In Vivo Administration of Phosphatidic Acid, a Direct Alcohol Target Rescues Fetal Growth Restriction and Maternal Uterine Artery Dysfunction in Rat FASD Model.

Fetal growth restriction is a hallmark of Fetal Alcohol Syndrome (FAS) and is accompanied by maternal uterine circulatory maladaptation. FAS is the most severe form of Fetal Alcohol Spectrum Disorder (FASD), a term for the range of conditions that can develop in a fetus when their pregnant mother consumes alcohol. Alcohol exerts specific direct effects on lipids that control fundamental developmental processes. We previously demonstrated that direct in vitro application of phosphatidic acid (PA, the simplest phospholipid and a direct target of alcohol exposure) to excised uterine arteries from alcohol-exposed rats improved vascular function, but it is unknown if PA can rescue end organ phenotypes in our FASD animal model. Pregnant Sprague-Dawley rats (n = 40 total dams) were gavaged daily from gestational day (GD) 5 to GD 19 with alcohol or maltose dextrin, with and without PA supplementation, for a total of four unique groups. To translate and assess the beneficial effects of PA, we hypothesized that in vivo administration of PA concomitant with chronic binge alcohol would reverse uterine artery dysfunction and fetal growth deficits in our FASD model. Mean fetal weights and placental efficiency were significantly lower in the binge alcohol group compared with those in the control (p < 0.05). However, these differences between the alcohol and the control groups were completely abolished by auxiliary in vivo PA administration with alcohol, indicating a reversal of the classic FAS growth restriction phenotype. Acetylcholine (ACh)-induced uterine artery relaxation was significantly impaired in the uterine arteries of chronic in vivo binge alcohol-administered rats compared to the controls (p < 0.05). Supplementation of PA in vivo throughout pregnancy reversed the alcohol-induced vasodilatory deficit; no differences were detected following in vivo PA administration between the pair-fed control and PA alcohol groups. Maximal ACh-induced vasodilation was significantly lower in the alcohol group compared to all the other treatments, including control, control PA, and alcohol PA groups (p < 0.05). When analyzing excitatory vasodilatory p1177-eNOS, alcohol-induced downregulation of p1177-eNOS was completely reversed following in vivo PA supplementation. In summary, these novel data utilize a specific alcohol target pathway (PA) to demonstrate a lipid-based preventive strategy and provide critical insights important for the development of translatable interventions.

Phosphatidic Acid Promoting the Generation of Interleukin-17A Producing Double-Negative T Cells by Enhancing mTORC1 Signaling in Lupus.

OBJECTIVE: The goal was to investigate the role and intracellular regulatory mechanisms of double-negative T (DNT) cells in the pathogenesis of systemic lupus erythematosus (SLE). METHODS: DNT cells were assessed in murine models, patients with SLE, and controls using flow cytometry (FCM). DNT cells from either resiquimod (R848) or vehicle-treated C57BL/6 (B6) mice were cultured with B cells from R848-treated mice to explore functions. Differential mechanistic target of rapamycin (mTOR) pathway signaling in DNT cells measured using FCM and quantitative polymerase chain reaction was validated by rapamycin inhibition. Candidate lipid metabolites detected using liquid chromatography with electrospray ionization mass spectrometry/mass spectrometry were functionally assessed in DNT cell cultures. RESULTS: DNT cells were markedly increased in both spontaneous and induced mouse lupus models and in patients with SLE. Expanded DNT cells from R848-treated B6 mice produced elevated interleukin (IL)-17A and IgG with increased germinal center B (GCB) cells. Expansion of DNT cells associated with activation of mTORC1 pathway that both IL-17A levels and the number of DNT cells exhibited dose-dependent reduction with rapamycin treatment. Lipidomics studies revealed differential patterns of lipid metabolites in T cells of R848-treated mice. Among candidate metabolites, elevated phosphatidic acid (PA) that was partially controlled by phospholipase D2 increased the expression of the mTORC1 downstream target p-S6 and positively expanded IL-17A-producing DNT cells. Similarly, elevated proportions of circulating DNT cells in patients with SLE correlated with disease activity and proteinuria, and IL-17A secretion was elevated after in vitro PA stimulation. CONCLUSION: The accumulation of PA in T cells could activate the mTORC1 pathway, promoting DNT cell expansion and IL-17A secretion, resulting in GCB cell abnormalities in lupus.

2-Carba cyclic phosphatidic acid regulates blood coagulation and fibrinolysis system for repair after brain injury.

Effective blood coagulation prevents inflammation and neuronal loss after brain injury. 2-Carba-cyclic phosphatidic acid (2ccPA), a biotherapeutic for brain injury, inhibits blood extravasation resulting from blood-brain barrier breakdown. However, the hemostasis mechanism of 2ccPA remains unclear. We determined the effects of 2ccPA-injection on blood coagulation and fibrinolysis using a needle-induced brain injury model. 2ccPA suppressed the expression of platelet degranulation-related genes. Immediately after brain injury, 2ccPA increased CD41+ platelet aggregation around the lesions and promoted fibrin aggregation. Additionally, 2ccPA supported fibrinolysis by upregulating plasminogen activator expression. These results suggest the acute effects of 2ccPA on brain hemostasis.

AGK Potentiates Arterial Thrombosis by Affecting Talin-1 and αIIbß3-Mediated Bidirectional Signaling Pathway.

BACKGROUND: AGK (acylglycerol kinase) was first identified as a mitochondrial transmembrane protein that exhibits a lipid kinase function. Recent studies have established that AGK promotes cancer growth and metastasis, enhances glycolytic metabolism and function fitness of CD8+ T cells, or regulates megakaryocyte differentiation. However, the role of AGK in platelet activation and arterial thrombosis remains to be elaborated. METHODS: We performed hematologic analysis using automated hematology analyzer and investigated platelets morphology by transmission electron microscope. We explored the role of AGK in platelet activation and arterial thrombosis utilizing transgenic mice, platelet functional experiments in vitro, and thrombosis models in vivo. We revealed the regulation effect of AGK on Talin-1 by coimmunoprecipitation, mass spectrometry, immunofluorescence, and Western blot. We tested the role of AGK on lipid synthesis of phosphatidic acid/lysophosphatidic acid and thrombin generation by specific Elisa kits. RESULTS: In this study, we found that AGK depletion or AGK mutation had no effect on the platelet average volumes, the platelet microstructures, or the expression levels of the major platelet membrane receptors. However, AGK deficiency or AGK mutation conspicuously decreased multiple aspects of platelet activation, including agonists-induced platelet aggregation, granules secretion, JON/A binding, spreading on Fg (fibrinogen), and clot retraction. AGK deficiency or AGK mutation also obviously delayed arterial thrombus formation but had no effect on tail bleeding time and platelet procoagulant function. Mechanistic investigation revealed that AGK may promote Talin-1Ser425 phosphorylation and affect the αIIbß3-mediated bidirectional signaling pathway. However, AGK does not affect lipid synthesis of phosphatidic acid/lysophosphatidic acid in platelets. CONCLUSIONS: AGK, through its kinase activity, potentiates platelet activation and arterial thrombosis by promoting Talin-1 Ser425 phosphorylation and affecting the αIIbß3-mediated bidirectional signaling pathway.

Maturation of the malarial phosphatidylserine decarboxylase is mediated by high affinity binding to anionic phospholipids.

Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (Kd) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.

Effects of 2-carba-cyclic phosphatidic acid derivatives on IL-1ß-stimulated human chondrocytes.

Osteoarthritis (OA) is a common joint disease characterized by the breakdown of subchondral bone and cartilage damage, most often affecting middle-aged and elderly people. Although the etiology of OA is still unknown, some reports suggest that inflammatory factors such as interleukin (IL)- 1ß mediate the progression of OA. To investigate the effect of IL-1ß and the possibility of treatment for OA, we applied 2-carba-cyclic phosphatidic acid (2ccPA) and its derivatives on human chondrocytes. 2ccPA is a synthesized phospholipid derived from a bioactive phospholipid mediator: cyclic phosphatidic acid (cPA). It has been previously reported that 2ccPA exhibits anti-inflammatory and chondroprotective effects in an OA animal model. 2ccPA and its ring-opened body (ROB) derivative significantly suppressed IL-1ß-induced upregulation of IL-6, matrix metalloproteinase-13, and cyclooxygenase-2, as well as the degradation of type II collagen and aggrecan. However, the other two derivatives, namely the deacylated and ring-opened deacylated bodies, showed little effect on an IL-1ß-exposed human chondrosarcoma cell-line. These data suggest that the intactness of 2ccPA and ROB is essential for anti-inflammatory effects on OA. Collectively, this study provides evidence that 2ccPA and ROB would be novel therapeutic agents for OA.

Molecular targets of endothelial phosphatidic acid regulating major depressive disorder.

Major depressive disorder (MDD) is a severe disease of unknown pathogenesis with a lifetime prevalence of ~10%. Therapy requires prolonged treatment that often fails. We have previously demonstrated that ceramide levels in the blood plasma of patients and in mice with experimental MDD are increased. Neutralization of blood plasma ceramide prevented experimental MDD in mice. Mechanistically, we demonstrated that blood plasma ceramide accumulated in endothelial cells of the hippocampus, inhibited phospholipase D (PLD) and thereby decreased phosphatidic acid in the hippocampus. Here, we demonstrate that phosphatidic acid binds to and controls the activity of phosphotyrosine phosphatase (PTP1B) in the hippocampus and thus determines tyrosine phosphorylation of a variety of cellular proteins including TrkB. Injection of PLD, phosphatidic acid, or inhibition of PTP1B abrogated MDD and normalized cellular tyrosine phosphorylation, including phosphorylation of TrkB and neurogenesis in the hippocampus. Most importantly, these treatments also rapidly normalized behavior of mice with experimental MDD. Since phosphatidic acid binds to and inhibits PTP1B, the lack of phosphatidic acid results in increased activity of PTP1B and thereby in reduced tyrosine phosphorylation of TrkB and other cellular proteins. Thus, our data indicate a novel pathogenetic mechanism of and a rapidly acting targeted treatment for MDD.

Interaction of alcohol & phosphatidic acid in maternal rat uterine artery function.

Alcohol has been demonstrated to impair maternal uterine arterial adaptations in Fetal Alcohol Spectrum Disorder (FASD) animal models. However, the exact mechanism remains inconclusive. We hypothesized that phosphatidic acid (PA), a direct target of alcohol metabolism, would alleviate alcohol-induced vascular dysfunction of the maternal uterine artery. Mean fetal weight, and crown-rump length of the alcohol administered rats were ~9 % and 7.6 % lower than the pair-fed control pups, respectively. Acetylcholine (Ach)-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P < 0.05). Supplementation of 10-5 M PA reversed alcohol-induced vasodilatory deficit; no difference was detected after PA treatment between pair-fed control and alcohol groups (P = 0.37). There was a significant interaction between PA concentrations and alcohol exposure (PA X Alcohol effect, P < 0.0001). Pair-wise comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats, with % relaxation significantly improved at PA concentrations > 10-7 M (P < 0.05). Alcohol significantly reduced vasodilatory P-Ser1177 endothelial nitric oxide synthase (eNOS) levels in the uterine artery (↓90.7 %; P = 0.0029). PA treatment significantly reversed P-Ser1177 eNOS level in alcohol uterine arteries (153.7 %↑; P = 0.005); following ex vivo PA, there was no difference in P-Ser1177 eNOS levels between Control and Alcohol. Neither alcohol treatment nor PA affected total eNOS levels. Our data provide the first evidence of the interaction of alcohol and PA in rat maternal uterine artery vascular function and demonstrates PA's relationship with the eNOS system. Overall, the current study demonstrates that PA may be a promising therapeutic molecule of interest in alcohol-related gestational vascular dysfunction.

Phosphatidylcholine Enhances Homeostasis in Peach Seedling Cell Membrane and Increases Its Salt Stress Tolerance by Phosphatidic Acid.

Salt stress is a major adverse abiotic factor seriously affecting fruit tree growth and development. It ultimately lowers fruit quality and reduces yield. Phosphatidylcholine (PC) is an important cell membrane component that is critical for cell structure and membrane stability maintenance. In this study, we found that the addition of external PC sources significantly increased the tolerance of one-year-old peach trees, Prunus persica (L.) Batsch., to salt stress and attenuated their damage. The effect of exogenous application of 200 mg/L PC exerted the most significant positive effect. Its use caused seedling leaf stomatal opening, contributing to normal gas exchange. Moreover, beneficial effects were exerted also to the root system, which grew normally under salt stress. Meanwhile, phospholipase D activity in the cell was promoted. The production of phosphatidic acid (PA) was enhanced by increased decomposition of phospholipids; PA serves as a secondary messenger involved in plant biological process regulation and the reduction in the reactive oxygen species- and peroxide-induced damage caused by salt stress. The possible mechanism of action is via promoted plant osmotic regulation and tolerance to salt stress, reducing salt stress-induced injury to plants.

Lipid headgroup and side chain architecture determine manganese-induced dose dependent membrane rigidification and liposome size increase.

Metal ion-membrane interactions have gained appreciable attention over the years resulting in increasing investigations into the mode of action of toxic and essential metals. More work has focused on essential ions like Ca or Mg and toxic metals like Cd and Pb, whereas this study investigates the effects of the abundant essential trace metal manganese with model lipid systems by screening zwitterionic and anionic glycerophospholipids. Despite its essentiality, deleterious impact towards cell survival is known under Mn stress. The fluorescent dyes Laurdan and diphenylhexatriene were used to assess changes in membrane fluidity both in the head group and hydrophobic core region of the membrane, respectively. Mn-rigidified membranes composed of the anionic phospholipids, phosphatidic acid, phosphatidylglycerol, cardiolipin, and phosphatidylserine. Strong binding resulted in large shifts of the phase transition temperature. The increase was in the order phosphatidylserine > phosphatidylglycerol > cardiolipin, and in all cases, saturated analogues > mono-unsaturated forms. Dynamic light scattering measurements revealed that Mn caused extensive aggregation of liposomes composed of saturated analogues of phosphatidic acid and phosphatidylserine, whilst the mono-unsaturated analogue had significant membrane swelling. Increased membrane rigidity may interfere with permeability of ions and small molecules, possibly disrupting cellular homeostasis. Moreover, liposome size changes could indicate fusion, which could also be detrimental to cellular transport. Overall, this study provided further understanding into the effects of Mn with biomembranes, whereby the altered membrane properties are consequential to the proper structural and signalling functions of membrane lipids.

The effects of phosphatidic acid on performance and body composition - a scoping review.

Phosphatidic acid (PA) is a lipid mediator proposed to increase muscle protein synthesis via direct stimulation of the mammalian target of rapamycin (mTOR) and may act as an anabolic supplemental aid. Evidence on the effectiveness of PA as an anabolic supplement is equivocal. We aimed to systematically assess the effect of PA on performance and body composition. Due to the small number of studies, this is a scoping review. A comprehensive search was performed in Pubmed, SPORTDiscus and Web of Science, from the 1 January 2010 to the 31 August 2020. Our search retrieved 2009 articles, which when filtered, resulted in six studies, published between 2012 and 2019, which were analysed further. Five studies were performed in adult male populations and one in an elderly male population. From these, three studies suggested no effect of PA on lean body mass , while the remaining showed a possible positive effect (body composition and performance improvements). In one of these, the supplement included other potentially anabolic substances, precluding an isolated effect of PA. After a thorough analysis of the studies included, the evidence does not support the supplementation with PA to increase performance or improve body composition in young or elderly men.

Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro.

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA6) was predominantly expressed in HDLECs, a siRNA experiment against LPA6 attenuated the cPA-mediated tube formation. A synthetic LPA1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and ß-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA6/ROCK and LPA1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and ß-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.

Development of a selective fluorescence-based enzyme assay for glycerophosphodiesterase family members GDE4 and GDE7.

Lysophosphatidic acid (LPA) is a lipid mediator that regulates various processes, including cell migration and cancer progression. Autotaxin (ATX) is a lysophospholipase D-type exoenzyme that produces extracellular LPA. In contrast, glycerophosphodiesterase (GDE) family members GDE4 and GDE7 are intracellular lysophospholipases D that form LPA, depending on Mg2+ and Ca2+, respectively. Since no fluorescent substrate for these GDEs has been reported, in the present study, we examined whether a fluorescent ATX substrate, FS-3, could be applied to study GDE activity. We found that the membrane fractions of human GDE4- and GDE7-overexpressing human embryonic kidney 293T cells hydrolyzed FS-3 in a manner almost exclusively dependent on Mg2+ and Ca2+, respectively. Using these assay systems, we found that several ATX inhibitors, including α-bromomethylene phosphonate analog of LPA and 3-carbacyclic phosphatidic acid, also potently inhibited GDE4 and GDE7 activities. In contrast, the ATX inhibitor S32826 hardly inhibited these activities. Furthermore, FS-3 was hydrolyzed in a Mg2+-dependent manner by the membrane fraction of human prostate cancer LNCaP cells that express GDE4 endogenously but not by those of GDE4-deficient LNCaP cells. Similar Ca2+-dependent GDE7 activity was observed in human breast cancer MCF-7 cells but not in GDE7-deficient MCF-7 cells. Finally, our assay system could selectively measure GDE4 and GDE7 activities in a mixture of the membrane fractions of GDE4- and GDE7-overexpressing human embryonic kidney 293T cells in the presence of S32826. These findings allow high-throughput assays of GDE4 and GDE7 activities, which could lead to the development of selective inhibitors and stimulators as well as a better understanding of the biological roles of these enzymes.

The neuroprotective function of 2-carba-cyclic phosphatidic acid: Implications for tenascin-C via astrocytes in traumatic brain injury.

We examined the mechanism how 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, regulates neuronal apoptosis in traumatic brain injury (TBI). First, we found 2ccPA suppressed neuronal apoptosis after the injury, and increased the immunoreactivity of tenascin-C (TN-C), an extracellular matrix protein by 2ccPA in the vicinity of the wound region. 2ccPA increased the mRNA expression levels of Tnc in primary cultured astrocytes, and the conditioned medium of 2ccPA-treated astrocytes suppressed the apoptosis of cortical neurons. The neuroprotective effect of TN-C was abolished by knockdown of TN-C. These results indicate that 2ccPA contributes to neuroprotection via TN-C from astrocytes in TBI.

The SAC1 phosphatase domain of synaptojanin-1 is activated by interacting with polyunsaturated fatty acid-containing phosphatidic acids.

Although there are many phosphatidic acid (PA) molecular species based on its fatty acyl compositions, their interacting partners have been poorly investigated. Here, we identified synaptojanin-1 (SYNJ1), Parkinson's disease-related protein that is essential for regulating clathrin-mediated synaptic vesicle endocytosis via dually dephosphorylating D5 and D4 position phosphates from phosphatidylinositol (PI) (4,5)-bisphosphate, as a 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-PA-binding protein. SYNJ1 failed to substantially associate with other acidic phospholipids. Although SYNJ1 interacted with 18:0/20:4-PA in addition to 18:0/22:6-PA, the association of the enzyme with 16:0/16:0-, 16:0/18:1-, 18:0/18:0-, or 18:1/18:1-PA was not considerable. 18:0/20:4- and 18:0/22:6-PAs bound to SYNJ1 via its SAC1 domain, which preferentially hydrolyses D4 position phosphate. Moreover, 18:0/20:4- and 18:0/22:6-PA selectively enhanced the D4-phosphatase activity, but not the D5-phosphatase activity, of SYNJ1.

Enhancement of Cornified Envelope-Related Gene and Protein Expression by Carba Cyclic Phosphatidic Acid in Normal Human Epidermal Keratinocytes.

The aim of this study was to examine the effects of carba cyclic phosphatidic acid (ccPA) on cornified envelope (CE) formation and keratinocyte differentiation. ccPA-treated keratinocytes showed higher mRNA and protein levels of differentiation markers and CE components than untreated cells. These results suggest that ccPA could serve as therapeutic targets for treating skin barrier dysfunction because of their roles in upregulating genes and proteins associated with CE formation and keratinocyte differentiation.

Lysophosphatidic acid receptors 2 and 3 regulate erythropoiesis at different hematopoietic stages.

Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.

Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development.

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. Here we show that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. We propose that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes.

Expression of TNF­α and IL­ß can be suppressed via the PPAR­Î³/mTOR signaling pathway in BV­2 microglia: A potential anti­inflammation mechanism.

Currently, microglia are considered as crucial factors in suppressing inflammatory reactions, but the specific molecular mechanism remains unknown. To elucidate whether peroxisome proliferator­activated receptor­Î³ (PPAR­Î³) can inhibit neuroinflammatory cytokine expression via the mTOR signal pathway, the BV­2 cell line was incubated with lipopolysaccharide (10 mM/ml) to induce an inflammatory injury. PPAR­Î³ was activated by rosiglitazone, and was inhibited by GW9662. The mTOR signal pathway was activated by phosphatidic acid (P.A.), while it was inhibited by rapamycin. Western blotting and reverse transcription­quantitative PCR were used to evaluate the expression levels of PPAR­Î³/mTOR signal pathway related proteins and neuroinflammatory cytokines, including NF­κB, tumor necrosis factor (TNF)­α and interleukin (IL)­1ß. When treated with P.A., the expression levels of phosphorylated (p)mTOR and p­ribosomal protein S6 kinase (pS6K) were significantly increased and the expression levels of TNF­α and IL­1ß were significantly lower. However, the expression of PPAR­Î³ was similar in P.A. treated cells and cells treated with rapamycin. When PPAR­Î³ was activated, pmTOR and pS6K protein expression levels were significantly decreased, and the mRNA expression levels of TNF­α and IL­1ß were significantly reduced, but this inhibition could be alleviated by administrating GW9662. Collectively, it was indicated that the mTOR signal pathway may be located downstream of PPAR­Î³. Furthermore, neuroinflammatory reactions could be inhibited via the activation of PPAR­Î³ by suppressing the mTOR signal pathway in microglia.

Multiple basic amino acid residues contribute to phosphatidic acid-mediated inhibition of rice potassium channel OsAKT2.

Anionic phospholipid phosphatidic acid (PA) behaves as an important second messenger involved in many cellular processes, such as development, cytoskeletal dynamics, vesicle trafficking, and stress response. Recently, it was reported that PA can directly bind with the rice Shaker K+ channel OsAKT2 to inhibit its channel activity. Two adjacent arginine residues (R644 and R645) in ANK domain were identified as a PA-binding site essential to the PA-mediated inhibition of OsAKT2. However, there may be still other PA-binding sites unidentified in OsAKT2. Here, using a PA biosensor (PAleon), we found that the exogenous PA treatment significantly increased the PA level at the plasma membrane of Xenopus oocytes which were used to express OsAKT2 for electrophysiological assays. As reported previously, exogenous PA markedly inhibited OsAKT2 K+ currents. Replacement of two adjacent basic residues (R190 and K191) in the S4 voltage sensor by glycine completely abolished the time-dependent K+ currents of OsAKT2, but this variant was insensitive to PA treatment. In addition, we also identified other two adjacent arginines (R755 and R756) located in the cytosolic domain as a PA-binding site, which were also essential to the PA-mediated inhibition of OsAKT2. These results provide a more comprehensive understanding of the PA-K+ channel interaction mechanism. Combining the findings here with the previous study, we propose that multiple basic residues (R190/K191, R644/R645, and R755/R756) in different domains of OsAKT2 contribute to PA-mediated regulation of OsAKT2.