scholarly journals Mitogenic Action of Calcium-Sensing Receptor on Rat Calvarial Osteoblasts

Endocrinology ◽  
2004 ◽  
Vol 145 (7) ◽  
pp. 3451-3462 ◽  
Author(s):  
Naibedya Chattopadhyay ◽  
Shozo Yano ◽  
Jacob Tfelt-Hansen ◽  
Paul Rooney ◽  
Deepthi Kanuparthi ◽  
...  
Keyword(s):  
Dominant Negative ◽  
Calcium Sensing Receptor ◽  
Jnk Pathway ◽  
Early Growth Response ◽  
Calcium Sensing ◽  
Mitogenic Action ◽  
Igf I ◽  
Extracellular Factor ◽  
Post Harvesting ◽  
Early Growth Response 1

Abstract The parathyroid calcium-sensing receptor (CaR) plays a nonredundant role in systemic calcium homeostasis. In bone, Ca2+o, a major extracellular factor in the bone microenvironment during bone remodeling, could potentially serve as an extracellular first messenger, acting via the CaR, that stimulates the proliferation of preosteoblasts and their differentiation to osteoblasts (OBs). Primary digests of rat calvarial OBs express the CaR as assessed by RT-PCR, Northern, and Western blot analysis, and immunocolocalization of the CaR with the OB marker cbfa-1. Real-time PCR revealed a significant increase in CaR mRNA in 5- and 7-d cultures compared with 3-d cultures post harvesting. High Ca2+o did not affect the expression of CaR mRNA during this time but up-regulated cyclin D (D1, D2, and D3) genes, which are involved in transition from the G1 to the S phase of the cell cycle, as well as the early oncogenes, c-fos and early growth response-1; high Ca2+o did not, however, alter IGF-I expression, a mitogenic factor for OBs. The high Ca2+o-dependent increase in the proliferation of OBs was attenuated after transduction with a dominant-negative CaR (R185Q), confirming that the effect of high Ca2+o is CaR mediated. Stimulation of proliferation by the CaR involves the Jun-terminal kinase (JNK) pathway, as high Ca2+o stimulated the phosphorylation of JNK in a CaR-mediated manner, and the JNK inhibitor SP600125 abolished CaR-induced proliferation. Our data, therefore, show that the parathyroid/kidney CaR expressed in rat calvarial OBs exerts a mitogenic effect that involves activation of the JNK pathway and up-regulation of several mitogenic genes.

scholarly journals A novel mutation in the calcium-sensing receptor in a French family with familial hypocalciuric hypercalcaemia

2011 ◽  
Vol 165 (2) ◽  
pp. 359-363 ◽  
Author(s):  
Abdallah Al-Salameh ◽  
Filomena Cetani ◽  
Elena Pardi ◽  
Carmen Vulpoi ◽  
Peggy Pierre ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Novel Mutation ◽  
Dominant Negative ◽  
Calcium Handling ◽  
Dominant Negative Effect ◽  
Base Substitution ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Extensive Evaluation ◽  
Negative Effect

ObjectiveThe calcium-sensing receptor (CASR) has an important role in calcium homoeostasis by controlling PTH secretion and renal calcium handling. Inactivating mutations in the CASR gene (HGNC ID: 1514) cause familial hypocalciuric hypercalcaemia (FHH). We present a case of FHH patient to describe a novel mutation in the CASR.Subjects and methodsA 34-year-old patient was referred because of recurrent hypercalcaemia after resection of two hyperplastic parathyroids. Extensive evaluation found elevated PTH and low calcium/creatinine clearance ratio. One of her three children had high serum calcium concentrations. Genetic studies were performed by PCR amplification of CASR coding exons and direct sequencing of PCR products. Transient transfection of the wild-type (WT) CASR and the mutant CASR into COS-7 was performed to assess functional impact of the mutation and the capacity of either protein to mediate increases in cellular levels of inositol phosphates (IPs).ResultsCASR sequencing found a previously undescribed heterozygous base substitution, determining a change of threonine to isoleucine at codon 550 (p.T550I) in the sixth exon. In contrast to those transfected with WT CASR, which showed a five- to eightfold increase in total IPs at high levels of calcium, COS-7 cells transfected with the (p.T550I) mutant showed no increase confirming to the inactivating nature of the mutation. COS-7 cells co-transfected with the WT and the (p.T550I) mutant showed an intermediate response suggesting a possible dominant negative effect.ConclusionThis case report presents a not-yet-described mutation in the cysteine-rich region of the CASR extracellular domain, a mutation with a possible dominant negative effect.

The extracellular calcium-sensing receptor reciprocally regulates the secretion of BMP-2 and the BMP antagonist Noggin in colonic myofibroblasts

2007 ◽  
Vol 292 (3) ◽  
pp. G753-G766 ◽  
Author(s):  
Dinithi Peiris ◽  
Ivan Pacheco ◽  
Craig Spencer ◽  
R. John MacLeod
Keyword(s):  
Conditioned Medium ◽  
Intestinal Barrier ◽  
Human Colon ◽  
Transient Transfection ◽  
Extracellular Calcium ◽  
Dominant Negative ◽  
Rt Pcr ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Morphogenetic Protein

To understand whether postprandial extracellular Ca2+ (Cao2+) changes were related to intestinal epithelial homeostasis, we performed array analysis on extracellular calcium-sensing receptor (CaSR)-expressing colonic myofibroblasts (18Co cells) and observed increases in bone morphogenetic protein (BMP)-2 transcripts. The present experiments demonstrated that regulated secretion of BMP-2 occurs in response to CaSR activation of these cells and revealed a new property of BMP-2 on the intestinal barrier. Activation by Cao2+, spermine, GdCl3, or neomycin sulfate of 18Co cells or primary isolates of myofibroblasts from the normal human colon stimulated both the synthesis (RT-PCR) and secretion (ELISA) of BMP-2. Transient transfection with short interfering RNA against CaSR completely inhibited BMP-2 secretion. Transient transfection with dominant negative CaSR (R185Q) increased the EC50 of Cao2+ (5.7 vs. 2.3 mM). Upregulation of BMP-2 transcript and secretion occurring within 3 h of CaSR activation was prevented by actinomycin D. CaSR-mediated BMP-2 synthesis and secretion required phosphatidylinositol 3-kinase activation (as assessed by phospho-Akt generation). Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells accelerated restitution in wounded postconfluent Caco-2 cells. Exogenous BMP-2 and conditioned medium from CaSR-stimulated 18Co cells increased the transepithelial resistance of low- and high-resistance T-84 epithelial monolayers. CaSR stimulation of T-84 epithelia and colonic myofibroblasts downregulated the BMP family antagonist Noggin, as assessed by RT-PCR and Western blot analysis. Together, our data suggest that the CaSR mediates the effective concentration of BMP-2 in the intestine, which leads to enhanced repair and barrier development.

scholarly journals Transcriptional Response to Calcium-Sensing Receptor Stimulation

Endocrinology ◽  
2012 ◽  
Vol 153 (10) ◽  
pp. 4716-4728 ◽  
Author(s):  
Gerald Thiel ◽  
Andrea Lesch ◽  
Anja Keim
Keyword(s):  
Gene Transcription ◽  
Ternary Complex ◽  
Dominant Negative ◽  
Signaling Cascade ◽  
Dominant Negative Mutant ◽  
Calcium Sensing Receptor ◽  
Receptor Stimulation ◽  
Calcium Sensing ◽  
Negative Mutant ◽  
Stimulation Of

Abstract Elevated extracellular Ca2+ concentrations stimulate the G-protein coupled receptor calcium-sensing receptor. Here we show that this stimulation induces the expression of biologically active early growth response protein 1 (Egr-1), a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor Ets-like protein-1 (Elk-1), a key transcriptional regulator of serum response element-driven gene transcription, prevented Egr-1 expression, indicating that Elk-1 or related ternary complex factors connect the intracellular signaling cascade elicited by activation of calcium-sensing receptors with transcription of the Egr-1 gene. These data were corroborated by the fact that stimulation of calcium-sensing receptors increased the transcriptional activation potential of Elk-1. In addition, activator protein-1 (AP-1) transcriptional activity was significantly elevated after the stimulation of calcium-sensing receptors. The expression of a dominant-negative mutant of Elk-1 reduced c-Fos expression and prevented the up-regulation of AP-1 activity as a result of calcium-sensing receptor stimulation, indicating that ternary complex factors control both Egr-1- and AP-1-regulated transcription. In addition, AP-1 activity was reduced after the expression of a dominant-negative mutant of c-Jun in cells expressing an activated calcium-sensing receptor. Stimulus-transcription coupling leading to the up-regulation of Egr-1 and AP-1 controlled transcription in cells expressing calcium-sensing receptors required the protein kinases Raf and ERK, whereas the overexpression of MAPK phosphatase-1 interrupted the signaling cascade connecting calcium-sensing receptor stimulation with transcription of Egr-1 and AP-1 controlled genes. The fact that calcium-sensing receptor stimulation activates the transcription factors Egr-1, Elk-1, and AP-1 indicates that regulation of gene transcription is an integral part of calcium-sensing receptor induced signaling.

NFAT regulates calcium-sensing receptor-mediated TNF production

2006 ◽  
Vol 290 (5) ◽  
pp. F1110-F1117 ◽  
Author(s):  
Huda Ismail Abdullah ◽  
Paulina L. Pedraza ◽  
Shoujin Hao ◽  
Karin D. Rodland ◽  
John C. McGiff ◽  
...  
Keyword(s):  
Extracellular Fluid ◽  
Dominant Negative ◽  
Peptide Ligand ◽  
Thick Ascending Limb ◽  
Dependent Manner ◽  
Calcium Sensing Receptor ◽  
Activated T Cells ◽  
Calcium Sensing ◽  
Dependent Activity ◽  
Calcineurin Activity

Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

Calcium receptor is functionally expressed in rat neonatal ventricular cardiomyocytes

2006 ◽  
Vol 290 (3) ◽  
pp. H1165-H1171 ◽  
Author(s):  
Jacob Tfelt-Hansen ◽  
Jakob Lerche Hansen ◽  
Sanela Smajilovic ◽  
Ernest F. Terwilliger ◽  
Stig Haunso ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Messenger Rna ◽  
Cardiac Development ◽  
Inositol Phosphate ◽  
Tritiated Thymidine ◽  
Extracellular Calcium ◽  
Dominant Negative ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Ventricular Cardiomyocytes

Both intra- and extracellular calcium play multiple roles in the physiology and pathophysiology of cardiomyocytes, especially in stimulus-contraction coupling. The intracellular calcium level is closely controlled through the concerted actions of calcium channels, exchangers, and pumps; however, the expression and function(s) of the so-called calcium-sensing receptor (CaR) in the heart remain less well characterized. The CaR is a seven-transmembrane receptor, which, in response to noncovalent binding of extracellular calcium, activates intracellular effectors, including G proteins and extracellular signal-regulated kinases (ERK1/2). We have shown that cultured neonatal cardiomyocytes express the CaR messenger RNA and the CaR protein. Furthermore, increasing concentrations of extracellular calcium and a type II CaR activator “calcimimetic” caused inositol phosphate (IP) accumulation, downregulated tritiated thymidine incorporation, and supported ERK1/2 phosphorylation, suggesting that the CaR protein is functionally active. Interestingly, the calcimimetic induced a more rapid ERK1/2 phosphorylation than calcium and left-shifted the IP concentration-response curve for extracellular calcium, supporting the hypothesis that CaR is functionally expressed in cardiac myocytes. This notion was underscored by studies using a virus containing a dominant-negative CaR construct, because this protein blunted the calcium-induced IP response. In conclusion, we have shown that the CaR is functionally expressed in neonatal ventricular cardiomyocytes and that the receptor activates second messenger pathways, including IP and ERK, and decreases DNA synthesis. A specific calcium-sensing receptor on cardiac myocytes could play a role in regulating cardiac development, function, and homeostasis.

Calcium-sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells

2003 ◽  
Vol 285 (2) ◽  
pp. E329-E337 ◽  
Author(s):  
J. Tfelt-Hansen ◽  
R. J. MacLeod ◽  
N. Chattopadhyay ◽  
S. Yano ◽  
S. Quinn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
P38 Mapk ◽  
Leydig Cells ◽  
Actinomycin D ◽  
Dominant Negative ◽  
Calcium Sensing Receptor ◽  
Pkc Inhibitor ◽  
Calcium Sensing ◽  
Parathyroid Hormone Related Peptide ◽  
First Time

Elevated extracellular calcium ([Ca2+]o) and other agonists potentially acting via the calcium-sensing receptor (CaR) increase parathyroid hormone-related peptide (PTHrP) release from H-500 Leydig cells. Here, we provide strong evidence for the CaR's involvement by using a dominant negative CaR that attenuates high [Ca2+]o-induced PTHrP release. This effect is likely transcriptional, because high [Ca2+]oupregulates the PTHrP transcript, an effect that is abolished by actinomycin D. Regulation of PTHrP release by the CaR involves activation of PKC as well as ERK1/2, p38 MAPK, and JNK pathways. However, we show for the first time that high [Ca2+]o-induced activation of the stress-activated protein kinase SEK1 is PKC independent, because there is an additive effect of a PKC inhibitor in combination with the JNK inhibitor on [Ca2+]o-stimulated PTHrP release. Furthermore, high [Ca2+]o, in a PKC-independent fashion, induces phosphorylation of ERK1/2, SEK1, p38 MAPK, and its downstream transcription factor ATF-2. We conclude that CaR regulation of PTHrP release in H-500 cells involves activation of PKC as well as the ERK1/2, p38 MAPK, and JNK pathways.

scholarly journals Calcium-sensing receptor signaling pathways in medullary thick ascending limb cells mediate COX-2-derived PGE2 production: functional significance

2008 ◽  
Vol 295 (4) ◽  
pp. F1082-F1089 ◽  
Author(s):  
Huda Ismail Abdullah ◽  
Paulina L. Pedraza ◽  
John C. McGiff ◽  
Nicholas R. Ferreri
Keyword(s):  
Ion Transport ◽  
Dominant Negative ◽  
Prostaglandin E ◽  
Thick Ascending Limb ◽  
Calcium Sensing Receptor ◽  
Activated T Cells ◽  
Calcium Sensing ◽  
Medullary Thick Ascending Limb ◽  
Cox 2

We determined the functional implications of calcium-sensing receptor (CaR)-dependent, Gq- and Gi-coupled signaling cascades, which work in a coordinated manner to regulate activity of nuclear factor of activated T cells and tumor necrosis factor (TNF)-α gene transcription that cause expression of cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) synthesis by rat medullary thick ascending limb cells (mTAL). Interruption of Gq, Gi, protein kinase C (PKC), or calcineurin (CaN) activities abolished CaR-mediated COX-2 expression and PGE2 synthesis. We tested the hypothesis that these pathways contribute to the effects of CaR activation on ion transport in mTAL cells. Ouabain-sensitive O2 consumption, an in vitro correlate of ion transport in the mTAL, was inhibited by ∼70% in cells treated for 6 h with extracellular Ca2+ (1.2 mM), an effect prevented in mTAL cells transiently transfected with a dominant negative CaR overexpression construct (R796W), indicating that the effect was initiated by stimulation of the CaR. Pretreatment with the COX-2-selective inhibitor, NS-398 (1 μM), reversed CaR-activated decreases in ouabain-sensitive O2 consumption by ∼60%, but did not alter basal levels of ouabain-sensitive O2 consumption. Similarly, inhibition of either Gq, Gi, PKC, or CaN, which are components of the mechanism associated with CaR-stimulated COX-2-derived PGE2 synthesis, reversed the inhibitory effects of CaR on O2 consumption without affecting basal O2 consumption. Our findings identified signaling elements required for CaR-mediated TNF production that are integral components regulating mTAL function via a mechanism involving COX-2 expression and PGE2 production.

Involvement of calcium-sensing receptor in osteoblastic differentiation of mouse MC3T3-E1 cells

2005 ◽  
Vol 288 (3) ◽  
pp. E608-E616 ◽  
Author(s):  
Mika Yamauchi ◽  
Toru Yamaguchi ◽  
Hiroshi Kaji ◽  
Toshitsugu Sugimoto ◽  
Kazuo Chihara
Keyword(s):  
Alkaline Phosphatase ◽  
Osteoblastic Differentiation ◽  
Northern Blotting ◽  
Dominant Negative ◽  
Type I ◽  
Calcium Sensing Receptor ◽  
Type I Procollagen ◽  
Calcium Sensing ◽  
High Calcium ◽  
Matrix Production

We have previously shown that the extracellular calcium-sensing receptor (CaR) is expressed in various bone marrow-derived cell lines and plays an important role in stimulating their proliferation and chemotaxis. It has also been reported that the CaR modulates matrix production and mineralization in chondrogenic cells. However, it remains unclear whether the CaR plays any role in regulating osteoblast differentiation. In this study, we found that mineralization of the mouse osteoblastic MC3T3-E1 cells was increased when the cells were exposed to high calcium (2.8 and 3.8 mM) or a specific CaR activator, NPS-R467 (1 and 3 μM). Next, we stably transfected MC3T3-E1 cells with either a CaR antisense vector (AS clone) or a vector containing the inactivating R185Q variant of the CaR (DN clone) that has previously been shown to exert a dominant negative action. Alkaline phosphatase activities were decreased compared with controls in both the AS and DN clones. However, the levels of type I procollagen and osteopontin mRNA in the AS clone, as detected by Northern blotting, were almost the same as in the controls. On the other hand, the expression of osteocalcin, which is expressed at a later stage of osteoblastic differentiation, was significantly reduced in both the AS and DN clones. Mineralization was also decreased in both clones. In conclusion, this study showed that the abolition of CaR function results in diminishing alkaline phosphatase activity, osteocalcin expression, and mineralization in mouse osteoblastic cells. This suggests that the CaR may be involved in osteoblastic differentiation.

Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective Wnt signaling in colon cancer cells

2007 ◽  
Vol 293 (1) ◽  
pp. G403-G411 ◽  
Author(s):  
R. John MacLeod ◽  
Madeline Hayes ◽  
Ivan Pacheco
Keyword(s):  
Colon Cancer ◽  
Wnt Signaling ◽  
Extracellular Calcium ◽  
Dominant Negative ◽  
Orphan Receptor ◽  
Chimeric Antibody ◽  
Calcium Sensing Receptor ◽  
Sirna Duplex ◽  
Calcium Sensing ◽  
Ht 29

To understand the role of the colonic extracellular calcium-sensing receptor (CaSR) in calcium chemoprotection against colon cancer, we activated the CaSR with 5 mM Ca2+ on HT-29 cells, an adenocarcinoma cell line. High Ca2+ stimulated the upregulation (as assessed by RT-PCR) and the secretion of Wnt5a (assessed by Western blot), a noncanonical Wnt family member. Inhibiting CaSR activity with a short interfering RNA (siRNA) duplex against the CaSR reduced CaSR protein and prevented the secretion of Wnt5a. Dominant negative CaSR (R185Q) or siRNA blocked the high Ca2+-mediated inhibition of the β-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of β-catenin reporter was prevented by dominant negative ubiquitin ligase seven in absentia homolog 2 (Siah2). In low-calcium medium, overexpressing Wnt5a increased Siah2 amplicons and protein. Inducing the expression of full-length adenomatous polyposis coli (APC) prevented CaSRmediated increases of Siah2 and Wnt5a. Overexpressing the receptor tyrosine kinase-like orphan receptor 2 (Ror2) increased Wnt5a and CaSR-mediated inhibition of TOPflash. Conditioned medium from Wnt5a-transfected cells added to HT-29 cells in low-Ca2+ medium inhibited the β-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca2+ medium increased the inhibition of β-catenin reporter caused by recombinant Wnt5a protein compared with addition of Wnt5a protein alone. Our findings demonstrate that APC status plays a key role as a determinant of Wnt5a secretion and suggest that CaSR-mediated secretion of Wnt5a will inhibit defective Wnt signaling in APC-truncated cells in an autocrine manner.

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