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Background: Previous studies have demonstrated that cytosolic phospholipase A2α (cPLA2α) is required for NOX2 NADPH oxidase activation in human and mouse phagocytes. Moreover, upon stimulation, cPLA2α translocates to the plasma membranes by binding to the assembled oxidase, forming a complex between its C2 domain and the PX domain of the cytosolic oxidase factor, p47phox in human phagocytes. Intravenous administration of antisense against cPLA2α that significantly inhibited its expression in mouse peritoneal neutrophils and macrophages also inhibited superoxide production, in contrast to cPLA2α knockout mice that showed normal superoxide production. The present study aimed to determine whether there is a binding between cPLA2α-C2 domain and p47phox-PX in mouse macrophages, to further support the role of cPLA2α in oxidase regulation also in mouse phagocytes. Methods and results: A significant binding of mouse GST-p47phox-PX domain fusion protein and cPLA2α in stimulated mouse phagocyte membranes was demonstrated by pull-down experiments, although lower than that detected by the human p47phox-PX domain. Substituting the amino acids Phe98, Asn99, and Gly100 to Cys98, Ser99, and Thr100 in the mouse p47phox-PX domain (present in the human p47phox-PX domain) caused strong binding that was similar to that detected by the human p47phox-PX domain Conclusions: The binding between cPLA2α-C2 and p47phox-PX domains exists 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, Ser 99, and Thr100in the p47phox-PX domain that facilitate the binding to cPLA2α. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Colitis, an inflammation of the colon, is a well-characterized massive tissue injury. Cytosolic phospholipase A2α (cPLA2α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA2α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA2α and NF-κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP-R14, ICAM-1, COX-2, iNOS upregulation and LTB4 and TNF-α secretion. Prevention of cPLA2α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA2α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF-κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA2α upregulation in inflammation and development of murine colitis. [ABSTRACT FROM AUTHOR]

Amyloid-β peptides generated by proteolysis of the β-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2α (cPLA2α) plays a role in elevated APP protein expression induced by aggregated amyloid-β1-42 (Aβ) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aβ for 24 h and in cortical neuron of human amyloid-β1-42 brain infused mice. Prevention of cPLA2α up-regulation and its activity by oligonucleotide antisense against cPLA2α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2α in the signals leading to APP induction, increased cPLA2α expression and activity induced by Aβ was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aβ. Inhibition of elevated cPLA2α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aβ which coincided with cPLA2α dependent activation of CREB in the cortex of Aβ brain infused mice. Our results show that accumulation of Aβ induced elevation of APP protein expression mediated by cPLA2α, PGE2 release, and CREB activation via protein kinase A pathway. [ABSTRACT FROM AUTHOR]

In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A2α (cPLA2α) in this process. The 3-day HFD caused a significant upregulation of cPLA2α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA2α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD--induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA2α induction in the liver, mice were injected intraperitoneally with anti--intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA2α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD--induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA2α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding. [ABSTRACT FROM AUTHOR]

Cytosolic phospholipase A (cPLA) plays an important role in the development of several inflammatory diseases. The aim of the present study is to determine whether inhibition of cPLA expression, using specific antisense oligonucleotides against cPLA (antisense), is efficient in reducing inflammation after its development. Two mouse models of inflammation were included in the study: thioglicolate peritonitis and collagen-induced arthritis (CIA). The antisense was found to be specific and efficient in inhibiting cPLA expression and NADPH oxidase activity ex vivo in peritoneal phagocytes. Immunoblotting and immunohistochemistry analysis showed a significant elevation in cPLA expression in the inflamed joints of collagen-induced arthritis mice localized in cell infiltrate, chondrocytes and the surrounding skin and skeletal muscle. Similarly, the cPLA metabolite, leukotriene B, accumulated in the peritoneal cavity of mice with peritonitis. Inhibition of elevated cPLA expression after development of inflammation by intravenous administration of antisense resulted in a dramatic reduction in inflammation and a significant reduction in neutrophils recruitment to the site of inflammation in both mouse models of inflammation. Our results demonstrate the critical role of cPLA for the duration of inflammation and suggest that inhibition of cPLA expression by antisense oligonucleotides may serve as an efficient treatment of inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction.Methods: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction.Results: Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation.Conclusions: Our results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS. [ABSTRACT FROM AUTHOR]

Colitis, an inflammation of the colon, is a well-characterized massive tissue injury. Cytosolic phospholipase A2 α (cPLA2 α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA2 α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA2 α and NF-κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP-R14, ICAM-1, COX-2, iNOS upregulation and LTB4 and TNF-α secretion. Prevention of cPLA2 α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA2 α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF-κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA2 α upregulation in inflammation and development of murine colitis., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amyloid-β peptides generated by proteolysis of the β-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2 α (cPLA2 α) plays a role in elevated APP protein expression induced by aggregated amyloid-β1-42 (Aβ) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2 α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aβ for 24 h and in cortical neuron of human amyloid-β1-42 brain infused mice. Prevention of cPLA2 α up-regulation and its activity by oligonucleotide antisense against cPLA2 α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2 α in the signals leading to APP induction, increased cPLA2 α expression and activity induced by Aβ was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2 ) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aβ. Inhibition of elevated cPLA2 α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aβ which coincided with cPLA2 α dependent activation of CREB in the cortex of Aβ brain infused mice. Our results show that accumulation of Aβ induced elevation of APP protein expression mediated by cPLA2 α, PGE2 release, and CREB activation via protein kinase A pathway., (© 2014 International Society for Neurochemistry.)