Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 mRNA were detected in LPMC. CRF receptor type 2β mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.     

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.     

Microcalcifications,calcium-sensing receptor,and cancer

Calcium stones and calculi are observed in numerous human tissues. They are the result of deposition of calcium salts and are due to high local calcium concentrations. Prostatic calculi are usually classified as endogenous or extrinsic stones. Endogenous stones are commonly caused by obstruction of the prostatic ducts around an enlarged prostate resulting from benign prostatic hyperplasia or from chronic inflammation. The latter occurs mainly around the urethra and is generally caused by reflux of urine into the prostate. Calcium concentrations higher than in the plasma at sites of infection may induce the chemotactic response that eventually leads to recruitment of inflammatory cells. The calcium sensing receptor (CaSR) may be crucial for this recruitment as its expression and activity are increased by cytokines such as IL-6 and high extracellular calcium concentrations, respectively. The links between calcium calculi, inflammation, calcium supplementation, and CaSR functions in prostate cancer patients will be discussed in this review.     

Immunocytochemical localization of the extracellular calcium-sensing receptor in normal and malignant human large intestinal mucosa.

We identified the parathyroid type Ca(2+)-sensing receptor (CaR) in normal human colon mucosa and in cancerous lesions at the mRNA and protein level. Polymerase chain reaction produced an amplification product from reverse-transcribed large intestinal RNA which corresponded in size and length to a 537-bp sequence from exon 7 of the CaR gene. With a specific antiserum against its extracellular domain, the CaR could be detected by immunostaining in normal human colon mucosa in cells preferentially located at the crypt base. The CaR protein was also expressed in tumors of the large bowel in all 20 patients examined. However, the great majority of CaR-positive cells in the adenocarcinomas inspected were confined to more differentiated areas exhibiting glandular-tubular structures. Poorly or undifferentiated regions were either devoid of specific immunoreactivity or contained only isolated CaR-positive cells. In the normal mucosa and in glandular-tubular structures of cancerous lesions, the CaR was exclusively expressed in chromogranin A-positive enteroendocrine cells and in only a small fraction of PCNA-positive cells.     

Extracellular calcium-sensing receptor transactivates the epidermal growth factor receptor by a triple-membrane-spanning signaling mechanism

Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.     

Biochemistry, physiology and pathophysiology of the extracellular calcium-sensing receptor

Calcium (Ca(2+)) has long been recognized as a physiologically indispensable ion owing to its numerous intra- and extracellular roles. More recently, it has become apparent that extracellular calcium (Ca(2+)(o)) also serves as an extracellular first messenger following the cloning of a Ca(2+)(o)-sensing receptor (CaR) that belongs to the superfamily of G protein-coupled receptors (GPCR). The CaR probably functions as a dimer in performing its central role of "sensing" minute alterations in Ca(2+)(o) and adjusting the secretion of parathyroid hormone (PTH) so as to normalize Ca(2+)(o) through the actions of PTH on the effector elements of the mineral ion homeostatic system (e.g., kidney, bone and intestine). Several inherited human conditions are caused by inactivating or activating mutations of this receptor, and mice have been generated with targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in patients with these conditions and in CaR-deficient mice have proven the physiological importance of the CaR in mineral ion homeostasis. An accumulating body of evidence, however, suggests that the CaR also plays numerous roles outside the realm of systemic mineral ion homeostasis. The receptor regulates processes such as cellular proliferation and differentiation, secretion, membrane polarization and apoptosis in a variety of tissues/cells. Finally, the availability of specific "calcimimetic", allosteric CaR activators - which are currently in clinical trials - will probably have therapeutic implications for diseases caused by malfunction of the CaR in tissues not only within, but also outside, the mineral ion homeostatic system.     

pH Sensing by the calcium-sensing receptor

The calcium-sensing receptor (CaR) is activated by small changes in the ionic extracellular calcium concentration (Ca(o)) within the physiological range, allowing the parathyroid gland to regulate serum Ca(o); however, the CaR is also distributed in a number of other tissues where it may sense other endogenous agonists and modulators. CaR agonists are polycationic molecules, and our previous studies suggest that charged residues in the extracellular domain of the CaR are critical for receptor activation through electrostatic interactions. Therefore, pH could also potentially modulate CaR activation by its polycationic agonists. Changes in the concentration of extracellular H(+) substantially altered the activation of the CaR by Ca(o) and other CaR agonists. The effects of external pH on the CaR's sensitivity to its agonists were observed for both acidic and basic deviations from physiological pH of 7.4, with increases in pH rendering the receptor more sensitive to activation by Ca(o) and decreases in pH producing the converse effect. At pH values more acidic than 5.5, CaR sensitivity to its agonists showed some recovery. Changes in the intracellular pH could not account for the effects of external pH on CaR sensitivity to its agonists. Other G-protein-coupled receptors, which are endogenously expressed in human embryonic kidney 293 cells, showed little change in activity with alterations in external pH or effects opposite those found for the CaR. Extracellular pH directly alters the CaR in the case of Ca(o) and Mg(o) activation; however, the charges on many organic and inorganic agonists are pH-dependent. Activating CaR mutations show reduced pH(o) modulation, suggesting a molecular mechanism for increased CaR activity at physiological pH(o). Several CaR-expressing tissues, including regions of the stomach, the kidney, bone, and the brain, could potentially use the CaR as a sensor for pH and acid-base status.     

Gammadelta T cell receptor repertoire in blood and colonic mucosa of rhesus macaques

Although their precise roles are not well defined, gammadelta T lymphocytes are recognized as regular components of immune responses. These cells express a limited T cell receptor repertoire and they can be stimulated by soluble ligands without conventional processing and presentation by major histocompatibility antigens. Progress in this area has been limited by the substantial differences between murine and human gammadelta T cells and the lack of knowledge about these cells in nonhuman primates. We used molecular analysis of T cell receptor diversity to characterize gammadelta T cell populations from peripheral blood and colon of rhesus macaques (Macaca mulatta). The gammadelta T cell receptor diversity was limited and distinct for these tissue compartments, particularly in the TCRGV2 family. Furthermore, the TCRDV1 + subset of peripheral blood gammadelta T cells showed signs of progressive oligoclonalization as a function of age. Similar observations have been reported for human tissue samples and our results validate rhesus macaques as an appropriate animal model for studying primate gammadelta T cell populations.     

Calcium, magnesium, and growth control in the WI-38 human fibroblast cell

WI-38 and SV40WI-38 cells have been synchronized using centrifugal elutriation. This technique allows for the rapid harvesting of early G1 phase cells from exponentially growing populations of both the normal and transformed cell. Using these cells, as well as WI-38 cells synchronized by serum deprivation, we have examined the effects of extracellular Ca and Mg levels on the progression of cells through G1 phase. A differential sensitivity to both Ca and Mg deprivation is observed between normal and transformed cells. The WI-38 cell requires higher levels of both ions for traversal of G1 phase and for continued proliferation as compared to the transformed cell. The temporal nature of the Ca and Mg requirements for the WI-38 cell has been examined during G1 phase. Ca is strictly required during early and late G1 phase, but not necessarily throughout mid-G1. An early as well as a late G1 Ca requirement is also found in serum-stimulated WI-38 cells. In contrast, the Mg requirement of WI-38 cells does not appear to be temporally well-defined. Mg appears to be a permissive factor, required throughout G1 phase rather than at certain prescribed intervals. On the basis of these data, it seems unlikely that these two cations exert their effects on cell growth entirely through a common competitive mechanism. Ca would appear to be involved in early serum or growth factor-mediated G1 events and later pre-S-phase events, as suggested in previous studies on other cell lines.     

Biased agonism of the calcium-sensing receptor

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.     

The calcium-sensing receptor in taste tissue

Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.     

The role of the calcium-sensing receptor in cancer

The extracellular calcium-sensing receptor (CaR) is a versatile sensor of small, polycationic molecules ranging from Ca2+ and Mg2+ through polyarginine, spermine, and neomycin. The sensitivity of the CaR to changes in extracellular Ca2+ over the range of 0.05-5 mM positions the CaR as a key mediator of cellular responses to physiologically relevant changes in extracellular Ca2+. For many cell types, including intestinal epithelial cells, breast epithelial cells, keratinocytes, and ovarian surface epithelial cells, changes in extracellular Ca2+ concentration over this range can switch the cellular behaviour from proliferation to terminal differentiation or quiescence. As cancer is predominantly a disease of disordered balance between proliferation, differentiation, and apoptosis, disruptions in the function of the CaR could contribute to the progression of neoplastic disease. Loss of the growth suppressing effects of elevated extracellular Ca2+ have been demonstrated in parathyroid hyperplasias and in colon carcinoma, and have been correlated with changes in the level of CaR expression. Activation of the CaR has also been linked to increased expression and secretion of PTHrP (parathyroid hormone-related peptide), a primary causal factor in hypercalcemia of malignancy and a contributor to metastatic processes involving bone. Although mutation of the CaR does not appear to be an early event in carcinogenesis, loss or upregulation of normal CaR function can contribute to several aspects of neoplastic progression, so that therapeutic strategies directed at the CaR could potentially serve a supportive function in cancer management.     

Regeneration of colonic mucosa in the rat

As part of a study of ulcer formation and healing, regeneration of colonic mucosa in rats was studied following placement of a surgical lesion. Alterations in mucosubstances and connective tissue were examined and their possible significance discussed. The sequence of events in healing was: (1) The mucosa adjacent to the lesion tipped into the lesioned area. The crypts in this mucosa became lined with cells which contained no mucus and had no striated borders. Later in the experimental period, these undifferentiated cells gave rise to cells containing carboxymucins. Cells containing sulfomucin, neutral mucin, or having striated borders arose from the carboxymucin cells. (2) An epithelial ledge of undifferentiated cells migrated onto a sulfated glycosaminoglycan, fibrous interface between necrotic and living tissue in the lesion. (3) Crypt formation began with the appearance of intraepithelial anlagen. (4) Crypts lengthened by a process of epithelial-connective tissue proliferation from the base of the crypt upwards. Following completion of connective tissue regeneration, crypts formed by invading the reestablished lamina propria. (5) The first mucous cells in the ledge contained carboxymucins. As crypt formation occurred, these cells gave rise to typical columnar absorptive cells, to cells containing sulfomucins, and to cells containing neutral mucins. (6) Lengthening of crypts ceased following the appearance of a sulfated acid glycosaminoglycan—collagenous layer deep in the submucosa.     

Beta-arrestin- and G protein receptor kinase-mediated calcium-sensing receptor desensitization

Extracellular calcium rapidly controls PTH secretion through binding to the G protein-coupled calcium-sensing receptor (CASR) expressed in parathyroid glands. Very little is known about the regulatory proteins involved in desensitization of CASR. G protein receptor kinases (GRK) and beta-arrestins are important regulators of agonist-dependent desensitization of G protein-coupled receptors. In the present study, we investigated their role in mediating agonist-dependent desensitization of CASR. In heterologous cell culture models, we found that the transfection of GRK4 inhibits CASR signaling by enhancing receptor phosphorylation and beta-arrestin translocation to the CASR. In contrast, we found that overexpression of GRK2 desensitizes CASR by classical mechanisms as well as through phosphorylation-independent mechanisms involving disruption of Galphaq signaling. In addition, we observed lower circulating PTH levels and an attenuated increase in serum PTH after hypocalcemic stimulation in beta-arrestin2 null mice, suggesting a functional role of beta-arrestin2-dependent desensitization pathways in regulating CASR function in vivo. We conclude that GRKs and beta-arrestins play key roles in regulating CASR responsiveness in parathyroid glands.     

Diseases associated with the extracellular calcium-sensing receptor

The human calcium-sensing receptor (CaSR) is a 1078 amino acid cell surface protein, which is predominantly expressed in the parathyroids and kidney, and is a member of the family of G protein-coupled receptors. The CaSR allows regulation of parathyroid hormone (PTH) secretion and renal tubular calcium reabsorption in response to alterations in extracellular calcium concentrations. The human CaSR gene is located on chromosome 3q21.1 and loss-of-function CaSR mutations have been reported in the hypercalcaemic disorders of familial benign (hypocalciuric) hypercalcaemia (FHH, FBH or FBHH) and neonatal severe primary hyperparathyroidism (NSHPT). However, some individuals with loss-of-function CaSR mutations remain normocalcaemic. In addition, there is genetic heterogeneity amongst the forms of FHH. Thus, the majority of FHH patients have loss-of-function CaSR mutations, and this is referred to as FHH type 1. However, in one family, the causative gene for FHH is located on 19p13, referred to as FHH type 2, and in another family it is located on 19q13, referred to as FHH type 3. Gain-of-function CaSR mutations have been shown to result in autosomal dominant hypocalcaemia with hypercalciuria (ADHH) and Bartter's syndrome type V. CaSR auto-antibodies have been found in FHH patients who did not have loss-of-function CaSR mutations, and in patients with an acquired form (i.e. autoimmune) of hypoparathyroidism. Thus, abnormalities of the CaSR are associated with three hypercalcaemic and three hypocalcaemic disorders.     

Structure-function relationship of the extracellular calcium-sensing receptor

The extracellular calcium-sensing receptor (CaR) originally cloned from bovine parathyroid gland is a G protein-coupled receptor. The physiological relevance of the cloned CaR for sensing and regulating the extracellular calcium concentration has been established by identifying hyper- and hypocalcemic disorders resulting from inactivating and activating mutations, respectively, in the CaR. The cloned CaR has been stably or transiently expressed in human embryonic kidney cells and significant progress has been made in elucidating its regulation and activation process using physiological, biochemical and molecular biological methods. A large collection of naturally occurring CaR mutations offers a valuable resource for studies aimed at understanding the structure-function relationships of the receptor, including functional importance of CaR dimerization. In turn, characterization of these naturally occurring mutations has clarified the pathogenesis of clinical conditions involving abnormalities in the CaR, such as familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.     

Interaction of the calcium-sensing receptor and filamin, a potential scaffolding protein

In many cases, the biologic responses of cells to extracellular signals and the specificity of the responses cannot be explained solely on the basis of the interactions of known signaling proteins. Recently, scaffolding and adaptor proteins have been identified that organize signaling proteins in cells and that contribute to the nature and specificity of signaling pathways. In an effort to identify proteins that might organize the signaling system(s) activated by the extracellular Ca(2+) receptor (CaR), we used a bait construct representing the intracellular C terminus of the human CaR and the yeast two hybrid system to screen a human kidney cDNA library. We identified a clone representing the C-terminal 1042 amino acids (aa) of the cytoskeletal protein filamin (ABP-280). Analysis of truncation and deletion constructs of the CaR C terminus and the filamin cDNA clone demonstrated that the CaR and filamin interact via regions containing aa 907-997 of the CaR C terminus and aa 1566-1875 of filamin. Interaction of the two proteins in mammalian HEK-293 cells was demonstrated by co-immunoprecipitation and colocalization of them using immunofluorescence microscopy. The functional importance of their interaction was documented by transiently expressing the CaR in M2 melanoma cells that lack filamin, or in A7 melanoma cells that stably express filamin, and demonstrating that the CaR activated ERK only in the presence of filamin. Co-expression of the CaR with a peptide derived from the region of the CaR C terminus that interacts with filamin reduced the ability of the CaR to activate p42ERK in a dose-dependent manner, but did not inhibit the ability of the ET(A) receptor to activate ERK. The fact that filamin interacts with the CaR and other cell signaling proteins including mitogen-activated protein kinases and small GTPases, indicates that it may act as a scaffolding protein to organize cell signaling systems involving the CaR.