A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

Early upregulation of cytosolic phospholipase A2α in motor neurons is induced by misfolded SOD1 in a mouse model of amyotrophic lateral sclerosis

Background Amyotrophic lateral sclerosis (ALS) is a fatal multifactorial neurodegenerative disease characterized by the selective death of motor neurons. Cytosolic phospholipase A2 alpha (cPLA2α) upregulation and activation in the spinal cord of ALS patients has been reported. We have previously shown that cPLA2α upregulation in the spinal cord of mutant SOD1 transgenic mice (SOD1G93A) was detected long before the development of the disease, and inhibition of cPLA2α upregulation delayed the disease’s onset. The aim of the present study was to determine the mechanism for cPLA2α upregulation. Methods Immunofluorescence analysis and western blot analysis of misfolded SOD1, cPLA2α and inflammatory markers were performed in the spinal cord sections of SOD1G93A transgenic mice and in primary motor neurons. Over expression of mutant SOD1 was performed by induction or transfection in primary motor neurons and in differentiated NSC34 motor neuron like cells. Results Misfolded SOD1 was detected in the spinal cord of 3 weeks old mutant SOD1G93A mice before cPLA2α upregulation. Elevated expression of both misfolded SOD1 and cPLA2α was specifically detected in the motor neurons at 6 weeks with a high correlation between them. Elevated TNFα levels were detected in the spinal cord lysates of 6 weeks old mutant SOD1G93A mice. Elevated TNFα was specifically detected in the motor neurons and its expression was highly correlated with cPLA2α expression at 6 weeks. Induction of mutant SOD1 in primary motor neurons induced cPLA2α and TNFα upregulation. Over expression of mutant SOD1 in NSC34 cells caused cPLA2α upregulation which was prevented by antibodies against TNFα. The addition of TNFα to NSC34 cells caused cPLA2α upregulation in a dose dependent manner. Conclusions Motor neurons expressing elevated cPLA2α and TNFα are in an inflammatory state as early as at 6 weeks old mutant SOD1G93A mice long before the development of the disease. Accumulated misfolded SOD1 in the motor neurons induced cPLA2α upregulation via induction of TNFα.

Singapore Grouper Iridovirus Disturbed Glycerophospholipids Homeostasis: Cytosolic Phospholipase A2 Was Essential for Virus Replication

Singapore grouper iridovirus (SGIV), belonging to genus Ranavirus, family Iridoviridae, causes great economic losses in the aquaculture industry. Previous studies demonstrated the lipid composition of intracellular unenveloped viruses, but the changes in host-cell glyceophospholipids components and the roles of key enzymes during SGIV infection still remain largely unknown. Here, the whole cell lipidomic profiling during SGIV infection was analyzed using UPLC-Q-TOF-MS/MS. The lipidomic data showed that glycerophospholipids (GPs), including phosphatidylcholine (PC), phosphatidylserine (PS), glycerophosphoinositols (PI) and fatty acids (FAs) were significantly elevated in SGIV-infected cells, indicating that SGIV infection disturbed GPs homeostasis, and then affected the metabolism of FAs, especially arachidonic acid (AA). The roles of key enzymes, such as cytosolic phospholipase A2 (cPLA2), 5-Lipoxygenase (5-LOX), and cyclooxygenase (COX) in SGIV infection were further investigated using the corresponding specific inhibitors. The inhibition of cPLA2 by AACOCF3 decreased SGIV replication, suggesting that cPLA2 might play important roles in the process of SGIV infection. Consistent with this result, the ectopic expression of EccPLA2α or knockdown significantly enhanced or suppressed viral replication in vitro, respectively. In addition, the inhibition of both 5-LOX and COX significantly suppressed SGIV replication, indicating that AA metabolism was essential for SGIV infection. Taken together, our results demonstrated for the first time that SGIV infection in vitro disturbed GPs homeostasis and cPLA2 exerted crucial roles in SGIV replication.

Characterization the binding of cytosolic phospholipase A2 alpha and NOX2 NADPH oxidase in mouse macrophages

Background: Previous studies have demonstrated that Cytosolic phospholipase A2a (cPLA2a) is absolutely required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2a translocates to the plasma membranes of by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the oxidase cytosolic factor, p47phox in human phagocytes. Intravenous administration of an antisense against cPLA2a that significantly inhibited its expression in mouse peritoneal neutrophil and macrophages also inhibited superoxide production, in contrast to cPLA2a knockout mice that showed normal superoxide production. The aim of the present study was to determine whether there is a binding between cPLA2a-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2a in oxidase regulation also in mouse phagocytes. Methods and Results: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2a in stimulated mouse phagocyte membranes was demonstrated by pull down experiments, although lower than that detected by human p47phox-PX domain. Substituting the amino acids Phe98, Asn99 and Gly100 to Cys98 Ser99 and Thr100 in mouse p47phox-PX domain (that are present in human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain. Conclusions: the binding between cPLA2a-C2 and p47phox-PX domains exist in mouse macrophages and is not unique to human phagocytes. The binding between the two proteins is lower in the mice probably due to the absence of amino acids Cys98 Ser99 and Thr100 in p47phox-PX domain that facilitate the binding to cPLA2a.

CDK5 mediated phosphorylation of cytosolic phospholipase A2 regulates its activity and neuroinflammation in Parkinsons Disease

Hyperactivation of cyclin-dependent kinase 5 (CDK5) by p25, contributes to neuroinflammation causing neurodegeneration in Parkinsons Disease (PD) and Alzheimer diseases (AD). However, the mechanism by which CDK5 induces neuroinflammation in the PD brain is largely unexplored. Here, we show that CDK5 phosphorylates cytosolic phospholipase A2 (cPLA2) at Thr-268 and Ser-505 sites lead to its activation and generation of eicosanoid products. Mutational studies using site-directed mutagenesis and molecular simulations show that the architecture of the protein changes upon each single-point mutation. Interestingly, double-mutations also led to severe decline in the activity of cPLA2 and disruption of its translocation to the plasma membrane. Further, the brain lysates of transgenic PD mouse models show hyperactivation of CDK5 resulting in enhanced phosphorylation of Thr-268 and Ser-505 of cPLA2 and its heightened activity confirming the findings observed in the cell culture model of PD. These phosphorylation sites of cPLA2 and CDK5 could be explored as the future therapeutic targets against neuroinflammation in PD. Further, conjoint transcriptomic analysis of the publicly available human PD datasets strengthens the hypothesis that genes of the arachidonic acid, prostaglandin synthesis and inflammatory pathways are significantly upregulated in the case of the PD patients as compared to that of healthy controls.

Cytosolic Phospholipase A2 is Required for Fexofenadine′s Therapeutic Effects Against Inflammatory Bowel Disease in Mice

Inflammatory Bowel Disease (IBD) is an autoimmune condition with complicated pathology and diverse clinical signs. TNFα is believed to play a crucial role in the pathogenesis of IBD. We recently identified fexofenadine, a well-known antagonist of histamine H1 receptor, as a novel inhibitor of TNFα signaling. Additionally, cytosolic phospholipase A2 (cPLA2) was isolated as a binding target of fexofenadine, and fexofenadine-mediated anti-TNF activity relied on cPLA2 in vitro. The objective of this study is to determine whether fexofenadine is therapeutic against chemically-induced murine IBD model and whether cPLA2 and/or histamine H1 receptor is important for fexofenadine’s anti-inflammatory activity in vivo by leveraging various genetically modified mice and chemically induced murine IBD models. Both dextran sulfate sodium- and 2, 4, 6-trinitrobenzene sulfonic acid-induced murine IBD models revealed that orally delivered fexofenadine was therapeutic against IBD, evidenced by mitigated clinical symptoms, decreased secretions of the proinflammatory cytokine IL-6 and IL-1β, lowered intestinal inflammation, and reduced p-p65 and p-IĸBα. Intriguingly, Fexofenadine-mediated protective effects against IBD were lost in cPLA2 deficient mice but not in histamine H1 receptor-deficient mice. Collectively, these findings demonstrate the therapeutic effects of over-the-counter drug Fexofenadine in treating DSS-induced IBD murine and provide first in vivo evidence showing that cPLA2 is required for fexofenadine’s therapeutic effects in murine IBD model and probably other inflammatory and autoimmune diseases as well.

Abstract MP63: Cytochrome P450 1B1 Mediated Inhibition Of Group IV Cytosolic Phospholipase A 2 α In Paraventricular Nucleus Protects Female Mice From Angiotensin II-Induced Hypertension, Increased Renal Sympathetic Activity And Proteinuria

Recently we showed that 2-methoxyestradiol (2-ME), an estrogen (E2) metabolite generated by CYP1B1 (cytochrome P450 1B1) in the paraventricular nucleus (PVN), protects female mice from Ang (angiotensin) II-induced hypertension and increased renal sympathetic activity. We also demonstrated that group IV cPLA 2 α (cytosolic phospholipase A 2 α) in the brain contributes to Ang II-induced hypertension in male mice. This study was conducted to determine the contribution of central cPLA 2 α and its relationship to CYP1B1 in Ang II-induced hypertension in female mice. cPLA 2 α knockdown in the PVN by transduction with adenovirus (Ad)-cPLA 2 α-short hairpin (sh)RNA (200 nL, bilaterally, 1.0х10 12 pfu/mL) but not its control Ad-scrambled (Scr)-shRNA (2.5х10 11 pfu/mL) in ovariectomized (OVX) wild-type ( cPLA 2 α +/+ / Cyp1b1 +/+ , n=8/group) and intact cPLA 2 α +/+ / Cyp1b1 -/- (n=12/group) female mice attenuated the effect of Ang II (700 ng/kg/min, subcutaneous, osmotic pump, 2 weeks) to increase the systolic blood pressure (SBP, mmHg) as measured by tail-cuff (Day 12: 129±3 vs 168±7 and 119±3 vs 172±5, respectively, P<0.05). Moreover, reconstitution of cPLA 2 α in the PVN by transduction with Ad-cPLA 2 α-DNA (1.1х10 12 pfu/mL) but not its control Ad-GFP-DNA (1.0х10 11 pfu/mL) in OVX- cPLA 2 α -/- / Cyp1b1 +/+ mice (n=4/group) restored the effect of Ang II to increase SBP (Day 12: 163±7 vs 124±4, P<0.05). Furthermore, Ad-cPLA 2 α-shRNA but not Ad-Scr-shRNA transduction in the PVN in OVX- cPLA 2 α +/+ / Cyp1b1 +/+ and intact cPLA 2 α +/+ / Cyp1b1 -/- mice reduced and Ad-cPLA 2 α-DNA but not Ad-GFP-DNA transduction in the PVN in OVX- cPLA 2 α -/- / Cyp1b1 +/+ mice restored the effect of Ang II to increase the renal sympathetic activity as indicated by urinary norepinephrine level (ng/mL, 324±36 vs 707±94, 359±49 vs 979±70, 690±44 vs 421±43, respectively, n=4/group, P<0.05) and proteinuria (mg/24 hour, 4±1 vs 10±0.4, 3±0.4 vs 7±1, 9±0.8 vs 3±0.7, respectively, n=4/group, P<0.05). These data suggest that E2-CYP1B1 derived metabolite 2-ME protects against Ang II-induced hypertension, renal sympathetic activity, and proteinuria by inhibiting cPLA 2 α activity in the PVN. Thus, 2-ME and/or agents inhibiting cPLA 2 α activity could be useful for treating hypertension and its pathogenesis in females.