Huang Alice, Binmahfouz Lenah, Hancock Dale P, Anderson Paul H, Ward Donald T, Conigrave Arthur D
School of Life and Environmental Sciences, Charles Perkins Centre (D17), University of Sydney, NSW 2006Australia.
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK.
J Endocr Soc. 2021 Jun 5;5(9):bvab057. doi: 10.1210/jendso/bvab057. eCollection 2021 Sep 1.
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.
25-羟维生素D 1α-羟化酶（由[基因名称未给出]编码）催化1,25-二羟维生素D的合成，根据组织不同，它受到细胞外钙（Ca²⁺）的负调控或正调控。然而，钙传感器和潜在机制尚不清楚。我们使用稳定表达钙敏感受体（CaSR）的HEK-293细胞（HEK-CaSR细胞），测试了钙敏感受体是否介导Ca²⁺对1α-羟化酶的依赖性调控。在与CYP27B1 - （萤火虫）荧光素酶和对照荧光素酶报告基因共转染的HEK-CaSR细胞中，而非对照HEK-293细胞中，Ca²⁺浓度从0.5mM增加到3.0mM会导致萤火虫荧光素酶活性以及mRNA和蛋白质水平增加2至3倍。令人惊讶的是，当Ca²⁺≥5.0mM时，萤火虫荧光素酶被特异性抑制，表明存在双相Ca²⁺调控。如正负调节剂所揭示的，两个阶段均由CaSR介导。然而，Ca²⁺诱导萤火虫荧光素酶和内源性CYP27B1 mRNA水平简单单调增加，表明高Ca²⁺的抑制作用是转录后水平的。使用抑制剂和CaSR C末端突变体T888A的研究确定了蛋白激酶C（PKC）、T888的磷酸化以及细胞外调节蛋白激酶（ERK）在高Ca²⁺依赖性抑制萤火虫荧光素酶中的作用。PKC和ERK的阻断消除了Ca²⁺刺激的萤火虫荧光素酶，表明PKC或ERK足以刺激CYP27B1启动子。一个关键的CCAAT框（-74 bp至-68 bp），其在PKC和ERK下游受到调控，对于基础转录和Ca²⁺介导的转录上调都是必需的。CaSR介导Ca²⁺依赖性的1α-羟化酶转录上调，并且确定了一种额外的CaSR介导的机制，通过该机制Ca²⁺可以促进荧光素酶以及可能的1α-羟化酶降解。
