The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.     

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium-sensing receptor (CaR) in the stringent control of extracellular Ca(2+) concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca(2+) regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr(-/-) mice lacking the full-length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca(2+)-signaling properties. The absence of the full-length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down-regulated in the epidermis of Casr(-/-) mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full-length CaR is required for the normal terminal differentiation of keratinocytes.     

The extracellular calcium-sensing receptor and cell-cell signaling in epithelia

In multicellular organisms, cells are crowded together in organized communities, surrounded by an interstitial fluid of extremely limited volume. Local communication between adjacent cells is known to occur through gap junctions in cells that are physically connected, or through the release of paracrine signaling molecules (e.g. ATP, glutamate, nitric oxide) that diffuse to their target receptors through the extracellular microenvironment. Recent evidence hints that calcium ions may possibly be added to the list of paracrine messengers that allow cells to communicate with one another. Local fluctuations in extracellular [Ca2+] can be generated as a consequence of intracellular Ca2+ signaling events, owing to the activation of Ca2+ influx and efflux pathways at the plasma membrane. In intact tissues, where the interstitial volumes between cells are much smaller than the cells themselves, this can result in significant alterations in external [Ca2+]. This article will explore emerging evidence that these extracellular [Ca2+] changes can be detected by the extracellular calcium-sensing receptor (CaR) on adjacent cells, forming the basis for a paracrine signaling system. Such a mechanism could potentially provide CaR-expressing cells with the means to sense the Ca2+ signaling status of their neighbors, and expand the utility of the intracellular Ca2+ signal to a domain outside the cell.     

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

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 Ca(2+) on HT-29 cells, an adenocarcinoma cell line. High Ca(2+) 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 Ca(2+)-mediated inhibition of the beta-catenin reporter TOPflash. The CaSR/Wnt5a inhibition of beta-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-Ca(2+) medium inhibited the beta-catenin reporter. This inhibition was blocked dose responsively by Frizzled-8/Fc chimeric antibody. Overexpression of Ror2 in HT-29 cells in low-Ca(2+) medium increased the inhibition of beta-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.     

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

The extracellular calcium-sensing receptor is expressed in rat microglia and modulates an outward K+ channel

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that "senses" extracellular calcium ions (Ca2+o) as an extracellular first messenger. In this report, we have shown that the CaR is expressed in primary cultures of microglial cells derived from rat brain as assessed by RT-PCR using four CaR-specific primer pairs followed by sequencing of the amplified products, by northern blot analysis using a CaR-specific probe, as well as by immunocytochemistry and western analysis utilizing a specific polyclonal anti-CaR antiserum. In addition, raising Ca2+o from 0.75 to 3.0 mM or addition of the polycationic CaR agonist neomycin or a "calcimimetic" CaR activator (R-467; NPS Pharmaceuticals) increased the open state probability (Po) of a Ca(+)-activated K+ channel having a unitary conductance of 84+/-4 pS, indicating that the channel is modulated by the CaR. Therefore, our data strongly suggest that a functional CaR is expressed in cultured rat microglia, similar to that in parathyroid gland and kidney, which could potentially play an important role(s) in regulating microglial function.     

The calcium-sensing receptor acts as a modulator of gastric acid secretion in freshly isolated human gastric glands

Gastric acid secretion is activated by two distinct pathways: a neuronal pathway via the vagus nerve and release of acetylcholine and an endocrine pathway involving gastrin and histamine. Recently, we demonstrated that activation of H(+)-K(+)-ATPase activity in parietal cells in freshly isolated rat gastric glands is modulated by the calcium-sensing receptor (CaSR). Here, we investigated if the CaSR is functionally expressed in freshly isolated gastric glands from human patients undergoing surgery and if the CaSR is influencing histamine-induced activation of H(+)-K(+)-ATPase activity. In tissue samples obtained from patients, immunohistochemistry demonstrated the expression in parietal cells of both subunits of gastric H(+)-K(+)-ATPase and the CaSR. Functional experiments using the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein and measurement of intracellular pH changes allowed us to estimate the activity of H(+)-K(+)-ATPase in single freshly isolated human gastric glands. Under control conditions, H(+)-K(+)-ATPase activity was stimulated by histamine (100 microM) and inhibited by omeprazole (100 microM). Reduction of the extracellular divalent cation concentration (0 Mg(2+), 100 microM Ca(2+)) inactivated the CaSR and reduced histamine-induced activation of H(+)-K(+)-ATPase activity. In contrast, activation of the CaSR with the trivalent cation Gd(3+) caused activation of omeprazole-sensitive H(+)-K(+)-ATPase activity even in the absence of histamine and under conditions of low extracellular divalent cations. This stimulation was not due to release of histamine from neighbouring enterochromaffin-like cells as the stimulation persisted in the presence of the H(2) receptor antagonist cimetidine (100 microM). Furthermore, intracellular calcium measurements with fura-2 and fluo-4 showed that activation of the CaSR by Gd(3+) led to a sustained increase in intracellular Ca(2+) even under conditions of low extracellular divalent cations. These experiments demonstrate the presence of a functional CaSR in the human stomach and show that this receptor may modulate the activity of acid-secreting H(+)-K(+)-ATPase in parietal cells. Furthermore, our results show the viability of freshly isolated human gastric glands and may allow the use of this preparation for experiments investigating the physiological regulation and properties of human gastric glands in vitro.     

The general protein-export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12

Pullulanase is an extracellular, cell surface-anchored lipoprotein produced by Gram-negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF and secY) are all required for processing of the prepullulanase signal peptide to occur. This implies that pullulanase crosses the cytoplasmic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide-bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.     

The calcium-sensing receptor stimulates JNK in MDCK cells

The calcium-sensing receptor (CaR) stimulates ERK1 in rat fibroblasts, but its effect on other MAP kinases is not known. We used a model of renal distal tubule, the MDCK cell, to determine the effects of CaR stimulation on Jun kinase (JNK) activity. Stimulation of the CaR with 5 mM Ca(2+) resulted in a time-dependent increase in JNK activity. Activation of JNK occurred preferentially with stimulation on the basal surface relative to the apical surface. Basal administration of the CaR agonist gadolinium (30 microm) also stimulated JNK activity. Pertussis toxin blocked the ability of both CaR agonists to stimulate JNK, indicating that the effect was mediated through G(ialpha) class G proteins. Finally, we used confocal microscopy to determine that the CaR was located predominantly on the basal surface. These studies demonstrate for the first time that the CaR stimulates JNK activity.     

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.     

Calcium-sensing receptor mediates phenylalanine-induced cholecystokinin secretion in enteroendocrine STC-1 cells

Intraluminal L-phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK-producing enteroendocrine cells sense Phe is unknown. The calcium-sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK-producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK-producing enteroendocrine cell line STC-1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose-dependent CCK secretion and intracellular Ca2+ mobilization in STC-1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe-induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT-PCR in STC-1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC-1 cells.     

Receptor type for cholecystokinin on isolated intestinal muscle cells of the guinea pig

Smooth muscle cells isolated from the longitudinal muscle layer of guinea pig ileum were used to determine the presence and type of cholecystokinin/gastrin receptor mediating contraction. This was accomplished with a series of cholecystokinin and gastrin agonists (CCK-8, des(SO3)CCK-8, gastrin-17, des(SO3)gastrin-17 and pentagastrin) and antagonists (glutaramic acid derivatives CR 1392, CR 1409, CR 1505 and proglumide). The order of potency of agonists based on EC50 values derived from concentration-response curves was: CCK-8 greater than des(SO3)CCK-8 greater than gastrin-17 greater than des(SO3)gastrin-17. The inhibitory dissociation constant (Ki) for the antagonist CR 1505 derived from Schild plots was the same whether sulfated CCK-8 or desulfated gastrin-17 was used as agonist (4.47 +/- 0.76 versus 4.68 +/- 0.78 nM). Pentagastrin acted as a partial agonist and inhibited partially the response to CCK-8. The Ki values determined for all antagonists with pentagastrin as agonist were similar to those with CCK-8 as agonist. The order of potency of agonists and the independence of Ki values from the type of agonist used implied that CCK and gastrin interact with one receptor type; the receptor is more sensitive to CCK-8 but is minimally influenced by sulfation of the tyrosine residue. In this respect, the receptor appears to be distinct from the CCK receptor on gallbladder muscle cells and pancreatic acinar cells.     

Proteolytic processing of reovirus is required for adherence to intestinal M cells.

Reovirus adheres specifically to apical membranes of mouse intestinal M cells and exploits M-cell transepithelial transport activity to enter Peyer's patch mucosa, where replication occurs. Proteolytic conversion of native reovirus to intermediate subviral particles (ISVPs) occurs in the intestine, but it is not known whether conversion is essential for interaction of virus with M cells. We tested the capacity of native virions, ISVPs, and cores (that lack outer capsid proteins) to bind to intestinal epithelial cells in vivo and found that only ISVPs adhered to M cells. Thus, intraluminal conversion of native reovirus to ISVPs is a prerequisite for M-cell adherence, and outer capsid proteins unique to ISVPs (either sigma 1 or products of mu 1) mediate interaction of virus with M-cell apical membranes.     

Signaling through extracellular signal-regulated kinase is required for spermatogonial proliferative response to stem cell factor

In vitro addition of stem cell factor (SCF) to c-kit-expressing A(1)-A(4) spermatogonia from prepuberal mice stimulates their progression into the mitotic cell cycle and significantly reduces apoptosis in these cells. SCF addition results in a transient activation of extracellular signal-regulated kinases (Erk)1/2 as well as of phosphatidylinositol 3-kinase (PI3K)-dependent Akt kinase. These events are followed by a rapid re-distribution of cyclin D3, which becomes predominantly nuclear, whereas its total cellular amount does not change. Nuclear accumulation of cyclin D3 is coupled to transient activation of the associated kinase activity, assayed using the retinoblastoma protein (Rb) as a substrate. These events were followed by a transient accumulation of cyclin E, stimulation of the associated histone H1-kinase activity, a delayed accumulation of cyclin A2, and Rb hyper-phosphorylation. All the events associated with SCF-induced cell cycle progression are inhibited by the addition of either a PI3K inhibitor or a mitogen-activated protein-kinase kinase (MEK) inhibitor, indicating that both MEK and PI3K are essential for c-kit-mediated proliferative response. On the contrary, the anti-apoptotic effect of SCF is not influenced by the separate addition of either MEK or PI3K inhibitors. Thus, SCF effects on mitogenesis and survival in c-kit expressing spermatogonia rely on different signal transduction pathways.     

Odorant response of isolated olfactory receptor cells is blocked by amiloride

Summary Olfactory receptor cells were isolated from the nasal mucosa ofRana esculenta and patch clamped. Best results were obtained with free-floating cells showing ciliary movement. 1)On-cell mode: Current records were obtained for up to 50 min. Under control conditions they showed only occasional action potentials. The odorants cineole, amyl acetate and isobutyl methoxypyrazine were applied in saline by prolonged superfusion. At 500 nanomolar they elicited periodic bursts of current transients arising from cellular action potentials. The response was rapidly, fully and reversibly blocked by 50 m amiloride added to the odorant solution. With 10 m amiloride, the response to odorants was only partially abolished. 2)Whole-cell mode: Following breakage of the patch, the odorant response was lost within 5 to 15 min. Prior to this, odorants evoked a series of slow transient depolarizations (0.1/sec, 45 mV peak to peak) which reached threshold and thus elicited the periodic discharge of action potentials. These slow depolarizing waves were reversibly blocked by amiloride, which stabilized the membrane voltage between –80 and –90 mV. We conclude that amiloride inhibits chemosensory transduction of olfactory receptor cells, probably by blocking inward current pathways which open in response to odorants.     

Correct folding of alpha-lytic protease is required for its extracellular secretion from Escherichia coli

alpha-Lytic protease is a bacterial serine protease of the trypsin family that is synthesized as a 39-kD preproenzyme (Silen, J. L., C. N. McGrath, K. R. Smith, and D. A. Agard. 1988. Gene (Amst.). 69: 237-244). The 198-amino acid mature protease is secreted into the culture medium by the native host, Lysobacter enzymogenes (Whitaker, D. R. 1970. Methods Enzymol. 19:599-613). Expression experiments in Escherichia coli revealed that the 166-amino acid pro region is transiently required either in cis (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325) or in trans (Silen, J. L., and D. A. Agard. 1989. Nature (Lond.). 341:462-464) for the proper folding and extracellular accumulation of the enzyme. The maturation process is temperature sensitive in E. coli; unprocessed precursor accumulates in the cells at temperatures above 30 degrees C (Silen, J. L., D. Frank, A. Fujishige, R. Bone, and D. A. Agard. 1989. J. Bacteriol. 171:1320-1325). Here we show that full-length precursor produced at nonpermissive temperatures is tightly associated with the E. coli outer membrane. The active site mutant Ser 195----Ala (SA195), which is incapable of self-processing, also accumulates as a precursor in the outer membrane, even when expressed at permissive temperatures. When the protease domain is expressed in the absence of the pro region, the misfolded, inactive protease also cofractionates with the outer membrane. However, when the folding requirement for either wild-type or mutant protease domains is provided by expressing the pro region in trans, both are efficiently secreted into the extracellular medium. Attempts to separate folding and secretion functions by extensive deletion mutagenesis within the pro region were unsuccessful. Taken together, these results suggest that only properly folded and processed forms of alpha-lytic protease are efficiently transported to the medium.