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, 188 (11), 2605-2616

Adult Hepatocytes Are Hedgehog-Responsive Cells in the Setting of Liver Injury: Evidence for Smoothened-Mediated Activation of NF-κB/Epidermal Growth Factor Receptor/Akt in Hepatocytes That Counteract Fas-Induced Apoptosis

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Adult Hepatocytes Are Hedgehog-Responsive Cells in the Setting of Liver Injury: Evidence for Smoothened-Mediated Activation of NF-κB/Epidermal Growth Factor Receptor/Akt in Hepatocytes That Counteract Fas-Induced Apoptosis

Ying Wang et al. Am J Pathol.

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.

Figures

Figure 1
Figure 1
Liver-specific deletion of Smo enhances Fas-induced liver injury. A: Schematic diagram showing breeding strategy to generate liver-specific deletion of Smo. Smoflox/flox:Alb-Cre+/− mice were used as Smo LKO mice; their matched littermates Smoflox/flox:Alb-Cre−/− and Smoflox/wt:Alb-Cre−/− were used as controls. B: Survival rates of Smo LKO and control mice. The mice were intraperitoneally injected with 0.30 μg/g of body weight Jo2. The Smo LKO mice exhibited significantly higher mortality (five of seven mice died within 5 hours, two of seven mice survived; mortality rate = 71%) compared with the control mice (one of seven mice died at 7 hours, six of seven mice survived; mortality rate = 14%) (P = 0.01). C: Gross and microscopic images of livers. Smo LKO and control mice were intraperitoneally injected with 0.30 μg/g of body weight Jo2, and the animals were sacrificed 3 hours after Jo2 injection. Although the control livers appeared normal under gross examination, the Smo LKO livers turned dark red because of hemorrhage. Histologic examination showed more prominent liver tissue damage in Smo LKO livers compared with the control livers (hematoxylin and eosin stain). D: Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels increase in Smo LKO mice compared with control mice. Data are expressed as means ± SD (D). n = 7 per group (B); n = 6 per group (C). ∗∗P < 0.01. Original magnification, ×100 (C). WT, wild type.
Figure 2
Figure 2
Smo deletion enhances Fas-induced caspase activation. A:Left panels show terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis of liver tissues from Smo LKO and control mice (sacrificed 3 hours after Jo2 injection). Right panel shows quantification of TUNEL-positive hepatocytes. B: Left panels show immunohistochemical stain for cleaved caspase-3. Right panel shows quantification of cleaved caspase-3–positive hepatocytes. C: Western blot analysis for caspases 3, 7, 8, and 9 and poly (ADP-ribose) polymerase (PARP) in the liver tissues. D: Hh signaling pathway is activated in the control mice but not in Smo LKO mice after Jo2 injection. Real-time quantitative RT-PCR analysis for Smo, Ptch1, and Gli1 mRNAs in the liver tissues. Data are expressed as means ± SD (A, B, and D). P < 0.05, ∗∗P < 0.01. Original magnification, ×100 (A and B). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type.
Figure 3
Figure 3
The impact of Smo deletion on the NF-κB/epidermal growth factor receptor (EGFR)/Akt pathway. Smo LKO and control mice were intraperitoneally injected with 0.30 μg/g of body weight Jo2. The mice were sacrificed 3 hours after Jo2 injection. A: The liver tissue lysates were subjected to Western blot analysis for indicated molecules. B: The liver nuclear extracts were processed for DNA-protein pull-down analysis. 4E-BP1, 4E-binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type.
Figure 4
Figure 4
Smo deletion enhances Fas-induced apoptosis in hepatocytes. Primary hepatocytes were isolated from Smo LKO and control mice. The cells were treated with Jo2 (0.50 μg/mL) plus cycloheximide (CHX; 10 μg/mL) for 4 hours. A:Left panels show representative Hoechst staining. Right panel shows quantitative analysis for apoptotic cells under Hoechst staining. B: Caspase-3/7 and caspase-8 activities. C: Primary hepatocytes were isolated from Smo LKO and control mice, and the cells were treated with 0.50 μg/mL Jo2 plus 10 μg/mL CHX for 2 or 4 hours. Real-time quantitative RT-PCR analysis was performed to determine the mRNA levels of Smo, Ptch1, and Gli1. D: Western blot analysis to detect the levels of indicated proteins in hepatocytes with indicated treatments. Data are expressed as means ± SD (A–C). ∗∗P < 0.01. Original magnification, ×200 (A). 4E-BP1, 4E-binding protein 1; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type.
Figure 5
Figure 5
Overexpression of NF-κB prevents Fas-induced liver injury in Smo LKO mice. A: NF-κB overexpression prevents Fas-induced liver tissue injury in Smo LKO mice. Twenty-four hours after hydrodynamic tail vein injection of NF-κB or control plasmid, the mice were intraperitoneally injected with Jo2 (0.30 μg/g of body weight). Three hours after Jo2 injection, the mice were sacrificed and the liver tissues were obtained and processed for histologic evaluation (hematoxylin and eosin staining). B: Serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) analysis. C: Western blot analysis for poly (ADP-ribose) polymerase (PARP), caspase-8, and caspase-3. D: The efficiency of NF-κB expression in mice receiving tail vein hydrodynamic injection of NF-κB expression plasmid (20 μg/mL). E: Illustration of hepatocyte Hh signaling in Fas-induced liver injury. Data are expressed as means ± SD (B). ∗∗P < 0.01. Original magnification, ×100 (A). EGFR, epidermal growth factor receptor; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type.
Supplemental Figure S1
Supplemental Figure S1
Liver-specific Smo KO exacerbates lipopolysaccharide (LPS)/D-galactosamine (GalN)–induced liver injury. Wild-type (WT) and Smo LKO mice were intraperitoneally injected with LPS (30 ng/g body weight) plus D-GalN (800 μg/g body weight). A: Survival curve of mice after LPS/GalN administration (P < 0.05, log-rank test). B: The mice were sacrificed at 4 or 7 hours after LPS/GalN injection. Representative images of hematoxylin and eosin staining. C: Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Data are expressed as means ± SD (C). n = 6 (A). ∗∗P < 0.01. Original magnification, ×100 (B).
Supplemental Figure S2
Supplemental Figure S2
Liver-specific Smo KO exacerbates lipopolysaccharide (LPS)/D-galactosamine (GalN)–induced liver apoptosis. Wild-type (WT) and Smo LKO mice were intraperitoneally injected with LPS (30 ng/g body weight) plus D-GalN (800 μg/g body weight). The mice were sacrificed at 4 or 7 hours after LPS/GalN injection. A: Immunohistochemical staining of cleaved caspase-3. B: Quantification of cleaved caspase-3–positive hepatocytes. C: Western blot analysis to detect poly (ADP-ribose) polymerase (PARP) and caspase cleavage at 4 and 7 hours after LPS/GalN injection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Data are expressed as means ± SD (B). ∗∗P < 0.01. Original magnification, ×100 (A).

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