Macrophage RAGE activation is proinflammatory in NASH.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Modulating sphingosine 1-phosphate receptor signaling skews intrahepatic leukocytes and attenuates murine nonalcoholic steatohepatitis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid associated with nonalcoholic steatohepatitis (NASH). Immune cell-driven inflammation is a key determinant of NASH progression. Macrophages, monocytes, NK cells, T cells, NKT cells, and B cells variably express S1P receptors from a repertoire of 5 receptors termed S1P1 - S1P5. We have previously demonstrated that non-specific S1P receptor antagonism ameliorates NASH and attenuates hepatic macrophage accumulation. However, the effect of S1P receptor antagonism on additional immune cell populations in NASH remains unknown. We hypothesized that S1P receptor specific modulation may ameliorate NASH by altering leukocyte recruitment. A murine NASH model was established by dietary feeding of C57BL/6 male mice with a diet high in fructose, saturated fat, and cholesterol (FFC) for 24 weeks. In the last 4 weeks of dietary feeding, the mice received the S1P1,4,5 modulator Etrasimod or the S1P1 modulator Amiselimod, daily by oral gavage. Liver injury and inflammation were determined by histological and gene expression analyses. Intrahepatic leukocyte populations were analyzed by flow cytometry, immunohistochemistry, and mRNA expression. Alanine aminotransferase, a sensitive circulating marker for liver injury, was reduced in response to Etrasimod and Amiselimod treatment. Liver histology showed a reduction in inflammatory foci in Etrasimod-treated mice. Etrasimod treatment substantially altered the intrahepatic leukocyte populations through a reduction in the frequency of T cells, B cells, and NKT cells and a proportional increase in CD11b+ myeloid cells, polymorphonuclear cells, and double negative T cells in FFC-fed and control standard chow diet (CD)-fed mice. In contrast, FFC-fed Amiselimod-treated mice showed no changes in the frequencies of intrahepatic leukocytes. Consistent with the improvement in liver injury and inflammation, hepatic macrophage accumulation and the gene expression of proinflammatory markers such as Lgals3 and Mcp-1 were decreased in Etrasimod-treated FFC-fed mice. Etrasimod treated mouse livers demonstrated an increase in non-inflammatory (Marco) and lipid associated (Trem2) macrophage markers. Thus, S1P1,4,5 modulation by Etrasimod is more effective than S1P1 antagonism by Amiselimod, at the dose tested, in ameliorating NASH, likely due to the alteration of leukocyte trafficking and recruitment. Etrasimod treatment results in a substantial attenuation of liver injury and inflammation in murine NASH.

A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity.

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

Circulating extracellular vesicles are a biomarker for NAFLD resolution and response to weight loss surgery.

There is increasing interest in the development of minimally invasive biomarkers for the diagnosis and prognosis of NAFLD via extracellular vesicles (EV). Plasma EVs were isolated by differential ultracentrifugation and quantified by nanoparticle tracking analysis from pre (n = 28) and post (n = 28) weight loss patients. In the pre weight loss group 22 had NAFLD. Nanoplasmon enhanced scattering (nPES) of gold nanoparticles conjugated to hepatocyte-specific antibodies was employed to identify hepatocyte-specific EVs. Complex lipid panel and targeted sphingolipids were performed. Logistic regression analysis was used to identify predictors of NAFLD. Plasma levels of EVs and hepatocyte-derived EVs are dynamic and decrease following NAFLD resolution due to weight loss surgery. Hepatocyte-derived EVs correlate with steatosis in NAFLD patients and steatosis and inflammation in NASH patients. Plasma levels of small EVs correlate with EV sphingolipids in patients with NASH. Hepatocyte-derived EVs measured by the nPES assay could serve as a point-of-care test for NAFLD.

Circulating Extracellular Vesicles Carrying Sphingolipid Cargo for the Diagnosis and Dynamic Risk Profiling of Alcoholic Hepatitis.

BACKGROUND AND AIMS: Alcoholic hepatitis (AH) is diagnosed by clinical criteria, although several objective scores facilitate risk stratification. Extracellular vesicles (EVs) have emerged as biomarkers for many diseases and are also implicated in the pathogenesis of AH. Therefore, we investigated whether plasma EV concentration and sphingolipid cargo could serve as diagnostic biomarkers for AH and inform prognosis to permit dynamic risk profiling of AH subjects. APPROACH AND RESULTS: EVs were isolated and quantified from plasma samples from healthy controls, heavy drinkers, and subjects with end-stage liver disease (ESLD) attributed to cholestatic liver diseases and nonalcoholic steatohepatitis, decompensated alcohol-associated cirrhosis (AC), and AH. Sphingolipids were quantified by tandem mass spectroscopy. The median plasma EV concentration was significantly higher in AH subjects (5.38 × 1011 /mL) compared to healthy controls (4.38 × 1010 /mL; P < 0.0001), heavy drinkers (1.28 × 1011 /mL; P < 0.0001), ESLD (5.35 × 1010 /mL; P < 0.0001), and decompensated AC (9.2 × 1010 /mL; P < 0.0001) disease controls. Among AH subjects, EV concentration correlated with Model for End-Stage Liver Disease score. When EV counts were dichotomized at the median, survival probability for AH subjects at 90 days was 63.0% in the high-EV group and 90.0% in the low-EV group (log-rank P value = 0.015). Interestingly, EV sphingolipid cargo was significantly enriched in AH when compared to healthy controls, heavy drinkers, ESLD, and decompensated AC (P = 0.0001). Multiple sphingolipids demonstrated good diagnostic and prognostic performance as biomarkers for AH. CONCLUSIONS: Circulating EV concentration and sphingolipid cargo signature can be used in the diagnosis and differentiation of AH from heavy drinkers, decompensated AC, and other etiologies of ESLD and predict 90-day survival permitting dynamic risk profiling.

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

BACKGROUND & AIMS: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis. METHODS: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1αΔhep) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4µ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses. RESULTS: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation. CONCLUSIONS: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.

Characterization of Cellular Sources and Circulating Levels of Extracellular Vesicles in a Dietary Murine Model of Nonalcoholic Steatohepatitis.

Circulating extracellular vesicles (EVs) are a novel and emerging biomarker for nonalcoholic steatohepatitis (NASH). It has been demonstrated that total circulating EVs and hepatocyte-derived EVs are elevated in male mice with diet-induced NASH. How hepatocyte-derived EVs change over time and other cellular sources of EVs in NASH have not been determined. Our objective was to define the quantitative evolution of hepatocyte-derived, macrophage-derived, neutrophil-derived, and platelet-derived EVs in male and female mice with dietary NASH. Fluorescently labeled antibodies and a nanoscale flow cytometer were used to detect plasma levels of EVs. Asialoglycoprotein receptor 1 (ASGR1) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) are markers of hepatocyte-derived EVs; galectin 3 is a marker of macrophage-derived EVs; common epitope on lymphocyte antigen 6 complex, locus G/C1 (Ly-6G and Ly-6C) is a marker of neutrophil-derived EVs; and clusters of differentiation 61 (CD61) is a marker of platelet-derived EVs. Nonalcoholic fatty liver disease activity score (NAS) was calculated using hematoxylin and eosin-stained liver sections, and magnetic resonance imaging (MRI) was used for measurement of the fat fraction and elastography. Hepatocyte-derived EVs increased in both male and female mice at 12 and 10 weeks of feeding, respectively, and remained elevated at 24 weeks in both male and female mice and at 48 weeks in male mice and 36 weeks in female mice. Macrophage- and neutrophil-derived EVs were significantly elevated at 24 weeks of dietary feeding concomitant with the histologic presence of inflammatory foci in the liver. In fat-, fructose-, and cholesterol- (FFC) fed male mice, platelet-derived EVs were elevated at 12, 24, and 48 weeks, whereas in female mice, platelet derived EVs were significantly elevated at 24 weeks. Hepatocyte-, macrophage- and neutrophil-derived EVs correlated well with the histologic NAS. Conclusion: Circulating cell-type-specific EVs may be a novel biomarker for NASH diagnosis and longitudinal follow up.

Prediction of nonalcoholic fatty liver disease (NAFLD) activity score (NAS) with multiparametric hepatic magnetic resonance imaging and elastography.

OBJECTIVES: To investigate the use of MR elastography (MRE)-derived mechanical properties (shear stiffness (|G*|) and loss modulus (G″)) and MRI-derived fat fraction (FF) to predict the nonalcoholic fatty liver disease (NAFLD) activity score (NAS) in a NAFLD mouse model. METHODS: Eighty-nine male mice were studied, including 64 training and 25 independent testing animals. An MRI/MRE exam and histologic evaluation were performed. Pairwise, nonparametric comparisons and multivariate analyses were used to evaluate the relationships between the three imaging parameters (FF, |G*|, and G″) and histologic features. A virtual NAS score (vNAS) was generated by combining three imaging parameters with an ordinal logistic model (OLM) and a generalized linear model (GLM). The prediction accuracy was evaluated by ROC analyses. RESULTS: The combination of FF, |G*|, and G″ predicted NAS > 1 with excellent accuracy in both training and testing sets (AUROC > 0.84). OLM and GLM predictive models misclassified 3/54 and 6/54 mice in the training, and 1/25 and 1/25 in the testing cohort respectively, in distinguishing between "not-NASH" and "definite-NASH." "Borderline-NASH" prediction was poorer in the training set, and no borderline-NASH mice were available in the testing set. CONCLUSION: This preliminary study shows that multiparametric MRI/MRE can be used to accurately predict the NAS score in a NAFLD animal model, representing a promising alternative to liver biopsy for assessing NASH severity and treatment response. KEY POINTS: • MRE-derived liver stiffness and loss modulus and MRI-assessed fat fraction can be used to predict NAFLD activity score (NAS) in our preclinical mouse model (AUROC > 0.84 for all NAS levels greater than 1). • The overall agreement between the histological-determined NASH diagnosis and the imaging-predicted NASH diagnosis is 80-92%. • The multiparametric hepatic MRI/MRE has great potential for noninvasively assessing liver disease severity and treatment efficacy.

StAR-related lipid transfer domain 11 (STARD11)-mediated ceramide transport mediates extracellular vesicle biogenesis.

Extracellular vesicles are important carriers of cellular materials and have critical roles in cell-to-cell communication in both health and disease. Ceramides are implicated in extracellular vesicle biogenesis, yet the cellular machinery that mediates the formation of ceramide-enriched extracellular vesicles remains unknown. We demonstrate here that the ceramide transport protein StAR-related lipid transfer domain 11 (STARD11) mediates the release of palmitate-stimulated extracellular vesicles having features consistent with exosomes. Using palmitate as a model of lipotoxic diseases and as a substrate for ceramide biosynthesis in human and murine liver cell lines and primary mouse hepatocytes, we found that STARD11-deficient cells release fewer extracellular vesicles. Moreover, STARD11 reciprocally regulated exosome ceramide enrichment and cellular ceramide depletion. We further observed that in STARD11 knockout cells intracellular ceramide accumulates and that this apparent inability to transfer cellular ceramide into extracellular vesicles reduces cellular viability. Using endogenous markers, we uncovered structural and functional colocalization of the endoplasmic reticulum (ER), STARD11, and multivesicular bodies. This colocalization increased following palmitate treatment, suggesting a functional association that may mediate ceramide trafficking from the ER to the multivesicular body. However, the size and number of multivesicular bodies were comparable in WT and STARD11-knockout cells. In conclusion, we propose a model of how STARD11 mediates ceramide trafficking in palmitate-treated cells and stimulates exosome biogenesis.

Deletion of endoplasmic reticulum stress-responsive co-chaperone p58IPK protects mice from diet-induced steatohepatitis.

AIM: Activation of PKR-like endoplasmic reticulum kinase (PERK), an endoplasmic reticulum stress sensor, is a feature of non-alcoholic steatohepatitis (NASH), yet regulators of PERK signaling remain undefined in this context. The protein p58IPK regulates PERK; however, its role in NASH has not been examined. The aim of this study was to assess the in vivo role of p58IPK in the pathogenesis of dietary NASH. METHODS: Parameters of hepatocyte cell death, liver injury, inflammation, fibrosis, indirect calorimetry and PERK activation were assessed in p58IPK knockout (p58ipk-/- ) mice and their wild-type littermate controls. All animals were fed a diet enriched in fat, fructose, and cholesterol (FFC) for 20 weeks. RESULTS: Activation of PERK was attenuated in FFC-fed p58ipk-/- mice. Accordingly, FFC-fed p58ipk-/- mice showed a reduction in hepatocyte apoptosis and death receptor expression, with a significant reduction in serum alanine transaminase values. Correspondingly, macrophage accumulation and fibrosis were significantly lower in FFC-fed p58ipk-/- mice. CONCLUSION: We have shown that, in an in vivo dietary NASH model, p58IPK mediates hepatocyte apoptosis and liver injury, likely through PERK phosphorylation. In the absence of p58IPK , PERK phosphorylation and NASH are attenuated. Inhibition of hepatic p58IPK could be a future target for NASH therapy.

Hepatocyte-Derived Lipotoxic Extracellular Vesicle Sphingosine 1-Phosphate Induces Macrophage Chemotaxis.

Background: The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Methods: Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P1 receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P1 receptor on intrahepatic leukocytes and hepatic expression of S1P1 receptor, respectively. Results: Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P1 receptor toward lipotoxic EVs. S1P1 receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Conclusions:Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P1 receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.

Inhibition of sphingosine 1-phosphate signaling ameliorates murine nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disorder, wherein proinflammatory lipids, such as ceramide and its derivative sphingosine 1-phosphate (S1P), contribute to macrophage-associated liver inflammation. For example, we have previously demonstrated a role for S1P in steatotic hepatocyte-derived S1P-enriched extracellular vesicles in macrophage chemotaxis in vitro. Therefore, we hypothesized that FTY720, an S1P antagonist, would ameliorate NASH by inhibiting proinflammatory monocyte chemotaxis. To test our hypothesis, NASH was established in C57BL/6 male mice by feeding a diet high in fructose, saturated fat, and cholesterol for 22 wk. Then mice received daily intraperitoneal injections of FTY720 for 2 wk before analysis of liver injury, inflammation, and fibrosis. FTY720-treated mice with NASH demonstrated improved liver histology with a significant reduction in hepatocyte ballooning and inflammatory foci. Hepatomegaly was reversed, and liver triglycerides were reduced following FTY720 administration to mice with NASH. Correspondingly, serum ALT levels, hepatic inflammatory macrophage accumulation, and the expression of Ly6C in recruited myeloid cells was reduced in FTY720-treated mice. Hepatic collagen accumulation and expression of α-smooth muscle actin were significantly lowered as well. Body composition, energy consumption and utilization, and hepatic sphingolipid composition remained unchanged following FTY720 administration. FTY720 ameliorates murine nonalcoholic steatohepatitis. Reduction in liver injury and inflammation is associated with a reduction in hepatic macrophage accumulation, likely due to dampened recruitment of circulating myeloid cells into the liver. Nonalcoholic steatohepatitis may be a novel indication for the therapeutic use of FTY720.NEW & NOTEWORTHY There are no approved pharmacologic therapies for nonalcoholic steatohepatitis (NASH), the leading cause of chronic liver disease worldwide. This study describes the use of FTY720, a novel small molecule, for the amelioration of NASH in a mouse model. We demonstrate that 2-wk administration of FTY720 to mice with NASH led to a reduction in liver injury, inflammation, and fibrosis. These data provide a preclinical rationale for studying this drug in human NASH.

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner.

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.

Mmu-miR-615-3p regulates lipoapoptosis by inhibiting C/EBP homologous protein.

Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.

C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.

Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop(+/+)) and knock-out (Chop(-/-)) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop(-/-) mice developed greater liver injury, inflammation, and fibrosis than Chop(+/+) mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop(+/+) and Chop(-/-) were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop(+/+) and Chop(-/-) macrophages, Chop(-/-) macrophages were resistant to its lipotoxicity. Chop(-/-) mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop(-/-) and Chop(+/+) mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2.

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.

Cellular and subcellular localization of the neuron-specific plasma membrane calcium ATPase PMCA1a in the rat brain.

Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca(2+) extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a "housekeeping" function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca(2+) transients.

Calmodulin-like protein upregulates myosin-10 in human keratinocytes and is regulated during epidermal wound healing in vivo.

Epidermal wound healing is required for normal skin barrier function. Cell motility is a key factor in the ability of keratinocytes to heal epithelial damage. Calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein that is regulated during terminal keratinocyte differentiation. CLP is a specific light chain of unconventional myosin-10 (Myo10) and its expression increases filopodial length, filopodial number, and Myo10-dependent cell motility in vitro. However, the effects of CLP expression on keratinocyte motility are unknown. Here we used cultured human keratinocytes to study the role of CLP in regulating Myo10 and the effects of Myo10 and CLP on cell migration. CLP and Myo10 expression were correlated in vitro and required for keratinocyte motility in wound-healing assays. We examined the localization of CLP in wounded skin by immunohistochemistry and found an upregulation and peripheral localization of CLP in the basal and suprabasal cells adjacent to and immediately over the wound bed in vivo. The results suggest that increased CLP expression and CLP-mediated Myo10 function are important for skin differentiation and wound reepithelialization.

Interaction with the IQ3 motif of myosin-10 is required for calmodulin-like protein-dependent filopodial extension.

Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.

Calmodulin-like protein increases filopodia-dependent cell motility via up-regulation of myosin-10.

Human calmodulin-like protein (CLP) is an epithelial-specific protein that is expressed during cell differentiation but down-regulated in primary cancers and transformed cell lines. Using stably transfected and inducible HeLa cell lines, we found that CLP expression did not alter the proliferation rate and colony-forming potential of these cells. However, remarkable phenotypic changes were observed in CLP-expressing compared with control cells. Soft agar colonies of CLP-expressing cells had rough boundaries, with peripheral cells migrating away from the colony. Cells expressing CLP displayed a striking increase in the number and length of myosin-10-positive filopodia and showed increased mobility in a wound healing assay. This increase in wound healing capacity was prevented by small interference RNA-mediated down-regulation of myosin-10. Fluorescence microscopy and Western blotting revealed that CLP expression results in up-regulation of its target protein, myosin-10. This up-regulation occurs at the protein level by stabilization of myosin-10. Thus, CLP functions by increasing the stability of myosin-10, leading to enhanced myosin-10 function and a subsequent increase in filopodial dynamics and cell migration. In stratified epithelia, CLP may be required during terminal differentiation to increase myosin-10 function as cells migrate toward the upper layers and establish new adhesive contacts.