Involvement of the Jak/STAT pathway in platelet-activating factor receptor signaling
Platelet-activating factor (PAF) is a potent phospholipid mediator involved in a variety of pathophysiological events, such as inflammation, asthma, cardiovascular disease, reproduction, and cerebral ischemia. PAF activates multiple signaling pathways and triggers a diverse array of biological actions by interacting with a specific receptor that belongs to the G-protein coupled receptor family (GPCR). Initially we were interested in determining whether PAF could activate the Jak/STAT pathway. This signaling cascade is known to be stimulated by cytokines and growth factors, however, recent reports indicate that it is also implicated in GPCR-mediated signal transduction. We found that PAF induced rapid Jak2 and Tyk2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 and in COS-7 cells transiently transfected with PAFR and Tyk2 or Jak2 cDNAs. Tyk2 activation in MonoMac-1 cells was rapid and declined to basal levels after 5 min of PAF treatment, while Jak2 phosphorylation was sustained for a longer period. In COS-7 cells, transiently expressing PAFR and Jaks, Jak2 tyrosine phosphorylation was comparable to that in MonoMac-1 cells, whereas the increase in the level of tyrosine phosphorylation of Tyk2 was lower. In MonoMac-1 cells, PAF-induced activation of kinases was followed by transient tyrosine phosphorylation of STAT1, 2, 3 and sustained phosphorylation of STAT5 and subsequent STAT1 and STAT3 translocation to the nucleus. In a reconstituted system, PAF-mediated STAT1 and STAT3 nuclear translocation required the presence of Tyk2. PAF is known to upregulate its own receptor in certain cells. To explore the role of the Jak/STAT pathway in regulation of PAFR transcription, we used a PAFR promoter 1 construct that consists of several putative STAT-binding sites. Our results showed that Tyk2 was obligatory for PAF-stimulated PAFR promoter 1 activation. To study the mechanisms of Tyk2-dependent PAFR transcription we used mutants with impaired G-protein coupling and C-terminal deletion mutants of the receptor. We also created minigene constructs encoding the intracellular loops of PAFR and investigated their ability to inhibit signaling. We found that mutant receptors D63N, D289A, Y293A, which do not couple to G-proteins, were capable of inducing Tyk2-dependent PAFR promoter activation. We also determined that the PAFR second intracellular loop and the C-terminus of the receptor are important for Tyk2-dependent-PAFR transcription.