Arylsulfatases and neuraminidases modulate engagement of CCR5 by chemokines by removing key electrostatic interactions.

The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident 'source enzyme, and a 'sink' activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation 'sink' enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate 'sink' enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase 'sink' enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization.

Mir-21 Suppression Promotes Mouse Hepatocarcinogenesis.

The microRNA 21 (miR-21) is upregulated in almost all known human cancers and is considered a highly potent oncogene and potential therapeutic target for cancer treatment. In the liver, miR-21 was reported to promote hepatic steatosis and inflammation, but whether miR-21 also drives hepatocarcinogenesis remains poorly investigated in vivo. Here we show using both carcinogen (Diethylnitrosamine, DEN) or genetically (PTEN deficiency)-induced mouse models of hepatocellular carcinoma (HCC), total or hepatocyte-specific genetic deletion of this microRNA fosters HCC development-contrasting the expected oncogenic role of miR-21. Gene and protein expression analyses of mouse liver tissues further indicate that total or hepatocyte-specific miR-21 deficiency is associated with an increased expression of oncogenes such as Cdc25a, subtle deregulations of the MAPK, HiPPO, and STAT3 signaling pathways, as well as alterations of the inflammatory/immune anti-tumoral responses in the liver. Together, our data show that miR-21 deficiency promotes a pro-tumoral microenvironment, which over time fosters HCC development via pleiotropic and complex mechanisms. These results question the current dogma of miR-21 being a potent oncomiR in the liver and call for cautiousness when considering miR-21 inhibition for therapeutic purposes in HCC.

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

S100A11/ANXA2 belongs to a tumour suppressor/oncogene network deregulated early with steatosis and involved in inflammation and hepatocellular carcinoma development.

OBJECTIVE: Hepatocellular carcinoma (HCC) development occurs with non-alcoholic fatty liver disease (NAFLD) in the absence of cirrhosis and with an increasing incidence due to the obesity pandemic. Mutations of tumour suppressor (TS) genes and oncogenes (ONC) have been widely characterised in HCC. However, mounting evidence indicates that non-genomic alterations of TS/ONC occur early with NAFLD, thereby potentially promoting hepatocarcinogenesis in an inflammatory/fibrotic context. The aim of this study was to identify and characterise these alterations. DESIGN: The proteome of steatotic liver tissues from mice spontaneously developing HCC was analysed. Alterations of TSs/ONCs were further investigated in various mouse models of NAFLD/HCC and in human samples. The inflammatory, fibrogenic and oncogenic functions of S100A11 were assessed through in vivo, in vitro and ex-vivo analyses. RESULTS: A whole set of TSs/ONCs, respectively, downregulated or upregulated was uncovered in mice and human with NAFLD. Alterations of these TSs/ONCs were preserved or even exacerbated in HCC. Among them, overexpression of S100A11 was associated with high-grade HCC and poor prognosis. S100A11 downregulation in vivo significantly restrains the development of inflammation and fibrosis in mice fed a choline/methionine-deficient diet. Finally, in vitro and ex-vivo analyses revealed that S100A11 is a marker of hepatocyte de-differentiation, secreted by cancer cells, and promoting cell proliferation and migration. CONCLUSION: Cellular stress associated with NAFLD triggers non-genomic alterations of a whole network of TSs/ONCs fostering hepatocarcinogenesis. Among those, overexpression of the oncogenic factor S100A11 promotes inflammation/fibrosis in vivo and is significantly associated with high-grade HCC with poor prognosis.

Exercise Improves Outcomes of Surgery on Fatty Liver in Mice: A Novel Effect Mediated by the AMPK Pathway.

OBJECTIVE: To investigate whether exercise improves outcomes of surgery on fatty liver, and whether pharmacological approaches can substitute exercising programs. SUMMARY OF BACKGROUND DATA: Steatosis is the hepatic manifestation of the metabolic syndrome, and decreases the liver's ability to handle inflammatory stress or to regenerate after tissue loss. Exercise activates adenosine monophosphate-activated kinase (AMPK) and mitigates steatosis; however, its impact on ischemia-reperfusion injury and regeneration is unknown. METHODS: We used a mouse model of simple, diet-induced steatosis and assessed the impact of exercise on metabolic parameters, ischemia-reperfusion injury and regeneration after hepatectomy. The same parameters were evaluated after treatment of mice with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Mice on a control diet served as age-matched controls. RESULTS: A 4-week-exercising program reversed steatosis, lowered insulin levels, and improved glucose tolerance. Exercise markedly enhanced the ischemic tolerance and the regenerative capacity of fatty liver. Replacing exercise with AICAR was sufficient to replicate the above benefits. Both exercise and AICAR improved survival after extended hepatectomy in mice challenged with a Western diet, indicating protection from resection-induced liver failure. CONCLUSIONS: Exercise efficiently counteracts the metabolic, ischemic, and regenerative deficits of fatty liver. AICAR acts as an exercise mimetic in settings of fatty liver disease, an important finding given the compliance issues associated with exercise. Exercising, or its substitution through AICAR, may provide a feasible strategy to negate the hepatic consequences of energy-rich diet, and has the potential to extend the application of liver surgery if confirmed in humans.

Activation of the oncogenic miR-21-5p promotes HCV replication and steatosis induced by the viral core 3a protein.

BACKGROUND & AIMS: miR-21-5p is a potent oncogenic microRNA targeting many key tumour suppressors including phosphatase and tensin homolog (PTEN). We recently identified PTEN as a key factor modulated by hepatitis C virus (HCV) to promote virion egress. In hepatocytes, expression of HCV-3a core protein was sufficient to downregulate PTEN and to trigger lipid droplet accumulation. Here, we investigated whether HCV controls PTEN expression through miR-21-5p-dependent mechanisms to trigger steatosis in hepatocytes and to promote HCV life cycle. METHODS: MiR-21-5p expression in HCV-infected patients was evaluated by transcriptome meta-analysis. HCV replication and viral particle production were investigated in Jc1-infected Huh-7 cells after miR-21-5p inhibition. PTEN expression and steatosis were assessed in HCV-3a core protein-expressing Huh-7 cells and in mouse primary hepatocytes having miR-21-5p inhibited or genetically deleted respectively. HCV-3a core-induced steatosis was assessed in vivo in Mir21a knockout mice. RESULTS: MiR-21-5p expression was significantly increased in hepatic tissues from HCV-infected patients. Infection by HCV-Jc1, or transduction with HCV-3a core, upregulated miR-21-5p expression and/or activity in Huh-7 cells. miR-21-5p inhibition decreased HCV replication and release of infectious virions by Huh-7 cells. HCV-3a core-induced PTEN downregulation and steatosis were further prevented in Huh-7 cells following miR-21-5p inhibition or in Mir21a knockout mouse primary hepatocytes. Finally, steatosis induction by AAV8-mediated HCV-3a core expression was reduced in vivo in Mir21a knockout mice. CONCLUSION: MiR-21-5p activation by HCV is a key molecular step, promoting both HCV life cycle and HCV-3a core-induced steatosis and may be among the molecular changes induced by HCV-3a to promote carcinogenesis.

PTEN Down-Regulation Promotes ß-Oxidation to Fuel Hypertrophic Liver Growth After Hepatectomy in Mice.

In regenerating liver, hepatocytes accumulate lipids before the major wave of parenchymal growth. This transient, regeneration-associated steatosis (TRAS) is required for liver recovery, but its purpose is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is a key inhibitor of the protein kinase B/mammalian target of rapamycin axis that regulates growth and metabolic adaptations after hepatectomy. In quiescent liver, PTEN causes pathological steatosis when lost, whereas its role in regenerating liver remains unknown. Here, we show that PTEN down-regulation promotes liver growth in a TRAS-dependent way. In wild-type mice, PTEN reduction occurred after TRAS formation, persisted during its disappearance, and correlated with up-regulated ß-oxidation at the expense of lipogenesis. Pharmacological modulation revealed an association of PTEN with TRAS turnover and hypertrophic liver growth. In liver-specific Pten-/- mice shortly after induction of knockout, hypertrophic regeneration was accelerated and led to hepatomegaly. The resulting surplus liver mass was functional, as demonstrated by raised survival in a lethal model of resection-induced liver failure. Indirect calorimetry revealed lipid oxidation as the primary energy source early after hepatectomy. The shift from glucose to lipid usage was pronounced in Pten-/- mice and correlated with the disappearance of TRAS. Partial inhibition of ß-oxidation led to persisting TRAS in Pten-/- mice and abrogated hypertrophic liver growth. PTEN down-regulation may promote ß-oxidation through ß-catenin, whereas hypertrophy was dependent on mammalian target of rapamycin complex 1. CONCLUSION: PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

Omega-3 Fatty Acids Protect Fatty and Lean Mouse Livers After Major Hepatectomy.

OBJECTIVE: The aim of this study was to assess the effect of Ω3 fatty acids (Ω3FA) on fatty and lean liver in hepatic surgery. BACKGROUND: The global spread of energy-dense diets has led to an endemic rise in fatty liver disease and obesity. Besides metabolic pathologies, steatosis enhances hepatic sensitivity to ischemia reperfusion (I/R) and impedes liver regeneration (LR). Steatosis limits the application of liver surgery, still the main curative option for liver cancer. Ω3FA are known to reverse steatosis, but how these lipids affect key factors defining surgical outcomes-that is, I/R, LR, and liver malignancy-is less clear. METHODS: We established a standardized mouse model of high fat diet (HFD)-induced steatosis followed by Ω3FA treatment and the subsequent assessment of Ω3FA effects on I/R, LR, and liver malignancy (n = 5/group), the latter through a syngeneic metastasis approach. Fatty liver outcomes were compared with lean liver to assess steatosis-independent effects. Nonparametric statistics were applied. RESULTS: Ω3FA reversed HFD-induced steatosis and markedly protected against I/R, improved LR, and prolonged survival of tumor-laden mice. Remarkably, these beneficial effects were also observed in lean liver, albeit at a smaller scale. Notably, mice with metastases in fatty versus lean livers were associated with improved survival. CONCLUSIONS: Ω3FA revealed multiple beneficial effects in fatty and lean livers in mice. The improvements in I/R injury, regenerative capacity, and oncological outcomes await confirmatory studies in humans.

Stress-activated miR-21/miR-21* in hepatocytes promotes lipid and glucose metabolic disorders associated with high-fat diet consumption.

OBJECTIVE: miR-21 is an oncomir highly upregulated in hepatocellular carcinoma and in early stages of liver diseases characterised by the presence of steatosis. Whether upregulation of miR-21 contributes to hepatic metabolic disorders and their progression towards cancer is unknown. This study aims at investigating the role of miR-21/miR-21* in early stages of metabolic liver disorders associated with diet-induced obesity (DIO). DESIGN: Constitutive miR-21/miR-21* knockout (miR21KO) and liver-specific miR-21/miR-21* knockout (LImiR21KO) mice were generated. Mice were then fed with high-fat diet (HFD) and alterations of the lipid and glucose metabolism were investigated. Serum and ex vivo explanted liver tissue were analysed. RESULTS: Under normal breeding conditions and standard diet, miR-21/miR-21* deletion in mice was not associated with any detectable phenotypic alterations. However, when mice were challenged with an obesogenic diet, glucose intolerance, steatosis and adiposity were improved in mice lacking miR-21/miR-21*. Deletion of miR-21/miR-21* specifically in hepatocytes led to similar improvements in mice fed an HFD, indicating a crucial role for hepatic miR-21/miR-21* in metabolic disorders associated with DIO. Further molecular analyses demonstrated that miR-21/miR-21* deletion in hepatocytes increases insulin sensitivity and modulates the expression of multiple key metabolic transcription factors involved in fatty acid uptake, de novo lipogenesis, gluconeogenesis and glucose output. CONCLUSIONS: Hepatic miR-21/miR-21* deficiency prevents glucose intolerance and steatosis in mice fed an obesogenic diet by altering the expression of several master metabolic regulators. This study points out miR-21/miR-21* as a potential therapeutic target for non-alcoholic fatty liver disease and the metabolic syndrome.

MicroRNAs in fatty liver disease.

Overweight and obesity, insulin resistance and diabetes, chronic alcoholism, as well as infection by specific genotypes of hepatitis C viruses are all associated with an excessive and chronic ectopic accumulation of fat in the liver (steatosis). If the underlining causes of steatosis development are not resolved, progression toward more severe liver diseases such as inflammation, fibrosis, and cirrhosis can then occur with time. These hepatic metabolic and histological disorders are commonly referred to as fatty liver disease (FLD) and result from multiple deregulated molecular mechanisms controlling hepatic homeostasis. Among these mechanisms, deregulation of a whole network of small noncoding RNAs called microRNAs (miRNAs), which regulate gene expression at a posttranscriptional level, critically contributes to the development and progression of FLD. Specific miRNAs secreted in body fluids are also emerging as useful biomarkers of FLD and therapeutic targeting of miRNAs is currently being evaluated. The authors discuss recent findings highlighting the role and complexity of miRNA regulatory networks, which critically contribute to the development of FLD. As well, the potential therapeutic perspectives for FLD that our understanding of hepatic miRNA biology offers is considered.

The mouse interleukin (Il)33 gene is expressed in a cell type- and stimulus-dependent manner from two alternative promoters.

GenBank entries for mouse Il33 reveal the existence of two transcripts, Il33a and Il33b, with different 5'UTRs but coding for the same protein. We investigated expression of these transcripts in different mouse organs and cell types in basal and inflammatory conditions. Il33a and Il33b mRNAs start with different noncoding first exons, transcribed from different promoter regions, which both contain a consensus TATA-like sequence. Constitutive Il33a mRNA expression was detected in mouse stomach, lung, spleen, and brain, whereas basal Il33b mRNA expression was observed only in the stomach. Expression of both transcripts increased after systemic LPS administration. In vitro, we observed high constitutive expression of Il33 transcripts in MEFs. Constitutive Il33a mRNA expression was observed also in BMDCs, where it was preferentially increased in response to poly(I:C), whereas LPS increased levels of Il33a and Il33b mRNA. In contrast, BMMs and Raw 264.7 cells did not express Il33 mRNA constitutively, and LPS stimulation selectively induced expression of Il33b mRNA in these cells. Our data indicate that the Il33 gene is expressed from two alternative promoters in the mouse and that the relative expression of Il33a and Il33b transcripts is cell type- and stimulus-dependent.