TY - JOUR
T1 - Calcium-Sensing Receptors Control CYP27B1-Luciferase Expression
T2 - Transcriptional and Posttranscriptional Mechanisms
AU - Huang, Alice
AU - Binmahfouz, Lenah
AU - Hancock, Dale P
AU - Anderson, Paul H
AU - Ward, Donald T
AU - Conigrave, Arthur D
N1 - © The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.
PY - 2021/9/1
Y1 - 2021/9/1
N2 - 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyzes the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca2+ (Ca2+ o) depending on the tissue. However, the Ca2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca2+ o-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-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca2+ o 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 Ca2+ o ≥ 5.0mM, demonstrating biphasic Ca2+ o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca2+ o induced simple monotonic increases in firefly luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca2+ o 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 Ca2+ o-dependent suppression of firefly luciferase. Blockade of both PKC and ERK1/2 abolished Ca2+ o-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 Ca2+ o-mediated transcriptional upregulation. The CaSR mediates Ca2+ o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca2+ o can promote luciferase and possibly 1α-hydroxylase breakdown.
AB - 25-hydroxyvitamin D 1α-hydroxylase (encoded by CYP27B1), which catalyzes the synthesis of 1,25-dihydroxyvitamin D3, is subject to negative or positive modulation by extracellular Ca2+ (Ca2+ o) depending on the tissue. However, the Ca2+ sensors and underlying mechanisms are unidentified. We tested whether calcium-sensing receptors (CaSRs) mediate Ca2+ o-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-Photinus pyralis (firefly) luciferase and control Renilla luciferase, an increase in Ca2+ o 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 Ca2+ o ≥ 5.0mM, demonstrating biphasic Ca2+ o control. Both phases were mediated by CaSRs as revealed by positive and negative modulators. However, Ca2+ o induced simple monotonic increases in firefly luciferase and endogenous CYP27B1 mRNA levels, indicating that the inhibitory effect of high Ca2+ o 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 Ca2+ o-dependent suppression of firefly luciferase. Blockade of both PKC and ERK1/2 abolished Ca2+ o-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 Ca2+ o-mediated transcriptional upregulation. The CaSR mediates Ca2+ o-dependent transcriptional upregulation of 1α-hydroxylase and an additional CaSR-mediated mechanism is identified by which Ca2+ o can promote luciferase and possibly 1α-hydroxylase breakdown.
U2 - 10.1210/jendso/bvab057
DO - 10.1210/jendso/bvab057
M3 - Article
C2 - 34337274
SN - 2472-1972
VL - 5
JO - Journal of the Endocrine Society
JF - Journal of the Endocrine Society
IS - 9
M1 - bvab057
ER -