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

Alice Huang; Lenah Binmahfouz; Dale P Hancock; Paul H Anderson; Donald T Ward; Arthur D Conigrave

doi:10.1210/jendso/bvab057

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

J Endocr Soc. 2021 Jun 5;5(9):bvab057. doi: 10.1210/jendso/bvab057. eCollection 2021 Sep 1.

Authors

Alice Huang¹, Lenah Binmahfouz^{2

3}, Dale P Hancock¹, Paul H Anderson⁴, Donald T Ward², Arthur D Conigrave¹

Affiliations

¹ School of Life and Environmental Sciences, Charles Perkins Centre (D17), University of Sydney, NSW 2006Australia.
² Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK.
³ Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
⁴ Clinical and Health Sciences, Health and Biomedical Innovation, University of South Australia, Adelaide, SA, 5001, Australia.

Abstract

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 ERK_1/2 abolished Ca²⁺ _o-stimulated firefly luciferase, demonstrating that either PKC or ERK_1/2 is sufficient to stimulate the CYP27B1 promoter. A key CCAAT box (-74 bp to -68 bp), which is regulated downstream of PKC and ERK_1/2, 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.

Keywords: 1αOHase; CYP27B1; ERK1/2; PKC; Thr-888 phosphorylation; calcium-sensing receptor; protein breakdown; protein synthesis; transcription.