Recently, a novel head-to-tail linear type of ubiquitination was shown to play an essential role in nuclear factor-κB (NF-κB) activation [1
]. The linear ubiquitin assembly complex (LUBAC) conjugates the linear polyubiquitin chain on NF-κB essential modulator (NEMO) and thereby activates the canonical NF-κB pathway through IκB kinase (IKK) complex activation [2
]. Currently, LUBAC, which has a molecular weight that is approximately 600 kDa, is assumed to be comprised of SHANK-associated RH domain interacting protein in postsynaptic density (SHARPIN), the longer isoform of heme-oxidized IRP2 ligase-1 (HOIL-1L) and HOIL-1L interacting protein (HOIP) [3
]. Although both HOIL-1L and HOIP possess E3 ligase activity, it is HOIP that serves as an E3 ligase for linear polyubiquitination. SHARPIN and HOIL-1L bind to HOIP via their ubiquitin-like (UBL) domain and have thus far been regarded as being important for the stability of LUBAC [3
]. Previous reports indicate that NF-κB transcriptional activation leads to increased releases of several inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β in Kupffer cells as observed in non-alcoholic steatohepatitis (NASH) livers [6
]. On the other hand, NF-κB activity also reportedly promotes the survival of hepatocytes via the inductions of anti-apoptosis factors such as inhibitors of apoptosis protein (IAPs), including X-linked IAP [10
]. Thus, NF-κB is involved in both inflammation and cell survival.
In this study, first, mice were treated with either carbon tetrachloride (CCl4
) or acetaminophen (APAP), and SHARPIN expression was revealed to be significantly downregulated in the livers of these acute hepatic injury models. We previously demonstrated that LUBAC formation is severely impaired mainly via markedly reduced SHARPIN expression in the NASH livers of mice fed a methionine choline deficient (MCD) diet for eight weeks [12
]. In contrast to the MCD diet- or high-fructose diet-induced NASH model [13
or APAP-induced liver injury occurred within a surprisingly short period (i.e., just 24 h after treatment), which accompanied a marked reduction of LUBAC formation.
Thus, we subsequently attempted to investigate the pathological significance of reduced LUBAC expression in the liver as well as in Hepa1-6 hepatoma cells by infection with the corresponding short-hairpin RNA (shRNA) adenoviruses. Importantly, livers deficient in SHARPIN or HOIP exhibited marked death of hepatocytes, which may have resulted in severe inflammation and fibrosis. Herein, we present data suggesting that reduction of LUBAC is involved in the development of CCl4 and APAP-induced hepatocyte death.
To our knowledge, this is the first study to demonstrate the expressions of individual LUBAC components, especially SHARPIN, to be significantly decreased in mouse livers in response to treatment with CCl4
or APAP, agents widely used to create liver injury models. In contrast to the NASH model produced by eight weeks of MCD diet feeding [12
], it should be noted that CCl4
or APAP-induced reduction of LUBAC formation occurred within just 24 h after treatment with these agents. Given that there were no significant decreases in their component mRNA levels, we can reasonably speculate that enhanced degradation would be the most likely mechanism underlying the observed reductions, though further study is necessary to clarify this issue.
APAP overdosage results in the formation of N
-benzoquinone imine (NAPQI), which covalently binds to glutathione (GSH) and depletes hepatic anti-oxidative capacity. Uncontrolled oxidative stress leads to hepatocyte cell death and the appearance of damage associated molecular patterns, a process which in turn activates Kupffer cells and results in the release of inflammatory cytokines and subsequent infiltration of neutrophils and macrophages [20
]. Hepatotoxicity caused by CCl4
is also mediated via oxidative stress exerted by its degraded metabolites, trichloromethyl (CCl3
) and trichloromethyl peroxyl (CCl3
), both of which are unstable radicals [21
We speculated that not only the toxicities of these agents themselves but also impaired LUBAC formation may contribute to the aggravation of liver injury. In fact, it was clearly shown that hepatic knockdown of either SHARPIN or HOIP by adenoviral transfer of the corresponding shRNA into mice produced severe inflammation and fibrosis accompanied by hepatocyte death, features typically observed in both drug-induced hepatitis and NASH livers. Similar results were obtained using the Hepa1-6 cell line. Knockdown of any one of the LUBAC components in Hepa1-6 cells induced apoptosis in the presence of TNFα-stimulation (Figure 4
a). One difference from the data obtained with mouse livers was that no increase in caspase cleavage was apparent at baseline (i.e., without TNFα-stimulation) in Hepa1-6 cells (Figure 4
a). This finding is in good agreement with those of a previous study showing that HOIP deficient cells tend to form complex-II, a cell death complex which involves RIP1, RIP3, FADD, cFLIP, and caspase-8, rendering HOIP deficient cells susceptible to cell death in response to TNFα stimulation [18
In contrast, either SHARPIN or HOIP knockdown by adenoviral transfer of the corresponding shRNA into mouse livers was sufficient for rapid induction of massive hepatocyte death together with inflammation and fibrosis, with no additional stimulation being necessary. Gerlach et al. [5
] reported the presence of nodular lymphocyte aggregates in the livers of cpdm
mice (mice with a spontaneous Sharpin
gene mutation), but these aggregates were absent when the Tnf
gene was further knocked out in cpdm
mice. In the mouse system, gut-derived bacterial endotoxins, such as lipopolysaccharide, have been regarded as an endogenous inducer of hepatic inflammation which exerts its effects by stimulating TNFα release from Kupffer cells, which is assumed to be a factor contributing to the progression from simple steatosis to NASH [22
]. In addition to such endogenous inflammatory stimuli, it is possible that adenovirus infection, as used in our experiments, may contribute to apoptosis by functioning as a trigger, when HOIP or SHARPIN expression is suppressed. Thus, it is reasonable to speculate that LUBAC deficiency would lower the threshold of apoptosis and render hepatocytes susceptible to apoptosis, even when exposed to minor inflammatory stimuli such as gut-derived lipopolysaccharide, which in turn leads to inflammatory cell recruitment and hepatic stellate cell activation, thereby inducing inflammation and fibrosis in the liver [16
]. It is noteworthy that the expression of IL-6, which is known to play a protective role against CCl4
-induced hepatocyte apoptosis and fibrosis [25
] and is reportedly important in the regenerative response to partial hepatectomy [26
], was upregulated in the livers of both L-SHARPIN KD and L-HOIP KD mice concomitantly with other inflammatory cytokines (Figure 2
e and Figure 3
d), an observation ruling out the possibility that the enhancements of hepatic apoptosis and fibrosis in these mice were mediated via impaired IL-6 expression.
Two hepatic LUBAC-deficient murine models, L-SHARPIN KD mice and L-HOIP KD mice, were investigated to elucidate the role of LUBAC in the liver. Liver inflammation and fibrosis accompanied by hepatocyte apoptosis were observed in both of these KD murine models. l
-SHARPIN KD mice showed a milder phenotype than l
-HOIP KD mice, however, judging from serum ALT (Figure 2
f and Figure 3
e) and liver histology. Since the suppressive effects of our shRNA adenoviruses for HOIP and SHARPIN were both partial, these phenotypic differences are difficult to explain. Nevertheless, intriguingly, HOIL-1L expression was significantly upregulated on both the mRNA and the protein level in the livers of L-SHARPIN KD mice (Figure 2
a,b). Thus, one possibility is that this upregulated HOIL-1L prevented apoptotic cell death to some degree by increasing residual LUBAC activity, but further study is necessary to clarify this issue.
Recent studies suggest circulating microRNAs, including miR-122, as potential biomarkers of drug-induced acute liver injuries [28
]. Alteration of protein expression patterns in the liver in response to APAP administration has been also investigated using a proteomic approach [30
]. It is intriguing whether these changes in microRNAs and protein expression patterns are observed in L-SHARPIN KD and L-HOIP KD mice as well, which also awaits further investigation.
In conclusion, to our knowledge, this is the first report showing that LUBAC formation, particularly the SHARPIN expression level, is reduced in the livers of mice with toxic agent-induced hepatitis. In addition, our findings strongly suggest impairments and/or reductions of LUBAC component functions to be a factor exacerbating hepatocyte death, in addition to the direct toxicity of the agent itself.
4. Materials and Methods
4.1. Animals and Experimental Protocols
C57BL6/J male mice, at seven to eight weeks of age, were fed normal chow diet. To prepare the mouse model with toxic agent-induced fulminant hepatitis, mice were intraperitoneally injected with 5 mL/kg CCl4
(8% in corn oil) or 300 mg/kg APAP (dissolved in warm saline) after being fasted for 20 h and were then sacrificed 24 h after the injection [15
]. To reduce the hepatic expression levels of SHARPIN or HOIP, mice were injected via the tail vein with recombinant adenovirus encoding short-hairpin (sh) RNA for SHARPIN, HOIP, or LacZ as a control, and were sacrificed seven days after the injection. Liver-specific knockdown was confirmed by subjecting tissue lysates to Western blotting (Figure S1
). HOIL-1L shRNA adenovirus was not used for the mouse experiments, since this virus suppressed the expression of HOIL-1L in vitro but not in mouse livers. The animals were handled in accordance with the Guidelines for the Care and Use of Experimental Animals published by Hiroshima University (Hiroshima, Japan), and the experimental protocols were approved by the Institutional Review Board of Hiroshima University (identification code: A16-35).
4.2. Histological Studies
Livers were fixed with 10% formaldehyde and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin or picrosirius red. For TUNEL staining, either the Apoptag Peroxidase In Situ Apoptosis Detection Kit (Merck Millipore, Darmstadt, Germany) or the DeadEnd Colorimetric Apoptosis Detection System (Promega Corporation, Madison, WI, USA) was used according to the manufacturers’ instructions.
4.3. Serum Investigations
Whole blood samples were obtained from the heart and serum was collected after centrifugation at 1000× g for 30 min. Alanine aminotransferase (ALT) was assayed with the Transaminase CII Test (Wako, Osaka, Japan).
4.4. Cell Culture and Cell Death Assay
Hepa1-6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 °C in 5% CO2 in air. At sub-confluence, Hepa1-6 cells were infected with LacZ, SHARPIN, HOIL-1L, or HOIP shRNA expressing adenoviruses and stimulated with 10 ng/mL TNFα for 8 or 24 h. Three days after the transduction, total lysates were prepared and then subjected to immunoblotting. Quantitative cell death assays were performed by staining dead cells with trypan blue. Two days after adenoviral infection, Hepa1-6 cells were stimulated with 10 ng/mL TNFα for 24 h. Cells were trypsinized, resuspended in culture medium, and stained by adding a 1/10 volume of 0.5% trypan blue. In the experiment designed to investigate the effect of APAP in vitro, Hepa1-6 cells were stimulated with 5 or 20 mM APAP for 24 h and total lysates were subjected to immunoblotting.
Antibodies were purchased from Cell Signaling Technology (cleaved caspase-3, GAPDH) and Santa Cruz (actin, α-tubulin). The antibodies against SHARPIN, HOIL-1L, and HOIP were prepared as previously described [12
4.6. Western Blotting
Livers were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM etylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM NaF, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated on ice for 30 min and then centrifuged at 15,000 rpm for 10 min and subsequently for 30 min at 4 °C. After adjusting the protein concentrations, the supernatants were mixed and boiled with sample buffer. Samples were electrophoresed with SDS (sodium dodecyl sulfate) -polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membranes, and subjected to immunoblotting using Supersignal West Pico Substrate (Thermo Scientific, Waltham, MA, USA) or ImmunoStar LD (Wako).
4.7. RNAi Interference
For the individual knockdowns of mouse SHARPIN, HOIL-1L, and HOIP, we generated each shRNA expressing recombinant adenoviruses using the BLOCK-iTTM U6 RNAi Entry Vector Kit and the BLOCK-iTTM Adenoviral RNAi Expression System (Invitrogen). The top and bottom strands used to generate each of the shRNA expressing recombinant adenoviruses are as follows: SHARPIN shRNA, top strand 5′-CACCGCGGAAGCTGCAATTGATAGCCGAAGCTATCAATTGCAGCTTCCGC-3′, bottom strand 5′-AAAAGCGGAAGCTGCAATTGATAGCTTCGGCTATCAATTGCAGCTTCCGC-3′; HOIL-1L shRNA, top strand 5′-CACCGCCTATCTCTACCTGCTGTCACGAATGACAGCAGGTAGAGATAGGC-3′, bottom strand 5′-AAAAGCCTATCTCTACCTGCTGTCATTCGTGACAGCAGGTAGAGATAGGC-3′; HOIP shRNA, top strand 5′-CACCGGTCTTCTCAGCTCTCCAATACGAATATTGGAGAGCTGAGAAGACC-3′, bottom strand 5′-AAAAGGTCTTCTCAGCTCTCCAATATTCGTATTGGAGAGCTGAGAAGACC-3′.
4.8. Real-Time PCR (Polymerase Chain Reaction)
Total RNA from the liver was isolated using Sepazol reagent (Nacalai Tesque, Kyoto, Japan) and first-strand cDNA was obtained using the Verso cDNA Synthesis Kit (Thermo Scientific), according to the manufacturers’ instructions. Real-time PCR was performed using the CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) with SYBR Premix Ex Taq (Takara-Bio, Kusatsu, Japan). The primers used are shown in Table 1
4.9. Gel Filtration
Gel filtration was performed to detect LUBAC components forming the LUBAC complex, which could be eluted in fractions of higher molecular weight and thereby distinguished from those existing as monomers. Livers were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM PMSF, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail. After adding an equal volume of lysis buffer containing 300 mM NaCl, lysates were centrifuged at 15,000 rpm for 30 min. The supernatants were subjected to gel filtration using Superdex 200 10/300 GL (GE Healthcare, Chicago, IL, USA) and fractionated at 1 mL/min using ÄKTA explorer 10S (GE Healthcare). Proteins were precipitated with trichloroacetate and acetone from the collected samples, mixed and boiled with sample buffer, and subjected to western blotting.
4.10. Statistical Analysis
Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan). Values are presented as means ± S.E. We used student’s unpaired t-test when comparing two groups, and one-way ANOVA or the Kruskal-Wallis test for multiple comparisons. We considered p < 0.05 to indicate a statistically significant difference.