Effects of Orai3-Mediated Store-Operated Calcium Entry on Parathyroid Hormone Release in Secondary Hyperparathyroidism

Secondary hyperparathyroidism (SHPT) is a common complication of chronic kidney disease, is characterized by elevated parathyroid hormone (PTH) secretion and Hypocalcemia. Orai3 is a highly selective calcium (Ca2+) channel that plays important roles in tumor development, cardiovascular disease, and autoimmune diseases; however, its role in SHPT is unclear. In the present study, RNA sequencing and western blot assays were used to detect the expression levels of Orai3 in parathyroid tissue from patients with SHPT and from individuals without SHPT. Ca2+ imaging was used to detect the effect of Orai3 channels on Ca2+ signaling in parathyroid gland cells. Enzyme-linked immunosorbent assays were used to detect changes in PTH release. Orai3 knockout rats were used to detect the effect of decreased Orai3 expression on serum PTH levels. We found that the expression of Orai3 in parathyroid tissue obtained from patients with SHPT was signicantly higher than that in patients without SHPT. Knockdown of Orai3 in parathyroid cells by transfection with Orai3-specic small inhibitor RNA inhibited store-operated Ca2+ entry (SOCE) in parathyroid cells. Inhibition of SOCE or knockdown of Orai3 signicantly inhibited PTH release in parathyroid cells. PTH levels in the blood of Orai3 knockout rat were signicantly reduced. Therefore, Orai3 expression and Orai3-mediated Ca2+ signaling may be a mechanism underlying PTH release, and Orai3 may play a role in the development of SHPT.


Introduction
Secondary hyperparathyroidism (SHPT), a common complication of chronic kidney disease (CKD), is characterized by elevated parathyroid hormone (PTH) secretion and parathyroid hyperplasia. Moderate (stage 3 and stage 4) chronic kidney disease affects more than 8% of Americans [1]. The prevalence of SHPT in CKD stage 3 is 71.3%, and 79.7% in CKD stage 4, indicating that CKD is frequently complicated by SHPT, leading to increased risk of morbidity and mortality [2][3][4](!!! INVALID CITATION !!! ; Komaba et al. 2010). Hypocalcemia continuously stimulates PTH secretion and leads to hyperplasia of the parathyroid gland [5] as well as to unbalanced bone remodeling, soft tissue and vascular calci cation, and increased risk of cardiovascular events and all-cause mortality [6]. Understanding the molecular and cellular mechanisms underlying SHPT should thus improve treatment strategies and patients' prognosis.
PTH levels are inversely correlated with intracellular calcium (Ca 2+ ) levels. Stimulation of certain cellsurface receptors induces Ca 2+ release from the endoplasmic reticulum (ER). Decreases in ER Ca 2+ content trigger the accumulation of the ER Ca 2+ sensor protein stromal interaction molecule 1 (STIM1) into puncta located close to the plasma membrane. This accumulated STIM1 activates Orai family proteins, allowing Ca 2+ entry to re ll the ER [7]. Store-operated Ca 2+ entry (SOCE), which occurs through STIM proteins located in the ER and Orai proteins located on the cell membrane, is an important mediator of extracellular Ca 2+ entry into cells. Three subfamilies of Orai proteins (Orai1, Orai2, and Orai3) form the pores of highly Ca 2+ -selective store-operated channels (SOCs) [8,9]. Orai1, Orai2, and Orai3 have been identi ed as being structural elements in Ca 2+ release-activated Ca 2+ channels that re ll the sarcoplasmic/ER Ca 2+ stores after ER Ca 2+ -store depletion [10]. Orai protein mediation of Ca 2+ signaling is involved in a variety of physiological and cellular processes as well as in some pathological processes, including cardiovascular disorders, cancer, and neurodegenerative diseases [11]. Of the three identi ed Orai protein subtypes, Orai3 is receiving increased attention. Orai3-mediated SOCE has been shown to contribute to invasion and colony formation in an estrogen receptor-positive breast cancer cell line [12][13][14]. Orai3-mediated SOCE has also been studied in adult normal and hypertrophied cardiomyocytes [15]. In addition, the decreased redox sensitivity of effector human T helper lymphocytes correlates with increased expression of Orai3 [16]. However, it is unknown whether Orai3 is involved in the regulation of Ca 2+ signaling in parathyroid cells or in the release of PTH.
In the present study, we rst evaluated RNA sequencing data to assess whether Orai3 expression levels differed in the parathyroid glands of patients with vs. without SHPT. We then explored the potential importance of Orai3 in Ca 2+ signaling and in the release of PTH by using immunohistochemistry analyses, Ca 2+ imaging experiments, small interfering RNA (siRNA) techniques, enzyme-linked immunosorbent assays (ELISAs), and Orai3 knockout rats.

Materials And Methods
Participants and tissue specimen collection Parathyroid tissue specimens for the control group were obtained from patients with unilateral thyroid micropapillary carcinoma via a tracheoesophageal groove lymph node dissection. Parathyroid tissue specimens for the experimental group were obtained from patients with SHPT and renal failure via parathyroidectomy. All specimens were collected at the First A liated Hospital of Anhui Medical University. Patients or their families provided written informed consent prior to the specimen collection process. This study was approved by the Ethics Committee of Anhui Medical University (Anhui Medical Ethics approval No. 20190381). All experimental procedures conformed to the requirements of the Declaration of Helsinki and of clinical management.
Tissue culture and transfection of human primary parathyroid cells The parathyroid tissue was repeatedly rinsed with sterile phosphate-buffered saline (PBS) prior to the removal of the fatty tissue envelope and connective tissue. The parathyroid tissue was cut into blocks of 2 mm 3 . The blocks were treated with collagenase (2 g/L) in a 37 °C water bath for 90 min. The resulting suspension was ltered through sterile gauze and centrifuged at 1000 × g for 5 min at room temperature. The collected cell pellets were resuspended in DMEM/F12 medium with 10% fetal bovine serum and placed in 6-well culture plates incubated at 37 °C, 5% CO 2 , and 95% relative humidity. The cells were transiently transfected with human Orai3-speci c siRNA using Lipofectamine 3000 and Opti-MEM (Invitrogen; Carlsbad, CA, USA). The sequence (5′-GGGUCAAGUUUGUGCCCAUdTdT-3′) was designed by and obtained from Biomics Biotechnologies.

Western blot assay
Western blot assays were used to evaluate the protein levels and the transfection e ciency of Orai3 siRNA. The protein extract prepared using RIPA Lysis Buffer (P0013B, Strong; Beyotime; Shanghai, China) was centrifuged at 4 °C and 12000 × g for 20 min. The supernatant was mixed with SDS-PAGE Sample Loading Buffer (P0015L; Beyotime; Shanghai, China) using a ratio of 4:1. The protein was separated by 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene uoride membranes (Millipore; Burlington, MA, USA) and blocked with 5% skim milk at room temperature for 1 h. The membranes were incubated with antibodies against Orai3 (ProteinTech Group, Chicago, Illinois, USA) or β-tubulin or GAPDH (Biosharp; Hefei, China) overnight at 4 °C. The membranes were washed with a PBS Tween 20 (PBST) solution (0.05% Tween 20) and incubated with the secondary antibody. After being rinsed three times in PBST buffer, the membranes were visualized with an ECL light emitting system (Shanghai Peiqing Technology Co., Ltd; Shanghai, China). The optical density (OD) of each blot was analyzed using the software ImageJ (National Institutes of Health; Bethesda, MD, USA) and is presented herein as a relative OD value.

Ratiometric calcium imaging
The cytosolic Ca 2+ concentration ([Ca 2+ ] i ) was measured as in our previous report [17]. In brief, primary parathyroid cells were loaded with 10 μM Fluo-8/AM in Ca 2+ -free PBS, which contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM glucose, 0.2 mM EGTA, and 5 mM HEPES, pH 7.4, at 37 °C for 20 min. The cells were treated with ATP (4 μM) for 10 min to consume intracellular Ca 2+ stores. Ca 2+ in ux was initiated by applying 1 mM extracellular Ca 2+ . The uorescence was recorded using a Leica TCS SP5 confocal laser scanning microscope system. The change in [Ca 2+ ] i is presented as a ratio of the uorescence intensity following experimental treatment (F1) relative to the uorescence intensity at baseline (F1/F0) for Fluo-8/AM.

PTH assay
The collected cell supernatant or serum was processed according to the instructions included with the ELISA kit (Elabscience Biotechnology Co., Ltd; Wuhan, China). In brief, each sample was placed in four or ve wells, appropriately diluted, added to the enzyme plate, and blocked at 37 ℃ for 90 min. The reaction solution was discarded and the plate was rinsed ve times with rinsing solution. The enzyme-labeled antibody was incubated with cells at 37 °C for 60 min. The substrate was added and the cells were incubated in the dark at room temperature for 15 min. Once the color was satisfactorily developed, the solution to stop the reaction (50 μL) was added, and the OD value was measured at a wavelength of 450 nm using a microplate reader. The OD value was positively correlated with the PTH concentration, and the PTH concentration in the sample was calculated using a standard curve. The changes in PTH release were evaluated under the different experimental treatment conditions. The data presented are from at least three independent experiments.
Orai3 knockout rats Statistical analysis GraphPad Prism software, version 5.0 (GraphPad Software; San Diego, California) was used to analyze the experimental results of Mann-Whitney tests (two-tailed). The experimental data are expressed as mean ± standard error of the mean (SEM). Two-sided values of P < 0.05 were considered statistically signi cant.

Results
Expression level of Orai3 is increased in SHPT We identi ed Orai3 transcripts in parathyroid tissue from three control patients and from three patients with SHPT. The generated clustergram describes the genes differentially expressed between the two groups (Figure 1a), and the heatmap shows that Orai3 was signi cantly increased in SHPT compared with normal tissue (Figure 1b). We used western blot assays to detect expression levels of Orai3 in three pairs of parathyroid tissues from control individuals and patients with SHPT. The results showed that compared with normal tissue, the expression of Orai3 protein in SHPT was increased (Figure 1c, d). These results suggested that Orai3 was potentially one of the proteins in Ca 2+ channels affecting [Ca 2+ ] i . SOCE is decreased in Orai3-de cient cells ATP acts on inositol 1,4,5-trisphosphate (IP 3 ) to activate Ca 2+ release from the ER. By blocking sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA), thapsigargin (TG) passively depletes Ca 2+ from the ER and prevents SERCA from lling the ER with Ca 2+ , thereby activating SOCE to allow extracellular Ca 2+ in ux. To explore the Ca 2+ entry mechanism in parathyroid cells, we used TG or ATP to deplete Ca 2+ from the ER and activate SOCE. BTP2 abolishes TG-or ATP-induced Ca 2+ release and SOCE. Here, parathyroid gland cells were treated with ATP (100 μM) or TG (2 μM) in a Ca 2+ -free saline solution for 10 min, after which 1 mM Ca 2+ was added to the extracellular solution ( Figure 2). Ca 2+ release was activated by ATP or TG. The subsequent Ca 2+ entry into parathyroid cells in the presence of extracellular Ca 2+ was inhibited by the SOCE inhibitor BTP2. This result indicated that SOCE is a mechanism regulating Ca 2+ entry into parathyroid cells.
Many studies have shown that STIM1 and Orai1 proteins are involved in SOCE. To assess whether Orai3 contributes to SOCE in parathyroid gland cells, we transfected primary cultured parathyroid gland cells with Orai3-speci c siRNA to suppress Orai3 expression. Compared with cell transfected with scrambled RNA (control), ATP-induced Ca 2+ release was not altered in cells transfected with siRNA; however, the subsequently activated SOCE was inhibited by approximately 50% after Orai3 knockdown (Figure 3). These ndings suggest that Orai3 is a crucial component mediating SOCE in parathyroid cells.

Effect of Orai3 on PTH release from human parathyroid cells
The experimental results to this point indicated that the Orai3 channel mediated extracellular Ca 2+ in ux in human parathyroid cells and that the activation of these Orai3-mediate Ca 2+ channels caused an increase in [Ca 2+ ] i . In order to further examine the effect of [Ca 2+ ] i on PTH release in human parathyroid cells, primary cultured human parathyroid cells were seeded in 96-well plates at an appropriate density and subjected for 12 h to one of four treatments: application of DMSO (control) or of the SOCE inhibitor BTP2 (10 μM) or transfection with scrambled siRNA or Orai3 siRNA. The cell medium was collected and analyzed using an ELISA kit to detect the level of the PTH release from the cells into the cell medium after the treatments (Figure 4a). The results indicated that treatment with the SOCE inhibitor BTP2 or transfection with Orai3 siRNA inhibited PTH release from human parathyroid cells, suggesting that Orai3mediated SOCE participated in PTH release.

Blood PTH levels in Orai3 knockout rats
The latter aforementioned experiments studied the effect of [Ca 2+ ] i changes on PTH release from parathyroid gland cells at the cellular level. In this experiment, we assessed PTH release at the animal level by using an ELISA assay commonly used for clinical detection of PTH levels to assess PTH levels in blood derived from the tail vein of Orai3 knockout and control rats. (Figure 4b). We found that the PTH level in blood obtained from Orai3 knockout rats was signi cantly lower than that obtained from control rats. This result provided additional evidence for our ndings at the level of the cell that Orai3 participates in PTH release.

Discussion
In the present study, we investigated the role of Orai3 in the parathyroid gland. Through RNA-sequencing (RNA-seq) analysis and western blot assays, we found that Orai3 was highly expressed in the parathyroid glands of patients with SHPT. In parathyroid cells, SOCE was activated by ATP or TG but was inhibited by BTP2. SOCE was also inhibited after Orai3 knockdown, resulting in decreased PTH secretion. Therefore, we propose for the rst time, to our knowledge, that Orai3 regulates extracellular Ca 2+ in ux in parathyroid gland cells and mediates PTH secretion.
SHPT is characterized by parathyroid hyperplasia, with abnormal increases in the synthesis and secretion of PTH. SHPT pathogenesis includes decreased calcitriol production due to elevated serum levels of broblast growth factor 23 (FGF23) to maintain phosphate homeostasis, which eventually leads to hypocalcemia. The principal stimulus of PTH secretion is the extracellular Ca 2+ level, which affects Ca 2+sensitive receptors. Activation of these receptors can inhibit the secretion of PTH that is mediated by intracellular signaling pathways [18]. However, the intracellular signaling pathways associated with the pathogenesis of SHPT remain uncertain. In parathyroid cells, extracellular Ca 2+ evokes the mobilization of intracellular Ca 2+ and Ca 2+ in ux, which regulate the secretion of PTH. The Ca 2+ receptor couples to phospholipase C (PLC), eliciting rapid increases in IP 3 and intracellular Ca 2+ [19]. Indeed, PLC mediates breakdown of phosphatidylinositol-4,5-bisphosphate into two second messengers, diacylglycerol and IP 3 [20]. IP 3 can activate SOCE with the depletion of ER Ca 2+ stores [21]. ATP acts on IP 3 to activate Ca 2+ release from the ER. Here, we found that TG-or ATP-induced Ca 2+ release was inhibited by BTP2 in parathyroid cells, suggesting that SOCE may also be involved in the secretion of PTH.
Our RNA-Seq experiments showed that Orai3 levels were substantially higher than Orai1 and Orai2 levels in SHPT. Numerous studies have shown that the various homologous proteins, the subtypes of Orai and their mediated SOCE, are closely related to many diseases, such as autoimmune diseases, cardiac hypertrophy and arrhythmia, atherosclerosis, muscle pain, and various cancers [22]. Orai3 has also been shown to mediate SOCE in breast cancer cells and to be an estrogen receptor-regulated channel [23]. In our study, we found that SOCE was inhibited by knockdown of Orai3, resulting in a decrease in PTH secretion. These results indicated that Orai3-mediated Ca 2+ signaling may be one underlying mechanism of increased PTH secretion in patients with SHPT. It has been reported that PTH is a powerful stimulator of FGF23 synthesis and release and that SOCE stimulates FGF23 transcription [24]. Therefore, the increase in FGF23 levels may be an adaptive response to Orai3-mediated SOCE and PTH levels.
Another major characteristic of SHPT is parathyroid hyperplasia. Phosphate accumulation, increased FGF23 levels, decreased vitamin D activity, and hypocalcemia are persistent stimulants of parathyroid hyperplasia and increased PTH secretion [25,26]. Calcitriol, a metabolite of vitamin D, inhibits the cell cycle regulator c-MYC, inhibits transforming growth factor α, and induces p21 to act as cell cycle inhibitor [27]. In breast cancer cells, Orai3 can also change the cell proliferation process through the c-MYC pathway [23]. The effect of Orai3 on the c-MYC pathway and its potential involvement in the dysfunction of parathyroid hyperplasia warrant further investigation.

Conclusions
We found that the expression level of Orai3 in parathyroid tissue from patients with SHPT was signi cantly higher than that in individuals without SHPT. We also found that extracellular Ca 2+  Data availability Some or all data generated or used during the study are available from the corresponding author by request.
Compliance with ethical standards Con ict of interest Authors have no con ict of interest to disclose.
Ethical approval All procedures performed in this study involving patients and animals were in accordance with the ethical standards of the Ethics Committee of Anhui Medical University.
Consent to participate Written informed consent was signed by all patients or legal guardians.
Consent for publication All authors have read the manuscript and authorized the submission for publication.