Neutral ceramidase-active site inhibitor chemotypes and binding modes.

Ceramides impact a diverse array of biological functions and have been implicated in disease pathogenesis. The enzyme neutral ceramidase (nCDase) is a zinc-containing hydrolase and mediates the metabolism of ceramide to sphingosine (Sph), both in cells and in the intestinal lumen. nCDase inhibitors based on substrate mimetics, for example C6-urea ceramide, have limited potency, aqueous solubility, and micelle-free fraction. To identify non-ceramide mimetic nCDase inhibitors, hit compounds from an HTS campaign were evaluated in biochemical, cell based and in silico modeling approaches. A majority of small molecule nCDase inhibitors contained pharmacophores capable of zinc interaction but retained specificity for nCDase over zinc-containing acid and alkaline ceramidases, as well as matrix metalloprotease-3 and histone deacetylase-1. nCDase inhibitors were refined by SAR, were shown to be substrate competitive and were active in cellular assays. nCDase inhibitor compounds were modeled by in silico DOCK screening and by molecular simulation. Modeling data supports zinc interaction and a similar compound binding pose with ceramide. nCDase inhibitors were identified with notably improved activity and solubility in comparison with the reference lipid-mimetic C6-urea ceramide.

Mass Spectrometry and Pharmacological Approaches to Measuring Cooption and Reciprocal Activation of Receptor Tyrosine Kinases.

Receptor tyrosine kinases (RTKs) can show extensive crosstalk, directly and indirectly. Elucidating RTK crosstalk remains an important goal in the clinical combination of anti-cancer therapies. Here, we present mass spectrometry and pharmacological approaches showing the hepatocyte growth factor receptor (MET)-promoting tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and other membrane receptors in MET-amplified H1993 NSCLC cells. Conversely, in H292 wt-EGFR NSCLC cells, EGFR promotes the tyrosine phosphorylation of MET. Reciprocal regulation of the EGFR and insulin receptor (IR) was observed in the GEO CRC cells, where inhibition of the EGFR drives tyrosine phosphorylation of the insulin receptor. Similarly, in platelet-derived growth factor receptor (PDGFR)-amplified H1703 NSCLC cells, inhibition of the EGFR promotes the tyrosine phosphorylation of the PDGFR. These RTK interactions are used to illustrate basic principles applicable to other RTK signaling networks. More specifically, we focus on two types of RTK interaction: (1) co-option of one RTK by another and (2) reciprocal activation of one receptor following the inhibition of a distinct receptor.

Measurement of neutral ceramidase activity in vitro and in vivo.

Neutral ceramidase is a hydrolase of ceramide that has been implicated in multiple biologic processes, including inflammation and oncogenesis. Ceramides and other sphingolipids, belong to a family of N-acyl linked lipids that are biologically active in signaling, despite their limited structural functions. Ceramides are generally pro-apoptotic, while sphingosine and sphingosine-1-phosphate (S1P) exert proliferative and pro-oncogenic effects. Ceramidases are important regulators of ceramide levels that hydrolyze ceramide to sphingosine. Thus, ceramidase inhibition significantly increases the quantities of ceramide and its associated signaling. To better understand the function of ceramide, biochemical and cellular assays for enzymatic activity were developed and validated to identify inhibitors of human neutral ceramidase (nCDase). Here we review the measurement of nCDase activity both in vitro and in vivo.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening.

There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC50 and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).

Neutral ceramidase: Advances in mechanisms, cell regulation, and roles in cancer.

Extensive research conducted in the last three decades has identified the roles for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) as key regulators of cellular homeostasis, growth and death. One of the major groups of enzymes in the ceramide pathway, ceramidases, converts ceramide into sphingosine and fatty acids, with sphingosine being further metabolized to S1P. Thus, these enzymes play important roles in the network controlling the functions associated with these bioactive sphingolipids. Among the family of ceramidases, neutral ceramidase (nCDase), which is named according to its optimal pH for catalytic activity, has received increased attention in the last decade. The goal of this review is to provide a brief background on bioactive sphingolipids and the ceramidases. We then describe more recent advances on nCDase, specifically the resolution of its crystal structure and understanding its roles in cell biology and physiology.

Functions of neutral ceramidase in the Golgi apparatus.

Ceramidases hydrolyze ceramides into sphingosine and fatty acids, with sphingosine being further metabolized into sphingosine-1-phosphate (S1P); thus, ceramidases control the levels of these bioactive sphingolipids in cells and tissues. Neutral ceramidase (nCDase) is highly expressed in colorectal tissues, and a recent report showed that nCDase activity is involved in Wnt/ß-catenin signaling. In addition, the inhibition of nCDase decreases the development and progression of colorectal tumor growth. Here, to determine the action of nCDase in colorectal cancer cells, we focused on the subcellular localization and metabolic functions of this enzyme in HCT116 cells. nCDase was found to be located in both the plasma membrane and in the Golgi apparatus, but it had minimal effects on basal levels of ceramide, sphingosine, or S1P. Cells overexpressing nCDase were protected from the cell death and Golgi fragmentation induced by C6-ceramide, and they showed reduced levels of C6-ceramide and higher levels of S1P and sphingosine. Furthermore, compartment-specific metabolic functions of the enzyme were probed using C6-ceramide and Golgi-targeted bacterial SMase (bSMase) and bacterial ceramidase (bCDase). The results showed that Golgi-specific bCDase also demonstrated resistance against the cell death stimulated by C6-ceramide, and it catalyzed the metabolism of ceramides and produced sphingosine in the Golgi. Targeting bSMase to the Golgi resulted in increased levels of ceramide that were attenuated by the expression of nCDase, also supporting its ability to metabolize Golgi-generated ceramide. These results are critical in understanding the functions of nCDase actions in colorectal cancer cells as well as the compartmentalized pathways of sphingolipid metabolism.

AKT as a key target for growth promoting functions of neutral ceramidase in colon cancer cells.

Despite advances in the field, colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Research into bioactive sphingolipids over the past two decades has played an important role in increasing our understanding of the pathogenesis and therapeutics of CRC. In the complex metabolic network of sphingolipids, ceramidases (CDases) have a key function. These enzymes hydrolyze ceramides into sphingosine (SPH) which in turn is phosphorylated by sphingosine kinases (SK) 1 and 2 to generate sphingosine-1 phosphate (S1P). Importantly, we have recently shown that inhibition of neutral CDase (nCDase) induces an increase of ceramide in colon cancer cells which decreases cellular growth, increases apoptosis and modulates the WNT/ß-catenin pathway. We have also shown that the deletion of nCDase protected mice from the onset and progression of colorectal cancer in the AOM carcinogen model. Here, we demonstrate that AKT is a key target for the growth suppressing functions of ceramide. The results show that inhibition of nCDase activates GSK3ß through dephosphorylation, and thus is required for the subsequent phosphorylation and degradation of ß-catenin. Our findings show that inhibition of nCDase also inhibits the basal activation status of AKT, and we further establish that a constitutively active AKT (AKT T308D, S473D; AKTDD) reverses the effect of nCDase on ß-catenin degradation. Functionally, the AKTDD mutant is able to overcome the growth suppressive effects of nCDase inhibition in CRC cells. Moreover, nCDase inhibition induces a growth delay of xenograft tumors from control cells, whereas xenograft tumors from constitutively active AKT cells become resistant to nCDase inhibition. Taken together, these results provide important mechanistic insight into how nCDase regulates cell proliferation. These findings demonstrate a heretofore unappreciated, but critical, role for nCDase in enabling/maintaining basal activation of AKT and also suggest that nCDase is a suitable novel target for colon cancer therapy.

Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Bioactive sphingolipids are modulators of immune processes and their metabolism is often dysregulated in ulcerative colitis, a major category of inflammatory bowel disease (IBD). While multiple axes of sphingolipid metabolism have been investigated to delineate mechanisms regulating ulcerative colitis, the role of acid ceramidase (AC) in intestinal inflammation is yet to be characterized. Here we demonstrate that AC expression is elevated selectively in the inflammatory infiltrate in human and murine colitis. To probe for mechanistic insight into how AC up-regulation can impact intestinal inflammation, we investigated the selective loss of AC expression in the myeloid population. Using a model of intestinal epithelial injury, we demonstrate that myeloid AC conditional knockout mice exhibit impairment of neutrophil recruitment to the colon mucosa as a result of defective cytokine and chemokine production. Furthermore, the loss of myeloid AC protects from tumor incidence in colitis-associated cancer (CAC) and inhibits the expansion of neutrophils and granulocytic myeloid-derived suppressor cells in the tumor microenvironment. Collectively, our results demonstrate a tissue-specific role for AC in regulating neutrophilic inflammation and cytokine production. We demonstrate novel mechanisms of how granulocytes are recruited to the colon that may have therapeutic potential in intestinal inflammation, IBD, and CAC.-Espaillat, M. P., Snider, A. J., Qiu, Z., Channer, B., Coant, N., Schuchman, E. H., Kew, R. R., Sheridan, B. S., Hannun, Y. A., Obeid, L. M. Loss of acid ceramidase in myeloid cells suppresses intestinal neutrophil recruitment.

Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.

Ceramidases, roles in sphingolipid metabolism and in health and disease.

Over the past three decades, extensive research has been able to determine the biologic functions for the main bioactive sphingolipids, namely ceramide, sphingosine, and sphingosine 1-phosphate (S1P) (Hannun, 1996; Hannun et al., 1986; Okazaki et al., 1989). These studies have managed to define the metabolism, regulation, and function of these bioactive sphingolipids. This emerging body of literature has also implicated bioactive sphingolipids, particularly S1P and ceramide, as key regulators of cellular homeostasis. Ceramidases have the important role of cleaving fatty acid from ceramide and producing sphingosine, thereby controlling the interconversion of these two lipids. Thus far, five human ceramidases encoded by five different genes have been identified: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). These ceramidases are classified according to their optimal pH for catalytic activity. AC, which is localized to the lysosomal compartment, has been associated with Farber's disease and is involved in the regulation of cell viability. Neutral ceramidase, which is localized to the plasma membrane and primarily expressed in the small intestine and colon, is involved in digestion, and has been implicated in colon carcinogenesis. ACER1 which can be found in the endoplasmic reticulum and is highly expressed in the skin, plays an important role in keratinocyte differentiation. ACER2, localized to the Golgi complex and highly expressed in the placenta, is involved in programed cell death in response to DNA damage. ACER3, also localized to the endoplasmic reticulum and the Golgi complex, is ubiquitously expressed, and is involved in motor coordination-associated Purkinje cell degeneration. This review seeks to consolidate the current knowledge regarding these key cellular players.

Role of neutral ceramidase in colon cancer.

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of ß-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.

Distinct roles for hematopoietic and extra-hematopoietic sphingosine kinase-1 in inflammatory bowel disease.

Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.

Glucolipotoxicity impairs ceramide flow from the endoplasmic reticulum to the Golgi apparatus in INS-1 ß-cells.

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to ß-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of ß-cells exposed to glucolipotoxicity.

Hippocampal lipoprotein lipase regulates energy balance in rodents.

Brain lipid sensing is necessary to regulate energy balance. Lipoprotein lipase (LPL) may play a role in this process. We tested if hippocampal LPL regulated energy homeostasis in rodents by specifically attenuating LPL activity in the hippocampus of rats and mice, either by infusing a pharmacological inhibitor (tyloxapol), or using a genetic approach (adeno-associated virus expressing Cre-GFP injected into Lpl (lox/lox) mice). Decreased LPL activity by either method led to increased body weight gain due to decreased locomotor activity and energy expenditure, concomitant with increased parasympathetic tone (unchanged food intake). Decreased LPL activity in both models was associated with increased de novo ceramide synthesis and neurogenesis in the hippocampus, while intrahippocampal infusion of de novo ceramide synthesis inhibitor myriocin completely prevented body weight gain. We conclude that hippocampal lipid sensing might represent a core mechanism for energy homeostasis regulation through de novo ceramide synthesis.

Sphingolipids in colon cancer.

Colorectal cancer is one of the major causes of death in the western world. Despite increasing knowledge of the molecular signaling pathways implicated in colon cancer, therapeutic outcomes are still only moderately successful. Sphingolipids, a family of N-acyl linked lipids, have not only structural functions but are also implicated in important biological functions. Ceramide, sphingosine and sphingosine-1-phosphate are the most important bioactive lipids, and they regulate several key cellular functions. Accumulating evidence suggests that many cancers present alterations in sphingolipids and their metabolizing enzymes. The aim of this review is to discuss the emerging roles of sphingolipids, both endogenous and dietary, in colon cancer and the interaction of sphingolipids with WNT/ß-catenin pathway, one of the most important signaling cascades that regulate development and homeostasis in intestine. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.

Role of palmitate-induced sphingoid base-1-phosphate biosynthesis in INS-1 ß-cell survival.

Sphingoid base-1-phosphates represent a very low portion of the sphingolipid pool but are potent bioactive lipids in mammals. This study was undertaken to determine whether these lipids are produced in palmitate-treated pancreatic ß cells and what role they play in palmitate-induced ß cell apoptosis. Our lipidomic analysis revealed that palmitate at low and high glucose supplementation increased (dihydro)sphingosine-1-phosphate levels in INS-1 ß cells. This increase was associated with an increase in sphingosine kinase 1 (SphK1) mRNA and protein levels. Over-expression of SphK1 in INS-1 cells potentiated palmitate-induced accumulation of dihydrosphingosine-1-phosphate. N,N-dimethyl-sphingosine, a potent inhibitor of SphK, potentiated ß-cell apoptosis induced by palmitate whereas over-expression of SphK1 significantly reduced apoptosis induced by palmitate with high glucose. Endoplasmic reticulum (ER)-targeted SphK1 also partially inhibited apoptosis induced by palmitate. Inhibition of INS-1 apoptosis by over-expressed SphK1 was independent of sphingosine-1-phosphate receptors but was associated with a decreased formation of pro-apoptotic ceramides induced by gluco-lipotoxicity. Moreover, over-expression of SphK1 counteracted the defect in the ER-to-Golgi transport of proteins that contribute to the ceramide-dependent ER stress observed during gluco-lipotoxicity. In conclusion, our results suggest that activation of palmitate-induced SphK1-mediated sphingoid base-1-phosphate formation in the ER of ß cells plays a protective role against palmitate-induced ceramide-dependent apoptotic ß cell death.

Glycogen synthase kinase 3 involvement in the excessive proinflammatory response to LPS in patients with decompensated cirrhosis.

BACKGROUND & AIMS: In decompensated cirrhosis, the early innate immune response to the Toll-like receptor 4 (TLR4) agonist, lipopolysaccharides (LPS), is characterized by a hyper-production of pro-inflammatory cytokines and hypo-production of the anti-inflammatory cytokine IL-10. In LPS-stimulated non-cirrhotic immune cells, the constitutively active glycogen synthase kinase (GSK) 3 favors pro- vs. anti-inflammatory cytokines, by acting on gene induction. However, in these cells, TLR4 dampens its own pro-inflammatory response by inducing early (within minutes) AKT-mediated phosphorylation of GSK3ß (one of two GSK3 isoforms) on Ser9. Phosphorylation of GSK3ß (Ser9) inhibits its activity, decreases pro-inflammatory cytokines, and increases IL-10. Thus, we investigated the role of GSK3 in LPS-induced cytokine production by peripheral blood mononuclear cells (PBMCs) or monocytes from patients with advanced cirrhosis and normal subjects. METHODS: Cells were pre-incubated with or without GSK3 inhibitor (SB216763 or lithium chloride) for 1h and then stimulated with LPS. Cytokine production was assessed at mRNA and secreted proteins levels, by real-time RT-PCR at 1h and ELISA at 20 h, respectively. GSK3ß phosphorylation was assessed using Western blotting. RESULTS: In cirrhotic and normal PBMCs pretreated with GSK3 inhibitors, LPS-induced production of pro-inflammatory proteins TNF-α and IL-12p40 was significantly decreased while that of IL-10 was increased. LPS-induced, AKT-mediated phosphorylation of GSK3ß on Ser9 found in normal monocytes, was abolished in cirrhotic cells. CONCLUSIONS: GSK3 is involved in the early TLR4-mediated pro-inflammatory response in patients with decompensated cirrhosis. This was associated with a defect in AKT-mediated GSK3ß phosphorylation resulting in unrestricted 'pro-inflammatory' activity of the enzyme.

NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon.

The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/beta-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/beta-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/beta-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/beta-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector beta-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/beta-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

Ex vivo effects of high-density lipoprotein exposure on the lipopolysaccharide-induced inflammatory response in patients with severe cirrhosis.

UNLABELLED: High-density lipoproteins (HDL) are known to neutralize lipopolysaccharide (LPS). Because patients with cirrhosis have lower HDL levels, this may contribute to LPS-induced ex vivo monocyte overproduction of proinflammatory cytokines. However, the effects of HDL on cytokine production by monocytes from patients with cirrhosis have never been studied. The aim of this study was to determine the effects of HDL on LPS-induced proinflammatory cytokine production in whole blood and isolated monocytes from patients with severe cirrhosis and controls. Plasma levels of HDL and cytokines were determined. The effects of reconstituted HDL (rHDL) on LPS-induced cytokine production in whole blood were assessed by cytokine array and on tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) production in isolated monocytes. Plasma HDL levels were significantly lower in patients with cirrhosis than in controls. Plasma levels of TNF-alpha and IL-6 were significantly higher in patients with cirrhosis than in controls. Incubation of rHDL with whole blood prevented LPS-induced TNF-alpha and IL-6 overproduction in patients with cirrhosis. LPS-induced TNF-alpha production and CD14 expression were significantly more marked in cirrhotic monocytes than in control monocytes, and both decreased significantly after rHDL incubation. LPS-induced down-regulation of scavenger receptor, class B, type I (SR-BI) expression was abolished in cirrhotic monocytes. CONCLUSIONS: This study shows that rHDL abolishes the LPS-induced overproduction of proinflammatory cytokines in whole blood from patients with severe cirrhosis. These results were confirmed in isolated monocytes from these patients. This suggests that administration of rHDL might be a useful strategy for the treatment of cirrhosis to limit LPS-induced cytokine overproduction.