SMPD1 Research Articles

Genome-Wide Gene-by-Smoking Interaction Study of Chronic Obstructive Pulmonary Disease.

Risk of chronic obstructive pulmonary disease (COPD) is determined by both cigarette smoking and genetic susceptibility, but little is known about gene-by-smoking interactions. We performed a genome-wide association analysis of 179,689 controls and 21,077 COPD cases from UK Biobank subjects of European ancestry recruited from 2006 to 2010, considering genetic main effects and gene-by-smoking interaction effects simultaneously (2-degrees-of-freedom (df) test) as well as interaction effects alone (1-df interaction test). We sought to replicate significant results in COPDGene (United States, 2008-2010) and SpiroMeta Consortium (multiple countries, 1947-2015) data. We considered 2 smoking variables: 1) ever/never and 2) current/noncurrent. In the 1-df test, we identified 1 genome-wide significant locus on 15q25.1 (cholinergic receptor nicotinic β4 subunit, or CHRNB4) for ever- and current smoking and identified PI*Z allele (rs28929474) of serpin family A member 1 (SERPINA1) for ever-smoking and 3q26.2 (MDS1 and EVI1 complex locus, or MECOM) for current smoking in an analysis of previously reported COPD loci. In the 2-df test, most of the significant signals were also significant for genetic marginal effects, aside from 16q22.1 (sphingomyelin phosphodiesterase 3, or SMPD3) and 19q13.2 (Egl-9 family hypoxia inducible factor 2, or EGLN2). The significant effects at 15q25.1 and 19q13.2 loci, both previously described in prior genome-wide association studies of COPD or smoking, were replicated in COPDGene and SpiroMeta. We identified interaction effects at previously reported COPD loci; however, we failed to identify novel susceptibility loci.

A Murine Model Of Kidney Injury: The role of SMPDL3b In Radiation And Cisplatin-Induced Nephrotoxicity

Cisplatin is widely used as a chemotherapeutic agent and a known radiosensitizer. The current study aimed to investigate the role of lipid-modulating enzyme sphingomyelin phosphodiesterase acid-like 3B (SMPDL3b) in cisplatin toxicity. Nephrotoxicity is dose-limiting toxicity. Cultured oocytes were treated with rituximab mAb (100 μg/ml) or IgG (100 μg/ml) 30 minutes before irradiation with 0-8 Gy, with or without Cisplatin (2μM). In other experiments, 10–14-week old C57BL/6 male and female mice were given bilateral kidney X-irradiation (4Gyx1, 6 Gyx5, and 2Gyx24) with or without Cisplatin (6mg/kg, IP). Animals received rituximab mAb (RTX) or IgG (50mg/kg; IV) 30 minutes before radiation treatment. Functional kidney parameters (serum BUN, hematocrit, and creatinine), histopathological assessments (oil red staining, H&E, Periodic Acid-Schiff, and TEM) and biochemical changes were studied at baseline, 10- and 20-weeks post-chemo-radiotherapy. Glomerular filtration rate (GFR) was estimated at 10- and 20-weeks post-radiation using FITC-sinistrin based transdermal monitor and iSMAART dynamic contrast-enhanced (DCE) imaging. RT and concurrent Cisplatin (2 μM) produced time-dependent apoptosis in wild-type human podocytes, causing an 85% increase in caspase 3 activity 2h post-RT (8Gy). Different doses of RT, 1x4Gy, 1x10Gy, and 1x14Gy increased glomerular base membrane (GBM) thickness in irradiated mice significantly (100%, 150%, and 200% respectively). Cisplatin and RT (1x4Gy) increased GBM thickness from 225nm to 425nm, respectively (p = 0.01). The eGFR values changed significantly from 1010±214 to 718±205, 687±175, 508±56, 596±180, and 639±221 mL/min after x4Gy, 1x10Gy, 1x14Gy, 5x6Gy, and 24x2Gy, respectively. RTX pretreatment improved eGFR and other functional parameters (serum BUN, hematocrit, serum creatinine, and urine albumin/creatinine ratio (ACR) significantly post chemoradiotherapy. RTX pretreatment protected podocytes cytoskeletal remodeling in vitro and foot process effacement in vivo. SMPDL3b expression and podocytes count significantly decreased post chemoradiotherapy. RTX ameliorated SMPDL3b down-regulation, and improved podocytes count significantly. This study shows that RTX pretreatment protects podocytopathy in vitro and chemoradiation induced kidney injury in vivo. SMPDL3b may be an important therapeutic target for radiation-induced kidney injuries in cancer patients.

Human adipocyte differentiation and composition of disease-relevant lipids are regulated by miR-221-3p

MicroRNA-221-3p (miR-221-3p) is associated with both metabolic diseases and cancers. However, its role in terminal adipocyte differentiation and lipid metabolism are uncharacterized. miR-221-3p or its inhibitor was transfected into differentiating or mature human adipocytes. Triglyceride (TG) content and adipogenic gene expression were monitored, global lipidome analysis was carried out, and mechanisms underlying the effects of miR-221-3p were investigated. Finally, cross-talk between miR-221-3p expressing adipocytes and MCF-7 breast carcinoma (BC) cells was studied, and miR-221-3p expression in tumor-proximal adipose biopsies from BC patients analyzed. miR-221-3p overexpression inhibited terminal differentiation of adipocytes, as judged from reduced TG storage and gene expression of the adipogenic markers SCD1, GLUT4, FAS, DGAT1/2, AP2, ATGL and AdipoQ, whereas the miR-221-3p inhibitor increased TG storage. Knockdown of the predicted miR-221-3p target, 14-3-3γ, had similar antiadipogenic effects as miR-221-3p overexpression, indicating it as a potential mediator of mir-221-3p function. Importantly, miR-221-3p overexpression inhibited de novo lipogenesis but increased the concentrations of ceramides and sphingomyelins, while reducing diacylglycerols, concomitant with suppression of sphingomyelin phosphodiesterase, ATP citrate lyase, and acid ceramidase. miR-221-3p expression was elevated in tumor proximal adipose tissue from patients with invasive BC. Conditioned medium of miR-221-3p overexpressing adipocytes stimulated the invasion and proliferation of BC cells, while medium of the BC cells enhanced miR-221-3p expression in adipocytes. Elevated miR-221-3p impairs adipocyte lipid storage and differentiation, and modifies their ceramide, sphingomyelin, and diacylglycerol content. These alterations are relevant for metabolic diseases but may also affect cancer progression.

Sphingomyelin Phosphodiesterase Acid-like 3B (SMPDL3b) Affects Podocyte Lipid Metabolism and Lipid Droplets Formation

Sphingomyelin Phosphodiesterase Acid-like 3B (SMPDL3b) Affects Podocyte Lipid Metabolism and Lipid Droplets Formation

Sphingomyelinase Disables Inactivation in Endogenous PIEZO1 Channels

SummaryEndogenous PIEZO1 channels of native endothelium lack the hallmark inactivation often seen when these channels are overexpressed in cell lines. Because prior work showed that the force of shear stress activates sphingomyelinase in endothelium, we considered if sphingomyelinase is relevant to endogenous PIEZO1. Patch clamping was used to quantify PIEZO1-mediated signals in freshly isolated murine endothelium exposed to the mechanical forces caused by shear stress and membrane stretch. Neutral sphingomyelinase inhibitors and genetic disruption of sphingomyelin phosphodiesterase 3 (SMPD3) cause PIEZO1 to switch to profoundly inactivating behavior. Ceramide (a key product of SMPD3) rescues non-inactivating channel behavior. Its co-product, phosphoryl choline, has no effect. In contrast to ceramide, sphingomyelin (the SMPD3 substrate) does not affect inactivation but alters channel force sensitivity. The data suggest that sphingomyelinase activity, ceramide, and sphingomyelin are determinants of native PIEZO gating that enable sustained activity.

Podocyte sphingomyelin phosphodiesterase acid-like 3b decreases among children with idiopathic nephrotic syndrome.

Sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b), a regulator of the cytoskeleton, is expressed on podocytes. Recent reports present evidence that it is directly targeted by rituximab in the treatment of intractable nephrotic syndrome. However, the implications of SMPDL-3b for treatment of paediatric-onset idiopathic nephrotic syndrome (INS) remain unclear. This study aimed to investigate the level of expression of SMPDL-3b in urine, serum, and biopsy specimens and explore its implications in treatment of patients with INS. Levels of urinary SMPDL-3b among 31 patients (20 in remission and 11 in relapse) with INS were analysed by dot blotting. For reference of precise quantitative analysis, we examined urinary excretion of SMPDL-3b from 10 patients with INS by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in both remitted and relapsed status. The levels of serum SMPDL-3b among 20 patients (13 in remission and 7 in relapse or onset) with INS were also measured using enzyme-linked immunosorbent assay. Further, the immunoreactivity of SMPDL-3b in the biopsy specimens obtained from patients with INS was compared with those from patients with proteinuric IgA nephropathy, lupus nephritis, and non-proteinuric controls. Urinary excretion of SMPDL-3b in patients with INS was significantly decreased in relapse cases compared with cases of remission and other types of proteinuric glomerular disease or controls by both dot blotting and LC-MS/MS method. On the other hand, serum SMPDL-3b level in INS was not different between cases of remission and relapse. Glomerular immunoreactivity of SMPDL-3b in patient with INS in remission was almost the same level to that of control. The expression of SMPDL-3b on podocytes is specifically decreased in paediatric-onset INS and its urinary excretion level reflects such conditions.

Construction of a lipid metabolism-related and immune-associated prognostic signature for hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the most lethal malignancies. We aimed to identify a robust lipid metabolism‐related signature associated with the HCC microenvironment to improve the prognostic prediction of HCC patients.MethodsWe analyzed the gene expression profiles of lipid metabolism from Molecular Signatures Database and information of patients from The Cancer Genome Atlas. Gene set variation analysis (GSVA), gene set enrichment analysis (GSEA), and principal component analysis (PCA) were employed for functional annotation. Quantitative real‐time polymerase chain reaction (qRT‐PCR) was employed to verify the expression of model genes in HCC and adjacent tissues.ResultsAs a result, a lipid metabolism‐related signature consisting of acyl‐CoA synthetase long‐chain family member 6 (ACSL6), lysophosphatidylcholine acyltransferase 1, phospholipase A2 group 1B, lecithin‐cholesterol acyltransferase (LCAT), and sphingomyelin phosphodiesterase 4 (SMPD4) was identified among HCC patients. Lysophosphatidylcholine acyltransferase 1, PLA2G1B, and SMPD4 were proved significantly high expression while ACSL6 and LCAT were remarkably low expression in our 15 pairs of matched HCC and normal tissues by qRT‐PCR. Under different conditions, the overall survival (OS) of patients in low‐risk group was prolonged than that in high‐risk group. Moreover, the as‐constructed signature was an independent factor, which was remarkably associated with gender, histologic grade, and platelet level of HCC patients. In addition, the receiver operating characteristic (ROC) curve analysis confirmed the good potency of the model. Functional enrichment analysis further revealed that lower fatty acid (FA) oxidation and higher infiltration of immunocytes were detected in patients from the high‐risk group compared with those in the low‐risk group.ConclusionsOur findings indicate that the lipid metabolism‐related signature shows prognostic significance for HCC.

Phosphodiesterase SMPDL3B Gene Expression as Independent Outcome Prediction Marker in Localized Prostate Cancer.

Current outcome prediction markers for localized prostate cancer (PCa) are insufficient. The impact of the lipid-modifying Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) in PCa is unknown. Two cohorts of patients with PCa who underwent radical prostatectomy (n = 40, n = 56) and benign prostate hyperplasia (BPH) controls (n = 8, n = 11) were profiled for SMPDL3B expression with qRT-PCR. Publicly available PCa cohorts (Memorial Sloane Kettering Cancer Centre (MSKCC; n = 131, n = 29 controls) and The Cancer Genome Atlas (TCGA; n = 497, n = 53 controls)) served for validation. SMPDL3B’s impact on proliferation and migration was analyzed in PC3 cells by siRNA knockdown. In both cohorts, a Gleason score and T stage independent significant overexpression of SMPDL3B was seen in PCa compared to BPH (p < 0.001 each). A lower expression of SMPDL3B was associated with a shorter overall survival (OS) (p = 0.005) in long term follow-up. A SMPDL3B overexpression in PCa tissue was confirmed in the validation cohorts (p < 0.001 each). In the TCGA patients with low SMPDL3B expression, biochemical recurrence-free survival (p = 0.011) and progression-free interval (p < 0.001) were shorter. Knockdown of SMPDL3B impaired PC3 cell migration but not proliferation (p = 0.0081). In summary, SMPLD3B is highly overexpressed in PCa tissue, is inversely associated with localized PCa prognosis, and impairs PCa cell migration.

Rs1542705–67,992,843-1,050,239 represents a novel informative haplotype at the SMPD1 locus in the Iranian population

Niemann-Pick disease (NPD) refers to a group of lysosomal storage diseases that cause abnormal metabolism of lipids. Direct sequencing and deletion analysis is used to detect mutations and sequence variations in the SMPD1 gene. However, due to technical difficulties, linkage analysis of polymorphic markers such as single nucleotide polymorphism (SNP) has been used in heterozygous carrier detection and prenatal diagnosis of the disease in families with an affected individual. The SNP markers usually show a population-based haplotype frequency and heterozygosity. A large number of SNP markers have been introduced for the SMPD1 gene. In the present study, rs1542705, rs67992843and rs1050239 genetic markers located in the SMPD1 gene region were genotyped using ARMS PCR technique in 188 unrelated control individuals and 11 family trios in the Iranian population. The allele frequency, degree of heterozygosity and Hardy-Weinberg equilibrium (HWE) were estimated using the GENEPOP program. The estimation of haplotype frequency and linkage disequilibrium for the unrelated individuals was estimated using the PowerMarker software. The observed degree of heterozygosity for rs1542705, rs6799843and rs1050239 were 39.36, 46.68 and 28.19, respectively. The result showed all markers were in linkage disequilibrium. Consequently, T-G-G, T-C-G, C-G-G, C-C-G, T-C-A, T-G-A, C-G-A informative haplotypes in population level were identified by using a combination of these three markers. Therefore, 1,542,705–6,799,843-1,050,239 represents a novel informative haplotype at the SMPD1 locus in the Iranian population, which could be suggested as appropriate markers for linkage analysis and identification of heterozygous carriers as well as prenatal diagnosis of NPD in the Iranian families with affected children.

Characterization of the small molecule ARC39, a direct and specific inhibitor of acid sphingomyelinase in vitro

Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM’s catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.

Human alkaline ceramidase 2 promotes the growth, invasion, and migration of hepatocellular carcinoma cells via sphingomyelin phosphodiesterase acid-like 3B.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid‐like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment.

Niemann-Pick disease type-B: a unique case report with compound heterozygosity and complicated lipid management

BackgroundNiemann-Pick disease (NPD) is a rare autosomal recessive hereditary disease characterized by deficient activity of acid sphingomyelinase.Case presentationWe present a case of NPD type B with a unique compound heterozygosity for SMPD1 (NM_000543.4:c.[84delC];[96G > A]) in which both mutations that induce an early stop codon are located before the second in-frame initiation codon. The clinical presentation of the patient is compatible with NPD type B. She was initially diagnosed of Gaucher Disease, but her altered lipid profile led to a clinical suspicion of NPD.Combined high doses of atorvastatin and ezetimibe were given to treat the severe hypercholesterolemia.ConclusionsThe pharmacological management of the lipid profile in these patients is important. A unique compound mutation in SMPD1 gene is described.

Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α

Abstract Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether the nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase2 activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of IL-1b and MCP-1. However, inhibition aSMase activity did not attenuate CD11c expression and secretion of IL-1b and MCP-1. Phosphorylation of JNK, p38 and NF-κB resulting from TNF-α stimulation was also attenuated by the inhibition of nSMase. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. Our human data show that SMPD3 gene expression was not only found to be elevated in obese individuals but also positively corelate with TNF-α elevated expression. These findings indicate that nSMase acts, in a part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.

SMPD1 variants do not have a major role in rapid eye movement sleep behavior disorder

Mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene were reported to be associated with Parkinson’s disease and dementia with Lewy bodies. In the current study, we aimed to evaluate the role of SMPD1 variants in isolated rapid eye movement sleep behavior disorder (iRBD). SMPD1 and its untranslated regions were sequenced using targeted next-generation sequencing in 959 iRBD patients and 1287 controls from European descent. Our study reports no statistically significant association of SMPD1 variants and iRBD. It is hence unlikely that SMPD1 plays a major role in iRBD.

Novel mutations in the SMPD1 gene in Jordanian children with Acid sphingomyelinase deficiency (Niemann-Pick types A and B)

Acid sphingomyelinase (ASM) deficiency (ASMD) is a spectrum that includes Niemann–Pick disease (NPD) types A (NPD A) and B (NPD B). ASMD is characterized by intracellular accumulation of unesterified cholesterol and gangliosides within the endosomal–lysosomal system. It is caused by different mutations in SMPD1 gene that result in reduction or complete absence of acid sphingomyelinase activity in the cells. Herein, four unrelated consanguineous families with two NPD A and three NPD B patients were assessed for their genotypes via sequencing of the SMPD1 gene and their acid sphingomyelinase enzymatic activity. Among the eight identified mutations, three were novel and reported for the first time in Jordanian families (c.120_131delGCTGGCGCTGGC or c.132_143delGCTGGCGCTGGC, c.1758T > G, and c.1344T > A). All the patients displayed ASM activity lower than 1.3 µmol/l/h (P < 0.001). Genotyping and enzymatic assessment might play a significant role in disease identification in people at risk to facilitate genetic counseling in the future.

Modulation of radiation-induced damage of human glomerular endothelial cells by SMPDL3B.

The intracellular molecular pathways involved in radiation-induced nephropathy are still poorly understood. Glomerular endothelial cells are key components of the structure and function of the glomerular filtration barrier but little is known about the mechanisms implicated in their injury and repair. The current study establishes the response of immortalized human glomerular endothelial cells (GEnC) to ionizing radiation (IR). We investigated the role of sphingolipids and the lipid-modifying enzyme sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) in radiation-induced GEnC damage. After delivering a single dose of radiation, long and very-long-chain ceramide species, and the expression levels of SMPDL3b were elevated. In contrast, levels of ceramide-1-phosphate (C1P)dropped in a time-dependent manner although mRNA and protein levels of ceramide kinase (CERK) remained stable. Treatment with C1P or knocking down SMPDL3b partially restored cell survival and conferred radioprotection. We also report a novel role for the NADPH oxidase enzymes (NOXs), namely NOX1, and NOX-derived reactive oxygen species (ROS) in radiation-induced GEnC damage. Subjecting cultured endothelial cells to radiation was associated with increased NOX activity and superoxide anion generation. Silencing NOX1 using NOX1-specific siRNA mitigated radiation-induced oxidative stress and cellular injury. In addition, we report a novel connection between NOX and SMPDL3b. Treatment with the NOX inhibitor, GKT, decreased radiation-induced cellular injury and restored SMPDL3b basal levels of expression. Our findings indicate the importance of SMPDL3b as a potential therapeutic target in radiation-induced kidney damage.

Amelioration of Podocytopathy by Smpd1 Gene Deletion in Podocyte‐Specific Asah1 Gene Knockout Mice

Podocyte‐specific deletion of Asah1 (AC gene code in mouse) gene has been found to produce podocytopathy and nephrotic syndrome in mice (Asah1fl/fl/PodoCre mice). However, the mechanism underlying the pathogenesis of podocytopathy in these mice remains unknown. To test whether ceramide accumulation contributes to podocyte injury in Asah1fl/fl/PodoCre mice, Asah1fl/fl/PodoCre mice were mated with Smpd1−/− mice (Smpd1 is mouse gene coding acid sphingomyelinase), which produced mice lacking both Smpd1 and Asah1 gene in podocytes, namely, Smpd1−/−/Asah1fl/fl/PodoCre mice. Using LC‐MS/MS, ceramide accumulation was detected in isolated glomeruli of Asah1fl/fl/PodoCre mice, and Smpd1 gene knockout blocked glomerular ceramide accumulation Asah1fl/fl/PodoCre mice. Correspondingly, severe proteinuria and albuminuria in Asah1fl/fl/PodoCre mice were ameliorated by Smpd1 gene deletion. By immunofluorescence staining, we found reduced glomerular podocin and increased glomerular desmin in Asah1fl/fl/PodoCre mice, which was confirmed using Western blot analysis in isolated glomeruli from these mice. All these podocyte injuries in Asah1fl/fl/PodoCre mice were substantially blocked by cross breeding with Smpd1 gene knockout mice. Under transmission electron microscope, Asah1fl/fl/PodoCre mice were found to have foot process effacement and microvillus formation in podocytes, which were also reversed by deletion of Smpd1 gene. These results suggest that AC deficiency induces ceramide accumulation in mouse glomeruli which is critical to the development of podocytopathy and nephrotic syndrome. Blockade of ceramide production via ASM may attenuate ceramide accumulation and consequent podocytopathy due to AC gene deficiency.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.

Lysosome‐MVB Encountering and Inflammatory Exosome Release Controlled by Acid Sphingomyelinase in Podocytes during Hyperhomocysteinemia

Previous studies have demonstrated that overexpression of acid sphingomyelinase (ASM) enhances NLRP3 inflammasome activation and podocyte‐derived inflammatory exosome release during hyperhomocysteinemia (hHcy). However, the mechanism underlying the enhancement of inflammatory exosome release remains unclear. The present study tested a hypothesis that overexpression of ASM blocks lysosome‐MVB encountering via inhibition of lysosome trafficking, leading to enhanced inflammatory exosome release from podocytes during hHcy. Nanoparticle tracking analysis showed a largely increased urinary exosomes in WT/WT mice with hHcy, but not in Smpd1−/− (ASM gene knockout) mice. In Smpd1trg/Podocre (podocyte‐specific ASM gene overexpression) mice, this increase in urinary exosomes was not only during hHcy, but also on the ND. Increased exosomes were podocytes‐derived by detection of podocin as podocyte marker and CD63 as exosome marker. Biochemical analyses showed that NLRP3 products, IL‐1β and IL‐18 significantly increased in hHcy in WT/WT and Smpd1trg/Podocre mice, but not in Smpd1−/− mice. In vitro, we found that Hcy stimulation enhanced release of exosomes out of podocytes from WT/WT and Smpd1trg/Podocre mice, but it had no effect on exosome release from podocytes of Smpd1−/− mice. Exosomes were isolated with ultracentrifugation and purified with exosome purification kit from podocyte culture medium. It was found that Hcy increased CD63 in purified exosomes from WT/WT and Smpd1trg/Podocre podocytes, but it had no effect in Smpd1−/− podocytes. ELISA analysis showed that these increased exosomes contain a lot of IL‐1β and IL‐18 when podocytes were stimulated by Hcy. Super‐resolution microscopy showed the interaction of lysosomes with MVBs. Lysosomes encountered and fused to MVBs much less in podocytes from WT/WT and Smpd1trg/Podocre mice when they were treated with Hcy. In Smpd1−/− podocytes, Hcy failed to change the lysosome‐MVB encounters and interactions. ML‐SA1 as a cell‐permeable lysosome trafficking stimulator via activation of lysosomal TRPML1 channel markedly increased lysosomes movement toward nucleus. Hcy significantly reduced lysosomes movements to nucleus and overexpression of Smpd1 gene enhanced the effect of Hcy. In Smpd1−/− podocytes, ML‐SA1‐induced lysosome movement toward nucleus remained unchanged during Hcy treatment. These results suggest that ASM‐mediated sphingolipid signaling controls lysosome function determining inflammatory exosome release from podocytes during hHcy.Support or Funding InformationThis study was supported by NIH grants DK54927 and DK102539.

Differential regulation of microRNA-15a by radiation affects angiogenesis and tumor growth via modulation of acid sphingomyelinase

Activation of acid sphingomyelinase (SMPD1) and the generation of ceramide is a critical regulator of apoptosis in response to cellular stress including radiation. Endothelial SMPD1 has been shown to regulate tumor responses to radiation therapy. We show here that the SMPD1 gene is regulated by a microRNA (miR), miR-15a, in endothelial cells (ECs). Standard low dose radiation (2 Gy) upregulates miR-15a and decreases SMPD1 levels. In contrast, high dose radiation (10 Gy and above) decreases miR-15a and increases SMPD1. Ectopic expression of miR-15a decreases both mRNA and protein levels of SMPD1. Mimicking the effects of high dose radiation with a miR-15a inhibitor decreases cell proliferation and increases active Caspase-3 & 7. Mechanistically, inhibition of miR-15a increases inflammatory cytokines, activates caspase-1 inflammasome and increases Gasdermin D, an effector of pyroptosis. Importantly, both systemic and vascular-targeted delivery of miR-15a inhibitor decreases angiogenesis and tumor growth in a CT26 murine colorectal carcinoma model. Taken together, our findings highlight a novel role for miR mediated regulation of SMPD1 during radiation responses and establish proof-of-concept that this pathway can be targeted with a miR inhibitor.

Sphingomyelin phosphodiesterase 3 methylation and silencing in oral squamous cell carcinoma results in increased migration and invasion and altered stress response.

Neutral sphingomyelinase 2 (nSMase2), the product of the sphingomyelin phosphodiesterase 3 (SMPD3) gene, catalyzes the hydrolysis of sphingomyelin to ceramide. Ceramide acts on various signaling pathways to influence cell proliferation, survival, and stress response. Altered levels of sphingolipids and ceramides have been reported in various cancer types, including oral squamous cell carcinoma (OSCC). OSCC patients exhibit a poor 5-year survival rate of 50%, a figure that has remained stagnant for decades. To overcome this requires a better understanding of the molecular events driving this disease. The molecular analysis of the oral cavity reported here has identified the SMPD3 promoter region as a site of frequent hypermethylation and downregulation in pre-malignant and malignant tissues as compared with healthy control tissues. While lentivirus-induced overexpression of SMPD3 in cell models of oral dysplasia and OSCC did not significantly alter proliferation, it did decrease migration and invasion and increased resistance to the epidermal growth factor receptor (EGFR) inhibitor erlotinib. These results suggest that SMPD3 downregulation is a common event in OSCC progression and may promote the spread of tumor cells.