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* Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York, USA;
Department of Medicine II, Medical Faculty at Mannheim, University of Heidelberg, Mannheim, Germany;
Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas, USA;
National Institute on Alcohol Abuse and Alcoholism, National Institute of Health, Bethesda, Maryland, USA;
|| Nathan Kline Institute for Psychiatric Research, Orangeburg, New York, USA; and
¶ Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, New York, USA
1Correspondence: Columbia University, Russ Berrie Pavillion, Rm. 415, 1150 St. Nicholas Ave, New York, NY, USA 10032. E-mail: rfs2102{at}columbia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ROS • 2-AG • HSC • hepatocyte • fibrosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
but can also occur independently of these receptors (3
4
5
6
7)
. Endocannabinoids were initially described to play major roles in the central nervous system where they regulate food intake, emotions, pain perception, and sleep (8
9
10)
. More recent studies have shown that endocannabinoids are also involved in the regulation of inflammation, cell death, and peripheral lipogenesis (11
12
13)
. 2-Arachidonoyl glycerol (2-AG) is the best characterized and most potent agonist among the monoacylglycerol class of endocannabinoids. The levels of 2-AG are the highest among all endocannabinoids in humans (14)
. 2-AG evokes different responses in different cell types that include proliferation, growth arrest, decreased cell contractility, cell death, and anti-inflammatory pathways (15
16
17
18)
.
Recently, it has been demonstrated that endocannabinoids are involved in the regulation of fibrogenesis in the liver. CB2–/– mice show increased fibrogenesis in response to CCl4 injection, whereas CB1–/– mice have decreased hepatic fibrogenesis (19
, 20)
. However, the mechanisms by which endocannabinoids regulate liver injury and fibrogenesis are not well characterized. Although it has been suggested that endocannabinoids promote the resolution of hepatic fibrosis by inducing cell death in HSCs, the main fibrogenic cell type in the liver, it remains elusive which endocannabinoids are involved in regulation of hepatic fibrogenesis (6
, 19
20
21)
. Although we and others have shown that anandamide (AEA) induces cell death in hepatic stellate cells (HSCs) (6
, 21)
, the hepatic levels of AEA in vivo are much lower than those of other endocannabinoids, e.g., 2-AG, suggesting less prominent role for this endocannabinoid in the regulation of hepatic fibrogenesis.
In this study, we investigate the effects of 2-AG, the most abundant endocannabinoid in vertebrate animals, on primary HSCs and hepatocytes. 2-AG efficiently induces apoptotic cell death in HSCs but not in hepatocytes. Cell death induced by 2-AG depends on the mitochondrial generation of reactive oxygen species (ROS), suggesting that different levels of antioxidants in HSCs and hepatocytes contribute to their different susceptibility toward 2-AG.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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or CB2–/– mice (23)
(25–30 g, n=4 each, all mice in pure C57Bl/6 background) followed by a Nycodenz (Axis-Shield, Oslo, Norway) two-layer discontinuous density gradient centrifugation as described previously (6)
. Purity of human, rat, and mouse HSCs was consistently >95%, as determined vitamin A fluorescence 2 days after isolation. Rat or mouse HSCs were activated on uncoated plastic tissue culture dishes and used between day 7 and 14 after isolation without any passaging. Human HSCs were cultured under the same condition but passaged up to five times. Primary rat hepatocytes were isolated from male Sprague-Dawley rats (225–250 g, n=10) by collagenase perfusion and cultured as described previously (6)
. All human tissues were obtained by qualified medical staff, with donor consent and the approval of the Ethics Committee of Columbia University. All animal procedures were performed under the guidelines set by the Columbia University Institutional Animal Care and Use Committee and are in accordance with those set by the National Institutes of Health.
Cell treatment and detection of cell death
Hepatocytes were kept in serum-free hormonally defined medium for 12 h before experiments (6)
. HSCs were serum-starved with serum-free Dulbecco's modified Eagle's medium for 12 h. Cells were treated either with 2-AG (Cayman Chemicals, Ann Arbor, MI, USA) or vehicle (ethanol; 0.1% final concentration), or actinomycin D (Sigma, St. Louis, MO, USA) plus recombinant murine or human TNF-
(R&D Systems, Minneapolis, MN, USA). Where indicated, cells were pretreated with the cell-permeable antioxidants glutathione-ethylester (GSH-EE; Sigma), Trolox (Calbiochem-EMD Biosciences, La Jolla, CA, USA), NADPH oxidase inhibitor diphenylene iodonium (DPI; Sigma), 
-glutamyl cysteine synthase inhibitor DL-buthionine-(S,R)-sulfoximine (BSO; Sigma), CB1 antagonist SR141716, CB2 antagonist SR144528 (Sanofi-Synthélabo, Montpellier, France), MGL inhibitor URB 754 (24)
, or cholesterol depletor methyl-ß-cyclodextrin (MCD; Sigma). Cell death was measured by LDH release into the culture medium according to the manufacturer's instructions (Roche, Indianapolis, IN, USA) and visualized by propidium iodide fluorescence (PI; Sigma). Apoptosis was visualized by fluorescent microscopy using an annexin V/PI-staining kit (Roche) according to the manufacturer's instructions.
Hepatic injury and fibrosis models
Hepatic injury and fibrosis in a cholestatic model were induced in mice by ligating the common bile duct as described previously (21
, 25)
. Control animals underwent sham operation. Acute hepatic injury was induced in a model by intraperitoneal injection of CCl4 (0.5 µl/g body weight) dissolved in an equal volume of sterile olive oil (26)
. Controls received oil only. Mice were euthanized 3, 7, or 21 days after BDL or after 1 injection of CCl4.
Adenoviral infection
Adenoviruses expressing monoacylglycerol lipase (MLP; a gift from Dr. Daniele Piomelli, Irvine, CA, USA) or GFP have been previously described (21
, 27)
. Adenoviruses were expanded in 293 cells as described previously (21)
. HSCs were infected with adenoviruses at a multiplicity of infection of 250 particles/cell for 12 h. After further 12 h, cells underwent treatment with 2-AG.
Measurement of hepatic endocannabinoid levels
The levels of the endocannabinoids AEA and 2-AG were measured by liquid chromatography/mass spectrometry according to Wang et al. (28)
in liver tissue from male mice 3 (n=4), 7 (n=3), and 21 days (n=7) after bile duct ligation (BDL) or sham operation (n=6), as well as in liver tissue from CCl4-treated (n=3) mice 3 days after injection or controls (n=4).
Detection of ROS
Serum-starved HSCs (2x104 cells/well) or hepatocytes (5x104 cells/well) plated in 24-well-plates (2 cm2 well area, BD Biosciences, San Jose, CA, USA) were loaded with 4 µM of the redox-sensitive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes, Eugene, OR, USA) for 30 min at 37°C, washed, and stimulated with agonists. ROS formation was measured for the indicated time in a multiwell fluorescence plate reader (Fluostar Optima, BMG) using excitation and emission filters of 485 and 535 nm, respectively.
Confocal microscopy
For detection of mitochondrial ROS production, confocal microscopy was carried out as described previously (29)
. Briefly, HSCs (1x105 cells) were plated on 40 mm diameter glass cover slips. The cells were loaded with 4µM CM-H2DCFDA (Molecular Probes) and 500 nM of the mitochondrial fluorescent dye tetramethyl rhodamine methylester (TMRM; Molecular Probes) for 30 min, washed, and treated with 2-AG or vehicle only. For staining of nuclei, we used Hoechst 33258 (10 µg/ml; Molecular Probes). ROS production was visualized by a Zeiss LSM 510 confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany).
Quantitative real time-PCR analysis
RNA was isolated from serum-starved activated primary rat HSCs (day 7) and primary rat hepatocytes using the TRIzol method (Invitrogen, Carlsbad, CA, USA). After DNase treatment, RNA was reverse transcribed using random hexamer primers. Real time PCR was performed for 40 cycles of 15 s at 95°C and 60 s at 60°C using an ABI 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) as described previously (30)
. Each sample was measured in duplicate, and quantification was performed by comparing the Ct values of each sample to a standard curve. Probes and primers for rat MGL and 18S were designed by ABI. MGL mRNA levels were normalized to 18S and are expressed as fold induction.
Western blot analysis
Electrophoresis of protein extracts and subsequent blotting was performed as described (31)
. Blots were incubated with anti-caspase 3 or anti-PARP antibodies (both Cell Signaling Technologies, Beverly, MA, USA) at a dilution of 1:1000 overnight at 4°C. After incubation with secondary horseradish-peroxidase conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the bands were visualized by the enhanced chemiluminescence light method (Amersham Biosciences, Piscataway, NJ, USA) and exposed to a chemiluminescence imager (Image Station 2000R, Eastman Kodak Co., New Haven, CT, USA). Blots were reprobed with anti-actin mouse antibody (MP Biomedicals, Irvine, CA, USA) to demonstrate equal loading.
Statistical analysis
All data represent the mean of at least three independent experiments ± SEM, if not otherwise stated. For the determination of statistical significance, unpaired Student's t-tests were performed using SigmaStat (SPSS, Chicago, IL, USA). P values of <0.05 were considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 19
20
21)
. We found that 2-AG induced cell death in activated rat HSCs starting at concentrations of 5 µM and induced almost 80% cell death after 18 h of treatment with 25 µM 2-AG (Fig. 1
A). In contrast, primary rat hepatocytes showed no sign of cell death even at 100 µM 2-AG. To determine whether 2-AG-induced cell death was apoptotic or necrotic, we investigated 2-AG-treated HSC for the presence of cleaved caspase 3 and the 89 kDa PARP cleavage product, two hallmarks of apoptotic cell death. We found that 2-AG induced caspase 3- and PARP-cleavage starting 8 h after treatment (Fig. 1B
). To confirm this data, HSCs were stained with a combination of annexin V, which binds phosphatidylserine in the outer membrane leaflet of apoptotic cells and PI, which indicates cell membrane rupture in necrotic cells. After 2-AG treatment, cells showed strong annexin V staining, but no PI staining consistent with apoptotic cell death (Fig. 1C
). In contrast, rat hepatocytes did not show any signs of cell death after 2-AG treatment such as caspase 3 cleavage, PARP cleavage, annexin V or PI staining (Fig. 1B, C
).
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2-AG-induced cell death in HSCs is dependent on ROS generation
After confirming that 2-AG also induces marked cell death in human HSCs (Fig. 2
A), we analyzed whether 2-AG treatment promotes ROS formation, which we had previously shown to be involved in AEA-induced cell death in HSCs (6)
. After 2-AG stimulation, HSCs generated extremely high amounts of ROS in a dose-dependent manner with substantial induction of ROS formation at submicromolar doses of 2-AG (Fig. 2B
). It has been demonstrated that NADPH oxidase induces ROS production in HSCs in response to several mediators (32)
. To investigate whether 2-AG-induced ROS was mediated by NADPH oxidase, we treated HSCs with the NADPH inhibitor DPI and analyzed ROS generation in response to 2-AG. Although the antioxidants Trolox and GSH-EE significantly reduced 2-AG-mediated caspase-3 cleavage, cell death, and ROS production, we detected no reduction of cell death and only a minor reduction of ROS production after DPI pretreatment (Fig. 2C, D
). Since DPI is not an entirely specific inhibitor of NADPH oxidase, we additionally measured 2-AG-induced ROS production and cell death in HSCs isolated from mice that are deficient in p47phox, an essential subunit of NADPH oxidase. Similar to the results obtained with DPI, we could not detect any difference in 2-AG-induced cell death or ROS production in HSCs from p47phox –/– mice (Fig. 2E, F
). To provide further evidence for ROS as key mediator of cytotoxic 2-AG effects, we tested the effects of GSH depletion on 2-AG-induced cell death. We found a small, but significant increase, in ROS production and cell death in HSCs that had been depleted of GSH by BSO pretreatment (Fig. 2G, H
), confirming our hypothesis that ROS mediate cytotoxic 2-AG signals.
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Mitochondria are the predominant source of ROS after 2-AG stimulation
Since mitochondria are known to be capable of generating high amounts of ROS in response to cytotoxic stimuli, we monitored mitochondrial ROS production by confocal microscopy. For this purpose, we loaded HSCs with TMRM, which labels polarized mitochondria, and the redox-sensitive dye CM-H2-DCFDA. Five minutes after stimulating HSCs with 2-AG, we observed a speckled pattern of ROS production that largely colocalized with TMRM-labeled mitochondria (Fig. 3
) but not with membrane-bound NADPH oxidase. After 15 min, DCFDA fluorescence showed a diffusely cytoplasmic and nuclear pattern, suggesting that ROS leaked from the mitochondria.
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2-AG-induced cell death in HSCs is not mediated by CB1 or CB2 receptors, but depends on membrane cholesterol
Next, we investigated which cannabinoid receptor mediates HSC death in response to 2-AG using both pharmacologic and genetic approaches. 2-AG-induced cell death was neither blocked by SR141716, a specific antagonist of CB1, nor by SR144528, a specific antagonist of CB2 (Fig. 4
A). Moreover, pretreatment of HSCs with these CB receptor antagonists before 2-AG exposure did not decrease the amount of 2-AG-induced ROS (Fig. 4B
). Accordingly, activated primary HSCs isolated from wildtype, CB1–/– and CB2–/– mice were equally susceptible toward 2-AG-induced cell death (Fig. 4E
). We had previously shown that membrane cholesterol depletion prevented AEA-induced ROS production in HSCs (6)
. When cells were preincubated with the membrane cholesterol depletor MCD, 2-AG-induced ROS production was almost completely blocked and cell death was significantly inhibited (Fig. 4A, C, D
), suggesting that 2-AG and AEA employ similar mechanisms to induce cytotoxic signals, i.e., interaction with cholesterol, at the cell membrane (6)
.
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Intracellular glutathione levels determines 2-AG induced cell death in hepatocytes
We have previously shown that different responses of HSCs and hepatocytes to the endocannabinoid AEA are in part caused by expression levels of the andamide-degrading enzyme fatty acid amide hydrolase. To test whether a difference of 2-AG degradation contributes to the different 2-AG sensitivity of HSCs and hepatocytes, we first measured the expression of the 2-AG degrading enzyme MGL. However, we found no difference of MGL mRNA expression between HSCs and hepatocytes (Fig. 5
A). Next, we overexpressed MGL in HSCs and hepatocytes by adenoviral infection. MGL overexpression was confirmed by quantitative real-time PCR (data not shown). Adenoviral overexpression of MGL did not alter cell death in HSCs or hepatocytes (Fig. 5B, C
) nor did the MGL inhibitor URB754 sensitize hepatocytes to 2-AG-induced cell death (Fig. 5D
), suggesting that MGL is indeed not involved in determining the sensitivity toward 2-AG-mediated cell death. The strong induction of mitochondrial ROS generation in HSCs but not hepatocytes suggested that the resistance of hepatocytes to 2-AG might instead be caused by more powerful antioxidant defense systems. When we treated hepatocytes with 2-AG, we indeed observed only a minor induction of ROS generation. Since glutathione is a crucial antioxidant defense system in the mitochondria and its levels are 
10-fold higher in hepatocytes than in HSCs (21)
, we determined whether GSH depletion would sensitize hepatocytes to 2-AG-induced cell death. After pretreatment with BSO, we indeed observed not only a significant induction of cell ROS production in hepatocytes but also induction of cell death at concentrations of 25 µM and 100 µM 2-AG (Fig. 5E, F
). Hepatocytes treated with BSO plus 2-AG displayed a cell morphology typical of necrosis and were all PI-positive (Fig. 5E
). Accordingly, there was no caspase 3- or PARP-cleavage after 2-AG, suggesting that 2-AG induces necrosis and not apoptosis in GSH-depleted hepatocytes (Fig. 5G, H
).
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Hepatic levels of 2-AG are significantly increased during hepatic injury and fibrogenesis
Since the selective induction of apoptosis in HSCs is believed to contribute to the resolution of hepatic fibrosis (33
, 34)
, we analyzed whether hepatic 2-AG levels are elevated during the hepatic wound healing response. For this purpose, mice either underwent ligation of the common bile duct or were treated with a single injection of CCl4. In both models, we observed a significant, 3-fold elevation of 2-AG (Fig. 6
A, B). Hepatic levels of 2-AG in the injured liver were in the micromolar range, suggesting that 2-AG might indeed induce HSC apoptosis under these conditions in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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36
37)
. However, the underlying disease cannot be cured in the majority of patients with hepatic fibrosis. Thus, additional therapeutic approaches are required to inhibit hepatic fibrogenesis. Recently, it was demonstrated that the resolution of hepatic fibrosis correlates with an increase in HSC apoptosis (33)
. Moreover, the active induction of HSC apoptosis, e.g., by gliotoxin or sulfasalazin, accelerates the resolution of hepatic fibrosis (38
, 39)
. Based on these findings, current concepts to treat hepatic fibrosis include targeting of antiapoptotic and profibrogenic pathways in hepatocytes and HSCs, respectively, as well as selective induction of apoptosis in activated HSCs (40)
.
Our study demonstrates that 2-AG induces apoptosis in primary activated HSCs at tissue concentrations that occur during hepatic fibrogenesis in vivo. In contrast to AEA, which predominantly induces necrosis in HSCs (6)
, cell death induced by 2-AG displayed characteristic features of apoptosis such as caspase 3- and PARP-cleavage as well as positive annexin V membrane staining. Our results are similar to a previous study that demonstrated cytotoxic effects of 2-AG in hepatic myofibroblasts (19)
. We report for the first time that 2-AG induces ROS production in target cells and provide two lines of evidence that ROS play an essential role in mediating cell death after 2-AG stimulation: 1) Cell death was blocked by pretreatment with antioxidants such as GSH-EE or Trolox; and 2) Glutathione depletion sensitized cells to 2-AG-induced cell death. These results are consistent with a previous study demonstrating a reduction of 2-AG-induced growth inhibition by antioxidants (41)
but differ from another study that postulated prevention of ROS formation by 2-AG (42)
. However, this study only investigated the effects of 2-AG on H2O2-induced cytoskeletal changes and Ca2+ mobilization, and the effects of 2-AG on ROS production were not quantified. In contrast to the strong ROS induction in HSCs, we observed only minor amounts of ROS in response to 2-AG in primary hepatocytes. Accordingly, hepatocytes were resistant to 2-AG-induced cell death suggesting that 2-AG may be a selective inducer of HSC apoptosis. When depleted of GSH, hepatocytes became sensitive to 2-AG-induced cell death, suggesting that the previously reported higher levels of GSH in hepatocytes are responsible for their resistance to endocannabinoids such as 2-AG and AEA (21)
. However, considering the high amount of 2-AG required to induce cell death in GSH-depleted hepatocytes (starting at 20 µM) and the much lower concentrations of 2-AG found during fibrogenesis in vivo (up to 2.25 µM), it is not very likely that 2-AG causes significant hepatocellular injury.
To further characterize this novel 2-AG-induced death pathway, we investigated the role of CB receptors in 2-AG induced cell death and attempted to determine the source of ROS production in 2-AG stimulated HSCs. Pharmacologic as well as genetic evidence from this study suggest that 2-AG does not mediate cell death through a CB-receptor-dependent pathway. Instead we found that 2-AG induces cell death in HSCs through a pathway that requires the presence of membrane cholesterol, which has previously been shown to be required for AEA-mediated cannabinoid receptor-independent cell death in several cell types including primary HSCs (3
, 5
, 6)
. Thus, it is possible that 2-AG and AEA share a common upstream mechanism for the induction of cell death in HSCs, but differ in their downstream signals. Recent studies have suggested that endocannabinoids such as AEA diffuse into the outer leaflet of the cell membrane before interacting with its targets (43)
. It is conceivable that membrane cholesterol is required either for endocannabinoids to enter the cell membrane or that its targets require lipid-rich microdomains for signaling. 2-AG is a more potent inducer of ROS than AEA. Using confocal microscopy, we demonstrated mitochondria as the main source of ROS production after 2-AG stimulation. Mitochondria are known to be involved in the regulation of apoptosis and necrosis, being both a target of cell death pathways as well as initiators and amplifiers of cell death. At least two components of the permeability transition pore complex (PTPC) such as the adenine nucleotide transporter, the voltage-dependent anion channel and cyclophilin D are targets of ROS (44
, 45)
. An increase of ROS may trigger oxidation of these proteins and opening of the PTPC, leading to mitochondrial permeability transition (MPT), permitting the influx or efflux of any molecule with the molecular weight of 1500 Da (46)
. Under many conditions, the MPT is deleterious to mitochondrial physiology and can lead to cytochrome c release, caspase activation, and cell death (47)
. Our data suggest that 2-AG stimulation leads to mitochondrial ROS generation which then triggers caspase activation and apoptosis. Although our study shows that ROS induces cell death in HSCs, other studies have proposed ROS as mediators of HSC activation and proliferation (25
, 48)
. This apparent contradiction is explained by fact that proproliferative and activating effects of ROS in HSCs are mediated by low-level generation, mostly mediated through NADPH oxidase. In contrast, our study detects high level ROS production after 2-AG treatment that does not depend on NADPH oxidase as shown by pharmacologic and genetic approaches. Thus, ROS production in HSCs may result in different and even opposite biological effects depending on the amount of ROS generation and their localization, i.e., cytosolic, membrane-associated, or mitochondrial.
Our study detected elevated levels of 2-AG in two mouse models of hepatic fibrogenesis, suggesting that 2-AG may indeed act as a potential endogenous antifibrogenic mediator. Compared to hepatic levels of AEA (data not shown), 2-AG not only shows a higher degree of induction but also reaches tissue levels close to those that induce cell death in vitro. 2-AG being lipophilic, its local concentration at the cell membrane is likely higher and well within the range found to cause apoptosis in HSC. Like AEA, 2-AG does not induce cell death in hepatocytes unless they are severely depleted of GSH. In contrast to 2-AG, other endogenous mediators such as TNF- and FasL induce cell death not only in HSCs (49
, 50)
, but also in hepatocytes (51
, 52)
, and are therefore not good candidates for antifibrotic therapy. Moreover, members of the TNF family are also potent inducers of inflammation, which may also be unfavorable for the treatment of hepatic fibrogenesis, whereas 2-AG shows strong antiinflammatory properties under most physiological conditions (53)
. Thus, increasing the levels of 2-AG during fibrogenesis may represent a strategy to promote HSC cell death and resolution of hepatic fibrosis.
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ACKNOWLEDGMENTS |
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Received for publication November 22, 2006. Accepted for publication March 8, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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