Potential conflict of interest: Nothing to report.
Human immunodeficiency virus (HIV) protease inhibitors (HIV PIs) are the core components of highly active antiretroviral therapy, which has been successfully used in the treatment of HIV-1 infection in the past two decades. However, benefits of HIV PIs are compromised by clinically important adverse effects, such as dyslipidemia, insulin resistance, and cardiovascular complications. We have previously shown that activation of endoplasmic reticulum (ER) stress plays a critical role in HIV PI–induced dys-regulation of hepatic lipid metabolism. HIV PI–induced hepatic lipotoxicity is closely linked to the up-regulation of CCAAT enhancer binding protein homologous protein (CHOP) in hepatocytes. To further investigate whether CHOP is responsible for HIV PI–induced hepatic lipotoxicity, C57BL/6J wild-type (WT) or CHOP knockout (CHOP−/−) mice or the corresponding primary mouse hepatocytes were used in this study. Both in vitro and in vivo studies indicated that HIV PIs (ritonavir and lopinavir) significantly increased hepatic lipid accumulation in WT mice. In contrast, CHOP−/− mice showed a significant reduction in hepatic triglyceride accumulation and liver injury, as evidenced by hematoxylin and eosin and Oil Red O staining. Real-time reverse-transcriptase polymerase chain reaction and immunoblotting data showed that in the absence of CHOP, HIV PI–induced expression of stress-related proteins and lipogenic genes were dramatically reduced. Furthermore, tumor necrosis factor alpha and interleukin-6 levels in serum and liver were significantly lower in HIV PI–treated CHOP−/− mice, compared to HIV PI–treated WT mice. Conclusion: Taken together, these data suggest that CHOP is an important molecular link of ER stress, inflammation, and hepatic lipotoxicity, and that increased expression of CHOP represents a critical factor underlying events leading to hepatic injury. (HEPATOLOGY 2013)
Human immunodeficiency virus (HIV) protease inhibitors (HIV PIs), as the key components of highly active antiretroviral therapy (HAART), have been successfully used to reduce the morbidity and mortality of HIV-infected patients during the last two decades. However, long-term use of HIV PI therapy is compromised by serious metabolic side effects, such as dyslipidemia, insulin resistance (IR), and cardiovascular complications. 1-3 Previous studies suggest that HIV PI–induced endoplasmic reticulum (ER) stress response and subsequent activation of unfolded protein response (UPR) represent important cellular signaling mechanisms of HIV PI–induced metabolic syndromes. 4-10
Emerging evidence indicates that ER stress contributes to the pathogenesis of metabolic and various other liver diseases. 11-13 CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP), also known as growth-arrest– and DNA-damage–inducible gene 153, is a major transcription factor involved in ER stress-mediated apoptosis. 14 Recent studies have shown that CHOP is an important regulator of lipotoxicity and oxidative damage in various types of cells. 15-20 In addition, our previous studies demonstrated that activation of ER stress and up-regulation of CHOP expression are closely linked to HIV PI–induced inflammatory response and foam-cell formation in macrophages. 5, 7, 8 It also has been reported that CHOP deficiency improved beta-cell ultrastructure and promoted cell survival in both genetic and diet-induced mouse models of IR, 19 and that ER stress/CHOP/Bax-mediated apoptosis in macrophages contributed to the instability of atherosclerotic plaques. 17 However, whether CHOP is central in HIV PI–induced hepatic lipotoxicity has not been fully investigated.
The aim of the present study was to examine the role of CHOP in HIV PI–induced dys-regulation of hepatic lipid metabolism and to further identify the potential underlying mechanisms. Wild-type (WT) C57BL/6J mice and CHOP−/− mice as well as isolated mouse primary hepatocytes (MPH) were used to determine whether the absence of CHOP could prevent HIV PI–induced dyslipidemia and hepatic lipotoxicity. The results indicate that CHOP not only contributed to HIV PI–induced dyslipidemia and hepatic lipid accumulation, but also to HIV PI–induced inflammatory response. The identification of a key role of CHOP in ER stress-mediated hepatic lipotoxicity provides novel insights into potential avenues to develop therapeutic strategies for treatment of ER stress-mediated hepatic lipotoxicity associated with drug-induced liver injury and various metabolic diseases.
Materials and Methods
Antibodies against CHOP, activating transcription factor 4 (ATF4), X-box-binding protein 1 (XBP-1), lamin B, horseradish peroxidase (HRP)-conjugated donkey antigoat immunoglobulin G (IgG), and HRP-conjugated goat antirabbit IgG were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibody against β-actin was from Calbiochem (San Diego, CA). Bio-Rad protein assay reagent, Criterion XT Precast Gel, 30% Acrylamide/Bis-acrylamide mix, TEMED, and Precision Plus Protein Kaleidoscope standards were obtained from Bio-Rad Laboratories (Hercules, CA). High-capacity complementary DNA (cDNA) Reverse Transcription kits were from Applied Biosystems (Foster City, CA). The SV Total RNA Isolation System was from Promega (Madison, WI). The ApoAlert Annexin V kit was from BD Bisociences (San Jose, CA). L-type triglycyeride (TG) H, and Cholesterol E kits were from Wako Diagnostics (Richmond, VA). O.C.T gel was from Sakura Finetek (Torrance, CA). Mouse tumor necrosis factor alpha (TNF-α) and mouse interleukin (IL)-6 enzyme-linked immunosorbent assay (ELISA) Max Set Deluxe Kits were from BioLegend (San Diego, CA). The DeadEnd Colorimetric terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) System was from Promega. All other chemical reagents were from Sigma-Aldrich (St. Louis, MO).
C57BL/6J and CHOP knockout mice (CHOP−/–; back-crossed at least 12 generations to the C57BL/6J background) were obtained from the Jackson Laboratory (Bar Harbor, ME). All experiments and procedures involving mice were approved by the institutional animal care and use committee of Virginia Commonwealth University (Richmond, VA) and were conducted in accord with the Declaration of Helsinki, the Guide for the Care and Use of Animals (National Academies Press, Washington, DC, 1996), and all applicable regulations.
To examine the effect of CHOP on HIV PI–induced lipid accumulation in liver, male WT and CHOP−/− mice (8 weeks old) were randomly assigned to four groups (n = 5): (1) control; (2) amprenavir; (3) ritonavir; and (4) lopinavir. Mice were fed with a standard diet and gavaged daily with control solution (0.2% CMC-Na) or individual HIV PIs at a dose of 50 mg/kg for 4 weeks. All mice were housed under identical conditions in an aseptic facility and given free access to water and food. Mice were weighed daily to adjust drug intake. At the end of each time period, mice were fasted for 16 hours and blood samples and liver tissues were collected and analyzed.
Isolation and Culture of Mouse Primary Hepatocytes.
Primary hepatocytes were isolated from C57BL/6J (WT) and CHOP−/− mice (male, 8 weeks old) and cultured as described previously. 6 Cells were cultured in serum-free Williams' E medium containing dexamethasone (0.1 μM), penicillin (100 units/mL), and thyroxine (1 μM). Cells were incubated from 12 to 24 hours in a 5% CO2 environment at 37°C before additions were made to culture medium. HIV PIs (amprenavir, lopinavir, and ritonavir) were dissolved in dimethyl sulfoxide and were added directly to culture medium (final concentrations: 15-50 μM) and incubated for 24 hours.
Western Blot Analysis.
Total lysate proteins were prepared from liver tissue using radioimmunoprecipitation assay (RIPA) reagent (20 mM of Tris-HCl, 150 mM of NaCl, 2 mM of ethylenedimainetetraacetic acid, 20 mM of NaF, 1 mM of NaVO4, 1% NP40, and 0.1% sodium dodecyl sulfate [SDS]; pH 8.0). Protein concentration was determined using Bio-Rad protein assay reagent. Total lysate proteins (100 μg) were resolved on 10% Criterion XT precast gels or 10% SDS/polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membranes. Immunoreactive bands were detected as described previously. 7 Density of immunoblotting bands was analyzed using Bio-Rad Image Lab computer software.
Analysis of Apoptosis by Annexin V and Propidium Iodine Staining.
Mouse primary hepatocytes (MPH) were treated with HIV PIs (25 μM) for 24 hours and stained with Annexin V/fluorescein isothiocyanate (FITC) and propidium iodide using the BD ApoAlert Annexin V kit. Annexin V/propidium-iodide–stained cells were visualized under fluorescence microscopy with a ×40 objective using a dual-filter set for FITC and rhodamine, as described previously. 6, 7
Real-Time Quantitative Polymerase Chain Reaction.
Total cellular RNA was isolated from mouse primary hepatocytes or liver tissues using the SV Total RNA Isolation System. Total RNA (2 μg) was used for first-strand cDNA synthesis using the High-Capacity cDNA Archive kit. Messenger RNA (mRNA) levels of key genes involved in hepatic lipid metabolism, such as fatty acid synthase (FAS), cholesterol 7-α-hydroxylase (CYP7A1), sterol 27-hydroxylase (CYP27A1), sterol regulatory element-binding protein 1 and 2 (SREBP-1/2), 3-hydroxy-3-methylglutaryl-CoA (coenzyme A) reductase (HMG-coAR), and C/EBP beta (C/EBP-β), were quantified using specific primers for each gene (Table 1 in Supporting Materials). iQ SYBR Green Supermix was used as a fluorescent dye to detect the presence of double-stranded DNA. mRNA values for each gene were normalized to internal control glyceraldehyde-3-phospahte dehydrogenase mRNA. The ratio of normalized mean value for each treatment group to vehicle control group was calculated.
Measurement of TG.
MPH isolated from WT and CHOP−/− mice were plated on 60-mm plates overnight and then treated with vehicle control or HIV PIs for 24 hours. At the end of the treatment, cells were washed with phosphate-buffered saline (PBS) twice and harvested with 300 μL of RIPA buffer (described above). Mouse sera were collected at the end of the in vivo study. Liver tissues were homogenized in RIPA buffer. The amount of TG was measured by using the Wako TG assay kit (Wako Diagnostics).
ELISA of Cytokines.
TNF-α and IL-6 levels in MPH, serum, and liver tissue were determined by ELISA using mouse TNF-α and mouse IL-6 ELISA Max Set Deluxe Kits, as described previously. 8 Total protein concentrations of viable cell pellets and liver tissues were determined using the Bio-Rad Protein Assay reagent. Total amounts of TNF-α and IL-6 in hepatocytes and liver tissues were normalized to total protein amounts.
Liver tissue sections were collected and fixed in 4% paraformaldehyde in 0.1 M of PBS at room temperature overnight. Regions of specimens were standardized for all mice. Paraffin-embedded tissue sections (≤5 μm) were stained with hematoxylin and eosin (H&E), according to standard techniques. Images were taken using a Motic BA200 microscope (Motic Instruments, Inc, Baltimore, MD). Samples were examined in a blind manner to evaluate the presence of steatosis, inflammation, and fibrosis, as described previously. 21
Oil Red O Staining.
MPH were treated with HIV PIs for 24 hours. The intracellular lipid was stained with Oil Red O, as described previously. 21 Liver tissue sections were collected and covered with O.C.T gel and kept at −80°C. Frozen sections of mouse liver tissue (≤10 μm) were fixed in 3.7% formaldehyde for 10 minutes and rinsed with PBS and 60% isopropanol, followed by staining with 0.5% Oil Red O in 60% 2-propanol for 15 minutes. After washing with distilled water, nuclei were stained with hematoxylin for 2 minutes and rinsed thoroughly with distilled water. Images were taken using a microscope equipped with an image recorder under a ×40 lens.
To detect apoptosis in liver tissue, 5-μm sections were deparaffinized and rehydrated through washes with graded concentrations of ethanol. Tissue was pretreated with proteinase K (20 μg/mL) for 15 minutes at room temperature, followed by incubation in 3% H2O2 in PBS for 5 minutes at room temperature to quench endogenous peroxidase activity. Apoptotic cells were detected using the DeadEnd Colorimetric TUNEL System, following the manufacturer's protocol (Promega). Control stains were obtained by processing, in parallel, duplicate sections, omitting only the TnT enzyme.
All in vitro experiments were repeated at least three times, and results are expressed as the mean ± standard error. For in vivo studies, one-way analysis of variance was used to analyze differences between different treatments. Statistics were performed using GraphPad Pro (GraphPad Software Inc., San Diego, CA). A probability (P) of less than 0.05 was considered statistically significant.
Effect of CHOP on HIV PI–Induced Apoptosis in MPH.
We have previously reported that HIV PIs induced ER stress and activated UPR in primary rat hepatocytes. We also showed that HIV PI–induced UPR activation is linked to cell apoptosis and tissue injury. 6, 7, 22 Ritonavir and lopinavir are the most commonly used HIV PIs in the clinic and significantly activated UPR in various cell types, including hepatocytes. Similar to our findings in rat primary hepatocytes, both ritonavir and lopinavir dose dependently activated UPR in MPH (Fig. 1 in Supporting Materials). Our previous studies also showed that amprenavir did not induce ER stress or activate UPR. 6, 7, 22 Therefore, we used amprenavir as a negative control for our studies. To determine whether CHOP expression contributed to HIV PI–induced apoptosis in hepatocytes, we isolated MPH from WT and CHOP−/− mice and treated with individual HIV PIs for 24 hours. In WT MPH, both ritonavir and lopinavir induced cell apoptosis, but not amprenavir (Fig. 1A). However, in CHOP−/− MPH, both ritonavir and lopinavir failed to induce apoptosis (Fig. 1B). We also observed that viability of CHOP−/− MPH was higher than that of WT MPH during isolation (data not shown). These results suggest that CHOP may be a key player in HIV PI–induced apoptosis in MPH.
Effect of CHOP on HIV PI–Induced Lipid Accumulation in MPH.
We have shown that both ritonavir and lopinavir markedly induced CHOP expression and lipid accumulation in hepatocytes and macrophages. 6, 7 To determine whether HIV PIs induce lipid accumulation through CHOP, we treated both WT and CHOP−/− MPH with individual HIV PIs (15 or 25 μM) for 24 hours and intracellular lipid was stained using Oil Red O, as described previously. 21 Both ritonavir and lopinavir dose dependently increased intracellular lipid accumulation in WT MPH, but amprenavir had no effect (Fig. 2A). In contrast, ritonavir- and lopinavir-induced intracellular lipid accumulation was significantly reduced in CHOP−/− MPH (Fig. 2B). We further quantified cellular TG levels. In WT MPH, ritonavir and lopinavir dose dependently increased TG levels, which were markedly inhibited in the absence of CHOP (Fig. 3). Basal levels of TG in CHOP−/− MPH were much lower than that in WT MPH.
Effect of CHOP on HIV PI–Induced Dys-regulation of Key Genes Involved in Hepatic Lipid Metabolism in MPH.
To further identify the cellular mechanisms underlying CHOP-mediated lipid accumulation in hepatocytes, we examined the expression of key genes involved in cholesterol and fatty acid metabolism in HIV PI–treated WT and CHOP−/− MPH by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Ritonavir- and lopinavir-induced increase of SREBP-1, SREBP-2, FAS, HMG-CoAR and C/EBP-β was blunted in CHOP−/− MPH (MPH) (Fig. 4). In addition, HIV PI–induced inhibition of CYP7A1, the rate-limiting enzyme involved in bile acid synthesis, was reversed in CHOP−/− MPH. Western blotting analysis further confirmed that ritonavir- and lopinavir-induced increase of protein expression levels of SREBP-1 and SREBP-2 in WT mouse primary hepatocytes was blocked in CHOP−/− MPH (Fig. 2 in Supporting Materials). These results suggest that CHOP contributes to HIV PI–induced increase of cholesterol synthesis and inhibition of bile acid synthesis in hepatocytes.
Effect of CHOP on HIV PI–Induced Hepatic Lipotoxicity In Vivo.
To further determine whether CHOP deficiency has a protective effect against HIV PI–induced dys-regulation of lipid metabolism and hepatic lipotoxicity in vivo, C57/BL6 WT and CHOP−/− mice were treated daily with individual HIV PIs for 4 weeks. The serum lipid profile was determined. Ritonavir and lopinavir, but not amprenavir, significantly increased serum TG levels in WT mice, which were diminished in CHOP−/− mice (Fig. 5A). Similarly, ritonavir and lopinavir induced the increase of TG in livers of WT mice, but not in livers of CHOP−/− mice (Fig. 5B). H&E and Oil Red O staining results indicated that HIV PI–induced hepatic lipid accumulation was significantly reduced in CHOP−/− mice (Fig. 6A,B). TUNEL assay further indicated that HIV PI–induced hepatic injury was markedly reduced in CHOP−/− mice (Fig. 6C). mRNA expression levels of key genes involved in hepatic lipid metabolism were further analyzed by real-time RT-PCR. HIV PI–induced increase of mRNA expression of SREBP-1, SREBP-2, FAS, HMG-CoAR, and C/EBP-β was inhibited in CHOP−/− mice. Similarly, HIV PI–induced down-regulation of CYP7A1 was also reversed in CHOP−/− mice.
Effect of CHOP on HIV PI–Induced Inflammatory Response In Vivo.
It is well-known that inflammation plays a critical role in numerous human diseases, including metabolic, cardiovascular, and various liver diseases. 23-28 ER stress has been identified as an important inducer of inflammation and is closely linked to various inflammatory diseases. 29-31 Our previous studies showed that HIV PI–induced ER stress activation contributed to the increase of the expression of inflammatory cytokines, TNF-α and IL-6, in macrophages. 5, 8 We also reported that HIV PI–induced ER stress and CHOP expression contributed to disruption of intestinal epithelial integrity. 9 To determine whether CHOP expression also induces inflammation response in vivo, we measured TNF-α and IL-6 levels in serum and liver by ELISA. Ritonavir and lopinavir, but not amprenavir, significantly increased TNF-α and IL-6 levels in serum and livers of WT mice (Fig. 8). In contrast, ritonavir and lopinavir failed to increase TNF-α and IL-6 levels in serum and livers of CHOP−/− mice. These results indicated that CHOP deficiency diminished HIV PI–induced inflammatory response in vivo.
HAART has been well documented to markedly reduce the morbidity and mortality of HIV-1-infected patients during the last two decades. However, HAART, especially HIV PI–associated adverse metabolic effects, such as dyslipidemia, IR, and cardiovascular risk, have raised major concerns in the clinic. 1, 32-35 During last decade, a lot of effort has been put into identifying the potential underlying mechanisms of HIV PI–induced metabolic disorders. Studies from our laboratory and others have identified that activation of ER stress contributes greatly to HIV PI–associated dyslipidemia, hepatic injury, and cardiovascular complications. 4, 6-10 Our current studies show that CHOP is a key player in HIV PI–induced hepatic lipotoxicity. Both in vitro and in vivo animal studies indicated that knocking out CHOP not only prevented HIV PI–induced dys-regulation of lipid metabolism and hepatic injury, but also reduced HIV PI–induced inflammatory response.
The ER is a central organelle of eukaryotic cells, which plays a critical role in lipid synthesis, protein folding, and protein maturation. Conditions interfering with the function of the ER are collectively called ER stress. The ER has evolved highly specific signaling pathways (termed UPR) to sense cellular stress. 36, 37 Recent advances in both basic and clinical studies shed light on mechanistic complexities and on the role of UPR in numerous diseases, including metabolic diseases, cardiovascular diseases, nonalcoholic fatty liver diseases, alcoholic fatty liver diseases, and drug-induced liver diseases. 11, 37-44 We have previously shown that HIV PIs induced ER stress and activated UPR in macrophages, hepatocytes, and intestinal epithelial cells. 6, 7, 9 Our studies and studies from others also showed that HIV PIs disrupted lipid metabolism and induced hepatic injury. 4, 6, 45 However, the exact linkage between UPR and HIV PI–mediated hepatic injury remains unclear. CHOP is a major transcription factor involved in ER stress-mediated apoptosis. 46-48 Recent studies have shown that CHOP plays multifunctional roles in various diseases and contributes to ER stress-mediated cellular and tissue injury as well as induction of inflammatory response. 18, 19, 47-50 Studies done by Ji et al. showed that deletion of CHOP remarkably reduced hepatocellular apoptosis in response to alcohol feeding, but had no effect on alcohol-induced hepatic steatosis. 18 It has been reported that CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury. 49 In addition, CHOP deletion reduces oxidative stress, improves beta-cell function, and promotes cell survival in multiple mouse models of diabetes. 19 Recent studies further showed that CHOP-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques. 17
In the present study, both ritonavir and lopinavir, the most commonly used HIV PIs in the clinic, dose dependently activated UPR, significantly induced apoptosis, and increased lipid accumulation in WT MPH, but not in CHOP−/− MPH. Amprenavir, which does not induce activation of UPR, had no effect on lipid accumulation. Similarly, in vivo mouse studies showed that ritonavir- and lopinavir-induced increase of serum TG and hepatic lipid accumulation were also blunted in CHOP−/− mice (Figs. 5 and 6). Hepatic steatosis is the result of dys-regulation of lipid metabolism caused by increased lipid uptake, increased de novo lipid synthesis, and reduced lipid oxidation and metabolism. Further analysis of the effect of HIV PIs on key genes involved in lipid metabolism indicated that CHOP plays an important role in regulating the expression of SREBP-1, SREBP-2, HMG-CoAR, FAS, and CYP7A1. In WT MPH and livers, both ritonavir and lopinavir increased the expression of SREBP-1, SREBP-2, HMG-CoAR, FAS, and C/EBP-β and reduced CYP7A1 expression. However, in the absence of CHOP, ritonavir and lopinavir had no effect on the expression of SREBP-1, SREBP-2, HMG-CoAR, FAS, and C/EBP-β, but increased CYP7A1 expression. Recent studies demonstrated that CHOP knockdown delayed palmitate-mediated cell death in hepatocytes, but had limited effect against methionine-choline–deficient diet (MCD)-induced liver injury. 50 Soon et al. reported that in a MCD mouse model of fatty liver disease, liver injury is not dependent upon the activation of UPR. CHOP deficiency did not alleviate, and in fact worsened, MCD-mediated liver injury. 51 However, deletion of C/EBP-β significantly reduced MCD-induced hepatic TG accumulation and decreased liver injury. 52 Our current studies suggest that HIV PI–induced hepatic lipotoxicity is dependent on UPR activation and CHOP expression. We also noticed that HIV PI–induced c-Jun N-terminal kinase activation was partially reduced in livers of CHOP−/− mice (data not shown). However, the exact molecular and cellular mechanisms underlying CHOP-mediated dys-regulation of hepatic lipid metabolism remain to be identified. CHOP may directly regulate the transcription of key genes involved in cholesterol and lipid metabolism or indirectly regulate lipid metabolism through activating other signaling pathways in hepatocytes. Recent advances in the field of autophagy study have identified that the regulation of autophagy and lipid metabolism are interrelated. 53, 54 It also has been shown that activation of ER stress is closely linked to the autophagy-signaling pathway. However, whether HIV PI–induced ER stress and CHOP activation has any effect on hepatic autophagy activity remains to be further studied and is our ongoing project.
Inflammation is an important contributor to hepatic lipotoxicity. 25, 28 Our previous studies have reported that HIV PI–induced expression of the inflammatory cytokines, TNF-α and IL-6, is linked to ER stress and UPR activation in macrophages, 5, 8 and that HIV PI-induced CHOP expression plays a role in dysfunction of the intestinal barrier. 9 In this study, we further demonstrated that HIV PI–induced systemic inflammatory response was also significantly reduced in CHOP−/− mice. Our studies suggest that the ER stress-mediated signaling pathways are emerging as a potential site for the intersection of inflammation and metabolic disease. 55
In summary, together with our previous findings, our studies demonstrated that HIV PI–induced activation of UPR, especially the up-regulation of CHOP, represents an important molecular mechanism underlying HIV PI–associated dyslipidemia, inflammation, and hepatic lipotoxicity. Our studies suggest that targeting UPR-signaling pathways is a promising, novel approach for reducing HAART-induced metabolic complications in HIV patients. Our studies also provide important information for the future development of new anti-HIV drugs with fewer metabolic side effects.
The authors thank the National Institutes of Health AIDS Research and Reference Reagent Program for providing lopinavir and ritonavir and GlaxoSmithKline for providing amprenavir.