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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.035048

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Ligand modulation of REV-ERB function resets the peripheral circadian clock in a phasic manner

David Ray^2,

Andrew Loudon^1,

¹ Faculty of Life Sciences, A. V. Hill Building, Oxford Road, Manchester M13 9PT, UK
² Medical and Human Sciences, A. V. Hill Building, Oxford Road, Manchester M13 9PT, UK
³ GlaxoSmithKline, 5 Moore Drive, P.O. Box 13398, Research Triangle Park, NC 27709, USA
⁴ GlaxoSmithKline, Discovery Biology, Respiratory CEDD, GSK Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK

View larger version (16K): [in this window] [in a new window]   Fig. 1. Identification of a ligand that activates recruitment of NCoR to REV-ERB. (A) Functional EC50 determination of the REV-ERB ligand. Intensities of the FRET signals following addition of the compound at different concentrations are quantified and normalized to the basal level. Data are shown as mean ± s.e.m. for three independent experiments. (B) Effects of the REV-ERB ligand on Pai1 transcription. Rat-1 cells were transiently transfected with the Pai1::Luc reporter construct. 48 hours later, cells were treated with the compound or DMSO (as control) for 1 hour or 24 hours. Cell lysates were then prepared and bioluminescence recorded using a luminometer. Four replicates were analyzed for each treatment group, and experiments were performed three times. Results are shown as mean ± s.e.m. *P<0.05, Student's t-test.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Rhythmic transcription profiles and protein expression of core clock genes in Rat-1 cells. (A) Rat-1 fibroblasts that were stably transfected with different reporter constructs (Bmal1::Luc, Per2::Luc and Rev-erb::Luc) were synchronized by treatment with 200 nM dexamethasone (Dex) for 1 hour. Cells were then switched to recording medium and bioluminescence (photon counts per minute) was recorded using photon-multiplier tubes. (B) Phase relationships with respect to synchronization were depicted after the data were de-trended by 24 hour moving average. (C) Damped oscillation of Bmal1::Luc Rat-1 cells (synchronized by 50% horse serum) can be re-initiated by medium change, treatment with Dex, or a combination of both. As expected, Dex treatment induced a phase-delay compared with medium-change alone. Arrow shows the time of addition of Dex and/or medium change. (D) Representative western blot (WB) of REV-ERB in Rat-1 cells synchronized by 200 nM Dex and harvested at 4-hour intervals. The time points indicated are relevant to the time of Dex treatment. This experiment was repeated three times.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Phase-resetting effects on the molecular oscillators by the REV-ERB ligand. Individual cultures of confluent Rat-1 cells stably transfected with Per2::Luc reporter, or primary lung fibroblasts from PER2::Luc mice were synchronized by Dex for 1 hour and monitored using PMT. Cells were then treated with the compound (10 µM, red) at a single phase point for each culture dish. Control cultures were treated with DMSO (black). (A,B,D,E) Representative traces of PMT real-time recording. Compound addition is indicated by vertical arrows and phase changes by horizontal arrows. (C,F) Ligand-induced phase-response curves (PRCs). For the PRC estimates, actual phase changes were plotted against circadian time, with the peak of PER2 expression taken as CT12. Data shown are representative of three independent experiments. Negative values indicate phase delays, whereas positive values refer to phase advances.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Bi-directional phase shifts induced by the REV-ERB ligand on Bmal1::Luc Rat-1 cells or PER2::Luc lung slices. Individual cultures of confluent Rat-1 cells stably transfected with Bmal1::Luc reporter or ectopic lung slices from PER2::Luc mice were synchronized and subject to PMT recording. Cells or tissue slices were then treated with the compound (10 µM, red) or DMSO (black) at the indicated circadian time. Representative phase changes (horizontal arrow) after compound treatment (indicated by a vertical arrow) are shown in A and B (for Bmal1::Luc Rat-1), and D and E (for PER2::Luc lung slices). (C,F) Phase changes following the compound treatment are shown as mean ± s.e.m. n=3 for Bmal1::Luc Rat-1 cells, n=5 for the lung slices.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Hypothesis for the mode of action of the ligand. Application of REV-ERB ligand to the circadian cycle of REV-ERB protein (A, green) causes the early decline of Bmal1 levels (black). At phase X in A (red box), this leads to an advance in the next Bmal1 cycle (red in B), as demonstrated in Fig. 4B. At phase Y (blue box), a delay results from the same treatment (blue in B), as seen in Fig. 4A.

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