracts Fas-induced hepatocyte apoptosis. The hedgehog (Hh) signaling pathway plays an important role in embryonic development and in the regulation of a variety of cellular functions, including proliferation, survival, migration, differentiation, and maintenance of progenitor cells.1Omenetti A. Choi S. Michelotti G. Diehl A.M. Hedgehog signaling in the liver.J Hepatol. 2011; 54: 366-373Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar Although the Hh pathway is quiescent in normal liver, it becomes reactivated as a repair mechanism in acute and chronic liver injuries and diseases.1Omenetti A. Choi S. Michelotti G. Diehl A.M. Hedgehog signaling in the liver.J Hepatol. 2011; 54: 366-373Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar Although healthy livers express low levels of Hh ligands,2Jung Y. McCall S.J. Li Y.X. Diehl A.M. Bile ductules and stromal cells express hedgehog ligands and/or hedgehog target genes in primary biliary cirrhosis.Hepatology. 2007; 45: 1091-1096Crossref PubMed Scopus (105) Google Scholar, 3Omenetti A. Popov Y. Jung Y. Choi S.S. Witek R.P. Yang L. Brown K.D. Schuppan D. Diehl A.M. The hedgehog pathway regulates remodelling responses to biliary obstruction in rats.Gut. 2008; 57: 1275-1282Crossref PubMed Scopus (114) Google Scholar, 4Omenetti A. Yang L. Li Y.X. McCall S.J. Jung Y. Sicklick J.K. Huang J. Choi S. Suzuki A. Diehl A.M. Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation.Lab Invest. 2007; 87: 499-514Crossref PubMed Scopus (155) Google Scholar liver injury enhances the production of Hh ligands from several types of resident cells (including hepatocytes, cholangiocytes, hepatic stellate cells, natural killer T cells, and sinusoidal endothelial cells); this process is mediated by various growth factors and cytokines as well as by cytotoxic and apoptotic stresses. The produced Hh ligands are released into the local environment, where they activate the receptors on Hh-responsive cells (including progenitor cells, hepatic stellate cells, and biliary epithelial cells), which mediates liver tissue remodeling and repair. For example, Hh ligands can serve as viability and proliferative factors for liver epithelial progenitors, which are important for liver tissue repair.5Sicklick J.K. Li Y.X. Melhem A. Schmelzer E. Zdanowicz M. Huang J. Caballero M. Fair J.H. Ludlow J.W. McClelland R.E. Reid L.M. Diehl A.M. Hedgehog signaling maintains resident hepatic progenitors throughout life.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G859-G870Crossref PubMed Scopus (181) Google Scholar, 6Fleig S.V. Choi S.S. Yang L. Jung Y. Omenetti A. VanDongen H.M. Huang J. Sicklick J.K. Diehl A.M. Hepatic accumulation of hedgehog-reactive progenitors increases with severity of fatty liver damage in mice.Lab Invest. 2007; 87: 1227-1239Crossref PubMed Scopus (75) Google Scholar, 7Jung Y. Witek R.P. Syn W.K. Choi S.S. Omenetti A. Premont R. Guy C.D. Diehl A.M. Signals from dying hepatocytes trigger growth of liver progenitors.Gut. 2010; 59: 655-665Crossref PubMed Scopus (135) Google Scholar Hh ligands also promote the growth and viability of hepatic stellate cells/myofibroblasts and contribute to liver fibrosis and cirrhosis.1Omenetti A. Choi S. Michelotti G. Diehl A.M. Hedgehog signaling in the liver.J Hepatol. 2011; 54: 366-373Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 8Syn W.K. Choi S.S. Liaskou E. Karaca G.F. Agboola K.M. Oo Y.H. Mi Z. Pereira T.A. Zdanowicz M. Malladi P. Chen Y. Moylan C. Jung Y. Bhattacharya S.D. Teaberry V. Omenetti A. Abdelmalek M.F. Guy C.D. Adams D.H. Kuo P.C. Michelotti G.A. Whitington P.F. Diehl A.M. Osteopontin is induced by hedgehog pathway activation and promotes fibrosis progression in nonalcoholic steatohepatitis.Hepatology. 2011; 53: 106-115Crossref PubMed Scopus (202) Google Scholar, 9Chen Y. Choi S.S. Michelotti G.A. Chan I.S. Swiderska-Syn M. Karaca G.F. Xie G. Moylan C.A. Garibaldi F. Premont R. Suliman H.B. Piantadosi C.A. Diehl A.M. Hedgehog controls hepatic stellate cell fate by regulating metabolism.Gastroenterology. 2012; 143: 1319-1329.e1-11Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 10Bohinc B.N. Diehl A.M. Mechanisms of disease progression in NASH: new paradigms.Clin Liver Dis. 2012; 16: 549-565Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 11Syn W.K. Agboola K.M. Swiderska M. Michelotti G.A. Liaskou E. Pang H. Xie G. Philips G. Chan I.S. Karaca G.F. Pereira Tde A. Chen Y. Mi Z. Kuo P.C. Choi S.S. Guy C.D. Abdelmalek M.F. Diehl A.M. NKT-associated hedgehog and osteopontin drive fibrogenesis in non-alcoholic fatty liver disease.Gut. 2012; 61: 1323-1329Crossref PubMed Scopus (184) Google Scholar, 12Choi S.S. Syn W.K. Karaca G.F. Omenetti A. Moylan C.A. Witek R.P. Agboola K.M. Jung Y. Michelotti G.A. Diehl A.M. Leptin promotes the myofibroblastic phenotype in hepatic stellate cells by activating the hedgehog pathway.J Biol Chem. 2010; 285: 36551-36560Crossref PubMed Scopus (145) Google Scholar Considering these mechanistic aspects, it is not surprising that Hh pathway activation has been shown to play a role in liver regeneration during partial hepatectomy13Ochoa B. Syn W.K. Delgado I. Karaca G.F. Jung Y. Wang J. Zubiaga A.M. Fresnedo O. Omenetti A. Zdanowicz M. Choi S.S. Diehl A.M. Hedgehog signaling is critical for normal liver regeneration after partial hepatectomy in mice.Hepatology. 2010; 51: 1712-1723Crossref PubMed Scopus (143) Google Scholar and contribute to the pathogenesis of several liver diseases, including nonalcoholic fatty liver disease,14Guy C.D. Suzuki A. Zdanowicz M. Abdelmalek M.F. Burchette J. Unalp A. Diehl A.M. Hedgehog pathway activation parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease.Hepatology. 2012; 55: 1711-1721Crossref PubMed Scopus (148) Google Scholar, 15Syn W.K. Oo Y.H. Pereira T.A. Karaca G.F. Jung Y. Omenetti A. Witek R.P. Choi S.S. Guy C.D. Fearing C.M. Teaberry V. Pereira F.E. Adams D.H. Diehl A.M. Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.Hepatology. 2010; 51: 1998-2007Crossref PubMed Scopus (204) Google Scholar alcoholic liver disease,16Jung Y. Brown K.D. Witek R.P. Omenetti A. Yang L. Vandongen M. Milton R.J. Hines I.N. Rippe R.A. Spahr L. Rubbia-Brandt L. Diehl A.M. Accumulation of hedgehog-responsive progenitors parallels alcoholic liver disease severity in mice and humans.Gastroenterology. 2008; 134: 1532-1543Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar and hepatocellular carcinoma.17Wang Y. Han C. Lu L. Magliato S. Wu T. Hedgehog signaling pathway regulates autophagy in human hepatocellular carcinoma cells.Hepatology. 2013; 58: 995-1010Crossref PubMed Scopus (132) Google Scholar, 18Sicklick J.K. Li Y.X. Jayaraman A. Kannangai R. Qi Y. Vivekanandan P. Ludlow J.W. Owzar K. Chen W. Torbenson M.S. Diehl A.M. Dysregulation of the hedgehog pathway in human hepatocarcinogenesis.Carcinogenesis. 2006; 27: 748-757Crossref PubMed Scopus (238) Google Scholar, 19Patil M.A. Zhang J. Ho C. Cheung S.T. Fan S.T. Chen X. Hedgehog signaling in human hepatocellular carcinoma.Cancer Biol Ther. 2006; 5: 111-117Crossref PubMed Scopus (89) Google Scholar, 20Huang S. He J. Zhang X. Bian Y. Yang L. Xie G. Zhang K. Tang W. Stelter A.A. Wang Q. Zhang H. Xie J. Activation of the hedgehog pathway in human hepatocellular carcinomas.Carcinogenesis. 2006; 27: 1334-1340Crossref PubMed Scopus (199) Google Scholar, 21Pereira Tde A. Witek R.P. Syn W.K. Choi S.S. Bradrick S. Karaca G.F. Agboola K.M. Jung Y. Omenetti A. Moylan C.A. Yang L. Fernandez-Zapico M.E. Jhaveri R. Shah V.H. Pereira F.E. Diehl A.M. Viral factors induce hedgehog pathway activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma.Lab Invest. 2010; 90: 1690-1703Crossref PubMed Scopus (96) Google Scholar, 22Villanueva A. Newell P. Chiang D.Y. Friedman S.L. Llovet J.M. Genomics and signaling pathways in hepatocellular carcinoma.Semin Liver Dis. 2007; 27: 55-76Crossref PubMed Scopus (500) Google Scholar, 23Philips G.M. Chan I.S. Swiderska M. Schroder V.T. Guy C. Karaca G.F. Moylan C. Venkatraman T. Feuerlein S. Syn W.K. Jung Y. Witek R.P. Choi S. Michelotti G.A. Rangwala F. Merkle E. Lascola C. Diehl A.M. Hedgehog signaling antagonist promotes regression of both liver fibrosis and hepatocellular carcinoma in a murine model of primary liver cancer.PLoS One. 2011; 6: e23943Crossref PubMed Scopus (135) Google Scholar Therefore, delineation of Hh actions in liver cells will help further understand the pathogenesis of liver diseases and provide new therapeutic implications. The Hh signaling pathway is activated on the binding of Hh ligands (Sonic Hh, Indian Hh, and Desert Hh) to their receptor, Ptch. In the absence of Hh ligands, Ptch inhibits Smoothened (Smo), a seven-pass membrane protein that belongs to the G-protein–coupled receptor superfamily.24Alcedo J. Ayzenzon M. Von Ohlen T. Noll M. Hooper J.E. The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal.Cell. 1996; 86: 221-232Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar Binding of Hh ligands to Ptch causes activation of Smo, which subsequently activates the Gli family of transcription factors, leading to expression of Hh target genes. Activation of transcription by the Glis is referred to as the canonical Hh signaling pathway, whereas those not requiring Smo or those downstream of Smo that do not require the Gli transcription faction are collectively referred to as the noncanonical Hh signaling pathway.25Shevde L.A. Samant R.S. Nonclassical hedgehog-GLI signaling and its clinical implications.Int J Cancer. 2014; 135: 1-6Crossref PubMed Scopus (34) Google Scholar, 26Robbins D.J. Fei D.L. Riobo N.A. The hedgehog signal transduction network.Sci Signal. 2012; 5: re6Crossref PubMed Scopus (309) Google Scholar In the liver, the nonparenchymal cells (including hepatic stellate cells and natural killer T cells) and cholangiocytes are known to produce Hh ligands in the setting of liver injury. The adult healthy hepatocytes produce a low amount of Hh ligands,27Matz-Soja M. Hovhannisyan A. Gebhardt R. Hedgehog signalling pathway in adult liver: a major new player in hepatocyte metabolism and zonation?.Med Hypotheses. 2013; 80: 589-594Crossref PubMed Scopus (26) Google Scholar although dying or ballooning hepatocytes are known to produce large amounts of Shh ligands.7Jung Y. Witek R.P. Syn W.K. Choi S.S. Omenetti A. Premont R. Guy C.D. Diehl A.M. Signals from dying hepatocytes trigger growth of liver progenitors.Gut. 2010; 59: 655-665Crossref PubMed Scopus (135) Google Scholar, 28Rangwala F. Guy C.D. Lu J. Suzuki A. Burchette J.L. Abdelmalek M.F. Chen W. Diehl A.M. Increased production of sonic hedgehog by ballooned hepatocytes.J Pathol. 2011; 224: 401-410Crossref PubMed Scopus (123) Google Scholar The Hh-responsive cells in the liver include progenitor cells, hepatic stellate cells, and biliary epithelial cells, whereas normal adult hepatocytes are considered to be nonresponsive to Hh ligands.1Omenetti A. Choi S. Michelotti G. Diehl A.M. Hedgehog signaling in the liver.J Hepatol. 2011; 54: 366-373Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar However, the latter viewpoint has been challenged by a recent study showing that there is cilia-independent but Smo-dependent signaling in mature healthy hepatocytes and that the Hh signaling is detectable in healthy mature hepatocytes.29Matz-Soja M. Aleithe S. Marbach E. Bottger J. Arnold K. Schmidt-Heck W. Kratzsch J. Gebhardt R. Hepatic hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels.Cell Commun Signal. 2014; 12: 11Crossref PubMed Scopus (37) Google Scholar To date, the status and impact of Hh signaling in hepatocytes, particularly in the setting of liver injury, remain largely unknown. The current study was designed to determine the role of Hh signaling in hepatocytes in the setting of Fas-induced liver injury. We developed a novel mouse model with liver-specific knockout of Smo, and the animals were subjected to Fas-induced liver injury to document the status of Hh signaling and the extent of liver injury. Our findings provide novel evidence for an important role of hepatocyte Hh signaling, which counteracts Fas-induced liver injury. Homozygous floxed Smo (Smoflox/flox; Smotm2Amc/J) mice and albumin (Alb)-Cre transgenic [B6.Cg-Tg (Alb-cre) 21Mgn/J] mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Smoflox/flox mice were bred to Alb-cre mice and the offspring carried a floxed Smo allele and Alb-cre (Smoflox/wt:Alb-Cre+/−). Then, Smoflox/wt:Alb-Cre+/− mice were bred with Smoflox/flox mice to obtain Smoflox/flox:Alb-Cre+/−; Smoflox/flox:Alb-Cre−/−; Smoflox/wt:Alb-Cre+/−; and Smoflox/wt:Alb-Cre−/− mice (Figure 1A). We used Smoflox/flox:Alb-Cre+/− mice as liver-specific knockout of Smo (Smo LKO); their matched littermates Smoflox/flox:Alb-Cre−/− and Smoflox/wt:Alb-Cre−/− were used as control mice. Eight-week–old male mice were used in this study. The mice were kept at 22°C under a 12-hour light/dark cycle and received food and water freely. All of the breeding and handling of the mice and all experimental procedures were approved by the Institutional Animal Care and Use Committee of Tulane University (New Orleans, LA). To determine the survival rate, 8-week–old male Smo LKO and control mice were injected intraperitoneally with 0.30 μg/g of body weight Jo2 (anti-Fas antibody; BD Bioscience, Franklin Lakes, NJ) (Jo2 was prepared in a sterile 1× Dulbecco's phosphate-buffered saline; Sigma-Aldrich, St. Louis, MO); after Jo2 injection, the mice were observed continuously for up to 24 hours to assess mortality. For other experiments, the mice were injected intraperitoneally with 0.30 μg/g of body weight Jo2 and sacrificed at 3 hours after Jo2 injection; blood was collected from mouse orbital sinus according to the standard procedure; liver tissue samples were collected for histology analysis. Eight-week–old male Smo LKO and control mice were anesthetized with an i.p. injection of ketamine/xylazine (80/10 mg/kg; Sigma-Aldrich). Bioseb's Mouse Tail Illuminator (Bioseb Company, Vitrolles, France) was provided by the Central Animal Facility at Tulane University Health Sciences Center. The anesthetized mouse was placed into the restraining tube. Tail vein injections were performed by using a 27-gauge needle (BD, Franklin Lakes, NJ). Each mouse was injected with 10% volume/body weight of EGFP empty control plasmid (20 μg/mL; Addgene, Cambridge, MA) or EGFP P65 plasmid (20 μg/mL; Addgene) within 10 seconds. Twenty-four hours after tail vein injection, the mice were intraperitoneally administered Jo2, 0.30 μg/g of body weight. Three hours after Jo2 injection, the mice were sacrificed. Blood and liver tissue samples were collected for further analyses. As previously described,30Chen W. Han C. Zhang J. Song K. Wang Y. Wu T. miR-150 deficiency protects against FAS-induced acute liver injury in mice through regulation of AKT.PLoS One. 2015; 10: e0132734Google Scholar blood samples were centrifuged at 800 × g for 15 minutes, and the sera were collected and then stored at −80°C. Serum alanine aminotransferase and aspartate aminotransferase activities were measured with an automatic analyzer at the Department of Clinical Chemistry, Tulane University Hospital. The liver tissue samples were fixed with 10% buffered formalin and embedded in paraffin. Sections (5 μm thick) were affixed to slides, deparaffinized, and stained with hematoxylin and eosin. Light microscopy was performed to assess the morphologic changes. Detection of apoptotic cells in liver tissue was performed by In Situ Cell Death Detection Kit, Fluorescein (Roche, Indianapolis, IN), according to the manufacturer's protocol. Paraffin-embedded liver tissue sections were dewaxed in xylene twice (5 minutes each time), hydrated in 100% ethanol for 2 minutes, and washed in decreasing concentrations of ethanol (95%, 90%, 80%, and 70%). The tissue sections were incubated for 30 minutes at room temperature with Proteinase K working solution (20 μg/mL in 10 mmol/L Tris/HCl, pH 7.4 to 8). The slides were rinsed twice with 1× phosphate-buffered saline (Sigma-Aldrich). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction mixture (50 μL) was added to each sample (50 μL label solution was used for negative control). The slides were incubated in a humidified atmosphere for 60 minutes at 37°C in the dark. After rinsing with 1× phosphate-buffered saline for three times, the slides were directly analyzed under a fluorescence microscope using a wavelength at 450 to 500 nm. Paraffin-embedded liver tissue sections were stained with cleaved caspase-3 antibody (Biocare Medical, Pike Lane Concord, CA), which specifically recognizes the large fragment (17/19 kDa) of activated caspase-3. The liver tissue sections were deparaffinized by immersing sections in xylene for 10 minutes and hydrated through ethanol to running water. Sections were processed for heat-induced epitope retrieval. After blocking with Biocare's Peroxidazed 1 (Biocare Medical) for 5 minutes, the sections were incubated for 1 hour at room temperature with rabbit polyclonal antibody against cleaved caspase-3. After washing three times in tris-buffered saline, the sections were incubated with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:10,000; Abcam, Cambridge, MA) for 1 hour at room temperature. After washing in tris-buffered saline, diaminobenzidine–horseradish peroxidase substrate was then added for color development. Total RNA was extracted from liver tissues and hepatocytes using Trizol (Invitrogen, Grand Island, NY). For first-strand cDNA synthesis, 1 μg total RNA was reverse transcribed using Qiagen miScript reverse transcription kit (Qiagen, Valencia, CA). Primers were synthesized at IDT (Coralville, IA) as follows: Smo, 5′-ACCTATGCCTGGCACACTTC-3′ (forward) and 5′-GTGAGGACAAAGGGGAGTGA-3′ (reverse); Ptch1, 5′-CTGTCAAGGTGAATGGAC-3′ (forward) and 5′-GGGGTTATTCTGTAAAAGG-3′ (reverse); Gli1, 5′-GCTGTCGGAAGTCCTATT-3′ (forward) and 5′-ACTGGCATTGCTAAAGG-3′ (reverse); and 18S, 5′-CGCTTCCTTACCTGGTTGAT-3′ (forward) and 5′-GAGCGACCAAAGGAACCATA-3′ (reverse). The miScript SYBR Green PCR Kit (Qiagen) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA) were used. The data were analyzed by using CFX Manager software version 3.1 (Bio-Rad), and the results were normalized to 18S ribosomal RNA. Hepatocytes were isolated from male Smo LKO and control mice by an adaptation of the calcium two-step collagenase perfusion technique, as described previously.31Li W.C. Ralphs K.L. Tosh D. Isolation and culture of adult mouse hepatocytes.Methods Mol Biol. 2010; 633: 185-196Crossref PubMed Scopus (195) Google Scholar Cells were plated onto collagen-coated 10-cm dishes (BD Biosciences, San Jose, CA) at a density of 3 × 106 cells. The cultures were maintained in Williams' Medium E medium (Invitrogen) supplemented with Hepatocyte Maintenance Supplement Pack (Invitrogen), 10% fetal calf serum (Sigma-Aldrich), 2 mmol/L l-glutamine (Invitrogen), and antibiotic-antimycotic (Invitrogen). After 2 hours to allow for attachment, the hepatocyte cultures were washed with 1× phosphate-buffered saline and then incubated for 4 hours with media containing 0.50 μg/mL Jo2 plus 10 μg/mL cycloheximide (Sigma-Aldrich). Western blot analysis was performed according to standard procedures. Primary antibodies against caspase-3, caspase-7, caspase-8, cleaved caspase-8, caspase-9, cleaved caspase-9, poly (ADP-ribose) polymerase, NF-κB, p-NF-κB (Ser536), epidermal growth factor receptor (EGFR), Akt, p-Akt (Ser473), 4E-binding protein 1, and p-4E-binding protein 1 were obtained from Cell Signaling Technology (Beverly, MA); primary antibody against glyceraldehyde-3-phosphate dehydrogenase was obtained from Ambion (Austin, TX); goat anti-rabbit or goat anti-mouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). Results were analyzed by LI-COR Odyssey (Lincoln, NE). Nuclear extract was obtained using CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich). Specifically, 100 mg liver tissue was homogenized in 500 μL 1× Lysis Buffer (containing 1 mmol/L dithiothreitol and protease inhibitor cocktail) using the PT 1200 E Homogenizer (Kinematica, Bohemia, NY) until >90% of the cells are broken and nuclei are visualized under a microscope. The samples were then centrifuged at 11,000 × g for 20 minutes at 4°C. The supernatant was transferred to a fresh tube (collected as cytoplasmic fraction). The crude nuclei pellets were resuspended in 0.67× packed cell volume of Extraction Buffer (containing 1 mmol/L dithiothreitol and protease inhibitor cocktail), followed by homogenization using a 27-gauge needle (BD) on ice. The samples were shaken gently for 30 minutes and then centrifuged at 20,000 × g for 5 minutes at 4°C; the supernatant was collected as nuclear extract. Biotinylated and nonbiotinylated EGFR primers were synthesized at IDT. Probes for DNA-protein pull-down assays were generated by annealing biotinylated (or nonbiotinylated) EGFR primers as follows: 5′-end biotinylated EGFR forward primer (5′-biotin-GGAACGCCCC-3′) and 5′-end biotinylated EGFR reverse primer (5′-biotin-GGGGCGTTCC-3′). The primers were diluted to a concentration of 2 μg/μL; 45 μL of each primer was mixed with 10 μL annealing buffer (10 mmol/L HEPES, pH 7.9, 100 mmol/L KCL, 5 mmol/L MgCl2, 10% glycerol, 1 mmol/L dithiothreitol, and 0.5% NP40). The solution was incubated at 95°C for 2 minutes, followed by slowly cooling to room temperature (the nonbiotinylated EGFR forward and reverse primers were annealed through the same process). For DNA-protein pull-down assay, a total of 2 μg nuclear protein was incubated with 1 μg annealed biotin-labeled EGFR probe and 10 μg poly (deoxyinosinic-deoxycytidylic) acid sodium (Sigma-Aldrich) in 500 μL HKMG buffer (10 mmol/L HEPES, pH 7.9, 100 mmol/L KCL, 5 mmol/L MgCl2, 10% glycerol, 1 mmol/L dithiothreitol, and 0.5% NP40) on a rocker platform (VWR, Radnor, PA) at 4°C overnight. As negative control, a total of 2 μg nuclear protein was incubated with 1 μg annealed biotin-labeled EGFR probe, 20 μg annealed non–biotin-labeled EGFR probe, and 10 μg poly (deoxyinosinic-deoxycytidylic) acid sodium (Sigma-Aldrich) in 500 μL HKMG buffer at 4°C overnight. Protein A/G PLUS-Agarose beads (20 μL; Santa Cruz Biotechnology, Dallas, TX) were then added, and the samples were incubated at 4°C for 1 hour. The immunoprecipitates were collected by centrifugation at 1000 × g for 5 minutes at 4°C. The pellets were washed twice with 1 mL 1× phosphate-buffered saline by centrifugation at 1000 × g for 5 minutes at 4°C. After a final wash, the pellet was resuspended in 20 μL 1× electrophoresis sample buffer (Bio-Rad), and the samples were subjected to SDS-PAGE and Western blot analysis. Data are presented as means ± SD. Differences between two groups were determined by a two-tailed t-test. Kaplan-Meier survival analysis (log-rank) was used for mortality analysis. The statistical analyses were performed using Sigma Stat software version 3.5 (Systat Software, Inc., San Jose, CA). P < 0.05 was considered statistically significant. To produce mice with Smo LKO, Smoflox/wt:Alb-Cre+/− mice were crossed with Smoflox/flox mice (Figure 1A). The resulting Smoflox/flox:Alb-Cre+/− offsprings were used as Smo LKO mice; their matched littermates (Smoflox/flox:Alb-Cre−/− and Smoflox/wt:Alb-Cre−/−) were used as controls. To assess whether liver-specific deletion of Smo would affect Fas-induced acute liver injury, 8-week–old male Smo LKO and matched control mice were injected intraperitoneally with a single dose of the anti-Fas antibody Jo2 (0.30 μg/g of body weight), and the animals were closely monitored for survival. The Smo LKO mice exhibited significantly higher mortality compared with the control mice (P = 0.01) (Figure 1B). To further document the extent of liver injury, separate groups of Smo LKO and matched control mice were subjected to Jo2 treatment (at the same dose), and the animals were sacrificed at 3 hours to obtain blood and liver tissue samples. Although the livers of control mice appeared normal on gross examination, the livers of Smo LKO mice turned dark red (Figure 1C). Histologic examination of the liver tissues under hematoxylin and eosin staining revealed more prominent parenchymal hemorrhage and more hepatocyte apoptosis in the livers of Smo LKO mice compared with the livers of control mice (Figure 1C). Accordingly, the levels of serum aspartate aminotransferase and alanine aminotransferase in Jo2-treated Smo LKO mice were significantly higher than in Jo2-treated control mice (Figure 1D). These findings demonstrate that Smo deletion in hepatocytes enhances Fas-induced liver injury. Activation of Fas by its ligand (or antibody Jo2) is known to cause the formation of the death-inducing signaling complex, which recruits and activates caspase-8, leading to subsequent activation of caspase-3/7 and caspase-9; such caspase cascade activation finally results in apoptosis. Therefore, apoptotic parameters were further analyzed in the liver tissues harvested from Jo2-treatd Smo LKO and control mice (3 hours after Jo2 injection). The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling–positive hepatocytes in Smo KO livers was significantly higher compared with control livers (Figure 2A). Under immunohistochemical staining for cleaved caspase-3, the Smo KO livers showed a higher percentage of cleaved caspase-3–positive hepatocytes and stronger staining intensity compared with the control livers (Figure 2B). Under Western blot analysis, the Smo KO livers showed more prominent cleavage of caspases 3/7, 8, and 9 and poly (ADP-ribose) polymerase compared with the control livers (Figure 2C). These findings indicate more prominent caspase activation in the liver tissues from Jo2-treated Smo LKO mice compared with Jo2-treated control mice. Although Hh signaling is inactive in normal adult livers, this pathway becomes activated during acute and chronic liver injuries, manifested by enhanced production of Hh ligands and their target genes4Omenetti A. Yang L. Li Y.X. McCall S.J. Jung Y. Sicklick J.K. Huang J. Choi S. Suzuki A. Diehl A.M. Hedgehog-mediated mesenchymal-epithelial interactions modulate hepatic response to bile duct ligation.Lab Invest. 2007; 87: 499-514Crossref PubMed Scopus (155) Google Scholar, 7Jung Y. Witek R.P. Syn W.K. Choi S.S. Omenetti A. Premont R. Guy C.D. Diehl A.M. Signals from dying hepatocytes trigger growth of liver progenitors.Gut. 2010; 59: 655-665Crossref PubMed Scopus (135) Google Scholar, 13Ochoa B. Sy" @default.
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• W2896226433 title "Adult Hepatocytes Are Hedgehog-Responsive Cells in the Setting of Liver Injury" @default.
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• W2896226433 doi "https://doi.org/10.1016/j.ajpath.2018.07.018" @default.
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