Autonomous and Self-sustained Circadian Oscillators Displayed in Human Primary Parathyroid Cell Culture Drive Parathormone Secretion

Abstract

Abstract Background Most cells in our body possess circadian oscillators controlling diurnal rhythmicity of body metabolism. The role of molecular clocks in regulating the function and disfunction of human parathyroid gland (PG), responsible for calcium homeostasis, has not been unraveled. Hyperparathyroidism is a common endocrine pathology, characterized by an unadapted PTH secretion to calcium levels. Aims Here we aimed at characterizing molecular makeup of circadian clocks in human primary parathyroid cell culture (HPPCC), and at establishing differential transcriptional patterns of normal and pathological PGs. Methods RNA extracted from normal, adenomatous, and hyperplastic PG tissues was subjected to RNA sequencing (RNAseq) analysis. The rim of normal PG tissue was dissected from parathyroid adenoma specimens based on the higher autofluorescence intensity assessed by the near infra-red imaging. HPPCCs were established from adenomatous and hyperplastic PG biopsies. Efficient circadian clock disruption was achieved in HPPCCs by small interfering RNA-mediated knockdown of CLOCK. The functionality of circadian clock machinery was assessed by continuous recording of circadian bioluminescence introduced via lentivectors and paralleled with around-the-clock measurement of PTH secretion using perfusion system. Results The RNAseq analyses revealed transcriptional signatures that distinguished between pathological and normal PGs, and among adenomatous and hyperplastic PGs. We report, for the first time, robust anti-phasic circadian rhythmicity of Per2-luciferase and Bmal1-luciferase reporters in HPPCCs synchronized in vitro. Strikingly, we observed circadian rhythmicity of PTH secretion by parathyroid adenoma cells. Moreover, clock disruption in PG adenoma cells has an impact on transcription of functional genes. Conclusion Our data indicate presence of cell-autonomous molecular clock in human PG cells paralleled with circadian rhythmic PTH secretion and provide large-scale transcriptional pattern of parathyroid adenoma and hyperplastic tissues.