Calcium-sensing Receptor, a Potential Biomarker Revealed by Large-scale Public Databases and Experimental Verification in Metastatic Breast Cancer.

INTRODUCTION: Breast cancer (BC) is a common cancer characterized by a high molecular heterogeneity. Therefore, understanding its biological properties and developing effective treatments for patients with different molecular features is imperative. Calcium-sensing receptor (CaSR) has been implicated in several regulatory functions in various types of human cancers. However, its underlying pathological mechanism in BC progression remains elusive. METHODS: We utilized The Cancer Genome Atlas and Gene Expression Omnibus databases to explore the function of CaSR in the metastasis of BC. Gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Gene Set Enrichment Analysis of biological processes and cell signaling pathways revealed that CaSR could be activated or inhibited. Importantly, quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to verify the gene expression of the CaSR. Wound healing and transwell assays were conducted to assess the effect of CaSR on the migration of BC cells. RESULTS: We demonstrated that CaSR expression in metastatic BC was higher than that in non-metastatic BC. It is the first time that database information has been used to reveal the biological process and molecular mechanism of CaSR in BC. Moreover, the CaSR expression in normal breast epithelial cells was notably less compared to that in BC cells. The activation of CaSR by Cinacalcet (a CaSR agonist) significantly enhanced the migration of BC cells, whereas NPS-2143 (a CaSR antagonist) treatment dramatically inhibited these effects. CONCLUSION AND FUTURE PERSPECTIVE: Bioinformatics techniques and experiments demonstrated the involvement of CaSR in BC metastasis. Our findings shed new light on the receptor therapy and molecular pathogenesis of BC, and emphasize the crucial function of CaSR, facilitating the metastasis of BC.

New insights into renal calcium-sensing receptor activation.

PURPOSE OF REVIEW: Activation of the calcium-sensing receptor (CASR) in the parathyroid gland suppresses the release of parathyroid hormone (PTH). Furthermore, activation of the renal CASR directly increases the urinary excretion of calcium, by inhibiting transepithelial calcium transport in the nephron. Gain-of-function mutations in the CASR gene lead to autosomal dominant hypocalcemia 1 (ADH1), with inappropriately low PTH levels and hypocalcemia, indicative of excessive activation of the parathyroid CASR. However, hypercalciuria is not always observed. The reason why the manifestation of hypercalciuria is not uniform among ADH1 patients is not well understood. RECENT FINDINGS: Direct activation of the CASR in the kidney has been cumbersome to study, and an indirect measure to effectively estimate the degree of CASR activation following chronic hypercalcemia or genetic gain-of-function CASR activation has been lacking. Studies have shown that expression of the pore-blocking claudin-14 is strongly stimulated by the CASR in a dose-dependent manner. This stimulatory effect is abolished after renal Casr ablation in hypercalcemic mice, suggesting that claudin-14 abundance may gauge renal CASR activation. Using this marker has led to unexpected discoveries regarding renal CASR activation. SUMMARY: These new studies have informed on renal CASR activation thresholds and the downstream CASR-regulated calcium transport mechanisms.

Rare genetic disorders that impair parathyroid hormone synthesis, secretion, or bioactivity provide insights into the diagnostic utility of different parathyroid hormone assays.

PURPOSE OF REVIEW: Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays. RECENT FINDINGS: Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test. SUMMARY: Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD.

Absence of calcium-sensing receptor basal activity due to inter-subunit disulfide bridges.

G protein-coupled receptors naturally oscillate between inactive and active states, often resulting in receptor constitutive activity with important physiological consequences. Among the class C G protein-coupled receptors that typically sense amino-acids and their derivatives, the calcium sensing receptor (CaSR) tightly controls blood calcium levels. Its constitutive activity has not yet been studied. Here, we demonstrate the importance of the inter-subunit disulfide bridges in maintaining the inactive state of CaSR, resulting in undetectable constitutive activity, unlike the other class C receptors. Deletion of these disulfide bridges results in strong constitutive activity that is abolished by mutations preventing amino acid binding. It shows that this inter-subunit disulfide link is necessary to limit the agonist effect of amino acids on CaSR. Furthermore, human genetic mutations deleting these bridges and associated with hypocalcemia result in elevated CaSR constitutive activity. These results highlight the physiological importance of fine tuning the constitutive activity of G protein-coupled receptors.

Functional evaluation of a novel nonsense variant of the calcium-sensing receptor gene leading to hypocalcemia.

OBJECTIVE: The calcium-sensing receptor (CASR) gene encodes a G protein-coupled receptor crucial for calcium homeostasis. Gain-of-function CASR variants result in hypocalcemia, while loss-of-function variants lead to hypercalcemia. This study aims to assess the functional consequences of the novel nonsense CASR variant [c.2897_2898insCTGA, p.(Gln967*) (Q967*)] identified in adolescent patient with chronic hypocalcemia, a phenotype expected for a gain-of-function variants. DESIGN AND METHODS: To functionally characterize the Q967* mutant receptor, both wild-type (WT) and mutant CASR were transiently transfected into HEK293T cells and calcium-sensing receptor (CaSR) protein expression and functions were comparatively evaluated using multiple read-outs. RESULTS: Western blot analysis revealed that the CaSR mutant protein displayed a lower molecular weight compared with the WT, consistent with the loss of the last 122 amino acids in the intracellular domain. Mitogen-activated protein kinase activation and serum responsive element luciferase assays demonstrated that the mutant receptor had higher baseline activity than the WT. Extracellular-signal-regulated kinase/c-Jun N-terminal kinase phosphorylation, however, remained consistently high in the mutant, without significant modulations following exposure to increasing extracellular calcium (Ca2+o) levels, suggesting that the mutant receptor is more sensitive to Ca2+o compared with the WT. CONCLUSIONS: This study provides functional validation of the pathogenicity of a novel nonsense CASR variant, resulting in an abnormally hyperfunctioning protein consistent with the patient's phenotype. Functional analyses indicate that mutant receptor is constitutively active and poorly sensitive to increasing concentrations of extracellular calcium, suggesting that the cytoplasmic tail may contain elements regulating signal transduction.

The calcium-sensing receptor has only a parathyroid hormone-dependent role in the acute response of renal phosphate transporters to phosphate intake.

The kidney controls systemic inorganic phosphate (Pi) levels by adapting reabsorption to Pi intake. Renal Pi reabsorption is mostly mediated by sodium-phosphate cotransporters NaPi-IIa (SLC34A1) and NaPi-IIc (SLC34A3) that are tightly controlled by various hormones including parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). PTH and FGF23 rise in response to Pi intake and decrease NaPi-IIa and NaPi-IIc brush border membrane abundance enhancing phosphaturia. Phosphaturia and transporter regulation occurs even in the absence of PTH and FGF23 signaling. The calcium-sensing receptor (CaSR) regulates PTH and FGF23 secretion, and may also directly affect renal Pi handling. Here, we combined pharmacological and genetic approaches to examine the role of the CaSR in the acute phosphaturic response to Pi loading. Animals pretreated with the calcimimetic cinacalcet were hyperphosphatemic, had blunted PTH levels upon Pi administration, a reduced Pi-induced phosphaturia, and no Pi-induced NaPi-IIa downregulation. The calcilytic NPS-2143 exaggerated the PTH response to Pi loading but did not abolish Pi-induced downregulation of NaPi-IIa. In mice with a dominant inactivating mutation in the Casr (CasrBCH002), baseline NaPi-IIa expression was higher, whereas downregulation of transporter expression was blunted in double CasrBCH002/PTH knockout (KO) transgenic animals. Thus, in response to an acute Pi load, acute modulation of the CaSR affects the endocrine and renal response, whereas chronic genetic inactivation, displays only subtle differences in the downregulation of NaPi-IIa and NaPi-IIc renal expression. We did not find evidence that the CaSR impacts on the acute renal response to oral Pi loading beyond its role in regulating PTH secretion.NEW & NOTEWORTHY Consumption of phosphate-rich diets causes an adaptive response of the body leading to the urinary excretion of phosphate. The underlying mechanisms are still poorly understood. Here, we examined the role of the calcium-sensing receptor (CaSR) that senses both calcium and phosphate. We confirmed that the receptor increases the secretion of parathyroid hormone involved in stimulating urinary phosphate excretion. However, we did not find any evidence for a role of the receptor beyond this function.

Identification and characterization of a novel CASR mutation causing familial hypocalciuric hypercalcemia.

Context: Although a monoallelic mutation in the calcium-sensing receptor (CASR) gene causes familial hypocalciuric hypercalcemia (FHH), the functional characterization of the identified CASR mutation linked to the clinical response to calcimimetics therapy is still limited. Objective: A 45-year-old male presenting with moderate hypercalcemia, hypocalciuria, and inappropriately high parathyroid hormone (PTH) had a good response to cinacalcet (total serum calcium (Ca2+) from 12.5 to 10.1 mg/dl). We identified the genetic mutation and characterized the functional and pathophysiological mechanisms, and then linked the mutation to calcimimetics treatment in vitro. Design: Sanger sequencing of the CASR, GNA11, and AP2S1 genes was performed in his family. The simulation model was used to predict the function of the identified mutant. In vitro studies, including immunoblotting, immunofluorescence, a cycloheximide chase study, Calbryte™ 520 Ca2+ detection, and half-maximal effective concentration (EC50), were examined. Results: This proband was found to carry a de novo heterozygous missense I554N in the cysteine-rich domain of CASR, which was pathogenic based on the different software prediction models and ACGME criteria. The simulation model showed that CASR I554N mutation decreased its binding energy with Ca2+. Human CASR I554N mutation attenuated the stability of CASR protein, reduced the expression of p-ERK 1/2, and blunted the intracellular Ca2+ response to gradient extracellular Ca2+ (eCa2+) concentration. The EC50 study also demonstrated the correctable effect of calcimimetics on the function of the CASR I554N mutation. Conclusion: This novel CASR I554N mutation causing FHH attenuates CASR stability, its binding affinity with Ca2+, and the response to eCa2+ corrected by therapeutic calcimimetics.

A novel mouse model for familial hypocalciuric hypercalcemia (FHH1) reveals PTH-dependent and independent CaSR defects.

The Calcium-sensing receptor (CaSR) senses extracellular calcium, regulates parathyroid hormone (PTH) secretion, and has additional functions in various organs related to systemic and local calcium and mineral homeostasis. Familial hypocalciuric hypercalcemia type I (FHH1) is caused by heterozygous loss-of-function mutations in the CaSR gene, and is characterized by the combination of hypercalcemia, hypocalciuria, normal to elevated PTH, and facultatively hypermagnesemia and mild bone mineralization defects. To date, only heterozygous Casr null mice have been available as model for FHH1. Here we present a novel mouse FHH1 model identified in a large ENU-screen that carries an c.2579 T > A (p.Ile859Asn) variant in the Casr gene (CasrBCH002 mice). In order to dissect direct effects of the genetic variant from PTH-dependent effects, we crossed CasrBCH002 mice with PTH deficient mice. Heterozygous CasrBCH002 mice were fertile, had normal growth and body weight, were hypercalcemic and hypermagnesemic with inappropriately normal PTH levels and urinary calcium excretion replicating some features of FHH1. Hypercalcemia and hypermagnesemia were independent from PTH and correlated with higher expression of claudin 16 and 19 in kidneys. Likewise, reduced expression of the renal TRPM6 channel in CasrBCH002 mice was not dependent on PTH. In bone, mutations in Casr rescued the bone phenotype observed in Pth null mice by increasing osteoclast numbers and improving the columnar pattern of chondrocytes in the growth zone. In summary, CasrBCH002 mice represent a new model to study FHH1 and our results indicate that only a part of the phenotype is driven by PTH.

Calcium/calcimimetic via calcium-sensing receptor ameliorates cholera toxin-induced secretory diarrhea in mice.

BACKGROUND: Enterotoxins produce diarrhea through direct epithelial action and indirectly by activating the enteric nervous system. Calcium-sensing receptor (CaSR) inhibits both actions. The latter has been well documented in vitro but not in vivo. The hypothesis to be tested was that activating CaSR inhibits diarrhea in vivo. AIM: To determine whether CaSR agonists ameliorate secretory diarrhea evoked by cholera toxin (CTX) in mice. METHODS: CTX was given orally to C57BL/6 mice to induce diarrhea. Calcium and calcimimetic R568 were used to activate CaSR. To maximize their local intestinal actions, calcium was administered luminally via oral rehydration solution (ORS), whereas R568 was applied serosally using an intraperitoneal route. To verify that their actions resulted from the intestine, effects were also examined on Cre-lox intestine-specific CaSR knockouts. Diarrhea outcome was measured biochemically by monitoring changes in fecal Cl- or clinically by assessing stool consistency and weight loss. RESULTS: CTX induced secretory diarrhea, as evidenced by increases in fecal Cl-, stool consistency, and weight loss following CTX exposure, but did not alter CaSR, neither in content nor in function. Accordingly, calcium and R568 were each able to ameliorate diarrhea when applied to diseased intestines. Intestinal CaSR involvement is suggested by gene knockout experiments where the anti-diarrheal actions of R568 were lost in intestinal epithelial CaSR knockouts (villinCre/Casrflox/flox) and neuronal CaSR knockouts (nestinCre/Casrflox/flox). CONCLUSION: Treatment of acute secretory diarrheas remains a global challenge. Despite advances in diarrhea research, few have been made in the realm of diarrhea therapeutics. ORS therapy has remained the standard of care, although it does not halt the losses of intestinal fluid and ions caused by pathogens. There is no cost-effective therapeutic for diarrhea. This and other studies suggest that adding calcium to ORS or using calcimimetics to activate intestinal CaSR might represent a novel approach for treating secretory diarrheal diseases.

Alterations in gene expression and microbiome composition upon calcium-sensing receptor deletion in the mouse esophagus.

The calcium-sensing receptor (CaSR), a G protein-coupled receptor, regulates Ca2+ concentration in plasma by regulating parathyroid hormone secretion. In other tissues, it is reported to play roles in cellular differentiation and migration and in secretion and absorption. We reported previously that CaSR can be conditionally deleted in the mouse esophagus. This conditional knockout (KO) (EsoCaSR-/-) model showed a significant reduction in the levels of adherens and tight junction proteins and had a marked buildup of bacteria on the luminal esophageal surface. To further examine the role of CaSR, we used RNA sequencing to determine gene expression profiles in esophageal epithelia of control and EsoCaSR-/-mice RNA Seq data indicated upregulation of gene sets involved in DNA replication and cell cycle in EsoCaSR-/-. This is accompanied by the downregulation of gene sets involved in the innate immune response and protein homeostasis including peptide elongation and protein trafficking. Ingenuity pathway analysis (IPA) demonstrated that these genes are mapped to important biological networks including calcium and Ras homologus A (RhoA) signaling pathways. To further explore the bacterial buildup in EsoCaSR-/- esophageal tissue, 16S sequencing of the mucosal-associated bacterial microbiome was performed. Three bacterial species, g_Rodentibacter, s_Rodentibacter_unclassified, and s_Lactobacillus_hilgardi were significantly increased in EsoCaSR-/-. Furthermore, metagenomic analysis of 16S sequences indicated that pathways related to oxidative phosphorylation and metabolism were downregulated in EsoCaSR-/- tissues. These data demonstrate that CaSR impacts major pathways of cell proliferation, differentiation, cell cycle, and innate immune response in esophageal epithelium. The disruption of these pathways causes inflammation and significant modifications of the microbiome.NEW & NOTEWORTHY Calcium-sensing receptor (CaSR) plays a significant role in maintaining the barrier function of esophageal epithelium. Using RNA sequencing, we show that conditional deletion of CaSR from mouse esophagus causes upregulation of genes involved in DNA replication and cell cycle and downregulation of genes involved in the innate immune response, protein translation, and cellular protein synthesis. Pathway analysis shows disruption of signaling pathways of calcium and actin cytoskeleton. These changes caused inflammation and esophageal dysbiosis.

Exploring Adaptive Phenotypes for the Human Calcium-Sensing Receptor Polymorphism R990G.

Rainforest hunter-gatherers from Southeast Asia are characterized by specific morphological features including a particularly dark skin color (D), short stature (S), woolly hair (W), and the presence of steatopygia (S)-fat accumulation localized in the hips (DSWS phenotype). Based on previous evidence in the Andamanese population, we first characterized signatures of adaptive natural selection around the calcium-sensing receptor gene in Southeast Asian rainforest groups presenting the DSWS phenotype and identified the R990G substitution (rs1042636) as a putative adaptive variant for experimental follow-up. Although the calcium-sensing receptor has a critical role in calcium homeostasis by directly regulating the parathyroid hormone secretion, it is expressed in different tissues and has been described to be involved in many biological functions. Previous works have also characterized the R990G substitution as an activating polymorphism of the calcium-sensing receptor associated with hypocalcemia. Therefore, we generated a knock-in mouse for this substitution and investigated organismal phenotypes that could have become adaptive in rainforest hunter-gatherers from Southeast Asia. Interestingly, we found that mouse homozygous for the derived allele show not only lower serum calcium concentration but also greater body weight and fat accumulation, probably because of enhanced preadipocyte differentiation and lipolysis impairment resulting from the calcium-sensing receptor activation mediated by R990G. We speculate that such differential features in humans could have facilitated the survival of hunter-gatherer groups during periods of nutritional stress in the challenging conditions of the Southeast Asian tropical rainforests.

Mg2+ supplementation treats secretory diarrhea in mice by activating calcium-sensing receptor in intestinal epithelial cells.

Cholera is a global health problem with no targeted therapies. The Ca2+-sensing receptor (CaSR) is a regulator of intestinal ion transport and a therapeutic target for diarrhea, and Ca2+ is considered its main agonist. We found that increasing extracellular Ca2+ had a minimal effect on forskolin-induced Cl- secretion in human intestinal epithelial T84 cells. However, extracellular Mg2+, an often-neglected CaSR agonist, suppressed forskolin-induced Cl- secretion in T84 cells by 65% at physiological levels seen in stool (10 mM). The effect of Mg2+ occurred via the CaSR/Gq signaling that led to cAMP hydrolysis. Mg2+ (10 mM) also suppressed Cl- secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide by 50%. In mouse intestinal closed loops, luminal Mg2+ treatment (20 mM) inhibited cholera toxin-induced fluid accumulation by 40%. In a mouse intestinal perfusion model of cholera, addition of 10 mM Mg2+ to the perfusate reversed net fluid transport from secretion to absorption. These results suggest that Mg2+ is the key CaSR activator in mouse and human intestinal epithelia at physiological levels in stool. Since stool Mg2+ concentrations in patients with cholera are essentially zero, oral Mg2+ supplementation, alone or in an oral rehydration solution, could be a potential therapy for cholera and other cyclic nucleotide-mediated secretory diarrheas.

Cinacalcet Reverses Short QT Interval in Familial Hypocalciuric Hypercalcemia Type 1.

CONTEXT: Familial hypocalciuric hypercalcemia type 1 (FHH-1) defines an autosomal dominant disease, related to mutations in the CASR gene, with mild hypercalcemia in most cases. Cases of FHH-1 with a short QT interval have not been reported to date. OBJECTIVE: Three family members presented with FHH-1 and short QT interval (<360 ms), a condition that could lead to cardiac arrhythmias, and the effects of cinacalcet, an allosteric modulator of the CaSR, in rectifying the abnormal sensitivity of the mutant CaSR and in correcting the short QT interval were determined. METHODS: CASR mutational analysis was performed by next-generation sequencing and functional consequences of the identified CaSR variant (p.Ile555Thr), and effects of cinacalcet were assessed in HEK293 cells expressing wild-type and variant CaSRs. A cinacalcet test consisting of administration of 30 mg cinacalcet (8 Am) followed by hourly measurement of serum calcium, phosphate, and parathyroid hormone during 8 hours and an electrocardiogram was performed. RESULTS: The CaSR variant (p.Ile555Thr) was confirmed in all 3 FHH-1 patients and was shown to be associated with a loss of function that was ameliorated by cinacalcet. Cinacalcet decreased parathyroid hormone by >50% within two hours, and decreases in serum calcium and increases in serum phosphate occurred within 8 hours, with rectification of the QT interval, which remained normal after 3 months of cinacalcet treatment. CONCLUSION: Our results indicate that FHH-1 patients should be assessed for a short QT interval and a cinacalcet test used to select patients who are likely to benefit from this treatment.

Distinct and targetable role of calcium-sensing receptor in leukaemia.

Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa2+] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa2+] and response to [eCa2+] differ between leukaemias. CaSR influences the location of MLL-AF9+ acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9+ AML versus BCR-ABL1+ leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, ß-catenin and c-MYC signaling and intracellular levels of [Ca2+] in MLL-AF9+ AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa2+] cues from the BMM.

Pan-cancer analysis reveals the prognostic gene CASR suppresses tumor progression and epithelial-mesenchymal transition in renal clear cell carcinoma.

Calcium-sensing receptor (CASR), primarily found in the parathyroid gland and other tissues, plays a crucial role in sensing and regulating extracellular calcium, which was also aberrantly expressed in human tumors. Nevertheless, a comprehensive analysis of CASR in pan-cancer has yet to be conducted. To gain a better understanding of CASR in pan-cancer, data profiles on CASR cancers were collected from TCGA database. The expression level, clinical significance, prognostic value, and potential mechanisms of CASR in pan-cancer were analyzed via multiple public databases. The functional assays were conducted using human kidney renal clear cell carcinoma (KIRC) cell lines, clinical samples, and nude mice. Our research revealed that the abnormal expression of CASR was found in a variety of tumors. The expression and mutation of CASR were significantly associated with tumor prognosis and stage. Pathway analyses suggested that CASR was involved in the epithelial-mesenchymal transition (EMT) progress. Besides, CASR expression was correlated with immune inhibitory genes and immunotherapy in cancers. Particularly in KIRC, we established that CASR mRNA and protein levels were downregulated in clinical samples and cell lines. Moreover, a Cox regression analysis revealed that CASR was an independent prognostic factor in both TCGA-KIRC samples and clinical samples from our center. In vitro and in vivo experiments revealed that blocking CASR with lentivirus could suppress tumor growth and invasion, and EMT progress in KIRC cells. In summary, our study provides a comprehensive bioinformatic analysis of CASR in pan-cancer, offering deeper insights into its function and the EMT mechanism in KIRC, warranting further investigation.

[MiR-301a-5p modulates parathyroid hormone secretion in secondary hyperparathyroidism possibly by regulating calcium-sensing receptor].

OBJECTIVE: To explore the miRNAs that down- regulate calcium-sensing receptor (CaSR) in secondary hyperparathyroidism (SHPT) and their effects on parathyroid hormone (PTH) secretion. METHODS: Whole transcriptome sequencing was performed for 6 normal parathyroid tissue samples and 11 SHPT parathyroid tissue samples. Based on bioinformatic prediction, we screened out 7 candidate miRNAs that regulate CaSR, among which the most likely miRNA for CaSR regulation was identified by double luciferase test. We detected the differential expression of miR-301a-5p and CaSR mRNA in SHPT and normal parathyroid tissue using qRT-PCR, and analyzed the correlation between their expressions and serum PTH levels of the patients. Western blotting was used to detect the expression of CaSR protein in primary SHPT parathyroid cells transfected with miR-301a-5p mimics or inhibitors, and the level of PTH in the supernatant of the cell culture was determined. RESULTS: Among the preliminarily selected 7 miRNAs that potentially regulate CaSR (miR-15a-5p, miR-15b-5p, miR- 16- 5p, miR- 221- 3p, miR- 222- 3p, miR- 301a- 5p and miR- 503- 5p), miR- 301a-5p was significantly upregulated in SHPT compared with normal parathyroid tissue (P < 0.05), and its expression appeared to be positively correlated with PTH level, but this correlation was not statistically significant (P > 0.05); The expression of CaSR mRNA was significantly downregulated in SHPT (P < 0.05), and its expression tended to inversely correlate with the patient's PTH level, but the correlation was not statistically significant (P > 0.05). In primary culture of SHPT parathyroid cells, miR-301a-5p overexpression caused a significant decrease of CaSR protein expression (P < 0.05), and conversely, inhibition of miR-301a-5p expression increased the expression of CaSR protein (P < 0.05). Although miR-301a-5p overexpression did not significantly affect PTH secretion of the cells (P > 0.05), inhibition of iR-301a-5p expression strongly increased the secretion of PTH (P < 0.05). CONCLUSION: MiR-301a-5p affects PTH secretion in SHPT possibly by regulating the expression of CaSR.

Case Report: Calcium sensing receptor gene gain of function mutations: a case series and report of 2 novel mutations.

Autosomal dominant hypocalcemia (ADH1) is a genetic disorder characterized by low serum calcium and low or inappropriately normal levels of parathyroid hormone. The disease is caused by a heterozygous activating mutation of the calcium-sensing receptor (CaSR) gene, encoding a G-Protein-coupled cell membrane sensor of extracellular calcium concentration mainly expressed by parathyroid glands, renal tubules, and the brain. ADH1 has been linked to 113 unique germline mutations, of which nearly 96% are missense mutations. There is often a lack of a clear genotype/phenotype correlation in the reported literature. Here, we described a case series of 6 unrelated ADH1 probands, each one bearing a gain-of-function CaSR mutation, and two children of one of these cases, matching our identified mutations to the same ones previously reported in the literature, and comparing the clinical and biochemical characteristics, as well as the complication profile. As a result of these genetic and clinical comparisons, we propose that a genotype/phenotype correlation may exist because our cases showed similar presentation, characteristics, and severity, with respect to published cases with the same or similar mutations. We also contend that the severity of the presentation is highly influenced by the specific CaSR variant. These findings, however, require further evaluation and assessment with a systematic review.

The Induction of Parathyroid Cell Differentiation from Human Induced Pluripotent Stem Cells Promoted Via TGF-α/EGFR Signaling.

The parathyroid gland plays an essential role in mineral and bone metabolism. Cultivation of physiological human parathyroid cells has yet to be established and the method by which parathyroid cells differentiate from pluripotent stem cells remains uncertain. Therefore, it has been hard to clarify the mechanisms underlying the onset of parathyroid disorders, such as hyperparathyroidism. In this study, we developed a new method of parathyroid cell differentiation from human induced pluripotent stem (iPS) cells. Parathyroid cell differentiation occurred in accordance with embryologic development. Differentiated cells, which expressed the parathyroid hormone, adopted unique cell aggregation similar to the parathyroid gland. In addition, these differentiated cells were identified as calcium-sensing receptor (CaSR)/epithelial cell adhesion molecule (EpCAM) double-positive cells. Interestingly, stimulation with transforming growth factor-α (TGF-α), which is considered a causative molecule of parathyroid hyperplasia, increased the CaSR/EpCAM double-positive cells, but this effect was suppressed by erlotinib, which is an epidermal growth factor receptor (EGFR) inhibitor. These results suggest that TGF-α/EGFR signaling promotes parathyroid cell differentiation from iPS cells in a similar manner to parathyroid hyperplasia.

Leucine and arginine enhance milk fat and milk protein synthesis via the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways.

BACKGROUND AND AIMS: Amino acids (AAs) not only constitute milk protein but also stimulate milk synthesis through the activation of mTORC1 signaling, but which amino acids that have the greatest impact on milk fat and protein synthesis is still very limited. In this study, we aimed to identify the most critical AAs involved in the regulation of milk synthesis and clarify how these AAs regulate milk synthesis through the G-protein-coupled receptors (GPCRs) signaling pathway. METHODS: In this study, a mouse mammary epithelial cell line (HC11) and porcine mammary epithelial cells (PMECs) were selected as study subjects. After treatment with different AAs, the amount of milk protein and milk fat synthesis were detected. Activation of mTORC1 and GPCRs signaling induced by AAs was also investigated. RESULTS: In this study, we demonstrate that essential amino acids (EAAs) are crucial to promote lactation by increasing the expression of genes and proteins related to milk synthesis, such as ACACA, FABP4, DGAT1, SREBP1, α-casein, ß-casein, and WAP in HC11 cells and PMECs. In addition to activating mTORC1, EAAs uniquely regulate the expression of calcium-sensing receptor (CaSR) among all amino-acid-responsive GPCRs, which indicates a potential link between CaSR and the mTORC1 pathway in mammary gland epithelial cells. Compared with other EAAs, leucine and arginine had the greatest capacity to trigger GPCRs (p-ERK) and mTORC1 (p-S6K1) signaling in HC11 cells. In addition, CaSR and its downstream G proteins Gi, Gq, and Gßγ are involved in the regulation of leucine- and arginine-induced milk synthesis and mTORC1 activation. Taken together, our data suggest that leucine and arginine can efficiently trigger milk synthesis through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 pathways. CONCLUSION: We found that the G-protein-coupled receptor CaSR is an important amino acid sensor in mammary epithelial cells. Leucine and arginine promote milk synthesis partially through the CaSR/Gi/mTORC1 and CaSR/Gq/mTORC1 signaling systems in mammary gland epithelial cells. Although this mechanism needs further verification, it is foreseeable that this mechanism may provide new insights into the regulation of milk synthesis.

CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence.

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence (PTHrP87-139 ), and their role in the proliferation behaviors of the superficial zone cells. A gain- and loss-of-function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87-139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone-related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4-CreERT2 ) in the tamoxifen-inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87-139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4-expressing superficial zone cells, in which process CaSR acted as an up-streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87-139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.