Regulatory role of cytosolic phospholipase A(2) alpha in the induction of CD40 in microglia

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 A(2) alpha (cPLA(2)α) 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 cPLA(2)α 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 cPLA(2)α in the events leading to CD40 protein induction. RESULTS: Addition of LPS or IFNγ caused a significant upregulation of cPLA(2)α and of CD40, while prevention of cPLA(2)α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA(2)α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA(2)α detected by its phosphorylated form, while addition of IFNγ induced cPLA(2)α activation at a later time scale (4 h). The activation of cPLA(2)α is mediated by ERK activity. Suppression of cPLA(2)α 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 cPLA(2)α activation, suggesting cPLA(2)α is located upstream to NOX2 and NF-κB. The activation of cPLA(2) by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA(2)α 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 cPLA(2)α inhibitors, indicating that STAT1α is not under cPLA(2)α regulation. CONCLUSIONS: Our results show for the first time that cPLA(2) 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 cPLA(2)α may serve as a pivotal amplifier of the inflammatory response in the CNS.