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Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates cell proliferation, survival and migration and plays an essential role in angiogenesis and lymphocyte trafficking. S1P levels in the circulation and tissues are tightly regulated for proper cell functioning, and dysregulation of this system may contribute to the pathophysiology of certain human diseases. Sphingosine phosphate lyase (SPL) irreversibly degrades S1P and thereby acts as a gatekeeper that regulates S1P signaling by modulating intracellular S1P levels and the chemical S1P gradient that exists between lymphoid organs and circulating blood and lymph. However, SPL also generates biochemical products that may be relevant in human disease. SPL has been directly implicated in various physiological and pathological processes, including cell stress responses, cancer, immunity, hematopoietic function, muscle homeostasis, inflammation and development.
This review will summarize the current knowledge of SPL structure, function, regulation, its involvement in various disease states, and currently available small molecules known to modulate SPL activity.
This review provides he evidence that SPL presents itself as a potential target for pharmacological manipulation for the treatment of malignant, autoimmune, inflammatory and other diseases.
Sphingosine 1-phosphate (S1P) is a bioactive metabolite of sphingolipid metabolism. S1P has been shown to promote cell survival, proliferation and migration, ischemia preconditioning and plays an essential role in angiogenesis and lymphocyte trafficking1–3. S1P exerts its functions either by acting as an intracellular second messenger4 or by activating its cognate G protein-coupled receptors (GPCR). Five GPCRs that respond specifically to S1P ligand have been identified (designated S1P1 to S1P5) and they show differential expression patterns depending on tissue and cell type 5, 6. In mammals, S1P1, S1P2, and S1P3 are expressed ubiquitously, whereas S1P4 is restricted to lymphoid tissues and lung and S1P5, to brain and skin. Activation of S1P receptors stimulates downstream signaling pathways, including those involving mitogen activated protein kinases (MAPK), phosphoinositide-3 kinase, cyclic AMP and other downstream mediators6, 7. The effects of S1P signaling through these receptors include cytoskeletal rearrangements, cell proliferation and migration, invasion, vascular development, platelet aggregation, and lymphocyte trafficking1,8. In contrast to the well characterized receptor-mediated effects of S1P, much less is known about the intracellular actions of S1P. It has been suggested that intracellular actions of S1P can regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli in a receptor-independent manner1, 4. Because of the importance of S1P, considerable interest has developed in understanding the function and regulation of the enzymes involved in its metabolism.
S1P is formed from sphingosine in a reaction catalyzed by sphingosine kinases (Sphk)9, 10. Once formed, S1P phosphatases and nonspecific lipid hydrolases can dephosphorylate S1P11. Alternatively, S1P can be irreversibly degraded by sphingosine phosphate lyase (SPL), which catalyzes the cleavage of the carbon chain at positions 2–3, leading to the formation of a long-chain aldehyde (hexadecenal) and phosphoethanolamine 12–14. SPL plays an important role in development, reproduction, cell survival, stress responses, the maintenance of tissue integrity, lymphocyte trafficking and tumorigenesis15–22. Here, we summarize the current understanding of SPL function, regulation and its role in physiology and disease. We further hypothesize how SPL might serve as a pharmacological target for cancer therapy, immune modulation and therapeutic intervention for other diseases.
SPL catalyzes the final step of sphingolipid catabolism. The substrates of SPL are phosphorylated long chain bases that include phosphorylated sphingosine, dihydrosphingosine, sphingadiene, and phytosphingosine as well as dihydrosphingosine-1-phosphonate. SPL shows specificity towards the D(+)-erythro isomer of its substrates23. SPL is a pyridoxal 5′-phosphate-dependent enzyme and its lysine residue (Lys-353) is an active site that forms an internal Schiff base with pyridoxal 5′-phosphate, facilitating the cleavage of S1P to hexadecenal and phosphoethanolamine24. Dysregulation of paracrine and autocrine S1P signaling through its receptors, as well as potentially receptor-independent effects of intracellular S1P and abnormal accumulation of other sphingolipid intermediates appear to be responsible for many of the effects associated with SPL inhibition, as described at length below (Figure 1). However, product depletion may also be a consideration. For example, a lack of phosphoethanolamine production was accountable for the lack of viability and differentiation of Leishmania major spl-mutants, as the supplementation of ethanolamine completely reversed these defects22. The products of the SPL reaction can also serve as precursors to phospholipid metabolism25, and products of the SPL reaction were found to promote F9 embryonal carcinoma cell proliferation through an S1P-independent mechanism26. Therefore, SPL exerts effects on pathophysiological processes through mechanisms involving both the substrate and products of the reaction.
The first SPL gene to be cloned was DPL1 (dihydrosphingosine phosphate lyase). Its identification in yeast facilitated the identification of functional SPL homologs within in the genomes of Caenorhabditis elegans, Dictyostelium discoideum, Leishmania major, Drosophila melanogaster, Mus musculus, Homo sapiens and recently in the plants12, 13, 16, 17, 22, 27, 28.
The human SPL gene, sphingosine phosphate lyase 1 (Sgpl1), encodes a protein of 568 amino acids with a predicted molecular mass of 63.5 kDa14. Human SPL displays 84% amino acid identity and 91% similarity to the mouse ortholog. Immunofluorescence and subcellular fractionation studies revealed that SPL is predominantly localized within the endoplasmic reticulum (ER)18, 23, 24. Based on structural modeling and biochemical characterization, SPL is predicted to be a single-pass type III membrane protein. SPL contains one transmembrane segment close to the N-terminus. Its N-terminal domain resides in the ER lumen, whereas its large C-terminal domain which contains the catalytic site is exposed to the cytosol14. Several conserved amino acid residues have been mapped to the C-terminal domain of human SPL, including a 20 amino-acid stretch spanning positions 344–364, which contains predicted cofactor binding lysine residues at position K353 and K359. Site-directed mutagenesis of human SPL revealed the importance of two conserved cysteine residues at positions C218 and C317, which are required for proper enzymatic activity14. Interestingly, recombinant SPL lacking the transmembrane domain and the ER luminal sequence was fully active in vitro when expressed in bacteria14. Conversely, a mutagenesis study conducted on the yeast ortholog Dpl1p revealed the important role of the N-terminal domain29. The luminal domain of Dpl1p appears to be required to maintain protein stability, and a Dpl1p NΔ57 mutant lacking the entire luminal domain was completely inactive when evaluated using an in vivo complementation assay in yeast29. Dpl1p has been shown to form higher order complexes that are required for its in vivo function. Polar amino acid residues in the transmembrane domain of Dpl1p play an important role in protein oligomerization29.
SPL is expressed in many mammalian tissues to variable degrees. In mice and rats, SPL activity/expression is highest in the small intestine, colon, thymus, spleen, and harderian gland, whereas moderate expression is observed in liver, kidney, lung, stomach and testis. SPL expression is lowest in the heart, skeletal muscle and brain, with the exception of the olfactory mucosal epithelium, where the enzyme is highly enriched14, 30 (Our unpublished observations). SPL expression is low in lymphocytes and absent in erythrocytes and platelets, which are major sources of plasma S1P31, 32. SPL is expressed in some inflammatory cells, but its activity in macrophages, monocytes, dendritic cells and neutrophils has not been characterized to date.
SPL expression seems to be high in tissues characterized by rapid cell turnover. This is exemplified by the pronounced expression and activity of SPL in intestinal epithelial cells, which are renewed every 12 hours. High SPL expression in intestinal epithelial cells suggests that it may play an important role in catabolizing dietary sphingolipids33. SPL may also be required to maintain low S1P levels in the cells at the villus tips, facilitating cell death and tissue turnover in response oxidative stress and as a mechanism of gut immunity. SPL expression is high in the olfactory mucosa, a unique neuronal tissue that is subject to high rates of apoptosis due to inhaled toxic-induced cell damage. Interestingly, olfactory mucosa is unique among adult neuronal tissues for its ability to sustain continuous neurogenesis30. The high expression of SPL in the thymus might be required to maintain low tissue S1P levels compared to the surrounding plasma. The S1P concentration gradient between thymus and the plasma enables T cell egress from thymus into the circulation20.
There is evidence that SPL activity is regulated at multiple levels, including epigenetic, transcriptional and post-translational. GATA family transcription factors were shown to regulate SPL expression in nematode and human cells at the transcriptional level34. SPL was also identified as an immediate early gene and transcriptional target of platelet-derived growth factor (PDGF)35. PDGF signaling plays a role in embryonic development, cell proliferation, cell migration and angiogenesis and has been linked to several diseases including atherosclerosis, fibrosis and tumorigenesis36. Furthermore, an SPL knockout mouse demonstrated phenotypes consistent with PDGF signaling abnormalities, including vascular developmental abnormalities, skeletal defects, and defects of cell migration37. Analysis of the 5′ flanking region of the SPL gene suggests that other mechanisms of transcriptional control may be involved in the regulation of SPL expression. In addition, Sgpl1 has also been identified as a microRNA (mir-125b) target in prostate cancer cells38. A comparative genomics approach to the analysis of immune system genes identified common cis-elements, including early growth response factor, zinc-binding protein factor, and GC-box factor motifs in the promoters of Sgpl1 and other thymic-enriched genes39. Sgpl1 was also shown along with other pro-apoptotic genes to be a downstream target of mSin3A, a core component of a co-repressor complex involved in embryonic development and in regulating cellular functions such as cell cycle progression, DNA replication, DNA repair, apoptosis and mitochondrial metabolism 40.
In addition to transcriptional regulation, post-translational regulation of human SPL has also been suggested by the finding of SPL in a screen for nitrosylated proteins. SPL is predicted to be nitrosylated on tyrosine residues Y356 and Y36641. By altering the charge of the tyrosine residues, nitrosylation may interfere with enzyme-substrate binding. Multiple phosphorylation sites are also predicted by protein sequence analysis, but these findings have not been verified experimentally42. A recent study has demonstrated that Dpl1p forms higher order complexes, and oligomerization of Dpl1p is required for its in vivo function29. However, to date formation of high order complexes by human SPL has not been tested.
The involvement of S1P in diverse essential cellular processes necessitates precise control and regulation of its intracellular and extracellular levels. The importance of SPL in modulating the levels of S1P, other sphingolipid intermediates and cell fate is likely to contribute to tissue homeostasis. Alternatively, its dysregulation could contribute to the pathophysiology of disease (Figure 2). In support of this notion, genetically modified mice lacking SPL expression in all tissues demonstrate stunted growth and early mortality with defects reported in the kidney, bone and vasculature37, as well as myeloid cell hyperplasia, and significant lesions in the lung, heart, urinary tract43. The potential role of SPL in specific disease states is described below.
Sphingolipids regulate various aspects of cell growth, proliferation, and cell death44. Ceramide, the backbone of all higher order sphingolipids from which S1P derives, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. Ceramide generation is considered to be a key mechanism by which chemotherapeutic agents induce apoptosis in cancer cells. On the other hand, S1P promotes cell growth, migration, tumor angiogenesis, invasion and metastasis. Consistent with their key roles in the regulation of cancer growth and therapy, genetic changes acquired by malignant cells that result in either a reduction in ceramide generation and/or an accentuation of S1P generation are implicated in the development of resistance to drug-induced apoptosis and escape from cell death45. A dynamic balance between ceramide and S1P is, thus, maintained within the cells, contributing to the determination of cellular outcome in response to stress. SPL has the ability to shift the balance towards cell death by attenuating the proliferative S1P signal.
A potential role of SPL in regulating cell fate and stress responses has been investigated in several model systems. For example, an insertional mutagenesis study revealed that mutations in the Dictyostelium sglA gene, which encodes SPL, confer resistance to the anticancer drug cisplatin46. Conversely, overexpression of SPL enhanced the sensitivity of Dictyostelium cells to the drug47. SPL has also been shown to sensitize mammalian cells to various stressful stimuli including serum starvation and DNA damage. Enforced expression of SPL in malignant and non-transformed human cells sensitizes these cells to platinum-based chemotherapy drugs (cisplatin and carboplatin), daunorubicin and etoposide18, 21, 48. The ability of SPL to potentiate cell death requires the actions of p53 and p38 MAPK signaling pathways. Conversely, knockdown of endogenous SPL expression in HEK293 cells by siRNA results in diminished apoptosis in etoposide-treated cells21. Furthermore, elevation in Sgpl1 mRNA levels has been associated with testicular degeneration caused by increased apoptosis due to leptin deficiency in mice49. Conversely, SPL deficiency can also lead to cell death, as evidenced by increased apoptosis observed in the reproductive organs of Drosophila Sply null mutant50. The sphingolipid metabolite responsible for this effect is unknown, although these tissues show a profound accumulation of long-chain bases including Δ4,6-sphingadienes, which are found to promote apoptosis in Drosophila cell lines19,51. Interestingly, it has been proposed that accumulation of intracellular S1P above a certain threshold can also induce apoptosis52. Cerebellar granule neurons isolated from SPL-deficient mice showed a similar elevation in the intracellular S1P levels upon treatment with either sphingosine or exogenous S1P. However, only S1P addition to the cells induces apoptosis52.
Consistent with a role for S1P in various processes associated with tumorigenesis, the genes regulating S1P metabolism are altered in variety of human cancers44, 53. Sphingosine kinase 1 (SK1), which encodes the main enzyme responsible for S1P synthesis, functions as an oncogene54 and is regulated in a variety of solid tumors, such as colorectal, breast, stomach, lung, ovary, uterus and kidney 55, 56. SPL expression and activity are downregulated during intestinal tumorigenesis in the ApcMin/+ mouse model of intestinal tumorigenesis, as well as in human colon cancer specimen compared with normal adjacent tissues21. Alterations of Sgpl1 expression in several types of cancers have been demonstrated by cDNA microarrays. Sgpl1 is repressed during myc-mediated β-cell tumorigenesis in mice and is re-expressed upon Myc deactivation57. Moreover, Myc binds to the Sgpl1 promoter in Burkitt’s lymphoma cells and negatively regulates its expression58. Fatty acid synthase (FAS) is upregulated in a wide variety of cancers, and is considered a potential metabolic oncogene by virtue of its ability to enhance tumor cell survival in certain cancers, especially those with the lipogenic phenotype59. Knockdown of FAS in mammary carcinoma cells upregulates the expression of Sgpl160. Furthermore, Sgpl1 is among a set of genes downregulated in metastatic tumor tissues compared to primary tumors from the same patients61. These findings suggest that SPL may act as tumor suppressor and may be involved in cancer surveillance pathways. Presumably SPL would act by preventing intracellular S1P accumulation, whereas loss of SPL expression or activity might promote tumorigenesis through activation of S1P-mediated signaling. Although, the role of SPL in tumor suppression has not been established in vivo, such a notion is consistent with a study wherein administration of a monoclonal S1P antibody substantially reduced tumor progression and angiogenesis in murine xenograft and allograft models62. However, upregulation of SPL expression has been observed in ovarian cancer63. Sgpl1 was also identified in a group of genes whose expression is upregulated in ovarian tumors that were resistant to chemotherapy64. Lysophospholipids, including S1P and lysophosphatidic acid, are elevated in malignant ovarian tissues and malignant ascites and stimulate tumor cell proliferation65. Whether induction of SPL is a causative factor or, conversely, a manifestation of tissue responses to high levels of lysophospholipids remain to be determined. Interestingly, S1P signaling is radioprotective to oocytes and ovarian function and has been proposed as a therapeutic strategy for preventing reproductive failure in cancer patients66, 67.
Future studies are warranted to address whether SPL expression is causally related to intestinal tumorigenesis and other forms of neoplasia. If SPL downregulation in intestinal adenomas is a reversible phenomenon, it might be exploited as an adjuvant approach to enhance the therapeutic response of tumor cells to DNA-damaging agents. Conversely, SPL might also be a target for pharmacological intervention to transiently raise S1P levels as a way of protecting reproductive organs and potentially other organ functions in patients receiving radio- and chemotherapy66.
S1P signaling is essential for immune cell trafficking and plays a role in immune surveillance, cytokine secretion, immune cell differentiation and allergic responses68,7. Importantly, a recent study demonstrated that the immune system is exquisitely sensitive to partial loss of SPL activity, suggesting that activation of the S1P pathway is more stringently controlled in the immune system than the non-lymphoid environment43. Interest in the physiological role of S1P signaling in the immune system developed in last decade after it was recognized that the potent immunosuppressive drug FTY720 functions as a sphingosine analog69. Currently, FTY720 is under clinical trials for the treatment of allograft rejection in renal transplant patients and for multiple sclerosis. FTY720 is rapidly phosphorylated in vivo by Sphk270,71, and the phosphorylated compound acts as a ligand for four of the five known S1P receptors69. Although FTY720-phosphate is an S1P1 receptor agonist, chronic exposure to the drug induces internalization and degradation of the S1P1 receptor, resulting in functional antagonism8. FTY720 treatment deprives thymocytes and lymphocytes of an S1P signal that stimulates their egress from thymus and secondary lymphoid tissues8. The majority of circulating lymphocytes are, thus, sequestered in lymph nodes, causing lymphopenia. FTY720 has been shown to inhibit SPL activity, which raises the possibility that SPL inhibition might contribute to the immunological effects of the drug72.
S1P levels in the blood and lymph are high (0.1–1.0 μM) compared with those in most tissues, which maintain baseline levels in the range of 0.5 to 75 pmol/mg2. This difference establishes an S1P concentration gradient between circulatory fluids (lymph and blood) and lymphoid organs, which is tightly maintained by SPL activity in the tissues. This S1P gradient is essential for lymphocyte egress from lymphoid organs20. Inhibition of SPL activity by a food colorant tetrahydroxybutylimidazole (THI) or by reducing hematopoietic cell SPL expression through RNA interference-mediated knockdown prevents lymphocyte egress from thymus and secondary lymphoid organs20. Inhibition of SPL by THI raised the bioavailable S1P levels ~100–1000-fold in thymus and secondary lymphoid organs without altering plasma S1P levels, thereby causing a disruption of the S1P gradient. The critical role of SPL in lymphocyte trafficking was recently confirmed in a study showing that genetically modified mice lacking Sgpl1 expression exhibit lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes43. Furthermore, humanized knock-in mice lacking murine Sgpl1 expression but harboring one or two alleles of human Sgpl1 also exhibited immune defects. Replacement of the human SPL gene in the Sgpl1 null background resulted in SPL expression at 10–20% of normal mouse SPL levels, yet failed to restore normal T-cell development and trafficking. These data suggest that immune functions including lymphocyte trafficking are exquisitely sensitive to alterations in SPL activity43.
Apart from its role lymphocyte trafficking, S1P signaling plays a role in cell proliferation, survival and differentiation of lymphocytes7. S1P treatment enhances the survival of B and T cells73, inhibits both homoeostatic proliferation and T-cell receptor-induced proliferation of T cells 74, and inhibits cytokine production 75. Importantly, it has recently been demonstrated that genetic ablation of Sgpl1 in mice hampers B and T cell development in addition to lymphocyte egress43. Sgpl1 knockout (Sgpl1−/−) and humanized knock-in mice harboring one human allele (Sgpl1H/−) mice showed severe hypocellularity and increased apoptosis of lymphocytes within the thymic cortex, as well as a paucity of T-cells in the splenic periarteriolar lymphoid sheaths and paracortical areas of lymph nodes. It was suggested that excess tissue S1P levels in Sgpl1−/− mice may interfere with lymphocyte-stromal interactions. SPL deficiency also caused the vacuolization of thymic epithelial and stromal cells required for T-cell selection and maturation43.
Multiple sclerosis (MS) is an autoimmune condition in which autoreactive T-cells migrate across the blood-brain barrier and attack myelin sheaths, leading to demyelination. Clinical manifestations include visual loss, extra-ocular movement disorders, paresthesias, loss of sensation, weakness, dysarthria, spasticity, ataxia, and bladder dysfunction. The usual pattern is one of recurrent attacks followed by partial recovery but acute fulminating and chronic progressive forms also occur76. Based on the observations in the SPL knockout mouse models, targeting SPL to reduce T-cell circulation might be an effective treatment for MS and other autoimmune diseases. Many studies have shown that FTY720 is highly effective in animal models of MS77, 78, and phase II clinical trials in patients with relapsing MS are encouraging79. Patients with relapsed MS who were treated FTY720 showed reduced number of gadolinium-enhanced lesions detected on magnetic resonance imaging scan. Moreover, 70% of patients with MS, treated with FTY720 were relapse-free even after three years, showing the advantage over current treatments for MS that reduce the relapse rate by only 30%. In addition, only transient and relatively mild side effects such as nasopharyngitis, dyspnea, headache, diarrhea, and nausea were observed79. Thus, strategies targeting SPL may provide alternative or adjuvant therapy for MS and other autoimmune diseases.
Type 1 diabetes is a Th1-mediated autoimmune disease that results in destruction of pancreatic β cells. S1P and its analogs show protective effects on islet β-cells against cytokine-induced cell death80. Treatment of a nonobese diabetic (NOD) mouse model of autoimmune type 1 diabetes with FTY720 has been shown to either prevent the onset of diabetes or cure spontaneous diabetes81. In addition, CD4+ T-cells from diabetic NOD mice are highly activated and secrete twofold more ineterferon-γ and interleukin-17 than nondiabetic lymphocytes. S1P or a S1P1 receptor agonist inhibit CD4+ T-cell activation through regulation of hypoxia-inducible factor 1 α short isoform, resulting in decreased cytokine production82. The role of SPL in type I diabetes has not been investigated but could potentially be a therapeutic target in this widespread disease.
There is evidence that S1P signaling plays a role in inflammation, particularly in relation to secretion and function of pro-inflammatory cytokines TNF-α and interleukin-1. SphK1 is activated by TNF-α stimulation in monocytes and contributes to subsequent intracellular signaling, degranulation, cytokine production and activation of nuclear factor-κB in these cells83. Furthermore, knockdown studies revealed that both S1P and SphK1 are required for TNF-α-mediated induction of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and activation of endothelial nitric oxide synthase84. Concomitantly, knockdown of SPL or S1P phosphatase augmented COX-2 induction, leading to enhanced generation of PGE2. The S1P1 receptor was found to be expressed in rheumatoid arthritis (RA) synovium, and S1P signaling enhanced synoviocyte proliferation85. Inhibition of SphK1 by N, N-dimethylsphingosine reduced the levels of inflammatory cytokines in peripheral blood mononuclear cells from RA patients and inhibited disease severity, articular inflammation and joint destruction in a murine model of RA85. An anti-inflammatory function of S1P has also been reported86, wherein pretreatment of mouse macrophages inhibited LPS-induced cytokine production. Expression of SPL in lymphocytes is low, but other cells in inflammatory infiltrates express SPL21. This raises the possibility that SPL might regulate S1P levels and inflammatory cell functions.
Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease often associated with autoimmune diseases. Increased Sgpl1 expression has been reported in the skin of patients affected by AD87. This finding might be related to inflammatory changes in the skin associated with the disease. Alternatively, changes in sphingolipid metabolism, including SPL upregulation, might be responsible for a breakdown in the skin barrier function, because the stratum corneum is enriched in ceramides that provide much of the barrier function of the epidermis88. Interestingly, we have found significant expression Sgpl1 in the sebaceous glands of normal murine skin (our unpublished results). Migration of Langerhans cells from the skin to the lymph node is an essential step in the pathogenesis of allergic contact dermatitis. Therefore, inhibition of Langerhans cells-migration could be a promising strategy to improve AD. Interestingly, in a murine model of the disease, topical application of S1P and FTY720 inhibited the inflammatory reaction in the elicitation phase allergic contact dermatitis through inhibition of dendritic cell migration89. These studies suggest that dysregulation of the S1P/SPL axis could be important in mediating multiple aspects of this disease and, conversely, may serve as an avenue for therapeutic intervention.
S1P signaling through S1P1 receptor is essential for vascular maturation during embryogenesis and contributes to pathophysiology of vascular diseases such as atherosclerosis, vascular permeability and tumor angiogenesis90. Platelets were widely considered as the major source of plasma S1P levels31. However, recent findings show that erythrocytes and vascular endothelium are also major contributors of plasma S1P32,91. Moreover, laminar shear stress downregulates the expression of SPL and S1P phosphatase in vascular endothelial cells. Furthermore, knockdown or overexpression of Sgpl1 expression in endothelial cells directly affects intracellular S1P levels and subsequent release into the extracellular compartment, implicating a role for SPL in maintenance of vascular homeostasis91.
Disruption of vascular barrier integrity increases permeability to fluid and solutes and is the central pathophysiologic mechanism of many inflammatory disease processes, including inflammation, atherogenesis, sepsis and acute lung injury92. S1P regulates endothelial barrier function by activating S1P1 receptor which triggers intracellular signaling that promotes adherans junction integrity and cytoskeleton organization93. Treatment of mice with an S1P1 receptor antagonist compromises vascular integrity in the skin and lungs94. In addition, administration of intravenous S1P reduces acute lung injury caused by combined intrabronchial endotoxin administration or high tidal volume mechanical ventilation95. Furthermore, Sphk1 plays a crucial role in regulating endothelial barrier function in lungs96. In comparison to wild-type mice, Sphk1-deficient mice show markedly enhanced pulmonary edema formation in response to LPS and protease-activating receptor-1 activation, and endothelial barrier function is restored upon exogenous S1P treatment96. Conversely, alveolar epithelial cells express S1P3 receptors, and airway administration of S1P disrupts epithelial cell tight junctions, leading to pulmonary edema97. Interestingly, SPL-deficient mice showed lesions in lungs and alveoli that contained variable sized irregularly shaped polygonal flakes and clumps of smooth homogenous material accompanied by increased numbers of alveolar macrophages43. It was postulated that respiratory distress caused by pulmonary lesions was the cause of the reduced life span of SPL knockout mice. It might be possible that in the absence of SPL, high levels of extracellular S1P may play a direct role in the development of pulmonary lesions by compromising the integrity of the pulmonary epithelial barrier43, 97.
Angiogenesis and vascular maturation are pivotal for wound healing. When tissues are wounded, damaged blood vessels recruit and activate platelets that release soluble mediators of vascular dilatation, permeability and cell proliferation. Endothelial cells also participate in wound healing through enhancement of cell proliferation, blood coagulation and angiogenesis98. Diabetes impairs wound healing by impairing neo-vascularization, migration and proliferation of endothelial, keratinocytes and fibroblasts99. Treatment of diabetic mice with S1P accelerated cutaneous wound healing100. In addition, S1P2 receptor is essential for wound healing response to acute liver injury101. Furthermore, embryonic fibroblast cells from SPL null mice demonstrate defects in migration in vitro37, 102. These findings suggest the possibility that SPL could be a useful therapeutic target for diabetic wound healing.
There is growing evidence that S1P regulates skeletal muscle differentiation and regeneration, myoblastic cell contraction and survival of cardiac myocyte103, 104. Striated muscle possesses the full enzymatic machinery to generate S1P and expresses the transcripts of S1P receptors105. Further, S1P acts as a trophic factor of skeletal muscle106. Drosophila Sply mutants lacking SPL accumulate sphingolipid intermediates and develop a progressive myopathy in the thoracic muscles needed to power flight17, 19. Although Sgpl1 expression in the skeletal muscle is low at baseline, its expression is increased 3-fold in the gastrocnemius dystrophin-deficient mice107. These studies suggest that SPL expression may be dynamically regulated in muscle and predict a role for SPL in muscle regeneration.
Cardiac myocytes predominantly express S1P1 receptors, and activation of this receptor inhibits cAMP formation and antagonizes adrenergic receptor-mediated contractibility105. Expression of S1P3 receptors is low on cardiac myocytes. These receptors were shown to be responsible for mediating the bradycardic effect of S1P agonists108, 109. Cardiac smooth muscle cells express S1P2 receptors, activation of which can induce coronary vasoconstriction110. Intriguingly, levels of circulatory S1P are abnormally high in patients with coronary artery disease111. Conversely, activation of S1P signaling plays a cardioprotective role in response to acute ischemia/perfusion injury3, 112. Sphk1 knockout mice demonstrate increased ischemia/reperfusion injury and respond poorly to ischemic pre- and post-conditioning 3, 113. However, the role of SPL in the cardiovascular system is largely unknown. SPL null mice developed cardiac lesions characterized by patchy to diffuse expansion of the interstitium by numerous variably sized vacuoles and vacuolated mesenchymal cells separated by cadiomyocytes43. The physiological consequence of these changes has not been determined, but these findings suggest that SPL expression is important in cardiac tissue, even though baseline expression is very low. As in skeletal muscle, SPL may be induced in response to injury, a concept that will require further investigation.
Inappropriate induction of apoptosis has been proposed to underlie the progressive neuronal attrition associated with various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and other neurological disorders characterized by the gradual loss of specific populations of neurons. Alterations in sphingolipid metabolism have been reported in Alzheimer’s disease patients114, 115. Accumulation of ceramide and reduction in S1P levels in the brains of Alzheimers’s patients shift sphingolipid metabolism in the direction of cell death. Consistent with this is the finding of upregulation in the mRNA expression levels of S1P degrading enzymes, SPL and phosphatidic acid phosphatase type 2B and ceramide synthase in the different regions of brain of Alzheimer’s disease patients114. Additionally, upregulation of SPL expression was correlated with the progression of clinical dementia114. Furthermore, two single nucleotide polymorphisms were detected in the Sgpl1 gene in late-onset Alzheimer’s disease patients, which suggests that variation in Sgpl1 expression and/or function may confer susceptibility to late-onset Alzheimer’s disease116.
Modulation of SPL activity through the use of small molecule inhibitors appears to hold promise for therapeutic purposes, particularly for immunomodulation in the treatment of autoimmune disorders and for transplantation, as well as for enhancement of wound healing and protection of normal tissues from various insults such as radiation, hypoxia and ischemia. Different pharmacological categories of SPL inhibitors have been described. First are the substrate analogs, including 1-desoxydihydrosphingosine-1-phosphonate117 and the 2D,3L-isomer of DHS1P and 2-vinyldihydrosphingosine-1-phosphate117, 118. The second group of SPL inhibitors includes pyridoxal 5′-phosphate analogs or compounds that inhibit the binding of the cofactor. Deoxypyridoxine acts as a competitive inhibitor of the enzyme and semicarbazide, cyanide, and bisulfite also inhibit the enzyme activity. The ceramide analog and dihydroceramide desaturase inhibitor N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide (GT11) also inhibits SPL activity, but it appears to do so only in vivo, suggesting that a metabolite of the compound may be the active inhibitor119. FTY720 is a modest inhibitor of SPL activity both in vivo and in vitro72. New and more specific agonists and antagonists of S1P receptor subtypes are also being developed, but their effects on SPL activity have not been reported. THI is a caramel food colorant that has been shown to inhibit SPL activity in vivo, and inhibition of the enzyme by THI causes potent immunomodulatory effects20. Finally, SPL activity is also inhibited by divalent metal ions such as Ca2+ and Zn2+ 23.
FTY720 has shown promise in preventing rejection with both standard and reduced cyclosporine exposure. However, the results of large multicenter clinical trials with FTY720 in renal transplant patients have been disappointing120–122. Phase III clinical trial studies not only failed to show an advantage of FTY720 over standard immunosuppressive drugs but also demonstrate lower creatine clearance and increased risk of macular edema. FTY720 was associated with higher incidence of bradycardia and respiratory disorders122. Thus, there remains a need for additional approaches to modulating S1P-mediated effects on the immune system for therapeutic purposes. Despite the number of small molecules reported to inhibit SPL, none is specific and non-cytotoxic. However, Lexicon Pharmaceutical, Inc., The Woodlands, Texas, has developed an orally-delivered small molecule inhibitor of SPL, LX2931, which has completed Phase I clinical trial for the treatment of rheumatoid arthritis (Lexicon Pharmaceuticals, Inc. http://www.lexicon-genetics.com/pipeline/lx2931-lx2932.html). Preclinical studies with LX2931 showed a consistent reduction in circulating lymphocyte counts in multiple species. In addition, LX2931 reduced joint inflammation and prevented arthritic destruction of joints in mouse and rat models of arthritis 123, 124. These findings strongly suggest that SPL inhibitors could be a powerful addition to the arsenal of immunomodulatory therapy for autoimmune diseases.
Alterations of S1P signaling and metabolism induce marked effects on various organ systems. S1P exerts its effects either by activating its cognate receptors or by initiating intracellular signaling pathways. There is growing evidence that S1P plays an important role in angiogenesis, immune function, inflammatory responses, wound healing, muscle regeneration, tumor invasion and metastasis. S1P levels in tissues and in the circulation are tightly maintained, as would be expected of a potent signaling molecule with such diverse functions. SPL appears to be an important gatekeeper for regulating sphingolipid homeostasis and S1P signaling.
SPL can exert its effects on physiological processes by depleting its substrate (S1P) or by forming the products (phosphoethanolamine and hexadecenal). The fate and importance of the trans-2-hexadecenal and phosphoethanolamine produced in SPL reaction are not well understood. Phosphoethanolamine produced in the SPL reaction regulates viability and differentiation of Leishmania major. Thus, SPL may be a target for anti-leishmanial antibiotic therapy. In addition, products of SPL reaction have shown to promote mammalian cell proliferation. However, the product responsible for these effects is not known. Due to the reactivity of the trans-2-hexadecenal, it is possible that it may react with other cellular components, including DNA and proteins and influence cellular function and may contribute to the some of the effects of SPL upregulation. Studies addressing this will be important next steps in understanding the full impact of SPL on physiology and disease.
Future studies should also address whether SPL downregulation is causally related to intestinal tumorigenesis and other forms of neoplasia, and whether mutations of SPL are present in cancer specimens. Tumorigenesis studies with SPL null mice have been limited due to their reduced life span. The development of tissue-specific knockout mice and cell lines with inducible SPL expression or knockdown would provide an invaluable set of models with which to directly address whether SPL is an anti-oncogene. If SPL downregulation in cancer is a reversible phenomenon, it might be exploited as an adjuvant approach to enhance the therapeutic response of tumor cells to DNA-damaging agents. Conversely, SPL blockade might be used to transiently raise S1P levels in tissues for a variety of therapeutic purposes, such as for protecting reproductive function in patients that receive cytotoxic therapy and radiation, to enhance wound healing or stimulate heart muscle survival after ischemic insult.
SPL holds great promise as a therapeutic target for designing novel immunosuppressant drug for the prevention of allograft rejection, multiple sclerosis and treatment of T-lymphocyte-driven inflammatory skin diseases, such as lupus erythematosus, psoriasis, and atopic dermatitis. Clinical trials with LX2931 for rheumatoid arthritis appear promising. Identifying additional regulatory mechanisms that control SPL expression might reveal novel ways to pharmacologically reactivate this enzyme. Mechanisms responsible for transcriptional and posttranslational control of SPL are beginning to emerge. It is noteworthy that Sgpl1 has been identified as a target for a microRNA mir-125b in prostate cancer cells. While this remains to be validated, microRNAs present a unique small molecule approach to regulating gene expression for therapeutic purposes, especially in cancer. Similarly, postranslational modifications such as phosphorylation, nitrosylation and glycosylation of SPL are intriguing possibilities for pharmacological modulation. The topology and the catalytic site of the enzyme have been determined, but its tertiary structure has not been fully elucidated. Studies of SPL structure including its crystallization should expand our understanding of the molecular requirements for enzyme activity, facilitating the design of more specific and potent inhibitors. Our current knowledge of SPL’s expression patterns and the phenotypes associated with its pharmacological modulation or genetic deletion in rodents, human pathogens and model organisms together illustrate the important role of SPL in physiology and its potential impact upon human disease. Advances in the molecular and biochemical understanding of SPL regulation and function should lead to the development of novel therapeutic strategies against human cancers, immunological disorders, vascular and neurodegenerative diseases.
This work was supported by National Institutes of Health grants CA77528, CA129438 and GM66594 (JDS)