Janus Kinase 3 Activity Is Necessary for Phosphorylation of Cytosolic Phospholipase A2 and Prostaglandin E2 Synthesis by Macrophages Infected with Francisella tularensis Live Vaccine Strain

Abstract

Francisella tularensis, the causative agent of tularemia, modulates the host immune response to gain a survival advantage within the host. One mechanism of immune evasion is the ability of F. tularensis to induce the synthesis of the small lipid mediator prostaglandin E2 (PGE 2 ), which alters the host T cell response making the host more susceptible to Francisella growth. PGE 2 is synthesized by a tightly regulated biosynthetic pathway following stimulation. The synthesis of PGE 2 begins with the liberation of arachidonic acid (AA) from membrane phospholipids by cytosolic phospholipase A2 (cPLA 2 ). AA is subsequently converted to the unstable intermediate PGH 2 by cyclooxygenase-2 (COX-2), and PGH 2 undergoes an isomerization reaction to generate PGE 2 . Our objective was to identify F. tularensis-activated host signaling pathways that regulate the activity of the enzymes in the PGE 2 -biosynthetic pathway. In this study, we show that cPLA 2 , p38 mitogen-activated protein kinase (MAPK), and Janus kinase 3 (JAK3) signaling are necessary for F. tularensis-induced PGE 2 production. Inhibition of JAK3 activity reduced the phosphorylation of cPLA 2 and COX-2 protein levels. In addition, JAK3 regulates cPLA 2 phosphorylation independent of transcription. Moreover, p38 MAPK activity is required for F. tularensis-induced COX-2 protein synthesis, but not for the phosphorylation of cPLA 2 . This research highlights a unique signaling axis in which JAK3 and p38 MAPK regulate the activity of multiple enzymes of the PGE 2 -biosynthetic pathway in macrophages infected with F. tularensis.

Differential regulation of Janus kinase 3 (JAK3) in bovine preovulatory follicles and identification of JAK3 interacting proteins in granulosa cells

“…JAK3 has been shown to phosphorylate insulin receptor substrate-1 (IRS-1), IRS-2, PI3K/Akt and focal adhesion kinase (FAK) [38], which contain SH2 or other phospho-tyrosine-binding domains. Other studies have used Drosophila to investigate the JAK/STAT pathway in ovarian cell migration, sex determination and stem-cell maintenance [39, 40], and in macrophages infected with F. tularensis, JAK3 is involved in the phosphorylation of p38MAPK [41]. Herein, we observed that JAK3 strongest expression was found in the dominant follicle, which is active and growing while in the ovulatory follicle, following the endogenous LH surge or hCG injection, JAK3 expression was dramatically reduced.…”

Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

“…PGH 2 is isomerized to PGE 2 by microsomal prostaglandin E synthase (mPGES1). We previously demonstrated that F. tularensis-induced PGE 2 synthesis by macrophages requires cPLA 2 , COX-2, and mPGES1 (8,18), yet we observed a temporal disconnect between cPLA 2 activation and the time at which increases in COX-2 protein and PGE 2 synthesis are observed. AA release and utilization by COX-2 are highly coupled events, and this apparent disconnect between cPLA 2 activation and the increase in COX-2 protein led us to hypothesize that an alternative pathway utilizing other phospholipases was required for supplying AA to the inducible PGE 2 biosynthetic pathway in F. tularensis-infected macrophages.…”

“…We have previously demonstrated the requirement of cPLA 2 for F. tularensis LVS-induced PGE 2 synthesis; however, the observed temporal disconnect between cPLA 2 activation and detectable PGE 2 synthesis suggested that cPLA 2 was not supplying AA that was being converted into PGE 2 in these infected macrophages (18). Our results demonstrating that cPLA 2 inhibition only partially blocks late AA release or that the addition of exogenous AA does not restore PGE 2 biosynthesis by cPLA 2 -inhibited infected macrophages would support this model.…”

“…We also established that the activities of the major enzymes in the inducible PG biosynthetic pathway (cPLA 2 , COX-2, and mPGES1) are indispensable for F. tularensis LVS-induced PGE 2 biosynthesis (8,18). However, there is a temporal disconnect between the activation of cPLA 2 (via phosphorylation) and COX-2 protein increases during F. tularensis LVS infection.…”

“…We previously demonstrated that Ser505 cPLA 2 phosphorylation peaks and returns to baseline (2 hours after inoculation) before either an increase in COX-2 protein (ϳ4 h after inoculation) or detectable biosynthesis of PGE 2 (ϳ10 to 12 h after inoculation) in F. tularensis LVS-infected macrophages (18). Since cPLA 2 phosphorylation was not measured more than 2 h after inoculation in our previous study, the possibility for a biphasic pattern of cPLA 2 phosphorylation that corresponds with PGE 2 biosynthesis still remains.…”