Biomedical subjects

Sheldon Milstien

Publications and source records attributed to Sheldon Milstien.

At least 37 records · Page 2Linked to original sources

PubMed2004

Aminoacylase 1 is a sphingosine kinase 1-interacting protein.

Sphingosine kinase type 1 (SphK1) and its product sphingosine-1-phosphate have been shown to promote cell growth and inhibit apoptosis of tumor cells. In an effort to further understand the regulation of SphK1, we used a yeast two-hybrid screen to find SphK1-interacting proteins. One of these was identified as aminoacylase 1 (Acy1), a metalloenzyme that removes amide-linked acyl groups from amino acids and may play a role in regulating responses to oxidative stress. Both the C-terminal fragment found in the two-hybrid screen and full-length Acy1 co-immunoprecipitate with SphK1. Though both C-terminal and full-length proteins slightly reduce SphK1 activity measured in vitro, the C-terminal fragment inhibits while full-length Acy1 potentiates the effects of SphK1 on proliferation and apoptosis. Interestingly, Acy1 induces redistribution of SphK1 as observed by immunocytochemistry and subcellular fractionation. Collectively, our data suggest that Acy1 physically interacts with SphK1 and may influence its physiological functions.

Amidohydrolases↗

PubMed2004

Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis.

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF. To determine whether the inhibitory effect on EGF-induced migration was because of decreased S1P or increased ceramide as a consequence of acylation of increased sphingosine by ceramide synthase, we used fumonisin B1, a specific inhibitor of ceramide synthase. Although fumonisin B1 blocked ceramide production and increased sphingosine, it did not reverse the negative effect of SPP-1 expression on EGF- or S1P-induced chemotaxis. EGF activated the epidermal growth factor receptor to the same extent in SPP-1-expressing cells, yet ERK1/2 activation was impaired. In agreement, PD98059, an inhibitor of the ERK-activating enzyme MEK, decreased EGF-stimulated migration. We next examined the possibility that intracellularly generated S1P might be involved in activating a G protein-coupled S1P receptor important for EGF-directed migration. Treatment with pertussis toxin to inactivate Galpha(i) suppressed EGF-induced migration. Moreover, expression of regulator of G protein signaling 3, which inhibits S1P receptor signaling and completely prevented ERK1/2 activation mediated by S1P receptors, not only reduced migration toward S1P but also markedly reduced migration toward EGF. Collectively, these results suggest that metabolism of S1P by SPP-1 is important for EGF-directed cell migration.

Blotting, Western↗

PubMed2004

Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis.

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

Animals↗

PubMed2004

Modulation of adaptive immune responses by sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) has long been recognized as a mediator of a variety of cell functions. A growing body of evidence has accumulated demonstrating its role in cell migration and as a mediator of growth factor-induced events. In recent years, it has become apparent that S1P also mediates many cytokine and chemokine functions. Cells of the immune system function and migrate in response to a complex network of cytokines and chemokines, and the outcome is determined by the interplay of the effects of these molecules on the target cell. S1P may be a bona fide component of these networks and influence the responses of cells to these immune modulators.

Animals↗

PubMed2004

The fate of intravenously administered tetrahydrobiopterin and its implications for heterologous gene therapy of phenylketonuria.

Tetrahydrobiopterin (BH(4)) is a required cofactor for the enzymatic activity of phenylalanine hydroxylase (PAH) and is synthesized de novo from GTP in several tissues. Heterologous expression of PAH in tissues other than liver is a potential novel therapy for human phenylketonuria that is completely dependent upon BH(4) supply in the PAH-expressing tissue. Previous experiments with liver PAH-deficient transgenic mice that expressed PAH in skeletal muscle demonstrated transient correction of hyperphenylalaninemia only with hourly parenteral BH(4) administration. In this report, the fate of intravenously administered BH(4) is examined. The conclusions are that (1) BH(4) administered intravenously is rapidly taken up by liver and kidney, and (2) uptake of BH(4) into muscle is relatively low. The levels of BH(4) achieved in skeletal muscle following IV injection are only 10% of the amount expected were BH(4) freely and equally distributed across all tissues. The half-life of BH(4) in muscle is approximately 30 min, necessitating repeated injections to maintain muscle BH(4) content sufficient to support phenylalanine hydroxylation. The efficacy of heterologous muscle-directed gene therapy for the treatment of PKU will likely be limited by the BH(4) supply in PAH-expressing muscle.

Animals↗

PubMed2003

The immunosuppressant FTY720 is phosphorylated by sphingosine kinase type 2.

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.

Animals↗

PubMed2003

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner.

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.

Animals↗

PubMed2003

A specific ceramide kinase assay to measure cellular levels of ceramide.

Human ceramide kinase was recently cloned and characterized. Recombinant ceramide kinase is highly active and ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of ceramide levels in biological samples by conversion to ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol kinase method. Using recombinant ceramide kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain ceramides in cellular lipid extracts. This new ceramide kinase assay is more specific than the commonly used diacylglycerol kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol kinase which is usually present at higher concentrations than ceramide and can interfere with ceramide phosphorylation, is completely inactive with ceramide kinase. Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of ceramide kinase reactions.

Biological Assay↗

PubMed2003

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation, yet promotes growth and survival independent of G protein-coupled receptors.

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.

Animals↗

PubMed2003

Sphingosine kinase type 2 is a putative BH3-only protein that induces apoptosis.

There are two isoforms of sphingosine kinase (SphK) that catalyze the formation of sphingosine 1-phosphate, a potent sphingolipid mediator. Whereas SphK1 stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of sphingosine 1-phosphate receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-219, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

3T3 Cells↗

PubMed2003

Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity.

In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of tetrahydrobiopterin and restoration of eNOS enzymatic activity.

Animals↗

PubMed2003

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric vasculature of cirrhotic rats.

In cirrhosis, arterial vasodilation and the associated hemodynamic disturbances are most prominent in the mesenteric circulation, and its severity has been linked to bacterial translocation (BT) and endotoxemia. Synthesis of nitric oxide (NO), the main vasodilator implicated, is dependent on the essential cofactor tetrahydrobiopterin (BH(4)). The key enzyme involved in BH(4) synthesis is GTP-cyclohydrolase I (GTPCH-I), which is stimulated by endotoxin. Therefore, we investigated GTPCH-I activity and BH(4) biosynthesis in the mesenteric vasculature of cirrhotic rats with ascites, as well as their relationship with BT and endotoxemia, serum NO, and mean arterial pressure (MAP). GTPCH-I activity and BH(4) content in mesenteric vasculature was determined by high-performance liquid chromatography. BT was assessed by standard bacteriologic culture of mesenteric lymph nodes (MLNs). Serum endotoxin was measured by a kinetic turbidimetric limulus amebocyte lysate assay, and serum NO metabolite (NOx) concentrations were assessed by chemiluminescence. BT was associated with local lymphatic and systemic appearance of endotoxin and was accompanied by increases in serum NOx levels. GTPCH-I activity and BH(4) content in mesenteric vasculature were both increased in animals with BT and correlated significantly (r = 0.69, P <.01). Both GTPCH-I activity and BH(4) levels significantly correlated with serum endotoxin and NOx levels (r = 0.69 and 0.54, 0.81 and 0.53, P <.05). MAP (a marker of systemic vasodilatation) correlated with endotoxemia (r = 0.58, P <.03) and with GTPCH-I activity (r = 0.69, P <.01). In conclusion, in cirrhotic animals BT appears to lead to endotoxemia, stimulation of GTPCH-I, increased BH(4) synthesis, and further enhancement of vascular NO production that leads to aggravation of vasodilatation.

Animals↗

PubMed2003

Pleiotropic actions of sphingosine-1-phosphate.

Sphingosine-1-phosphate (SIP) is a bioactive sphingolipid metabolite that regulates diverse cellular responses including, growth, survival, cytoskeleton rearrangements and movement. SIP plays an important role during development, particularly in vascular maturation and has been implicated in pathophysiology of cancer, wound healing, and atherosclerosis. This review summarizes the evidence showing that signaling induced by SIP is complex and involves both intracellular and extracellular actions. The intracellular effects of SIP remain speculative awaiting the identification of specific targets whereas the extracellular effects of SIP are clearly mediated through the activation of five specific G protein coupled receptors, called S1P1-5. Recent studies demonstrate that intracellular generated SIP can act in a paracrine or autocrine manner to activate its cell surface receptors.

Apoptosis↗

PubMed2003

Sphingosine-1-phosphate: an enigmatic signalling lipid.

The evolutionarily conserved actions of the sphingolipid metabolite, sphingosine-1-phosphate (S1P), in yeast, plants and mammals have shown that it has important functions. In higher eukaryotes, S1P is the ligand for a family of five G-protein-coupled receptors. These S1P receptors are differentially expressed, coupled to various G proteins, and regulate angiogenesis, vascular maturation, cardiac development and immunity, and are important for directed cell movement.

Animals↗

PubMed2002

Sphingosine kinase, sphingosine-1-phosphate, and apoptosis.

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.

Animals↗

PubMed2002

Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells.

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha. SPHK1 also conferred a growth advantage, as determined by proliferation and growth in soft agar, which was estrogen dependent. While both ERK and Akt have been implicated in MCF-7 cell growth, SPHK1 stimulated ERK1/2 but had no effect on Akt. Surprisingly, parental growth of MCF-7 cells was only weakly stimulated by S1P or dihydro-S1P, ligands for the S1P receptors which usually mediate growth effects. When injected into mammary fat pads of ovariectomized nude mice implanted with estrogen pellets, MCF-7/SPHK1 cells formed more and larger tumors than vector transfectants with higher microvessel density in their periphery. Collectively, our results suggest that SPHK1 may play an important role in breast cancer progression by regulating tumor cell growth and survival.

Adenocarcinoma↗

PubMed2002

Sphingosine-1-phosphate: dual messenger functions.

The sphingolipid metabolite sphingosine-1-phosphate (S1P) is a serum-borne lipid that regulates many vital cellular processes. S1P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S1P. It has been suggested that S1P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S1P has been challenging, but there is ample evidence to suggest that the balance between S1P and ceramide and/or sphingosine levels in cells is an important determinant of cell fate.

Animals↗

PubMed2002

Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

3T3 Cells↗