Furthermore, the PGD2 concentration in rat cerebrospinal fluid shows a circadian shift coupled to the sleep wake cycle. To confirm whether 15d PGJ2 is an authentic endogenous entrain ment factor, we examined the expression profiles of clock genes in NIH3T3 fibroblast cells stimulated by 15d PGJ2. Per2 and Bmal1 BET bromodomain inhibitor expression patterns were examined by quantitative real time RT PCR at 4 h intervals for duration of 56 h and rhythmic expressions were observed when treated for 1 h with 15d PGJ2 and with high concentration serum, but not when treated with DMSO as a control. Moreover, phases of Per2 and Bmal1 mRNA expression triggered by 15d PGJ2 treatment were antiphasic with respect to each other, which is consistent with those trig gered by serum and with previously reported expression profiles.
Taken together, these results demonstrate that 15d PGJ2 can act as an in vitro entrainment factor for circadian clocks. PGD2 and other prostaglandins and prostanoids examined in this study showed no rhythmic fluctuation of luciferase activity. The facts that 15d PGJ2s precursor PGD2 has been recognized as the most potent endogenous sleep promoting sub stance and that the PGD2 concentration in rat cerebrospi nal fluid shows a circadian change coupled to the sleep wake cycle, have led to the hypothesis that 15d PGJ2 may act as an endogenous circadian entrainment factor in vivo. It would be interesting to see the effects of 15d PGJ2 in vivo. However, it should be noted that the endogenous concentration of 15d PGJ2 is extremely low, com pared with the one used for the in vitro screening.
15d PGJ2 up regulates Cry1, Cry2, and Ror mRNA expressions To examine which clock genes are induced by 15d PGJ2 treatment, we systematically quantified the expression levels of the canonical clock genes. After the isolation of total RNA at 1 h intervals from NIH3T3 cells stimulated by 15d PGJ2, quantitative real time RT PCR was per formed using primers for Per1, Per2, Per3, Bmal1, Npas2, Cry1, Cry2, Dec1, Dec2, E4bp4, Dbp, and Ror by using low density arrays. Unexpectedly, stimulation with 15d PGJ2 did not affect a transient Per1 and Per2 mRNA accu mulation, although both Per genes are known to be transiently accumulated by the various stimuli of entrainment. On the other hand, we for the first time found that 15d PGJ2 induced accumulation of Cry1 and Cry2 transcripts, as well as Ror mRNA, which is consistent with a previous report.
Entrainment triggered by 15d PGJ2 is independent of PPAR signaling pathway We next sought to identify entrainment signaling path ways triggered by 15d PGJ2. Since 15d PGJ2 has been known to be a natural ligand of the peroxisome prolifera tor activated receptor, AV-951 we assessed whether the clock gene expression triggered by 15d PGJ2 is dependent on the PPAR mediated signaling pathway.
No related posts.