Calcium-sensing Receptor Mediated Control of CYP27B1 Promoter-dependent Gene & Protein Expression: Complex Extracellular Ca2+ Concentration Dependence

Abstract

Changes in extracellular Ca2+ (Ca2+o) differentially modulate 25-hydroxyvitamin D3 1α-hydroxylase (1αOHase; encoded by CYP27B1) mRNA and protein levels in cell types including the renal proximal tubule (inhibitory), parathyroid, and skeletal osteoblasts (stimulatory) to control 1,25-dihydroxyvitamin D3 synthesis. We hypothesised that the calcium-sensing receptor (CaSR) mediated Ca2+o concentration-dependent control of 1αOHase, either directly through Ca2+o, or through the local production of parathyroid hormone related peptide (PTHrP). To investigate promoter activity, I transfected a firefly luciferase reporter gene under the control of the 1501 bp human CYP27B1 promoter into HEK-293 cells that stably expressed the CaSR (HEK-CaSR cells) and measured luciferase activities from cells exposed to various Ca2+o concentrations. CYP27B1 promoter-controlled luciferase expression exhibited a biphasic Ca2+o-dependent response in luciferase activity and protein that peaked with a 2-fold increase from basal levels at around 3.0 mM Ca2+o in HEK-CaSR cells. This response was absent in HEK-293 cells and was shifted to the left or right in the presence of the CaSR positive allosteric modulator, cinacalcet, or negative allosteric modulator, NPS-2143, respectively, indicating that both the stimulatory and inhibitory phases were CaSR-mediated. Firefly luciferase and 1αOHase mRNA levels obtained from quantitative RT-PCR exhibited a monophasic Ca2+o-dependent increase and suggests that the stimulatory phase arises from increased mRNA expression, whereas the inhibitory phase arises from reduced protein levels. Inhibitor and mutational studies suggested that the stimulatory phase was dependent on Gq/11 signalling, whereas the inhibitory phase requires MEK and PKC-dependent phosphorylation of the crucial T888 site of the CaSR's C-terminal tail.