Suppression of interleukin-2 secretion by 15 deoxy-prostaglandin J2-glycerol, a putative cyclooxygenase-2 metabolite of 2-arachidonyl glycerol, involves the activation of peroxisome proliferator activated receptor gamma

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative capable of suppressing interleukin (IL)-2 production by activated T cells. 2-AG-mediated IL-2 suppression is dependent on cyclooxygenase-2 (COX-2) metabolism and peroxisome proliferator activated receptor gamma (PPARgamma) activation. The overall goal of this project was to determine the mechanism of IL-2 suppression by 2-AG by testing the following hypothesis: 15d-PGJ2-G, a putative COX-2 metabolite of 2-AG, suppresses IL-2 secretion, in part, by the activation of PPARgamma and involves modulation of nuclear factor of activated T cells (NFAT) activity. NFAT is a critical transcription factor in IL-2 gene transcription. Because COX-2 metabolism of 2-AG is important for IL-2 suppression, the effect of 2-AG on COX-2 and PPARgamma mRNA expression was investigated. In both activated Jurkat cells and primary murine splenocytes, COX-2 protein expression was upregulated (as early as 2 h and 4 h respectively after activation). 2-AG treatment decreased the upregulation of COX-2 mRNA following T cell activation which suggests negative feedback limiting COX-2 mediated metabolism of 2-AG. PPARgamma mRNA expression was increased upon activation and 2-AG treatment produced a modest decrease in PPARgamma mRNA expression. 15d-PGJ2-G bound PPARgamma-LBD in a PPARgamma competitive binding assay. 15d-PGJ 2-G treatment suppressed IL-2 production by activated Jurkat cells, which was partially attenuated when pretreated with T0070907. Concordant with IL-2 suppression, 15d-PGJ2-G treatment decreased nuclear factor of activated T cells (NFAT) transcriptional activity in transiently transfected Jurkat cells. Interestingly, T0070907 alone markedly increased NFAT reporter activity suggesting the existence of endogenous PPARgamma activation and modulation of NFAT. 15d-PGJ2-G treatment decreased PMA/Io-stimulated NFAT DNA binding to the human IL-2 promoter and nuclear NFAT2 accumulation. Interestingly, 15d-PGJ2-G treatment increased nuclear active HDM2 (human homolog of the oncoprotein and E3 ubiquitin ligase murine double minute 2 -- MDM2) expression whereas there was no change in the expression of glycogen synthase kinase 3 " (GSK-3"), both of which regulate NFAT. 15d-PGJ 2-G and other PPARgamma agonists, such as rosiglitazone (RGZ) and ciglitazone (CGZ), decreased PMA/Io-mediated elevation in intracellular calcium in activated Jurkat cells. Surprisingly, the PPARgamma antagonists, T0070907 and GW9662, also decreased the PMA/Io-mediated elevation in intracellular calcium in activated Jurkat cells. In addition, the presence of T0070907 plus 15d-PGJ2-G (and other PPARgamma agonists such as RGZ and CGZ) produced an additive decrease in PMA/Io-mediated elevation of intracellular calcium, suggesting that 15d-PGJ2-G effects on calcium might be PPARgamma independent. Collectively, our findings suggest that 15d-PGJ 2-G activates PPARgamma to decrease nuclear NFAT2 accumulation (via active HDM2) leading to decreased NFAT transcriptional activity eventually causing IL-2 suppression in activated T cells.