The period circadian clock 2 gene responds to glucocorticoids and regulates osteogenic capacity

Abstract

Introduction: The central regulatory system that generates biological rhythms is regulated by circadian clock genes expressed by cells in the suprachiasmatic nucleus. Signals from this system are converted to adrenocortical hormones through the sympathetic nervous system and transmitted to peripheral organs. Another system releases glucocorticoids (GCs) in response to stress through the HPA-axis. Here we investigated the second messenger GC, which is shared by these systems and influences the expression of circadian clock genes of cells of the musculoskeletal system and in viable bone tissue.

Methods: We used mouse-derived cell lines, which differentiate into osteoblasts (MC3T3-E1, C2C12, and 10T1/2) as well as primary cultures of mouse osteoblasts to determine the expression levels of circadian clock genes that respond to GC. Mice (mPer2^m/m) with an inactivating mutation in the period circadian clock 2 gene (Per2) exhibit marked dysrhythmia. Here we compared the bone morphologies of mPer2^m/m mice with those of wild-type (WT) mice.

Results: The expression of major circadian clock genes was detected in each cell line, and their responsiveness to GC was confirmed. We focused on Per2, a negative regulator of the circadian clock and found that a Per2-loss-of-function mutation increased the proliferative capacity of osteoblasts. Treatment of mutant mice with slow-release GC and bisphosphonate affected the maturation of bone tissue, which reflects a tendency to retard calcification.

Conclusion: Our investigations of the mechanisms that regulate circadian rhythm function in tissues of the musculoskeletal system that respond to the stress hormone GC, reveal that Per2 is required for the maturation of bone tissue. Thus, the influences of the systems that control circadian rhythms and the responses to stress by regenerating tissue used for regenerative medicine must be considered and studied in greater detail.

Keywords: ACTH, adrenocorticotropic hormone; ASPS, advanced sleep phase syndrome; BMSCs, bone marrow stem cells; BV/TV, bone volume/tissue volume; CRH, corticotropin-releasing hormone; Circadian rhythm; ES/BS, Eroded surface/ Bone surface; G.P.Th, growth plate thickness; Glucocorticoids; HPA-axis, hypothalamic-pituitary-adrenal-axis; MS/OS, Mineralizing surface/Osteoid surface; OS/BS, Osteoid surface/ Bone surface; OV/BV, Osteoid volume/ Bone volume; OV/TV, Osteoid volume/Tissue volume; Period circadian clock 2 gene; Second messenger; Tb.Th, Trabecular thickness.

Fig. 1

Analysis of the expression of circadian clock genes in mouse cell lines treated with dexamethasone (Dex). A. RT-PCR analysis of mRNAs encoding Clock proteins expressed by the C2C12, MC3T3-E1, and 10T1/2 cell lines. Cultures were treated with 0–10³ nM Dex.

Fig. 2

Analysis of the expression of circadian clock genes in primary cultures of osteoblasts of wild-type (WT) and mPer2^m/m mice treated with Dex. Top panel. Agarose gel electrophoresis of Clock gene expression in primary osteoblast (pOB) cultures treated with different concentrations of Dex. Bottom panel. Quantitation of mRNA levels normalized to those of Gapdh mRNA (*p < 0.05).

Fig. 3

Analysis of the proliferation of osteoblast. A. Proliferation of pOBs of wild-type (WT) and mPer2^m/m mice (*p < 0.05). B. Proliferation of MC3T3-E1 cells in the presence and absence of 100 nM each of Dex and zoledronic acid (ZA). Growth medium without supplements (black line), with Dex (blue line), and Dex plus ZA (red line). C. Proliferation of pOBs of WT and mPer2^m/m mice after 8 days in culture in the absence or presence (100 nM each) of Dex, ZA, and Dex plus ZA (*p < 0.05).

Fig. 4

Morphometric analysis of the fibulae of WT and mPer2^m/m treated with ZA. ZA was administered daily for 21 days to WT and mPer2^m/m mice injected with slow-release pellets containing prednisolone (PSL). A. μ-CT 3D images of fibulae. Left: sagittal view (upper panel) and axial view (lower panel) of WT mice demonstrating the usual cortical bone and trabeculae. Right: sagittal view (upper panel) and axial view (lower panel) of mPer2^m/m mice revealing more thicker cortical bone and smaller trabeculae than those of WT. B. Morphometric analysis of the fibulae of WT and mPer2^m/m mice treated with ZA (*p < 0.05).