Abstract
25-hydroxyvitamin D 1 alpha-hydroxylase (encoded by CYP27B1), which catalyzes the synthesis of 1,25-dihydroxyvitamin D-3, is subject to negative or positive modulation by extracellular Ca2+ (Ca-o(2+)) depending on the tissue. However, the Ca2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca-o(2+)-dependent control of 1 alpha-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-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca-o(2+) 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-o(2+) >= 5.0mM, demonstrating biphasic Ca-o(2+) control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca-o(2+) induced simple monotonic increases in firefly luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca-o(2+) 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)(1/2) in high Ca-o(2+)-dependent suppression of firefly luciferase. Blockade of both PKC and ERK1/2 abolished Ca-o(2+)-stimulated firefly luciferase, demonstrating that either PKC or ERK1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (-74 bp to -68 bp), which is regulated downstream of PKC and ERK1/2, was required for both basal transcription and Ca-o(2+)-mediated transcriptional upregulation. The CaSR mediates Ca-o(2+)-dependent transcriptional upregulation of 1 alpha-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca-o(2+) can promote luciferase and possibly 1 alpha-hydroxylase breakdown.