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1.  Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts[S] 
Journal of Lipid Research  2014;55(7):1298-1309.
In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifically, fibroblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK−/− mice) and their wild-type littermates (CERK+/+) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fibroblasts from CERK+/+ mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK−/− fibroblasts. This observed difference was reflected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the inflammatory stage to the peak of the fibroplasia stage (e.g., proliferation and migration of fibroblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fibroblasts isolated from CERK−/−. As the proper migration of fibroblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing response of wounds.
doi:10.1194/jlr.M048207
PMCID: PMC4076082  PMID: 24823941
wound healing; ceramide-1-phosphate; lipidomics
2.  Structural Stereochemistry of Androstene Hormones Determines Interactions with Human Androgen, Estrogen, and Glucocorticoid Receptors 
DHEA, 17α-AED, 17β-AED, and 17β-AET exhibit strong biological activity that has been attributed to androgenic, estrogenic, or antiglucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17β-AED, and 17β-AET for their ability to activate the human AR, ER, and GR and determine the relative androgenicity, estrogenicity, and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERβ may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR, only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors and their biological effects.
doi:10.1155/2013/203606
PMCID: PMC3981456  PMID: 24729874
3.  Characterization of lysophosphatidic acid subspecies produced by autotaxin using a modified HPLC ESI-MS/MS method 
Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a modified HPLC ESI-MS/MS method with enhanced sensitivity was developed, which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Specifically, autotaxin overexpression induced an increase in the 16:0, 18:2, 18:1, 18:0, and 20:4 subspecies of LPA, but not the 22:6 LPA subspecies. Lastly, this HPLC ESI-MS/MS method was cross-validated via biological assays previously utilized to assay LPA levels. Hence, this HPLC ESI-MS/MS method will allow researchers to measure in vitro LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.
doi:10.1039/C1AY05459G
PMCID: PMC3956132  PMID: 24648853
4.  Nonvesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids 
Nature  2013;500(7463):463-467.
Phosphorylated sphingolipids [ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P)] have emerged as key regulators of cell growth, survival, migration, and inflammation1–5. C1P (Fig. 1a) produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production3,6–9, which contributes to disease pathogenesis in asthma/airway hyper-responsiveness, cancer, atherosclerosis, and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking, and presentation of C1P to specific cellular sites. Vesicular trafficking is likely10 but nonvesicular mechanisms for C1P sensing, transfer, and presentation remain unexplored11,12. Moreover, the molecular basis for selective recognition and binding among signaling lipids with phosphate headgroups, namely C1P, phosphatidic acid (PA) or their lyso-derivatives, remains unclear. Herein, an ubiquitously-expressed lipid transfer protein (CPTP) is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding via a novel surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated ‘sandwich’ topology identifies CPTP as the prototype for a new GLTP-fold13 subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi/TGN, nucleus, and plasma membrane. RNAi-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
doi:10.1038/nature12332
PMCID: PMC3951269  PMID: 23863933
5.  The flip-flop HuR: part of the problem or the solution in fighting cancer? 
Chronic inflammation has long been appreciated to play a critical role in tumor development and maintenance. Among the mechanisms involved in coordinating the initiation and resolution of inflammation are those responsible for modifying mRNA stability and/or translation. Several studies have linked the RNA-binding protein HuR, which increases mRNA stability, with malignant transformation. However, in this issue of the JCI, Yiakouvaki et al. compellingly demonstrate in mice that increased HuR activity in myeloid cells has a protective role in the onset of pathologic intestinal inflammation (i.e., colitis) and colitis-associated cancer (CAC). These observations highlight the need to understand the roles of HuR in distinct cell populations in vivo and suggest that enhancing HuR activity may be of clinical benefit in protecting against pathologic inflammation and cancer.
doi:10.1172/JCI61677
PMCID: PMC3248312  PMID: 22201677
6.  Systems-Level Lipid Analysis Methodologies for Qualitative and Quantitative Investigation of Lipid Signaling Events During Wound Healing 
Advances in Wound Care  2013;2(9):538-548.
Objective
Accumulating evidence implicates a prominent role for lipid signaling molecules in the regulation of wound healing. These lipids regulate hemostasis, onset and resolution of inflammation, migration and proliferation cells, angiogenesis, epithelialization, and remodeling of collagen. The objective of this overview is to demonstrate the applicability of systems level lipid analyses to identify and quantify lipid involved in events leading to wound healing.
Approach
Current advances in liquid chromatography coupled to tandem mass spectrometry have provided the means for carrying out quantitative and qualitative analysis of lipids at a systems level. This emerging field is collectively referred to as lipidomics and its potential in wound healing research is largely ignored.
Results
While comprehensive applications of lipidomics in wound healing are limited, studies carried out by the authors as well as others demonstrate distinct changes in the lipidome during the wound healing process.
Innovation
Until recently, investigations into lipids were limited to the study of a few lipids at a time. Lipidomics approaches provide the capability to quantitatively and qualitatively assay almost the full complement of lipid signaling circuits at the same time. This allows obtaining a system level understanding of changes to the entire lipidome during the wound healing process.
Conclusion
The technology provides promising approach to understanding new signaling pathways based on lipids involved in wound healing. The understanding gained from such studies has the potential for the development of novel lipid based treatment strategies to promote wound healing.
doi:10.1089/wound.2012.0402
PMCID: PMC3816989  PMID: 24527363
7.  Metallofullerene-Nanoplatform-Delivered Interstitial Brachytherapy Improved Survival in a Murine Model of Glioblastoma Multiforme 
Bioconjugate chemistry  2012;23(9):1873-1880.
Fullerenes are used across scientific disciplines because of their diverse properties gained by altering encapsulated or surface bound components. In this study, the recently developed theranostic agent based on a radiolabeled functionalized metallofullerene (177Lu-DOTA-f-Gd3N@C80) was synthesized with high radiochemical yield and purity. The efficacy of this agent was demonstrated in two orthotopic xenograft brain tumor models of glioblastoma multiforme (GBM). A dose-dependent improvement in survival was also shown. The in vivo stability of the agent was verified through dual label measurements of biological elimination from the tumor. Overall, these results provide evidence that nanomaterial platforms can be used to deliver effective interstitial brachytherapy.
doi:10.1021/bc300206q
PMCID: PMC3448337  PMID: 22881865
fullerene; nanomedicine; brachytherapy; glioblastoma; metallofullerene
8.  OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2 
BMC Cancer  2013;13:256.
Background
Lapatinib is characterized as an ErbB1/ErbB2 dual inhibitor and has recently been approved for the treatment of metastatic breast cancer. In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies.
Methods
In the present studies, estrogen receptor (ER) positive and ER negative breast cancer cells were genetically manipulated to up- or downregulate eIF2-alpha, its phospho-mutant, Nck1, or Nck2, then treated with OSU-03012, lapatinib or the combination and assayed for cytotoxicity/cytostaticity using clonogenic assays.
Results
Treatment of breast cancer cell lines with lapatinib and OSU-03012 (a small molecule derivative of the Cox-2 inhibitor celecoxib) induced synergistic cytotoxic/cytostatic effects. This combination therapy corresponded to an increase in the phosphorylation of eIF2-α at serine51 and a decrease in Nck1 expression. Ectopic expression of phospho-mutant eIF2-α (Ser51Ala) or downregulation of eIF2-α in addition to downregulation of the eIF2-α kinase PERK inhibited the synergistic and cytotoxic effects. Furthermore, ectopic expression of Nck1, but not Nck2 abolished the decrease in cell viability observed in combination-treated cells. Downregulation of Nck1 failed to “rescue” the ablation of the cytotoxic/cytostatic effects by the phospho-mutant of eIF2-α (Ser51Ala) demonstrating that Nck1 downregulation is upstream of eIF2-α phosphorylation in the anti-survival pathway activated by lapatinib and OSU-03012 treatment. Finally, co-immunoprecipitation assays indicated that eIF2-α dissociates from the Nck1/PP1 complex after OSU-03012 and lapatinib co-treatment.
Conclusions
These data indicate that OSU-03012 and lapatinib co-treatment is an effective combination therapy, which functions to enhance cell killing through the Nck1/eIF2 complex. Hence, this complex is a novel target for the treatment of metastatic breast cancer.
doi:10.1186/1471-2407-13-256
PMCID: PMC3674920  PMID: 23706161
Breast cancer; Lapatinib; Combination therapy; Nck; eIF2-alpha
9.  The proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA 
Molecular Cancer Research  2012;10(5):660-669.
Two splice variants derived from the BCL-x gene via alternative 5′ splice site selection (5′SS) are pro-apoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L). Previously, our laboratory demonstrated that apoptotic signaling pathways regulated the alternative 5′SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive pro-survival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by utilizing a number of small molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μM) or the pan-PKC inhibitor GÖ6983 (25 μM) decreased the Bcl-x(L)/Bcl-x(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/Bcl-x(s) mRNA ratio. Additional studies demonstrated that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/Bcl-x(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Lastly, forced expression of Bcl-x(L) “rescued” the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.
doi:10.1158/1541-7786.MCR-11-0363
PMCID: PMC3356487  PMID: 22522453
Bcl-x; Bcl-x(L); Bcl-x(s); alternative splicing; non-small cell lung cancer; protein kinase C iota; SAP155
10.  SRSF1 (SRp30a) regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells 
Molecular cancer research : MCR  2011;9(7):889-900.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the four exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.
doi:10.1158/1541-7786.MCR-11-0061
PMCID: PMC3140550  PMID: 21622622
ceramide; non-small cell lung cancer; RNA trans-factor; tumor repressor; oncogene; ASF/SF2; SRp30a; SRSF1; chemotherapy; erlotinib; daunorubicin; cisplatinum; paclitaxel
11.  A ceramide analog inhibits cPLA2 activity and consequent PGE2 formation in LPS-stimulated macrophages 
Immunology letters  2010;135(1-2):136-143.
Prostaglandin E2 (PGE2) is an important mediator of the inflammatory response. Phospho-ceramide analogue-1 (PCERA-1), a synthetic phospholipid-like molecule, was previously reported to modulate pro- and anti-inflammatory cytokine production. We show here that PCERA-1 inhibited LPS-stimulated PGE2 production in RAW264.7 macrophages, without affecting COX-2 expression. Furthermore, PCERA-1 efficiently suppressed arachidonic acid (AA) release in response to LPS. The dephosphorylated derivative of PCERA-1, ceramide analogue-1 (CERA-1), mimicked the inhibitory effect of PCERA-1 on AA release and PGE2 production in macrophages. Inhibition of PGE2 production by CERA-1 was completely rescued by addition of exogenous AA. Importantly, PCERA-1 and ceramide-1-phosphate (C1P) stimulated the enzymatic activity of cPLA2α in an in-vitro assay, whereas CERA-1 and ceramide inhibited both basal and C1P-stimulated cPLA2α activity. Collectively, these results indicate that CERA-1 suppresses AA release and subsequent PGE2 production in LPS-stimulated macrophages by direct interaction with cPLA2, and suggest that ceramide may similarly counteract C1P effect on cPLA2 activity in cells. The suppression of PGE2 production is suggested to contribute to the anti-inflammatory action of PCERA-1.
doi:10.1016/j.imlet.2010.10.014
PMCID: PMC3026855  PMID: 21040745
phospholipase A2; arachidonic acid; prostaglandin E2; LPS; ceramide; ceramide-1-phosphate
12.  Antihypertensive Treatment Differentially Affects Vascular Sphingolipid Biology in Spontaneously Hypertensive Rats 
PLoS ONE  2011;6(12):e29222.
Background
We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A2, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A2. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR.
Methods and Findings
For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20–25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A2. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction.
Conclusion
In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.
doi:10.1371/journal.pone.0029222
PMCID: PMC3240660  PMID: 22195025
13.  Alternative splicing of Caspase 9 is modulated by the PI3K/Akt pathway via phosphorylation of SRp30a 
Cancer research  2010;70(22):9185-9196.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathological classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, EGFR overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of PI3K or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227 and 234. Taken together, our findings demonstrate that oncogenic factors activating the PI3Kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.
doi:10.1158/0008-5472.CAN-10-1545
PMCID: PMC3059118  PMID: 21045158
SRp30a; alternative splicing; erlotinib; Akt
14.  Ceramide Kinase Regulates the Production of Tumor Necrosis Factor α (TNFα) via Inhibition of TNFα-converting Enzyme* 
The Journal of Biological Chemistry  2011;286(50):42808-42817.
Background: Pro-TNFα is transformed into the active/soluble form through proteolysis by TNFα-converting enzyme (TACE).
Results: Genetic ablation of ceramide kinase induces an increase in TACE activity and secreted TNFα.
Conclusion: Ceramide 1-phosphate (C1P) negatively regulates the activity of TACE.
Significance: The TACE/C1P interaction is a viable drug target for the treatment of heart disease and sepsis.
Tumor necrosis factor α (TNFα) is a well known cytokine involved in systemic and acute inflammation. In this study, we demonstrate that ceramide 1-phosphate (C1P) produced by ceramide kinase (CERK) is a negative regulator of LPS-induced TNFα secretion. Specifically, bone marrow-derived macrophages isolated from CERK knock-out mice (CERK−/−) generated higher levels of TNFα than the wild-type mice (CERK+/+) in response to LPS. An increase in basal TNFα secretion was also observed in CERK−/− murine embryonic fibroblasts, which was rescued by re-expression of wild-type CERK. This effect was due to increased secretion and not transcription. The secretion of TNFα is regulated by TNFα-converting enzyme (TACE also known as ADAM17), and importantly, the activity of TACE was higher in cell extracts from CERK−/− as compared with wild type. In vitro analysis also demonstrated that C1P is a potent inhibitor of this enzyme, in stark contrast to ceramide and sphingosine 1-phosphate. Furthermore, TACE specifically bound C1P with high affinity. Finally, several putative C1P-binding sites were identified via homology throughout the protein sequence of TACE. These results indicate that C1P produced by CERK has a negative effect on the processing/secretion of TNFα via modulation of TACE activity.
doi:10.1074/jbc.M111.310169
PMCID: PMC3234830  PMID: 22009748
Lipids; Lipid-binding Protein; Lipid Synthesis; Sphingolipid; Tumor Necrosis Factor (TNF)
15.  Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide 
PLoS ONE  2011;6(7):e21817.
Background
Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function.
Methods and Findings
In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p<0.05). Imaging mass spectrometry and immunohistochemistry indicated that these contractions were most likely mediated by ceramide and dependent on iPLA2, cyclooxygenase-1 and thromboxane synthase. Expression levels of these enzymes were higher in SHR vessels. In concurrence, infusion of dimethylsphingosine caused a marked rise in blood pressure in anesthetized SHR (42±4%; n = 7), but not in WKY (−12±10%; n = 6). Lipidomics analysis by mass spectrometry, revealed elevated levels of ceramide in arterial tissue of SHR compared to WKY (691±42 vs. 419±27 pmol, n = 3–5 respectively, p<0.05). These pronounced alterations in SHR sphingolipid biology are also reflected in increased plasma ceramide levels (513±19 pmol WKY vs. 645±25 pmol SHR, n = 6–12, p<0.05). Interestingly, we observed similar increases in ceramide levels (correlating with hypertension grade) in plasma from humans with essential hypertension (185±8 pmol vs. 252±23 pmol; n = 18 normotensive vs. n = 19 hypertensive patients, p<0.05).
Conclusions
Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone.
doi:10.1371/journal.pone.0021817
PMCID: PMC3139577  PMID: 21818267
16.  Exogenous and endogenous ceramides elicit volume-sensitive chloride current in ventricular myocytes 
Cardiovascular Research  2009;86(1):55-62.
Aims
Because ceramide accumulates in several forms of cardiovascular disease and ceramide-induced apoptosis may involve the volume-sensitive Cl− current, ICl,swell, we assessed whether ceramide activates ICl,swell.
Methods and results
ICl,swell was measured in rabbit ventricular myocytes by whole-cell patch clamp after isolating anion currents. Exogenous C2-ceramide (C2-Cer), a membrane-permeant short-chain ceramide, elicited an outwardly rectifying Cl− current in both physiological and symmetrical Cl− solutions that was fully inhibited by DCPIB, a specific ICl,swell blocker. In contrast, the metabolically inactive C2-Cer analogue C2-dihydroceramide (C2-H2Cer) failed to activate Cl− current. Bacterial sphingomyelinase (SMase), which generates endogenous long-chain ceramides as was confirmed by tandem mass spectrometry, also elicited an outwardly rectifying Cl− current that was inhibited by DCPIB and tamoxifen, another ICl,swell blocker. Bacterial SMase-induced current was partially reversed by osmotic shrinkage and fully suppressed by ebselen, a scavenger of reactive oxygen species. Outward rectification with physiological and symmetrical Cl− gradients, block by DCPIB and tamoxifen, and volume sensitivity are characteristics that identify ICl,swell. Insensitivity to C2-H2Cer and block by ebselen suggest involvement of ceramide signalling rather than direct lipid-channel interaction.
Conclusion
Exogenous and endogenous ceramide elicited ICl,swell in ventricular myocytes. This may contribute to persistent activation of ICl,swell and aspects of altered myocyte function in cardiovascular diseases associated with by ceramide accumulation.
doi:10.1093/cvr/cvp399
PMCID: PMC2836262  PMID: 20008476
Cl channel; Ceramide; Sphingomyelinase; ICl,swell; VRAC
17.  hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing 
The Journal of Clinical Investigation  2010;120(11):3923-3939.
Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non–small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.
doi:10.1172/JCI43552
PMCID: PMC2964989  PMID: 20972334
18.  Ceramide-Activated Protein Phosphatase (CAPP) involvement in insulin resistance via Akt, SRp40, and RNA splicing in L6 skeletal muscle cells 
Endocrinology  2006;148(3):1359-1366.
Elevated TNFα levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of PKCβII alternative splicing through phosphorylation of SRp40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFα attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase (CAPP). Western blot analysis of L6 cell lysates demonstrated impaired insulin–stimulated phosphorylation of Akt, SRp40, and GSK3β in response to TNFα and the short chain C6 ceramide analog. TNFα increased serine/threonine phosphatase activity of PP1 in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by LC-MS showed consistent increases in C24:1 and C16 ceramides. Effects of TNFα and C6 on insulin–stimulated phosphorylation of GSK3β were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFα abolished insulin-mediated inclusion of the PKCβII exon. Collectively, our work demonstrates a role for PP1-like CAPP in mediating TNFα effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.
doi:10.1210/en.2006-0750
PMCID: PMC2664306  PMID: 17158207
TNFα; Ceramide activated protein phosphatase (CAPP); Akt; SR proteins; PKCβII; alternative splicing
19.  The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x 
The Journal of Cell Biology  2007;176(7):929-939.
The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.
doi:10.1083/jcb.200701005
PMCID: PMC2064079  PMID: 17371836
20.  Metabolic Gene Remodeling and Mitochondrial Dysfunction in Failing Right Ventricular Hypertrophy due to Pulmonary Arterial Hypertension 
Circulation. Heart failure  2012;6(1):136-144.
Background
Right ventricular dysfunction (RVD) is the most frequent cause of death in patients with pulmonary arterial hypertension. Whereas abnormal energy substrate utilization has been implicated in the development of chronic left heart failure, data describing such metabolic remodeling in RVD remain incomplete. Thus, we sought to characterize metabolic gene expression changes and mitochondrial dysfunction in functional and dysfunctional RV hypertrophy.
Methods and Results
Two different rat models of RV hypertrophy were studied. The model of RVD (SU5416/hypoxia) exhibited a significantly decreased gene expression of PPAR-gamma coactivator-1 alpha (PGC-1α), PPAR-α and ERR-α. The expression of multiple PCG-1α target genes required for fatty acid oxidation (FAO) was similarly decreased. Decreased PGC-1α expression was also associated with a net loss of mitochondrial protein and oxidative capacity. Reduced mitochondrial number was associated with a downregulation of TFAM and other genes required for mitochondrial biogenesis. Electron microscopy demonstrated that in RVD tissue, mitochondria had abnormal shape and size. Lastly, respirometric analysis demonstrated that mitochondria isolated from RVD-tissue had a significantly reduced ADP-stimulated (state 3) rate for complex I. Conversely, functional RV hypertrophy in the pulmonary artery banding (PAB) model showed normal expression of PGC-1α, whereas the expression of FAO genes was either preserved or unregulated. Moreover, PAB-RV tissue exhibited preserved TFAM expression and mitochondrial respiration despite elevated RV pressure-overload.
Conclusions
Right ventricular dysfunction, but not functional RV hypertrophy in rats, demonstrates a gene expression profile compatible with a multilevel impairment of fatty acid metabolism and significant mitochondrial dysfunction, partially independent of chronic pressure-overload.
doi:10.1161/CIRCHEARTFAILURE.111.966127
PMCID: PMC3790960  PMID: 23152488
pulmonary heart disease; metabolism; pressure; fatty acids; mitochondria

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