Calreticulin Represses E-cadherin Gene Expression in Madin-Darby Canine Kidney Cells via Slug
2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês
10.1074/jbc.m607240200
ISSN1083-351X
AutoresYasushi Hayashida, Yoshishige Urata, Eiji Muroi, Takaaki Kono, Yasuyoshi Miyata, Koichiro Nomata, Hiroshi Kanetake, Takahito Kondo, Yoshito Ihara,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCalreticulin (CRT) is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum. In mammals, the expression level of CRT differs markedly in a variety of organs and tissues, suggesting that CRT plays a specific role in each cell type. In the present study, we focused on CRT functions in the kidney, where overall expression of CRT is quite low, and established CRT-overexpressing kidney epithelial cell-derived Madin-Darby canine kidney cells by gene transfection. We demonstrated that, in CRT-overexpressing cells, the morphology was apparently changed, and the original polarized epithelial cell phenotype was destroyed. Furthermore, CRT-overexpressing cells showed enhanced migration through Matrigel®-coated Boyden chamber wells, compared with controls. E-cadherin expression was significantly suppressed at the protein and transcriptional levels in CRT-overexpressing cells compared with controls. On the other hand, the expression of mesenchymal protein markers, such as N-cadherin and fibronectin, was up-regulated. We also found that the expression of Slug, a repressor of the E-cadherin promoter, was up-regulated by overexpression of CRT through altered Ca2+ homeostasis, and this led to enhanced binding of Slug to the E-box element in the E-cadherin promoter. Thus, we conclude that CRT regulates the epithelial-mesenchymal transition-like change of cellular phenotype by modulating the Slug/E-cadherin pathway through altered Ca2+ homeostasis in cells, suggesting a novel function of CRT in cell-cell interaction of epithelial cells. Calreticulin (CRT) is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum. In mammals, the expression level of CRT differs markedly in a variety of organs and tissues, suggesting that CRT plays a specific role in each cell type. In the present study, we focused on CRT functions in the kidney, where overall expression of CRT is quite low, and established CRT-overexpressing kidney epithelial cell-derived Madin-Darby canine kidney cells by gene transfection. We demonstrated that, in CRT-overexpressing cells, the morphology was apparently changed, and the original polarized epithelial cell phenotype was destroyed. Furthermore, CRT-overexpressing cells showed enhanced migration through Matrigel®-coated Boyden chamber wells, compared with controls. E-cadherin expression was significantly suppressed at the protein and transcriptional levels in CRT-overexpressing cells compared with controls. On the other hand, the expression of mesenchymal protein markers, such as N-cadherin and fibronectin, was up-regulated. We also found that the expression of Slug, a repressor of the E-cadherin promoter, was up-regulated by overexpression of CRT through altered Ca2+ homeostasis, and this led to enhanced binding of Slug to the E-box element in the E-cadherin promoter. Thus, we conclude that CRT regulates the epithelial-mesenchymal transition-like change of cellular phenotype by modulating the Slug/E-cadherin pathway through altered Ca2+ homeostasis in cells, suggesting a novel function of CRT in cell-cell interaction of epithelial cells. Calreticulin (CRT) 3The abbreviations used are: CRT, calreticulin; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; BiP, immunoglobulin heavy chain-binding protein; CNX, calnexin; EMSA, electrophoretic mobility shift assay; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; RT, reverse transcription; TBS, Tris-buffered saline; TRPV, transient receptor potential vanilloid receptor; EBSS, Earle's balanced salt solution. 3The abbreviations used are: CRT, calreticulin; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester; BiP, immunoglobulin heavy chain-binding protein; CNX, calnexin; EMSA, electrophoretic mobility shift assay; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; RT, reverse transcription; TBS, Tris-buffered saline; TRPV, transient receptor potential vanilloid receptor; EBSS, Earle's balanced salt solution. is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER) (1Michalak M. Corbett E.F. Masaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (671) Google Scholar) and known to influence many biological processes, such as the regulation of Ca2+ homeostasis (2Michalak M. Robert J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (371) Google Scholar), intercellular or intracellular signaling (3Johnson S. Michalak M. Opas M. Eggleton P. Trends. Cell Biol. 2001; 11: 122-129Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 4Gardai S.J. McPhillips K.A. Frasch S.C. Janssen W.J. Starefeldt A. Murphy-Ullrich J.E. Bratton D.L. Oldenborg P.A. Michalak M. Henson P.M. Cell. 2005; 123: 321-334Abstract Full Text Full Text PDF PubMed Scopus (1037) Google Scholar), gene expression (5Krause K.H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), glycoprotein folding (6Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar), and nuclear transport (7Holaska J.M. Black B.E. Love D.C. Hanover J.A. Leszyk J. Paschal B.M. J. Cell Biol. 2001; 152: 127-140Crossref PubMed Scopus (223) Google Scholar). The biological significance of CRT was revealed by the finding that CRT-deficient mice die in the embryonic stage due to impaired development of cardiac and neural tissues (8Mesaeli N. Nakamura K. Zvaritch E. Dickie P. Dziak E. Krause K.H. Opas M. MacLennan D.H. Michalak M. J. Cell Biol. 1999; 144: 857-868Crossref PubMed Scopus (424) Google Scholar, 9Rauch F. Prud'homme J. Arabian A. Dedhar S. St-Arnaud R. Exp. Cell Res. 2000; 256: 105-111Crossref PubMed Scopus (114) Google Scholar). CRT is expressed in rat embryos, especially in the heart, but its expression is significantly suppressed after birth (10Imanaka-Yoshida K. Amitani A. Ioshii S.O. Koyabu S. Yamakado T. Yoshida T. J. Mol. Cell. Cardiol. 1996; 28: 553-562Abstract Full Text PDF PubMed Scopus (31) Google Scholar). On the other hand, CRT-overexpressing transgenic mice are born alive, but suffer a complete heart block and sudden death after birth (11Nakamura K. Robertson M. Liu G. Dickie P. Nakamura K. Guo J.Q. Duff H.J. Opas M. Kavanagh K. Michalak M. J. Clin. Invest. 2001; 107: 1245-1253Crossref PubMed Scopus (110) Google Scholar). We also found that overexpression of CRT enhanced sensitivity to apoptosis in myocardial H9c2 cells undergoing differentiation in response to retinoic acid (12Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) or in cells exposed to stress caused by hydrogen peroxide (13Ihara Y. Urata Y. Goto S. Kondo T. Am. J. Physiol. 2006; 290: C208-C221Crossref PubMed Scopus (40) Google Scholar), suggesting the importance of CRT in the pathophysiology of myocardial cells. These findings indicate that CRT expression plays a vital role in the development and physiology of cardiac cells. Despite its general importance in cell physiology, CRT is differentially expressed in various organs and tissues in mammals, showing a characteristic expression pattern. For example, CRT levels are low in the kidney and heart, compared with the pancreas and liver, in both bovine and rat tissues (14Waisman D.M. Salimath B.P. Anderson M.J. J. Biol. Chem. 1985; 260: 1652-1660Abstract Full Text PDF PubMed Google Scholar, 15Michalak M. Milner R.E. Burns K. Opas M. Biochem. J. 1992; 285: 681-692Crossref PubMed Scopus (410) Google Scholar). This characteristic distribution of CRT suggests specific functions in each organ or tissue. In this study, we focused on the function of CRT in kidney epithelial cells, because CRT levels are quite low in these cells compared with other cell types such as liver cells (14Waisman D.M. Salimath B.P. Anderson M.J. J. Biol. Chem. 1985; 260: 1652-1660Abstract Full Text PDF PubMed Google Scholar). To investigate the functional effects of CRT overexpression in kidney epithelial cells, we chose Madin-Darby canine kidney (MDCK) cells to establish stable CRT-overexpressing cell lines by gene transfection. MDCK cells are derived from canine kidney and have a well polarized epithelial cell phenotype, maintaining the normal characteristics and functions of renal efferent duct epithelial cells (16Rodriguez-Boulan E. Nelson W.J. Science. 1989; 245: 718-725Crossref PubMed Scopus (815) Google Scholar). The results showed apparent changes in the morphology of CRT-overexpressing MDCK cells and destruction of the polarized epithelial cell phenotype. Furthermore, overexpression of CRT repressed E-cadherin gene expression through up-regulation of its repressor, Slug, via altered Ca2+ homeostasis in MDCK cells. The results suggest a novel function of CRT related to an epithelial-mesenchymal transition-like change of cellular phenotype. Materials—Antibodies against CRT, calnexin (CNX), binding protein (BiP), and ERp57 were purchased from Stressgen (Victoria, BC, Canada). Mouse antibodies against E-cadherin and fibronectin were obtained from BD Biosciences, and rabbit antibodies against β-catenin and pancadherin were obtained from Sigma. Antibodies against Slug, SIP1/ZEB2, transient receptor potential vanilloid receptor (TRPV) 5, TRPV6, and polycystin 2 were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was from Chemicon (Temecula, CA). Anti-Cu,Zn-superoxide dismutase antibody was kindly provided by Dr. K. Suzuki (Hyogo College of Medicine, Japan). Peroxidase-conjugated secondary antibodies against IgG of rabbit, mouse, and goat were from Dako (Glostrup, Denmark). All other reagents used in the study were of high grade and obtained from Sigma and Wako Pure Chemicals (Osaka, Japan). Cell Lines and Culture—MDCK cells were obtained from American Type Culture Collection (NBL-2). The expression vector for mouse CRT cDNA was constructed as described previously (12Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Expression vectors for CRT-gene expression and the control were introduced into MDCK cells using Lipofectamine 2000 reagent (Invitrogen) in accordance with the instructions provided by the manufacturer. Stable gene transfectants were generated after selection with 500 μg/ml G418. Two independent clones expressing high levels of CRT protein were isolated from CRT gene transfectants and used in the study. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in an atmosphere of 5% CO2 and 95% air. Subcellular Fractionation—Cultured cells were harvested and homogenized with homogenization buffer (10 mm Hepes, pH 7.0, 0.25 m sucrose, 2 mm EGTA, and protease inhibitors (20 μm phenylmethylsulfonyl fluoride, 50 μm pepstatin, and 50 μm leupeptin)) by using a homogenizer of the Potter-Elvehjem type. Subcellular fractionation was performed at 4 °C according to the method of Hogeboom (17Hogeboom G.H. Methods Enzymol. 1955; 1: 16-19Crossref Scopus (823) Google Scholar) with a modification. The homogenates were centrifuged at 2,000 × g for 10 min, and the post-nuclear supernatant was again centrifuged at 8,000 × g for 20 min at 4 °C. The post-lysosome supernatant was ultracentrifuged at 55,000 × g for 2 h at 4°C in a Beckman SW41TI rotor (Beckman Instruments). The resulting supernatant contains the soluble cytosolic fraction, and the microsomal pellet represents the ER membrane and lumen proteins as well as Golgi membranes. The pellet was dissolved in lysis buffer (20 mm Tris-HCl (pH 7.2), 130 mm NaCl, and 1% Nonidet P-40, including protease inhibitors), and used as a microsomal fraction. Immunoblot Analysis—Cells were harvested and lysed in the lysis buffer. The lysate was sonicated on ice for 10 min intermittently, and then solubilized samples were prepared after centrifugation at 10,000 × g for 10 min at 4 °C. Protein samples were electrophoresed on 7.5 or 10% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS, 10 mm Tris-HCl (pH 7.5) and 0.15 m NaCl) and incubated at room temperature for 2 h with the primary antibody in TBS containing 0.05% Tween 20. The blots were coupled with peroxidase-conjugated secondary antibodies, washed, and then developed using the ECL detection kit (Amersham Biosciences) according to the instructions recommended by the manufacturer. Immunofluorescence Microscopy—Cells (5 × 105 per ml) were grown on Lab Tek chamber slides (Nalgen Nunc International, Naperville, IL) for 24 h. They were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.2) and permeabilized for 10 min with PBS containing 1% Triton X-100. The cells were then blocked with 1% bovine serum albumin in PBS, incubated with the antibody for 1 h, and washed with PBS containing 1% bovine serum albumin. The immunoreactive primary antibodies were visualized with fluorescein isothiocyanate-conjugated anti-rabbit immunoglobulins (Cappel), anti-mouse immunoglobulins (Dako), or rhodamine-conjugated anti-rabbit immunoglobulins (Cappel). After being washed, stained cells were mounted in the Vectashield medium, visualized under a Carl Zeiss LSM5 microscope (Carl Zeiss, Jena, Germany), and analyzed using PASCAL analytic software. Invasion Assays—A cell invasion assay was carried out using Boyden chambers (Transwell chambers) as described previously (18Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8754-8758Crossref PubMed Scopus (255) Google Scholar) with a slight modification. In brief, Transwell chambers, equipped with 8-μm Matrigel®-coated filters (24-well format, BD Biosciences), were rehydrated, and suspensions of 1 × 105 cells in 200 μl of Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum were plated in the upper compartment of the chamber. Serum-free medium (800 μl) was placed in the lower compartment. After 24 h at 37 °C, noninvasive cells on the upper surface of the filters were removed completely by wiping with a cotton swab. The filters were then fixed with 4% paraformaldehyde in PBS and stained with 0.01% crystal violet. Cells on the lower surface were photographed under a microscope (magnification × 100), and enumerated. The data were expressed as the mean ± S.D. of assays performed in triplicate for each filter. Cell Proliferation Assays—Cell proliferation assays were performed as described previously (12Kageyama K. Ihara Y. Goto S. Urata Y. Toda G. Yano K. Kondo T. J. Biol. Chem. 2002; 277: 19255-19264Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The proliferation of cultured cells was evaluated by measuring attached live cells photometrically after staining with crystal violet. Cells were seeded onto 96-well plates at a density of 3000 cells per well in 100 μlof medium. After culturing for the times indicated in the text, cells were fixed with 4% paraformaldehyde in PBS, washed three times, and stained with 0.01% crystal violet at room temperature for 20 min. After an extensive wash with water, each well was dried. The stained cells were dissolved in 100 μl of 10% SDS and 0.1 m HCl, and cell numbers were estimated by measuring the absorbance at 570 nm using a microplate reader. Reverse Transcription-PCR Analysis—Total RNA was isolated from cultured cells (i.e. MDCK, NIH3T3, PC3, and LNCaP) or rat tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany). A specific system for the amplification of mRNA was used: an mRNA-selective PCR kit (AMV, TaKaRa Biomedicals, Shiga, Japan). One microgram of total RNA extracted from cells was used as a template. PCR products were obtained after 30–35 cycles of amplification with an annealing temperature of 55–65 °C. The primer sequences used were as follows: canine E-cadherin (GenBank™ accession number XM_536807, fragment size 497 bp), forward primer (5′-GGC ATT CTC GGA GGA ATC CTC GC-3′), and reverse primer (5′-CCA TAC ATG TCC GCC AGC TTC-3′); mouse Snail (M95604, 258 bp), forward primer (5′-GGA CTC TCT CCT GGT ACC CCA AGT GCG GCC G-3′), and reverse primer (5′-CCT TGG CCA CCG AGA GCC TGG CCA GCT GC-3′); canine Slug (XM_544069, 454 bp), forward primer (5′-CAG CTC (G/A)GG AGC (G/A)TA CAG CCC C-3′), reverse primer (5′-TAA CCA GGG TCT GGA AAA CGC C-3′); mouse δEF1 (NM011546, 570 bp), forward primer (5′-GCT CCC TGT GCA GTT ACA CCT TTG CAT ACA G-3′), and reverse primer (5′-GCA CCA CAC CCT GAG GAG AAC TGG TTG CCT G-3′); mouse SIP1 (AF033116, 401 bp), forward primer (5′-GCT ACG ACC ATA CCC AGG AC-3′), and reverse primer (5′-TCT CGC CCG AGT GCA GCC-3′); mouse E12/E47 (BC006860, 620 bp), forward primer (5′-AGT GAC CTC CTG GAC TTC AGC ATG ATG TTC CCG CT-3′), and reverse primer (5′-GGG TGC AGG CTG CCA TCT GCC ACG TAG AAG GGG G-3′); human TWIST (BC036704, 436 bp), forward primer (5′-CTG AGC AAC AGC GAG GAA GA-3′), and reverse primer (5′-CTG GTA GAG GAA GTC GAT GT-3′); canis GAPDH (NM_001003142, 979 bp), forward primer (5′-GGT CGG AGT CAA CGG ATT TGG C-3′), and reverse primer (5′-CAT GTA GAC CAT GAG GTC CAC CAC-3′); rat TRPV5 (NM_053787, 411 bp) primer (5′-CCG AGG ATT CCA GAT GCT GGG-3′), and reverse primer (5′-CTC TCC AGC ATC ACT GTG GTG-3′); and rat TRPV6 (NM_053686, 419 bp) primer (5′-GCT CGC CAG ATC CTG GAC CAG-3′), and reverse primer (5′-CGC ATC ACC ATG GTC ACC AGC-3′). The PCR products were subcloned into pCRII (Invitrogen). The nucleotide sequences were confirmed by sequencing with an ALFexpress II system (Amersham Biosciences). Northern Blot Analysis—The cDNA fragments for E-cadherin, Snail, Slug, δEF1/ZEB1, SIP1/ZEB2, E12/E47, TWIST, and GAPDH generated by RT-PCR were labeled with [α-32P]dCTP (Amersham Biosciences) using a Random Primer DNA Labeling Kit (TaKaRa Biomedicals, Japan). The isolation of cytoplasmic RNA and Northern blotting were essentially performed as described previously (19Yasuoka C. Ihara Y. Ikeda S. Miyahara Y. Kondo T. Kohno S. J. Biol. Chem. 2004; 279: 51182-51192Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Isolated RNAs (10 μg) were electrophoresed on a 1% agarose gel containing 0.6 m formaldehyde, transferred to a nylon membrane, and then hybridized with 32P-labeled probes. Autoradiographed membranes were analyzed using the BAS5000 bioimage analyzer (Fuji Photo Film, Japan). Electrophoretic Mobility Shift Assays—EMSA for the E-boxes were performed as described previously (19Yasuoka C. Ihara Y. Ikeda S. Miyahara Y. Kondo T. Kohno S. J. Biol. Chem. 2004; 279: 51182-51192Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) with a slight modification. Oligonucleotide probes were labeled with [γ-32P]ATP using T4 polynucleotide kinase, and then annealed to double-stranded oligonucleotides. Specific oligonucleotides for E-boxes were prepared according to the nucleotide sequence of the canine E-cadherin gene promoter (GenBank™ accession numbers AF330162 and AF330163). Oligonucleotides used were as follows: E-box A probe, 5′-CCG CCC GCC GCA GGT GCA GCC GCA G-3′; E-box B probe, 5′-CTC GCG GCT CAC CTG GCG GCC GGA C-3′; and E-box C probe, 5′-GGC GCT GCG GGC ACC TGT GAT TC-3′ (bold letters indicate consensus sequences for the E-box). Binding reactions were carried out in 15 μl of reaction mixture (25 mm Tris-HCl (pH 7.0), 6.25 mm MgCl2, 0.5 mm dithiothreitol, and 10% glycerol) containing 10 μg of nuclear extract and 25 ng of labeled oligonucleotides. For the supershift assay, anti-Slug and anti-SIP1 antibodies were added to the reaction mixture during the 30-min binding reaction. Promoter Reporter Assays—The promoter of human E-cadherin (–178 to +66 bp: GenBank™ accession number L34545) was isolated from the genomic DNA of A549 cells and amplified by PCR using Pfu turbo DNA polymerase (Stratagene). The primer sequences used were as follows (20Ohkubo T. Ozawa M. J. Cell Sci. 2004; 117: 1675-1685Crossref PubMed Scopus (281) Google Scholar): forward primer (5′-ACT CCA GGC TAG AGG GTC A-3′) and reverse primer (5′-TGG AGC GGG CTG GAG TCT-3′). The PCR product was subcloned into pGL3-Basic vector (KpnI-PstI site, Promega, Madison, WI). PCR-based site-directed mutagenesis was used for the generation of reporter gene constructs with E-box mutations by using a QuikChange site-directed mutagenesis kit (Stratagene), resulting in a mutation in the E-box element from 5′-CANNTG-3′ to 5′-AANNTA-3′ (sense strand). Each vector was transiently transfected into MDCK cells using Lipofectamine 2000 (Invitrogen) as described above. Twenty-four hours after the transfection, luciferase activities were assayed with cellular extracts using a Dual Luciferase Reporter Assay System (Promega) and were normalized to pRL activity. Assays for Release and Uptake of Ca2+ in the Cell—For the 45Ca2+ release assay, cells were cultured for 48 h with medium containing 45Ca2+ (1 μCi/ml). After a wash with Ca2+-free Earle's balanced salt solution (EBSS, Invitrogen) containing 3 mm EGTA, cells were detached from the culture plates with trypsinization buffer (0.25% trypsin and 0.02% EDTA in EBSS), and the cell suspensions were preincubated in Ca2+-free EBSS at 37 °C for 3 min, and sequentially stimulated with thapsigargin (0.1 μm), ionomycin (2 μm), and monensin (2 μm). The cell suspensions were collected 5 min after the addition of each reagent and centrifuged. The radioactivity released from the cells was measured in the supernatant. Cell pellets were lysed, and protein amounts were determined using a BCA assay kit (Pierce). 45Ca2+ release was expressed as the cpm subtracted from those recovered in the preceding collection, and normalized to the protein in the corresponding cell pellets. The uptake of Ca2+ was measured radiometrically using the Millipore filtration technique as described previously (13Ihara Y. Urata Y. Goto S. Kondo T. Am. J. Physiol. 2006; 290: C208-C221Crossref PubMed Scopus (40) Google Scholar) with a slight modification. The cells were washed with 45Ca2+ uptake buffer, which consisted of EBSS supplemented with 0.1 mm CaCl2, and cultured for specific periods in 45Ca2+ uptake buffer containing 45Ca2+ (5 μCi/ml). Cells were detached from the culture plates by trypsinization buffer, and the cell suspension was filtered through a 0.45-μm nitrocellulose filter (Bio-Rad) under vacuum. The filters were rinsed twice with 0.5 ml of washing buffer (10 mm Hepes (pH 7.4), 150 mm KCl, 2 mm EGTA, and 2.5 mm MgCl2). 45Ca2+ uptake was calculated by measuring the radioactivity and standardized using protein concentrations. Measurement of Cytoplasmic-free Ca2+—The cytoplasmic free Ca2+ concentration, [Ca2+]i, was measured with a dual-excitation wavelength fluorescence microscope using Fura-2. Cultured cells on quartz-bottom dishes were loaded with 5 μm Fura-2 tetra(acetoxymethyl)ester (Fura-2-AM, Dojindo, Kumamoto, Japan) for 20 min in EBSS with 2 mm CaCl2 in the presence of 0.01% pluronic acid F-127. After four washes with EBSS, Fura-2 fluorescence was determined at 37 °C using an IX71 inverted research microscope (Olympus, Tokyo, Japan) and a FURA ratiometric imaging system operating at an emission wavelength of 505 nm with an excitation wavelength of 340 and 380 nm. The FURA ratiometric imaging system includes filters (Chroma Technology Corp., Rockingham, VT) switched by filter wheels (Sutter Instrument Company, Novato, CA) and a MicroMax camera (Roper Scientific, Tucson, AZ) controlled by SlideBook software. To measure the change in [Ca2+]i during store-operated Ca2+ influx, Fura-2-labeled cells were washed with Ca2+-free EBSS, then stimulated with thapsigargin (5 μm) followed by re-addition of Ca2+ (2 mm). Stimulated calcium release was calculated as the change in the excitation ratio from baseline integrated over 800 s of stimulation with thapsigargin or Ca2+. The maximal signal (Rmax) was obtained by adding ionomycin at a final concentration of 4 μm. The minimal signal (Rmin) was then obtained by adding EGTA at a final concentration of 10 mm, followed by Tris-free base to a final concentration of 30 mm, to increase the pH to 8.3. R is the ratio (F1/F2) of the fluorescence of Ex 340 nm, Em 505 nm (F1) to that of Ex 380 nm, Em 505 nm (F2). The actual calcium concentration was calculated as Kd × (R – Rmin)/(Rmax – R) × Sf2/Sb2 with the Kd equal to 224 nm (21Grynkiewcz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Google Scholar). Sf2/Sb2 is the ratio of Fura-2 fluorescence at 380 nm in Ca2+-free and Ca2+-replete medium, respectively. Overexpression of CRT Causes Morphological Change in MDCK Cells—Canine renal epithelial MDCK cells were transfected with the expression vector for CRT cDNA to obtain cell lines overexpressing CRT (MDCK-CRT1 and -CRT2). The expression level of CRT was examined by immunoblot analysis in the gene-transfected cells using specific antibodies as described under "Experimental Procedures." Fig. 1A shows that the expression of CRT was increased in the overexpressers to ∼3-fold the levels in the parental (MDCK-WT) and mock-transfected MDCK (MDCK-Control) cells. The transfection had no apparent effect on the expression of molecular chaperones in the ER, such as BiP, ERp57, and cytosolic GAPDH. However, the expression of CNX, another membrane-bound ER homologue of CRT, showed a slight decrease in the CRT-overexpressing cells. Cell morphology was examined in control and CRT gene-transfected cells by phase-contrast microscopy (Fig. 1B). MDCK cells are known to grow in colonies of adherent cells (22Cano A. Perez-Moreno M.A. Rodoligo I. Locascio A. Blanco M.J. Barrio M.G. Portillo F. Nietro M.A. Nat. Cell Biol. 2000; 2: 76-83Crossref PubMed Scopus (2910) Google Scholar, 23Battle E. Sancho E. Franci C. Dominguez D. Monfar M. Baulida J. Herreros A.G. Nat. Cell Biol. 2000; 2: 84-89Crossref PubMed Scopus (2166) Google Scholar). Overexpression of CRT (MDCK-CRT1 and CRT2) caused an apparent morphological change with a fibroblastoid-like phenotype and loss of cell-cell contacts, although there was no remarkable morphological change in mock-transfected cells compared with parental cells. Intracellular localization of CRT was characterized by immunofluorescence microscopy in control and CRT-overexpressing cells (Fig. 2A). Under conditions in which cellular membranes were permeabilized by Triton X-100, strong immunoreactivity for CRT showed a perinuclear localization and a vesicular pattern in CRT-overexpressing cells, although the immunoreactive signal was weak in controls. No significant increase in the cell surface expression of CRT was observed in the CRT-overexpressing cells under conditions without Triton X-100 treatment. To investigate whether the cytosolic localization of CRT was increased in the gene-transfected cells, control and MDCKCRT1 cells were lysed and fractionated by centrifugation to separate cytosolic and microsomal fractions as described under "Experimental Procedures." As shown in Fig. 2B, overexpressed CRT was present in the microsomal but not cytosolic fraction. CNX and Cu,Zn-superoxide dismutase were detected as marker proteins for microsomes and the cytosol, respectively. Similar results were also obtained with MDCK-CRT2 cells (data not shown). Together, these results indicate that overexpression of CRT did not influence the localization of CRT in the ER of MDCK cells.FIGURE 2A, intracellular localization of E-cadherin was evaluated in control and CRT gene-transfected MDCK cells using indirect immunofluorescence microscopy with specific antibodies (magnification, ×200). Data represent three independent experiments. B, control and CRT gene-transfected MDCK cells were lysed and fractionated by ultracentrifugation to separate cytosolic and microsomal fractions as described under "Experimental Procedures." The expression of CRT, CNX, and Cu,Zn-superoxide dismutase in each fraction was examined by immunoblot analysis using specific antibodies. Ms, microsomes; Cy, cytosol. Data represent three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overexpression of CRT Enhances Cellular Migration in MDCK Cells—To investigate whether the altered morphology in CRT-overexpressing cells affected cellular functions, a cell invasion assay was performed using a modified Boyden chamber as described under "Experimental Procedures." Cells were seeded on Transwell filters that had been coated with extracellular matrix components, including laminin, fibronectin, and proteoglycans (i.e. Matrigel®-coated polycarbonate membrane). After 24 h, the numbers of cells that had migrated through the filters were estimated by enumerating stained cells. There was a significant increase in cell motility through a Matrigel®-coated polycarbonate membrane in CRT-overexpressing cells compared with controls (Fig. 3, A and B). To investigate whether the enhanced migration of CRT-overexpressing cells was due to an increase in cell growth, cell proliferation was examined in control and CRT gene-transfected cells as described under "Experimental Procedures." However, the results showed that cell growth was suppressed in CRT-overexpressing cells compared with controls (Fig. 3C). These results indicate that the incre
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