Calcium-Sensing Receptors Control CYP27B1-Luciferase Expression: Transcriptional and Posttranscriptional Mechanisms.

25-hydroxyvitamin D 1α-hydroxylase (encoded by ), which catalyzes the synthesis of 1,25-dihydroxyvitamin D, is subject to negative or positive modulation by extracellular Ca (Ca ) depending on the tissue. However, the Ca sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca -dependent control of 1α-hydroxylase using HEK-293 cells stably expressing the CaSR (HEK-CaSR cells). In HEK-CaSR cells, but not control HEK-293 cells, cotransfected with reporter genes for CYP27B1- (firefly) luciferase and control luciferase, an increase in Ca from 0.5mM to 3.0mM induced a 2- to 3-fold increase in firefly luciferase activity as well as mRNA and protein levels. Surprisingly, firefly luciferase was specifically suppressed at Ca ≥ 5.0mM, demonstrating biphasic Ca control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca induced simple monotonic increases in firefly luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca was posttranscriptional. Studies with inhibitors and the CaSR C-terminal mutant T888A identified roles for protein kinase C (PKC), phosphorylation of T888, and extracellular regulated protein kinase (ERK) in high Ca -dependent suppression of firefly luciferase. Blockade of both PKC and ERK abolished Ca -stimulated firefly luciferase, demonstrating that either PKC or ERK is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (-74 bp to -68 bp), which is regulated downstream of PKC and ERK, was required for both basal transcription and Ca -mediated transcriptional upregulation. The CaSR mediates Ca -dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca can promote luciferase and possibly 1α-hydroxylase breakdown.