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

Organelle stress and Liver injuries often occur in human immunodeficiency virus (HIV) infected patients under anti-HIV therapies, yet few molecular off-targets of anti-HIV drugs have been identified in the liver. Here, we found through total RNA sequencing that the transcription of a host protease Ras converting CAAX endopeptidase 1 (RCE1) was altered in HepG2 cells treated with anti-HIV protease inhibitors, ritonavir and lopinavir. Levels of RCE1 protein were inhibited in HepG2 and primary mouse hepatocytes and in the liver of mice treated with the anti-HIV drugs, which were accompanied with inhibition of two potential substrates of RCE1, small GTP binding protein Rab13 and Rab18, which are with a common CAAX motif and known to regulate the ER-Golgi traffic or lipogenesis. Neither Rce1 transcription nor RCE1 protein level was inhibited by Brefeldin A, which is known to interfere with the ER-Golgi traffic causing Golgi stress. Knocking down Rce1 with RNA interference increased ritonavir and lopinavir-induced cell death as well as expression of Golgi stress response markers, TFE3, HSP47 and GCP60, in both primary mouse hepatocytes and mouse liver, and deteriorated alcohol-induced alanine aminotransferase (ALT) and fatty liver injury in mice. In addition, overexpressing Rab13 or Rab18 in primary human hepatocytes reduced partially the anti-HIV drugs and alcohol-induced Golgi fragmentation, Golgi stress response, and cell death injury. Conclusion: We identified a mechanism linking a host protease and its substrates, small guanosine triphosphate-binding proteins, to the anti-HIV drug-induced Golgi dysfunction, organelle stress response, and fatty liver injury.

Effects of combined alcohol and anti-HIV drugs on cellular stress responses in primary hepatocytes and hepatic stellate and kupffer cells.

BACKGROUND: Certain anti-HIV drugs alone or in combination are often associated with liver damages, which are frequently worsened by alcohol consumption. We previously found an endoplasmic reticulum (ER) stress mechanism for the drug- and alcohol-induced hepatic injuries in animal models and in vitro hepatocytes. However, it is unknown whether anti-HIV drugs and alcohol induce similar cellular stress responses and injuries in liver nonparenchymal cells. METHODS: Primary mouse hepatocytes (PMH), kupffer cells (KC), and hepatocellular stellate cells (HSC) were freshly isolated from mouse liver and treated with DMSO, stress-inducing pharmaceutical agents, alcohol alone, or in combination with antiviral ritonavir (RIT), lopinavir (LOP), or efavirenz (EFV). Expression of cellular stress markers, protein colocalization, and cell death were analyzed with immunoblotting, immunocytochemistry, and positive double staining with Sytox green and Hoechst blue, respectively. RESULTS: Expression of the ER stress markers of BiP, CHOP, and SERCA and the autophagy marker LC3 was significantly changed in PMH in response to combined alcohol, RIT, and LOP, which was companied by increased cell death compared with control. In contrast, although pharmaceutical agents induced ER stress and cell death, no significant ER stress or cell death was found in KC treated with alcohol, RIT, LOP, and EFV singly or in combination. In HSC, alcohol, RIT, LOP, or EFV induced BiP, but not CHOP, SERCA, or cell death compared with vehicle control. Further in PMH, RIT and LOP or in combination with alcohol-induced dose-dependent inhibition of ß-actin. Inhibition of ß-actin by RIT and LOP was companied with an inhibited nuclear expression of the antioxidant response regulator Nrf2 and reduced GST downstream of Nrf2. Ascorbic acid treatment reduced the alcohol-, RIT-, and LOP-induced cell death. CONCLUSIONS: The data suggest for the first time that sensitivities of hepatocytes and nonparenchymal cells to alcohol and anti-HIV drugs in vitro are different in terms of cellular stress response and cell death injury. Oxidative stress mediated by Nrf2 contributes to the alcohol- and drug-induced toxicity in the hepatocytes.

Not all lacrimal epithelial cells are created equal-heterogeneity of the rabbit lacrimal gland and differential secretion.

AIMS: To test the hypotheses that some epithelial cells in the rabbit lacrimal gland (LG) are mucin-secreting cells that are also particularly rich in aquaporin 5 (AQP5) and sodium potassium ATPase ß(1) subunit (NKAß(1)), LG-secreted mucins contribute to the total mucin pool in tear film, and that the rabbit LG is a heterogenic gland where proteins secreted in response to different agonists are varied. MATERIALS AND METHODS: LGs were obtained from adult female rabbits and processed for paraffin sections for hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS), mucicarmine, and Alcian blue (pH 0.4, 1.0, and 2.5) for the detection of mucins. Serial sections were used for immunohistochemistry (IHC) and PAS. LG lysates and fluids were assayed by dot blot for detection of mucins, and by SDS-PAGE to detect differences in protein profiles of LG fluids stimulated by different agonists. RESULTS: HE staining demonstrated that the LG is a heterogeneous gland where most epithelial cells are serous, while all duct cells are mucous cells. Some acini and individual acinar cells within serous acini are also mucous or seromucous cells and these cells are particularly rich in AQP5 and NKAß(1). Dot blot assay showed the presence of mucins in the LG fluids. The protein profiles of LG fluids from pilocarpine, phenylephrine, and isoproterenol varied significantly, particularly in the mid range. CONCLUSIONS: Our data indicated that the rabbit LG is a heterogeneous gland that is composed of both serous and mucin-secreting cells, and mucins produced by the LG contribute to the mucin pool in the tear film. The heterogeneity of the rabbit LG supports the notion of differential secretion, i.e. the volume and composition of the LG fluids vary depending on various circumstances in the ocular surface and the body's needs.

Increased expression of cathepsins and obesity-induced proinflammatory cytokines in lacrimal glands of male NOD mouse.

PURPOSE: Lacrimal glands (LGs) of male NOD mice, a model of Sjögren's syndrome (SjS), exhibit immune cell infiltration and lipid deposition. The mechanism of SjS was further investigated by characterizing gene expression profiles of NOD mouse LGs in comparison with those of healthy control mice. Differentially expressed genes were further investigated at the protein level to correlate changes in location and abundance with development of disease. METHODS: Microarray followed by real-time RT-PCR was conducted to compare the gene expression in 12-week-old male NOD mouse LG relative to that in matched BALB/c mouse LG. Immunofluorescence and Western blot analyses were used to localize and quantify proteins of interest. Enzymatic assays measured catalytic activity of cathepsins. RESULTS: Cathepsin H (Ctsh), S (Ctss), and Z (Ctsz) and proinflammatory factors, including tumor necrosis factor (Tnf), interleukin 6 (Il6), and interleukin 1 beta (Il1b), were upregulated at the mRNA level. Increased cathepsin S immunofluorescence was detected in lysosomes and secretory vesicle-like organelles in LG acinar cells and CD68-positive infiltrating macrophages in NOD mouse LG. Cathepsin S (CATS) and cathepsin H (CATH) activities were significantly higher in NOD mouse LG lysate than in control lysates, and CATS was also significantly elevated in NOD mouse tears. CONCLUSIONS: Expression of CATS and CATH increases in parallel with proinflammatory cytokines during the development of autoimmune inflammatory disease in the NOD mouse disease model. Tear CATS may represent a biomarker for diagnosis of dacryoadenitis in SjS.

Altered expression of genes functioning in lipid homeostasis is associated with lipid deposition in NOD mouse lacrimal gland.

Functional atrophy and accompanying lymphocytic infiltration and destruction of the lacrimal gland (LG) are characteristics of Sjögren's Syndrome (SjS). The male NOD mouse is an experimental model for the autoimmune exocrinopathy that develops in the LG of SjS patients. Acinar cells in LG of male NOD mice aged 3-4 months were previously shown to accumulate lipid droplets. In the current study, analysis of lipid components revealed that the accumulated lipids were mostly cholesteryl esters (CE). Gene expression microarray analysis followed by real-time RT-PCR revealed alterations in the expression of several genes involved in lipid homeostasis in LG of 12-week-old male NOD mice relative to matched BALB/c controls. A series of upregulated genes including apolipoprotein E, apolipoprotein F, hepatic lipase, phosphomevalonate kinase, ATP-binding cassette D1 and ATP-binding cassette G1 were identified. Comparison of liver mRNAs to LG mRNAs in BALB/c and NOD mice revealed that the differential expressions were LG-specific. Gene expression profiles were also characterized in LGs of female mice, younger mice and immune-incompetent NOD SCID mice. Investigation of the cellular distribution of Apo-E and Apo-F proteins suggested that these proteins normally coordinate to mediate lipid efflux from the acinar cells but that dysfunction of these processes due to missorting of Apo-F may contribute to CE deposition. Finally, the initiation and extent of lipid deposition were correlated with lymphocytic infiltration in the LG of male NOD mice. We propose that impaired lipid efflux contributes to lipid deposition, an event that may contribute to the development and/or progression of dacryoadenitis in the male NOD mouse.