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.