Cyclooxygenases and UCP1 Figure 2. Indomethacin prevents isoproterenol-induced UCP1 expression in Rb-negative adipocytes. Rb-negative mouse embryo fibroblasts were induced to differentiate as described in experimental procedures. Differentiated cells were treated with vehicle or isoproterenol (100 nM) and 9-cis-retinoic acid (1 mM) for 24 h. Indomethacin (1 mM) was included when indicated in the figure. A and D. RNA was isolated and expressions of genes were measured by RT-qPCR in duplicates and normalized to TBP. C. The levels of prostaglandin E2, F2a and 6-keto-prostaglandin F1a were determined in cell medium using ELISA kits after 24 h. B and E. Proteins were harvested and expressions of COX1, COX2 and UCP1 were measured by Western blotting. The bars represent mean 6 standard error. The experiments were performed in triplicates and performed 3–5 times. * indicates statistical significant difference (p,0.05). doi:10.1371/journal.pone.0011391.g002 adipocytes, but not in WT-1 cells and primary interscapular brown adipocytes, we again used Rb2/2 adipocytes as a model system for ‘‘brite’’ adipocytes. To investigate the relative importance of COX1 and COX2 activities in mediating induction of UCP1 expression in such cells, we treated Rb2/2 adipocytes with isoproterenol/9-cis retinoic acid in the absence and presence of selective COX1 and COX2 inhibitors. As shown in Figure 4A, selective inhibition of COX1 and COX2 with SC560 or NS398, respectively, partially prevented UCP1 induction, whereas a combination of these inhibitors or treatment with the non-selective inhibitor indomethacin fully prevented UCP1 induction. Accord- ingly, activities of both COX1 and COX2 seem necessary for full UCP1 induction. To further examine the relative importance of COX1 and COX2 for prostaglandin synthesis and UCP1 expression, these enzymes were retrovirally expressed both singly and in combi- nation in Rb2/2 MEFs (Figure 4B). The cells were induced to differentiate, and on day 8, the medium was replaced by fresh medium, which was harvested 24 h later and analyzed for PGE2 content. The level of PGE2 was higher when the cells were transduced with COX2 alone or in combination with COX1, than with COX1 alone (Figure 4C). These results, together with the fact that PGE2 formation in adipose tissue in COX2 KO mice is significantly lower than in COX1 KO mice [48], point to COX2 expression as being of major importance for PGE2 production. In accordance with this, forced expression of COX1 alone was unable to induce UCP1 expression (Figure 4D). However, UCP1 expression was significantly induced by forced expression of COX2 alone or in combination with COX1 (Figure 4D). Increased expression of UCP1 was accompanied with increased expression of PGC1a, Dio2, Cox8b, Eva1 and Cidea, as well as reduced expression of RIP140, but not 4E-BP1 (Figure 4D). Cold-induced UCP1 expression is attenuated in iWAT in COX2 KO mice To confirm the importance of COX2 for UCP1 induction in iWAT, wild-type and COX2 KO mice were challenged with a cold environment after warm acclimation. The wild-type mice defended their body temperature better than the COX2 KO mice (Figure 5A). The COX2 KO mice develop severe nephropathy and are susceptible to peritonitis in early life [49]; therefore, KO and wild-type littermates 6 weeks of age were used in this experiment. Unfortunately, we were unable to collect sufficient amounts of iWAT from these young mice to detect UCP1 or COX by Western blotting. However, as expected, cold-induced UCP1 mRNA expression was attenuated in iWAT in COX2 KO mice (Figure 5B). Cold-induced expression of Dio2 and Cidea was also attenuated in iWAT in the COX2 KO mice and PGC1a expression also tended to be attenuated (Figure 5B). Moreover, the cold-induced reduction of RIP140 expression was prevented in the COX2 KO mice (Figure 5B). Expression of Cox8b, Eva1 and 4E BP1 was, however, not significantly different in iWAT from wild-type and COX2 KO mice, suggesting that inhibition of both COX1 and COX2 might be necessary to attenuate cold-induced changes in the expression of these genes. As expected, we observed no differences in UCP1 expression in iBAT in COX2 KO and wild-type mice, and surprisingly, cold-treated COX2 KO mice had significantly higher expression of PGC1a in iBAT than did wild-type mice (Figure 5B). PGE2 induces UCP1 expression via activation of the EP3/ EP4 receptors PGE2 is reported to mediate its action by interacting with four subtypes of PGE receptors, the EP1, EP2, EP3 and EP4 receptors [50], but may also bind to the prostaglandin F (FP) receptor with an PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11391