Parathyroid Hormone, But Not Melatonin, Resets The Bone Circadian Clock Naoki Okubo 1,2,3, Yoichi Minami 1,3, Hiroyoshi Fujiwara 2, Tatsuya Kunimoto 1,2,3, Toshihiro Hosokawa 1,2,3, Ryo Oda 2, Toshikazu Kubo 2,3, Kazuhiro Yagita 1. 1 Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2 Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan, 3 Department of Musculoskeletal chronobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. Disclosures: N. Okubo: None. Y. Minami: None. H. Fujiwara: None. T. Kunimoto: None. T. Hosokawa: None. R. Oda: None. T. Kubo: None. K. Yagita: None. Introduction: The circadian clock is an endogenous oscillator which generates approximately 24-hour biological cycles. At the molecular level, the circadian clock is composed of a set of clock genes, such as Per1, Per2, Cry1, Bmal1, and Clock, forming cell-autonomous transcription/translation feedback loops. Recently, we reported that bone tissues have the circadian clock [1]. In mammals, the master circadian clock locates in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is thought to send time-cues to the peripheral tissues including bones and synchronize the entire circadian clock. It remains poorly understood what the time-cues to bones are. Parathyroid hormone (PTH) and melatonin are reported to affect bone metabolism and these plasma levels show day-night variation. Therefore, we hypothesized that PTH and melatonin play a role as a time-cue to bones. In this study, we tested whether PTH and melatonin reset the bone circadian clock. Methods: We used PER2 Luc knock-in mice which carry Per2 gene fused with firefly luciferase gene [2]. Femurs were collected and cultured. Bioluminescence was measured every 20 minutes using a photomultitipler tube based real-time bioluminescence monitoring equipment. Cultured bones were synchronized with forskolin (10μM) for 1 hour and thereafter released into a fresh culture medium. PTH (1-34) solved in distilled water, melatonin solved in ethanol or their vehicles were added after PER2::Luc peak (41-hour after synchronization) or before PER2::Luc peak (53-hour after synchronization). Statistical analysis was performed by one-way ANOVA followed by Dunnett s test. Bone bioluminescence images were obtained every hour using a high-sensitivity charge-coupled device (CCD) camera-based microscopic image analyzer. PTH (1-34) and its vehicle were added at 41-hour after synchronization. All experiments were performed with approval from the Experimental Animal Committee, Kyoto Prefectural University of Medicine. Results: By real-time monitoring of bioluminescence, PTH administered at 41-hr after synchronization induced phase advance; in other words, the next peak occurred earlier than expected (Fig. 1A). Meanwhile, PTH administered at 53-hr after synchronization induced phase delay (Fig. 1B). When PTH at 10-11 M was administered at 41-hr after synchronization, we could not observe a significant phase shift. PTH induced a significant phase advance compared to vehicle control at 10-9 M (3.22 ±0.63 hours), and the largest phase advance was induced at 10-7 M (7.69 ±1.15 hours) (Fig.1C). When PTH was administered at 53-hr after synchronization, we could not observe a significant phase delay compared to vehicle control at 10-11 M, but significant phase delay at 10-9 M (-3.05 ±0.88 hours) and 10-7 M (-4.03 ±0.71 hours) (Fig.1D).
Unlike PTH, melatonin administered both at 41-hr and at 53-hr after synchronization failed to induce phase shift (Fig.2A-B). When vehicle control was administered at 41-hr after synchronization, the phase shift was -0.22 ±0.12 hours. By melatonin administration, the phase shift was -1.37 ±0.49 hours (10-8 M), and 0.78 ±0.65 hours (10-5 M), and no statistical significance was observed (Fig.2C). When vehicle was administered at 53-hr after synchronization, the induced phase shift was 1.03 ±0.81 hours. On the other hand, melatonin induced phase shifts were 0.76 ±0.40 hours (10-8 M) and 1.28 ±1.39 hours (10-5 M). No statistical significance was observed (Fig.2D).
PTH-induced phase shift was also observed by a high-sensitivity CCD camera-based microscopic image analyzer (Fig.3). Strong PER2::Luc signals were observed in the epiphyseal cartilage of the growth plate (Fig.3A). By PTH administration 41-hr after synchronization, the phase of the bioluminescence signal was
advanced. In contrast, vehicle administration slightly affected the circadian rhythm of the bioluminescence (Fig.3B-C). Discussion: In this study, we showed that the phase of the bone circadian clock was altered by PTH, but not melatonin in a time-dependent manner. These results suggest that PTH plays a role as the time-cue to bones and resets the bone circadian clock. By microscopic bioluminescence observation, we showed that the PTH-induced phase shift occurred in the epiphyseal cartilage of the growth plate. Recently, it was reported that one year of tereparatide (recombinant human PTH (1-34)) administration in the morning resulted in a larger increase in the bone mineral density than the evening application [3]. Our results suggest that teriparatide treatment resets the bone circadian clock, and that the timedependent effect of teriparatide treatment is caused via the bone circadian clock. The correlation between PTH and the bone circadian clock should be elucidated in further studies. Significance: In clinical practice, teriparatide is the anabolic drug for osteoporosis which is caused by the unbalance of bone metabolism. Our findings suggest that teriparatide treatment induces a phase shift of the bone circadian clock. Given that bone metabolism shows diurnal rhythms, our findings raised the possibility of chronotherapy with teriparatide. ORS 2015 Annual Meeting Poster No: 1452