Synthesis and biochemical investigation of scyphostatin analogues as inhibitors of neutral sphingomyelinase

The sphingolipid ceramide is considered to be an important intracellular mediator. However, many aspects of its action and the role of several different ceramide generating sphingomyelinases are still unclear. Recently, we reported on the synthesis of the first selective irreversible inhibitor of the neutral sphingomyelinase (N-SMase), as well as the identification of Manumycin A and some of its analogues as irreversible inhibitors of N-SMase. For the development of pharmacologically interesting competitive inhibitors of N-SMase, structure-activity studies are essential. Herein we show the synthesis and enzymatic investigation of two scyphostatin analogues 3a and 3b, revealing the importance of the primary hydroxy group in compound 2 for N-SMase inhibition.     

Transient mechanoactivation of neutral sphingomyelinase in caveolae to generate ceramide

The vascular endothelium acutely autoregulates blood flow in vivo in part through unknown mechanosensing mechanisms. Here, we report the discovery of a new acute mechanotransduction pathway. Hemodynamic stressors from increased vascular flow and pressure in situ rapidly and transiently induce the activity of neutral sphingomyelinase but not that acid sphingomyelinase in a time- and flow rate-dependent manner, followed by the generation of ceramides. This acute mechanoactivation occurs directly at the luminal endothelial cell surface primarily in caveolae enriched in sphingomyelin and neutral sphingomyelinase, but not acid sphingomyelinase. Scyphostatin, which specifically blocks neutral but not acid sphingomyelinase, inhibits mechano-induced neutral sphingomyelinase activity as well as downstream activation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) by increased flow in situ. We postulate a novel physiological function for neutral sphingomyelinase as a new mechanosensor initiating the ERK cascade and possibly other mechanotransduction pathways.     

Neutral sphingomyelinase: past, present and future

Sphingomyelin and its metabolic products are now known to have second messenger functions in a variety of cellular signaling pathways. At the epicenter of the sphingomyelin--cell signaling pathway is a family of phospholipases called sphingomyelinases. These enzymes cleave sphingomyelin to produce ceramide and phosphocholine. Ceramide in turn serves as a lipid second messenger that induces a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Neutral sphingomyelinase (N-SMase) is a Mg2+ sensitive enzyme that can be activated by a host of physiologically relevant and structurally diverse molecules like tumor necrosis factor-alpha (TNF-alpha), oxidized human low density lipoproteins (Ox-LDL), and several growth factors. Large amounts of ceramide accumulate in human fatty streaks and plaques along with Ox-LDL, growth factors, and proinflammatory cytokines in human atherosclerosis. A further role of ceramide and N-SMase in atherosclerosis was uncovered by the finding that Ox-LDL and TNF-alpha stimulated N-SMase activity. In turn, ceramide and/or a homolog serves as an important stress signaling molecule in signal transduction, which leads to apoptosis. Interestingly, an antibody against N-SMase can abrogate Ox-LDL and TNF-alpha induced apoptosis, and therefore may be useful for additional studies of apoptosis in experimental animals. Overexpression of recombinant human N-SMase in human aortic smooth muscle cells markedly stimulate apoptosis, presumably via the multioligomerization of the 'death domain'. Since plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke and heart failure. In contrast to these observations in human hepatocytes, TNF-alpha mediated N-SMase activation did not induce apoptosis. Rather it stimulated the maturation of sterol regulatory element (SRE) binding protein (SREBP-1). Moreover, a cell permeable ceramide was found to reconstitute the phenomenon above in a sterol-independent fashion. These findings provide alternate avenues for therapy of patients with hypercholesterolemia and atherosclerosis. The findings reported here suggests that N-SMase plays important cell regulatory roles and provide an exciting opportunity to further these findings to understand the pathophysiology of human disease states.     

Neutral sphingomyelinase 2 deficiency increases hyaluronan synthesis by up-regulation of Hyaluronan synthase 2 through decreased ceramide production and activation of Akt

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.     

Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide

Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.     

Content and structure of ceramide and sphingomyelin and sphingomyelinase activity in mouse hepatoma-22

The relative content of phosphatidylcholine is lower and that of sphingomyelin is higher in transplantable fast growing mouse hepatoma-22, thus decreasing their ratio approximately 2.5-fold versus normal liver. The ceramide content and the neutral sphingomyelinase activity is markedly higher (3- and 6.5-fold, respectively), whereas the acid sphingomyelinase activity is 4-fold lower in hepatoma-22 versus normal liver. The content of saturated fatty acids in ceramide and sphingomyelin of hepatoma-22 is higher than in normal liver. All sphingolipids of hepatoma-22 contain a considerable amount (25-37%) of sphinganine (dihydrosphingosine) along with sphingenine (sphingosine), whereas sphingolipids of normal liver contain predominantly sphingenine (over 95%). These results indicate that the activity of enzymes involved in sphingolipid biosynthesis and catabolism is disturbed in the transplantable mouse hepatoma-22 compared to normal liver.     

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.     

Integrins and mechanotransduction of the vascular myogenic response

This review summarizes what is currently known about the role of integrins in the vascular myogenic response. The myogenic response is the rapid and maintained constriction of a blood vessel in response to pressure elevation. A role for integrins in this process has been suggested because these molecules form an important mechanical link between the extracellular matrix and the vascular smooth muscle cytoskeleton. We briefly summarize evidence for a general role of integrins in mechanotransduction. We then describe the integrin subunit combinations known to exist in smooth muscle and the vascular wall matrix proteins that may interact with these integrins. We then discuss the effects of integrin-specific peptides and antibodies on vascular tone and on calcium entry mechanisms in vascular smooth muscle. Because integrin function is linked to the cytoskeleton, we discuss evidence for the role of the cytoskeleton in determining myogenic responsiveness. Finally, we analyze evidence that integrin-linked signaling pathways, such as those involving protein tyrosine phosphorylation cascades and mitogen-activated protein kinases, are required for myogenic tone.     

Identification,partial purification,and localization of a neutral sphingomyelinase in rabbit skeletal muscle: Neutral sphingomyelinase in skeletal muscle

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic -cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).     

Membrane depolarization and NADPH oxidase activation in aortic endothelium during ischemia reflect altered mechanotransduction

Vascular endothelial growth factor (VEGF) is considered to be important in promotion of capillary growth in skeletal muscles exposed to increased activity. We studied its interactions with nitric oxide (NO) by examining the expression of endothelial NO synthase (NOS), VEGF, and VEGF receptor-2 (VEGFR-2) proteins in relation to capillary growth in rat extensor digitorum longus muscles electrically stimulated for 2, 4, or 7 days with and without NOS inhibition by N(omega)-nitro-L-arginine (L-NNA, 3 mg/day). Stimulation increased all proteins from 2 days onward, concomitantly with capillary proliferation (labeling for proliferating cell nuclear antigen). Capillary-to-fiber ratio was elevated by 25% after 7 days. Concurrent oral administration of L-NNA did not affect the increase in endothelial NOS but depressed its activity, as shown by increased blood pressure and decreased arteriolar diameters in 2-day-stimulated muscles. NOS inhibition eliminated the increased expression of VEGFR-2 and VEGF proteins in muscles stimulated for 2 and 4 days but not for 7 days. However, it depressed capillary proliferation and the increase in C/F at all time points. We conclude that, in stimulated muscles, NO, generated by activation of neuronal NOS by muscle activity or endothelial NOS by increased blood flow and capillary shear stress, may increase capillary proliferation in the early stages of stimulation through upregulation of VEGFR-2 and VEGF. With longer stimulation, capillary growth appears to require NO, and high levels of VEGF and VEGFR-2 may be contributing to maintenance of the increased capillary bed.     

Plasma phospholipid transfer protein prevents vascular endothelium dysfunction by delivering alpha-tocopherol to endothelial cells.

alpha-tocopherol, the most potent antioxidant form of vitamin E, is mainly bound to lipoproteins in plasma and its incorporation into the vascular wall can prevent the endothelium dysfunction at an early stage of atherogenesis. In the present study, the plasma phospholipid transfer protein (PLTP) was shown to promote the net mass transfer of alpha-tocopherol from high density lipoproteins (HDL) and alpha-tocopherol-albumin complexes toward alpha-tocopherol-depleted, oxidized low density lipoproteins (LDL). The facilitated transfer reaction of alpha-tocopherol could be blocked by specific anti-PLTP antibodies. These observations indicate that PLTP may restore the antioxidant potential of plasma LDL at an early stage of the oxidation cascade that subsequently leads to cellular damages. In addition, the present study demonstrated that the PLTP-mediated net mass transfer of alpha-tocopherol can constitute a new mechanism for the incorporation of alpha-tocopherol into the vascular wall in addition to the previously recognized LDL receptor and lipoprotein lipase pathways. In ex vivo studies on rabbit aortic segments, the impairment of the endothelium-dependent arterial relaxation induced by oxidized LDL was found to be counteracted by a pretreatment with purified PLTP and alpha-tocopherol-albumin complexes, and both the maximal response and the sensitivity to acetylcholine were significantly improved. We conclude that PLTP, by supplying oxidized LDL and endothelial cells with alpha-tocopherol through a net mass transfer reaction may play at least two distinct beneficial roles in preventing endothelium damage, i.e., the antioxidant protection of LDL and the preservation of a normal relaxing function of vascular endothelial cells.     

Alpha7beta1-integrin regulates mechanotransduction and prevents skeletal muscle injury

Alpha7beta1-integrin links laminin in the extracellular matrix with the cell cytoskeleton and therein mediates transduction of mechanical forces into chemical signals. Muscle contraction and stretching ex vivo result in activation of intracellular signaling molecules that are integral to postexercise injury responses. Because alpha7beta1-integrin stabilizes muscle and provides communication between the matrix and cytoskeleton, the role of this integrin in exercise-induced cell signaling and skeletal muscle damage was assessed in wild-type and transgenic mice overexpressing the alpha7BX2 chain. We report here that increasing alpha7beta1-integrin inhibits phosphorylation of molecules associated with muscle damage, including the mitogen-activated protein kinases (JNK, p38, and ERK), following downhill running. Likewise, activation of molecules associated with hypertrophy (AKT, mTOR, and p70(S6k)) was diminished in mice overexpressing integrin. While exercise resulted in Evans blue dye-positive fibers, an index of muscle damage, increased integrin protected mice from injury. Moreover, exercise leads to an increase in alpha7beta1 protein. These experiments provide the first evidence that alpha7beta1-integrin is a negative regulator of mechanotransduction in vivo and provides resistance to exercise-induced muscle damage.     

Primary cilia in stem cells and neural progenitors are regulated by neutral sphingomyelinase 2 and ceramide

We show here that human embryonic stem (ES) and induced pluripotent stem cell–derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide—in particular, saturated, long-chain C_16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C_24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C_24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.     

Neutral sphingomyelinase: Localization in rat liver nuclei and involvement in regeneration/proliferation

We have studied the localization of neutral sphingomyelinase (N-SMase) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed.We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.Abbreviations N-SMase neutral sphingomyelinase - A-SMase acid sphingomyelinase     

Neutral sphingomyelinase 1 deficiency in the mouse causes no lipid storage disease

Sphingomyelin is a major lipid in the bilayer of subcellular membranes of eukaryotic cells. Different sphingomyelinases catalyze the initial step in the catabolism of sphingomyelin, the hydrolysis to phosphocholine and ceramide. Sphingomyelinases have been postulated to generate ceramide as a lipophilic second messenger in intracellular signaling pathways involved in cell proliferation, differentiation, or apoptosis. To elucidate the function of the first cloned Mg(2+)-dependent, neutral sphingomyelinase (nSMase 1) in sphingomyelin catabolism and its potential role in signaling processes in a genetic and molecular approach, we have generated an nSMase 1-null mutant mouse line by gene targeting. The nSMase 1-deficient mice show an inconspicuous phenotype and no accumulation or changed metabolism of sphingomyelin or other lipids, despite grossly reduced nSMase activity in all organs except brain. We also addressed the recent proposal that nSMase 1 possesses lysophospholipase C activity. The unaltered metabolism of lysophosphatidylcholine or lyso-platelet-activating factor excludes the proposed role of nSMase 1 as a lysophospholipase C.     

Neutral sphingomyelinase from human urine. Purification and preparation of monospecific antibodies

A neutral sphingomyelinase which cleaves phosphorylcholine from sphingomyelin at a pH optima of 7.4 was purified 440-fold to apparent homogeneity from normal human urine concentrate employing Sephadex G-75 column chromatography, preparative isoelectric focusing, and sphingosylphospholcholine CH-Sepharose column chromatography. The enzyme is composed of a single polypeptide whose apparent molecular weight is 92,000. Analytical isoelectric focusing revealed that the pI of this enzyme is 6.5. Purified neutral sphingomyelinase was devoid of beta-galactosidase and beta-N-acetylglucosaminidase activity originally present in the urine concentrate. The purified neutral sphingomyelinase (N-SMase) had low levels of phospholipase A1 and A2 activity when phosphatidylcholine was used as a substrate and detergents were included in the assay mixture. However, it had no phospholipase activity toward phosphatidylglycerol and sphingomyelin at pH 4.5 irrespective of the presence or absence of detergents. Monospecific polyclonal antibodies raised against N-SMase immunoprecipitated approximately 70% of N-SMase activity from urine, human kidney proximal tubular cells, and partially purified membrane-bound N-SMase from these cells. Western immunoblot assays revealed that the monospecific polyclonal antibody against urinary N-SMase recognized both the urinary N-SMase and the membrane-bound N-SMase. Because this enzyme is distinct biochemically and immunologically as compared to acid sphingomyelinase (EC 3.1.4.12), we would like to assign it an enzyme catalog number of EC 3.1.4.13. The availability of N-SMase and corresponding antibody will be useful in studying various aspects of this enzyme in biological systems.     

Flow-activated ion channels in vascular endothelium

The ability of vascular endothelial, cells (ECs) to respond to fluid mechanical forces associated with blood flow is essential for flow-mediated vasoregulation and arterial wall remodeling. Abnormalities in endothelial responses to flow also play a role in the development of atherosclerosis. Although our understanding of the endothelial signaling pathways stimulated by flow has greatly increased over the past two decades, the mechanisms by which ECs sense flow remain largely unknown. Activation of flow-sensitive ion channels is among the fastest known endothelial responses to flow; therefore, these ion channels have been proposed as candidate flow sensors. This review focuses on: 1) describing the various types of flow-sensitive ion channels that have been reported in ECs, 2) discussing the implications of activation of these ion channels for endothelial function, and 3) proposing candidate mechanisms for activation of flow-sensitive ion channels.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.