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Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.
Sphingosine 1-phosphate (S1P) is a lipid mediator with numerous biological functions. The term ‘S1P’ mainly refers to the sphingolipid molecule with a long-chain sphingoid base of 18 carbon atoms, d18:1 S1P. The enzyme serine palmitoyltransferase catalyses the first step of the sphingolipid de novo synthesis using palmitoyl-CoA as the main substrate. After further reaction steps, d18:1 S1P is generated. However, also stearyl-CoA or myristoyl-CoA can be utilised by the serine palmitoyltransferase, which at the end of the S1P synthesis pathway, results in the production of d20:1 S1P and d16:1 S1P respectively. We measured these S1P homologues in mice and renal tissue of patients suffering from renal cell carcinoma (RCC). Our experiments highlight the relevance of d16:1 S1P for the induction of connective tissue growth factor (CTGF) in the human renal clear cell carcinoma cell line A498 and human RCC tissue. We show that d16:1 S1P versus d18:1 and d20:1 S1P leads to the highest CTGF induction in A498 cells via S1P2 signalling and that both d16:1 S1P and CTGF levels are elevated in RCC compared to adjacent healthy tissue. Our data indicate that d16:1 S1P modulates conventional S1P signalling by acting as a more potent agonist at the S1P2 receptor than d18:1 S1P. We suggest that elevated plasma levels of d16:1 S1P might play a pro-carcinogenic role in the development of RCC via CTGF induction.
To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.
Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.
Sphingosine 1 phosphate (S1P) lyase (Sgpl1) catalyses the irreversible cleavage of S1P and thereby the last step of sphingolipid degradation. Loss of Sgpl1 in humans and mice leads to accumulation of sphingolipids and multiple organ injuries. Here, we addressed the role of hepatocyte Sgpl1 for regulation of sphingolipid homoeostasis by generating mice with hepatocyte-specific deletion of Sgpl1 (Sgpl1HepKO mice). Sgpl1HepKO mice had normal body weight, liver weight, liver structure and liver enzymes both at the age of 8 weeks and 8 months. S1P, sphingosine and ceramides, but not glucosylceramides or sphingomyelin, were elevated by ~1.5–2-fold in liver, and this phenotype did not progress with age. Several ceramides were elevated in plasma, while plasma S1P was normal. Interestingly, S1P and glucosylceramides, but not ceramides, were elevated in bile of Sgpl1HepKO mice. Furthermore, liver cholesterol was elevated, while LDL cholesterol decreased in 8-month-old mice. In agreement, the LDL receptor was upregulated, suggesting enhanced uptake of LDL cholesterol. Expression of peroxisome proliferator-activated receptor-γ, liver X receptor and fatty acid synthase was unaltered. These data show that mouse hepatocytes largely compensate the loss of Sgpl1 by secretion of accumulating sphingolipids in a specific manner into blood and bile, so that they can be excreted or degraded elsewhere.
Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.
Genes encoding endocannabinoid and sphingolipid metabolism pathways were suggested to contribute to the genetic risk towards attention deficit hyperactivity disorder (ADHD). The present pilot study assessed plasma concentrations of candidate endocannabinoids, sphingolipids and ceramides in individuals with adult ADHD in comparison with healthy controls and patients with affective disorders. Targeted lipid analyses of 23 different lipid species were performed in 71 mental disorder patients and 98 healthy controls (HC). The patients were diagnosed with adult ADHD (n = 12), affective disorder (major depression, MD n = 16 or bipolar disorder, BD n = 6) or adult ADHD with comorbid affective disorders (n = 37). Canonical discriminant analysis and CHAID analyses were used to identify major components that predicted the diagnostic group. ADHD patients had increased plasma concentrations of sphingosine-1-phosphate (S1P d18:1) and sphinganine-1-phosphate (S1P d18:0). In addition, the endocannabinoids, anandamide (AEA) and arachidonoylglycerol were increased. MD/BD patients had increased long chain ceramides, most prominently Cer22:0, but low endocannabinoids in contrast to ADHD patients. Patients with ADHD and comorbid affective disorders displayed increased S1P d18:1 and increased Cer22:0, but the individual lipid levels were lower than in the non-comorbid disorders. Sphingolipid profiles differ between patients suffering from ADHD and affective disorders, with overlapping patterns in comorbid patients. The S1P d18:1 to Cer22:0 ratio may constitute a diagnostic or prognostic tool.
Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood–brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
Progranulin deficiency in mice is associated with deregulations of the scavenger receptor signaling of CD36/SCARB3 in immune disease models, and CD36 is a dominant receptor in taste bud cells in the tongue and contributes to the sensation of dietary fats. Progranulin-deficient mice (Grn−/−) are moderately overweight during middle age. We therefore asked if there was a connection between progranulin/CD36 in the tongue and fat taste preferences. By using unbiased behavioral analyses in IntelliCages and Phenomaster cages we showed that progranulin-deficient mice (Grn−/−) developed a strong preference of fat taste in the form of 2% milk over 0.3% milk, and for diluted MCTs versus tap water. The fat preference in the 7d-IntelliCage observation period caused an increase of 10% in the body weight of Grn−/− mice, which did not occur in the wildtype controls. CD36 expression in taste buds was reduced in Grn−/− mice at RNA and histology levels. There were no differences in the plasma or tongue lipids of various classes including sphingolipids, ceramides and endocannabinoids. The data suggest that progranulin deficiency leads to a lower expression of CD36 in the tongue resulting in a stronger urge for fatty taste and fatty nutrition.