Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

Pseudomonas Aeruginosa Theft Biofilm Require Host Lipids of Cutaneous Wound.

OBJECTIVE: This work addressing complexities in wound infection, seeks to test the reliance of bacterial pathogen Pseudomonas aeruginosa (PA) on host skin lipids to form biofilm with pathological consequences. BACKGROUND: PA biofilm causes wound chronicity. Both CDC as well as NIH recognizes biofilm infection as a threat leading to wound chronicity. Chronic wounds on lower extremities often lead to surgical limb amputation. METHODS: An established preclinical porcine chronic wound biofilm model, infected with PA or Pseudomonas aeruginosa ceramidase mutant (PA ∆Cer ), was used. RESULTS: We observed that bacteria drew resource from host lipids to induce PA ceramidase expression by three orders of magnitude. PA utilized product of host ceramide catabolism to augment transcription of PA ceramidase. Biofilm formation was more robust in PA compared to PA ∆Cer . Downstream products of such metabolism such as sphingosine and sphingosine-1-phosphate were both directly implicated in the induction of ceramidase and inhibition of peroxisome proliferator-activated receptor (PPAR)δ, respectively. PA biofilm, in a ceram-idastin-sensitive manner, also silenced PPARδ via induction of miR-106b. Low PPARδ limited ABCA12 expression resulting in disruption of skin lipid homeostasis. Barrier function of the wound-site was thus compromised. CONCLUSIONS: This work demonstrates that microbial pathogens must co-opt host skin lipids to unleash biofilm pathogenicity. Anti-biofilm strategies must not necessarily always target the microbe and targeting host lipids at risk of infection could be productive. This work may be viewed as a first step, laying fundamental mechanistic groundwork, toward a paradigm change in biofilm management.

Sex Differences in Sphingosine-1-Phosphate Levels Are Dependent on Ceramide Synthase 1 and Ceramidase in Lung Physiology and Tumor Conditions.

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.

Detection of Bacterial Neutral Ceramidase in Diabetic Foot Ulcers with an Optimized Substrate and Chemoenzymatic Probes.

Ceramidases (CDases) are important in controlling skin barrier integrity by regulating ceramide composition and affording downstream signal molecules. While the functions of epidermal CDases are known, roles of neutral CDases secreted by skin-residing microbes are undefined. Here, we developed a one-step fluorogenic substrate, S-B, for specific detection of bacterial CDase activity and inhibitor screening. We identified a non-hydrolyzable substrate mimic, C6, as the best hit. Based on C6, we designed a photoaffinity probe, JX-1, which efficiently detects bacterial CDases. Using JX-1, we identified endogenous low-abundance PaCDase in a P. aeruginosa monoculture and in a mixed skin bacteria culture. Harnessing both S-B and JX-1, we found that CDase activity positively correlates with the relative abundance of P. aeruginosa and is negatively associated with wound area reduction in clinical diabetic foot ulcer patient samples. Overall, our study demonstrates that bacterial CDases are important regulators of skin ceramides and potentially play a role in wound healing.

Presence of Ceramidase Activity in Electronegative LDL.

Electronegative low-density lipoprotein (LDL(-)) is a minor modified fraction of human plasma LDL with several atherogenic properties. Among them is increased bioactive lipid mediator content, such as lysophosphatidylcholine (LPC), non-esterified fatty acids (NEFA), ceramide (Cer), and sphingosine (Sph), which are related to the presence of some phospholipolytic activities, including platelet-activating factor acetylhydrolase (PAF-AH), phospholipase C (PLC), and sphingomyelinase (SMase), in LDL(-). However, these enzymes' activities do not explain the increased Sph content, which typically derives from Cer degradation. In the present study, we analyzed the putative presence of ceramidase (CDase) activity, which could explain the increased Sph content. Thin layer chromatography (TLC) and lipidomic analysis showed that Cer, Sph, and NEFA spontaneously increased in LDL(-) incubated alone at 37 °C, in contrast with native LDL(+). An inhibitor of neutral CDase prevented the formation of Sph and, in turn, increased Cer content in LDL(-). In addition, LDL(-) efficiently degraded fluorescently labeled Cer (NBD-Cer) to form Sph and NEFA. These observations defend the existence of the CDase-like activity's association with LDL(-). However, neither the proteomic analysis nor the Western blot detected the presence of an enzyme with known CDase activity. Further studies are thus warranted to define the origin of the CDase-like activity detected in LDL(-).

Targeting Ceramides and Adiponectin Receptors in the Islet of Langerhans for Treating Diabetes.

Ceramides belong to the sphingolipid family and represent the central hub of the sphingolipid network. In obesity, oversupply of saturated fatty acids including palmitate raises ceramide levels which can be detrimental to cells. Elevated ceramides can cause insulin resistance, endoplasmic reticulum stress, and mitochondrial dysfunction. Studies over the last few decades have highlighted the role played by ceramides in pancreatic islet ß-cell apoptosis, especially under glucolipotoxic and inflammatory conditions. This review focuses on ceramides and adiponectin receptor signaling, summarizing recent advancements in our understanding of their roles in islet ß-cells and the discovery of zinc-dependent lipid hydrolase (ceramidase) activity of adiponectin receptors. The therapeutic potential of targeting these events to prevent islet ß-cell loss for treating diabetes is discussed.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

Mechanistic insights into ceramidase inhibitor LCL521-enhanced tumor cell killing by photodynamic and thermal ablation therapies.

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).

Targeted proteomic response to coffee consumption.

PURPOSE: Coffee is widely consumed and implicated in numerous health outcomes but the mechanisms by which coffee contributes to health is unclear. The purpose of this study was to test the effect of coffee drinking on candidate proteins involved in cardiovascular, immuno-oncological and neurological pathways. METHODS: We examined fasting serum samples collected from a previously reported single blinded, three-stage clinical trial. Forty-seven habitual coffee consumers refrained from drinking coffee for 1 month, consumed 4 cups of coffee/day in the second month and 8 cups/day in the third month. Samples collected after each coffee stage were analyzed using three multiplex proximity extension assays that, after quality control, measured a total of 247 proteins implicated in cardiovascular, immuno-oncological and neurological pathways and of which 59 were previously linked to coffee exposure. Repeated measures ANOVA was used to test the relationship between coffee treatment and each protein. RESULTS: Two neurology-related proteins including carboxypeptidase M (CPM) and neutral ceramidase (N-CDase or ASAH2), significantly increased after coffee intake (P < 0.05 and Q < 0.05). An additional 46 proteins were nominally associated with coffee intake (P < 0.05 and Q > 0.05); 9, 8 and 29 of these proteins related to cardiovascular, immuno-oncological and neurological pathways, respectively, and the levels of 41 increased with coffee intake. CONCLUSIONS: CPM and N-CDase levels increased in response to coffee intake. These proteins have not previously been linked to coffee and are thus novel markers of coffee response worthy of further study. CLINICAL TRIAL REGISTRY: http://www.isrctn.com/ISRCTN12547806.

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

Ceramidase critically affects GPVI-dependent platelet activation and thrombus formation.

Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700-sec. Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling.

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, Crassostrea virginica.

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2-3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4-4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.

Role of acid sphingomyelinase and IL-6 as mediators of endotoxin-induced pulmonary vascular dysfunction.

BACKGROUND: Pulmonary hypertension (PH) is frequently observed in patients with acute respiratory distress syndrome (ARDS) and it is associated with an increased risk of mortality. Both acid sphingomyelinase (aSMase) activity and interleukin 6 (IL-6) levels are increased in patients with sepsis and correlate with worst outcomes, but their role in pulmonary vascular dysfunction pathogenesis has not yet been elucidated. Therefore, the aim of this study was to determine the potential contribution of aSMase and IL-6 in the pulmonary vascular dysfunction induced by lipopolysaccharide (LPS). METHODS: Rat or human pulmonary arteries (PAs) or their cultured smooth muscle cells (SMCs) were exposed to LPS, SMase or IL-6 in the absence or presence of a range of pharmacological inhibitors. The effects of aSMase inhibition in vivo with D609 on pulmonary arterial pressure and inflammation were assessed following intratracheal administration of LPS. RESULTS: LPS increased ceramide and IL-6 production in rat pulmonary artery smooth muscle cells (PASMCs) and inhibited pulmonary vasoconstriction induced by phenylephrine or hypoxia (HPV), induced endothelial dysfunction and potentiated the contractile responses to serotonin. Exogenous SMase and IL-6 mimicked the effects of LPS on endothelial dysfunction, HPV failure and hyperresponsiveness to serotonin in PA; whereas blockade of aSMase or IL-6 prevented LPS-induced effects. Finally, administration of the aSMase inhibitor D609 limited the development of endotoxin-induced PH and ventilation-perfusion mismatch. The protective effects of D609 were validated in isolated human PAs. CONCLUSIONS: Our data indicate that aSMase and IL-6 are not simply biomarkers of poor outcomes but pathogenic mediators of pulmonary vascular dysfunction in ARDS secondary to Gram-negative infections.

Implication of sphingosine-1-phosphate signaling in diseases: molecular mechanism and therapeutic strategies.

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.

The Arabidopsis ceramidase AtACER functions in disease resistance and salt tolerance.

Ceramidases hydrolyze ceramide into sphingosine and fatty acids. In mammals, ceramidases function as key regulators of sphingolipid homeostasis, but little is known about their roles in plants. Here we characterize the Arabidopsis ceramidase AtACER, a homolog of human alkaline ceramidases. The acer-1 T-DNA insertion mutant has pleiotropic phenotypes, including reduction of leaf size, dwarfing and an irregular wax layer, compared with wild-type plants. Quantitative sphingolipid profiling showed that acer-1 mutants and the artificial microRNA-mediated silenced line amiR-ACER-1 have high ceramide levels and decreased long chain bases. AtACER localizes predominantly to the endoplasmic reticulum, and partially to the Golgi complex. Furthermore, we found that acer-1 mutants and AtACER RNAi lines showed increased sensitivity to salt stress, and lines overexpressing AtACER showed increased tolerance to salt stress. Reduction of AtACER also increased plant susceptibility to Pseudomonas syringae. Our data highlight the key biological functions of ceramidases in biotic and abiotic stresses in plants.

Sphingomyelin synthase-related protein SMSr is a suppressor of ceramide-induced mitochondrial apoptosis.

Cells synthesize ceramides in the endoplasmic reticulum (ER) as precursors for sphingolipids to form an impermeable plasma membrane. As ceramides are engaged in apoptotic pathways, cells would need to monitor their levels closely to avoid killing themselves during sphingolipid biosynthesis. How this is accomplished remains to be established. Here we identify SMSr (SAMD8), an ER-resident ceramide phosphoethanolamine (CPE) synthase, as a suppressor of ceramide-mediated cell death. Disruption of SMSr catalytic activity causes a rise in ER ceramides and their mislocalization to mitochondria, triggering a mitochondrial pathway of apoptosis. Blocking de novo ceramide synthesis, stimulating ceramide export from the ER or targeting a bacterial ceramidase to mitochondria rescues SMSr-deficient cells from apoptosis. We also show that SMSr-catalyzed CPE production, although essential, is not sufficient to suppress ceramide-induced cell death and that SMSr-mediated ceramide homeostasis requires the N-terminal sterile α-motif, or SAM domain, of the enzyme. These results define ER ceramides as bona fide transducers of mitochondrial apoptosis and indicate a primary role of SMSr in monitoring ER ceramide levels to prevent inappropriate cell death during sphingolipid biosynthesis.

Adiponectin inhibits tumor necrosis factor-α-induced vascular inflammatory response via caveolin-mediated ceramidase recruitment and activation.

RATIONALE: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. OBJECTIVE: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin's vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. METHODS AND RESULTS: Adiponectin significantly reduced tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression and attenuated TNFα-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin-induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNFα-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNFα-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. CONCLUSIONS: These results demonstrate for the first time that adiponectin inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

The roles of cutaneous lipids in host defense.

Lauric acid (C12:0) and sapienic acid (C16:1Δ6) derived from human sebaceous triglycerides are potent antimicrobials found at the human skin surface. Long-chain bases (sphingosine, dihydrosphingosine and 6-hydroxysphingosine) are also potent and broad-acting antimicrobials normally present at the skin surface. These antimicrobials are generated through the action of ceramidases on ceramides from the stratum corneum. These natural antimicrobials are thought to be part of the innate immune system of the skin. Exogenously providing these lipids to the skin may provide a new therapeutic option, or could potentially provide prophylaxis in people at risk of infection. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.