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Sphingolipids have been appreciated as bioactive lipids that regulate a diverse range of cellular responses [1, 2]. In recent years many efforts of researchers were made to improve our knowledge of sphingolipids in pathophysiological inflammation. Different studies demonstrated that cellular signaling in inflammatory processes is controlled by ceramide [3], sphingosine-1-phosphate (S1P) [4], ceramide-1-phosphate [5], and glycosphingolipids (such as lactosylceramide and GM3) [6]. The molecular mechanisms underlying this signaling have been extensively studied. This special issue is composed of ten articles including three research articles and seven review articles. These contributions review important discoveries and provide novel findings that support the multifaceted role of sphingolipids in inflammation. Dysregulated formation of several sphingolipids including S1P and ceramide has been implicated in inflammatory bowel disease (IBD). L. Abdel Hadi et al. describe sphingolipid metabolism and signaling in IBD and discuss the potential of sphingolipid-targeted molecules as therapeutic strategies for this disease. Metabolic disease, such as obesity and type 2 diabetes, is emerging as a major health crisis in many countries. High fat diet is a primary contributing factor for obesity and its related diseases. S. Choi and A. J. Snider review the evidence for sphingolipid metabolism and pathobiology in models of high fat diet. Glycosphingolipids cluster with sphingomyelin and cholesterol in plasma membranes, forming lipid microdomains (lipid rafts) considered as platforms for signal transduction. K. Iwabuchi et al. review the evidence for biological significance of lactosylceramide-enriched microdomains in immunological and inflammatory responses of neutrophils. They also overview the significance of ceramide species and its metabolites in biological functions. A. M. Bryan et al. discuss the findings pointing to the importance of sphingolipids in immune responses of macrophages and neutrophils to fungal infections. R. Ghidoni et al. review roles of sphingolipid in the pathobiology of lung inflammation. Three research articles discuss novel findings for S1P and its receptors. E. Moon et al. discover an involvement of S1P in stroke damage in initial and recurrent stroke models. A. Chumanevich et al. reveal that S1P/S1P receptor 2 axis promotes mast cell angiogenic potential. C. Zhao et al. demonstrate that the sphingolipid pathway controlling S1P levels is dysregulated in rheumatoid arthritis synovial fibroblasts. In addition, M. Aoki et al. and S. Mahajan-Thakur et al. review the evidence pointing to roles of S1P and its receptors in immune system and blood coagulation system. This special issue discusses the topics associated with sphingolipid metabolism and pathobiology in inflammation. The articles in this special issue not only provide novel findings in sphingolipid pathobiology, but also discuss the evidence collected from a large number of research articles, giving insight into drug discovery for inflammation-associated diseases.

We investigated the effects of fasudil, a Rho kinase inhibitor, in the endothelial dysfunction of aortas from spontaneously hypertensive rats (SHRs). SHRs were divided in three groups; intraperitoneally (i.p.) vehicle-treated SHRs (SHR), SHRs treated with fasudil 3 mg/kg i.p. (Fas3), and SHRs treated with fasudil 10 mg/kg i.p. (Fas10). Vehicle-treated Wistar rats were used as normo-tensive control group. After a six-week-treatment, blood pressure and heart rate were measured by the tail cuff method. Afterwards animals were sacrificed and aortas were examined in vitro by organ bath studies to evaluate the contraction and relaxation ability. Rho kinase activity, myosin light chain (MLC), phosphorylated MLC (phospho-MLC), eNOS, phospho-eNOS protein expression and eNOS mRNA levels were evaluated. SHR demonstrated a significant hypercontractility and impaired relaxation compared to the control. Fasudil 10mg/kg significantly corrected the hypercontractility, restored the relaxation, and significantly decreased the mean arterial blood pressure, while no change observed in the systolic blood pressure. Rho kinase activity was significantly higher in the SHR, and was significantly inhibited by the high dose of fasudil. There was a slight up-regulation in the MLC, and phospho-MLC protein levels in the SHR. eNOS and phospho-eNOS protein levels were significantly lower in the SHR, and this abnormality was significantly normalized by fasudil treatment. No significant difference was observed in the eNOS gene expression. This study suggests that fasudil by inhibiting the Rho kinase activity normalizes the eNOS expression and phosphorylation and ameliorates the endothelial dysfunction induced by hypertension in the SHR model.

Aims To investigate the participation of KATP channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis. Main methods Eight-week-old male Sprague–Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300 μg/kg intraperitoneally), 30 min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24 h period of reperfusion followed. Then, expressions of KIR6.1, KIR6.2, caspase-3, PARP, Fas, FasL, and KIR6.1 and KIR6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining. Key findings Expressions of KIR6.1 protein and mRNA were more than 10-fold of those of KIR6.2 protein and mRNA in the testis. IR significantly increased the expressions of KIR6.1 protein and mRNA as well as KIR6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of KIR6.2 expressions between any of the groups. Significance Treatment with cromakalim has a protective effect against IR-induced testicular damage via activating KATP channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR.

What's known on the subject? and What does the study add? Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both KIR6.1 and KIR6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder. OBJECTIVE • To investigate whether ATP-sensitive potassium (KATP) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat. MATERIALS AND METHODS • Eight-week-old male Sprague–Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg). • AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min. • After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of KATP channel pores were investigated using ELISA and real-time PCR methods, respectively. • The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium. RESULTS • The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group. • Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations. • The expressions of KIR6.1 and KIR6.2 mRNAs were significantly up-regulated by the induction of AURC. • Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of KIR6.1 and KIR6.2 mRNAs in the bladder compared with the AUR group. CONCLUSION • Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of KATP channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

Transferrin (Tf) endocytosis and recycling are essential for iron uptake and the regulation of cell proliferation. Tf and Tf receptor (TfR) complexes are internalized via clathrin-coated pits composed of a variety of proteins and lipids and pass through early endosomes to recycling endosomes. We investigated the role of sphingomyelin (SM) synthases (SMS1 and SMS2) in clathrin-dependent trafficking of Tf and cell proliferation. We employed SM-deficient lymphoma cells that lacked SMSs and that failed to proliferate in response to Tf. Transfection of SMS1, but not SMS2, enabled these cells to incorporate SM into the plasma membrane, restoring Tf-mediated proliferation. SM-deficient cells showed a significant reduction in clathrin-dependent Tf uptake compared with the parental SM-producing cells. Both SMS1 gene transfection and exogenous short-chain SM treatment increased clathrin-dependent Tf uptake in SM-deficient cells, with the Tf being subsequently sorted to Rab11-positive recycling endosomes. We observed trafficking of the internalized Tf to late/endolysosomal compartments, and this was not dependent on the clathrin pathway in SM-deficient cells. Thus, SMS1-mediated SM synthesis directs Tf-TfR to undergo clathrin-dependent endocytosis and recycling, promoting the proliferation of lymphoma cells.

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 Ser508 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., Special thanks to the Medical University of South Carolina Analytical Lipidomics Core (Dr. Jacek Bielawski, Barbara Rembiesa, Justin Snider, and Jason Pierce) and the Medical University of South Carolina Synthetic Lipidomics Core (Dr. Alicja Bielawska and Nalini Mayroo). We also express thanks to Dr. Leah Siskind (Medical University of South Carolina) for careful reading of this manuscript.

Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

Sphingolipids, components of cellular membranes in eukaryotic cells, have roles in the regulation of tumor growth, inflammation, angiogenesis, and immunity. We investigated the effects of dietary glucosylceramides, sphingolipids isolated from rice bran, on tumor growth of human head and neck squamous cell carcinoma.The tumor cell line SCCKN cells isolated from well-differentiated human head and neck cancer were subcutaneously inoculated into the right flank of NOD/SCID mice, to establish an SCCKN xenograft model. Rice bran glucosylceramides (300 mg/kg/day) were administered orally to the mice for 14 consecutive days.Dietary glucosylceramides significantly inhibited the growth of the xenograft tumor in comparison with the control group. The TUNEL stain revealed that treatment of mice with glucosylceramides increased the number of apoptotic cells in the implanted tumor tissues and that apoptosis induction was accompanied by the formation of active/cleaved caspase-3.These results suggest that dietary glucosylceramides possibly exert anti-tumor activity by inducing apoptosis of head and neck squamous cell carcinoma. Therefore, their potential usefulness in treatment and prevention of human head and neck squamous cell carcinoma warrants further investigation.

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-aSMaseWT were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C16-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-aSMaseWT 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-aSMaseS508A MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C16-ceramide. Taken together, these data support a crucial role for Ser508 in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.

Saposins A, B, C and D are derived from a common precursor, prosaposin (psap). The few patients with saposin C deficiency develop a Gaucher disease-like central nervous system (CNS) phenotype attributed to diminished glucosylceramide (GC) cleavage activity by acid beta-glucosidase (GCase). The in vivo effects of saposin C were examined by creating mice with selective absence of saposin C (C-/-) using a knock-in point mutation (cysteine-to-proline) in exon 11 of the psap gene. In C-/- mice, prosaposin and saposins A, B and D proteins were present at near wild-type levels, but the saposin C protein was absent. By 1 year, the C-/- mice exhibited weakness of the hind limbs and progressive ataxia. Decreased neuromotor activity and impaired hippocampal long-term potentiation were evident. Foamy storage cells were observed in dorsal root ganglion and there was progressive loss of cerebellar Purkinje cells and atrophy of cerebellar granule cells. Ultrastructural analyses revealed inclusions in axonal processes in the spinal cord, sciatic nerve and brain, but no excess of multivesicular bodies. Activated microglial cells and astrocytes were present in thalamus, brain stem, cerebellum and spinal cord, indicating regional pro-inflammatory responses. No storage cells were found in visceral organs of these mice. The absence of saposin C led to moderate increases in GC and lactosylceramide (LacCer) and their deacylated analogues. These results support the view that saposin C has multiple roles in glycosphingolipid (GSL) catabolism as well as a prominent function in CNS and axonal integrity independent of its role as an optimizer/stabilizer of GCase.

The MYC family of oncogenes encodes transcription factors that are master regulators of cell proliferation and is a central driver in many human cancers. Without obvious druggable domains, direct inhibition of MYC by small molecules has remained a challenge. An alternative strategy is to exploit the concept of synthetic lethality, where specific combinations of mutations lead to cell death, while each mutation alone is not essential for survival. Targeting these synthetic lethal interactions provides an opportunity to minimize toxicity by sparing normal cells without mutations. Given that MYC amplification is reported in approximately 40% of ovarian cancers and is a leading cause of gynecologic cancer-related deaths, identifying novel therapeutic targets for this disease is of urgent clinical importance. We previously utilized a high-throughput functional genomics approach to identify 149 synthetic lethal candidates with cMYC overexpression using isogenic human foreskin fibroblast cells. In this study, 45 genes were selected for further study based on druggability, involvement in cancer pathways, and differential toxicity. We focused on 23 genes based on their expression pattern in ovarian cancers, of which 9 genes were identified to exhibit strong synthetic lethality in ovarian cancer cells with cMYC overexpression. Casein kinase 1ε (CSNK1ε) was one of the top candidates and was therefore selected for subsequent validation and further analysis. Knock-down of CSNK1ε had minimal toxicity in human fibroblasts, suggesting the possibility of a good therapeutic window. Using qPCR and immunohistochemistry analysis of 55 frozen samples from ovarian cancer patients, we showed significant correlation of MYC and CSNK1ε expression. Moreover, analysis of publically available microarray data showed that patients with high CSNK1ε expression are associated with poorer survival. Next, we performed functional validation in two sets of ovarian cancer cell lines with either high-MYC expression (IGROV-1 and TOV112D) or low-MYC expression (CaOV3 and DOV13). Using RNAi and small molecule inhibitors (IC261, PF-670462, PF-4800567), we demonstrated that CSNK1ε inhibition reduced cell viability and attenuated the WNT and SHH target pathways specifically in cells with high-MYC expression. Treatment with the CSNK1ε specific inhibitor, PF-4800567, further revealed that upregulation of CSNK1δ could be a potential mechanism of resistance. Finally, the growth of TOV112D cells (high MYC) in a tumor xenograft model was significantly inhibited by daily treatment with IC61. These findings reveal a new strategy to target the undruggable MYC oncogene through synthetic lethal interactions in MYC-driven ovarian cancer. Targeting CSNK1ε offers a novel therapeutic approach to complement traditional chemotherapies currently used to treat this disease in the clinic. Citation Format: Goldie Y. L. Lui, Franz Schaub, Shaifali Agrawal, Kazuyuki Kitatani, Christopher J. Kemp, Masafumi Toyoshima, Carla Grandori. Casein kinase 1ε (CSNK1ε) is a synthetic lethal target in MYC-driven ovarian cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A28.

Activation of protein kinase C (PKC) by the phorbol ester (phorbol 12-myristate 13-acetate) induces ceramide formation through the salvage pathway involving, in part, acid β-glucosidase 1 (GBA1), which cleaves glucosylceramide to ceramide. Here, we examine the role of the GBA1-ceramide pathway, in regulating a pro-inflammatory pathway initiated by PKC and leading to activation of p38 and induction of interleukin 6 (IL-6). Inhibition of ceramide formation by fumonisin B1 or down-regulation of PKCδ potentiated PMA-induced activation of p38 in human breast cancer MCF-7 cells. Similarly, knockdown of GBA1 by small interfering RNAs or pharmacological inhibition of GBA1 promoted further activation of p38 after PMA treatment, implicating the GBA1-ceramide pathway in the termination of p38 activation. Knockdown of GBA1 also evoked the hyperproduction of IL-6 in response to 4β phorbol 12-myristate 13-acetate. On the other hand, increasing cellular ceramide with cell-permeable ceramide treatment resulted in attenuation of the IL-6 response. Importantly, silencing the δ isoform of the p38 family significantly attenuated the hyperproduction of IL-6. Reciprocally, p38δ overexpression induced IL-6 biosynthesis. Thus, the GBA1-ceramide pathway is suggested to play an important role in terminating p38δ activation responsible for IL-6 biosynthesis. Furthermore, the p38δ isoform was identified as a novel and predominant target of ceramide signaling as well as a regulator of IL-6 biosynthesis.

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway.

Tumor necrosis factor (TNF) family member, the receptor activator of NF-κB ligand (RANKL) is expressed on marrow stromal/osteoblast cells in response to several osteotropic factors and is critical for osteoclast precursor differentiation to form multinucleated osteoclasts, which resorb bone [Lacey et al., 1998; Hsu et al., 1999; Kong et al., 1999]. Enhanced levels of RANKL are associated with pathologic conditions such as Paget's disease of bone [Menaa et al., 2000; Neale et al., 2000] and multiple myeloma [Roux et al., 2002]. Several osteotropic factors such as l,25-(OH)2D3, parathyroid hormone (PTH), interleukin 1β (IL-1β), interleukin-11, and prostaglandin E2 (PGE2) induce osteoclast differentiation through enhanced expression of RANKL in marrow stromal/osteoblast cells [Nakashima et al., 2000; Lee et al., 2002], but the molecular mechanisms which regulate RANKL gene expression are unclear. It has been reported that IL-1β and TNF-α stimulate RANKL expression in human bone marrow stromal cells through activation of p38 MAP kinase pathway [Rossa et al., 2006]. In addition, fibroblast growth factor-2 (FGF-2) has been shown to induce RANKL production through COX-2-mediated prostaglandin synthesis and by suppressing osteoclastogenesis inhibitory factor in mouse osteoblastic cells [Nakagawa et al., 1999]. Similarly, lipopolysaccharide treatment increased the levels of RANKL expression through activation of Toll-like receptors in primary murine osteoblast cells [Kikuchi et al., 2001]. Furthermore, transforming growth factor β (TGF-β) has been shown to increase RANKL expression in activated T-cells by increasing anti-CD3 [Wang et al., 2002]. Recently, it has also been reported that PTH stimulates RANKL expression through cAMP/protein kinase A/CREB cascade [Fu et al., 2006]. We recently demonstrated that heat shock factor-2 (HSF-2) is a downstream target of FGF-2 signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells [Roccisana et al., 2004]. Heat shock proteins (HSP) are molecular chaperones expressed in cells in response to a variety of stimuli such as temperature and stimulation of membrane-bound receptors by hormones/cytokines and other chemical factors. Thus, HSP are an integral part of mammalian development [Christians et al., 2003]. Heat shock transcription factors (HSF), which binds to the heat shock responsive element (HSE), modulate expression of HSP and several other genes including TNF-α family [Mathew et al., 1998; Snoeckx et al., 2001]. Although multiple osteotropic factors including FGF-2 are known to modulate RANKL gene expression in the bone microenvironment, the transcriptional regulatory mechanisms operative in marrow stromal/preosteoblast cells are not well established. DACH1 is a human homolog of the Drosophila dachshund gene, which is a key regulator for organ development and is considered a cell fate determination factor. DACH1 contains a conserved domain (dachshund domain (DS)) in the N-terminal region that is structurally homologous with the Ski and Sno proto-oncogenes and interacts with the nuclear co-repressor NCoR to modulate transcription factor activity. We have previously reported that DACH1 represses TGF-β signaling through binding to Smad4 [Wu et al., 2003]. Two vertebrate homologues, Dach1 and Dach2 are partially functionally redundant, since Dach1−/− mice survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress [Davis et al., 2001]. DACH1 expression is lost in a subset of human breast cancers associated with poor prognosis. Reintroduction of DACH1 inhibits breast tumor cell proliferation and tumor growth through suppression of cyclin D1 [Wu et al., 2006]. DACH1 inhibits TGF-β signaling in ovarian cancer cells [Sunde et al., 2006]. These studies are part of a growing body of evidence that DACH1 may function as a tumor suppressor. Recent evidence indicates that DACH1 is associated with FGF signaling during limb and skeletal development and may regulate cell proliferation or differentiation [Horner et al., 2002]. Dach1 binds with Runx2/Cbfa1 and co-localizes with cyclin-dependent kinase inhibitor p27 (Kip1) and p57 (Kip2) at the growth plate region in chondrocytes [Horner et al., 2002]. In this study, we show that DACH1 negatively regulate RANKL gene expression in human bone marrow-derived stromal/preosteoblast cells and that FGF-2 signaling promotes HSF-2 binding to DACH1 to modulate RANKL gene expression in these cells.

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.

Fallopian tubal epithelium is a candidate for the origin of high-grade serous ovarian cancer. Transferrin-containing follicular fluid and/or retrograde menstrual blood are possible risk factors for carcinogenesis. Accumulation of DNA double-strand breaks (DNA-DSBs) in the fallopian tubal epithelium is considered to play an important role in the development of cancer. However, the mechanisms by which DNA-DSBs accumulate have not yet been fully elucidated. The hydroxyl radical, which is produced in a Fenton reaction catalyzed by an iron ion, serves as a potent DNA-DSB-inducing molecule, raising the potential of an iron ion transporter of transferrin in the formation of DNA-DSBs. We studied the potential involvement of transferrin in DNA damage and the development of ovarian cancer. Treatment with transferrin facilitated the formation of histone 2AX phosphorylated at Serine 139 (γH2AX), which is known as a DNA-DSB marker, in human fallopian tube secretory epithelial cells and A2780 ovarian cancer cells. Knockdown of transferrin receptor 1 (TfR1), but not transferrin receptor 2, suppressed the transferrin uptake and consequent formation of γH2AX. As hydroxyl radicals in reactive oxygen species (ROS) are involved in DNA-DSBs, the formation of ROS was determined. Treatment with TfR1-specific small interference RNAs significantly diminished transferrin-induced formation of ROS. Moreover, TfR1-dependent uptake of transferrin was revealed to augment the formation of DNA-DSBs in the presence of hydrogen peroxide, which served as a substrate for the Fenton reaction. An ex vivo study with murine fallopian tubes further demonstrated that transferrin treatment introduced DNA-DSBs in the fallopian tubal epithelium. Collectively, these data suggested that the transferrin-TfR1 axis accounts for the induction of DNA-DSBs that potentially lead to DNA damage/genome instability. These findings also suggested that exposure to transferrin initiates and promotes the development of ovarian cancer by aiding the accumulation of DNA-DSBs in the fallopian tubal epithelium.

Gaucher’s disease is caused by defects in acid β-glucosidase 1 (GBA1) and has been also proposed as an inflammatory disease. GBA1 cleaves glucosylceramide to form ceramide, an established bioactive lipid, and defects in GBA1 lead to aberrant accumulation in glucosylceramide and insufficient formation of ceramide. We investigated if the pro-inflammatory kinase p38 is activated in Gaucher’s disease, since ceramide has been proposed to suppress p38 activation. Three Gaucher’s disease mouse models were employed, and p38 was found to be activated in lung and liver tissues of all Gaucher’s disease mice. Most interestingly, neuronopathic Gaucher’s disease type mice, but not non-neuronopathic ones, displayed significant activation of p38 and up-regulation of p38-inducible proinflammatory cytokines in brain tissues. In addition, all type of Gaucher’s disease mice also showed increases in serum IL-6. As cellular signalling is believed to represent an in vivo inflammatory phenotype in Gaucher’s disease, activation of p38 and possibly its-associated formation of proinflammatory cytokines were assessed in fibroblasts established from neuronopathic Gaucher’s disease mice. In mouse Gaucher’s disease cells, p38 activation and IL-6 formation by TNF-α treatment were enhanced as compared to those of wild type. Furthermore, human fibroblasts from Gaucher’s disease patients also displayed increases in p38 activation and IL-6 formation as comparison to healthy counterpart. These results raise the potential that proinflammatory responses such as p38 activation and IL-6 formation are augmented in Gaucher’s disease.

It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 degrees C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.

Since prostanoids such as prostaglandin E2 play a pivotal role in modulating renal function, we investigated the involvement of ceramide in expression of secretory phospholipase A2 (sPLA2) and cyclooxygenase-2 (COX-2) in tumor necrosis factor-alpha (TNF-alpha)-stimulated mesangial cells. TNF-alpha stimulation increased ceramide generation in parallel with a decrease in sphingomyelin. Pretreatment with exogenous sphingomyelinase (SMase) dose-dependently enhanced TNF-alpha-stimulated increases in COX-2 protein and sPLA) activity. SMase also augmented TNF-alpha-mediated nuclear factor kappaB (NF-kappaB) activation. N-acetylcysteine (NAC), an antioxidant, completely inhibited the SMase-induced increase in sPLA2 activity, whereas NAC inhibited partially the activity stimulated with TNF-alpha alone. Under the conditions, NAC completely inhibited reactive oxygen species (ROS) production induced by SMase followed by TNF-alpha. These results suggest that ceramide elicits up-regulation of NF-kappaB through ROS production, which, in turn, leads to stimulation of COX-2 and sPLA2 expression. Therefore, ceramide may be implicated in the pathogenesis of renal abnormalities.

The involvement of cytosolic phospholipase A 2 (cPLA 2 ) and ceramide in the accumulation of cholesteryl ester induced by the uptake of oxidized low-density lipoproteins (oxLDL) in macrophages was investigated. Uptake of oxLDL by [ 3 H]oleic acid-labeled macrophages stimulated the formation of cholesteryl oleate, and this process was completely inhibited by a cPLA 2 inhibitor. Under the conditions, a time-dependent increase in ceramide was observed, while sphingomyelin levels were unaffected. The production of ceramide was completely inhibited by fumonisin B 1 , an inhibitor of the de novo synthesis of ceramide, and oxLDL-induced cholesteryl oleate formation was inhibited partially. Treatment of the cells with sphingomyelinase accelerated the formation of cholesteryl ester. Furthermore, sphingomyelinase or cell-permeable ceramide induced the release of oleic acid, and this was inhibited by a cPLA 2 inhibitor. These results suggest that activation of cPLA 2 is responsible for the formation of cholesteryl ester induced by the uptake of oxLDL in macrophages, and that de novo-synthesized ceramide is implicated, at least in part, in this process.

OBJECTIVES: Platinum-resistance is one of the most challenging difficulties in the treatment of ovarian cancer patients. To overcome this problem, we have explored a target molecule which can conquer platinum-resistance of ovarian cancer cells utilizing a functional genomics approach. MATERIALS AND METHODS: High-throughput functional siRNA screening was designed to target 6550 genes in cisplatin-resistant A2780 CP ovarian cancer cells. Cell viability was assessed by luminescent cell viability assay. After identifying a candidate molecule, cisplatin uptake was determined by atomic absorption spectrometry. DNA damages were determined by the western blotting and immunofluorescent staining using γH2AX antibodies. RESULTS: Through a functional screening, receptor tyrosine kinase TIE 1 was identified as a top candidate gene, of which inhibition give rise to enhancement of cisplatin sensitivity in ovarian cancer cells. Conversely, over-expression of TIE 1 gene significantly decreased susceptibility to cisplatin-induced cell death without affecting cisplatin uptake. DNA damages induced by cisplatin was significantly suppressed in TIE 1 over-expressed cells, raising novel potential mechanisms of TIE 1 in nucleotide excision repair system that removes chemicals adduct to DNA. In addition, over-expression of TIE 1 increased the expression of XPC, which is responsible for nucleotide excision repair. CONCLUSION: We have identified TIE 1 as a molecular target to overcome platinum-resistance in ovarian cancer cells. TIE 1 contribute platinum- resistance in ovarian cancer cells by promoting XPC-dependent DNA repairing system. Citation Format: Masumi Ishibashi, Masafumi Toyoshima, Mahy Egiz, Xuewei Zhang, Junko Minato, Shogo Shigeta, Kazuyuki Kitatani, Nobuo Yaegashi. INVOLVEMENT OF TIE–1 TYROSINE KINASE RECEPTOR IN CHEMO–RESISTANCE: POTENTIAL OF TIE1 AS A NOVEL THERAPEUTIC TARGET [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-091.

We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM–ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM–ceramide–CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.

We previously reported that dietary glucosylceramides show cancer-prevention activity in a mouse xenograft model of human head and neck cancer cells (SCCKN). However, the mechanism was unclear. Ceramides, metabolites of glucosylceramides, induce apoptotic cell death in various malignancies. Here, we investigated the inhibitory effects of dietary glucosylceramides on tumor growth in vivo and in vitro. SCCKN were subcutaneously inoculated into the right flanks of NOD/SCID mice. Mice were treated with or without dietary glucosylceramides (300 mg/kg) daily for 14 consecutive days after confirmation of tumor progression. Microvessel areas around the tumor were assessed by hematoxylin–eosin staining and immunohistochemistry of CD31, and, as markers for angiogenesis, protein levels of VEGF, VEGF receptor-2, and HIF-1α were assessed by Western blotting. Mass spectrometry was performed to measure the levels of sphingolipids in mouse serum after treatment with dietary glucosylceramides. Oral administration of glucosylceramides significantly decreased SCCKN growth in the xenograft model with inhibition of angioinvasion. In tumor-invasive areas, VEGF and HIF-1α in the tumor cells, and VEGF receptor-2 in endothelial cells decreased after treatment with dietary glucosylceramides. Dietary glucosylceramides increased serum levels of sphingosine-based ceramides as compared to the control. In SCCKN and UV♀2 cells, C6-ceramide suppressed the expressions of VEGF, VEGF receptor-2, and HIF-1α in vitro. These results suggest that dietary glucosylceramides trigger the de novo pathway of ceramide synthesis, indicating that sphingosine-based ceramide suppresses the growth of head and neck tumors through the inhibition of pro-angiogenic signals such as VEGF, VEGF receptor-2, and HIF-1α.

The effect of ceramide on Ca2+-dependent translocation of cytosolic phospholipase A2 (cPLA2) to membranes was studied. Pretreatment of platelets with sphingomyelinase or C6-ceramide (N-hexanoylsphingosine) led to apparent enhancement of Ca2+-ionophore A23187-stimulated arachidonic acid release but did not affect the cytosolic phospholipase A2 (cPLA2) activity. Under these conditions, the cPLA2 proteins in membranes increased significantly, compared with those by A23187 alone. Sphingomyelinase and C6-ceramide, but not C6-dihydroceramide, a control analog of C6-ceramide, also facilitated the Ca2+-dependent increase in the cPLA2 protein, as well as the activity, in membranes induced by addition of Ca2+ into platelet lysate. Protein kinase Cα, which possesses a Ca2+-dependent lipid binding domain, was increased in membranes in a Ca2+-dependent manner, but the increase was not accelerated by sphingomyelinase or C6-ceramide. These findings suggest that ceramide in membranes potentiates Ca2+-dependent cPLA2 translocation from cytosol to membranes, probably through modification of membrane phospholipid organization.

The implications of phospholipase D (PLD) in cytosolic phospholipase A2 (cPLA2) activation were studied in a mast cell line, RBL-2H3, upon stimulation with antigen. Antigen-stimulated prostaglandin D2 generation was apparently suppressed by ethanol with a concomitant decrease in phosphatidic acid (PA) formation. The prostaglandin D2 generation was also inhibited almost completely by methyl arachidonyl fluorophosphonate (MAFP), an inhibitor of cPLA2, but not by diacylglycerol lipase inhibitor. Furthermore, stimulation with antigen resulted in an increase in lysophosphatidic acid formation, which was suppressed by MAFP in parallel with an increase in PA formation. These results suggest that PA formed by the catalytic action of PLD is used as a substrate for cPLA2, thus PLD regulates cPLA2 activation in antigen-stimulated RBL-2H3 cells.

The effects of ceramide on agonist-stimulated phospholipase A2 (PLA2) activity were studied in platelets. Cell-permeable C6-ceramide (N-hexanoylsphingosine) exogenously added to platelet suspension enhanced U46619-stimulated arachidonic acid release and lysophosphatidylcholine production. Treatment of platelets with sphingomyelinase also led to an enhancement of the release. The enhanced arachidonic acid release by exogenous ceramide was completely inhibited by methyl arachidonyl fluorophosphonate, a cytosolic PLA2 inhibitor. However, U46619-stimulated PLA2 activity was not significantly potentiated by ceramide. These results suggest that enrichment of ceramide in membranes causes modification of intermolecular organization, leading to increased susceptibility of substrate phospholipids to PLA2.

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Objectives: High-grade serous ovarian cancer (HGSOC) is now believed to arise from fallopian tube epithelium (FTE), and p53 signatures are indicated to be the early oncogenic change in HGSOC. Although accumulation of DNA-double strand breaks (DNA-DSBs) are frequently observed in p53 signatures, the mechanism of DNA-DSBs formation in FTE has not been revealed yet. Hydroxyl radicals, which strongly induce DNA-DSBs, are the most active reactive oxygen species (ROS) in a living organism, and hydroxyl radicals are produced in a Fenton reaction catalyzed by free iron ions. Focusing on transferrin, which is a transporter of iron ion and exists abundantly in the follicular fluid or retrograde menstrual blood, we inspected the role of transferrin and transferrin receptor family in DNA-DSBs formation at FTE. Materials and methods: The expressions of transferrin receptor 1(TfR1) and 2 (TfR2) in human FTE were assessed by immunohistochemistry. Immortalized fallopian tube secretory epithelial cells (kindly provided by Dr. Ronny Drapkin, Dana-Farber Cancer institute) and A2780 ovarian cancer cells were cultured with holo-transferrin or vehicle, and the extent of DNA-DSBs was compared. γH2AX was adopted as a marker of DNA-DSBs. ROS were also measured to verify whether transferrin promotes a Fenton reaction. The involvement of TfR1 and TfR2 were assessed with siRNA knockdown strategy. Further, the ex vivo study were performed using murine fallopian tubes. Results: In immunohistochemistry, both TfR1 and TfR2 were ubiquitously positive in human FTE. Transferrin administration significantly increased the γH2AX expression in these cells and led ROS formation. In addition, transferrin treatment also amplified hydrogen peroxide-inducing γH2AX expression. TfR1 knockdown cancelled the uptake of transferrin, subsequent γH2AX expression and ROS formation but TfR2 knockdown didn't. Also we confirmed that transferrin treatment facilitated γH2AX formation in murine FTE ex vivo. Conclusion: We identified transferrin-TfR1 axis facilitates DNA-DSBs by promoting a Fenton reaction. It is possible that FTE exposed to the extracellular transferrin highly concentrated in the follicular fluid or retrograde menstrual blood is deeply involved with the carcinogenesis of HGSOC. Citation Format: Shogo Shigeta, Masafumi Toyoshima, Kazuyuki Kitatani, Masumi Ishibashi, Toshinori Usui, Nobuo Yaegashi. Transferrin facilitates the formation of DNA-double strand breaks via transferrin receptor 1 in fallopian tube epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A13.

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.

What's known on the subject? and What does the study add? Acute urinary retention (AUR) and catheterization for AUR (AURC) or drainage of the urine is a well established cause of bladder dysfunction. Previously, we reported that the induction of AURC significantly reduced contractile responses to both carbachol and KCl compared with a control group, and that this reduction was prevented by nicorandil and cromakalim in a dose-dependent manner; however, although we reported a possible beneficial effect of nicorandil and cromakalim on bladder dysfunction caused by AURC, its molecular mechanism is still unknown. Our study establishes that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via up-regulation of both K(IR)6.1 and K(IR)6.2 with a subsequent decrease in oxidative stress and decreased induction of apoptosis in the bladder.To investigate whether ATP-sensitive potassium (K(ATP)) channel openers prevent bladder injury after acute urinary retention (AUR) and subsequent catheterization for AUR (AURC) in the rat.Eight-week-old male Sprague-Dawley rats were divided into five groups: a sham-operated control group, an AUR group, and three AUR groups treated with: nicorandil (10 mg/kg); cromakalim (300 µg/kg); or glibenclamide (5 mg/kg). AUR was induced by intravesical infusion of 3.0 mL of saline via cystostomy with simultaneous clamping of the penile urethra and, after 30 min of AUR, the bladder was allowed to drain for 60 min. After the experimental period, bladder function was assessed using organ bath techniques (carbachol and KCl), and by measuring tissue levels of 8-isoprostane, a marker of oxidative stress. The participation levels of K(ATP) channel pores were investigated using ELISA and real-time PCR methods, respectively. The degree of apoptosis was estimated using the TUNEL method in the bladder smooth muscle and epithelium.The AURC group showed significantly decreased contractile responses to carbachol and KCl, and significant increases in tissue 8-isoprostane levels and apoptosis index in the epithelium compared with the control group. Nicorandil and cromakalim, but not glibenclamide, significantly prevented these AURC-induced alterations. The expressions of K(IR)6.1 and K(IR)6.2 mRNAs were significantly up-regulated by the induction of AURC. Nicorandil and cromakalim, but not glibenclamide, significantly up-regulated expressions of K(IR)6.1 and K(IR)6.2 mRNAs in the bladder compared with the AUR group.Our data indicate that nicorandil and cromakalim, but not glibenclamide, prevent AURC-induced bladder dysfunction via activation of K(ATP) channels, with a subsequent decrease in oxidative stress and decreased induction of apoptosis.

Resistance to death receptor ligands (such as FasL and TRAIL) and anticancer treatments is a hallmark of cancer cells. Ceramide, a biologically active sphingolipid, antagonizes cell growth and promotes apoptosis and non-apoptotic forms of cell death. The intracellular levels of ceramide are highly regulated via complex metabolic pathways. Sphingomyelin synthases (SMS) 1 and 2 convert ceramide to sphingomyelin (SM), a ubiquitous phospholipid in mammals. A growing body of evidence in the literature indicates that SMSs likely modulate hematological cell growth and sensitivity to stress-induced apoptosis. On one hand, complete and sustained inhibition of SMS activity is likely to alter membrane composition and properties through membrane SM depletion, perturbing intracellular signaling pathways and leukemia cell growth and conferring partial resistance to death receptor ligands. On the other hand, different patents & reports point to anti-apoptotic functions for SMSs. In patients with chemoresistant leukemia, a decreased intracellular ceramide level was associated with a higher SMS activity. Thus, SMSs and cofactors may constitute original pharmacological targets to treat leukemia.

Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. As thoroughly discussed elsewhere in this volume, ceramide, central metabolite of the sphingolipid pathway, plays key roles in a variety of cellular responses. Since the discovery of the bioactive function of ceramide, a growing number of tools and techniques have been and still are being developed in order to better decipher the complexity and implications of ceramide-mediated signaling. With this chapter it is our intention to provide new comers to the sphingolipid arena with a short overview of tools and techniques currently available for the study ofsphingolipid metabolism, with the focus on ceramide.

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-1β 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-1β, 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.

Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Of these, ceramide is a central metabolite and plays key roles in a variety of cellular responses, including regulation of cell growth, viability, differentiation, and senescence. Ceramide is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups. Sphingosine serves as the product of sphingolipid catabolism, and it is mostly salvaged through re-acylation, resulting in the generation of ceramide or its derivatives. This recycling of sphingosine is termed the “salvage pathway”, and recent evidence points to important roles for this pathway in ceramide metabolism and function. A number of enzymes are involved in the salvage pathway, and these include sphingomyelinases, cerebrosidases, ceramidases, and ceramide synthases. Recent studies suggest that the salvage pathway is not only subject to regulation, but it also modulates the formation of ceramide and subsequent ceramide-dependent cellular signals. This review focuses on the salvage pathway in ceramide metabolism, its regulation, its experimental analysis, and emerging biological functions.

Sustained activation of protein kinase C (PKC) isoenzymes alpha and betaII leads to their translocation to a perinuclear region and to the formation of the pericentrion, a PKC-dependent subset of recycling endosomes. In MCF-7 human breast cancer cells, the action of the PKC activator 4beta-phorbol-12-myristate-13-acetate (PMA) evokes ceramide formation, which in turn prevents PKCalpha/betaII translocation to the pericentrion. In this study we investigated the mechanisms by which ceramide negatively regulates this translocation of PKCalpha/betaII. Upon PMA treatment, HEK-293 cells displayed dual phosphorylation of PKCalpha/betaII at carboxyl-terminal sites (Thr-638/641 and Ser-657/660), whereas in MCF-7 cells PKCalpha/betaII were phosphorylated at Ser-657/660 but not Thr-638/641. Inhibition of ceramide synthesis by fumonisin B1 overcame the defect in PKC phosphorylation and restored translocation of PKCalpha/betaII to the pericentrion. To determine the involvement of ceramide-activated protein phosphatases in PKC regulation, we employed small interference RNA to silence individual Ser/Thr protein phosphatases. Knockdown of isoforms alpha or beta of the catalytic subunits of protein phosphatase 1 not only increased phosphorylation of PKCalpha/betaII at Thr-638/641 but also restored PKCbetaII translocation to the pericentrion. Mutagenesis approaches in HEK-293 cells revealed that mutation of either Thr-641 or Ser-660 to Ala in PKCbetaII abolished sequestration of PKC, implying the indispensable roles of phosphorylation of PKCalpha/betaII at those sites for their translocation to the pericentrion. Reciprocally, a point mutation of Thr-641 to Glu, which mimics phosphorylation, in PKCbetaII overcame the inhibitory effects of ceramide on PKC translocation in PMA-stimulated MCF-7 cells. Therefore, the results demonstrate a novel role for carboxyl-terminal phosphorylation of PKCalpha/betaII in the translocation of PKC to the pericentrion, and they disclose specific regulation of PKC autophosphorylation by ceramide through the activation of specific isoforms of protein phosphatase 1.

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4beta-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C(16)-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C(16)-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C(16)-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.

Sphingolipids are abundant components of cellular membranes in eukaryotic cells as well as potent signaling molecules. De novo sphingolipid biosynthesis begins with condensation of L-serine and palmitoyl-CoA, generating sphingoid bases, ceramide, and other species through a series of reactions. Ceramide can also be formed from free sphingoid bases, which has been termed the’ salvage’ pathway. Sphingolipids from both the de novo and salvage pathways increase with exposure of yeast or mammals to various stimuli such as Fas ligands, chemotherapeutic drugs, tumor necrosis factor-γ and heat stress, and then act as lipid second messengers mediating inflammatory responses, senescence, cell cycle arrest, apoptosis or stress responses. Therefore, generation of signaling molecules by de novo synthesis and/or salvage accounts not only for homeostasis, but also for several disorders resulting from aberrant sphingolipid accumulation or depletion.

The ability of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) to induce the major inflammatory mediator prostaglandin (PG) E(2) depends on the activation of two rate-limiting enzymes, phospholipase A(2) (PLA(2)) and cyclooxygenase 2 (COX-2). PLA(2) acts to generate arachidonic acid, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to PGE(2) production. However, less is known about the mechanisms that coordinate the regulation of these two enzymes. We have provided prior evidence that sphingosine kinase 1 and its bioactive lipid product sphingosine-1-phosphate (S1P) mediate the effects of cytokines on COX-2 induction, whereas ceramide kinase and its distinct product, ceramide-1-phosphate (C1P), are required for the activation and translocation of cPLA(2) (FASEB J 17:1411-1421. 2003; J Biol Chem 278:38206-38213, 2003; J Biol Chem 279:11320-11326, 2004). Herein, we show that these two pathways are independent but coordinated, resulting in synergistic induction of PGE(2). Moreover, the combination of both S1P and C1P recapitulates the temporal and spatial activation of cPLA(2) and with COX-2 seen IL-1beta. Taken together, the results provide, for the first time, a mechanism that assures the coordinate expression and activation in time and space of COX-2 and cPLA(2), assuring maximal production of PGE(2).

Sphingosine kinase-1 (SK1) has emerged as a key component of cytokine responses, including roles in apoptosis, yet the specific mechanisms by which cytokines regulate SK1 in the apoptotic responses have not been studied. In this study, we show that prolonged treatment of MCF-7 cells with tumor necrosis factor (TNF) induces a dose- and time-dependent decrease in SK1 protein. Inhibition of the upstream caspase 8 by IETD significantly rescued TNF effects on SK1, yet the caspase 7 inhibitor DEVD failed to have any effect, suggesting that the decline in SK1 occurs downstream of the initiator caspase but upstream of the effector caspase. In addition to caspase activation, TNF caused disruption of lysosomes with relocation of the cysteine protease cathepsin B into the cytosol. Down-regulation of cathepsin B using small interfering RNA significantly restored SK1 levels following exposure to TNF, suggesting that SK1 loss was dependent on cathepsin B activity. The regulation of SK1 by the lysosomal protease was further supported by the colocalization of SK1 with the lysosome and cathepsin B in cells and the loss of the colocalization following exposure to TNF. The ability of cathepsin B to regulate SK1 was further corroborated by an in vitro approach where recombinant cathepsin B cleaved SK1 at multiple sites to produce several cleavage fragments. Therefore, these studies show that SK1 down-regulation by TNF is dependent on the "lysosomal pathway" of apoptosis and specifically on cathepsin B, which functions as an SK1 protease in cells.

In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.

In the present study, the effect of ceramide on antigen-stimulated phosphorylation of extracellular signal-regulated kinase (ERK) in the mechanism responsible for regulating production of prostaglandin (PG) D 2 was investigated in the mast cell line, RBL-2H3 cells. Cell-permeable C 6 -ceramide ( N -hexanoylsphingosine) suppressed antigen-stimulated phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase. Ceramide also inhibited production of PGD 2 and an increase in the activity of cytosolic phospholipase A 2 (cPLA 2 ), whereas it did not influence the tyrosine phosphorylation of major cellular proteins in response to antigen. The ceramide-induced inhibition of ERK1/2 phosphorylation and of cPLA 2 activation was suppressed by orthovanadate, a tyrosine phosphatase inhibitor, but not by okadaic acid, a serine/threonine phosphatase inhibitor. Addition of ceramide to the lysate prepared from antigen-stimulated cells reduced the phosphorylated ERK1/2, and orthovanadate effectively prevented the reduction. These results suggest that ceramide accelerates the dephosphorylation of phosphorylated ERK1/2 via activation of a protein tyrosine phosphatase, thus preventing activation of cPLA 2 and production of PGD 2 .

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.

The effects of docosapentaenoic acid (DPA) on platelet aggregation and arachidonic acid metabolism were studied in comparison to those of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Collagen- or arachidonic acid-stimulated platelet aggregation was inhibited dose-dependently by n-3 fatty acids, among which DPA was the most potent inhibitor. These fatty acids inhibited U46619-induced aggregation but to almost the same extent. No effect of the acids on thrombin-induced aggregation was observed. Furthermore, these fatty acids suppressed thromboxane A2 formation by platelets which were exposed to collagen or thrombin, or by platelets to which arachidonic acid was added. In these experiments also, DPA was the most potent inhibitor, whereas DHA was the most effective inhibitor of cyclooxygenase-1 activity. DPA enhanced formation of 12-hydroxyeicosatetraenoic acid in response to collagen or from arachidonic acid by intact platelets, while the other two acids had less of an effect. These results suggest that DPA possesses potent activity for interfering with the cyclooxygenase pathway and accelerating the lipoxygenase pathway, thus inhibiting platelet aggregation most effectively.