Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Calcium-sensing receptor (CaSR) regulated gene transcription (#309)

Alice Huang 1 , Vimesh A Avlani 1 , Dale Hancock 1 , Rebecca S Mason 2 , Arthur D Conigrave 1
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. School of Medical Sciences, University of Sydney, Sydney, NSW, Australia

The CaSR is important for maintenance of whole body calcium homeostasis by regulation of calciotropic hormones in response to extracellular Ca2+ (Ca2+o). We hypothesised that the CaSR is involved in Ca2+o-dependent regulation of CYP27B1 transcription, and local calcitriol synthesis. To investigate this, CYP27B1 promoter-luciferase constructs were transfected into control HEK-293 cells, or HEK-293 cells that express the CaSR (HEK-CaSR). Luciferase activity and mRNA were then measured by Dual-Luciferase Reporter Assay and RT-PCR, respectively. In HEK-CaSR, there was a Ca2+o-dependent biphasic response in luciferase activity that peaked at around 3.0 mM Ca2+o. These responses were left shifted by cinacalcet (1.0 μM), and inhibited by NPS 2143 (1.0 μM). Preliminary data also showed a 2.7 fold increase of luciferase mRNA at 3.0 mM Ca2+o, similar to luciferase activity. Interestingly, the secondary inhibition at 5.0 mM Ca2+o was not observed at the mRNA level, suggesting there may be post-transcriptional regulation of luciferase expression.

As CaSR activation increases PTHrP levels in the kidneys, it is possible that CYP27B1 promoter activation at 3.0 mM Ca2+o is regulated through autocrine PTHrP signalling. We therefore investigated the effects of CaSR activation on PTHrP and PTH1R mRNA expression. As expected, PTHrP mRNA increased with increasing Ca2+o concentrations in HEK-CaSR cells, and was enhanced by 1.0 μM cinacalcet. However, there was around 75% reduction of PTH1R expression at 7.0 mM Ca2+o.

The major finding of this study is that the CaSR regulates Ca2+o-dependent transcription of PTHrP and PTH1R in a reciprocal manner. Ca2+o-dependent downregulation of PTH1R has been reported to be important in chondrocyte differentiation, possibly to limit cell signalling in response to elevated PTHrP [1]. Further investigation is required to determine whether this occurs in osteoblasts, parathyroid, and renal proximal tubules, and to identify its role on CYP27B1 regulation.

  1. Rodriguez, L., Cheng, Z., Chen, T.-H., Tu, C. & Chang, W. (2005) Extracellular Calcium and Parathyroid Hormone-Related Peptide Signaling Modulate the Pace of Growth Plate Chondrocyte Differentiation, Endocrinology. 146, 4597-4608.