title: A dual-color luciferase assay system reveals circadian resetting of cultured fibroblasts by co-cultured adrenal glands.
abstract: In mammals, circadian rhythms of various organs and tissues are synchronized by pacemaker neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus. Glucocorticoids released from the adrenal glands can synchronize circadian rhythms in other tissues. Many hormones show circadian rhythms in their plasma concentrations; however, whether organs outside the SCN can serve as master synchronizers to entrain circadian rhythms in target tissues is not well understood. To further delineate the function of the adrenal glands and the interactions of circadian rhythms in putative master synchronizing organs and their target tissues, here we report a simple co-culture system using a dual-color luciferase assay to monitor circadian rhythms separately in various explanted tissues and fibroblasts. In this system, circadian rhythms of organs and target cells were simultaneously tracked by the green-emitting beetle luciferase from Pyrearinus termitilluminans (ELuc) and the red-emitting beetle luciferase from Phrixothrix hirtus (SLR), respectively. We obtained tissues from the adrenal glands, thyroid glands, and lungs of transgenic mice that expressed ELuc under control of the promoter from a canonical clock gene, mBmal1. The tissues were co-cultured with Rat-1 fibroblasts as representative target cells expressing SLR under control of the mBmal1 promoter. Amplitudes of the circadian rhythms of Rat-1 fibroblasts were potentiated when the fibroblasts were co-cultured with adrenal gland tissue, but not when co-cultured with thyroid gland or lung tissue. The phases of Rat-1 fibroblasts were reset by application of adrenal gland tissue, whereas the phases of adrenal gland tissue were not influenced by Rat-1 fibroblasts. Furthermore, the effect of the adrenal gland tissue on the fibroblasts was blocked by application of a glucocorticoid receptor (GR) antagonist. These results demonstrate that glucocorticoids are strong circadian synchronizers for fibroblasts and that this co-culture system is a useful tool to analyze humoral communication between different tissues or cell populations.
authors.affiliation: Health Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Noguchi
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ikeda
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakajima
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002450:Cell Communication / Q000502:physiology; D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology*; D018920:Coculture Techniques / Q000379:methods; D003116:Color; D003344:Cortical Synchronization / Q000235:genetics / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008156:Luciferases / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013493:Suprachiasmatic Nucleus / Q000378:metabolism / Q000502:physiology
chemical_list: D056950:Period Circadian Proteins; D008156:Luciferases