Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury.

Neonatal mouse hearts have transient renewal capacity, which is lost in juvenile and adult stages. In neonatal mouse hearts, myocardial infarction (MI) causes an initial loss of cardiomyocytes. However, it is unclear which type of regulated cell death (RCD) occurs in stressed cardiomyocytes. In the current studies, we induced MI in neonatal and juvenile mouse hearts and showed that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrated that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine effects and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. The fibrogenic activity of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI.

Cardiomyocyte-fibroblast interaction regulates ferroptosis and fibrosis after myocardial injury.

Neonatal mouse hearts have transient renewal capacity which is lost in juvenile and adult hearts. After myocardial infarction (MI) in neonatal hearts, an initial loss of cardiomyocytes occurs but it is unclear through which type of regulated cell death (RCD). In the current studies, we induced MI in neonatal and juvenile mouse hearts, and show that ischemic cardiomyocytes primarily undergo ferroptosis, a non-apoptotic and iron-dependent form of RCD. We demonstrate that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine factors and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. Meanwhile, the fibrogenic role of CFs, typically considered detrimental to heart function, is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition, Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI.

Depletion of adipocyte sphingosine kinase 1 leads to cell hypertrophy, impaired lipolysis, and nonalcoholic fatty liver disease.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Accumulating data support a role for bioactive lipids as mediators of lipotixicity in cardiomyocytes. One class of these, the ceramides, constitutes a family of molecules that differ in structure and are synthesized by distinct enzymes, ceramide synthase (CerS)1-CerS6. Data support that specific ceramides and the enzymes that catalyze their formation play distinct roles in cell function. In a mouse model of diabetic cardiomyopathy, sphingolipid profiling revealed increases in not only the CerS5-derived ceramides but also in very long chain (VLC) ceramides derived from CerS2. Overexpression of CerS2 elevated VLC ceramides caused insulin resistance, oxidative stress, mitochondrial dysfunction, and mitophagy. Palmitate induced CerS2 and oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. Neither overexpression nor knockdown of CerS5 had any function in these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function. This concept was also supported by the observation that synthetic mitochondria-targeted ceramides led to mitophagy in a manner proportional to N-acyl chain length. Finally, blocking mitophagy exacerbated cell death. Taken together, our results support a model by which CerS2 and VLC ceramides have a distinct role in lipotoxicity, leading to mitochondrial damage, which results in subsequent adaptive mitophagy. Our data reveal a novel lipotoxic pathway through CerS2.-Law, B. A., Liao, X., Moore, K. S., Southard, A., Roddy, P., Ji, R., Szulc, Z., Bielawska, A., Schulze, P. C., Cowart, L. A. Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Defining a role for acid sphingomyelinase in the p38/interleukin-6 pathway.

Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid ß-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4ß-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid ß-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.

Sustained activation of protein kinase C induces delayed phosphorylation and regulates the fate of epidermal growth factor receptor.

It is well established that acute activation of members of the protein kinase C (PKC) family induced by activation of cellular receptors can transduce extracellular stimuli to intracellular signaling. However, the functions of sustained activation of PKC are not well studied. We have previously shown that sustained activation of classical PKC isoforms over 15-60 min induced the formation of the pericentrion, a subset of recycling endosomes that are sequestered perinuclearly in a PKC- and phospholipase D (PLD)-dependent manner. In this study, we investigated the role of this process in the phosphorylation of EGFR on threonine 654 (Thr-654) and in the regulation of intracellular trafficking and fate of epidermal growth factor receptor (EGFR). Sustained stimulation of the angiotensin II receptor induced translocation of the EGFR to the pericentrion, which in turn prevents full access of EGF to the EGFR. These effects required PKC and PLD activities, and direct stimulation of PKC with phorbol esters was sufficient to reproduce these effects. Furthermore, activation of PKC induced delayed phosphorylation of EGFR on Thr-654 that coincided with the formation of the pericentrion and which was dependent on PLD and endocytosis of EGFR. Thus, Thr-654 phosphorylation required the formation of the pericentrion. On the other hand, using a T654A mutant of EGFR, we find that the phosphorylation on Thr-654 was not required for translocation of EGFR to the pericentrion but was required for protection of EGFR from degradation in response to EGF. Taken together, these results demonstrate a novel role for the pericentrion in the regulation of EGFR phosphorylation, which in turn is important for the fates of EGFR.

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

Epidermal growth factor-induced cellular invasion requires sphingosine-1-phosphate/sphingosine-1-phosphate 2 receptor-mediated ezrin activation.

Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.

Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins.

Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476-3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.

Identification and characterization of protein phosphatase 2C activation by ceramide.

Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2Cγ/PPM1G. This was consistent with our findings that PP2Cγ 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2Cα, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2Cα by ceramide, phospho-p38δ was utilized as substrate. Indeed, PP2Cα induced the dephosphorylation of p38δ only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.

Protein phosphatase 1α mediates ceramide-induced ERM protein dephosphorylation: a novel mechanism independent of phosphatidylinositol 4, 5-biphosphate (PIP2) and myosin/ERM phosphatase.

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP(2)) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP(2) hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP(2) and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1α, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1α acted as dominant negative of the endogenous PP1α. Additional results showed that the ceramide mechanism of PP1α activation is largely independent of PIP(2) hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1α and plasma membrane ceramide.

Neutral sphingomyelinase 2 (nSMase2) is the primary neutral sphingomyelinase isoform activated by tumour necrosis factor-α in MCF-7 cells.

Activation of N-SMase (neutral sphingomyelinase) is an established part of the response of cytokines such as TNF (tumour necrosis factor)-α. However, it remains unclear which of the currently cloned N-SMase isoforms (nSMase1, nSMase2 and nSMase3) are responsible for this activity. In MCF-7 cells, we found that TNF-α induces late, but not early, increases in N-SMase activity, and that nSMase2 is the primary isoform activated, most likely through post-transcriptional mechanisms. Surprisingly, overexpression of tagged or untagged nSMase3 in multiple cell lines had no significant effect on in vitro N-SMase activity. Moreover, only overexpression of nSMase2, but not nSMase1 or nSMase3, had significant effects on cellular sphingolipid levels, increasing ceramide and decreasing sphingomyelin. Additionally, only siRNA (small interfering RNA) knockdown of nSMase1 significantly decreased basal in vitro N-SMase activity of MCF-7 cells, whereas nSMase2 but not nSMase3 siRNA inhibited TNF-α-induced activity. Taken together, these results identify nSMase2 as the major TNF-α-responsive N-SMase in MCF-7 cells. Moreover, the results suggest that nSMase3 may not possess in vitro N-SMase activity and does not affect cellular sphingolipid levels in the cell lines evaluated. On the other hand, nSMase1 contributes to in vitro N-SMase activity, but does not affect cellular sphingolipids much.

Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase.

Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser(508) as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706-35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.

A novel mechanism of lysosomal acid sphingomyelinase maturation: requirement for carboxyl-terminal proteolytic processing.

Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn(2+))-independent lysosomal aSMase (L-SMase) and a Zn(2+)-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn(2+)-activated proenzyme to a mature 65 kDa, Zn(2+)-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn(2+)-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M(r) as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.

Regulated secretion of acid sphingomyelinase: implications for selectivity of ceramide formation.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase(WT) were treated with inflammatory cytokines. Interleukin-1ß and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C(16)-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase(WT) exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1ß, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

Differential effects of ceramide and sphingosine 1-phosphate on ERM phosphorylation: probing sphingolipid signaling at the outer plasma membrane.

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.

Identification and characterization of murine mitochondria-associated neutral sphingomyelinase (MA-nSMase), the mammalian sphingomyelin phosphodiesterase 5.

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MA-nSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg(2+) and Mn(2+) and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.

Identification of a novel amidase motif in neutral ceramidase.

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

Identification of two RNA cis-elements that function to regulate the 5' splice site selection of Bcl-x pre-mRNA in response to ceramide.

Two splice variants derived from the BCL-x gene, proapoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L), are produced via alternative 5' splice site selection within exon 2 of Bcl-x pre-mRNA. In previous studies, our laboratory demonstrated that ceramide regulated this 5' splice site selection, inducing the production of Bcl-x(s) mRNA with a concomitant decrease in Bcl-x(L) correlating with sensitization to chemotherapy (Chalfant, C. E., Rathman, K., Pinkerman, R. L., Wood, R. E., Obeid, L. M., Ogretmen, B., and Hannun, Y. A. (2002) J. Biol. Chem. 277, 12587-12595). We have now identified several possible RNA cis-elements within exon 2 of Bcl-x pre-mRNA by sequence analysis. To study the possible roles of these RNA cis-elements in regulating the alternative 5' splice site selection of Bcl-x pre-mRNA, we developed a BCL-x minigene construct which conferred the same ratio of Bcl-x(L)/Bcl-x(s) mRNA as the endogenous Bcl-x and was responsive to ceramide treatment. Mutagenesis of either a purine-rich splicing enhancer or a pyrimidine tract element within exon 2 induced a change in the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to 1 and 0.23, thereby diminishing the selection of the Bcl-x(L) 5' splice site with a concomitant increase in Bcl-x(s) 5' splice site selection. Furthermore, mutagenesis of these cis-elements abolished the ability of ceramide to affect the 5' splice site selection. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-activating proteins to these regions. SDS-PAGE analysis of cross-linked RNA trans-activating factors with these RNA cis-elements revealed the binding of 215-, 120-, and 30-kDa proteins to the purine-rich element and 120- and 76-kDa proteins to the pyrimidine tract element. In addition, exogenous treatment of A549 cells with ceramide increased the formation of protein complexes with these RNA cis-elements. Therefore, we have identified two ceramide-responsive RNA cis-elements within exon 2 of Bcl-x pre-mRNA, and this is the first report of an RNA cis-element responsive to a bioactive lipid.

The structural requirements for ceramide activation of serine-threonine protein phosphatases.

The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C(6) ceramide activated the catalytic subunit of PP2A (PP2Ac) approximately 3-fold in a stereospecific manner. In contrast, saturation of the 4-5 double bond, producing D-erythro-dihydro C(6) ceramide, inhibited PP2Ac (IC(50) = 8.5 microM). Furthermore, phyto C(6) ceramide, D-erythro-dehydro C(6) ceramide, and D-erythro-cis-C(6) ceramide had no effect on PP2Ac activity. Modification of the sphingoid chain also abolished the ability of ceramide to activate PP2Ac. Further studies demonstrated the requirement for the amide group, the primary hydroxyl group, and the secondary hydroxyl group of the sphingoid backbone for activation of PP2Ac through the synthesis and evaluation of D-erythro-urea C(6) ceramide, L-erythro-urea C(6) ceramide, D-erythro-N-methyl C(6) ceramide, D-erythro-L-O-methyl C(6) ceramide, D-erythro-3-O-methyl C(6) ceramide, and (2S) 3-keto C(6) ceramide. None of these compounds induced significant activation of PP2Ac. Liposome binding studies were also conducted using analogs of D-erythro-C C(6) ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP. This study demonstrates strict structural requirements for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP. The studies also begin to define features that transform ceramide analogs into inhibitors of CAPP.