MO553: Impact of the Internal Parathyroid Clock on the Regulation of Parathyroid Gene Expression

Abstract

Abstract BACKGROUND AND AIMS Proper rhythms in metabolism, hormone secretion and activity are maintained by a molecular circadian clock. The central clock in central nervous system (CNS) receives light input via retina to regulate timing of activity. Peripheral clocks are found in a number of tissues regulating an estimated 8–10% of all expressed genes[1]. We have previously shown that an internal circadian clock operates in the parathyroid gland (PTG) which is disturbed in a rat model of chronic kidney disease (CKD)[2]. Here, we examined the PTG transcriptome of wildtype mice over 24-h and compared with our novel mouse strain with PTG-specific circadian clock knockdown (by PTG-specific knockdown of core circadian clock gene Bmal1) to investigate the role of the PTG clock. METHOD PTG-specific knockdown of Bmal1 was generated by crossing PTHcre-mice with Bmal1flox/flox mice (WT) creating PTHcre; Bmal1flox/flox mice (KD). PTGs of WT and KD were harvested at 4-h interval and 24-h gene expression was examined by RNAseq. Circadian rhythmicity was assessed by cosinor-analysis and JTK_CYCLE algorithm. Cluster analysis was performed using the Mfuzz package in R. The clusterProfiler R package was used for gene ontology enrichment and gene set enrichment analysis (GSEA) with FDR-adjusted P-value at < 0.05. RESULTS A total of 6.85% of all expressed genes were significantly rhythmic in the wildtype PTG, including parathyroid signature genes Casr, Vdr, Klotho, Fgfr1 and Gcm2. Rhythmic genes were divided into nine clusters based on similar expression profile, indicating common regulation. Gene ontology analysis revealed that five clusters showed significant enrichment of genes involved in regulating transcription (cluster 1), mitochondrial ATP synthesis by oxidative phosphorylation (cluster 2), secretion (cluster 3), peptide biosynthesis (cluster 4) and cell cycle (cluster 5) (Figure 1). KD mice had global dampening of rhythmicity of PTG circadian clock genes as well as parathyroid signature genes compared to WT and significant downregulation of genes involved in mitochondrial ATP synthesis at four consecutive timepoints compared to WT by GSEA analysis (Figure 2A + B). Gene ontology analysis of all downregulated genes across timepoints in KD mice revealed multiple interconnected enriched terms related to mitochondrial ATP synthesis (Figure 2C). CONCLUSION Dampened rhythm of PTG circadian clock genes also reduced rhythmicity of parathyroid signature genes and downregulated genes involved in mitochondrial function and ATP synthesis in four separate comparisons. This indicates importance of the internal PTG molecular clock for parathyroid function.