Hypomorphic expression of parathyroid Bmal1 disrupts the internal parathyroid circadian clock and increases parathyroid cell proliferation in response to uremia
Authors: Søren Egstrand, Maria Lerche Mace, Marya Morevati, Anders Nordholm, Lars Henning Engelholm, Jesper Skovhus Thomsen, Annemarie Brüel, Tally Naveh-Many, Yuliu Guo, Klaus Olgaard, Ewa Lewin
Journal: Kidney International (2022)

The molecular circadian clock is an evolutionary adaptation to anticipate recurring changes in the environment and to coordinate variations in activity, metabolism and hormone secretion. Parathyroid hyperplasia in uremia is a significant clinical challenge. Here, we examined changes in the transcriptome of the murine parathyroid gland over 24 hours and found a rhythmic expression of parathyroid signature genes, such as Casr, Vdr, Fgfr1 and Gcm2. Overall, 1455 genes corresponding to 6.9% of all expressed genes had significant circadian rhythmicity. Biological pathway analysis indicated that the circadian clock system is essential for the regulation of parathyroid cell function. To study this, a novel mouse strain with parathyroid gland-specific knockdown of the core clock gene Bmal1 (PTHcre;Bmal1flox/flox) was created. Dampening of the parathyroid circadian clock rhythmicity was found in these knockdown mice, resulting in abrogated rhythmicity of regulators of parathyroid cell proliferation such as Sp1, Mafb, Gcm2 and Gata3, indicating circadian clock regulation of these genes. Furthermore, the knockdown resulted in downregulation of genes involved in mitochondrial function and synthesis of ATP. When superimposed by uremia, these PTHcre;Bmal1flox/flox mice had an increased parathyroid cell proliferative response, compared to wild type mice. Thus, our findings indicate a role of the internal parathyroid circadian clock in the development of parathyroid gland hyperplasia in uremia. The molecular circadian clock is an evolutionary adaptation to anticipate recurring changes in the environment and to coordinate variations in activity, metabolism and hormone secretion. Parathyroid hyperplasia in uremia is a significant clinical challenge. Here, we examined changes in the transcriptome of the murine parathyroid gland over 24 hours and found a rhythmic expression of parathyroid signature genes, such as Casr, Vdr, Fgfr1 and Gcm2. Overall, 1455 genes corresponding to 6.9% of all expressed genes had significant circadian rhythmicity. Biological pathway analysis indicated that the circadian clock system is essential for the regulation of parathyroid cell function. To study this, a novel mouse strain with parathyroid gland-specific knockdown of the core clock gene Bmal1 (PTHcre;Bmal1flox/flox) was created. Dampening of the parathyroid circadian clock rhythmicity was found in these knockdown mice, resulting in abrogated rhythmicity of regulators of parathyroid cell proliferation such as Sp1, Mafb, Gcm2 and Gata3, indicating circadian clock regulation of these genes. Furthermore, the knockdown resulted in downregulation of genes involved in mitochondrial function and synthesis of ATP. When superimposed by uremia, these PTHcre;Bmal1flox/flox mice had an increased parathyroid cell proliferative response, compared to wild type mice. Thus, our findings indicate a role of the internal parathyroid circadian clock in the development of parathyroid gland hyperplasia in uremia. Translational StatementCircadian rhythms in activity, metabolism, and hormone secretion are maintained by a molecular circadian clock. Parathyroid proliferation is a clinically challenging complication of chronic kidney disease (CKD) leading to nodular hyperplasia and autonomous parathyroid hormone (PTH) secretion with increased morbidity and mortality. We examined a novel transgenic mouse model with parathyroid-specific disruption of circadian clock and found downregulation of genes involved in mitochondrial adenosine triphosphate synthesis and disturbed rhythmicity of parathyroid signature genes: Casr, Vdr, and Gcm2. In CKD, we found compromised compensatory PTH secretory response, hypocalcemia, and severely increased proliferation of the parathyroid glands. Thus, the circadian clock might represent a new therapeutic target in treatment of secondary hyperparathyroidism. Circadian rhythms in activity, metabolism, and hormone secretion are maintained by a molecular circadian clock. Parathyroid proliferation is a clinically challenging complication of chronic kidney disease (CKD) leading to nodular hyperplasia and autonomous parathyroid hormone (PTH) secretion with increased morbidity and mortality. We examined a novel transgenic mouse model with parathyroid-specific disruption of circadian clock and found downregulation of genes involved in mitochondrial adenosine triphosphate synthesis and disturbed rhythmicity of parathyroid signature genes: Casr, Vdr, and Gcm2. In CKD, we found compromised compensatory PTH secretory response, hypocalcemia, and severely increased proliferation of the parathyroid glands. Thus, the circadian clock might represent a new therapeutic target in treatment of secondary hyperparathyroidism. Circadian rhythms in activity, metabolism, hormone secretion, and cell cycle are maintained by a molecular circadian clock (CC) to anticipate the daily recurrent changes of conditions such as light-dark cycle and food availability. In mammals, the primary circadian pacemaker is the CC in the suprachiasmatic nucleus of the hypothalamus from where circadian regulation is conveyed via neural, endocrine, and metabolic pathways.1Dibner C. Schibler U. Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks.Annu Rev Physiol. 2010; 72: 517-549Crossref PubMed Scopus (1545) Google Scholar In addition to this central pacemaker, a CC has been demonstrated in some peripheral tissues, and we have for the first time recently shown that a CC operates in parathyroid glands (PTGs).2Egstrand S. Nordholm A. Morevati M. et al.A molecular circadian clock operates in the parathyroid gland and is disturbed in chronic kidney disease associated bone and mineral disorder.Kidney Int. 2020; 98: 1461-1475Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar Both central and peripheral CCs consist of the same set of circadian regulator genes and proteins, which are working in conjunction, each regulating the others' expression, finely tuned for a 24-hour rhythm of gene expression, translation, and degradation.1Dibner C. Schibler U. Albrecht U. The mammalian circadian timing system: organization and coordination of central and peripheral clocks.Annu Rev Physiol. 2010; 72: 517-549Crossref PubMed Scopus (1545) Google Scholar This is achieved by transcriptional-translational feedback loops, effectively generating a 24-hour cycle of both mRNA and protein levels. Brain-muscle Arnt-like protein 1 (BMAL1) is the key circadian regulator. BMAL1 combines with CLOCK to bind to E-box DNA sequences and induce transcription of several genes including those of the repressor genes: Per1-3 and Cry1-2.3Landgraf D. Wang L.L. Diemer T. et al.NPAS2 compensates for loss of CLOCK in peripheral circadian oscillators.PLoS Genet. 2016; 12e1005882Crossref PubMed Scopus (52) Google Scholar The PER and CRY proteins, which are under casein kinase 1 epsilon (CK1ε) regulation then interact with BMAL1/CLOCK to inhibit their own transcription. BMAL1 also induces transcription of members of the nuclear receptor subfamily 1, group D (REV-ERB) and RORs, which in turn regulate the transcription of the Bmal1 gene, resulting in a rhythmic expression of the Bmal1 gene. CC regulates an estimated 8% to 10% of all expressed genes in the peripheral tissues.4Storch K.F. Lipan O. Leykin I. et al.Extensive and divergent circadian gene expression in liver and heart.Nature. 2002; 417: 78-83Crossref PubMed Scopus (1223) Google Scholar,5Rey G. Cesbron F. Rougemont J. et al.Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver.PLoS Biol. 2011; 9e1000595Crossref PubMed Scopus (327) Google Scholar Maintaining extracellular calcium concentration at a nearly constant level is crucial for survival. The calcium regulating hormones—parathyroid hormone (PTH), calcitriol, fibroblast growth factor 23, klotho, and calcitonin—modulate the function of their target organs—bone, kidney, and intestine.6Peacock M. Calcium metabolism in health and disease.Clin J Am Soc Nephrol. 2010; 5: S23-S30Crossref PubMed Scopus (457) Google Scholar,7Downie M.L. Alexander R.T. Molecular mechanisms altering tubular calcium reabsorption.Pediatr Nephrol. 2022; 37: 707-718Crossref PubMed Scopus (5) Google Scholar PTH increases renal tubular reabsorption of calcium, augments the release of calcium from bone, and enhances synthesis of calcitriol in the renal proximal tubules, which then augments intestinal calcium absorption and renal calcium conservation.7Downie M.L. Alexander R.T. Molecular mechanisms altering tubular calcium reabsorption.Pediatr Nephrol. 2022; 37: 707-718Crossref PubMed Scopus (5) Google Scholar PTGs increase PTH secretion according to the functional demand.8Lewin E. Wang W. Olgaard K. 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Mun H.-C. et al.Phosphate acts directly on the calcium-sensing receptor to stimulate parathyroid hormone secretion.Nat Commun. 2019; 10: 4693Crossref PubMed Scopus (85) Google Scholar In chronic kidney disease (CKD), failure of the kidney to maintain calcium levels, produce calcitriol, excrete phosphate, and maintain bone homeostasis raise the demand on PTGs to increase PTH synthesis and secretion and secondary hyperparathyroidism (sHPT) with extreme parathyroid cell proliferation becomes the consequence.12Naveh-Many T. Silver J. Transcription factors that determine parathyroid development power PTH expression.Kidney Int. 2018; 93: 7-9Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar MafB, Gata3, and Gcm2 are indispensable transcription factors in the embryogenesis of PTGs13Peissig K. Condie B.G. Manley N.R. Embryology of the parathyroid glands.Endocrinol Metab Clin North Am. 2018; 47: 733-742Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and activate PTH secretion by interacting with Sp1 in adult mice.14Han S.-I. Tsunekage Y. Kataoka K. Gata3 cooperates with Gcm2 and MafB to activate parathyroid hormone gene expression by interacting with SP1.Mol Cel Endocrinol. 2015; 411: 113-120Crossref PubMed Scopus (30) Google Scholar Gcm2 and MafB have been shown to maintain parathyroid cell mass and proliferation and have been proposed to play a role in sHPT.15Kamitani-Kawamoto A. Hamada M. Moriguchi T. et al.MafB interacts with Gcm2 and regulates parathyroid hormone expression and parathyroid development.J Bone Miner Res. 2011; 26: 2463-2472Crossref PubMed Scopus (40) Google Scholar, 16Grigorieva I.V. Mirczuk S. 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Anraku A. et al.Gcm2 regulates the maintenance of parathyroid cells in adult mice.PLoS One. 2019; 14e0210662Crossref Scopus (16) Google Scholar We recently demonstrated that an internal CC operates in PTGs, and we found significant rhythmicity in expression of Gata3 and MafB that was abolished in sHPT.2Egstrand S. Nordholm A. Morevati M. et al.A molecular circadian clock operates in the parathyroid gland and is disturbed in chronic kidney disease associated bone and mineral disorder.Kidney Int. 2020; 98: 1461-1475Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar In the present study, RNA sequencing (RNAseq) analysis of the parathyroid transcriptome over 24 hours was performed, and the impact of the internal molecular parathyroid CC on parathyroid function was explored. Furthermore, the parathyroid cell proliferative response to CKD was examined in the context of disturbed CC. For that purpose, we created a parathyroid Bmal1 knockdown mouse strain and examined the CC in PTGs under normal conditions and in a model of uremic sHPT. A total of 604 animals was included in the study. For details, see the Supplementary Methods. A mouse strain with PTG-specific recombination of exon 8 of the Bmal1 (Arntl) gene was generated using the Cre-LoxP recombination system by crossing B6.129S4(Cg)-Arntltm1Weit/J mice in which LoxP sequences are inserted into the flanking regions of exon 8 of the Bmal1 gene (Bmal1flox/flox) with 129;FVB-Tg(PTH-cre)4167Slib/J (PTHcre).20Libutti S.K. Crabtree J.S. Lorang D. et al.Parathyroid gland-specific deletion of the mouse Men1 gene results in parathyroid neoplasia and hypercalcemic hyperparathyroidism.Cancer Res. 2003; 63: 8022-8028PubMed Google Scholar To visualize Cre expression, PTHcre;Bmal1flox/flox;Tmflox/flox mice were generated. For details, see the Supplementary Methods. Wheel revolutions were continuously counted and recorded every hour at standard 12:12 hour light-dark conditions for 24 hours. Then lighting was switched to constant darkness for 3 days and recording repeated for 24 hours. For details, see the Supplementary Methods. Male mice (n = 7) of each genotype had full-body X-ray image taken and femur and tibial length were measured. For details, see the Supplementary Methods. Male Bmal1flox/flox (n = 6) and PTHcre;Bmal1flox/flox (n = 6) mice were killed. The distal metaphysis and mid-diaphysis of left femora were scanned in a micro–computed tomography (μCT) scanner.21Bouxsein M.L. Boyd S.K. Christiansen B.A. et al.Guidelines for assessment of bone microstructure in rodents using micro-computed tomography.J Bone Miner Res. 2010; 25: 1468-1486Crossref PubMed Scopus (2698) Google Scholar For details, see the Supplementary Methods. Female PTHcre;Bmal1flox/flox (n = 16) and Bmal1flox/flox (n = 28) mice were studied at ZT4 (zeitgebertime [ZT], "time-giver", is time since light onset). For induction of hypocalcemia ethyleneglycol-bis-(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) was given i.p. For details, see the Supplementary Methods. Male PTHcre;Bmal1flox/flox (n = 48) and Bmal1flox/flox (n = 43) mice were fed an adenine and high-phosphorus diet.22Volovelsky O. Cohen G. Kenig A. et al.Phosphorylation of ribosomal protein S6 mediates mammalian target of rapamycin complex 1-induced parathyroid cell proliferation in secondary hyperparathyroidism.J Am Soc Nephrol. 2016; 27: 1091-1101Crossref PubMed Scopus (22) Google Scholar Control mice— PTHcre;Bmal1flox/flox (n = 61) and Bmal1flox/flox (n = 65)—were fed standard diet. For details, see the Supplementary Methods. PTGs from Bmal1flox/flox (n = 16), PTHcre;Bmal1flox/flox (n = 17), adenine Bmal1flox/flox (n = 8), and adenine PTHcre;Bmal1flox/flox (n = 9) were dissected under a stereomicroscope and placed on a micrometer. For details, see the Supplementary Methods. Protein was extracted from pooled PTG samples from 9 mice of each genotype and used for Western blotting. For details and antibodies, see the Supplementary Methods. Plasma urea, creatinine, phosphate, calcium, and PTH were analyzed. For details, see the Supplementary Methods. Total RNA was extracted from PTGs for reverse transcriptase polymerase chain reaction (PCR). For details and primer sequences, see Supplementary Methods and Supplementary Table S1. Fluorescence from tdTomato expression was detected using a 510- to 540-nm excitation fluorescence adaptor and a 600-nm long pass emission filter. For details, see the Supplementary Methods. Thyroparathyroid specimens were fixed in 10% formalin for immunostaining. For details, see the Supplementary Methods. Quantification of positive 3,3′-diaminobenzidine staining was performed using Qupath software.23Bankhead P. Loughrey M.B. Fernández J.A. et al.QuPath: open source software for digital pathology image analysis.Sci Rep. 2017; 7: 16878Crossref PubMed Scopus (1441) Google Scholar For details, see the Supplementary Methods. Samples of 4 PTGs pooled from 11-week-old male mice of 42 Bmal1flox/flox (n = 21) and 36 PTHcre;Bmal1flox/flox (n = 18) genotype at 4-hour intervals for 24 hours were snap frozen for RNAseq analysis.24Dobin A. Davis C.A. Schlesinger F. et al.STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2012; 29: 15-21Crossref PubMed Scopus (16741) Google Scholar,25Liao Y. Smyth G.K. Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics. 2013; 30: 923-930Crossref PubMed Scopus (7521) Google Scholar For details, see the Supplementary Methods. Statistical analyses were performed in R. Circadian rhythmicity was analyzed by cosinor regression analysis26Cornelissen G. Cosinor-based rhythmometry.Theor Biol Med Model. 2014; 11: 16Crossref PubMed Scopus (324) Google Scholar and JTK_CYCLE analysis.27Hughes M.E. Hogenesch J.B. Kornacker K. JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data sets.J Biol Rhythms. 2010; 25: 372-380Crossref PubMed Scopus (574) Google Scholar,28Wu G. Anafi R.C. Hughes M.E. et al.MetaCycle: an integrated R package to evaluate periodicity in large scale data.Bioinformatics. 2016; 32: 3351-3353Crossref PubMed Scopus (189) Google Scholar For JTK_CYCLE analysis, an adjusted P value with Bonferroni correction for multiple testing, <0.05 is considered significant. Welch 2-sample t-test or Mann-Whitney U test was used for comparing 2 groups. More than 2 groups were compared by 1-way analysis of variance with Tukey multiple comparison test or Kruskal-Wallis test with post hoc test after Dunn test with Bonferroni adjustment. Significance at P < 0.05. For details, see the Supplementary Methods. The study was approved by the Danish Animal Inspectorate (Reference no. 2017-15-0201-01214) and executed in accordance with national guidelines for laboratory animals. To investigate CC regulation of PTGs, the transcriptome of PTGs from 11-week-old male mice (Bmal1flox/flox) was examined at 4-hour intervals for 24 hours (n = 21). JTK_CYCLE analysis revealed 1455 rhythmic genes corresponding to 6.85% of all expressed genes. Gene ontology analysis showed that these rhythmic transcripts were significantly enriched in genes involved in circadian regulation of gene expression and in genes regulating mRNA metabolism, translation, and protein ubiquitination (Figure 1a). To investigate the coexpression of rhythmic genes, all rhythmic genes were normalized to a rhythm-adjusted mean (midline estimating statistic of rhythm [MESOR]) of 1 and then the fuzzy c-means algorithm was utilized to allocate rhythmic genes to clusters based on expression profile29Futschik M.E. Carlisle B. Noise-robust soft clustering of gene expression time-course data.J Bioinform Comput Biol. 2005; 3: 965-988Crossref PubMed Scopus (243) Google Scholar (Figure 1b, Supplementary Figure S1). Nine distinct cycling profiles were identified with significant enrichment of important parathyroid genes in a time-of-day–dependent manner. The largest cluster (1) encompassed 24.9% of all rhythmic genes, exhibited acrophase at ZT12 and showed enrichment of genes involved in transcription by histone modification, conversion of primary mRNA transcripts into mature mRNA, regulation of ribosomal RNA transcription, and protein deubiquitination and organization of the Golgi apparatus (Figure 1b). The second largest cluster (2) contained 17.0%, peaked at ZT0 and was enriched in genes important for mitochondrial adenosine triphosphate (ATP) synthesis by oxidative phosphorylation (Figure 1b). Cluster (3) encompassed 15.7%, peaked at ZT4, and was enriched in genes assigned to the secretory process (Figure 1b). Cluster (4) constituting of 12.4%, was enriched in genes for peptide biosynthesis and peaked at ZT20 (Figure 1b). A total of 6.7% of the rhythmic genes were assigned to cluster (5), with a prominent peak at ZT16, enriched by genes of cell cycle control, and containing the CC genes Per1, Per2, and Cry1 as well as Usp2, which regulate cell cycle by decreasing poly-ubiquitination–dependent degradation of cyclin D1 and modulate the CC by deubiquitination of PER1 and BMAL1.30Kitamura H. Hashimoto M. USP2-related cellular signaling and consequent pathophysiological outcomes.Int J Mol Sci. 2021; 22: 1209Crossref PubMed Scopus (10) Google Scholar Taken together, we found the 2 largest clusters of rhythmic genes to peak at the transitions between light and dark phases and to regulate mRNA processing and ATP synthesis, while genes related to protein biosynthesis and secretion peaked in the dark or light phase. Parathyroid signature genes (according to the Kyoto Encyclopedia of Genes and Genomes [KEGG] pathway "Parathyroid hormone synthesis, secretion and action") were plotted and rhythmicity assessed by cosinor analysis (Figure 2). Significant rhythmicity of the Casr gene and of genes encoding proteins of the downstream calcium-receptor signaling pathway—Gna11, Adcy3, Adcy5, Adcy6, Adcy8, Adcy9, Itpr3—encoding Gq/11 protein, adenylate cyclases, and inositol triphosphate receptor were demonstrated. Significant rhythmicity of Vdr and of genes encoding the nuclear receptor RXR, Rxra and Rxrg, which form heterodimers with VDR was found. Both Fgfr1 and its coreceptor Klotho exhibited diurnal variation along with genes of the downstream signaling path: Mapk1 and Egr1. Finally, significant rhythmicity of all 4 transcription factors Gcm2, Gata3, MafB, and Sp1 implicated in regulating PTH secretion and parathyroid proliferation was demonstrated. Chga, co-secreted with PTH, was rhythmic too, whereas Pth itself was not. To examine the importance of the internal parathyroid CC, mice with PTG-specific knockdown of Bmal1 were generated (Figure 3). PTHcre;Bmal1flox/flox mice were born at the predicted Mendelian frequency. The mice were viable and exhibited growth curves similar to littermates of Bmal1flox/flox genotype (n = 7 of each genotype) (Figure 4a). The PTGs had normal gross appearance, location, size (Figures 3a and 4b and d) and histology (Figure 4c).Figure 4Phenotype of the PTHcre;Bmal1flox/flox mice. Weight curves were similar for PTHcre;Bmal1flox/flox mice, PTHcre;Bmal1flox/+ mice and Bmal1flox/flox mice (a) (n = 7 per genotype). Cross-sectional area measurements of parathyroid glands from Bmal1flox/flox (n = 16) and PTHcre;Bmal1flox/flox mice (n = 17) are plotted in (b) and representative images of parathyroid glands from both genotypes placed on a micrometer are shown in (d); there was no significant difference between genotypes. Hematoxylin and eosin staining showed microscopically normal parathyroid glands of PTHcre;Bmal1flox/flox mice with location, cell area and nucleus area similar to Bmal1flox/flox mice (c). (a) Data are mean ± SEM. Bars = 50 μm (c) and 1 mm (d). NS, not significant; PTG, parathyroid gland. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fluorescence stereomicroscopy of PTHcre;Bmal1flox/flox;Tmflox/flox confirmed the expression of parathyroid Cre (Figure 3a). Western blotting of PTG tissue from PTHcre;Bmal1flox/flox showed 82% reduction in BMAL1 protein levels compared with those from Bmal1flox/flox (Figure 3c and d). Conclusively, these results indicate a successful alteration of the core CC protein, BMAL1. Furthermore, it was demonstrated that parathyroid Bmal1 knockdown had no manifest impact on PTG development, anatomical position, and histological appearance. PTGs from PTHcre;Bmal1flox/flox mice were harvested at 4-hour intervals for 24 hours and examined by RNAseq along with samples from littermates of the Bmal1flox/flox genotype. PTHcre;Bmal1flox/flox mice retained rhythmic expression of Bmal1 in parathyroids, but with a significantly dampened amplitude (Figure 5, Table 1). Clock and Npas2, required for heterodimerization and nuclear translocation of BMAL1, were significantly rhythmic in PTGs of Bmal1flox/flox mice by cosinor analysis (P = 0.033 and P = 0.0006), while rhythmicity was abolished in PTHcre;Bmal1flox/flox mice (P = 0.79 and P = 0.053) and Npas2 significantly upregulated (Figure 5, Table 1). Similarly the rhythmicity of Cry1 and Cry2 (P < 0.0001) of Bmal1flox/flox was abolished and both were significantly upregulated in PTHcre;Bmal1flox/flox (Figure 5, Table 1). Per genes were also affected in PTHcre;Bmal1flox/flox although to a lesser extent. Per1 lost significant rhythmicity by JTK_CYCLE analysis; Per2 was upregulated with a significantly decreased amplitude; and Per3 showed a phase-shift compared to Bmal1flox/flox mice (Figure 5, Table 1). Rev-Erbα and Rev-Erbβ showed significant rhythmicity in both genotypes, while expression was significantly downregulated with a decreased amplitude of Rev-Erbα and a significant phase-shift of Rev-Erbβ in PTHcre;Bmal1flox/flox (Figure 5, Table 1). Rora and Rorb were arrhythmic in both genotypes, whereas Rorc showed significant rhythmicity in Bmal1flox/flox mice, which was abolished in PTHcre;Bmal1flox/flox mice along with a decreased amplitude, upregulated expression level, and reduced best-fitting period (Figure 5, Table 1). Tef, Dbp, and Nfil3 are circadian regulators binding to D-box elements, and all had reduced amplitude of rhythmicity in PTHcre;Bmal1flox/flox mice where Tef and Nfil3 were significantly upregulated (Figure 5, Table 1). Usp2 showed significant rhythmicity in Bmal1flox/flox mice that was abrogated in PTHcre;Bmal1flox/flox mice (Figure 5, Table 1). CK1ε was constitutively expressed in both genotypes, as expected. These results were confirmed by quantitative PCR analysis of additional 29 samples from PTHcre;Bmal1flox/flox mice and 31 samples from Bmal1flox/flox mice (Supplementary Figure S2).Table 1Circadian parameters of circadian clock genes estimated by JTK cycle analysis in the parathyroids from Bmal1flox/flox and PTHcre;Bmal1flox/floxBmal1flox/floxMESORAmplitudeAcrophase (ZT)Period, hP Value (JTK)Bmal18.88 (8.67 to 9.09)0.94 (0.62 to 1.25)23.28 (22.12 to 0.44)24<0.0001Clock11.89 (11.85 to 11.94)0.08 (0.02 to 0.14)1.05 (21.97 to 4.13)240.074Npas26.25 (6.08 to 6.43)0.55 (0.30 to 0.80)0.72 (22.99 to 2.45)240.0017Per110.84 (10.71 to 10.97)0.43 (0.25 to 0.61)12.79 (11.22 to 14.36)240.0013Per210.07 (9.96 to 10.17)0.84 (0.70 to 0.99)14.71 (13.99 to 15.43)24<0.0001Per38.59 (8.47 to 8.72)0.77 (0.59 to 0.95)11.72 (10.88 to 12.56)24<0.0001Cry19.81 (9.76 to 9.85)0.61 (0.55 to 0.68)18.68 (18.28 to 19.08)24<0.0001Cry29.57 (9.49 to 9.64)0.41 (0.31 to 0.51)14.70 (13.66 to 15.74)24<0.0001Rev-Erbα7.30 (7.13 to 7.47)1.49 (1.24 to 1.74)8.04 (7.16 to 8.92)24<0.0001Rev-Erbβ11.15 (11.01 to 11.29)0.93 (0.72 to 1.14)10.13 (9.38 to 10.88)24<0.0001Rora10.52 (10.48 to 10.56)0.06 (0.00 to 0.12)11.21 (7.63 to 14.79)200.38Rorb3.61 (3.55 to 3.68)0.07 (−0.02 to 0.16)14.04 (8.35 to 19.73)201Rorc10.09 (9.99 to 10.19)0.89 (0.74 to 1.04)19.07 (18.45 to 19.69)24<0.0001Tef11.72 (11.60 to 11.85)0.64 (0.46 to 0.82)11.98 (10.94 to 13.02)24<0.0001Dbp9.56 (9.32 to 9.80)1.29 (0.92 to 1.65)10.08 (9.14 to 11.02)240.0003Nfil36.58 (6.45 to 6.72)0.66 (0.46 to 0.86)21.74 (20.74 to 22.74)240.0052CK1ε8.50 (8.40 to 8.59)0.05 (−0.08 to 0.18)15.80 (4.42 to 3.18)81Usp29.27 (9.16 to 9.37)0.39 (0.23 to 0.55)12.04 (10.55 to 13.53)24<0.0001PTHcre;Bmal1flox/floxMESORAmplitudeAcrophase (ZT)Period (h)P Value (JTK)Bmal18.81 (8.72 to 8.89)0.48 (0.35 to 0.61)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.26 (23.33 to 1.19)240.0004Clock11.97 (11.93 to 12.01)0.02 (−0.04 to 0.08)3.06 (16.75 to 13.37)121Npas27.85 (7.70 to 8.00)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.25 (0.04 to 0.47)2.05 (23.06 to 5.04)240.29Per110.82 (10.67 to 10.97)0.35 (0.13 to 0.58)12.77 (10.58 to 14.96)240.063Per210.49 (10.38 to 10.60)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.37 (0.21 to 0.52)aSignificant difference compared to Bmal1flox/flox (P < 0.05).15.57 (14.00 to 17.14)240.0066Per38.54 (8.45 to 8.63)0.60 (0.47 to 0.73)13.35 (12.60 to 14.09)aSignificant difference compared to Bmal1flox/flox (P < 0.05).24<0.0001Cry110.80 (10.73 to 10.87)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.05 (−0.05 to 0.15)aSignificant difference compared to Bmal1flox/flox (P < 0.05).5.39 (14.01 to 6.15)161Cry29.95 (9.88 to 10.02)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.15 (0.04 to 0.25)aSignificant difference compared to Bmal1flox/flox (P < 0.05).15.25 (12.66 to 17.84)240.13Rev-Erbα6.93 (6.78 to 7.08)aSignificant difference compared to Bmal1flox/flox (P < 0.05).0.83 (0.62 to 1.03)aSignificant difference compared to Bmal1flox/flox (P < 0.05).8.59 (7.59 to 9.59)240.0001Rev-Erbβ10.46 (10.34 to 10.58)aSignificant