Background: Clock genes are expressed in different peripheral organs. Results: Rhythmic expression was lost with Ihh in the growth plate from mice defective of BMAL1 with a small body size. Conclusion: Endochondral ossification is under the control by clock genes in chondrocytes. Significance: Peripheral clocks are a target for treating cartilaginous diseases relevant to abnormal postnatal chondrogenesis.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

Abstract.Clock genes are believed to play a pivotal role in the generation and oscillation of circadian rhythm as a central clock in the hypothalamic suprachiasmatic nucleus in the mammalian brain. In this study, mRNA expression was for the first time demonstrated with clock genes in both cultured murine microglia and microglial cell line BV-2 cells. Exposure to ATP transiently increased Period-1 (Per1) mRNA expression without affecting that of other clock genes in BV-2 cells, while a similarly transient increase was shown in Per1 mRNA expression in a manner sensitive to P2X7 purinergic receptor antagonists in cultured microglia exposed to ATP. In BV-2 cells transfected with a Per1 promoter luciferase reporter plasmid, ATP significantly increased luciferase activity in a manner sensitive to a P2X7-receptor antagonist. In both microglia and BV-2 cells, a significant increase by ATP was seen in the immunocytochemical fluorescence intensity of cells expressing Per1 protein, with mRNA expression of different P2 receptors including P2X7. Per1 siRNA significantly decreased the number of cells with processes in BV-2 cells exposed to ATP. These results suggest that ATP selectively promotes Per1 expression through gene transactivation after stimulation of P2X7 purinergic receptors in microglial cells.