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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Clinical Pharmacology, Departments of Medicine and Biochemistry and Molecular Pharmacology (S.K., G.M.P., F.J.S., G.S.F., I.R.-S., S.S., S.A.W.), and Department of Pathology, Anatomy and Cell Biology (M.M., G.H.), Thomas Jefferson University, Philadelphia, Pennsylvania
Received May 3, 2005; accepted June 1, 2005.
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Abstract |
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). Although the epidemiology of this disease remains poorly understood, there is an unexplained inverse relationship between the incidence of colorectal cancer and enterotoxigenic Escherichia coli (ETEC) infections (Ferlay et al., 2001). ETEC produces heat-stable enterotoxins (STs), a principle cause of secretory diarrhea in endemic populations, travelers, and agriculturally important animal herds (Thielman and Guerrant, 2004). These enterotoxins are plasmid-encoded small (
19 amino acids) peptides that bind to guanylyl cyclase C specifically expressed in intestinal epithelial cells (Lucas et al., 2000). Association of STs with the extracellular domain of guanylyl cyclase C activates the intracellular catalytic domain that converts GTP into cGMP. The second messenger cGMP, in turn, activates cGMP-dependent protein kinase II, which ultimately activates the cystic fibrosis transmembrane conductance regulator, a chloride channel, resulting in secretory diarrhea.
Beyond volume homeostasis, guanylyl cyclase C suppresses the proliferation of human colon carcinoma cells (Pitari et al., 2001, 2003) and the development of hyperproliferative adenoma in mouse models of intestinal neoplasia (Shailubhai et al., 2000). Moreover, the expression of the endogenous guanylyl cyclase C ligands guanylin and uroguanylin is invariably lost during tumor progression, and this loss of function represents one key mutational event underlying proliferation associated with carcinogenesis in the colon (Cohen et al., 1998; Shailubhai et al., 2000; Pitari et al., 2001, 2003; Steinbrecher et al., 2002). Similar to secretion, the suppression of colon cancer cell proliferation by guanylyl cyclase C is mediated by cGMP (Pitari et al., 2001, 2003). However, in contrast to volume homeostasis, the antiproliferative signal initiated by cGMP is propagated by Ca2+ influx through cyclic nucleotide-gated (CNG) channels (Pitari et al., 2003). This role for guanylyl cyclase C as a tumor suppressor likely contributes to the inverse association between colorectal cancer and ETEC infections (Shailubhai et al., 2000; Ferlay et al., 2001; Pitari et al., 2001, 2003), reflecting in part the longitudinal exposure to ST-producing bacteria in underdeveloped countries.
Whereas Ca2+ influx through CNG channels suppresses colon cancer cell proliferation (Pitari et al., 2001, 2003), Ca2+ also is a critical signaling element required for cell proliferation (Weiss et al., 2001), controlling gene expression, progression through the cell cycle, and DNA synthesis (Berridge et al., 1998). Additionally, Ca2+ plays a central role in signaling cascades that drive tumorigenesis and neoplastic progression (Cole and Kohn, 1994; Berridge et al., 1998) and inhibitors of Ca2+-dependent signaling suppress proliferation of cancer cells in vitro and in solid tumors in vivo (Cole and Kohn, 1994; Holmuhamedov et al., 2002). One source of Ca2+ that promotes proliferation is capacitative entry from the extracellular pool through store-operated Ca2+ channels (SOCs) (Golovina et al., 2001; Peng et al., 2003). In human colon cancer cells, capacitative Ca2+ entry through SOCs is required to support proliferation (Weiss et al., 2001; Peng et al., 2003).
Thus, capacitative Ca2+ entry through SOCs and guanylyl cyclase C-mediated cGMP production represent opposing limbs of a signaling system that regulates colon cancer cell proliferation. This is one example of a paradigm in which Ca2+ and cGMP mediate opposing (patho)physiological processes, including smooth muscle contractility, platelet aggregation, endocrine secretion, and visual phototransduction (Sargeant and Sage, 1994; Lucas et al., 2000; Andric et al., 2001). A hallmark of systems regulated by opposing Ca2+ and cGMP signals is the reciprocal regulation of their intracellular concentrations (Lucas et al., 2000). Molecular mechanisms that regulate intracellular [Ca2+] ([Ca2+]i) by cGMP are well described (Rink and Merritt, 1990; Sargeant and Sage, 1994; Berridge et al., 1998). In contrast, those by which Ca2+ regulates intracellular [cGMP] ([cGMP]i) have only recently been explored (Lucas et al., 2000; Andric et al., 2001; Kazerounian et al., 2002). Here, we demonstrate that capacitative Ca2+ entry through SOCs promotes the proliferation of and reciprocally inhibits guanylyl cyclase C-dependent antiproliferative signaling in human colon carcinoma cells. Reciprocal regulation of cGMP-dependent antiproliferative signaling reflects the ability of guanylyl cyclase C to serve as a functional sensor for Ca2+, which is conditionally coupled to allosteric inhibition of the catalytic domain by ligand activation.
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Materials and Methods |
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Cells. T84 human colon carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and grown in Dulbecco's modified Eagle's/Ham's F-12 medium with 10% (v/v) fetal bovine serum (BioWhittaker, Walkersville, MD), 2.5 mM glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin (BioWhittaker) in a humidified atmosphere of 5% CO2, unless otherwise indicated. Cells were fed with fresh medium every second day and split when subconfluent. Cells were employed for experiments at passages 10 through 40 after acquisition.
Preparation of Cell Membranes. T84 cell membranes were prepared as described previously (Parkinson and Waldman, 1996). Confluent cells in 75-cm2 cell culture flasks were homogenized in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethanesulfonyl fluoride (TEED buffer) and centrifuged at 100,000g for 60 min at 4°C. Resulting pellets were washed once with TEED containing 1 mM EGTA and twice with TEED containing 100 µM EGTA to remove adventitial Ca2+. Pellets were resuspended in TEED containing 100 µM EGTA at 2 mg protein/ml and stored at 4°C.
Guanylyl Cyclase. Guanylyl cyclase activity was quantified as described previously (Kazerounian et al., 2002). Briefly, 20 µg of membrane proteins was incubated for 5 min at 37°C in 100 µl of 50 mM Tris buffer, pH 7.5, which contained 500 µM IBMX, 15 mM creatine phosphate, 2.7 units of creatine phosphokinase, 1 mM Mg-GTP or MnGTP with 3 mM MgCl2 or MnCl2 in excess of nucleotide (unless otherwise indicated) in the presence or absence of 1 µM ST, and different concentrations of free Ca2+. In these studies, 1 µM ST was used since it is the concentration that produces maximum stimulation of guanylyl cyclase activity in intestinal cell membranes (Parkinson and Waldman, 1996; Pitari et al., 2001, 2003). Specific free Ca2+ and Mg2+ concentrations, buffered with 100 µM EGTA, were quantified employing the WinMax computer program (Kazerounian et al., 2002). In studies in which the free Mg2+ concentration was varied, total Mg2+ concentrations were always maintained above the free Ca2+ concentration (250 µM) to prevent Ca2+ chelation of GTP from becoming rate-limiting. Incubations at 37°C were initiated by the addition of enzyme, continued for 5 min, and terminated by adding 400 µl of 50 mM sodium acetate (pH 4.0) and boiling for 3 min. Enzyme activity was linear with respect to time and protein concentration under all conditions examined. Enzyme assays were performed in duplicate, and cGMP was quantified by radioimmunoassay in triplicate (Kazerounian et al., 2002). Analyses of enzyme kinetics were performed using Prism (GraphPad Software Inc., San Diego, CA) or Microsoft Excel (Microsoft, Redmond, WA).
Receptor Binding. Iodination of ST was performed as described previously (Hugues et al., 1991). T84 cell membranes (40 µg) were incubated with 100 pM 125I-ST in binding buffer containing 50 mM Tris-HCl, pH 7.6, 0.66 mM cystamine, 0.1% bacitracin, 450 mM NaCl, and 1 mM EDTA and incubated at 37°C for 120 min in the presence or absence of 1 µM excess of unlabeled ST. Free and membrane-associated radioactivity were separated by vacuum filtration employing Whatman GF/B glass microfiber filters (Whatman, Hillsboro, OR) presoaked in 0.3% polyethyleneimine. After applying samples, filters were washed three times with 5 ml of washing buffer (150 mM NaCl, 20 mM Na3PO4, pH 7.2, and 1 mM EDTA) to remove excess of unbound radioactivity. Radioactivity on filters was quantified in a Cobra II Gamma Counter (PerkinElmer Life and Analytical Sciences, Boston, MA).
Cyclic GMP Accumulation. Confluent T84 cells in 24-well plates were washed twice with 500 µl of Ca2+-free buffer containing 121 mM NaCl, 5 mM NaHCO3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 10 mM D-glucose, and 0.25% bovine serum albumin, pH 7.4, preincubated in the same buffer containing 1 mM IBMX for 30 min, and then treated in duplicate with ST and the indicated agents for an additional 10 min. Reactions were terminated by adding 500 µl of ice-cold 100% ethanol, each well was washed with 200 µl of ice-cold 100% ethanol, and resultant extracts were centrifuged at 12,000g x 15 min at 4°C. Supernates containing cGMP were evaporated in a Savant SVC-100H concentrator (Thermo Electron Corporation, Waltham, MA) and reconstituted with 50 mM sodium acetate, pH 4.0, and cGMP was quantified in each sample in triplicate by radioimmunoassay (Kazerounian et al., 2002).
Cell Proliferation. Exponentially growing cells (
70% confluent in 96-well/plates) were synchronized by serum starvation in Eagle's minimal essential medium for 48 h. In some incubations, proliferation was induced with 10 mM glutamine (Pitari et al., 2001) in Ca2+-free minimal essential medium containing experimental agents as indicated, and incubations continued for 24 h. Cell proliferation was quantified by cell number or [3H]thymidine incorporation into DNA after the addition of 0.2 µCi/well [methyl-3H]thymidine for the final 3 h (Pitari et al., 2001, 2003).
Fluorescence Calcium Imaging. T84 cells were plated on poly-D-lysine-coated coverslips at a density of 25,000 to 30,000 cells/cm2 and grown for 3 to 4 days to form a monolayer. Cells were incubated for 20 min at 37°C in extracellular buffer containing 121 mM NaCl, 5 mM NaHCO3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 2 mM CaCl2, 10 mM D-glucose, and 2% bovine serum albumin, pH 7.4, and then loaded with 5 µM Fura-2-AM in the presence of 200 µM sulfinpyrazone and 0.003% (w/v) pluronic acid for 30 min at 37°C. Sulfinpyrazone also was present during imaging measurements to minimize dye loss. Cells were then washed once with extracellular buffer containing 2 mM CaCl2 and 2% bovine serum albumin and twice with Ca2+-free extracellular buffer containing 0.25% bovine serum albumin. Imaging measurements were performed in Ca2+-free extracellular buffer containing 0.25% bovine serum albumin at 35°C. The coverslip was placed on an open slide chamber (Harvard Apparatus, Holliston, MA) mounted on the stage of an Olympus IX70 inverted microscope fitted with a 40x (UApo, numerical aperture 1.35; Olympus, Tokyo, Japan) oil-immersion objective camera (NU 200; Photometrics, Tucson, AZ) under computer control and a light source. Fura-2-loaded cells were alternately excited at 340 and 380 nm using a motorized digitally controlled filter wheel (Ludl Electronic Products Ltd., Hawthorne, NY). Fluorescence emissions at 510 nm were collected with a cooled CCD 12-bit digital camera (PXL; Photometrics), and images were analyzed using customized software (Csordas and Hajnoczky, 2001).
Fluorescent signals determined from regions of interest were employed to calculate Ca2+ concentrations using an in vitro calibration method (Szalai et al., 1999). In vitro calibration was performed in intracellular medium containing 120 mM KCl, 10 mM NaCl, 1 mM KH2PO4, and 20 mM Tris-HEPES at pH 7.2 with 2 mM MgATP and 150 mM KOH in the presence of 5 µM Fura-2/free acid (Szalai et al., 1999). [Ca2+]i was quantified by the following equation (eq. 1):
 | (1) |
Statistics. Results represent at least three separate experiments. Statistical significance was determined using paired Student's t tests (two-tailed) employing Microsoft Excel.
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; Weiss et al., 2001). Here, glutamine-induced proliferation of T84 human colon carcinoma cells, quantified by cell number (Fig. 1A) or thymidine incorporation (Fig. 1B), required extracellular Ca2+ (Fig. 1, A and B). Stimulation of T84 cell proliferation by extracellular Ca2+ was abrogated by 2-APB, a selective inhibitor of SOCs (Fig. 1, C and D) (Bootman et al., 2002). Alterations of T84 cell proliferation by 2-APB reflected inhibition of capacitative Ca2+ entry, since 2-APB was without effect on the release of Ca2+ from intracellular stores (Fig. 1E) (Ma et al., 2000). In contrast, signaling by guanylyl cyclase C mediates cytostasis of human colon carcinoma cells by inducing cGMP-dependent opening of cyclic nucleotide-gated channels and influx of extracellular calcium (Pitari et al., 2001, 2003). Indeed, the specific guanylyl cyclase C ligand ST inhibited T84 cell proliferation in a time-dependent fashion (Fig. 2A). ST-induced cytostasis of T84 cells was blocked by eliminating Ca2+ from the media and by L-cis-diltiazem, a selective inhibitor of cyclic nucleotide-gated channels (Fig. 2B). It is significant that the inhibition of SOCs by 2-ABP remarkably potentiated the antiproliferative effects of ST in a concentration-dependent fashion (Fig. 2C). These observations suggest that capacitative Ca2+ entry through SOCs, which is required for T84 cell proliferation, tonically opposes cytostasis induced by guanylyl cyclase C signaling.
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; Andric et al., 2001; Kazerounian et al., 2002). Here, regulation of guanylyl cyclase C signaling by Ca2+ influx through SOCs was explored. Thapsigargin, which inhibits the Ca2+-ATPase in the endoplasmic reticulum, increases [Ca2+]i by preventing its intracellular sequestration (Thastrup et al., 1989). In turn, depletion of intracellular Ca2+ stores by thapsigargin opens SOCs, permitting capacitative Ca2+ entry (Parekh and Penner, 1997). In Ca2+-free media, thapsigargin induced a transient increase in [Ca2+]i in T84 cells originating from intracellular stores (Fig. 3A) without altering accumulation of [cGMP]i induced by ST (Fig. 3B). In the absence of thapsigargin and the associated opening of SOCs, 10 mM extracellular Ca2+ did not alter [Ca2+]i (Fig. 3A) or the accumulation of [cGMP]i induced by ST (data not shown). In contrast, in media containing Ca2+, thapsigargin produced a sustained elevation in [Ca2+]i (Fig. 3A) and inhibited accumulation of [cGMP]i induced by ST (Fig. 3C). Indeed, in the presence of thapsigargin, increasing [Ca2+]ext coordinately elevated [Ca2+]i and inhibited ST-induced [cGMP]i accumulation with an EC50 of 1.1 ± 0.7 mM (Fig. 3, D and E). In this context, the steady-state concentrations of [Ca2+]ext in intestine (1–20 mM) underscore the physiological significance of reciprocal inhibition of guanylyl cyclase C signaling by capacitative Ca2+ entry through SOCs (Welberg et al., 1993; Whitfield et al., 1995). BAPTA-AM, a chelator of Ca2+ that can be loaded intracellularly, reversed the inhibition of ST-induced [cGMP]i accumulation by thapsigargin and extracellular Ca2+ (Fig. 3C). Similarly, 2-APB prevented elevations in [Ca2+]i (Fig. 4, A–C) and reversed the inhibition of ST-induced [cGMP]i accumulation mediated by thapsigargin and extracellular Ca2+ (Fig. 4D). Furthermore, UTP and carbachol deplete intracellular Ca2+ stores and increase capacitative Ca2+ entry through G protein-dependent production of inositol polyphosphates mediated by P2Y purinergic and muscarinic receptors, respectively (Fig. 5, A and B) (Luo et al., 2001). Capacitative Ca2+ entry induced by UTP and carbachol mimicked the effects of thapsigargin, inhibiting [cGMP]i accumulation induced by ST (Fig. 5C). Moreover, ionomycin, a Ca2+ ionophore that directly increases [Ca2+]i (Fig. 5, A and B) (Liu and Hermann, 1978), mimicked the ability of UTP, carbachol, and thapsigargin to elevate [Ca2+]i and inhibit [cGMP]i accumulation induced by ST (Fig. 4, B and C). Thus, opposition of ST-dependent cytostasis by SOCs reflects inhibition of guanylyl cyclase C signaling by capacitative Ca2+ entry required for proliferation.
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Opposition of ST-Induced Signaling by Capacitative Ca2+ Entry Reflects Conditional Sensitization of Guanylyl Cyclase C to Ca2+ Inhibition by Ligand Activation. Whereas mechanisms reciprocally regulating [Ca2+]i and [cGMP]i remain incompletely defined, an emerging paradigm suggests that guanylyl cyclases are conditional sensors of Ca2+, which allosterically inhibits the ligand-activated conformation (Lucas et al., 2000; Kazerounian et al., 2002). Here, Ca2+ inhibited ST-stimulated but not basal guanylyl cyclase C in T84 cell membranes when Mg2+ (Fig. 6, A and C) but not Mn2+ (Fig. 6B) served as the cation cofactor. In contrast, Ca2+ did not support basal or ligand-stimulated guanylyl cyclase C activity (data not shown). Ca2+ inhibited ST-stimulated guanylyl cyclase C in a concentration-dependent fashion, with a Ki of 10–5 M (Fig. 6D). It is noteworthy that the potency of Ca2+ to inhibit guanylyl cyclase C signaling in cell-free membranes was nearly identical to that observed in intact cells employing physiological [Ca2+]ext characteristics of intestine (Fig. 3, D and E) (Welberg et al., 1993; Whitfield et al., 1995). Inhibition of guanylyl cyclase C by Ca2+ did not reflect interference with ligand-receptor interaction (Fig. 7A) or receptor-effector coupling (Fig. 7B; Table 1). Rather, the Ka of ST for activating guanylyl cyclase C in the presence of Ca2+ was lower than in its absence, consistent with enhanced receptor-effector coupling (Table 1). It is significant that Ca2+ decreased the Km and Vmax of ST-activated guanylyl cyclase C, characteristics of an uncompetitive allosteric mechanism (Fig. 7; Table 2). Indeed, Ca2+ inhibited guanylyl cyclase C by competitive antagonism of free Mg2+ binding to divalent cation sites required for ligand activation (Fig. 8; Table 3). Thus, in human colon cancer cells, cytostatic signaling by ST serves as a switch that conditionally sensitizes guanylyl cyclase C to reciprocal inhibition by capacitative Ca2+ entry conducted by SOCs central to mitogenesis.
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). Neoplastic transformation in the colon partly reflects reduced expression of tumor suppressor genes such as guanylin and uroguanylin, which inhibit enterocyte proliferation by activating guanylyl cyclase C and increasing [cGMP]i (Cohen et al., 1998; Shailubhai et al., 2000; Pitari et al., 2001, 2003; Steinbrecher et al., 2002). Indeed, "replacement" of these endogenous hormones by longitudinal exposure to enterotoxigenic bacteria secreting ST may underlie the inverse epidemiological association between colorectal cancer (Ferlay et al., 2001) and ETEC infections (Prevention CfDCa, 2001). The present study revealed that guanylyl cyclase C is a conditional sensor of Ca2+ that allosterically inhibits ST-stimulated but not basal cGMP production. In cells, conditional inhibition of ST-stimulated guanylyl cyclase C is imposed by capacitative Ca2+ entry through SOCs, a key proliferative signaling mechanism generally (Golovina et al., 2001; Peng et al., 2003) and in colon cancer cells specifically (Weiss et al., 2001; Peng et al., 2003). Importantly, influx of extracellular Ca2+ through SOCs and the reciprocal inhibition of guanylyl cyclase C block the accumulation of [cGMP]i absolutely required for antiproliferative signaling by that receptor (Pitari et al., 2001, 2003). These observations support a model in which colorectal carcinogenesis, in part, reflects an imbalance between opposing cGMP- and Ca2+-mediated mechanisms in which antiproliferative signaling by guanylyl cyclase C is attenuated by reduced availability of endogenous ligands (Cohen et al., 1998; Shailubhai et al., 2000; Pitari et al., 2001, 2003; Steinbrecher et al., 2002; Bardelli et al., 2003) and increased inhibition by proliferative signaling through SOCs (Weiss et al., 2001; Peng et al., 2003). Moreover, they suggest that guanylyl cyclase C ligands, such as bacterial enterotoxins that exhibit exceptional specificity and potency (Hugues et al., 1991; Lucas et al., 2000), and inhibitors of SOCs represent a novel synergistic strategy for cGMP-directed therapy in patients with colorectal cancer.
Colon cancer cell proliferation is one example of a general paradigm in which Ca2+ and cGMP mediate opposing (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. The molecular mechanism by which Ca2+ directly regulates guanylyl cyclases and cGMP production has only recently been revealed (Andric et al., 2001; Kazerounian et al., 2002). Thus, nitric oxide-stimulated but not basal soluble guanylyl cyclase (sGC) is allo-sterically inhibited by direct binding of Ca2+ to high-(Ki = 10–8 M) and low-affinity (Ki = 10–5 M) binding sites (Kazerounian et al., 2002). Those with high affinity seem to be unique allosteric cation inhibitory sites. In contrast, those with low affinity are Mg2+-binding sites required for nitric oxide activation, and Ca2+ inhibits sGC at those sites by an uncompetitive kinetic mechanism in which Ca2+ antagonizes Mg2+ binding to those sites (Kazerounian et al., 2002). Similarly, the present study demonstrates that ST-stimulated but not basal guanylyl cyclase C is allosterically inhibited by Ca2+. Inhibition of guanylyl cyclase C is mediated by an uncompetitive kinetic mechanism in which Ca2+ antagonizes Mg2+ binding to sites with a Ki nearly identical to that of low-affinity sites of sGC (Kazerounian et al., 2002). Moreover, high- and low-affinity Ca2+-binding sites mediate allosteric inhibition of adenylyl cyclase types V and VI, and low-affinity sites exhibit kinetic characteristics that are nearly identical to those reported herein for guanylyl cyclase C (Guillou et al., 1999). Thus, allosteric inhibition by direct interaction of Ca2+ with nucleotide cyclases seems to be a generalized mechanism that coordinates the reciprocal regulation of intracellular concentrations of cyclic nucleotides and Ca2+ that mediate opposing physiological processes.
Guanylyl cyclases and cGMP are emerging as important regulators of proliferation, although the mechanisms that mediate that activity seem to be cell-specific. Cyclic GMP delays the G1/S transition in human vascular smooth muscle cells by decreasing cyclin D1 and cyclin-dependent kinase 4 activities following platelet-derived growth factor stimulation (Fukumoto et al., 1999). In addition, proliferation of glomerular mesangial cells by phorbol ester is blocked by cGMP-induced expression of the phosphatase MKP-1 (Sugimoto et al., 1996). In colorectal cancer cells, inhibition of proliferation is mediated by a signaling mechanism initiated by ligand interaction with guanylyl cyclase C (Shailubhai et al., 2000; Pitari et al., 2001, 2003). The resulting accumulation of [cGMP]i suppresses proliferation by opening CNG channels permitting the influx of extracellular Ca2+ (Pitari et al., 2003). Indeed, inhibition of human colon cancer cell proliferation by ST is reversed by L-cis-diltiazem and BAPTA-AM and requires Ca2+ in the media (Pitari et al., 2003).
Although extracellular Ca2+ is the downstream effector that inhibits proliferation by guanylyl cyclase C through CNG channels on the one hand and promotes proliferation and reciprocal inhibition of guanylyl cyclase C through SOCs on the other (Cole and Kohn, 1994; Berridge et al., 1998; Weiss et al., 2001; Peng et al., 2003), these opposing mechanisms represent discrete pathways for Ca2+ signaling. Thus, in human colon carcinoma cells, ST induces the influx of extracellular Ca2+ through cGMP-activated CNG channels, which produces elevations in [Ca2+]i that inhibit proliferation and open KCa channels in a concentration-dependent fashion (Pitari et al., 2003) but do not inhibit accumulation of [cGMP]i induced by ST, demonstrated herein. Indeed, in the absence of open SOCs, [Ca2+]ext up to 10 mM did not alter the accumulation of [cGMP]i induced by ST (see Results). These observations suggest that influx of extracellular Ca2+ into colon carcinoma cells through CNG channels is not coupled to allosteric inhibition of guanylyl cyclase C, in contrast to capacitative Ca2+ entry mediated by SOCs.
Divergent anti- and proproliferative Ca2+ signals through CNG and SOCs, respectively, may be resolved in human colon carcinoma cells through spatial organization and relative affinity. Guanylyl cyclase C in intestinal cells tightly associates with the brush-border cytoskeleton (Hakki et al., 1993) and may organize into signaling complexes with other molecules, including cGMP-dependent protein kinase and cystic fibrosis transmembrane conductance regulator, through postsynaptic density 95/disc-large/zona occludens domains (Scott et al., 2002). Moreover, guanylyl cyclase C possesses only low-affinity sites that mediate allosteric inhibition by Ca2+, in contrast to sGC, which possesses high- and low-affinity sites (Kazerounian et al., 2002). This combination of high- and low-affinity allosteric sites in sGC permits the regulation of [cGMP]i over the dynamic range of cytosolic [Ca2+]i that characterizes reciprocally regulated opposing processes such as contraction and relaxation in smooth muscle cells (Kazerounian et al., 2002). In contrast, the presence of low- but not high-affinity sites specifically permits guanylyl cyclase C to discriminate cytostatic Ca2+ signaling through CNG channels, which produce extremely small changes in [Ca2+]i (Pitari et al., 2003), from proliferative signaling by capacitative Ca2+ entry through SOCs, which can produce [Ca2+]i of 10–5 M (Fig. 3, D and E) (Kerschbaum and Cahalan, 1999).
These considerations suggest a model wherein CNG channels may be extrinsic to guanylyl cyclase C-containing complexes but form complexes with other signaling molecules such as those that mediate cytostasis and KCa channels (Davare et al., 2001). The influx of extracellular Ca2+ through cGMP-activated CNG channels induces signaling by molecules within the complex but is insufficient (Pitari et al., 2003) to occupy low-affinity inhibitory sites on guanylyl cyclase C extrinsic to those complexes. In contrast, SOCs may form complexes with guanylyl cyclase C, and capacitative Ca2+ entry through those channels inhibits ST-induced [cGMP]i accumulation by binding to low-affinity allosteric sites facilitated by close proximity of molecules in the complex. The spatial organization of guanylyl cyclase C and cation channels in brush-border membranes and its role in signal discrimination await precise identification of the complement of SOCs expressed in human intestinal epithelial cells (Davare et al., 2001; Yue et al., 2001). In this regard, it is noteworthy that in intestinal cells a selective calcium entry channel, CaT1, which is a member of the transient receptor potential family with pore properties of SOCs, is localized in brush-border membranes (Davare et al., 2001; Yue et al., 2001) where guanylyl cyclase C resides (Hakki et al., 1993).
In summary, proliferative signaling mediated by capacitative Ca2+ entry through SOCs opposes signaling by guanylyl cyclase C and its downstream effector cGMP. Reciprocal regulation of these opposing signaling mechanisms is mediated in part by conditional allosteric inhibition of ligand-stimulated guanylyl cyclase C by Ca2+. The balance of proproliferative and antiproliferative signals mediated by Ca2+ and guanylyl cyclase C, respectively, may regulate the gradient of proliferation along the crypt-villus axis in normal intestine (Whitfield et al., 1995). Furthermore, an imbalance in these opposing mechanisms that involve attenuation of cGMP-dependent antiproliferative signaling may contribute to mechanisms that underlie colorectal carcinogenesis (Pitari et al., 2003). Importantly, the combination of bacterial enterotoxins that serves as super agonists of guanylyl cyclase C (Pitari et al., 2001, 2003) and specific inhibitors of SOCs offers a novel paradigm for cGMP-directed therapy for the treatment and prevention of colorectal tumors.
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Footnotes |
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ABBREVIATIONS: ETEC, enterotoxigenic E. coli; ST, heat-stable enterotoxin; CNG, cyclic nucleotide-gated; SOC, store-operated calcium channel; BAPTA, 1,2-bis(o-amino-5,5'-dibromophenoxy)ethane-N,N,N',N'-tetraacetic acid; AM, acetoxymethyl ester; 2-APB, 2-aminoethoxydiphenylborate; IBMX, isobutylmethylxanthine; sGC, soluble guanylyl cyclase.
Address correspondence to: Dr. Scott A. Waldman, Division of Clinical Pharmacology, Thomas Jefferson University, 132 S. 10th St., 1170 Main, Philadelphia, PA 19107. E-mail: scott.waldman{at}jefferson.edu
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3 experiments performed in triplicate.
) and inhibition of [cGMP]i production (
) in response to increasing [Ca2+]ext. Net [Ca2+]i represents the integration of increases in [Ca2+]i above baseline between 15 and 25 min (area under the curve), as outlined under Materials and Methods. Results are the means ± S.E.M. from 
) or presence (
) of 1 mM Ca2+. Data presented in B were subjected to double reciprocal analysis (inset). C, Ca2+ inhibits guanylyl cyclase C by an uncompetitive kinetic mechanism. Guanylyl cyclase C activity in T84 cell membranes was quantified in incubations containing 1 µM ST, Mg-GTP (0.1–10 mM), and 3 mM excess Mg2+ in the absence (
) of 250 uM Ca2+. Data presented in A were subjected to double reciprocal analysis (B). Results represent