Biomedical subjects

Lucie Canaff

Publications and source records attributed to Lucie Canaff.

17 recordsLinked to original sources

PubMed2006

Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly.

Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.

Biophysical Phenomena↗

PubMed2006

A hypocalcemic child with a novel activating mutation of the calcium-sensing receptor gene: successful treatment with recombinant human parathyroid hormone.

CONTEXT: Persistent hypercalciuria, with the attendant risk of nephrocalcinosis and eventual renal failure, is common in hypoparathyroid patients, especially those with activating mutations of the calcium-sensing receptor (CASR) gene, being treated with oral calcium and calcitriol. Treatment with replacement PTH may be warranted, although this has yet to be evaluated in children. OBJECTIVES: The objectives of this study were to identify the cause of the disorder in a young hypocalcemic patient and to assess the efficacy of treatment of the patient with recombinant human PTH(1-34). SUBJECT: An infant presenting with hypocalcemia at 3 wk of age was studied. METHODS: CASR gene mutation analysis was performed on genomic DNA of the proband and family members. The patient was treated with twice-daily administration of recombinant human PTH(1-34) over a 17-month period. RESULTS: The proband was heterozygous for a de novo novel missense mutation (L727Q), on the border between transmembrane helix 4 and intracellular loop 2 of the CASR. When transiently expressed in a human embryonic kidney 293 cell line, the mutant receptor demonstrated a significant leftward shift in the extracellular calcium/intracellular signaling dose-response curve vs. that for the wild-type receptor [EC(50); mutant, 2.59 +/- 0.11 mm (mean +/- se) vs. wild-type, 3.78 +/- 0.12 mm, P < 0.001]. During treatment with PTH(1-34), the patient had no further serious hypocalcemic episodes, and his urinary calcium excretion declined remarkably. CONCLUSION: PTH should be evaluated further as a treatment of autosomal dominant hypocalcemia in young patients.

Calcium↗

PubMed2006

Targeted ablation of the chromogranin a (Chga) gene: normal neuroendocrine dense-core secretory granules and increased expression of other granins.

Chromogranin A (CgA), originally identified in adrenal chromaffin cells, is a member of the granin family of acidic secretory glycoproteins that are expressed in endocrine cells and neurons. CgA has been proposed to play multiple roles in the secretory process. Intracellularly, CgA may control secretory granule biogenesis and target neurotransmitters and peptide hormones to granules of the regulated pathway. Extracellularly, peptides formed as a result of proteolytic processing of CgA may regulate hormone secretion. To investigate the role of CgA in the whole animal, we created a mouse mutant null for the Chga gene. These mice are viable and fertile and have no obvious developmental abnormalities, and their neural and endocrine functions are not grossly impaired. Their adrenal glands were structurally unremarkable, and morphometric analyses of chromaffin cells showed vesicle size and number to be normal. However, the excretion of epinephrine, norepinephrine, and dopamine was significantly elevated in the Chga null mutants. Adrenal medullary mRNA and protein levels of other dense-core secretory granule proteins including chromogranin B, and secretogranins II to VI were up-regulated 2- to 3-fold in the Chga null mutant mice. Hence, the increased expression of the other granin family members is likely to compensate for the Chga deficiency.

Adrenal Medulla↗

PubMed2006

Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells.

PTH, via the PTH/PTH-related protein receptor type 1 that couples to both protein kinase A (PKA) and protein kinase C (PKC) pathways, and the canonical Wnt-beta-catenin signaling pathway play important roles in bone formation. In the present study we have examined the interaction between the PTH and Wnt signaling pathways in mouse osteoblastic MC3T3-E1 cells. PTH dose- and time-dependently increased the concentrations of beta-catenin. The PKA activator, forskolin, and the PKC activator, phorbol 12-myristate-13-acetate, as well as the PTH analog, [Nle(8,18),Tyr(34)]human PTH-(3-34)amide, all increased beta-catenin levels. Both H-89, a specific PKA inhibitor, and PKC inhibitors, staurosporine and calphostin C, antagonized PTH stimulation of beta-catenin levels. TGF-beta as well as transfection of the TGF-beta-signaling molecule, Smad3, enhanced beta-catenin levels, and this was antagonized by transfection of a dominant-negative Smad3. The transcriptional activity of transfected dominant-active beta-catenin was enhanced by PTH, an effect that was antagonized by cotransfection of a dominant-negative Smad3. PTH as well as LiCl(2), which mimics the effects of the Wnt-beta-catenin pathway, rescued the dexamethasone- and etoposide-induced apoptosis of osteoblastic cells. In conclusion, the data demonstrate that PTH stimulates osteoblast beta-catenin levels via Smad3, and that both PKA and PKC pathways are involved. The canonical Wnt-beta-catenin pathway is likely to be involved in the antiapoptotic actions of PTH by acting through Smad3 in osteoblasts.

Animals↗

PubMed2005

Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia.

The CASR, a cell surface glycoprotein expressed in parathyroid gland and kidney, is critical for maintaining extracellular calcium homeostasis. The inherited disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), are caused by inactivating mutations in the CASR gene. The CASR has an N-terminal, 19 amino acid signal peptide that is predicted to direct the nascent polypeptide chain, as it emerges from the ribosome, into the endoplasmic reticulum (ER). Here, we report the functional characterization of three CASR mutations identified in hypercalcemic/hyperparathyroid patients. The mutations, L11S, L13P and T14A, lie within the signal peptide hydrophobic core. When transiently transfected into kidney cells, L11S and L13P mutants demonstrated reduced intracellular and plasma membrane expression and signaling to the mitogen-activated protein kinase pathway in response to extracellular calcium relative to wild-type CASR and the T14A mutant. All mutant CASR RNAs translated into protein normally. In cotranslational processing assays, which test the functionality of the signal peptide in the early secretory pathway, the wild-type CASR and mutant T14A nascent polypeptides were targeted to microsomal vesicles, representing the ER, translocated into the vesicular lumen and underwent core N-glycosylation. In contrast, the L11S and L13P mutants failed to be inserted in the microsomes and undergo glycosylation. This is the first study examining the function of the CASR signal sequence and reveals that both L11S and L13P mutants are markedly impaired with respect to cotranslational processing, accounting for the observed parathyroid dysfunction.

Adult↗

PubMed2005

Calcium-sensing receptor gene transcription is up-regulated by the proinflammatory cytokine, interleukin-1beta. Role of the NF-kappaB PATHWAY and kappaB elements.

The calcium-sensing receptor (CASR) in parathyroid, thyroid, and kidney is essential for calcium homeostasis. Hypocalcemia is common in critically ill patients having increased circulating proinflammatory cytokines, although the causes are unknown. We hypothesized that the cytokines increase CASR expression and reduce the set point for parathyroid hormone suppression by extracellular calcium, leading to hypocalcemia and hypoparathyroidism. Here, we show in vivo in the rat that parathyroid, thyroid, and kidney CASR mRNA and protein increased after injection of interleukin-1beta. This was associated with decreased circulating parathyroid hormone, calcium, and 1,25-dihydroxyvitamin D levels. Interleukin-1beta stimulated endogenous CASR gene transcripts and transfected promoter reporter activity in human thyroid C-cells (TT cells) and kidney proximal tubule (HKC) cells. Cotransfection of NF-kappaB proteins enhanced activity of the reporter constructs, whereas cotransfection with inhibitor-kappaB or application of an NF-kappaB nuclear localization sequence peptide abrogated responsiveness to cytokine or NF-kappaB proteins. Mutagenesis of some, but not all, of the potential kappaB elements in the 5' part of the CASR gene led to loss of responsiveness to cytokine. These elements conferred cytokine responsiveness to a heterologous promoter, and in electrophoretic mobility shift assays, NF-kappaB complexes formed on the same three kappaB elements. In summary, the CASR gene has several functional kappaB elements that mediate its upregulation by proinflammatory cytokines and probably contribute to altered extracellular calcium homeostasis in the critically ill.

Animals↗

PubMed2005

Calcium sensing receptor in human colon carcinoma: interaction with Ca(2+) and 1,25-dihydroxyvitamin D(3).

Recent studies show that the human parathyroid calcium sensing receptor (CaSR) is expressed in human colon epithelium and functions to regulate epithelial proliferation and differentiation. In this study, we show that the cells of the colon crypt acquire CaSR expression as they differentiate and migrate towards the apex of the crypt. CaSR expression was weak in colon carcinomas with a more-differentiated histologic pattern, whereas CaSR expression was undetectable in less-differentiated tumors. We found that Ca(2+) and/or 1,25(OH)(2)D(3) stimulated CaSR promoter activity and CaSR protein expression in the human colon carcinoma CBS cells, which possessed a functional CaSR. Both agents concomitantly induced a series of changes in the CBS cells that influence proliferation and differentiation, but cellular responses to the two agents were not identical. Ca(2+) strongly induced E-cadherin expression and inhibited the expression of the nuclear transcription factor, TCF4. 1,25(OH)(2)D(3) was weaker in its effect on E-cadherin and was not able to inhibit TCF4 expression. 1,25(OH)(2)D(3) was as strong or stronger than Ca(2+) in its induction of the cyclin-dependent kinase inhibitors, P21 and p27. It is concluded that CaSR may function in the colon to regulate epithelial differentiation and that loss of CaSR expression may be associated with abnormal differentiation and/or malignant progression. Extracellular Ca(2+) and 1,25(OH)(2)D(3) are potential candidates involved in regulating CaSR expression in the colon and the chemopreventive actions of Ca(2+) and 1,25(OH)(2)D(3) in colon cancer may be mediated, in part, through the CaSR.

Calcitriol↗

PubMed2004

Functional characterization of calcium-sensing receptor codon 227 mutations presenting as either familial (benign) hypocalciuric hypercalcemia or neonatal hyperparathyroidism.

Familial benign hypocalciuric hypercalcemia (FBHH), in which calcium homeostasis is disordered, can be distinguished from mild primary hyperparathyroidism by the finding of a heterozygous loss-of-function mutation in the calcium-sensing receptor (CaSR). Here, we report a Polish kindred with FBHH, the proband of which had undergone an unsuccessful parathyroidectomy. Direct sequence analysis of exon 4 of her CASR gene identified a heterozygous R227Q mutation in the extracellular domain of the receptor. This mutation segregated with other affected family members. A de novo heterozygous R227L mutation had previously been identified in a case of neonatal hyperparathyroidism. We performed a functional analysis by transiently transfecting wild-type and mutant (R227Q, R227L) CaSRs in human embryonic kidney (HEK293) cells. Both mutant receptors were expressed at a similar level to that of the wild-type, demonstrated a 160-kDa molecular species consistent with having undergone full maturation, and were visualized on the cell surface. Although both mutants were impaired in their MAPK responses to increasing extracellular calcium concentrations relative to wild type, this was more marked for R227L (EC(50) = 9.7 mM) than R227Q (EC(50) = 7.9 mM) relative to wild type (EC(50) = 3.7 mM). When cotransfected with wild-type CaSR to mimic the heterozygous state, the curves for both R227Q and R227L were right shifted intermediate to the curves for wild type and the respective mutant. This differential responsiveness may account, in part, for the markedly different clinical presentation of the R227Q mutation, classic FBHH, vs. the neonatal hyperparathyroidism of the R227L mutation.

Amino Acid Substitution↗

PubMed2004

Menin is required for bone morphogenetic protein 2- and transforming growth factor beta-regulated osteoblastic differentiation through interaction with Smads and Runx2.

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation.

3T3 Cells↗

PubMed2004

Activin inhibits pituitary prolactin expression and cell growth through Smads, Pit-1 and menin.

Activin, a member of the TGFbeta superfamily, is a negative regulator of cell growth and prolactin (PRL) production in pituitary lactotrope cells. However, the mechanisms by which this growth factor exerts its growth-inhibitory and -repressive effect on PRL remain unclear. In this study, we show that activin negatively regulates PRL expression at the transcriptional level through the Smad pathway and the multiple endocrine neoplasia type 1 gene product, menin. Our results also demonstrate that the tumor suppressor menin is required for activin-induced growth arrest of somatolactotrope cells. Moreover, we show that activin represses transcription and expression of Pit-1, a pituitary transcription factor that is essential for maintenance and development of lactotrope cells. We defined two Pit-1 DNA-binding sites in the proximal region of the PRL promoter as critical for the activin-mediated inhibition. Together, our results highlight the Smad pathway and the tumor suppressor menin as key regulators of activin effects on PRL and Pit-1 expression, as well as on cell growth inhibition, and emphasize the critical role of activin in the regulation of pituitary function.

Activins↗

PubMed2004

Menin inactivation leads to loss of transforming growth factor beta inhibition of parathyroid cell proliferation and parathyroid hormone secretion.

Primary hyperparathyroidism is a common endocrine disorder caused by parathyroid gland enlargement and excessive parathyroid hormone (PTH) secretion. However, the precise mechanisms of tumorigenesis of the parathyroids are unknown. Here we have investigated the roles of transforming growth factor (TGF)-beta and menin, the product of the multiple endocrine neoplasia type 1 (Men1) gene, in the proliferation and PTH production of parathyroid cells from either patients with secondary hyperparathyroidism or Men1. TGF-beta was expressed in the parathyroid endocrine cells. Addition of TGF-beta to parathyroid cells from patients with secondary hyperparathyroidism inhibited their proliferation and PTH secretion. These responses to TGF-beta were lost when menin was specifically inactivated by antisense oligonucleotides. Moreover, TGF-beta did not affect the proliferation and PTH production of parathyroid cells from a Men1 patient. These results indicate that menin is required for TGF-beta action in the parathyroid. We conclude that TGF-beta is an important autocrine/paracrine negative regulator of parathyroid cell proliferation and PTH secretion and that loss of TGF-beta signaling due to menin inactivation contributes to parathyroid tumorigenesis.

Cell Division↗

PubMed2004

CASRdb: calcium-sensing receptor locus-specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia.

Familial hypocalciuric hypercalcemia (FHH) is caused by heterozygous loss-of-function mutations in the calcium-sensing receptor (CASR), in which the lifelong hypercalcemia is generally asymptomatic. Homozygous loss-of-function CASR mutations manifest as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism, which occur in infancy. Activating mutations in the CASR gene have been identified in several families with autosomal dominant hypocalcemia (ADH), autosomal dominant hypoparathyroidism, or hypocalcemic hypercalciuria. Individuals with ADH may have mild hypocalcemia and relatively few symptoms. However, in some cases seizures can occur, especially in younger patients, and these often happen during febrile episodes due to intercurrent infection. Thus far, 112 naturally-occurring mutations in the human CASR gene have been reported, of which 80 are unique and 32 are recurrent. To better understand the mutations causing defects in the CASR gene and to define specific regions relevant for ligand-receptor interaction and other receptor functions, the data on mutations were collected and the information was centralized in the CASRdb (www.casrdb.mcgill.ca), which is easily and quickly accessible by search engines for retrieval of specific information. The information can be searched by mutation, genotype-phenotype, clinical data, in vitro analyses, and authors of publications describing the mutations. CASRdb is regularly updated for new mutations and it also provides a mutation submission form to ensure up-to-date information. The home page of this database provides links to different web pages that are relevant to the CASR, as well as disease clinical pages, sequence of the CASR gene exons, and position of mutations in the CASR. The CASRdb will help researchers to better understand and analyze the mutations, and aid in structure-function analyses.

Database Management Systems↗

PubMed2003

Differential regulation of PHEX expression in bone and parathyroid gland by chronic renal insufficiency and 1,25-dihydroxyvitamin D3.

Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, a renal phosphate-wasting disorder associated with defective skeletal mineralization. PHEX is predominantly expressed in bones and teeth and in the parathyroid gland of patients with chronic renal failure and tertiary hyperparathyroidism. The purpose of the present study was to examine the effects of renal insufficiency and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the regulation of PHEX expression in rat tibia and parathyroid gland. In rats fed a high-phosphate (Pi) diet, nephrectomy elicited a significant increase in the serum parathyroid hormone (PTH) concentration that was associated with a significant increase in the abundance of PHEX mRNA and protein in the tibia and a significant increase in PHEX mRNA in the parathyroid gland. In contrast, 1,25(OH)2D3 administration to intact rats fed a control diet elicited a significant decrease in the serum PTH concentration that was accompanied by a significant decrease in PHEX mRNA and protein abundance in the tibia and a significant decrease in PHEX mRNA in the parathyroid gland. In addition, the increases in serum PTH levels and PHEX mRNA in the tibia and parathyroid gland in nephrectomized rats fed a high-Pi diet were blunted by 1,25(OH)2D3. Serum PTH concentration was positively and significantly correlated with tibial PHEX mRNA and protein abundance. In summary, we demonstrate that PHEX expression in the tibia and parathyroid gland is increased by chronic renal insufficiency and decreased by 1,25(OH)2D3 administration and suggest that PTH status may play an important role in mediating these changes in PHEX expression.

Animals↗

PubMed2003

Inactivation of menin, the product of the multiple endocrine neoplasia type 1 gene, inhibits the commitment of multipotential mesenchymal stem cells into the osteoblast lineage.

The physiological roles of menin, the product of the multiple endocrine neoplasia type 1 gene, are not known. Homozygous menin knockout mice exhibit cranial and facial hypoplasia. We, therefore, investigated the role of menin in the regulation of osteoblastic differentiation. Menin antisense oligonucleotides (AS-oligo) reduced endogenous menin expression in the C3H10T1/2 (10T1/2) mouse mesenchymal stem cells and antagonized alkaline phosphatase (ALP) activity and the expression of type I collagen, Runx2/cbfa1 (Runx2), and osteocalcin (OCN) induced by bone morphogenetic protein 2 (BMP-2). AS-oligo did not affect adipogenic markers (Oil red staining and PPARgamma expression) and chondrogenic markers (Alcian blue staining and type IX collagen) induced by BMP-2 in 10T1/2 cells. Menin co-immunoprecipitated with Smad1 and Smad5, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5. In osteoblastic MC3T3-E1 cells, AS-oligo affected neither BMP-2-stimulated ALP activity nor the expression of Runx2 and OCN. Stable inactivation of menin in MC3T3-E1 cells increased ALP activity, mineralization, and the expression of type I collagen and OCN. In 21-day cultures of MC3T3-E1 cells and BMP-2-treated 10T1/2 cells, endogenous menin expression increased up to day 14 and declined thereafter. These data indicate that menin inactivation specifically inhibits the commitment of pluripotent mesenchymal stem cells to the osteoblast lineage, mediated by menin and Smad1/5 interactions. Menin is important for both early differentiation of osteoblasts and inhibition of their later differentiation, and it might be crucial for intramembranous ossification.

3T3 Cells↗

PubMed2003

Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene.

De novo activating mutations in the calcium-sensing receptor (CASR) gene are a common cause of sporadic isolated hypoparathyroidism. Here, we describe a family in which two affected siblings were found to be heterozygous for a novel F788L mutation in the fifth transmembrane domain encoded by exon 7 of the CASR. Both parents and the third sibling were clinically unaffected and genotypically normal by direct sequencing of their leukocyte exon 7 PCR amplicons. However, the mother was revealed to be a mosaic for the mutation by sequence analysis of multiple subclones as well as denaturing HPLC of the CASR exon 7 leukocyte PCR product. A functional analysis of the mutation was performed by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. The mutant CASR was expressed at a similar level as the wild type. The F788L mutant produced a significant shift to the left relative to the wild-type CASR in the MAPK response to increasing extracellular calcium concentrations. This is the first report of mosaicism for an activating CASR mutation and suggests that care should be exercised in counseling for risks of recurrence in a situation where a de novo mutation appears likely.

Adolescent↗

PubMed2002

Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D.

The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased approximately 2-fold at 8 and 12 h after 1,25(OH)2D3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5'-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10(-8) m 1,25(OH)2D3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 bp) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)2D3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitro transcribed/translated vitamin D receptor and retinoid X receptor-alpha, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)2D3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)2D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression.

Alternative Splicing↗

PubMed2002

Identification and functional characterization of novel calcium-sensing receptor mutations in familial hypocalciuric hypercalcemia and autosomal dominant hypocalcemia.

Familial hypocalciuric hypercalcemia (FHH), neonatal severe hyperparathyroidism (NSHPT), and autosomal dominant hypocalcemia (ADH), in which calcium homeostasis is disordered, are associated with mutations in the calcium-sensing receptor (CASR). Six unrelated kindreds with FHH and/or NSHPT and two unrelated kindreds with ADH were studied. Direct sequence analysis of the exons of the CASR gene identified heterozygous mutations in six of the kindreds with FHH and in one of those with ADH. We performed functional analyses on the novel missense and insertion/frameshift mutants by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. All mutant receptors were expressed at a similar level to that of the wild type; however, whereas mutants R220W and A835T (the ADH mutant) were fully glycosylated and were visualized on the cell surface, glycosylation of mutants G549R and C850-851 ins/fs was impaired, resulting in reduced cell surface staining. In fura-2-loaded HEK293 cells expressing the wild-type or mutant receptors, the inactivating R220W mutant produced a significant shift to the right relative to the wild-type CASR in the cytosolic calcium response to increasing extracellular calcium concentrations and the G549R and C850-851 ins/fs mutants were without detectable activity. The activating A835T mutation resulted in a shift to the left in the cytosolic calcium response to extracellular calcium concentrations relative to the wild type. Our studies have identified novel CASR mutations that alter the function of the CASR in several different ways.

Calcium↗