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Emilie Degagné, Julie D Saba Children's Hospital Oakland Research Institute, Oakland, CA, USA Abstract: Inflammatory bowel disease (IBD) is a complex disease that involves unpredictable and destructive inflammation in the gastrointestinal tract resulting in gastrointestinal symptoms, infection, and tissue destruction, and which can be associated with an increased risk of colon cancer. The underlying cause of IBD involves disruption of the innate and adaptive immune mechanisms that maintain homeostasis between the gut mucosa and its environment. Elucidating how the homeostatic mechanisms controlling gut mucosal immunity and inflammation are disrupted in IBD represents the first steps to identifying novel therapeutic targets. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is enriched in the blood and lymph, and functions in innate and adaptive immunity. S1P signaling regulates inflammation via its impact on the trafficking, differentiation, and effector functions of bone marrow-derived immune cells. S1P also activates nuclear factor kappa B and signal transducer and activator of transcription 3 inflammatory pathways. S1P is generated by the ubiquitously expressed lipid kinase, sphingosine kinase (SphK)1 and its tissue-restricted homolog, SphK2. S1P is irreversibly degraded by S1P lyase, which is highly expressed in enterocytes. Recent studies targeting S1P metabolism and signaling have shown promise in preclinical models of IBD and have shed light on the mechanisms by which S1P signaling impacts IBD. The evidence suggests that targeting S1P signaling and metabolism may represent a novel strategy in treating IBD and it may reduce colon cancer risk by interrupting the progression from inflammation to carcinogenesis. Keywords: sphingolipids, sphingadienes, sphingosine phosphate lyase, sphingosine kinase, STAT3, S1PR

Sphingosine phosphate lyase insufficiency syndrome (SPLIS) is a genetic disease associated with renal, endocrine, neurological, skin and immune defects. SPLIS is caused by inactivating mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). SPL catalyzes the irreversible degradation of the bioactive sphingolipid sphingosine-1-phosphate (S1P), a key regulator of lymphocyte egress. The SPL reaction represents the only exit point of sphingolipid metabolism, and SPL insufficiency causes widespread sphingolipid derangements that could additionally contribute to immunodeficiency. Herein, we review SPLIS, the sphingolipid metabolic pathway, and various roles sphingolipids play in immunity. We then explore SPLIS-related immunodeficiency by analyzing data available in the published literature supplemented by medical record reviews in ten SPLIS children. We found 93% of evaluable SPLIS patients had documented evidence of immunodeficiency. Many of the remainder of cases were unevaluable due to lack of available immunological data. Most commonly, SPLIS patients exhibited lymphopenia and T cell-specific lymphopenia, consistent with the established role of the S1P/S1P1/SPL axis in lymphocyte egress. However, low B and NK cell counts, hypogammaglobulinemia, and opportunistic infections with bacterial, viral and fungal pathogens were observed. Diminished responses to childhood vaccinations were less frequently observed. Screening blood tests quantifying recent thymic emigrants identified some lymphopenic SPLIS patients in the newborn period. Lymphopenia has been reported to improve after cofactor supplementation in some SPLIS patients, indicating upregulation of SPL activity. A variety of treatments including immunoglobulin replacement, prophylactic antimicrobials and special preparation of blood products prior to transfusion have been employed in SPLIS. The diverse immune consequences in SPLIS patients suggest that aberrant S1P signaling may not fully explain the extent of immunodeficiency. Further study will be required to fully elucidate the complex mechanisms underlying SPLIS immunodeficiency and determine the most effective prophylaxis against infection., Competing Interests: Declaration of competing interest JDS is co-founder of Sphinxion Therapeutics Inc., which is developing gene therapy for individuals with SPLIS. JDS is an author on patent International Application Serial No. PCT/US2021/018613, “Adeno-Associated Viral (Aav)-Mediated SGPL1 Gene Therapy for Treatment of Sphingosine-1-Phosphate Lyase Insufficiency Syndrome (SPLIS)” published on 08/26/2021 and assigned publication number WO 2021/168140. All other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a recently recognized inborn error of metabolism associated with steroid-resistant nephrotic syndrome as well as adrenal insufficiency and immunological, neurological, and skin manifestations. SPLIS is caused by inactivating mutations in SGPL1, encoding the pyridoxal 5'phosphate-dependent enzyme sphingosine-1-phosphate lyase, which catalyzes the final step of sphingolipid metabolism. Some SPLIS patients have undergone kidney transplantation, and others have been treated with vitamin B6 supplementation. In addition, targeted therapies including gene therapy are in preclinical development. In anticipation of clinical trials, it will be essential to characterize the full spectrum and natural history of SPLIS. We performed a retrospective analysis of 76 patients in whom the diagnosis of SPLIS was established in a proband with at least one suggestive finding and biallelic SGPL1 variants identified by molecular genetic testing. The main objective of the study was to identify factors influencing survival in SPLIS subjects., Results: Overall survival at last report was 50%. Major influences on survival included: (1) age and organ involvement at first presentation; (2) receiving a kidney transplant, and (3) SGPL1 genotype. Among 48 SPLIS patients with nephropathy who had not received a kidney transplant, two clinical subgroups were distinguished. Of children diagnosed with SPLIS nephropathy before age one (n = 30), less than 30% were alive 2 years after diagnosis, and 17% were living at last report. Among those diagnosed at or after age one (n = 18), ~ 70% were alive 2 years after diagnosis, and 72% were living at time of last report. SPLIS patients homozygous for the SPL R222Q variant survived longer compared to patients with other genotypes. Kidney transplantation significantly extended survival outcomes., Conclusion: Our results demonstrate that SPLIS is a phenotypically heterogeneous condition. We find that patients diagnosed with SPLIS nephropathy in the first year of life and patients presenting with prenatal findings represent two high-risk subgroups, whereas patients harboring the R222Q SGPL1 variant fare better than the rest. Time to progression from onset of proteinuria to end stage kidney disease varies from less than one month to five years, and kidney transplantation may be lifesaving., (© 2024. The Author(s).)

Despite efforts to understand breast cancer biology, metastatic disease remains a clinical challenge. Identifying suppressors of breast cancer progression and mechanisms of transition to more invasive phenotypes could provide game changing therapeutic opportunities. Transcriptional deregulation is central to all malignancies, highlighted by the extensive reprogramming of regulatory elements that underlie oncogenic programs. Among these, super-enhancers (SEs) stand out due to their enrichment in genes controlling cancer hallmarks. To reveal novel breast cancer dependencies, we integrated the analysis of the SE landscape with master regulator activity inference for a series of breast cancer cell lines. As a result, we identified T - h elper-inducing Poxviruses and Zinc-finger ( PO Z)/ K rüppel-like factor (ThPOK, ZBTB7B ), a CD4 + cell lineage commitment factor, as a breast cancer master regulator that is recurrently associated with a SE. ThPOK expression is highest in luminal breast cancer but is significantly reduced in the basal subtype. Manipulation of ThPOK levels in cell lines shows that its repressive function restricts breast cancer cells to an epithelial phenotype by suppressing the expression of genes involved in the epithelial-mesenchymal transition (EMT), WNT/β-catenin target genes, and the pro-metastatic TGFβ pathway. Our study reveals ThPOK as a master transcription factor that restricts the acquisition of metastatic features in breast cancer cells.

Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-βH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Neuroblastoma is the most common extracranial malignant tumor of childhood, accounting for 15% of all pediatric cancer deaths. Despite significant advances in our understanding of neuroblastoma biology, five-year survival rates for high-risk disease remain less than 50%, highlighting the importance of identifying novel therapeutic targets to combat the disease. MYCN amplification is the most frequent and predictive molecular aberration correlating with poor outcome in neuroblastoma. N-Myc is a short-lived protein primarily due to its rapid proteasomal degradation, a potentially exploitable vulnerability in neuroblastoma. AF1q is an oncoprotein with established roles in leukemia and solid tumor progression. It is normally expressed in brain and sympathetic neurons and has been postulated to play a part in neural differentiation. However, no role for AF1q in tumors of neural origin has been reported. In this study, we found AF1q to be a universal marker of neuroblastoma tumors. Silencing AF1q in neuroblastoma cells caused proteasomal degradation of N-Myc through Ras/ERK and AKT/GSK3β pathways, activated p53 and blocked cell cycle progression, culminating in cell death via the intrinsic apoptotic pathway. Moreover, silencing AF1q attenuated neuroblastoma tumorigenicity in vivo signifying AF1q's importance in neuroblastoma oncogenesis. Our findings reveal AF1q to be a novel regulator of N-Myc and potential therapeutic target in neuroblastoma., (© 2024. The Author(s).)

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is an inborn error of metabolism caused by inactivating mutations in SGPL1 , the gene encoding sphingosine-1-phosphate lyase (SPL), an essential enzyme needed to degrade sphingolipids. SPLIS features include glomerulosclerosis, adrenal insufficiency, neurological defects, ichthyosis, and immune deficiency. Currently, there is no cure for SPLIS, and severely affected patients often die in the first years of life. We reported that adeno-associated virus (AAV) 9-mediated SGPL1 gene therapy (AAV-SPL) given to newborn Sgpl1 knockout mice that model SPLIS and die in the first few weeks of life prolonged their survival to 4.5 months and prevented or delayed the onset of SPLIS phenotypes. In this study, we tested the efficacy of a modified AAV-SPL, which we call AAV-SPL 2.0, in which the original cytomegalovirus (CMV) promoter driving the transgene is replaced with the synthetic "CAG" promoter used in several clinically approved gene therapy agents. AAV-SPL 2.0 infection of human embryonic kidney (HEK) cells led to 30% higher SPL expression and enzyme activity compared to AAV-SPL. Newborn Sgpl1 knockout mice receiving AAV-SPL 2.0 survived ≥ 5 months and showed normal neurodevelopment, 85% of normal weight gain over the first four months, and delayed onset of proteinuria. Over time, treated mice developed nephrosis and glomerulosclerosis, which likely resulted in their demise. Our overall findings show that AAV-SPL 2.0 performs equal to or better than AAV-SPL. However, improved kidney targeting may be necessary to achieve maximally optimized gene therapy as a potentially lifesaving SPLIS treatment.

Background: Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) or nephrotic syndrome type-14 is caused by biallelic mutations in SGPL1. Here, we conducted a systematic review to delineate the characteristics of SPLIS patients., Methods: A literature search was performed in PubMed, Web of Science, and Scopus databases, and eligible studies were included. For all patients, demographic, clinical, laboratory, and molecular data were collected and analyzed., Results: Fifty-five SPLIS patients (54.9% male, 45.1% female) were identified in 19 articles. Parental consanguinity and positive family history were reported in 70.9% and 52.7% of patients, respectively. Most patients (54.9%) primarily manifested within the first year of life, nearly half of whom survived, while all patients with a prenatal diagnosis of SPLIS (27.5%) died at a median [interquartile (IQR)] age of 2 (1.4-5.3) months (P = 0.003). The most prevalent clinical feature was endocrinopathies, including primary adrenal insufficiency (PAI) (71.2%) and hypothyroidism (32.7%). Kidney disorders (42, 80.8%) were mainly in the form of steroid-resistant nephrotic syndrome (SRNS) and progressed to end-stage kidney disease (ESKD) in 19 (36.5%) patients at a median (IQR) age of 6 (1.4-42.6) months. Among 30 different mutations in SGPL1, the most common was c.665G > A (p.Arg222Gln) in 11 (20%) patients. Twenty-six (49.1%) patients with available outcome were deceased at a median (IQR) age of 5 (1.5-30.5) months, mostly following ESKD (23%) or sepsis/septic shock (23%)., Conclusion: In patients with PAI and/or SRNS, SGPL1 should be added to diagnostic genetic panels, which can provide an earlier diagnosis of SPLIS and prevention of ESKD and other life-threatening complications., (© 2022. Children's Hospital, Zhejiang University School of Medicine.)

In 2017, an inborn error of metabolism caused by recessive mutations in SGPL1 was discovered. The disease features steroid-resistant nephrotic syndrome, adrenal insufficiency, and neurological defects. The latter can include sensorineural hearing loss, cranial nerve defects, peripheral neuropathy, abnormal brain development, seizures and/or neurodegeneration. SGPL1 encodes the pyridoxal-5'-phosphate (PLP) dependent enzyme sphingosine phosphate lyase (SPL), and the condition is now referred to as SPL insufficiency syndrome (SPLIS). SPL catalyzes the final step in the degradative pathway of sphingolipids in which the bioactive sphingolipid sphingosine-1-phosphate (S1P) is irreversibly degraded to a long chain aldehyde and phosphoethanolamine (PE). SPL guards the only exit point for sphingolipid metabolism, and its inactivation leads to accumulation of various types of sphingolipids which have biophysical roles in plasma membrane rafts and myelin, and signaling roles in cell cycle progression, vesicular trafficking, cell migration, and programmed cell death. In addition, the products of the SPL reaction have biological functions including regulation of autophagic flux, which is important in axonal and neuronal integrity. In this review, the neurological manifestations of SPLIS will be described, and insights regarding the neurological consequences of SPL insufficiency from the study of brain-specific SPL knockout mice and Drosophila SPL mutants will be summarized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Atreya and Saba.)

Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.

Sphingosine 1-phosphate lyase (SPL) is a critical component of sphingosine 1-phosphate (S1P) metabolism. SPL has been associated with several crucial cellular functions due to its role in S1P metabolism, but its role in viral infections is poorly understood. Studies show that SPL has an antiviral function against influenza A virus (IAV) by interacting with IKKɛ, promoting the type I interferon (IFN) innate immune response to IAV infection. However, a more recent study has revealed that IAV NS1 protein hampers this by triggering ubiquitination and subsequent degradation of SPL, which reduces the type I IFN innate immune response. In this study, we describe SPL, the type I IFN response, and known interactions between SPL and IAV.

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 ( Sgpl1 HepKO mice). Sgpl1 HepKO 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 Sgpl1 HepKO 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.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by a deficiency in sphingosine-1-phosphate lyase (SPL), the final enzyme in the sphingolipid degradative pathway. Inactivating mutations of SGPL1-the gene encoding SPL-lead to a deficiency of its downstream products, and buildup of sphingolipid intermediates, including its bioactive substrate, sphingosine-1-phosphate (S1P), the latter causing lymphopenia, a hallmark of the disease. Other manifestations of SPLIS include nephrotic syndrome, neuronal defects, and adrenal insufficiency, but their pathogenesis remains unknown. In this report, we describe the correlation between SGPL1 genotypes, age at diagnosis, and patient outcome. Vitamin B6 serves as a cofactor for SPL. B6 supplementation may aid some SPLIS patients by overcoming poor binding kinetics and promoting proper folding and stability of mutant SPL proteins. However, this approach remains limited to patients with a susceptible allele. Gene therapy represents a potential targeted therapy for SPLIS patients harboring B6-unresponsive missense mutations, truncations, deletions, and splice-site mutations. When Sgpl1 knockout (SPL KO ) mice that model SPLIS were treated with adeno-associated virus (AAV)-mediated SGPL1 gene therapy, they showed profound improvement in survival and kidney and neurological function compared to untreated SPL KO mice. Thus, gene therapy appears promising as a universal, potentially curative treatment for SPLIS., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in sphingosine-1-phosphate lyase 1 (SGPL1), which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first 2 years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1-KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated proinflammatory and profibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared with untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis, and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.

SGPL1 encodes sphingosine-1-phosphate lyase, the final enzyme of sphingolipid metabolism. In 2017, a condition featuring steroid-resistant nephrotic syndrome and/or adrenal insufficiency associated with pathogenic SGPL1 variants was reported. In addition to the main features of the disease, patients often exhibit a range of neurologic deficits. In a handful of cases, brain imaging results were described. However, high-quality imaging results and a systematic analysis of brain MR imaging findings associated with the condition are lacking. In this study, MR images from 4 new patients and additional published case reports were reviewed by a pediatric neuroradiologist. Analysis reveals recurring patterns of features in affected patients, including isolated callosal dysgenesis and prominent involvement of the globus pallidus, thalamus, and dentate nucleus, with progressive atrophy and worsening of brain lesions. MR imaging findings of abnormal deep gray nuclei, microcephaly, or callosal dysgenesis in an infant or young child exhibiting other typical clinical features of sphingosine-1-phosphate lyase insufficiency syndrome should trigger prompt genetic testing for SGPL1 mutations., (© 2020 by American Journal of Neuroradiology.)

Sphingosine-1-phosphate (S1P) lyase is a vitamin B6-dependent enzyme that degrades sphingosine-1-phosphate in the final step of sphingolipid metabolism. In 2017, a new inherited disorder was described caused by mutations in SGPL1, which encodes sphingosine phosphate lyase (SPL). This condition is referred to as SPL insufficiency syndrome (SPLIS) or alternatively as nephrotic syndrome type 14 (NPHS14). Patients with SPLIS exhibit lymphopenia, nephrosis, adrenal insufficiency, and/or neurological defects. No targeted therapy for SPLIS has been reported. Vitamin B6 supplementation has therapeutic activity in some genetic diseases involving B6-dependent enzymes, a finding ascribed largely to the vitamin's chaperone function. We investigated whether B6 supplementation might have activity in SPLIS patients. We retrospectively monitored responses of disease biomarkers in patients supplemented with B6 and measured SPL activity and sphingolipids in B6-treated patient-derived fibroblasts. In two patients, disease biomarkers responded to B6 supplementation. S1P abundance and activity levels increased and sphingolipids decreased in response to B6. One responsive patient is homozygous for an SPL R222Q variant present in almost 30% of SPLIS patients. Molecular modeling suggests the variant distorts the dimer interface which could be overcome by cofactor supplementation. We demonstrate the first potential targeted therapy for SPLIS and suggest that 30% of SPLIS patients might respond to cofactor supplementation., (© 2020 SSIEM.)

Sphingosine phosphate lyase (SPL) is the final enzyme in the sphingolipid degradative pathway, catalyzing the irreversible cleavage of long-chain base phosphates (LCBPs) to yield a long-chain aldehyde and ethanolamine phosphate (EP). SPL guards the sole exit point of sphingolipid metabolism. Its inactivation causes product depletion and accumulation of upstream sphingolipid intermediates. The main substrate of the reaction, sphingosine-1-phosphate (S1P), is a bioactive lipid that controls immune-cell trafficking, angiogenesis, cell transformation, and other fundamental processes. The products of the SPL reaction contribute to phospholipid biosynthesis and programmed cell-death activation. The main features of SPL enzyme activity were first described in detail by Stoffel et al. in 1969. The first SPL-encoding gene was cloned from budding yeast in 1997. Reverse and forward genetic strategies led to the rapid identification of other genes in the pathway and their homologs in other species. Genetic manipulation of SPL-encoding genes in model organisms has revealed the contribution of sphingolipid metabolism to development, physiology, and host-pathogen interactions. In 2017, recessive mutations in the human SPL gene SGPL1 were identified as the cause of a novel inborn error of metabolism associated with nephrosis, endocrine defects, immunodeficiency, acanthosis, and neurological problems. We refer to this condition as SPL insufficiency syndrome (SPLIS). Here, we share our perspective on the 50-year history of SPL from discovery to disease, focusing on insights provided by model organisms regarding the pathophysiology of SPLIS and how SPLIS raises the possibility of a hidden role for sphingolipids in other disease conditions., (Copyright © 2019 Saba.)

Sphingosine-1-phosphate lyase (SPL) is an intracellular enzyme that controls the final step in the sphingolipid degradative pathway, the only biochemical pathway for removal of sphingolipids. Specifically, SPL catalyzes the cleavage of sphingosine 1-phosphate (S1P) at the C2-3 carbon bond, resulting in its irreversible degradation to phosphoethanolamine (PE) and hexadecenal. The substrate of the reaction, S1P, is a bioactive sphingolipid metabolite that signals through a family of five G protein-coupled S1P receptors (S1PRs) to mediate biological activities including cell migration, cell survival/death/proliferation and cell extrusion, thereby contributing to development, physiological functions and - when improperly regulated - the pathophysiology of disease. In 2017, several groups including ours reported a novel childhood syndrome that featured a wide range of presentations including fetal hydrops, steroid-resistant nephrotic syndrome (SRNS), primary adrenal insufficiency (PAI), rapid or insidious neurological deterioration, immunodeficiency, acanthosis and endocrine abnormalities. In all cases, the disease was attributed to recessive mutations in the human SPL gene, SGPL1. We now refer to this condition as SPL Insufficiency Syndrome, or SPLIS. Some features of this new sphingolipidosis were predicted by the reported phenotypes of Sgpl1 homozygous null mice that serve as vertebrate SPLIS disease models. However, other SPLIS features reveal previously unrecognized roles for SPL in human physiology. In this review, we briefly summarize the biochemistry, functions and regulation of SPL, the main clinical and biochemical features of SPLIS and what is known about the pathophysiology of this condition from murine and cell models. Lastly, we consider potential therapeutic strategies for the treatment of SPLIS patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Neuroblastoma is a childhood malignancy that accounts for approximately 15% of childhood cancer deaths. Only 20-35% of children with metastatic neuroblastoma survive with standard therapy. Identification of more effective therapies is essential to improving the outcome of children with high-stage disease. Sphingadienes (SD) are growth-inhibitory sphingolipids found in natural sources including soy. They exhibit chemopreventive activity in mouse models of colon cancer, where they mediate cytotoxicity by inhibiting key pro-carcinogenic signaling pathways. In this study, the effect of SD on neuroblastoma was analyzed. Low micromolar concentrations of SD were cytotoxic to transformed and primary neuroblastoma cells independently of N-Myc amplification status. SD induced both caspase-dependent apoptosis and autophagy in neuroblastoma cells. However, only inhibition of caspase-dependent apoptosis protected neuroblastoma cells from SD-mediated cytotoxicity. SD also inhibited AKT activation in neuroblastoma cells as shown by reduced phosphorylated AKT levels. Pre-treatment with insulin attenuated SD-mediated cytotoxicity in vitro. SD-loaded nanoparticles (NP) administered parenterally to immunodeficient mice carrying neuroblastoma xenografts resulted in cytotoxic levels of SD in the circulation and significantly reduced tumor growth compared to vehicle-treated controls. Analysis of tumor extracts demonstrated reduced AKT activation in tumors of mice treated with SD-NP compared to controls treated with empty NP. Our findings indicate SD are novel potential chemotherapeutic agents that promote neuroblastoma cell death and reduce tumorigenicity in vivo.

Goal: The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population., Background: IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD., Methods: Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS., Results: Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls., Conclusions: A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

Increasing evidence suggests a crucial role of inflammation in cytokine-mediated β-cell dysfunction and death in type 1 diabetes mellitus, although the mechanisms are incompletely understood. Sphingosine 1-phosphate (S1P) is a multifunctional bioactive sphingolipid involved in the development of many autoimmune and inflammatory diseases. Here, we investigated the role of intracellular S1P in insulin-secreting INS1E cells by genetically manipulating the S1P-metabolizing enzyme S1P lyase (SPL). The expression of spl was down-regulated by cytokines in INS1E cells and rat islets. Overexpression of SPL protected against cytokine toxicity. Interestingly, the SPL overexpression did not suppress the cytokine-induced NFκB-iNOS-NO pathway but attenuated calcium leakage from endoplasmic reticulum (ER) stores as manifested by lower cytosolic calcium levels, higher expression of the ER protein Sec61a, decreased dephosphorylation of Bcl-2-associated death promoter (Bad) protein, and weaker caspase-3 activation in cytokine-treated (IL-1β, TNFα, and IFNγ) cells. This coincided with reduced cytokine-mediated ER stress, indicated by measurements of CCAAT/enhancer-binding protein homologous protein ( chop ) and immunoglobulin heavy chain binding protein ( bip ) levels. Moreover, cytokine-treated SPL-overexpressing cells exhibited increased expression of prohibitin 2 (Phb2), involved in the regulation of mitochondrial assembly and respiration. SPL-overexpressing cells were partially protected against cytokine-mediated ATP reduction and inhibition of glucose-induced insulin secretion. siRNA-mediated spl suppression resulted in effects opposite to those observed for SPL overexpression. Knockdown of phb2 partially reversed beneficial effects of SPL overexpression. In conclusion, the relatively low endogenous Spl expression level in insulin-secreting cells contributes to their extraordinary vulnerability to proinflammatory cytokine toxicity and may therefore represent a promising target for β-cell protection in type 1 diabetes mellitus., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Colorectal cancer (CRC) is a leading cause of cancer mortality. Diet has a significant influence on colon cancer risk. Identifying chemopreventive agents, dietary constituents, practices and/or diet supplements that promote gut health and reduce the incidence of intestinal neoplasias and CRC could significantly impact public health. Sphingadienes (SDs) are dietary sphingolipids found in plant-based food products. SDs are cytotoxic to colon cancer cells and exhibit chemopreventive properties. The aim of the present study was to develop a sensitive and robust ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantifying SDs in food products and biological samples. The assay was linear over a concentration range of 80nM to 50μM and was sensitive to a detection limit of 3.3nM. Post-extraction stability was 100% at 24h. SD content in soy oils was approximately 10nM. SDs were detected transiently in the plasma of adult mice 10min after gavage delivery of a 25mg/kg bolus and declined to baseline by 1h. SD uptake in the gut was maximal in the duodenum and peaked 1h after gavage delivery. Disappearance of SDs in the lower gastrointestinal tract suggests either rapid metabolism to yet unidentified products or potentially luminal export., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Macroautophagy/autophagy defects have been identified as critical factors underlying the pathogenesis of neurodegenerative diseases. The roles of the bioactive signaling lipid sphingosine-1-phosphate (S1P) and its catabolic enzyme SGPL1/SPL (sphingosine phosphate lyase 1) in autophagy are increasingly recognized. Here we provide in vitro and in vivo evidence for a previously unidentified route through which SGPL1 modulates autophagy in neurons. SGPL1 cleaves S1P into ethanolamine phosphate, which is directed toward the synthesis of phosphatidylethanolamine (PE) that anchors LC3-I to phagophore membranes in the form of LC3-II. In the brains of SGPL1 fl/fl/Nes mice with developmental neural specific SGPL1 ablation, we observed significantly reduced PE levels. Accordingly, alterations in basal and stimulated autophagy involving decreased conversion of LC3-I to LC3-II and increased BECN1/Beclin-1 and SQSTM1/p62 levels were apparent. Alterations were also noticed in downstream events of the autophagic-lysosomal pathway such as increased levels of lysosomal markers and aggregate-prone proteins such as APP (amyloid β [A4] precursor protein) and SNCA/α-synuclein. In vivo profound deficits in cognitive skills were observed. Genetic and pharmacological inhibition of SGPL1 in cultured neurons promoted these alterations, whereas addition of PE was sufficient to restore LC3-I to LC3-II conversion, and control levels of SQSTM1, APP and SNCA. Electron and immunofluorescence microscopy showed accumulation of unclosed phagophore-like structures, reduction of autolysosomes and altered distribution of LC3 in SGPL1 fl/fl/Nes brains. Experiments using EGFP-mRFP-LC3 provided further support for blockage of the autophagic flux at initiation stages upon SGPL1 deficiency due to PE paucity. These results emphasize a formerly overlooked direct role of SGPL1 in neuronal autophagy and assume significance in the context that autophagy modulators hold an enormous therapeutic potential in the treatment of neurodegenerative diseases.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1Δ yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS.

Sphingosine-1-phosphate (S1P) is a potent lipid signaling molecule that regulates pleiotropic biological functions including cell migration, survival, angiogenesis, immune cell trafficking, inflammation, and carcinogenesis. It acts as a ligand for a family of cell surface receptors. S1P concentrations are high in blood and lymph but low in tissues, especially the thymus and lymphoid organs. S1P chemotactic gradients are essential for lymphocyte egress and other aspects of physiological cell trafficking. S1P is irreversibly degraded by S1P lyase (SPL). SPL regulates lymphocyte trafficking, inflammation and other physiological and pathological processes. For example, SPL located in thymic dendritic cells acts as a metabolic gatekeeper that controls the normal egress of mature T lymphocytes from the thymus into the circulation, whereas SPL deficiency in gut epithelial cells promotes colitis and colitis-associated carcinogenesis (CAC). Recently, we identified a complex syndrome comprised of nephrosis, adrenal insufficiency, and immunological defects caused by inherited mutations in human SGPL1 , the gene encoding SPL. In the present article, we review current evidence supporting the role of SPL in thymic egress, inflammation, and cancer. Lastly, we summarize recent progress in understanding other SPL functions, its role in inherited disease, and SPL targeting for therapeutic purposes.

After undergoing positive and negative selection in the thymus, surviving mature T cells egress from the thymic parenchyma and enter the bloodstream to participate in adaptive immunity. Thymic egress requires signals mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that serves as the ligand for a family of G protein-coupled receptors (S1P1-5) expressed on many cell types, including T cells. In the final stage of their development, T cells upregulate S1P1 expression on the cell surface, which enables them to recognize and respond to a chemotactic S1P gradient that lures them into the bloodstream. The gradient is generated by an S1P source close to the site of egress combined with an S1P sink generated by the actions of S1P catabolic enzymes including S1P lyase (SPL), the only enzyme that irreversibly degrades S1P. The requisite contribution of SPL to thymic egress is demonstrated by the profound lymphopenia observed in SPL knockout (KO) mice and wild type mice treated with SPL inhibitors. SPL is robustly expressed in thymic epithelial cells (TECs), which make up the stromal reticular network of the thymus. However, TEC SPL was recently found to be dispensable for thymic egress. In contrast, deletion of SPL in dendritic cells (DCs) - which represent only a small percent of thymic stroma - disrupts the S1P gradient and blocks thymic egress. These recent observations identify DCs as homeostatic regulators of thymic export through the actions of SPL, thereby adding one more piece to the complex puzzle of how S1P signaling contributes to the regulation of T cell trafficking., Competing Interests: Conflict of Interest The author has no financial conflicts of interest related to this work.

T cell egress from the thymus is essential for adaptive immunity and involves chemotaxis along a sphingosine-1-phosphate (S1P) gradient. Pericytes at the corticomedullary junction produce the S1P egress signal, whereas thymic parenchymal S1P levels are kept low through S1P lyase (SPL)-mediated metabolism. Although SPL is robustly expressed in thymic epithelial cells (TECs), in this study, we show that deleting SPL in CD11c + dendritic cells (DCs), rather than TECs or other stromal cells, disrupts the S1P gradient, preventing egress. Adoptive transfer of peripheral wild-type DCs rescued the egress phenotype of DC-specific SPL knockout mice. These studies identify DCs as metabolic gatekeepers of thymic egress. Combined with their role as mediators of central tolerance, DCs are thus poised to provide homeostatic regulation of thymic export., (© 2016 Zamora-Pineda et al.)

A new technique for quantifying sphingosine phosphate lyase activity in biological samples is described. In this procedure, 2-hydrazinoquinoline is used to convert (2E)-hexadecenal into the corresponding hydrazone derivative to improve ionization efficiency and selectivity of detection. Combined utilization of liquid chromatographic separation and multiple reaction monitoring-mass spectrometry allows for simultaneous quantification of the substrate S1P and product (2E)-hexadecenal. Incorporation of (2E)- d5-hexadecenal as an internal standard improves detection accuracy and precision. A simple one-step derivatization procedure eliminates the need for further extractions. Limits of quantification for (2E)-hexadecenal and sphingosine-1-phosphate are 100 and 50fmol, respectively. The assay displays a wide dynamic detection range useful for detection of low basal sphingosine phosphate lyase activity in wild type cells, SPL-overexpressing cell lines, and wild type mouse tissues. Compared to current methods, the capacity for simultaneous detection of sphingosine-1-phosphate and (2E)-hexadecenal greatly improves the accuracy of results and shows excellent sensitivity and specificity for sphingosine phosphate lyase activity detection., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Colitis-associated colon cancer (CAC) is a pathological condition defined by the development of colon cancer in patients afflicted by Crohn's disease (CD) or ulcerative colitis (UC), two idiopathic diseases of the gut which together comprise the disease group called inflammatory bowel disease (IBD). When IBD involves the colon, affected patients face an increased risk of developing colon cancer compared to the general population. The phenomenon of CAC represents one of the most convincing forms of evidence linking the processes of inflammation, oxidative stress and carcinogenesis. A greater understanding of the molecular events driving CAC could reveal new strategies to treat IBD and reduce the incidence of CAC. Sphingosine-1-phosphate (S1P) is a bioactive lipid produced through degradation of endogenous and dietary mammalian sphingolipids containing the long chain base sphingosine. S1P signals through a family of five G protein-coupled receptors. In addition, it activates nuclear factor kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3), two transcriptional regulators that serve as master switches in inflammation and carcinogenesis. Through these and other mechanisms, a causal role for S1P in inflammatory conditions including colitis and CAC has been implicated. In contrast to S1P, dietary sphingolipids called sphingadienes derived from plant food sources cannot be converted to S1P and exhibit anti-inflammatory and chemopreventive activities, reducing colitis and CAC in mouse models. In this review, we summarize recent findings implicating S1P signaling and metabolism in the pathogenesis of IBD and CAC. The potential role of oxidative stress in modulating S1P is also discussed. Further, we propose the hypothesis that dietary sphingolipids may promote or prevent CAC depending on their ability to be converted to S1P.

Sphingosine-1-phosphate [S1P] is a potent bioactive sphingolipid molecule. In response to a stimulus, S1P is produced intracellularly by the action of two sphingosine kinases, and then it is exported to the extracellular environment or acts as an intracellular second messenger. S1P binds to its cognate G-protein coupled receptors, which are known as S1P receptors. There are five S1P receptors that have been identified in vertebrates. By activating S1P receptors, S1P controls a variety of physiological and pathological processes including cell migration, angiogenesis, vascular maturation, inflammation, and invasion, metastasis, and chemoresistance in cancer. S1P has emerged as a critical regulator of leukocyte migration and plays a central role in lymphocyte egress from the thymus and secondary lymphoid organs. In the current review article, we summarize the current understanding of the emigration of lymphocytes and other leukocytes from bone marrow, thymus and secondary lymphoid organs to the circulation, as well as the clinical implications of modulating the activity of the major S1P receptor, S1PR1.

Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL.

Signaling through the Hippo pathway controls major aspects of cell growth and proliferation. Focusing on the metabolic consequences of Hippo signaling, Mulvihill and colleagues in this issue of Chemistry & Biology employ a large scale, integrative approach and uncover downstream reorganization of cellular metabolism when the effector TAZ is upregulated, identifying new connections to lipid metabolism., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle differentiation, homeostasis, and satellite cell (SC) activation. By regulating S1P levels, SPL also controls SC recruitment and muscle regeneration, representing a potential therapeutic target for muscular dystrophy. We found that SPL is induced during myoblast differentiation. To investigate SPL's role in myogenesis at the cellular level, we generated and characterized a murine myoblast SPL-knockdown (SPL-KD) cell line lacking SPL. SPL-KD cells accumulated intracellular and extracellular S1P and failed to form myotubes under conditions that normally stimulate myogenic differentiation. Under differentiation conditions, SPL-KD cells also demonstrated delayed induction of 3 myogenic microRNAs (miRNAs), miR-1, miR-206, and miR-486. SPL-KD cells successfully differentiated when treated with an S1P1 agonist, S1P2 antagonist, and combination treatments, which also increased myogenic miRNA levels. SPL-KD cells transfected with mimics for miR-1 or miR-206 also overcame the differentiation block. Thus, we show for the first time that the S1P/SPL/S1P-receptor axis regulates the expression of a number of miRNAs, thereby contributing to myogenic differentiation.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P 2 expression. Inhibition of S1P 2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P 2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.

Sphingosine-1-phosphate lyase (SPL) catalyzes the degradation of sphingosine-1-phosphate (S1P), a bioactive lipid that controls cell proliferation, migration and survival. Mice lacking SPL expression exhibit developmental abnormalities, runting and death during the perinatal period, suggesting that SPL plays a role in mammalian development and adaptation to extrauterine life. We investigated the pattern of SPL expression in the mouse embryo and placenta from day 8 to day 18. Our findings reveal that SPL is expressed in the developing brain and neural tube, Rathke's pouch, first brachial arch, third brachial arch, optic stalk, midgut loops, and lung buds. Diffuse signal was high at E12, whereas a recognizable adult SPL pattern was evident by E15 and more intensely at E18, with strong expression in skin, nasal epithelium, intestinal epithelium, cartilage, thymus and pituitary gland. These findings suggest SPL may be involved in development of the mammalian central nervous system (CNS), anterior pituitary, trigeminal nerve, palate and facial bones, thymus and other organs. Our findings are consistent with the SPL expression pattern of the adult mouse and with congenital abnormalities observed in SPL mutant mice., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiological processes including angiogenesis, lymphocyte trafficking and development. In addition, S1P serves as a muscle trophic factor that enables efficient muscle regeneration. This is due in part to S1P's ability to activate quiescent muscle stem cells called satellite cells (SCs) that are needed for muscle repair. However, the molecular mechanism by which S1P activates SCs has not been well understood. Further, strategies for harnessing S1P signaling to recruit SCs for therapeutic benefit have been lacking. S1P is irreversibly catabolized by S1P lyase (SPL), a highly conserved enzyme that catalyzes the cleavage of S1P at carbon bond C(2-3), resulting in formation of hexadecenal and ethanolamine-phosphate. SPL enhances apoptosis through substrate- and product-dependent events, thereby regulating cellular responses to chemotherapy, radiation and ischemia. SPL is undetectable in resting murine skeletal muscle. However, we recently found that SPL is dynamically upregulated in skeletal muscle after injury. SPL upregulation occurred in the context of a tightly orchestrated genetic program that resulted in a transient S1P signal in response to muscle injury. S1P activated quiescent SCs via a sphingosine-1-phosphate receptor 2 (S1P2)/signal transducer and activator of transcription 3 (STAT3)-dependent pathway, thereby facilitating skeletal muscle regeneration. Mdx mice, which serve as a model for muscular dystrophy (MD), exhibited skeletal muscle SPL upregulation and S1P deficiency. Pharmacological SPL inhibition raised skeletal muscle S1P levels, enhanced SC recruitment and improved mdx skeletal muscle regeneration. These findings reveal how S1P can activate SCs and indicate that SPL suppression may provide a therapeutic strategy for myopathies. This article is part of a Special Issue entitled Advances in Lysophospholipid Research., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc(Min/+) mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active β-catenin and Wnt targets c-Myc and cyclin D1 in Apc(Min/+) mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear β-catenin localization and subsequent reduction in active-β-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3β in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3β using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-β-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc(Min/+) mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3β-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a β-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. These findings and the known chemistry of related α,β-unsaturated aldehydes raise the possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-β-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. Thus, our findings suggest that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Sphingosine-1-phosphate (S1P) is a bioactive lipid with diverse functions including the promotion of cell survival, proliferation and migration, as well as the regulation of angiogenesis, inflammation, immunity, vascular permeability and nuclear mechanisms that control gene transcription. S1P is derived from metabolism of ceramide, which itself has diverse and generally growth-inhibitory effects through its impact on downstream targets involved in regulation of apoptosis, senescence and cell cycle progression. Regulation of ceramide, S1P and the biochemical steps that modulate the balance and interconversion of these two lipids are major determinants of cell fate, a concept referred to as the "sphingolipid rheostat." There is abundant evidence that the sphingolipid rheostat plays a role in the origination, progression and drug resistance patterns of hematopoietic malignancies. The pathway has also been exploited to circumvent the problem of chemotherapy resistance in leukemia and lymphoma. Given the broad effects of sphingolipids, targeting multiple steps in the metabolic pathway may provide possible therapeutic avenues. However, new observations have revealed that sphingolipid signaling effects are more complex than previously recognized, requiring a revision of the sphingolipid rheostat model. Here, we summarize recent insights regarding the sphingolipid metabolic pathway and its role in hematopoietic malignancies.

A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.

The bioactive signaling molecule D-erythro-sphingosine-1-phosphate (S1P) is irreversibly degraded by the enzyme S1P lyase (SPL). The reaction of SPL with C18-S1P generates ethanolamine phosphate and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S1P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac1 and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S1P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes cardiomyocyte survival and contributes to ischemic preconditioning. S1P lyase (SPL) is a stress-activated enzyme responsible for irreversible S1P catabolism. We hypothesized that SPL contributes to oxidative stress by depleting S1P pools available for cardioprotective signaling. Accordingly, we evaluated SPL inhibition as a strategy for reducing cardiac ischemia-reperfusion (I/R) injury. We measured SPL expression and enzyme activity in murine hearts. Basal SPL activity was low in wild-type cardiac tissue but was activated in response to 50 min of ischemia (n = 5, P < 0.01). Hearts of heterozygous SPL knockout mice exhibited reduced SPL activity, elevated S1P levels, smaller infarct size, and increased functional recovery after I/R compared with littermate controls (n = 5, P < 0.01). The small molecule tetrahydroxybutylimidazole (THI) is a Federal Drug Administration-approved food additive that inhibits SPL. When given overnight at 25 mg/l in drinking water, THI raised S1P levels and reduced SPL activity (n = 5, P < 0.01). THI reduced infarct size and enhanced hemodynamic recovery in response to 50 min of ischemia and to 40 min of reperfusion in ex vivo hearts (n = 7, P < .01). These data correlated with an increase in MAP kinase-interacting serine/threonine kinase 1, eukaryotic translation initiation factor 4E, and ribosomal protein S6 phosphorylation levels after I/R, suggesting that SPL inhibition enhances protein translation. Pretreatment with an S1P₁ and S1P₃ receptor antagonist partially reversed the effects of THI. These results reveal, for the first time, that SPL is an ischemia-induced enzyme that can be targeted as a novel strategy for preventing cardiac I/R injury.

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.