Diesel Particulate Matter Permeation into Normal Human Skin and Intervention Using a Topical Ceramide Formulation.

INTRODUCTION: Diesel particulate matter (DPM) emitted from diesel engines is a major source of air pollutants. DPM is composed of elemental carbon, which adsorbs organic compounds including toxic polycyclic aromatic hydrocarbons (PAHs). The skin, as well as airways, is directly exposed to DPM, and association of atopic dermatitis, psoriasis flares, and premature skin aging with air pollutant levels has been documented. In skin, the permeation of DPM and DPM-adsorbed compounds is primarily blocked by the epidermal permeability barrier deployed in the stratum corneum. Depending upon the integrity of this barrier, certain amounts of DPM and DPM-adsorbed compounds can permeate into the skin. However, this permeation into human skin has not been completely elucidated. METHODS: We assessed the permeation of PAHs (adsorbed to DPM) into skin using ex vivo normal (barrier-competent) organ-cultured human skin after application of DPM. Two major PAHs, 2-methylnaphthalene and triphenylene, and a carcinogenic PAH, benzo(a)pyrene, all found in DPM, were measured in the epidermis and dermis using liquid chromatography electrospray ionization tandem mass spectrometry. In addition, we investigated whether a topical formulation can attenuate the permeation of DPM into skin. RESULTS: 2-Methylnaphthalene, triphenylene, and benzo(a)pyrene were recovered from the epidermis. Although these PAHs were also detected in the dermis after DPM application, these PAH levels were significantly lower than those found in the epidermis. We also demonstrated that a topical formulation that has the ability to form more uniform membrane structures can significantly suppress the permeation of PAHs adsorbed to DPM into the skin. CONCLUSION: Toxic compounds adsorbed by DPM can permeate even barrier-competent skin. Hence, barrier-compromised skin, such as in atopic dermatitis, psoriasis, and xerosis, is even more vulnerable to air pollutants. A properly formulated topical mixture that forms certain membrane structures on the skin surface can effectively prevent permeation of exogenous substances, including DPM, into skin.

The Epidermal Environment's Influence on the Dermal Environment in Response to External Stress.

INTRODUCTION: The outermost layer of the skin, the epidermis, is directly exposed to external stress (e.g., irradiation, allergens, and chemicals). Changes in epidermal conditions/environment in response to this stress could also influence conditions of the dermis, located directly beneath the epidermis. Yet, whether/how any epidermal environment changes in response to external stress affect dermal functions has not been completely clarified. METHODS: We employed ultraviolet irradiation B (UVB) (which hardly reaches the dermis) as a model of external stress. Human keratinocytes and human dermal fibroblasts were treated with UVB and conditioned medium of keratinocytes exposed to UVB (UVB-keratinocyte-M), respectively. We assessed (1) inflammatory cytokines and lipid mediators in keratinocytes; (2) matrix metalloprotease (MMP) levels and collagen degradation in fibroblasts; (3) ex vivo organ-cultured human skin was treated with UVB. MMP levels and collagen degradation were examined; (4) test whether the mixture of agent (agent cocktail) consisting of dihydroceramide, niacin amide, resveratrol, glucosyl hesperidin, and phytosterol ester that has been shown to improve skin barrier integrity can mitigate influence of UVB in skin; and (5) a pilot one-arm human clinical test to assess efficacy of formulation containing agent cocktail on stratum corneum hydration, skin elasticity, and wrinkle index. RESULTS: Inflammatory-cytokine and -lipid mediator production were increased in cultured keratinocytes treated with UVB, while matrix MMP-1, -3, and -9 production and collagen degradation were increased in fibroblasts incubated with UVB-keratinocyte-M. mRNA expression of COL1A1 (that codes type 1 collagen) levels was decreased in fibroblasts incubated with UVB-keratinocyte-M. The study using ex vivo organ-cultured human skin showed both MMP-1 and MMP-9 expression were increased in both epidermis and dermis and increased dermal collagen degradation following UVB irradiation. Increased MMP production and collagen degradation were attenuated by application of an agent cocktail. Finally, a pilot clinical study demonstrated that the formulation containing our agent cocktail likely has the ability to improve skin hydration, increase skin elasticity, and reduce the appearance of wrinkles. CONCLUSION: Epidermal changes in epidermal environment and conditions in response to external stress affect dermal conditions, and these negative effects of external stress on various skin layers can be pharmacologically mitigated.

A Lipid Mixture Enriched by Ceramide NP with Fatty Acids of Diverse Chain Lengths Contributes to Restore the Skin Barrier Function Impaired by Topical Corticosteroid.

INTRODUCTION: The stratum corneum (SC) is a skin barrier that consists of corneocytes, intercellular lipids, and corneodesmosomes. Ceramides are composed of sphingoid bases linked with various types of fatty acids (FAs), and they are an essential constituent of SC intercellular lipids. Among their subtypes, ceramide NP with a phytosphingosine base is especially important. Most of the previous studies on barrier recovery have focused on a specific ceramide with a single chain FA, not with diverse chain lengths. Skin barrier function is impaired by various factors, including topical corticosteroid. OBJECTIVE: We evaluated whether a lipid mixture enriched by ceramide NP with FAs of diverse chain lengths (CER [NP]*) can restore the skin barrier function impaired by topical corticosteroid. METHODS: Twenty-seven healthy adult male volunteers were recruited. Topical corticosteroid was applied on both volar forearms of volunteers. Then, the test cream containing a lipid mixture with CER (NP)* was applied on the left forearm, and a vehicle cream without a lipid mixture was applied on the right forearm of each subject. The functional parameters of the skin barrier were compared before and after the treatment. Epidermal differentiation markers, hyaluronic acid synthase 3 (HAS3), cytokine levels, and the lipid profiles in the SC were analyzed. RESULTS: The functional parameters of the skin barrier, such as barrier recovery rate, SC integrity, and SC hydration were significantly improved in the test cream-applied site compared to the vehicle cream-applied sites. Filaggrin and HAS3 levels were significantly higher in the sites applied with the test cream. Interleukin (IL)-1α levels were also significantly increased in these sites. IL-2, IL-6, IL-10, and IL-13 levels were significantly decreased in the test cream-applied sites. Lipid analyses showed that C18, C20, and total ceramide NP levels significantly increased in the sites where the test cream was applied. Also, C16, C18, C20, C24, and total ceramide NP levels were significantly elevated in the test cream-applied sites after acute barrier disruption. CONCLUSION: Our results demonstrate that a lipid mixture enriched by CER (NP)* could recover the barrier function impaired by topical corticosteroid.

Diesel Particulate Extract Accelerates Premature Skin Aging in Human Fibroblasts via Ceramide-1-Phosphate-Mediated Signaling Pathway.

Both intrinsic (i.e., an individual's body clock) and extrinsic factors (i.e., air pollutants and ultraviolet irradiation) accelerate premature aging. Epidemiological studies have shown a correlation between pollutant levels and aging skin symptoms. Diesel particle matter in particular leads to some diseases, including in the skin. Our recent study demonstrates that diesel particulate extract (DPE) increases apoptosis via increases in an anti-mitogenic/pro-apoptotic lipid mediator, ceramide in epidermal keratinocytes. Here, we investigated whether and how DPE accelerates premature skin aging using cultured normal human dermal fibroblasts (HDF). We first demonstrated that DPE increases cell senescence marker ß-galactosidase activity in HDF. We then found increases in mRNA and protein levels, along with activity of matrix metalloprotease (MMP)-1 and MMP-3, which are associated with skin aging following DPE exposure. We confirmed increases in collagen degradation in HDF treated with DPE. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is activated by DPE and results in increased ceramide production by sphingomyelinase activation in HDF. We identified that ceramide-1-phosphate (C1P) (produced from ceramide by ceramide kinase activation) activates MMP-1 and MMP-3 through activation of arachidonate cascade, followed by STAT 1- and STAT 3-dependent transcriptional activation.

Rosmarinic Acid, as an NHE1 Activator, Decreases Skin Surface pH and Improves the Skin Barrier Function.

Stratum corneum (SC) pH regulates skin barrier functions and elevated SC pH is an important factor in various inflammatory skin diseases. Acidic topical formulas have emerged as treatments for impaired skin barriers. Sodium proton exchanger 1 (NHE1) is an important factor in SC acidification. We investigated whether topical applications containing an NHE1 activator could improve skin barrier functions. We screened plant extracts to identify NHE1 activators in vitro and found Melissa officinalis leaf extract. Rosmarinic acid, a component of Melissa officinalis leaf extract, significantly increased NHE1 mRNA expression levels and NHE1 production. Immunofluorescence staining of NHE1 in 3D-cultured skin revealed greater upregulation of NHE1 expression by NHE1 activator cream, compared to vehicle cream. Epidermal lipid analysis revealed that the ceramide level was significantly higher upon application of the NHE1 activator cream on 3D-cultured skin, compared to application of a vehicle cream. In a clinical study of 50-60-year-old adult females (n = 21), application of the NHE1 activator-containing cream significantly improved skin barrier functions by reducing skin surface pH and transepidermal water loss and increasing skin hydration, compared to patients who applied vehicle cream and those receiving no treatment. Thus, creams containing NHE1 activators, such as rosmarinic acid, could help maintain or recover skin barrier functions.

Phenotypic overlap between atopic dermatitis and autism.

BACKGROUND: Autism, a childhood behavioral disorder, belongs to a large suite of diseases, collectively referred to as autism spectrum disorders (ASD). Though multifactorial in etiology, approximately 10% of ASD are associated with atopic dermatitis (AD). Moreover, ASD prevalence increases further as AD severity worsens, though these disorders share no common causative mutations. We assessed here the link between these two disorders in the standard, valproic acid mouse model of ASD. In prior studies, there was no evidence of skin involvement, but we hypothesized that cutaneous involvement could be detected in experiments conducted in BALB/c mice. BALB/c is an albino, laboratory-bred strain of the house mouse and is among the most widely used inbred strains used in animal experimentation. METHODS: We performed our studies in valproic acid (VPA)-treated BALB/c hairless mice, a standard mouse model of ASD. Mid-trimester pregnant mice received a single intraperitoneal injection of either valproic acid sodium salt dissolved in saline or saline alone on embryonic day 12.5 and were housed individually until postnatal day 21. Only the brain and epidermis appeared to be affected, while other tissues remain unchanged. At various postnatal time points, brain, skin and blood samples were obtained for histology and for quantitation of tissue sphingolipid content and cytokine levels. RESULTS: AD-like changes in ceramide content occurred by day one postpartum in both VPA-treated mouse skin and brain. The temporal co-emergence of AD and ASD, and the AD phenotype-dependent increase in ASD prevalence correlated with early appearance of cytokine markers (i.e., interleukin [IL]-4, 5, and 13), as well as mast cells in skin and brain. The high levels of interferon (IFN)γ not only in skin, but also in brain likely account for a significant decline in esterified very-long-chain N-acyl fatty acids in brain ceramides, again mimicking known IFNγ-induced changes in AD. CONCLUSION: Baseline involvement of both AD and ASD could reflect concurrent neuro- and epidermal toxicity, possibly because both epidermis and neural tissues originate from the embryonic neuroectoderm. These studies illuminate the shared susceptibility of the brain and epidermis to a known neurotoxin, suggesting that the atopic diathesis could be extended to include ASD.

N-Palmitoyl Serinol Stimulates Ceramide Production through a CB1-Dependent Mechanism in In Vitro Model of Skin Inflammation.

Ceramides, a class of sphingolipids containing a backbone of sphingoid base, are the most important and effective structural component for the formation of the epidermal permeability barrier. While ceramides comprise approximately 50% of the epidermal lipid content by mass, the content is substantially decreased in certain inflammatory skin diseases, such as atopic dermatitis (AD), causing improper barrier function. It is widely accepted that the endocannabinoid system (ECS) can modulate a number of biological responses in the central nerve system, prior studies revealed that activation of endocannabinoid receptor CB1, a key component of ECS, triggers the generation of ceramides that mediate neuronal cell fate. However, as the impact of ECS on the production of epidermal ceramide has not been studied, we here investigated whether the ECS stimulates the generation of epidermal ceramides in an IL-4-treated in vitro model of skin inflammation using N-palmitoyl serinol (PS), an analog of the endocannabinoid N-palmitoyl ethanolamine. Accordingly, an IL-4-mediated decrease in cellular ceramide levels was significantly stimulated in human epidermal keratinocytes (KC) following PS treatment through both de novo ceramide synthesis- and sphingomyelin hydrolysis-pathways. Importantly, PS selectively increases ceramides with long-chain fatty acids (FAs) (C22-C24), which mainly account for the formation of the epidermal barrier, through activation of ceramide synthase (CerS) 2 and Cer3 in IL-4-mediated inflamed KC. Furthermore, blockade of cannabinoid receptor CB1 activation by AM-251 failed to stimulate the production of total ceramide as well as long-chain ceramides in response to PS. These studies demonstrate that an analog of endocannabinoid, PS, stimulates the generation of specific ceramide species as well as the total amount of ceramides via the endocannabinoid receptor CB1-dependent mechanism, thereby resulting in the enhancement of epidermal permeability barrier function.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 µM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Increased Expression of 11ß-Hydroxysteroid Dehydrogenase Type 1 Contributes to Epidermal Permeability Barrier Dysfunction in Aged Skin.

Inactive cortisone is converted into active cortisol by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1). Excessive levels of active glucocorticoids could deteriorate skin barrier function; barrier impairment is also observed in aged skin. In this study, we aimed to determine whether permeability barrier impairment in the aged skin could be related to increased 11ß-HSD1 expression. Aged humans (n = 10) showed increased cortisol in the stratum corneum (SC) and oral epithelium, compared to young subjects (n = 10). 11ß-HSD1 expression (as assessed via immunohistochemical staining) was higher in the aged murine skin. Aged hairless mice (56-week-old, n = 5) manifested greater transepidermal water loss, lower SC hydration, and higher levels of serum inflammatory cytokines than the young mice (8-week-old, n = 5). Aged 11ß-HSD1 knockout mice (n = 11), 11ß-HSD1 inhibitor (INHI)-treated aged wild type (WT) mice (n = 5) and young WT mice (n = 10) exhibited reduced SC corticosterone level. Corneodesmosome density was low in WT aged mice (n = 5), but high in aged 11ß-HSD1 knockout and aged INHI-treated WT mice. Aged mice exhibited lower SC lipid levels; this effect was reversed by INHI treatment. Therefore, upregulation of 11ß-HSD1 in the aged skin increases the active-glucocorticoid levels; this suppresses SC lipid biosynthesis, leading to impaired epidermal permeability barrier.

A Newly Developed HPLC-UV/Vis Method Using Chemical Derivatization with 2-Naphthalenethiol for Quantitation of Sulforaphane in Rat Plasma.

Sulforaphane (SFN), a naturally occurring isothiocyanate, has received significant attention because of its ability to modulate multiple biological functions, including anti-carcinogenic properties. However, currently available analytical methods based on high-performance liquid chromatography (HPLC)-UV/Vis for the quantification of SFN have a number of limitations, e.g., low UV absorbance, sensitivity, or accuracy, due to the lack of a chromophore for spectrometric detection. Therefore, we here employed the analytical derivatization procedure using 2-naphthalenethiol (2-NT) to improve the detectability of SFN, followed by HPLC separation and quantification with UV/Vis detection. The optimal derivatization conditions were carried out with 0.3 M of 2-NT in acetonitrile with phosphate buffer (pH 7.4) by incubation at 37 °C for 60 min. Separation was performed in reverse phase mode using a Kinetex C18 column (150 mm × 4.6 mm, 5 µm) at a flow rate of 1 mL/min, with 0.1% formic acid as a mobile phase A, and acetonitrile/0.1% formic acid solution as a mobile phase B with a gradient elution, with a detection wavelength of 234 nm. The method was validated over a linear range of 10-2000 ng/mL with a correlation of determination (R2) > 0.999 using weighted linear regression analysis. The intra- and inter-assay accuracy (% of nominal value) and precision (% of relative standard deviation) were within ±10 and <15%, respectively. Moreover, the specificity, recovery, matrix effect, process efficiency, and short-term and long-term stabilities of this method were within acceptable limits. Finally, we applied this method for studying in vivo pharmacokinetics (PK) following oral administration of SFN at doses of 10 or 20 mg/kg. The Cmax (µg/mL), Tmax (hour), and AUC0-12h (µg·h/mL) of each oral dose were 0.92, 1.99, and 4.88 and 1.67, 1.00, and 9.85, respectively. Overall, the proposed analytical method proved to be reliable and applicable for quantification of SFN in biological samples.

Efficient Separation of Phytochemicals from Muehlenbeckia volcanica (Benth.) Endl. by Polarity-Stepwise Elution Counter-Current Chromatography and Their Antioxidant, Antiglycation, and Aldose Reductase Inhibition Potentials.

Muehlenbeckia volcanica (Benth.) Endl. (M. volcanica), native to South America, is a traditional Peruvian medicinal plant that has multi-therapeutic properties; however, no phytochemicals have been identified from it yet. In this study, a five-step polarity-stepwise elution counter-current chromatography (CCC) was developed using methanol/water (1:5, v/v) as the stationary phase and different ratios of n-hexane, ethyl acetate, and n-butanol as mobile phases to separate the compounds from the 70% methanol extract of M. volcanica, by which six compounds with a wide range of polarities were separated in a single run of CCC and were identified as gallic acid, protocatechuic acid, 4,4'-dihydroxy-3,3'-imino-di-benzoic acid, rutin, quercitrin, and quercetin. Then, two compounds from the fractions of stepwise elution CCC were separated using conventional high-speed CCC, pH-zone-refining CCC, and preparative high-performance liquid chromatography, and identified as shikimic acid and miquelianin. These compounds are reported from M. volcanica for the first time. Notably, except for shikimic acid, all other compounds showed anti-diabetic potentials via antioxidant, antiglycation, and aldose reductase inhibition. The results suggest that the polarity-stepwise elution CCC can be used to efficiently separate or fractionate compounds with a wide range of polarities from natural products. Moreover, M. volcanica and its bioactive compounds are potent anti-diabetic agents.

Inhibition of sphingosine 1-phosphate lyase activates human keratinocyte differentiation and attenuates psoriasis in mice.

Sphingosine 1-phosphate (S1P) lyase is an intracellular enzyme that catalyzes the irreversible degradation of S1P and has been suggested as a therapeutic target for the treatment of psoriasis vulgaris. Because S1P induces differentiation of keratinocytes, we examined whether modulation of S1P lyase and altered intracellular S1P levels regulate proliferation and differentiation of human neonatal epidermal keratinocyte (HEKn) cells. To identify the physiological functions of S1P lyase in skin, we inhibited S1P lyase in HEKn cells with an S1P lyase-specific inhibitor (SLI) and with S1P lyase 1 (SGPL1)-specific siRNA (siSGPL1). In HEKn cells, pharmacological treatment with the SLI caused G1 arrest by upregulation of p21 and p27 and induced keratin 1, an early differentiation marker. Similarly, genetic suppression by siSGPL1 arrested the cell cycle at the G1 phase and activated differentiation. In addition, enzyme suppression by siSGPL1 upregulated keratin 1 and differentiation markers including involucrin and loricrin. When hyperproliferation of HEKn cells was induced by interleukin (IL)-17 and IL-22, pharmacologic inhibition of S1P lyase by SLI decreased proliferation and activated differentiation of HEKn cells simultaneously. In addition, SLI administration ameliorated imiquimod-induced psoriatic symptoms including erythema, scaling, and epidermal thickness in vivo. We thus demonstrated that S1P lyase inhibition reduces cell proliferation and induces keratinocyte differentiation, and that inhibition may attenuate psoriasiform changes. Collectively, these findings suggest that S1P lyase is a modulating factor for proliferation and differentiation, and support its potential as a therapeutic target for psoriasis in human keratinocytes.

NADPH Oxidase-Mediated Activation of Neutral Sphingomyelinase Is Responsible for Diesel Particulate Extract-Induced Keratinocyte Apoptosis.

Human epidermis is positioned at the interface with the external environment, protecting our bodies against external challenges, including air pollutants. Emerging evidence suggests that diesel particulate extract (DPE), a major component of air pollution, leads to impairment of diverse cellular functions in keratinocytes (KC). In this study, we investigated the cellular mechanism underlying DPE-induced KC apoptosis. We first addressed cell death occurring in KC exposed to DPE, paralleled by increased activation of NADPH oxidases (NOXs) and subsequent ROS generation. Blockade of NOX activation with a specific inhibitor attenuated the expected DPE-induced KC apoptosis. In contrast, pre-treatment with a specific inhibitor of reactive oxygen species (ROS) generation did not reverse DPE/NOX-mediated increase in KC apoptosis. We next noted that NOX-mediated KC apoptosis is mainly attributable to neutral sphingomyelinase (SMase)-mediated stimulation of ceramides, which is a well-known pro-apoptotic lipid. Moreover, we found that inhibition of NOX activation significantly attenuated DPE-mediated increase in the ratio of ceramide to its key metabolite sphingosine-1-phosphate (S1P), an important determinant of cell fate. Together, these results suggest that activation of neutral SMase serves as a key downstream signal for the DPE/NOX activation-mediated alteration in ceramide and S1P productions, and subsequent KC apoptosis.

Cellular and Metabolic Basis for the Ichthyotic Phenotype in NIPAL4 (Ichthyin)-Deficient Canines.

Mutations in several lipid synthetic enzymes that block fatty acid and ceramide production produce autosomal recessive congenital ichthyoses (ARCIs) and associated abnormalities in permeability barrier homeostasis. However, the basis for the phenotype in patients with NIPAL4 (ichthyin) mutations (among the most prevalent ARCIs) remains unknown. Barrier function was abnormal in an index patient and in canines with homozygous NIPAL4 mutations, attributable to extensive membrane stripping, likely from detergent effects of nonesterified free fatty acid. Cytotoxicity compromised not only lamellar body secretion but also formation of the corneocyte lipid envelope (CLE) and attenuation of the cornified envelope (CE), consistent with a previously unrecognized, scaffold function of the CLE. Together, these abnormalities result in failure to form normal lamellar bilayers, accounting for the permeability barrier abnormality and clinical phenotype in NIPA-like domain-containing 4 (NIPAL4) deficiency. Thus, NIPAL4 deficiency represents another lipid synthetic ARCI that converges on the CLE (and CE), compromising their putative scaffold function. However, the clinical phenotype only partially improved after normalization of CLE and CE structure with topical ω-O-acylceramide because of ongoing accumulation of toxic metabolites, further evidence that proximal, cytotoxic metabolites contribute to disease pathogenesis.

ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex.

We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate-induced colitis in mice due to increased intestinal permeability.

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

Anti-inflammatory mechanism of exogenous C2 ceramide in lipopolysaccharide-stimulated microglia.

Ceramide is a major molecule among the sphingolipid metabolites which are produced in the brain and other organs and act as intracellular second messengers. Although a variety of physiological roles of ceramide have been reported in the periphery and central nervous systems, the role of ceramide in microglial activation has not been clearly demonstrated. In the present study, we examined the effects of exogenous cell permeable short chain ceramides on microglial activation in vitro and in vivo. We found that C2, C6, and C8 ceramide and C8 ceramide-1-phosphate inhibited iNOS and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. In addition, the administration of C2 ceramide suppressed microglial activation in the brains of LPS-exposed mice. By HPLC and LC/MS/MS analyses, we found that C2 ceramide on its own, rather than its modified form (i.e. ceramide-1-phosphate or long chain ceramides), mainly work by penetrating into microglial cells. Further mechanistic studies by using the most effective C2 ceramide among the short chain ceramides tested, revealed that C2 ceramide exerts anti-inflammatory effects via inhibition of the ROS, MAPKs, PI3K/Akt, and Jak/STAT pathways with upregulation of PKA and hemeoxygenase-1 expressions. Interestingly, we found that C2 ceramide inhibits TLR4 signaling by interfering with LPS and TLR4 interactions. Therefore, our data collectively suggests the therapeutic potential of short chain ceramides such as C2 for neuroinflammatory disorders such as Alzheimer's disease and Parkinson's disease.

Barrier Abnormalities in Type 1 Diabetes Mellitus: The Roles of Inflammation and Ceramide Metabolism.

Xerosis is a common sign of both type 1 and type 2 diabetes mellitus (DM), and patients with DM and mouse models for DM show a compromised epidermal permeability barrier. Barrier defects then allow the entry of foreign substances into the skin, triggering inflammation, infection, and worsening skin symptoms. Characterizing how barrier abnormalities develop in DM could suggest treatments for xerosis and other skin disease traits. Because the proper ratio, as well as proper bulk amounts, of heterogeneous ceramide species are keys to forming a competent barrier, we investigated how ceramide metabolism is affected in type 1 DM using a mouse model (induced by streptozotocin). Chronic inflammation, evident in the skin of mice with DM, leads to (i) decreased de novo ceramide production through serine racemase activation-mediated attenuation of serine palmitoyl transferase activity by D-serine; (ii) changes in ceramide synthase activities and expression that modify the ratio of ceramide molecular species; and (iii) increased ceramide-1-phosphate, a proinflammatory lipid mediator, that stimulates inflammatory cytokine expression (TNFα and IFN-γ). Together, chronic inflammation affects ceramide metabolism, which attenuates epidermal permeability barrier formation, and ceramide-1-phosphate could amplify this inflammation. Alleviation of chronic inflammation is a credible approach for normalizing barrier function and ameliorating diverse skin abnormalities in DM.

Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction.

The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.

Synthesis and biological evaluation of a polyyne-containing sphingoid base probe as a chemical tool.

The sphingolipid metabolites have emerged as a starting point for the development of novel therapeutics for many diseases. However, details of the functions and mechanisms of sphingolipids remain unknown. To better understand the roles of sphingolipids, chemical tools with unique biological and physicochemical properties are needed. In this regard, we previously reported the synthesis of sphingoid base analogues in which the carbon chains are restricted by triple bonds. Here, we have conjugated a fluorescent dye to the polyyne analogues of the sphingoid bases to generate optical probes. Like the parent polyyne-containing sphingoid base, the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled triyne-sphingosine inhibited cancer cell growth far more effectively than did the corresponding sphingosine. NBD-triyne-sphingosine was rapidly incorporated into the cells and displayed broad cytoplasmic distribution. According to the results of a flow cytometric analysis, cancer cells fed with NBD-triyne-sphingosine showed significantly increased fluorescence intensity compared with the NBD-sphingosine treated cells. The metabolism of NBD-triyne-sphingosine was somewhat different from that of NBD-sphingosine. These results indicated that the incorporated rigid polyyne moiety in the sphingoid base altered the physicochemical properties of the sphingolipid, thereby affecting its biological behavior. The higher antiproliferative activity in the SRB assay and the significantly higher fluorescence intensity observed in the flow cytometric analysis are some of the interesting and distinct aspects of NBD-triyne-sphingosine compared to standard NBD-sphingosine probes. Thus, it is believed that the fluorescently labeled polyyne-containing sphingoid base developed in this study will be a useful chemical tool in sphingolipid research.