Skewing cPLA2α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis.

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A2 (cPLA2) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA2 mutant lacking the C1P binding site (cPLA2αKI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA2αKI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA2α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Bioactive lipid mediators in plasma are predictors of preeclampsia irrespective of aspirin therapy.

There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk.

A rapid and adaptable lipidomics method for quantitative UPLC-mass spectrometric analysis of phosphatidylethanolamine and phosphatidylcholine in vitro, and in cells.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A2 (PLA2) enzymes. To examine the substrate utilization of PE and PC by PLA2, we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A2 (cPLA2α) coupled to the generation of lyso-PE (LPE) and lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA2 in vitro and in cells.

The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

Lactic Acid Inhibits Lipopolysaccharide-Induced Mast Cell Function by Limiting Glycolysis and ATP Availability.

Sepsis has a well-studied inflammatory phase, with a less-understood secondary immunosuppressive phase. Elevated blood lactate and slow lactate clearance are associated with mortality; however, regulatory roles are unknown. We hypothesized that lactic acid (LA) contributes to the late phase and is not solely a consequence of bacterial infection. No studies have examined LA effects in sepsis models in vivo or a mechanism by which it suppresses LPS-induced activation in vitro. Because mast cells can be activated systemically and contribute to sepsis, we examined LA effects on the mast cell response to LPS. LA significantly suppressed LPS-induced cytokine production and NF-κB transcriptional activity in mouse bone marrow-derived mast cells and cytokine production in peritoneal mast cells. Suppression was MCT-1 dependent and reproducible with sodium lactate or formic acid. Further, LA significantly suppressed cytokine induction following LPS-induced endotoxemia in mice. Because glycolysis is linked to inflammation and LA is a byproduct of this process, we examined changes in glucose metabolism. LA treatment reduced glucose uptake and lactate export during LPS stimulation. LA effects were mimicked by glycolytic inhibitors and reversed by increasing ATP availability. These results indicate that glycolytic suppression and ATP production are necessary and sufficient for LA effects. Our work suggests that enhancing glycolysis and ATP production could improve immune function, counteracting LA suppressive effects in the immunosuppressive phase of sepsis.

Lipidomics in translational research and the clinical significance of lipid-based biomarkers.

Lipidomics is a rapidly developing field of study that focuses on the identification and quantitation of various lipid species in the lipidome. Lipidomics has now emerged in the forefront of scientific research due to the importance of lipids in metabolism, cancer, and disease. Using both targeted and untargeted mass spectrometry as a tool for analysis, progress in the field has rapidly progressed in the last decade. Having the ability to assess these small molecules in vivo has led to better understanding of several lipid-driven mechanisms and the identification of lipid-based biomarkers in neurodegenerative disease, cancer, sepsis, wound healing, and pre-eclampsia. Biomarker identification and mechanistic understanding of specific lipid pathways linked to a disease's pathologies can form the foundation in the development of novel therapeutics in hopes of curing human disease.

Exogenous ceramide-1-phosphate (C1P) and phospho-ceramide analogue-1 (PCERA-1) regulate key macrophage activities via distinct receptors.

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.

Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts.

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.

The role of ceramide-1-phosphate in biological functions.

In mammalian cells, cermide-1-phosphate (C1P) is produced via the ATP-dependent mechanism of converting ceramide to C1P by the enzyme, ceramide kinase (CERK). CERK was first described as a calcium-stimulated lipid kinase that co-purified with brain synaptic vesicles, and to date, CERK is the only identified mammalian enzyme known to produce C1P in cells. C1P has steadily emerged as a bioactive sphingolipid involved in cell proliferation, macrophage migration, and inflammatory events. The recent generation of the CERK knockout mouse and the development of CERK inhibitors have furthered our current understanding of CERK-derived C1P in regulating biological processes. In this chapter, the history of C1P as well as the biological functions attributed to C1P are reviewed.

Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase.

Multiple reports have demonstrated a role for ceramide kinase (CERK) in the production of eicosanoids. To examine the effects of the genetic ablation of CERK on eicosanoid synthesis, primary mouse embryonic fibroblasts (MEFs) and macrophages were isolated from CERK(-/-) and CERK(+/+) mice, and the ceramide-1-phosphate (C1P) and eicosanoid profiles were investigated. Significant decreases were observed in multiple C1P subspecies in CERK-/- cells as compared to CERK(+/+) cells with overall 24% and 48% decreases in total C1P. In baseline experiments, the levels of multiple eicosanoids were significantly lower in the CERK(-/-) cells compared with wild-type cells. Importantly, induction of eicosanoid synthesis by calcium ionophore was significantly reduced in the CERK(-/-) MEFs. Our studies also demonstrate that the CERK(-/-) mouse has adapted to loss of CERK in regards to airway hyper-responsiveness as compared with CERK siRNA treatment. Overall, we demonstrate that there are significant differences in eicosanoid levels in ex vivo CERK(-/-) cells compared with wild-type counterparts, but the effect of the genetic ablation of CERK on eicosanoid synthesis and the serum levels of C1P was not apparent in vivo.

Folate stress induces apoptosis via p53-dependent de novo ceramide synthesis and up-regulation of ceramide synthase 6.

We have investigated the role of ceramide in the cellular adaptation to folate stress induced by Aldh1l1, the enzyme involved in the regulation of folate metabolism. Our previous studies demonstrated that Aldh1l1, similar to folate deficiency, evokes metabolic stress and causes apoptosis in cancer cells. Here we report that the expression of Aldh1l1 in A549 or HCT116 cells results in the elevation of C16-ceramide and a transient up-regulation of ceramide synthase 6 (CerS6) mRNA and protein. Pretreatment with ceramide synthesis inhibitors myriocin and fumonisin B1 or siRNA silencing of CerS6 prevented C16-ceramide accumulation and rescued cells supporting the role of CerS6/C16-ceramide as effectors of Aldh1l1-induced apoptosis. The CerS6 activation by Aldh1l1 and increased ceramide generation were p53-dependent; this effect was ablated in p53-null cells. Furthermore, the expression of wild type p53 but not transcriptionally inactive R175H p53 mutant strongly elevated CerS6. Also, this dominant negative mutant prevented accumulation of CerS6 in response to Aldh1l1, indicating that CerS6 is a transcriptional target of p53. In support of this mechanism, bioinformatics analysis revealed the p53 binding site 3 kb downstream of the CerS6 transcription start. Interestingly, ceramide elevation in response to Aldh1l1 was inhibited by silencing of PUMA, a proapoptotic downstream effector of p53 whereas the transient expression of CerS6 elevated PUMA in a p53-dependent manner indicating reciprocal relationships between ceramide and p53/PUMA pathways. Importantly, folate withdrawal also induced CerS6/C16-ceramide elevation accompanied by p53 accumulation. Overall, these novel findings link folate and de novo ceramide pathways in cellular stress response.

hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L.

Caspase-9 has two splice variants, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b, which are regulated by RNA trans-factors associated with exon 3 of caspase-9 pre-mRNA (C9/E3). In this study, we identified hnRNP U as an RNA trans-factor associated with C9/E3. Down-regulation of hnRNP U led to a decrease in the caspase-9a/9b mRNA ratio, demonstrating a novel enhancing function. Importantly, hnRNP U bound specifically to C9/E3 at an RNA cis-element previously reported as the binding site for the splicing repressor, hnRNP L. Phosphorylated hnRNP L interfered with hnRNP U binding to C9/E3, and our results demonstrate the importance of the phosphoinositide 3-kinase/AKT pathway in modulating the association of hnRNP U to C9/E3. Taken together, these findings show that hnRNP U competes with hnRNP L for binding to C9/E3 to enhance the inclusion of the four-exon cassette, and this splice-enhancing function is blocked by the AKT pathway via phosphorylation of hnRNP L.

Challenges in the Treatment of Triple Negative and HER2-Overexpressing Breast Cancer.

While the 5-year survival rate of breast cancer is at an all-time high of 90%, this disease remains the second most common cause of cancer-related death, surpassed only by lung cancer in the US. The reasons for this discrepancy stem from cancer subtypes which become resistant to current therapies. These subtypes: "Triple negative" and ErbB2-overexpressing, are discussed in this review.

Activation of p21-Dependent G1/G2 Arrest in the Absence of DNA Damage as an Antiapoptotic Response to Metabolic Stress.

The folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase, ALDH1L1), a metabolic regulator of proliferation, activates p53-dependent G1 arrest and apoptosis in A549 cells. In the present study, we have demonstrated that FDH-induced apoptosis is abrogated upon siRNA knockdown of the p53 downstream target PUMA. Conversely, siRNA knockdown of p21 eliminated FDH-dependent G1 arrest and resulted in an early apoptosis onset. The acceleration of FDH-dependent apoptosis was even more profound in another cell line, HCT116, in which the p21 gene was silenced through homologous recombination (p21(-/-) cells). In contrast to A549 cells, FDH caused G2 instead of G1 arrest in HCT116 p21(+/+) cells; such an arrest was not seen in p21-deficient (HCT116 p21(-/-)) cells. In agreement with the cell cycle regulatory function of p21, its strong accumulation in nuclei was seen upon FDH expression. Interestingly, our study did not reveal DNA damage upon FDH elevation in either cell line, as judged by comet assay and the evaluation of histone H2AX phosphorylation. In both A549 and HCT116 cell lines, FDH induced a strong decrease in the intracellular ATP pool (2-fold and 30-fold, respectively), an indication of a decrease in de novo purine biosynthesis as we previously reported. The underlying mechanism for the drop in ATP was the strong decrease in intracellular 10-formyltetrahydrofolate, a substrate in two reactions of the de novo purine pathway. Overall, we have demonstrated that p21 can activate G1 or G2 arrest in the absence of DNA damage as a response to metabolite deprivation. In the case of FDH-related metabolic alterations, this response delays apoptosis but is not sufficient to prevent cell death.

Acyl carrier protein-specific 4'-phosphopantetheinyl transferase activates 10-formyltetrahydrofolate dehydrogenase.

4'-Phosphopantetheinyl transferases (PPTs) catalyze the transfer of 4'-phosphopantetheine (4-PP) from coenzyme A to a conserved serine residue of their protein substrates. In humans, the number of pathways utilizing the 4-PP post-translational modification is limited and may only require a single broad specificity PPT for all phosphopantetheinylation reactions. Recently, we have shown that one of the enzymes of folate metabolism, 10-formyltetrahydrofolate dehydrogenase (FDH), requires a 4-PP prosthetic group for catalysis. This moiety acts as a swinging arm to couple the activities of the two catalytic domains of FDH and allows the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. In the current study, we demonstrate that the broad specificity human PPT converts apo-FDH to holoenzyme and thus activates FDH catalysis. Silencing PPT by small interfering RNA in A549 cells prevents FDH modification, indicating the lack of alternative enzymes capable of accomplishing this transferase reaction. Interestingly, PPT-silenced cells demonstrate significantly reduced proliferation and undergo strong G(1) arrest, suggesting that the enzymatic function of PPT is essential and nonredundant. Our study identifies human PPT as the FDH-modifying enzyme and supports the hypothesis that mammals utilize a single enzyme for all phosphopantetheinylation reactions.

Cytosporone E: racemic synthesis and preliminary antibacterial testing.

The antibiotic cytosporone E (isolated from the broth of the endophytic fungi CR 200 (Cytospora sp.) and CR 146 (Diaporthe sp.)) was synthesized as a racemic mixture. The key step in the synthesis is the Meyers ortho-alkylation of a chiral aromatic oxazoline. Preliminary antibiotic activity shows antibiosis against Gram-positive bacteria but not Gram-negative bacteria as previously reported.