Ritonavir and Lopinavir Suppress RCE1 and CAAX Rab Proteins Sensitizing the Liver to Organelle Stress and Injury

Liver Cell Cultures and Treatments

Three types of liver cells were used: HepG2, primary mouse hepatocytes (PMHs), and primary human hepatocytes (PHHs). Sources, catalog numbers, and culturing conditions for HepG2 and PMHs are described in the Supporting Information. PMHs were isolated by the USC Liver Cell Culture Core and cultured as reported previously.⁽¹⁵⁾ In some experiments, the 5C long-term culturing of PHHs or PMHs was conducted according to Xiang et al.⁽²³⁾ The liver cells were treated in fetal bovine serum–free medium for 6 hours with 20 nM of Brefeldin A, 85 mM of alcohol, 25 μg/mL of RTV, 25 μg/mL of LPV, RL, or other alcohol and drug combinations. DMSO (< 0.05%, wt/vol) or ethanol (<0.05%, vol/vol) in Dulbecco's modified Eagle's medium was used as vehicle control. After the treatments, a portion of the treated cells were washed with ice-cold phosphate-buffered saline (PBS) and prepared for double staining with Syntax green and Hoechst blue for cell death or immunohistochemistry for confocal microscopy of organelle (e.g., Golgi apparatus) morphology described previously.⁽²¹⁾ Other portions of the cells were detached with 0.05% trypsin–ethylene diamine tetraacetic acid solution for RNA isolation for total RNA-sequencing analysis and real-time polymerase chain reaction (RT-PCR) or for protein extraction for western blotting. In vitro experiments were repeated in triplicate.

Animal Models

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were grouped into six groups: groups 1, 2, 3, and 4 were given with isocaloric control diet, and groups 5 and 6 were given with 5% alcohol liquid diet (Dyets, Inc., Bethlehem, PA) for 10 days. During the 10-day period and from the fifth day, groups 2, 3, and 4 with the control diet were injected intraperitoneally with RTV (25 μg/g), LPV (25 μg/g), and RL (25 μg/g each), respectively, for 5 days, and group 6 with the alcohol diet were injected with RL (25 μg/g each) for 5 days. On the 11th day, groups 5 and 6 were gavaged with 30% alcohol in PBS (5 mg/g) and groups 1, 2, 3, and 4 were gavaged with equal volume of isocaloric maltose solution. The mice were euthanized for serum and liver tissues 8 hours after the gavage. All animals were treated in accordance with the Guide for Care and Use of Laboratory Animals, and the study was approved by the local animal care committee. Analyses for serum alanine aminotransferase (ALT) and liver triglycerides, liver histology by hematoxylin and eosin staining, and liver steatosis by Oil Red O staining were described previously.^{(15, 21, 24)}

Total RNA Sequencing

Total RNA was isolated from the HepG2 cells that were pooled from the same duplicated treatments using the SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian Kit (Clontech; Takara, Tokyo, Japan). The RNA sequencing (RNA-Seq) featuring Illumina's next-generation systems was conducted by the Molecular Genomics Core of USC. Subsequent total RNA-Seq data analysis was performed with the Partek Flow software, version 5.0 (Partek Inc., St. Louis, MO). A z-score of 1 is 1 SD above the mean. A score of 2 is 2 SDs above the mean. The Ingenuity Pathway Analysis (IPA) provided by Qiagen (Venmo, Netherlands) was applied to identify drug targets and predict downstream/upstream drug effects. The Core Analysis tabs were filtered with organelle stress responses that were specific to the anti-HIV drugs and alcohol.

Immunoblotting and RT-PCR

Methods for protein and RNA extractions, immunoblotting, and RT-PCR were described previously.⁽²¹⁾ All names and sources of the primary antibodies are listed in Supporting Table S1. For PCR analysis, about 30 mg of tissue was grinded in Qiazaol and extracted with the DirectZol RNA extraction kit (Zymo Research, Irvine, CA). All primers for Golgi and ER stress markers are listed in Supporting Table S2. Quantifications of the protein bands on the western blots were conducted with ImageJ (National Institutes of Health).

RNA Interference

Stealth small interfering RNAs (siRNAs) generated from the next-generation RNA interference chemistry were purchased from Thermo Fisher Scientific (Waltham, MA). Assay IDs for Ras converting CAAX endopeptidase 1 (RCE1) and ZMPSTE24 were HSS145393 and HSS115697, respectively. Briefly, siRNAs were diluted at 30 pmol/200 μL OptiMEM and mixed with 3 μL of Lipofectamine 3000 at room temperature for 15 minutes, to which 0.1 million of HepG2 cells in 1 mL of antibiotics-free complete medium was added. The transfected cells were incubated in 12-well plates coated with collagen I for 48 hours. siRNA negative control (ID# 4390844) and positive control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA (ID# AM4605) were used for optimizing the knocking-down conditions. The gene knockdown was checked by RT-PCR or western blotting. The subsequent drug/alcohol treatments of the transfected cells were the same as described previously. Short hairpin RNA (shRNA) expression vector (pAV-RCE1v1-shRNA; SH898883) for knocking down RCE1 in PMHs and adenovirus with cytomegalovirus (CMV) promoter-driven expression of RAB13 (VH806960) or Rab18 (VH800874) in PHHs were purchased from ViGene Biosciences (Rockville, MD). The shRNA sequences are listed in Supporting Table S3. For systemic in vivo shRNA delivery in mice, the shRNA sequences were cloned into an adeno-associated virus (AAV) vector under the control of a mouse albumin (ALB) promoter for liver expression (AAV-ALB-RCE1-shRNA). AAV8-ALB-shRNA or AAV8–ALB–scrambled RNA (scRNA) (control) was produced through the custom cloning and viral packaging service from ViGene Biosciences. Each mouse was injected intravenously with 3 × 10⁹ AAV in 100 μL PBS and were examined for subsequent drug and/or alcohol treatments 2 days after the injection.

Statistics

Data are presented as means ± SEM unless otherwise indicated. Statistical analyses were performed with GraphPad Prism 6 software using the one-way analysis of variance (ANOVA) for comparison of multiple groups and two-way ANOVA for comparison of trends between different treatments. The P values of 0.05 or less are considered significant.

Identification of Genes Responsive to Anti-HIV Protease Inhibitors and Alcohol by Total RNA-Seq

It was reported previously that anti-HIV PIs (e.g., RTV and LPV) induced Golgi fragmentation, ER stress and hepatic injury, which could be deteriorated by alcohol.^{(15, 21)} We assumed that there are specific off-target(s) of the anti-HIV drugs that are relevant to Golgi or organelle stress. To find the specific off-target(s), we conducted total RNA-seq on RNA samples from HepG2 cells treated with brefeldin A (positive control for Golgi dysfunction), alcohol, RL, or RL combined with alcohol (RLE). The output of RNA-seq was analyzed using the Qiagen software for IPA, which predicts downstream effects and identifies new drug targets. First, genes/factors that are not relevant to organelle (e.g., ER, Golgi, mitochondria, lysosome) stress and with z-scores of less than 1.5 were filtered out, which narrowed the number of the genes of interest down to 132 (Fig. 1A, the heatmap). Eighty-seven genes were up-regulated and 45 were down-regulated in response to the anti-HIV drug treatment. Second, genes/factors that were drug-responsive but were not further affected by alcohol exposure were filtered out, which narrowed down the genes of interest to 10. These genes are CHD2 (encodes the chromodomain DNA helicase binding protein 2 involved in chromatin remodeling), FOSL2 (FOS like 2, AP-1 transcription factor subunit), GP73 (hepatic Golgi protein 73), MAFK (one of the small Maf transcription factor proteins with basic region and leucine zipper [bZIP]), MKRN2 (E3 ubiquitin-protein ligase makorin-2), MT-ATP6 (mitochondrially encoded ATP synthase membrane subunit 6), OSGIN2 (oxidative stress induced growth inhibitor family member 2), RCE1, SIAT4A (Golgi-related sialyltransferase 4A), and ZFPL1 (zinc finger protein like 1) (Fig. 1, highlighted heatmaps).

The transcriptional expression of the 10 genes was further examined in PMHs treated with brefeldin, alcohol, RL, or RLE. The expression patterns of CHD2, FOSL2, GP73, MAFK, MT-ATP6, OSGIN2, or SIAT4A revealed by RT-PCR were not consistent with those in HepG2 revealed by the RNA-seq (data not show). The expression patterns of three genes, MKRN2, RCE1 and ZFPL1, were consistent with those by the RNA-seq (Fig. 1B). However, only the expression patterns of RCE1 were robustly and significantly consistent in both PMHs and HepG2.

Association of Inhibited RCE1 Protein With Alcohol and Anti-HIV Drug-Induced Golgi Stress and Injury

In HepG2 cells, treatment of RTV, LPV, or alcohol slightly reduced the protein levels of RCE1 compared with vehicle control (Fig. 2A and Supporting Table S4). Remarkable reductions of RCE1 were detected in RL or in the combination of RL and alcohol. Brefeldin, which inhibits protein trafficking from the Golgi to the ER,⁽²⁵⁾ did not inhibit RCE1, indicative of the specific inhibition of RCE1 protein by the anti-HIV drugs and alcohol. Messenger RNA (mRNA) of Rce1 was significantly increased by RL and was nearly doubled in the combination treatment with the drugs and alcohol (Fig. 2B). The feedback increase of Rce1 mRNA was associated with increased Golgi stress markers: TFE3, Hsp47, and GCP60. In contrast, no significant changes of Rce1 mRNA were detected in response to brefeldin, despite that it induced increase of the Golgi stress markers by more than 2-fold. To confirm that the drugs and alcohol-induced RCE1 reduction is accompanied with Golgi dysfunction and cell injury, Golgi morphology and cell death were examined. As expected, either alcohol or the drugs induced Golgi fragmentation in HepG2, and the Golgi fragmentation was much severe in the liver cells treated with the drugs combined with alcohol (Fig. 2C). In addition, near 30% cell death was detected in the HepG2 treated with the anti-HIV drugs and alcohol, which was comparable to the cell death induced by the positive-control brefeldin (Fig. 2D).

The drug and alcohol-induced reduction of RCE1, increase of Golgi stress (e.g., TFE3), Golgi fragmentation, and cell death were also detected in PMHs (Fig. 2E-G and Supporting Table S4).

In addition, in the liver of mice fed the anti-HIV drugs or alcohol alone, minor changes in the protein levels of RCE1 and fatty liver injury indicated by ALT and Oil Red O staining were detected (Fig. 3 and Supporting Table S5). Consistent with the in vitro results, reduction of RCE1 and increase of Golgi stress marker TFE3 were observed in the liver of mice treated with combinations of the drugs and alcohol, which were accompanied with greater than 10-fold increase of ALT and accumulation of fat. In contrast, brefeldin induced Golgi stress, increased ALT, and caused moderate fat accumulation without reducing RCE1 protein.

Effects of Knocking Down Rce 1 on Golgi Stress and Hepatic Injury

Knocking down Rce1 with RNA interference was conducted to determine further the role of Rce1 in the drug and alcohol-induced Golgi stress and cell injury. Expression of RCE1 protein in HepG2 was reduced by 70% by Rce1 siRNA. The reduction of RCE1 in HepG2 did not lead to apparent morphological differences from the control cells treated with scRNA in the absence of the drugs or alcohol. However, the RCE1 knocking down sensitized the drugs and alcohol-induced Golgi stress response (Fig. 4A). The Golgi stress markers, TFE3 (protein expression) and GCP60 (mRNA expression), were increased by 4-fold and 5-fold, respectively, in the cells with the RCE1 being knocked down and treated with the drugs and alcohol (Fig. 4A-C). In contrast, the effect of knocking down RCE1 on brefeldin-induced Golgi stress was not significant. In addition, the anti-HIV PIs were reported to inhibit STE24 overexpressed in yeast.⁽²⁶⁾ We confirmed that knocking down of STE24 had significant effects on the drug-induced Golgi stress response (Fig. 4D-F). Consequently, knocking down RCE1, or STE24 to a less extent, sensitized the cells to the drugs and alcohol-induced death injury (Fig. 4G).

shRNA for RCE1 was chosen to suppress RCE1 in the liver of mice to further address the role of the identified host proteases in the anti-HIV drugs and alcohol-induced hepatic Golgi stress and injury. Interestingly, compared to mice treated with scRNA and shRNA, knocking down RCE1 in the liver did not result in significant changes in TFE3 or GCP60 expression, liver histology, or ALT (Fig. 5). However, when the mice were challenged with the anti-HIV drugs, the Golgi stress response induced by TFE3 and GCP60 was significantly higher in the liver with RCE1 knocking down than without the knocking down (Fig. 5A,B). The drug-induced hepatic foamy and ballooning degenerations were more severe in the liver of mice treated with RCE1 siRNAs, which were accompanied with increased liver triglycerides and ALT (Fig. 5C-E), indicative of exacerbated damage to the liver by the anti-HIV drugs.

Association of CAAX Rab Proteins With RCE1 Expression and Golgi Stress in Liver Cells

From previous results, we know that disturbed ER-Golgi trafficking underlies the anti-HIV drug and alcohol-induced liver injury.⁽²¹⁾ The ER-Golgi traffic involves many effectors such as vesicle tethers, SNAREs, and membrane and motor proteins, which are coordinated by the Rab GTPases.^{(27, 28)} We assumed that there is a biochemical connection between RCE1 and the Rab proteins and examined protein expression of the Rab GTPases in the liver of mice treated with the anti-HIV drugs. Immunoblotting was conducted for 20 Rab proteins that are likely to regulate the ER-Golgi trafficking in mammalians: Rab1, Rab2, Rab6, Rab8, Rab10, Rab11, Rab12, Rab13, Rab14, Rab18, Rab19, Rab24, Rab31, Rab32, Rab33, Rab34, Rab36, Rab39, Rab42, and Rab43 (Fig. 6A and Supporting Figs. S1 and S2). Two Rab proteins, Rab13 and Rab18, were significantly suppressed in the presence of the anti-HIV drugs. Intriguingly, both Rab13 and Rab18 have a common CAAX motif, and their maturation is known to require proteolytic cleavage of the motif by RCE1 or the evolutionarily unrelated protease STE24.^{(29, 30)} Because prelamin A is the substrate of STE24,⁽³⁰⁾ Rab13 and/or Rab18 are potential substrates of RCE1. In support of this assumption, Rab13 overexpression in PHHs infected with adenovirus capable of CMV promoter-driven expression of Rab13 (Ad-CMV-Rab13) rescued the human liver cells from the drug-induced RCE1 inhibition, Golgi stress, and injury. Rab13 was increased by more than 40% in PHHs compared with the mock control (Fig. 6B,C and Supporting Table S6). The Rab13 overexpression in PHHs decreased the drugs and alcohol-induced GCP60 mRNA by 47%, as well as reduced the cell death by 67% in the presence of RCE1 inhibition (Fig. 6D,E). In contrast, brefeldin-induced Golgi stress and cell injury were not affected by the Rab13 overexpression in PHHs. Moreover, the drug-induced Golgi fragmentation was reduced in the human hepatocytes overexpressing Rab13 (Fig. 6F,G). Similar rescuable Golgi stress response and cell death were observed in the human hepatocytes overexpressing Rab18 (Supporting Fig. S3).