IGNOU-I2IT Centre of Excellence for Advanced Education and Research, Pune, Maharashtra, India
*Corresponding author: Dr. Sudhir Kumar School of Biotechnology, IGNOU-I2IT Centre of Excellence for Advanced Education and Research, P14, Rajiv Gandhi Infotech Park, Phase 1, Hinjewadi, Pune, Maharashtra-411057, India. E-mail: sudhirk@isquareit.ac.in; Contact number: +91-20-22933441
December 2013
Key words: CaSR, G protein-coupled receptor, signaling
Abstract
The calcium sensing receptor (CaSR) is a member of the largest family of cell surface receptors, the G protein-coupled receptors involved in calcium homeostasis. Studies identified a crucial role for the CaSR in systemic calcium homeostasis through its ability to sense small changes in circulating calcium concentration and to combine this information to intracellular signaling pathways that influence parathyroid hormone secretion. The CaSR is the target of small molecule allosteric modifiers, either activators, calcimimetics, or inhibitors, calcilytics. The presence of CaSR protein in tissues is not directly involved in regulating mineral ion homeostasis points to a role for the CaSR in other cellular functions including the control of cellular proliferation, differentiation and apoptosis. This review will provide a comprehensive exploration of the different aspects of tissue expression patterns, and will relate their impact on the functionality of the CaSR from a molecular perspective.
Calcium sensing receptor
The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) in the plasma membrane. The CaSR gene is located from base pair 121,902,529 to base pair 122,005,349 on long q arm of chromosome 3. It plays an essential role in calcium ion homeostasis, cellular proliferation, differentiation and apoptosis (Diez-Fraile et al., 2013). The CaSR is composed of three main regions: a large extracellular domain, where the interaction with the Ca2+ occurs, a seven transmembrane domain that is a common feature of all GPCRs, and a carboxyl-terminal intracellular tail, necessary for Ca2+ mediated activation of G proteins, cell surface expression and phospholipase C activation, among other functions (Masvidal et al., 2013). Calcium sensing receptor is activated by many molecules such as polyamines and l-amino acids. The extracellular calcium ion is the primary physiological ligand for CaSR. Binding of the ligand with the receptor induces the change in the conformation of the receptor, which leads to 'ligand biased signaling'. Alteration in CaSR expression and function are associated with cancer progression. Interestingly, the CaSR appears to act both as a tumour suppressor and an oncogene, depending on the condition (Woudstra et al., 1990). Reduced expression of the CaSR occurs in both parathyroid and colon cancers, leading to loss of the growth suppressing effect of high calcium ion. On the other hand, activation of the CaSR might facilitate metastasis to bone in breast and prostate cancer. A deeper understanding of the mechanisms driving CaSR signalling in different tissues, aided by a systems biology approach, will be instrumental in developing novel drugs that target the CaSR or its ligands in cancer (Brennan et al., 2013). Three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) were analyzed and assessed for genetic variants producing a less active receptor are associated with more aggressive disease course (Masvidal et al., 2013). Calcium (Ca2+) is a vital cation involved in diverse biologic processes ranging from bone formation and neurotransmission to hormone secretion and muscle contraction. The CaSR is expressed abundantly in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland. Tissues in which the CaSR is expressed where it is likely to play a mineral ion homeostatic role include, skeletal tissues (Chang et al., 2008), osteoclasts and their precursors (Mentaverri et al., 2006), osteoblasts and their precursors, osteocytes, chondrocytes (Liaw et al., 1992) and placenta (Ayerdi, 1978). Tissues in which the CASR is expressed at lower levels and is likely to play roles unrelated to mineral ion homeostasis include neurons and glia of the brain (Ruat et al., 1995), keratinocytes (Oda et al., 1998), vascular smooth muscle cells (Molostvov et al., 2007), hematopoietic stem cells (in blood and bone marrow) (Adams et al., 2006), stomach (Ray et al., 1997), intestine (Geibel et al., 2006), colon (Cheng et al., 2002), liver (Canaff et al., 2001), pancreas (Bruce et al., 1999), and others.
Disorders associated with CaSR
Both gain-of-function and loss-of-function mutations have been reported in human CaSR. Gain of function mutations cause autosomal-dominant hypocalcemia with hypercalciuria as well as a variant form of Bartter's syndrome (Vargas-Poussou et al., 2002). Heterozygosity for loss-of-function mutations causes familial hypocalciuric hypercalcemia (FHH), whereas loss-of-function mutations in both alleles cause neonatal severe hyperparathyroidism (NSHPT) (Brown et al., 1998). Population studies have suggested a possible role for CaSR variants in explaining human variation in serum Ca2+ and bone mineral density (BMD) (O'Seaghdha et al., 2010; Lorentzon et al., 2001). Various studies have suggested a role for Casr in the regulation of renal Ca2+ handling. Increasing extracellular Ca2+ concentration elicits a marked increase in urinary Ca2+ excretion, independently of any obvious changes in calcium-regulating hormones. There is abundant evidence that renal tubular Casr plays a role in the control of divalent cation reabsorption under both normal and pathologic conditions (Houillier and Paillard, 2003). Clinical studies in humans under PTH clamp conditions suggested that alterations in serum Ca2+ modulate excretion of Ca2+, magnesium (Mg2+), and sodium (Na+) through a CaSR dependent mechanism (el-Hajj Fuleihan et al., 1998). Recent PTH clamp studies in mice showed similar results (Kantham et al., 2009).
CaSR in bone
In bone, Ca2+, acting via the CaSR, stimulates recruitment and proliferation of preosteoblasts, their differentiation to mature osteoblasts, and synthesis and mineralization of bone proteins. Conversely Ca2+ inhibits the formation and activity and promotes apoptosis of osteoclasts, likely via the CaSR. These actions tend to mobilize skeletal Ca2+ during deficiency and retain it when Ca2+ is plentiful (Brown, 2013). Osteopontin, an acidic glycoprotein produced by osteoblasts is highly enriched at the endosteal surface and was also shown necessary for the HSC lodgment (Haylock and Nilsson, 2006; Nilsson et al., 2005). The endosteal niche has uniquely enriched calcium content relative to serum, CaSR expression has been demonstrated on HSCs, including the stem cell-enriched (lin-) Sca-1+c-Kit+ (LSK) population (Adams et al., 2006). Finally, in a series of elegant experiments using CaSR-/- mice, Adams et al. demonstrated that it is the mineral content of the niche dictates HSC localization via the CaSR (Adams et al., 2006). These mechanisms by which the CaSR dictates preferential localization of HSCs in the bone marrow endosteal region may provide additional insights for the fundamental interrelationship between the stem cell niche and stem cell fate (Theman and Collins, 2009).
CaSR in kidney
As the CaSR in the kidney controls calcium reabsorption and excretion and subsequently affects blood calcium concentration, agonists and antagonists of the CaSR could be used to control blood calcium concentration in patients who have lost their ability to regulate parathyroid hormone secretion (Houillier, 2013). Since extracellular Ca2+ is essential for the development of stable epithelial tight junctions (TJ), the endogenous CaSR is located at the basolateral pole of Madin-Darby Canine Kidney (MDCK) cells (Riccardi et al., 1998). It is co-distributed with β-catenin on the basolateral membrane. Switching MDCK cells from low calcium media to media containing the normal calcium concentration significantly increases CaSR expression at both the mRNA and protein levels. Exposure of MDCK cells maintained in low-Ca2+ conditions to the CaSR agonists neomycin, Gd3+ or R-568 causes the transient relocation of the tight junction components ZO-1 and occludin to sites of cell-cell contact, while inducing no significant changes in the expression of mRNAs encoding junction-associated proteins. CaSR stimulation also increases the interaction between ZO-1 and the F-actin-binding protein, I-afadin. This effect does not involve activation of the AMP-activated protein kinase. In contrast, CaSR inhibition by NPS-2143 significantly decreases ZO-1/I-afadin interaction and reduces ZO-1 deposition at the cell surface following a Ca2+ switch from 5 μM to 200 μM [Ca2+]e. Pre-exposure of MDCK cells to the cell-permeant Ca2+ chelator, BAPTA-AM, similarly prevents TJ-assembly caused by CaSR activation. Finally, stable transfection of MDCK cells with a cDNA encoding a human disease-associated gain-of-function mutant form of the CaSR increases these cells' transepithelial electrical resistance in comparison to expression of the wild-type human CaSR (Jouret et al., 2013). In addition, more work is needed to decipher the molecular mechanisms through which CaSR determines calcium transport in the loop of Henle.
CaSR in adipocytes
CaSR being expressed in human adipocytes and adipocyte progenitor cells opens the possibility to investigate the physiological implications and thus contributing a novel component for adipose tissue biology research. Presence of the CaSR along with plasma membrane markers in adipocyte sub fractions is consistent with a putative role as a plasma membrane receptor in the adipose cells. It is theoretically plausible to relate CaSR signaling to proliferation, differentiation, and metabolic activity of adipose cells. For example, activation of the receptor in the adipocyte is expected to trigger signaling cascades (Hobson et al., 2003; Ward et al., 2002), which have been described in relevant phenomena in adipocyte metabolism such as adipogenesis and lipogenesis. Moreover, an increase in cytosolic Ca2+ as a consequence of CaSR activation, as reported in parathyroid cells (Roussanne et al., 2001), human intestinal epithelial cell lines (Gama et al., 2007), keratinocytes (Tu et al., 2001), and antral gastrin cells, among others, would also influence adipogenesis (Bost et al., 2002; Pérez et al., 2004) and triglyceride storage in the adipocyte (Cifuentes et al., 2005). CaSR activation may interfere with the initial stages of adipocyte differentiation; however, these events do not seem to preclude adipogenesis from continuing. Even though adipogenesis (particularly in subcutaneous depots) is associated with insulin sensitivity and adequate adipose function, the implications of our findings in visceral adipocytes, especially in the context of inflamed AT and over nutrition, remain to be established (Villarroel et al., 2013). Adipocyte differentiation and adipogenesis are closely related to obesity and obesity-induced metabolic disorders. The calcium-sensing receptor (CaSR) has been reported to play an antilipolytic role in human adipocyte and regulate cell differentiation in many tissues. Scientists have observed that activation of CaSR significantly promoted adipocyte differentiation and adipogenesis in human adipocytes. Gene expression analysis revealed that the CaSR activation increased the transcription factor proliferator-activated receptor γ (PPARγ) and its downstream genes including CCAAT element binding protein α (C/EBPα), adipose fatty acid-binding protein (aP2), and lipoprotein lipase. The activity of glycerol-3-phosphate dehydrogenase was also increased after the stimulation of CaSR. In addition, levels of cyclic AMP and calcium which have been shown to regulate PPARγ gene expression were significantly affected by the activation of CaSR. These effects could be suppressed by CaSR small interfering RNA (CaSR-siRNA) (He et al., 2012). Many findings suggest that activation of CaSR promotes differentiation and adipogenesis in adipocytes, which might be achieved by up-regulating PPARγ and its downstream gene expressions. Therefore, CaSR in adipocytes may be involved in the pathogenesis of obesity by promoting adipocyte differentiation and adipogenesis.
CaSR in nervous system
The calcium sensing receptor (CaSR) is expressed by subpopulations of neuronal and glial cells through out the brain and is activated by extracellular calcium. During development, the CaSR regulates neuronal cell growth and migration as well as oligodendroglial maturation and function. Emerging evidence suggests that in nerve terminals, CaSR is implicated in synaptic plasticity and neurotransmission (Ruat and Traiffort, 2013). The highest level of CaSR expression is within the region of the brain known as the sub fornical organ, which, due to an absence of a blood-brain barrier, is exposed to systemic fluid (Yano et al., 2004). CaSR mediated current regulates the bursting of action potentials and that subsequent depolarizing after potentials of neurons of the subfornical organ canal so be modulated by the CaSR (Washburn et al., 2000). There is also abundant expression of the CaSR in the hippocampus and studies of neurons of the hippocampus have revealed that the CaSR mediated activation of calcium permeable, non selective cation channels can be induced by the CaSR agonist amyloid β peptide, which is excessively produced in patients with Alzheimer's disease (Ye et al., 1997). The expression of the CaSR within the nervous system is not limited to neuronal cells because the receptor has been detected in glial cells as well (Washburn et al., 2000) CaSR identified in oligodendrocytes and microglia have also been found to regulate calcium activated potassium channels (Chattopadhyay et al., 1998b). More recently, the CaSR has been found to be expressed in the tooth dental pulp, sensory axons, and trigeminal ganglion of rats, where it was shown to be involved in the regulation of blood flow (Magno et al., 2011). Very little is known about the role, if any, of the CaSR in brain tissue that has under gone trauma, but one study examining three different types of experimental brain lesions has shown that there is a delayed increase in CaSR mRNA 7 d after the initial injury (Mudò et al., 2009). Finally, evidence that the CaSR contributes to the pathogenesis of various brain disorders raises the possibility that pharmacological modulators of the CaSR may have therapeutic benefit.
CaSR in breast
The CaSR has been identified in both normal and malignant breast tissue by Northern analysis and immuno-histochemistry (Cheng et al., 1998). In normal breast cell lines, CaSR stimulation inhibits PTHrP secretion, whereas in breast cancer cell lines PTHrP secretion is increased by CaSR stimulation (VanHouten et al., 2004; Sanders et al., 2000). During lactation, activation of the CaSR in mammary epithelial cells downregulates parathyroid hormone-related protein (PTHrP) levels in milk and in the circulation, and increases calcium transport into milk. In contrast, in breast cancer cells the CaSR upregulates PTHrP production. A switch in G-protein usage underlies the opposing effects of the CaSR on PTHrP expression in normal and malignant breast cells. During lactation, the CaSR in normal breast cells coordinates a feedback loop that matches the transport of calcium into milk and maternal calcium metabolism to the supply of calcium. A switch in CaSR G-protein usage during malignant transformation converts this feedback loop into a feed-forward cycle in breast cancer cells that may promote the growth of osteolytic skeletal metastases (Kirchhoff and Geibel, 2006). Further investigations may lead to improved methodologies for clinical stem cell transplantation using pharmaceutical modulators of the CaR to enhance either stem cell engraftment to or mobilization from the bone marrow (Adams et al., 2006).
CaSR in gastrointestinal tract
The gastrointestinal tract consists of a system of organs designed to cope with the nutrient, electrolyte, and fluid absorption requirements of the body, as well as the secretion of excess electrolytes and fluids (Kirchhoff and Geibel, 2006). The CaSR has been identified in a number of the organs that constitute the gastrointestinal tract, including the esophagus, stomach, small intestine, and colon (Chattopadhyay et al., 1998a). Expression and function of the CaSR have been shown in some mammalian taste buds and basal cells of the esophagus. In human gastric mucous epithelial cells, stimulation of the CaSR, which is primarily expressed at the basolateral membrane, results in increases in both intracellular Ca2+ levels and the rate of proliferation (Rutten et al., 1999). In colonic myofibroblasts, stimulation of the CaSR up regulated the expression and secretion of bone morphogenetic protein-2 in a PI3K-dependent manner, but decreased the expression of the bone morphogenetic protein-2 antagonist, Noggin (Peiris et al., 2007). In addition, the CaSR mediates a novel paracrine relationship between myofibroblasts and overlying epithelial cells in the colon. Thus, CaSR activators stimulate secretion of Wnt5a from myofibroblasts and expression of the Wnt5a receptor Ror2 in epithelial cells. CaSR-mediated Wnt5a/Ror2 engagement stimulates epithelial differentiation and reduces expression of the receptor for tumor necrosis factor 1 (TNFR1). CaSR activators also modulate intestinal motility, inhibit Cl- secretion and stimulate Na+ absorption in both the small intestine and colon. Colonic epithelia from conditional and global CaSR knockout mice exhibit increased proliferation with increased Wnt/β-catenin signaling, demonstrating that the CaSR negatively modulates colonic epithelial growth (Macleod, 2009). The role of the CaSR in the regulation of cell proliferation and differentiation and results of several studies examining the preventive effects of a high calcium diet on colon cancer have led to the proposal that the use of therapeutic CaSR agonists may reduce the risk of colon cancer (Kirchhoff and Geibel, 2006).
CaSR in cancer
The CaSR has recently been documented to be expressed in a variety of benign tumor and malignancies, often at expression levels that differ from those in their healthy counterparts like in breast cancer, prostate cancer, as well as in cancers originating from organs involved in Ca2+ homeostasis, including colorectal cancer and parathyroid adenomas (Sarkar and Kumar, 2012). A recent examination of publicly available gene expression data identified a variety of types of GPCRs (Dores and Trejo, 2012), including protease activated receptor and receptors for various chemokines, adenosine 2B, neuropeptide, metabotropic glutamate, and CaSR that are overexpressed in diverse type of cancer (Rozengurt, 2007). Anti-proliferative effects of calcium in the colon are mediated, at least in part, via the calcium sensing receptor (CaSR), a vitamin D target gene. The expression of CaSR decreases during colorectal tumor progression and the mechanisms regulating its expression (Fetahu et al., 2013). The extracellular Ca2+-sensing receptor (CaSR) is a robust promoter of differentiation in colonic epithelial cells and functions as a tumor suppressor in colon cancer. CaSR mediates its biologic effects through diverse mechanisms. Loss of CaSR expression activates a myriad of stem cell-like molecular features that drive and sustain the malignant and drug-resistant phenotypes of colon cancer. This CaSR-null phenotype, however, is not irreversible and induction of CaSR expression in CaSR-null cells promotes cell death mechanisms and restores drug sensitivity. The CaSR also functions as a tumor suppressor in breast cancer and promotes cellular sensitivity to cytotoxic drugs. BRCA1 and CaSR functions intersect in breast cancer cells, and CaSR activation can rescue breast cancer cells from the deleterious effect of BRCA1 mutations (Singh et al., 2013). High Ca2+ produced, CaSR mediated stimulation of proliferation as well as resistance to apoptosis. Thus it not only enhances tumor growth but also increases invasiveness and metastasis (Cheng et al., 1998; Thomsen et al., 1998). The surface epithelium of the ovary is contiguous with the mesothelial lining of the peritoneum. Thus the ovarian surface epithelial (OSE) cells are actually derived from a mesodermal lineage (Auersperg et al., 1994). Increasing extracellular calcium from 0.2 mM to 5 mM has a marked proliferative response in normal ovarian surface epithelial cells (Saxena et al., 2012). CaSR may mediate the growth of human ovarian surface epithelial cells by extracellular calcium (Saxena et al., 2012). CaSR is neither a potent oncogene nor tumor supressor; it does play an important role in Ca2+ homeostasis, which indirectly maintains a balance between proliferation and differentiation in response to change in extra cellular calcium level. Hence there should be a balanced intake of calcium rich diet, like patients suffering from colon or ovarian cancer should increase the calcium intake, while patients with prostate or breast cancer should decrease the intake of calcium rich food. CaSR can be considered as a molecule that can either promote or prevent tumor growth depending on the type of cancer. Targeting of receptor based on the cancer may offer a cure in designing new therapies, which might complement the existing therapies.
Discussion
The CaSR responds to a various range of stimuli extending well beyond that merely of calcium and these stimuli can pilot to the commencement of an extensive variety of intra cellular signaling pathways that in turn are able to regulate a diverse array of biological processes. The CaSR has been recognized as a novel molecular player in the determination of cellular fate with a fundamental impact on proliferation, apoptosis and differentiation in a varied array of tissues. The use of selective CaSR drugs, together with anti-sense and conditional gene knock-out technology, will definitely be of benefit in further unraveling the role of CaSR in cellular fate. CaSR agonists may be useful in treating hypertensive states characterized by an in appropriately elevated renin concentration, as well, and may constitute a new approach for the prevention and treatment of numerous kidney disorders, such as diabetic nephropathy, through the above-mentioned nephroprotective effects. Calcimimetics could be useful in bone marrow transplants for stimulating homing, lodging, and engraftment of transplanted hematopoietic stem cells and progenitor cells.
Conclusion
Current findings have highlighted the variety of the CaSR because it has been observed to regulate an array of biological processes in a range of tissues. However, many of these conclusions, particularly those relating to the heart and nervous system, were drawn from in vitro studies, and further examination using in vivo based methods such as the generation of CaSR conditional knockouts is required to definitively demonstrate the receptor's role in these regulatory events.
Acknowledgement
We thank SoBT, IGNOU- I2IT Centre of Excellence for Advanced Education and Research, Pune, for their support. The authors declare no conflict of interest.