Characterization of the calcium-sensing receptor pathway

Functional studies were undertaken using HEK293 cells (ATCC^® CRL-1573™) that had been transfected to stably express calcium-sensing receptors (CaSRs) (HEK-CaSR cells). In addition, cells used for serum-response element (SRE) and nuclear factor or activated T-cells (NFAT) assays were stably transfected to express luciferase under the control of SRE (HEK-CaSR-SRE cells) or NFAT (HEK-CaSR-NFAT cells), respectively. Cells were transfected with 10 nM scrambled or DGKD siRNA (Qiagen) using lipofectamine RNAiMAX (Thermo Fisher Scientific) 72 h before experiments and maintained in DMEM-Glutamax media (Thermo Fisher Scientific) with 10% FBS (Gibco) and 400 μg/ml geneticin (Thermo Fisher Scientific) and 200 μg/ml hygromycin (Invitrogen) at 37 °C, 5% CO₂.

Successful knockdown of DGKD and maintenance of CaSR expression was confirmed via quantitative reverse transcriptase PCR (qRT-PCR) and western blot analyses. qRT-PCR analyses were performed in quadruplicate using Power SYBR Green Cells-to-CT™ Kit (Life Technologies), DGKD, CASR, PGK1, GAPDH, TUB1A, CDNK1B specific primers (Qiagen, Supplementary Table ¹¹), and a Rotor-Gene Q real-time cycler (Qiagen Inc, Valencia, CA). Samples were normalized to a geometric mean of four housekeeper genes: PGK1, GAPDH, TUB1A, CDNK1B. Western blot analyses were undertaken using anti-DGKD (SAB1300472; Sigma; 1:1000), anti-CaSR (5C10, ADD; ab19347; Abcam; 1:1000), and anti−α−Tubulin (T5168; Sigma; 1:2000). The western blots were visualized using an Immuno-Star Western C kit (Bio-Rad) on a Bio-Rad Chemidoc XRS + system and relative expression of DGKD and CaSR were quantified by denistometry using ImageJ software. Both the 140 kDa immature CaSR band and the 160 kDa glycosylated band were considered in densitometry calculations. Captured images are included in the Source Data File.

To perform SRE response assays, at 60 h post transfection HEK-CaSR-SRE cells were incubated in 0.05% fetal bovine serum media with 0.45 mM calcium for 12 h, reducing extracellular calcium concentration and thus inducing basal cellular CaSR-mediated responses whilst maintaining cellular viability. At 72 h the media was changed to varying concentrations of extracellular calcium (0.1–5 mM), with either 5 nM cinacalcet or equivalent volume of DMSO (final concentration of DMSO 0.0001%), and the cells were incubated for a further 4 h at 37 °C. Cinacalcet (AMG-073 HCL) was obtained from Cambridge Bioscience (catalog CAY16042) and dissolved in DMSO prior to use in in vitro studies. Cells were lysed and luciferase activity measured using Luciferase Assay System (Promega) on a PHERAstar microplate reader (BMG Labtech). Assays were performed in > 4 biological replicates (independently transfected wells, performed on at least 4 different days). Nonlinear regression of concentration-response curves was performed with GraphPad Prism for determinations of maximal response.

To perform ERK phosphorylation assays HEK-CaSR cells were seeded in 96-well plates at 60 h post transfection. Cells were fasted for 4 h in 0.1 mM extracellular calcium then stimulated for 4 min with varying concentrations of extracellular calcium (0.1–5 mM), and lysed in Alphascreen Surefire lysis buffer. Alphascreen Surefire ERK phosphorylation assays were performed on whole cell lysates, as reported, and the fluorescence signal measured using a PHERAStar microplate reader (BMG Labtech)⁵⁶. Assays were performed in > 4 biological replicates (independently transfected wells, performed on at least 4 different days). Nonlinear regression of concentration-response curves was performed with GraphPad Prism for determinations of maximal response.

To perform NFAT response assays, at 60 h post transfection HEK-CaSR-NFAT cells were incubated in 0.05% fetal bovine serum media with 0.45 mM calcium for 12 h, reducing extracellular calcium concentration and thus inducing basal cellular CaSR-mediated responses whilst maintaining cellular viability. At 72 h the media was changed to varying concentrations of extracellular calcium (0.1–5 mM) and the cells were incubated for a further 4 h at 37 °C. Cells were lysed and luciferase activity measured using Luciferase Assay System (Promega) on a PHERAstar microplate reader (BMG Labtech). Assays were performed in > 4 biological replicates (independently transfected wells, performed on at least 4 different days). Nonlinear regression of concentration-response curves was performed with GraphPad Prism for determinations of maximal response.

To measure intracellular calcium responses, at 60 h post transfection HEK-CaSR cells were plated in 96-well plates. At 70 h post transfection cells were washed with 100 μl Complete Imaging Buffer (150 mM NaCl, 2.6 mM KCl, 1.18 mM MgCl₂,10 mM HEPES, 0.1 mM CaCl2, pH 7.4), loaded with Fluo-4 dye (Complete Imaging Buffer supplemented with; 1 μM Fluo-4 AM, 0.01% pluronic F-127, 0.5% BSA), and incubated for 60 min at 37 °C. Cells were washed again before the addition of a further 100 μl of Complete Imaging Buffer and incubated for 30 min at room temperature in the dark. Using an automated system, calcium chloride was injected into wells to achieve extracellular calcium concentrations ranging from 0.05 to 5 mM. Control wells were injected with 10 μM ionomycin. Changes in intracellular calcium concentrations were recorded via detection of fluorescence for 30 s using a PHERAstar microplate reader (BMG Labtech) at 37 °C with an excitation filter of 485 nm and an emission filter of 520 nm. The peak mean fluorescence ratio of the transient response following each individual stimulus was measured using MARS data analysis software (BMG Labtech). Relative fluorescence units were normalized to the fluorescence stimulated by ionomycin to account for differences in cell number and loading efficiency and then further normalized to the maximum response observed for the cells treated with scrambled siRNA^57,58. Assays were performed using 4 biological replicates (independently transfected wells, performed on at least 4 different days). Nonlinear regression of concentration–response curves was performed with GraphPad Prism for determinations of maximal response.