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0The American Journal of Pathology, Vol. 182, No. 3, March 2013
The American Journal of
PATHOLOGY
ELSEVIER -
ajp.amjpathol.org
GASTROINTESTINAL, HEPATOBILIARY, AND PANCREATIC PATHOLOGY
Absence of Nogo-B (Reticulon 4B) Facilitates Hepatic Stellate Cell Apoptosis and Diminishes Hepatic Fibrosis in Mice
Keitaro Tashiro,* Ayano Satoh,t Teruo Utsumi,* Chuhan Chung,* and Yasuko Iwakiri*
From the Section of Digestive Diseases, * Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut; and the Graduate School of Natural Sciences,y Okayama University, Okayama, Japan
Accepted for publication November 26, 2012.
Address correspondence to Yasuko Iwakiri, Ph.D., Section of Digestive Diseases, Yale University School of Medicine, 1080 LMP, 333 Cedar St., New Haven, CT 06520. E-mail: yasuko.iwakiri@yale.edu.
Nogo-B (reticulon 4B) accentuates hepatic fibrosis and cirrhosis, but the mechanism remains unclear. The aim of this study was to identify the role of Nogo-B in hepatic stellate cell (HSC) apoptosis in cirrhotic livers. Cirrhosis was generated by carbon tetrachloride inhalation in wild-type (WT) and Nogo-A/B knockout (Nogo-B KO) mice. HSCs were isolated from WT and Nogo-B KO mice and cultured for activation and transformation to myofibroblasts (MF-HSCs). Human hepatic stellate cells (LX2 cells) were used to assess apoptotic responses of activated HSCs after silencing or overexpressing Nogo-B. Livers from cirrhotic Nogo-B KO mice showed significantly reduced fibrosis (P < 0.05) compared with WT mice. Apoptotic cells were more prominent in fibrotic areas of cirrhotic Nogo-B KO livers. Nogo-B KO MF-HSCs showed significantly increased levels of apoptotic markers, cleaved poly (ADP-ribose) polymerase, and caspase-3 and -8 (P < 0.05) compared with WT MF-HSCs in response to staurosporine. Treatment with tunicamycin, an endoplasmic reticulum stress inducer, increased cleaved caspase-3 and -8 levels in Nogo-B KO MF-HSCs compared with WT MF-HSCs (P < 0.01). In LX2 cells, Nogo-B knockdown enhanced apoptosis in response to staurosporine, whereas Nogo-B overexpression inhibited apoptosis. The absence of Nogo-B enhances apoptosis of HSCs in experimental cirrhosis. Selective blockade of Nogo-B in HSCs may represent a potential therapeutic strategy to mitigate liver fibrosis. (Am J Pathol 2013, 182: 786-795; http://dx.doi.org/10.m6/jMjpath.2012.11.032)
Liver fibrosis and its end-stage manifestation of cirrhosis represent clinical challenges worldwide. Hepatic stellate cell (HSC) activation is the cardinal feature that results in hepatic fibrosis. When stimulated by reactive oxygen species or cytokines in response to various hepatic insults, quiescent HSCs are transformed to myofibroblasts (MF-HSCs) that proliferate and secrete collagen.1-4 Studies have shown that apoptosis of activated HSCs can reverse fibrosis.5-13 Thus, the mechanisms that control MF-HSC apoptosis may represent potential therapeutic targets that result in reduced
fibrosis.14e16
Nogo-B, also known as reticulon 4B, is a member of the reticulon protein family that is localized primarily to the endoplasmic reticulum (ER).17,18 Four groups of reticulons (1, 2, 3, and 4) exist, and each has multiple isoforms. Reticulon 4 has three isoforms, Nogo-A, B, and C. The most recognized isoform, Nogo-A (200 kDa), a potent neural
Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2012.11.032
outgrowth inhibitor,19 21 is expressed mainly in the nervous system.22-24 Nogo-C (25 kDa) is highly expressed in the
17,18,22
differentiated muscle fibers and somewhat in the brain, however, its function remains unclear.
Nogo-B (55 kDa), a splice variant of Nogo-A, is expressed in most tissues and has been reported for its role in modulating endothelial and smooth muscle cellular responses after injury in a variety of organs/tissues, including blood vessels,25,26
27,28 29 30
lung, kidney, and liver. We previously showed that the absence of Nogo-B in a murine model blocks the progression of fibrosis/cirrhosis and the development of portal hyper-tension.30 Further, we showed that lack of Nogo-B decreases the levels of a-smooth muscle actin (a-SMA), a marker of
Supported by grants R01DK082600 and a Yale Liver Center Pilot Project Award (P30-34989) from the NIH (Y.I.), a Female Researcher Science grant from Shiseido Japan (A.S.), and a VA merit award (C.C.).
MF-HSCs, in murine cholestatic livers. These findings led us to hypothesize that absence of Nogo-B may increase the susceptibility of MF-HSCs to apoptosis, thereby reducing fibrosis/ cirrhosis in mice. In this study, we investigated the role of Nogo-B in MF-HSC apoptosis in vivo and in vitro.
Materials and Methods
Animals
All animal studies were approved by the Institutional Animal Care and Use Committees of Yale University and the Veterans Affairs Connecticut Health Care System. Studies were performed in adherence with the NIH Guide for the Care and Use of Laboratory Animals. Nogo-A/B knockout (Nogo-B KO) mice were a gift from Stephen Strittmatter (Yale University, New Haven, CT)31 and Mark Tessier-Lavigne (The Rockefeller University, New York, NY).32
Induction of Hepatic Fibrosis and Cirrhosis
Seven male Nogo-B KO and their littermate wild-type (WT) mice at the age of 1 month were exposed to carbon tetrachloride (CCLO by inhalation for 12 weeks.33,34 Phenobarbital (0.35 g/L) was added to the drinking water 3 days before CCl4 exposure to accentuate fibrosis/cirrhosis. Mice were placed in a gas chamber (60 x 40 x 20 cm) under a fume hood and exposed to CCl4 gas three times a week. The duration of CCl4 inhalation was 1 to 2 minutes for the first 3 weeks and was increased to 3 to 5 minutes thereafter. CCl4 exposure was stopped 5 to 7 days before the experiment. Phenobarbital was no longer added to the drinking water once CCl4 exposure ended. Age-matched untreated WT and Nogo-B KO mice were used as treatment controls. Liver samples were isolated and fixed in formalin or directly embedded in optimal cutting temperature compound.
Bile duct ligation also was performed in mice as described.30 Male Nogo-B KO and their littermate WT mice underwent bile duct ligation surgery at 2 months of age.35 Liver samples from these mice were collected 4 weeks after surgery and directly embedded in OCT for histologic analyses.
Sinus Red Staining
Histologic specimens were embedded in paraffin and cut into 6-mm—thick sections. Sections were deparaffinized by washing in xylene three times for 5 minutes each time, and rehydrated with 100%, 90%, and 70% ethanol for 5 minutes each time. After rinsing in distilled water for 5 minutes, sections were incubated in 0.1% Sinus Red solution (Sigma-Aldrich, St. Louis, MO) for 90 minutes, soaked in 0.5% acetic acid buffer, and dehydrated gradually with 70%, 90%, and 100% ethanol, and washed three times with xylene for 5 minutes each. Fibrosis was determined by calculating the percentage of Sinus red—positive area (ie, collagen-positive area) over the total area analyzed. ImageJ 1.43u software
(Wayne Rasband, NIH, Bethesda, MD) was used for image analysis of the entire liver sections. At least 20 images per liver section were taken randomly and used for the analyses.30
Hydroxyproline Assay
Hydroxyproline levels were measured as described.30 Briefly, frozen liver tissues were homogenized in 6 N HCl and heated at 110°C in a heating block for 20 hours. After cooling, the samples were filtered and neutralized with 2.2% NaOH in citrate acetate buffer. Chloramine-T solution (0.141 g chloramine-T, 2 mL H2O, 3 mL methoxyethanol, and 5 mL citrate acetate buffer) was added to neutralized homogenate, standard hydroxyproline solution, or citrate acetate buffer, and incubated for 20 minutes at room temperature. Then, perchloric acid was added to the reaction mixture and incubated for 20 minutes. Finally, dimethyl benzaldehyde solution (2 g dimethyl benzaldehyde in 10 mL methoxyethanol) was added and incubated at 60°C for 20 minutes. After cooling, the absorbance was measured at 550 nm by the Synergy 2 Multi-Mode Microplate reader (BioTek Instruments, Winooski, VT).
Quantitative Real-Time PCR Analysis
Quantitative real-time PCR analysis was performed as described.30 Briefly, the total RNA from approximately 50 mg of frozen mouse livers was isolated using TRIzol reagent (Sigma-Aldrich). One microgram ofthe total RNA was used as a template to synthesize cDNA using a Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics). Then, cDNA was diluted 5 times to be used as a real-time PCR template. Real-time PCR was performed using ABI 7500 SDS software version 1.3 (Applied Biosystems, Foster City, CA). TaqMan gene expression assays (Applied Biosystems) were used for measuring glyceraldehyde-3-phosphate dehydrogenase (Mm99999915_g1), collagen type Ia (Mm00801666_g1), and transforming growth factor-b>1 (Mm01178820_m1) gene expression.
Immunohistochemistry
Paraffin-embedded sections (6 mm thick) were deparaffinized and rehydrated as described earlier. Antigen retrieval was performed by placing sections in 10 mmol/L sodium citrate buffer (pH 6.0) then heated in a microwave, placed in a steamer for 30 minutes, and steadily cooled down on the bench top for 20 minutes. Sections then were treated with 3% hydrogen peroxidase diluted with methanol, followed by blocking with 5% donkey serum plus 1% bovine serum albumin in PBS. After blocking nonspecific biotin and avidin using a kit (avidin/biotin blocking kit; Vector Laboratories, Burlingame, CA), sections were incubated overnight at 4°C with cleaved caspase-3 antibody (rabbit, 1:100; Cell Signaling, Danvers, MA). After washing three times with Tris-buffered saline with 0.05% Tween 20 (TBST) for 5 minutes, sections were incubated with biotinylated anti-rabbit IgG (1:500; Jackson
ImmunoResearch Laboratories, West Grove, PA) and avidin-conjugated horseradish peroxidase (Vector Laboratories) for 30 minutes each at room temperature. After washing three times with TBST, sections were incubated with 3,3'-dia-minobenzidine substrate (Vector Laboratories) for color development, followed by counterstaining with hematoxylin. Sections then were dehydrated and mounted. Images were taken using a light microscope (Eclipse 80i; Nikon, Melville, NY) and analyzed by Image J 1.43u software.
Dual Staining of TUNEL and a-SMA
OCT-embedded frozen liver tissues were cut into 6-mm-thick sections and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature, followed by washing with PBS three times for 5 minutes each. Antigen retrieval was performed by placing sections in 10 mmol/L sodium citrate buffer at pH 6.0, then heated in a microwave, placed in a steamer for 30 minutes, and steadily cooled down on the bench top for 20 minutes. Sections were incubated with a blocking buffer including 5% donkey serum plus 1% bovine serum albumin in PBS-0.3% Triton X-100 for 1 hour, followed by treatment with mouse Ig blocking reagent (Vector Laboratories) for 1 hour at room temperature. Sections then were incubated with a mouse monoclonal anti-a-SMA (1:1000; Sigma-Aldrich) at room temperature for 1 hour. After washing three times with TBST for 5 minutes each, sections were incubated with Alexa Fluor 555 donkey anti-mouse IgG (1:500; Invitrogen, Grand Island, NY) for 30 minutes at room temperature. After these processes, TUNEL staining was performed using a commercial kit (In Situ Cell Death Detection Kit; Roche Diagnostics, Indianapolis, IN) for 1 hour at room temperature. After washing three times with TBST for 5 minutes each, sections were mounted with DAPI-containing media (Invitrogen). Images were taken with a fluorescent microscope (Eclipse E800; Nikon) and recorded using Open-lab3 software version 5.5.2 (PerkinElmer, Waltham, MA).
Isolation of HSCs
Primary HSCs were isolated from WT and Nogo-B KO mice by in situ perfusion of livers with pronase-collagenase, followed by density gradient centrifugation using Nycodenz (Histodenz; Sigma-Aldrich) density gradients as described.36,37 HSCs were cultured in Dulbecco's modified Eagle's medium with high glucose (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 1% L-glutamine in humidified air containing 5% CO2 at 37°C. HSCs were cultured for more than 14 days to fully transform to MF-HSCs.38
Treatment of MF-HSCs with STS, FAS Ligand, and Tunicamycin
MF-HSCs were seeded in 6-well tissue culture plates at a density of 2.0 x 105 cells per well and incubated in
humidified air containing 5% CO2 at 37°C overnight. Media was changed to serum-free conditions in DMEM for 24 hours, and MF-HSCs were treated with 1 mmol/L staurosporine (STS; EMD Millipore, Billerica, MA) for 0, 2, 4, 6, 8, and 10 hours to induce apoptosis. To determine whether apoptosis was Fas-receptor—dependent, MF-HSCs were treated with 50 ng/mL Fas ligand (Calbiochem) in the presence of 20 ng/mL cycloheximide (Calbiochem) for 10 hours. To induce ER stress, MF-HSCs were treated with tunicamycin at concentrations of 0, 0.5, 1, 2, 5, and 10 mmol/L for 24 hours.
Hepatocyte Isolation
Hepatocytes were isolated from WT and Nogo-B KO mice by collagenase perfusion as previously described39 with slight modifications. Briefly, cells were cultured on collagen-coated cell culture dishes or glass coverslips in Hepatocyte Maintenance Medium (Clonetics/Lonza, Basel, Switzerland) supplemented with Hepatocyte Maintenance Medium Sin-gleQuots (Clonetics/Lonza) and Matrigel (BD Biosciences, San Jose, CA). An initial coating density was 0.4 x 106/mL. On the following day, cells were replaced with medium without any supplementation and incubated for 24 hours. Cells then were subjected to experiments for STS treatment.
Western Blot Analysis
After drug treatment, cells were collected in a lysis buffer containing 50 mmol/L Tris-HCl, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA, 0.1% SDS, 0.1% deoxycholic acid, 1% (vol/vol) Nonidet P-40, 5 mmol/L sodium fluoride, 1 mmol/L sodium pyrophosphate, 1 mmol/L activated sodium vanadate, 0.32% protease inhibitor cocktail (Roche Diagnostics), and 0.027% Pefabloc (Roche Diagnostics). Lysates were centrifuged at 14,000 x g at 4°C for 10 minutes. Protein concentration was determined using a Lowry assay. An equal amount of protein (10 to 20 mg) from each sample was loaded onto SDS-PAGE gels and transferred to 0.2-mm nitrocellulose membranes (BioRad, Hercules, CA). After blocking with 5% nonfat dry milk in 0.1% TBST, membranes were probed with rabbit anti-Nogo serum (1761A, 1:10,000; a kind gift from Dr. William C. Sessa, Yale University, New Haven, CT), goat anti-Nogo (N-18, 1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti—heat shock protein 90 (1:1000; BD Biosciences), mouse anti-Bip (1:1000; BD Biosciences), rabbit anti—poly (ADP-ribose) polymerase (PARP) (1:1000; Cell Signaling), rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling), rabbit anti-cleaved caspase-8 (1:1000; Cell Signaling), rabbit anti— B-cell lymphoma-extra large (1:1000; Cell Signaling), mouse anti—caspase-9 (1:1000; Cell Signaling), or mouse anti— ß-actin (1:5000; Sigma-Aldrich). After washing three times with TBST for 10 minutes, membranes were incubated with fluorophore-conjugated secondary antibodies (either 680-nm or 800-nm emission). Detection and quantification of bands were performed using the Odyssey Infrared Imaging System (Li-Cor
Biotechnology, Lincoln, NE). Heat shock protein 90 and b-actin were used for loading controls.
TUNEL and Annexin V Staining
MF-HSCs were fixed with 4% paraformaldehyde in PBS for 1 hour at room temperature, followed by washing with PBS three times for 5 minutes each time. MF-HSCs then were incubated with a permeabilization buffer containing 0.1% Triton X-100 in 0.1% sodium citrate on ice for 5 minutes. TUNEL staining was performed using a commercial kit (In Situ Cell Death Detection Kit; Roche Diagnostics) by incubating MF-HSCs for 1 hour at room temperature. Recombinant DNase I (3 U/mL; Roche Diagnostics) was incubated for 10 minutes as a positive control before labeling procedures. The label solution conjugated with fluorescein alone was used as a negative control. After washing three times with PBS for 5 minutes each time, MF-HSCs were mounted with a mounting media containing DAPI (Invi-trogen). Annexin V staining was performed using Alexa Fluor 488—conjugated Annexin V/Dead Cell Apoptosis Kit (Life Technologies). Images were taken using a fluorescent microscope (Eclipse E800; Nikon) and recorded with Openlab3 software (PerkinElmer).
Nogo-B Knockdown in Human Hepatic Stellate Cell Line (LX2)
LX-2 cells were a kind gift from Dr. Scott L. Friedman (Mount Sinai School of Medicine, New York, NY).40 Cells were seeded on 6-well tissue culture plates at a density of 1.5 x 105 cells per well and incubated in humidified air containing 5% CO2 overnight at 37°C. Cells were trans-fected with 100 nmol/L Nogo-B small-interfering RNA in 750 mL Opti-MEM (Gibco, Invitrogen) with 2 mL Oligo-fectamine (Invitrogen) and incubated for 6 hours. Then, 750 mL of DMEM containing 20% FBS and 2% L-glutamine was added to each well. After 48 hours of incubation, these cells were starved in DMEM without FBS for 24 hours and treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours.
Nogo-B Overexpression in LX2
LX2 cells were seeded in 12-well tissue culture plates at a density of 1.0 x 105 cells/well and incubated in humidified air containing 5% CO2 overnight at 37°C. Cells were transfected with 0.5 mg of hemagglutinin-tagged Nogo-B plasmid (or pcDNA3 vector alone as a negative control) in 500 mL Opti-MEM (Gibco, Invitrogen) with 1.5 mL FuGENE 6
Figure 1 Lack of Nogo-B decreases liver fibrosis and increases apoptotic cells in fibrotic areas in mice. A: Representative images of Sirius Red staining. Inset corresponds to F. B: Percentages of Sirius Red—positive areas in cirrhotic livers from WT and Nogo-B KO mice. C: Hydroxyproline levels in control and cirrhotic livers isolated from WT and Nogo-B KO mice. D: Collagen 1a1 expression in control and cirrhotic livers isolated from WT and Nogo-B KO mice. E: Transforming growth factor-b1 (TGF-b1) expression in control and cirrhotic livers isolated from WT and Nogo-B KO mice (n = 7 per group). Values represent means ± SE. *P < 0.05, **P < 0.01. F: Immunohisto-chemistry of cleaved caspase-3 in cirrhotic livers isolated from WT and Nogo-B KO mice. Red arrowheads in the enlarged images (lower panel) indicate cleaved caspase-3—positive cells in the fibrotic area. Serial liver sections from the same mice were stained for Sirius Red and cleaved caspase-3. At least five images were taken for each group. G: Co-localization of apoptotic cells with aSMA in cirrhotic livers isolated from WT and Nogo-B KO mice. a-SMA is shown in green, apoptotic (TUNEL-positive) cells in red, and nucleistained with DAPI in blue. Arrows indicate apoptotic cells positive with a-SMA. Scale bars: 100 mm.
Figure 2 Lack of Nogo-B facilitates apoptosis of mouse MF-HSCs in vitro. MF-HSCs were treated with 1 mmol/LSTS for 16 hours to induce apoptosis. A: TUNEL staining (green, TUNEL-positive nuclei; blue, DAPI and percentages of TUNEL-positive nucleiin WT and Nogo-B KO MF-HSCs). Representative images are shown from three independent experiments. B: Annexin V staining (green, Annexin V-positive nuclei;blue, DAPI and percentages of Annexin V-positive nucleiin WT and Nogo-B KO MF-HSCs). The numbers of TUNEL- and Annexin V-positive nuclei(green) were divided by the total numbers of nuclei determined by DAPI staining (blue). At least five images were taken for each group. Representative images are shown from three independent experiments. Values represent means ± SE. **P < 0.01. Scale bars: 100 mm.
(Roche Diagnostics) and incubated for 6 hours. Then, 500 mL of DMEM containing 10% FBS and 1% L-glutamine was added to each well. After 48 hours of incubation, cells were starved in DMEM without FBS for 24 hours, followed by treatment with 100 nmol/L STS for 4 and 8 hours, respectively.
For immunocytochemistry, cells were washed with cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. After washing three times with PBS for 5 minutes each time, cells were incubated in a buffer containing 0.3% Triton X-100, 5% donkey serum,
and 1% bovine serum albumin in PBS at room temperature for 1 hour. After washing with PBS, cells were incubated with antibodies including rat anti-HA (1:1000; Roche Diagnostics) and rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling), which were diluted with 1% bovine serum albumin in PBS overnight at 4°C. Cells then were incubated with Alexa Fluor 488 donkey anti-rat IgG (1:500; Invi-trogen) or Alexa Fluor 588 donkey anti-rabbit IgG (1:500; Invitrogen) as a secondary antibody for 1 hour at room temperature. Cells were mounted with DAPI-containing
Figure 3 Markers of apoptosis are increased in mouse MF-HSCs that lack Nogo-B. MF-HSCs were treated with 1 mmol/LSTS for 0, 2, 4, 6, 8, and 10 hours. A: Representative Western blot analysis of apoptotic markers, including cleaved PARP;cleaved caspase-3, -8, and -9;and Bcl-xL. b-actin was used as a loading control. B: Quantification of Western blot analysis from at least three to five independent experiments. Values represent means ± SE. *P < 0.05, **P < 0.01.
Figure 4 Lack of Nogo-B does not influence hepatocyte apoptosis in response to STS. Hepatocytes were isolated from WT and Nogo-B KO mice and treated with 10 mmol/L STS for 0, 2, 4, 6, 8, and 10 hours. Heat shock protein 90 (Hsp90) was used as a loading control. Representative images are shown from two independent experiments.
media (Invitrogen). Images were taken by a fluorescent microscope (Eclipse E800; Nikon) and recorded using Openlab3 software (PerkinElmer).
Statistical Analysis
Data were expressed as means ± SE. Statistical differences among the mean values of multiple groups were determined using analysis of variance followed by the Student's t-test. P values < 0.05 were considered statistically significant.
Results
Lack of Nogo-B Reduces Fibrosis and Facilitates Apoptosis in Fibrotic Areas of the Mouse Cirrhotic Liver
Sirius Red staining was performed in cirrhotic livers isolated from WT and Nogo-B KO mice that underwent CCl4 inhalation for 12 weeks (Figure 1A). Liver fibrosis was reduced significantly in Nogo-B KO livers compared with WT livers (Figure 1, A and B) (P < 0.05). Consistently, hydroxyproline levels were significantly lower in Nogo-B KO livers than in WT livers (Figure 1C) (P < 0.05). Collagen 1 a1 expression also was significantly lower in Nogo-B KO livers (Figure 1D) (P < 0.05), whereas transforming growth factor-b1 expression did not differ significantly between the two groups (Figure 1E). Apoptotic cells, as indicated by cleaved caspase-3 immunolabeling, were seen in fibrotic regions of Nogo-B KO livers, whereas they were much less apparent in WT livers (Figure 1F). Nogo-B KO cirrhotic livers showed co-localization of TUNEL staining with a-SMA—positive cells, whereas TUNEL staining was not apparent in WT cirrhotic livers (Figure 1G and Supplemental Figure S1). These results suggest that the lack of Nogo-B reduces fibrosis and enhances apoptosis of activated HSCs in experimental cirrhosis.
Lack of Nogo-B Facilitates Apoptosis of MF-HSCs
The role of Nogo-B in apoptosis of MF-HSCs was examined using STS, an inducer of apoptosis.41—43 WT and Nogo-B KO MF-HSCs were treated with 1 mmol/L STS for 16 hours and examined for TUNEL and Annexin V labeling. Nogo-B KO MF-HSCs showed significantly higher percentages of TUNEL- and Annexin V—positive nuclei compared with WT MF-HSCs (Figure 2, A and B) (P < 0.01).
We also determined the levels of several apoptotic markers in WT and Nogo-B KO MF-HSCs after treatment with 1 mmol/L STS for 0, 2, 4, 6, 8, and 10 hours (Figure 3). Levels of cleaved PARP were increased in a time-dependent manner in both WT and Nogo-B KO MF-HSCs, but were significantly higher in Nogo-B KO MF-HSCs 4, 6, and 10 hours after STS treatment (P < 0.05 for 4 and 6 hours and P < 0.01 for 10 hours) (Figure 3B). Similarly, cleaved caspase-3 levels were increased significantly in Nogo-B KO MF-HSCs 4, 6, and 8 hours after STS treatment (P < 0.05 for 4 and 6 hours and P < 0.01 for 8 hours) (Figure 3B). The levels of cleaved caspase-8 also were increased significantly in Nogo-B KO MF-HSCs 4, 6, and 8 hours after STS treatment (P < 0.05) (Figure 3B). Cleaved caspase-9 and Bcl-xL levels did not differ between these two groups at all time points examined (Figure 3B). This may reflect the fact that Bcl-xL primarily acts on mitochondria and prevents apoptosis by inhibiting the activity of caspase-9.44,45 These results indicate that the lack of Nogo-B facilitates apoptosis of MF-HSCs in response to STS.
Lack of Nogo-B Does Not Influence Hepatocyte Apoptosis in Response to STS
To investigate the possibility that Nogo-B may modulate apoptosis in hepatocytes, hepatocytes from WT and Nogo-B
Figure 5 Knockdown of Nogo-B increases apoptosis in human hepatic stellate cells (LX2). LX2 cells with or without Nogo-B siRNA were treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours. Western blot analysis for apoptotic markers, including cleaved PARP, cleaved caspase-3 and -9, and Bcl-xL, was performed. b-actin was used as a loading control. Representative images are shown from two independent experiments.
KO mice were treated with STS (Figure 4). Hepatocytes required a higher dose of STS than MF-HSCs to induce apoptosis (10 versus 1 mmol/L, respectively). Moreover, there was no difference in the levels of apoptotic markers between WT and Nogo-B KO hepatocytes. These results indicate that the absence of Nogo-B preferentially sensitizes MF-HSCs rather than hepatocytes to apoptosis under conditions of experimental cirrhosis.
Knockdown of Nogo-B Increases Apoptosis in Human HSCs (LX2)
To confirm the role of Nogo-B in apoptosis in human MF-HSCs, LX2 cells were transfected with Nogo-B small-interfering RNA to suppress Nogo-B expression. We then tested apoptosis in those cells treated with 100 nmol/L STS for 0, 2, 4, 6, 8, and 10 hours (Figure 5). Nogo-B small-interfering RNA resulted in a 66% reduction of Nogo-B expression. Similar to the results in mouse MF-HSCs, the levels of cleaved PARP and caspase-3 were increased in LX2 cells treated with Nogo-B small-interfering RNA compared with controls, however, the levels of cleaved caspase-9 and Bcl-xL did not differ. Consistent with mouse MF-HSCs, knockdown of Nogo-B resulted in enhanced apoptosis in human MF-HSCs in response to STS.
Overexpression of Nogo-B Blocks Apoptosis in Human HSCs (LX2)
To determine the effect ofNogo-B overexpression on apoptosis of human MF-HSCs, LX2 cells were transfected with HA-tagged human Nogo-B plasmid and treated with 100 nmol/L STS for 8 hours. The transfection resulted in a sevenfold higher level of Nogo-B over the endogenous Nogo-B level found in nontransfected LX2 cells (data not shown). As shown in Figure 6, the majority of HA—Nogo-B—positive cells were
Figure 6 Overexpression of Nogo-B blocks apoptosis in human hepatic stellate cells (LX2). LX2 cells were transfected with HA-tagged human Nogo-B plasmid to overexpress Nogo-B. Cells were treated with 100 nmol/L STS for 8 hours. Representative immunofluorescence images of HA and cleaved caspase-3 in LX2 cells 8 hours after STS treatment (green, HA;red, cleaved caspase-3;blue, DAPI). Arrows indicate HA-Nogo-B-positive cells. Scale bars: 20 mm. Cleaved caspase-3-positive cells were counted in LX2 cells with and without Nogo-B overexpression and divided by the total number of nuclei. At least eight images were taken per group. Values represent means ± SE. **P < 0.01.
negative for cleaved caspase-3. In fact, the percentage of cleaved caspase-3epositive cells was significantly lower in HAeNogo-Bepositive cells than in nontransfected cells (4.2% versus 29.4%, respectively; P < 0.01). These results suggest that MF-HSCs become resistant to apoptosis with Nogo-B overexpression. These data also lend support to our earlier data that the lack of Nogo-B facilitates apoptosis of MF-HSCs.
Nogo-B Is Involved in ER Stress-Induced Apoptosis in MF-HSCs
To examine how Nogo-B influences apoptosis of MF-HSCs, WT and Nogo-B KO MF-HSCs were treated with tunica-mycin, an ER stress inducer, and Fas ligand, an inducer of apoptosis through the death receptor pathway. Tunicamycin treatment (0, 0.5, 1, 2, 5, and 10 mg/mL for 24 hours) generated significantly higher levels (P < 0.01) of cleaved caspase-3 and -8 in Nogo-B KO MF-HSCs than in WT MF-HSCs at virtually all concentrations examined, whereas the levels of Bip, a marker of the ER stress response, were similar in WT and Nogo-B KO MF-HSCs (Figure 7A). In contrast, Fas ligand treatment (50 ng/mL for 10 hours in the presence of 20 ng/mL cycloheximide) did not affect the levels of apoptotic markers (cleaved PARP and cleaved caspase-3 and -8) between WT and Nogo-B KO MF-HSCs (Figure 7B). In addition, levels of Bcl-xL, which is related to the mitochondrial pathway of apoptosis, were not different between WT and Nogo-B KO MF-HSCs in response to STS (Figure 3B). These results suggest that the higher degree of apoptosis observed in Nogo-B KO MF-HSCs is attributable in part to ER stress-induced apoptosis (Figure 7C).
Discussion
Experimental studies have shown that inducing HSC apoptosis may reduce hepatic fibrosis.46—50 In this study, we discovered that lack of Nogo-B reduces liver fibrosis and enhances apoptosis of myofibroblasts derived from HSCs (MF-HSCs, ie, activated HSCs) in mice that underwent CCU inhalation for 12 weeks. Enhanced apoptosis resulting from the absence of Nogo-B was recapitulated in vitro as well. Cultured myofibroblasts derived from HSCs of Nogo-B KO mice, as well as human hepatic stellate cells (LX2) with Nogo-B gene knockdown, showed a greater degree of apoptosis than their respective controls in response to an apoptotic stimulus. Furthermore, Nogo-B overexpression decreased the susceptibility of LX2 cells to apoptosis. These findings suggest that Nogo-B has an anti-apoptotic effect on MF-HSCs and that the enhanced apoptosis of MF-HSCs lacking Nogo-B may be responsible for the reduced fibrosis observed in the livers of Nogo-B KO mice.
We previously reported that Nogo-B levels are increased in fibrotic areas of human cirrhotic liver specimens as well as in mouse cholestatic models of fibrosis after bile duct
ligation. Nogo-B gene deletion blocks the progression of cirrhosis and portal hypertension, suggesting that Nogo-B promotes liver fibrosis. This profibrotic effect of Nogo-B was mediated through transforming growth factor-b/Smad2 signaling in myofibroblasts.30 This study confirms that Nogo-B facilitates fibrosis in the CCl4 model of cirrhosis and supports our earlier studies that showed a profibrotic role for Nogo-B in the liver. Previous work using Nogo-B KO mice in a kidney injury model observed no significant differences in tissue fibrosis.29 The length of injury and the different organ systems studied may account for these different outcomes.
We also examined pathways through which Nogo-B performs anti-apoptosis of MF-HSCs with a focus on ER stress, Fas ligand, and mitochondrial pathways. Because ER stress induces apoptosis in HSCs12 and Nogo-B regulates ER structure,17,18,28,51 we hypothesized that the anti-apoptotic effect of Nogo-B on MF-HSCs might be mediated by ER stress. In fact, treatment with tunicamycin significantly increased the levels of cleaved caspase-3 and -8 in Nogo-B KO MF-HSCs compared with their WT
counterparts. These facts indicate that the anti-apoptotic role of Nogo-B in MF-HSCs is caused, at least in part, by its involvement in ER stress (Figure 7C). Thus, lack of Nogo-B appears to increase the susceptibility of MF-HSCs to ER stress, thereby predisposing them to apoptosis.
In contrast, our study indicated that the anti-apoptotic role of Nogo-B in MF-HSCs is not likely via the mitochondrial pathway because the level of Bcl-xL, an indicator of mitochondria-mediated apoptosis, did not differ between WT and Nogo-B KO MF-HSCs in response to STS. Although we found significantly higher levels of cleaved caspase-8 in Nogo-B KO MF-HSCs in response to STS, caspase-8 is known to be activated through the ER stress pathway52 and/or the death-receptor pathway.53—55 In addition, treatment with Fas ligand, a pro-apoptotic death ligand, did not generate any difference in the level of cleaved caspase-8 between these two groups of MF-HSCs, ruling out a role for Fas-mediated cell death.
The regulation of HSC apoptosis by Nogo-B appears to involve complex processes. First, ER stress-induced apoptosis takes place through many known and unknown
Figure 7 Lack of Nogo-B increases ER stress-induced apoptosis in mouse MF-HSCs. A: MF-HSCs were treated with tunicamycin (0,0.5,1,2, 5, and 10 mg/mL)for 24 hours. A representative Western blot analysis from at least three to five independent experiments and quantification of cleaved caspase-3 and -8 levels is shown. Bip was used as an ER stress marker and b-actin was used as loading control. Values represent means ± SE. **P < 0.01. B: MF-HSCs were treated with 50 ng/mLFas ligand (FasL) for 10 hours in the presence of 20 ng/mLcycloheximide (CHX), and blotted for cleaved PARP, cleaved caspase-3 and -8, and Nogo-B. b-actin was used as loading control. C: A diagram of apoptotic pathways that includes a putative role of Nogo-B. At least three major apoptotic pathways exist, including the death receptor—, mitochondria-, and ER stress—mediated pathways. Nogo-B reduces MF-HSC apoptosis by protecting cells from ER stress. Bcl-xLis an anti-apoptotic molecule.
pathways involving various ligands, receptors, proteases, and so forth. Second, although Nogo-B resides in the ER and is known to regulate ER structure, the functional roles and mechanisms of Nogo-B in the ER are largely unknown. The current literature examining the role of Nogo-B in ER stress-induced apoptosis is extremely complicated. Nogo-B blocks ER stress-induced apoptosis of smooth muscle cells in the pulmonary artery, but not in smooth muscle cells of the carotid artery.28
The involvement of Nogo-B in apoptosis also has been reported in cancer cells. However, those reports also are conflicting. One study showed increased apoptosis in HeLa-derived D98/AH2 cells transiently transfected with Nogo-B.56 Another study also presented the pro-apoptotic effect of Nogo-B on CGL4 (a HeLa-derived cell line), SaOS-2 (an osteosarcoma cell line), MeWo (a melanoma cell line), HT-1080 (a fibrosarcoma cell line), and HFL (the immortalized, nonmalignant, human fibroblast cell line), by transiently transfecting them with Nogo-B.57 In contrast, Oertle et al58 documented that SaOS-2 stably transfected with Nogo-B does not differ in apoptotic effects from its control. These discrepancies may suggest that timing and length of Nogo-B overexpression are a key to induce apoptosis in certain cancer cells and that those cancer cells with stable overexpression of Nogo-B might develop an adaptive machinery that protects them from apoptosis. Overall, Nogo-B may function as a pro-apoptotic or anti-apoptotic protein, depending on the specific cell type. Further studies are needed to determine detailed mechanisms by which Nogo-B regulates apoptosis.
Apoptosis also can mediate profibrotic responses in the liver depending on the cell type involved. Hepatocyte apoptosis is thought to facilitate fibrosis by triggering and maintaining HSC activation in response to hepatic insults.59 To address this issue, we examined whether the levels of apoptotic markers (cleaved PARP, cleaved caspase-3 and-8, and Bcl-xL) in cultured WT and Nogo-B KO hepatocytes differed in response to STS. No differences in apoptotic activity were observed, indicating that the anti-apoptotic effect of Nogo-B is specific to MF-HSCs.
In conclusion, absence of Nogo-B specifically increases apoptotic responses of MF-HSCs, which is associated with a reduction in hepatic fibrosis. Therefore, Nogo-B may be a potential target for the treatment of liver fibrosis/ cirrhosis.
Acknowledgment
We thank Kathy M. Harry for hepatocyte isolation (Yale Liver Center Cell Isolation Core Facility).
Supplemental Data
Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2012.11.032.
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