Selective inhibition of human group IIA-secreted phospholipase A2 (hGIIA) signaling reveals arachidonic acid metabolism is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes.

Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.

A bifunctional role for group IIA secreted phospholipase A2 in human rheumatoid fibroblast-like synoviocyte arachidonic acid metabolism.

Human group IIA-secreted phospholipase A(2) (sPLA(2)-IIA) is an important regulator of cytokine-mediated inflammatory responses in both in vitro and in vivo models of rheumatoid arthritis (RA). However, treatment of RA patients with sPLA(2)-IIA inhibitors shows only transient benefit. Using an activity-impaired sPLA(2)-IIA mutant protein (H48Q), we show that up-regulation of TNF-dependent PGE(2) production and cyclooxygenase-2 (COX-2) induction by exogenous sPLA(2)-IIA in RA fibroblast-like synoviocytes (FLSs) is independent of its enzyme function. Selective cytosolic phospholipase A(2)-α (cPLA(2)-α) inhibitors abrogate TNF/sPLA(2)-IIA-mediated PGE(2) production without affecting COX-2 levels, indicating arachidonic acid (AA) flux to COX-2 occurs exclusively through TNF-mediated activation of cPLA(2)-α. Nonetheless, exogenous sPLA(2)-IIA, but not H48Q, stimulates both AA mobilization from FLSs and microparticle-derived AA release that is not used for COX-2-dependent PGE(2) production. sPLA(2)-IIA-mediated AA production is inhibited by pharmacological blockade of sPLA(2)-IIA but not cPLA(2)-α. Exogenous H48Q alone, like sPLA(2)-IIA, increases COX-2 protein levels without inducing PGE(2) production. Unlike TNF, sPLA(2)-IIA alone does not rapidly mobilize NF-κB or activate phosphorylation of p38 MAPK, two key regulators of COX-2 protein expression, but does activate the ERK1/2 pathway. Thus, sPLA(2)-IIA regulates AA flux through the cPLA(2)-α/COX-2 pathway in RA FLSs by up-regulating steady state levels of these biosynthetic enzymes through an indirect mechanism, rather than direct provision of substrate to the pathway. Inhibitors that have been optimized for their potency in enzyme activity inhibition alone may not adequately block the activity-independent function of sPLA(2)-IIA.