RESUMEN

We previously reported that a pathogenic abnormality in the barrier and water-holding functions of the stratum corneum (SC) in the skin of patients with atopic dermatitis (AD) is mainly attributable to significantly decreased levels of total ceramides in the SC. That decrease is mediated by the abnormal expression of a novel ceramide-reducing enzyme, sphingomyelin/glucosylceramide deacylase (SGDase), which is the ß-subunit (ASAH1b) of acid ceramidase. In this study, we determined whether mice overexpressing ASAH1b in their epidermis develop AD-like skin symptoms. We generated transgenic (TG) mice overexpressing ASAH1b, regulated by the involucrin promoter, to localize its expression in the upper epidermis. After hair removal using a depilatory cream containing glycolic acid, the TG mice without any visible skin inflammation at 8 weeks of age had increased levels of ASAH1b and decreased levels of SC ceramide, with disrupted barrier functions measured by trans-epidermal water loss compared to the wild-type (WT) mice. Interestingly, enzymatic assays revealed that SGDase activity was not detectable in the skin of the TG mice compared to WT mice. Immunological staining revealed that there was an increased expression level of IL-33 in the epidermis and an accumulation of macrophages in the dermis of TG mice compared to WT mice, which are phenotypic characteristics of AD, that were exacerbated by tape-stripping of the skin. In the skin of the TG mice, the mRNA levels of IL-5, CCL11, IL-22, CXCL10, and IFNγ were significantly upregulated compared to the WT mice, and tape-stripping significantly increased the mRNA levels of IL-4, IL-33, CXCL1, CXCL12, TLR9, and CD163 compared to WT mice. These findings strongly indicate that the skin of the depilatory cream-treated TG mice exists in an atopic dry skin condition that is highly sensitive to various environmental stimuli. The sum of our results suggests that ASAH1b itself, even in the absence of its enzymatic activity, is a major etiologic factor for atopic dry skin symptoms via an unknown mechanism.

Asunto(s)

Ceramidasa Ácida , Ceramidas , Dermatitis Atópica , Epidermis , Animales , Ratones , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/genética , Ceramidas/metabolismo , Dermatitis Atópica/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Modelos Animales de Enfermedad , Epidermis/metabolismo , Epidermis/patología , Ratones Transgénicos , Piel/metabolismo , Piel/patología

RESUMEN

Introduction: Advanced cutaneous melanoma is a skin cancer characterized by a poor prognosis and high metastatic potential. During metastatic spread, melanoma cells often undergo dedifferentiation toward an invasive phenotype, resulting in reduced expression of microphthalmia-associated transcription factor (MITF)-dependent melanoma antigens and facilitating immune escape. Tumor Necrosis Factor (TNF) is known to be a key factor in melanoma dedifferentiation. Interestingly, accumulating evidence suggests that TNF may play a role in melanoma progression and resistance to immunotherapies. Additionally, TNF has been identified as a potent regulator of sphingolipid metabolism, which could contribute to melanoma aggressiveness and the process of melanoma dedifferentiation. Methods: We conducted RNA sequencing and mass spectrometry analyses to investigate TNF-induced dedifferentiation in two melanoma cell lines. In vitro experiments were performed to manipulate sphingolipid metabolism using genetic or pharmacologic alterations in combination with TNF treatment, aiming to elucidate the potential involvement of this metabolism in TNF-induced dedifferentiation. Lastly, to evaluate the clinical significance of our findings, we performed unsupervised analysis of plasma sphingolipid levels in 48 patients receiving treatment with immune checkpoint inhibitors, either alone or in combination with anti-TNF therapy. Results: Herein, we demonstrate that TNF-induced melanoma cell dedifferentiation is associated with a global modulation of sphingolipid metabolism. Specifically, TNF decreases the expression and activity of acid ceramidase (AC), encoded by the ASAH1 gene, while increasing the expression of glucosylceramide synthase (GCS), encoded by the UGCG gene. Remarkably, knockdown of AC alone via RNA interference is enough to induce melanoma cell dedifferentiation. Furthermore, treatment with Eliglustat, a GCS inhibitor, inhibits TNF-induced melanoma cell dedifferentiation. Lastly, analysis of plasma samples from patients treated with immune checkpoint inhibitors, with or without anti-TNF therapy, revealed significant predictive sphingolipids. Notably, the top 8 predictive sphingolipids, including glycosphingolipids, were associated with a poor response to immunotherapy. Discussion: Our study highlights that ceramide metabolism alterations are causally involved in TNF-induced melanoma cell dedifferentiation and suggests that the evolution of specific ceramide metabolites in plasma may be considered as predictive biomarkers of resistance to immunotherapy.

Asunto(s)

Desdiferenciación Celular , Ceramidas , Resistencia a Antineoplásicos , Inhibidores de Puntos de Control Inmunológico , Melanoma , Factor de Necrosis Tumoral alfa , Humanos , Melanoma/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Ceramidas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/inmunología , Masculino , Glucosiltransferasas/metabolismo , Glucosiltransferasas/genética , Esfingolípidos/metabolismo , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/genética , Femenino , Persona de Mediana Edad , Anciano

RESUMEN

Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer therapeutics. Yet, the role of the Ac in immune responses, particularly anti-viral immunity, is not fully understood. To investigate the impact of Ac expression on various leukocyte populations, we generated a tamoxifen-inducible global knockout mouse model for the Ac (iAc-KO). Following tamoxifen administration to healthy mice, we extracted primary and secondary lymphoid organs from iAc-KO and wild-type (wt) littermates and subsequently performed extensive flow cytometric marker analysis. In addition, we isolated CD4+ T cells from the spleen and lymph nodes for sphingolipid profiling and restimulated them in vitro with Dynabeads™ Mouse T-activator CD3/CD28. Intracellular cytokine expression (FACS staining) was analyzed and secreted cytokines detected in supernatants. To study cell-intrinsic effects, we established an in vitro model for iAc-KO in isolated CD4+ T and B cells. For CD4+ T cells of iAc-KO versus wt mice, we observed reduced Ac activity, an increased ceramide level, and enhanced secretion of IFNγ upon CD3/CD28 costimulation. Moreover, there was a marked reduction in B cell and plasma cell and blast numbers in iAc-KO compared to wt mice. To study cell-intrinsic effects and in line with the 3R principles, we established in vitro cell culture systems for iAc-KO in isolated B and CD4+ T cells. Our findings pinpoint to a key role of the Ac in mature B and antibody-secreting cells and in IFNγ secretion by CD4+ T cells.

Asunto(s)

Ceramidasa Ácida , Linfocitos B , Linfocitos T CD4-Positivos , Interferón gamma , Ratones Noqueados , Animales , Ratones , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Interferón gamma/metabolismo , Recuento de Linfocitos , Ratones Endogámicos C57BL

RESUMEN

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is prone to metastasis and therapy resistance. Owing to its aggressive nature and limited availability of targeted therapies, TNBC is associated with higher mortality as compared to other forms of breast cancer. In order to develop new therapeutic options for TNBC, we characterized the factors involved in TNBC growth and progression. Here, we demonstrate that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in TNBC cells and is regulated via p53 and PI3K-AKT signaling pathways. Genetic knockdown or pharmacological inhibition of ASAH1 suppresses TNBC growth and progression. Mechanistically, ASAH1 inhibition stimulates dual-specificity phosphatase 5 (DUSP5) expression, suppressing the mitogen-activated protein kinase (MAPK) pathway. Furthermore, pharmacological cotargeting of the ASAH1 and MAPK pathways inhibits TNBC growth. Collectively, we unmasked a novel role of ASAH1 in driving TNBC and identified dual targeting of the ASAH1 and MAPK pathways as a potential new therapeutic approach for TNBC treatment.

Asunto(s)

Ceramidasa Ácida , Fosfatasas de Especificidad Dual , Sistema de Señalización de MAP Quinasas , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/genética , Fosfatasas de Especificidad Dual/metabolismo , Fosfatasas de Especificidad Dual/genética , Femenino , Línea Celular Tumoral , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Animales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones Desnudos , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Acid ceramidase (AC) is a lysosomal enzyme required to hydrolyze ceramide to sphingosine by the removal of the fatty acid moiety. An inherited deficiency in this activity results in two disorders, Farber Lipogranulomatosis and spinal muscular atrophy with myoclonic epilepsy, leading to the accumulation of ceramides and other sphingolipids in various cells and tissues. In addition to ceramide hydrolysis, several other activities have been attributed to AC, including a reverse reaction that synthesizes ceramide from free fatty acids and sphingosine, and a deacylase activity that removes fatty acids from complex lipids such as sphingomyelin and glycosphingolipids. A close association of AC with another important enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM), has also been observed. Herein, we used a highly purified recombinant human AC (rhAC) and novel UPLC-based assay methods to investigate the recently described deacylase activity of rhAC against three sphingolipid substrates, sphingomyelin, galactosyl- and glucosylceramide. No deacylase activities were detected using this method, although we did unexpectedly identify a significant ASM activity using natural (C-18) and artificial (Bodipy-C12) sphingomyelin substrates as well as the ASM-specific fluorogenic substrate, hexadecanoylamino-4-methylumbelliferyl phosphorylcholine (HMU-PC). We showed that this ASM activity was not due to contaminating, hamster-derived ASM in the rhAC preparation, and that the treatment of ASM-knockout mice with rhAC significantly reduced sphingomyelin storage in the liver. However, unlike the treatment with rhASM, this did not lead to elevated ceramide or sphingosine levels.

Asunto(s)

Ceramidasa Ácida , Esfingomielinas , Animales , Ratones , Cricetinae , Humanos , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Esfingomielinas/metabolismo , Esfingosina/metabolismo , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Ceramidas/metabolismo , Esfingolípidos/metabolismo , Ácidos Grasos

RESUMEN

Innate immune memory, also called "trained immunity," is a functional state of myeloid cells enabling enhanced immune responses. This phenomenon is important for host defense, but also plays a role in various immune-mediated conditions. We show that exogenously administered sphingolipids and inhibition of sphingolipid metabolizing enzymes modulate trained immunity. In particular, we reveal that acid ceramidase, an enzyme that converts ceramide to sphingosine, is a potent regulator of trained immunity. We show that acid ceramidase regulates the transcription of histone-modifying enzymes, resulting in profound changes in histone 3 lysine 27 acetylation and histone 3 lysine 4 trimethylation. We confirm our findings by identifying single-nucleotide polymorphisms in the region of ASAH1, the gene encoding acid ceramidase, that are associated with the trained immunity cytokine response. Our findings reveal an immunomodulatory effect of sphingolipids and identify acid ceramidase as a relevant therapeutic target to modulate trained immunity responses in innate immune-driven disorders.

Asunto(s)

Ceramidasa Ácida , Inmunidad Entrenada , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Histonas , Lisina , Esfingolípidos/genética , Inmunidad Innata

RESUMEN

Tuberous sclerosis complex (TSC) is characterized by multisystem, low-grade neoplasia involving the lung, kidneys, brain, and heart. Lymphangioleiomyomatosis (LAM) is a progressive pulmonary disease affecting almost exclusively women. TSC and LAM are both caused by mutations in TSC1 and TSC2 that result in mTORC1 hyperactivation. Here, we report that single-cell RNA sequencing of LAM lungs identified activation of genes in the sphingolipid biosynthesis pathway. Accordingly, the expression of acid ceramidase (ASAH1) and dihydroceramide desaturase (DEGS1), key enzymes controlling sphingolipid and ceramide metabolism, was significantly increased in TSC2-null cells. TSC2 negatively regulated the biosynthesis of tumorigenic sphingolipids, and suppression of ASAH1 by shRNA or the inhibitor ARN14976 (17a) resulted in markedly decreased TSC2-null cell viability. In vivo, 17a significantly decreased the growth of TSC2-null cell-derived mouse xenografts and short-term lung colonization by TSC2-null cells. Combined rapamycin and 17a treatment synergistically inhibited renal cystadenoma growth in Tsc2+/- mice, consistent with increased ASAH1 expression and activity being rapamycin insensitive. Collectively, the present study identifies rapamycin-insensitive ASAH1 upregulation in TSC2-null cells and tumors and provides evidence that targeting aberrant sphingolipid biosynthesis pathways has potential therapeutic value in mechanistic target of rapamycin complex 1-hyperactive neoplasms, including TSC and LAM.

Asunto(s)

Neoplasias Pulmonares , Esclerosis Tuberosa , Humanos , Ratones , Femenino , Animales , Esclerosis Tuberosa/tratamiento farmacológico , Proteínas Supresoras de Tumor/genética , Regulación hacia Arriba , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Ceramidasa Ácida/uso terapéutico , Neoplasias Pulmonares/patología , Sirolimus/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones Noqueados

RESUMEN

Bi-allelic mutations in GBA1, the gene that encodes ß-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson's disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD-DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD-DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

Asunto(s)

Enfermedad de Gaucher , Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Inhibidores mTOR , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Enfermedad de Gaucher/metabolismo , Neuronas Dopaminérgicas/metabolismo , Serina-Treonina Quinasas TOR/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mutación , Lisosomas/metabolismo

RESUMEN

To study the mechanism by which nonalcoholic fatty liver disease (NAFLD) contributes to vascular endothelial Nod-like receptor pyrin domain 3 (NLRP3) inflammasome activation and neointima hyperplasia, NAFLD was established in high-fat diet (HFD)-treated Asah1fl/fl/Albcre (liver-specific deletion of the acid ceramidase gene Asah1) mice. Compared with Asah1 flox [Asah1fl/fl/wild type (WT)] and wild-type (WT/WT) mice, Asah1fl/fl/Albcre mice exhibited significantly enhanced ceramide levels and lipid deposition on HFD in the liver. Moreover, Asah1fl/fl/Albcre mice showed enhanced expression of extracellular vesicle (EV) markers, CD63 and annexin II, but attenuated lysosome-multivesicular body fusion. All these changes were accompanied by significantly increased EV counts in the plasma. In a mouse model of neointima hyperplasia, liver-specific deletion of the Asah1 gene enhanced HFD-induced neointima proliferation, which was associated with increased endothelial NLRP3 inflammasome formation and activation and more severe endothelial damage. The EVs isolated from plasma of Asah1fl/fl/Albcre mice on HFD were found to markedly enhance NLRP3 inflammasome formation and activation in primary cultures of WT/WT endothelial cells compared with those isolated from WT/WT mice or normal diet-treated Asah1fl/fl/Albcre mice. These results suggest that the acid ceramidase/ceramide signaling pathway controls EV release from the liver, and its deficiency aggravates NAFLD and intensifies hepatic EV release into circulation, which promotes endothelial NLRP3 inflammasome activation and consequent neointima hyperplasia in the mouse carotid arteries.

Asunto(s)

Vesículas Extracelulares , Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Inflamasomas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Ratones Noqueados , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Células Endoteliales/metabolismo , Neointima/metabolismo , Técnicas de Inactivación de Genes , Hiperplasia , Hígado/metabolismo , Vesículas Extracelulares/metabolismo , Ceramidas , Dieta Alta en Grasa/efectos adversos , Ratones Endogámicos C57BL

RESUMEN

Farber disease (FD) is a rare monogenic lysosomal storage disorder caused by mutations in ASAH1 that results in a deficiency of acid ceramidase (ACDase) activity and the abnormal systemic accumulation of ceramide species, leading to multi-system organ failure involving neurological decline and retinopathy. Here we describe the effects of rAAV-mediated ASAH1 over-expression on the progression of retinopathy in a mouse model of FD (Asah1P361R/P361R) and its littermate controls (Asah1+/+ and Asah1+/P361R). Using a combination of non-invasive multimodal imaging, electrophysiology, post-mortem histology and mass spectrometry we demonstrate that ASAH1 over-expression significantly reduces central retinal thickening, ceramide accumulation, macrophage activation and limits fundus hyper-reflectivity and auto-fluorescence in FD mice, indicating rAAV-mediated over-expression of biologically active ACDase protein is able to rescue the anatomical retinal phenotype of Farber disease. Unexpectedly, ACDase over-expression in Asah1+/+ and Asah1+/P361R control eyes was observed to induce abnormal fundus hyper-reflectivity, auto-fluorescence and retinal thickening that closely resembles a FD phenotype. This study represents the first evidence of a gene therapy for Farber disease-related retinopathy. Importantly, the described gene therapy approach could be used to preserve vision in FD patients synergistically with broader enzyme replacement strategies aimed at preserving life.

Asunto(s)

Lipogranulomatosis de Farber , Enfermedades de la Retina , Ratones , Animales , Lipogranulomatosis de Farber/genética , Lipogranulomatosis de Farber/terapia , Lipogranulomatosis de Farber/metabolismo , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Ceramidas/metabolismo , Mutación , Enfermedades de la Retina/genética , Enfermedades de la Retina/terapia

RESUMEN

Lysosomal acid ceramidase (AC) has been reported to determine multivesicular body (MVB) fate and exosome secretion in different mammalian cells including coronary arterial endothelial cells (CAECs). However, this AC-mediated regulation of exosome release from CAECs and associated underlying mechanism remain poorly understood. In the present study, we hypothesized that AC controls lysosomal Ca2+ release through TRPML1 channel to regulate exosome release in murine CAECs. To test this hypothesis, we isolated and cultured CAECs from WT/WT and endothelial cell-specific Asah1 gene (gene encoding AC) knockout mice. Using these CAECs, we first demonstrated a remarkable increase in exosome secretion and significant reduction of lysosome-MVB interaction in CAECs lacking Asah1 gene compared to those cells from WT/WT mice. ML-SA1, a TRPML1 channel agonist, was found to enhance lysosome trafficking and increase lysosome-MVB interaction in WT/WT CAECs, but not in CAECs lacking Asah1 gene. However, sphingosine, an AC-derived sphingolipid, was able to increase lysosome movement and lysosome-MVB interaction in CAECs lacking Asah1 gene, leading to reduced exosome release from these cells. Moreover, Asah1 gene deletion was shown to substantially inhibit lysosomal Ca2+ release through suppression of TRPML1 channel activity in CAECs. Sphingosine as an AC product rescued the function of TRPML1 channel in CAECs lacking Asah1 gene. These results suggest that Asah1 gene defect and associated deficiency of AC activity may inhibit TRPML1 channel activity, thereby reducing MVB degradation by lysosome and increasing exosome release from CAECs. This enhanced exosome release from CAECs may contribute to the development of coronary arterial disease under pathological conditions.

Asunto(s)

Exosomas , Canales de Potencial de Receptor Transitorio , Ratones , Animales , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Exosomas/metabolismo , Células Endoteliales/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Esfingosina/metabolismo , Lisosomas/metabolismo , Ratones Noqueados , Mamíferos/metabolismo

RESUMEN

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

Asunto(s)

Ceramidasa Ácida , Tratamiento Farmacológico de COVID-19 , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Fluoxetina , Humanos , Pandemias , ARN , SARS-CoV-2

RESUMEN

Glioblastoma (GBM) remains one of the most aggressive cancers, partially due to its ability to migrate into the surrounding brain. The sphingolipid balance, or the balance between ceramides and sphingosine-1-phosphate, contributes to the ability of GBM cells to migrate or invade. Of the ceramidases which hydrolyze ceramides, acid ceramidase (ASAH1) is highly expressed in GBM samples compared to non-tumor brain. ASAH1 expression also correlates with genes associated with migration and focal adhesion. To understand the role of ASAH1 in GBM migration, we utilized shRNA knockdown and observed decreased migration that did not depend upon changes in growth. Next, we inhibited ASAH1 using carmofur, a clinically utilized small molecule inhibitor. Inhibition of ASAH1 by carmofur blocks in vitro migration of U251 (GBM cell line) and GBM cells derived from patient-derived xenografts (PDXs). RNA-sequencing suggested roles for carmofur in MAPK and AKT signaling. We found that carmofur treatment decreases phosphorylation of AKT, but not of MAPK. The decrease in AKT phosphorylation was confirmed by shRNA knockdown of ASAH1. Our findings substantiate ASAH1 inhibition using carmofur as a potential clinically relevant treatment to advance GBM therapeutics, particularly due to its impact on migration.

Asunto(s)

Ceramidasa Ácida , Glioblastoma , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Línea Celular Tumoral , Movimiento Celular , Ceramidas/metabolismo , Fluorouracilo , Glioblastoma/metabolismo , Humanos , Proteínas Proto-Oncogénicas c-akt , ARN Interferente Pequeño

RESUMEN

Accumulation of lipids and their metabolites induces lipotoxicity in diabetic cardiomyopathy. Lowering ceramide concentration could reduce the impact of metabolic damage to target organs. Adiponectin improves lipotoxicity through its receptors (AdiopRs), which have sequence homology with ceramidase enzymes. Therefore, cardioprotective role of AdipoR agonism by AdipoRon was investigated. Sixteen-week-old male db/m and db/db mice were fed a diet containing AdipoRon for four weeks. Phenotypic and metabolic profiles with associated cellular signaling pathways involved in lipid metabolism were investigated in the mice heart and human cardiomyocytes to establish treatment effect of AdipoRon. AdipoRon ameliorated insulin resistance, fibrosis, M1-dominant inflammation, and apoptosis in association with reduced accumulations of free fatty acid, triglycerides, and TLR4-related ceramide in the heart. This resulted in overall reduction in the level of oxidative stress which ameliorated cardiac hypertrophy and its function. AdipoRon increased the expression of AdipoR1 and AdipoR2 via pAMPK/FoxO1-induced Akt phosphorylation resulting from a decrease in PP2A level. It also increased acid ceramidase activity which reduced ceramide and increased sphingosine-1 phosphate levels in the heart of db/db mice and cultured human cardiomyocytes. Consistent upregulation of AdipoRs and their downstream regulatory pathways involving pAMPK/PPARα/PGC-1α levels led to lipid metabolism enhancement, thereby improving lipotoxicity-induced peroxisome biogenesis and oxidative stress. AdipoRon might control oxidative stress, inflammation, and apoptosis in the heart through increased AdipoR expression, acid ceramidase activity, and activation of AMPK-PPARα/PGC-1α and related downstream pathways, collectively improving cardiac lipid metabolism, hypertrophy, and functional parameters.

Asunto(s)

Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Proteínas Quinasas Activadas por AMP/metabolismo , Ceramidasa Ácida/metabolismo , Adiponectina/metabolismo , Animales , Ceramidas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Inflamación/complicaciones , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR alfa/metabolismo , Receptores de Adiponectina/agonistas

RESUMEN

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.

Asunto(s)

Enfermedad de Fabry/genética , Enfermedad de Fabry/metabolismo , Podocitos/enzimología , Podocitos/metabolismo , alfa-Galactosidasa/genética , alfa-Galactosidasa/metabolismo , Ceramidasa Ácida/metabolismo , Adulto , Línea Celular , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Transducción de Señal , Proteínas de Unión al GTP rab/metabolismo

RESUMEN

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy.

Asunto(s)

Ceramidasa Ácida/antagonistas & inhibidores , Terapia Genética/métodos , Enfermedades Mitocondriales/etiología , Estrés Oxidativo , Neoplasias Pancreáticas/terapia , ARN Interferente Pequeño/uso terapéutico , Ceramidasa Ácida/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Ceramidas/metabolismo , Dependovirus , Vectores Genéticos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Niemann-Pick type C (NPC) disease, a lysosomal storage disorder caused by defective NPC1/NPC2 function, results in the accumulation of cholesterol and glycosphingolipids in lysosomes of affected organs, such as liver and brain. Moreover, increase of mitochondrial cholesterol (mchol) content and impaired mitochondrial function and GSH depletion contribute to NPC disease. However, the underlying mechanism of mchol accumulation in NPC disease remains unknown. As STARD1 is crucial in intramitochondrial cholesterol trafficking and acid ceramidase (ACDase) has been shown to regulate STARD1, we explored the functional relationship between ACDase and STARD1 in NPC disease. Liver and brain of Npc1-/- mice presented a significant increase in mchol levels and STARD1 expression. U18666A, an amphiphilic sterol that inhibits lysosomal cholesterol efflux, increased mchol levels in hepatocytes from Stard1f/f mice but not Stard1ΔHep mice. We dissociate the induction of STARD1 expression from endoplasmic reticulum stress, and establish an inverse relationship between ACDase and STARD1 expression and LRH-1 levels. Hepatocytes from Npc1+/+ mice treated with U18666A exhibited increased mchol accumulation, STARD1 upregulation and decreased ACDase expression, effects that were reversed by cholesterol extraction with 2-hydroxypropyl-ß-cyclodextrin. Moreover, transfection of fibroblasts from NPC patients with ACDase, decreased STARD1 expression and mchol accumulation, resulting in increased mitochondrial GSH levels, improved mitochondrial functional performance, decreased oxidative stress and protected NPC fibroblasts against oxidative stress-mediated cell death. Our results demonstrate a cholesterol-dependent inverse relationship between ACDase and STARD1 and provide a novel approach to target the accumulation of cholesterol in mitochondria in NPC disease.

Asunto(s)

Enfermedad de Niemann-Pick Tipo C , Fosfoproteínas/metabolismo , Ceramidasa Ácida/metabolismo , Animales , Colesterol/metabolismo , Humanos , Lisosomas/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Estrés Oxidativo

RESUMEN

Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LEs)/lysosomes may control cargo selection, membrane biogenesis, and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal-CER-mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin-1 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt-related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22-α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl /SMCre mice had markedly increased co-localization of mTORC1 with lysosome-associated membrane protein-1 (Lamp-1) (lysosome marker) and decreased co-localization of vacuolar protein sorting-associated protein 16 (VPS16) (a multivesicular bodies [MVBs] marker) with Lamp-1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin-1 significantly reduced the co-localization of mTOR vs Lamp-1, increased lysosome-MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi -stimulation significantly increased p-mTOR expression in Asah1fl/fl /SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin-1 blocked Pi -induced calcium deposition and mineralization. siRNA mTOR and Torin-1 significantly reduce co-localization of mTORC1 with Lamp-1, increased VPS16 vs Lamp-1 co-localization in Pi -stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduces arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER-mTOR signaling may play a critical role for the control of lysosome-MVB interaction, exosome secretion and arterial stiffening during AMC.

Asunto(s)

Ceramidasa Ácida/metabolismo , Exosomas/metabolismo , Mamíferos/metabolismo , Miocitos del Músculo Liso/metabolismo , Osteogénesis/fisiología , Sirolimus/metabolismo , Animales , Aorta/metabolismo , Calcio/metabolismo , Ceramidas/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Vasos Coronarios/metabolismo , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cuerpos Multivesiculares/metabolismo , Análisis de la Onda del Pulso/métodos , Transducción de Señal/fisiología , Esfingolípidos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Calcificación Vascular/metabolismo

RESUMEN

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated ß-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

Asunto(s)

Ceramidasa Ácida/metabolismo , Senescencia Celular , Fibroblastos/citología , Fibroblastos/enzimología , Ceramidasa Ácida/genética , Línea Celular , Proliferación Celular/genética , Supervivencia Celular , Ceramidas/metabolismo , Silenciador del Gen , Humanos , Metaboloma , Biosíntesis de Proteínas/genética , Estabilidad del ARN/genética

RESUMEN

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [14C]ethanolamine, and revealed that overexpressed AC lowered the levels of 14C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.

Asunto(s)

Ceramidasa Ácida/metabolismo , Etanolaminas/metabolismo , Animales , Células HEK293 , Humanos , Hidrólisis , Masculino , Ratones