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Calcitonin Down-regulates E-cadherin Expression in Rodent Uterine Epithelium during Implantation

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m203555200

1083-351X

Quanxi Li, Jun Wang, D. Randall Armant, Milan K. Bagchi, Indrani C. Bagchi,

Endometriosis Research and Treatment

Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4–5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst. Previous studies indicated that calcitonin (CT), a peptide hormone involved in calcium homeostasis, is transiently expressed in the receptive rat and human endometrial epithelia within the window of implantation. Attenuation of uterine CT expression using antisense methods severely impaired implantation in the rat. The molecular pathway of CT in the pregnant uterus, however, remains unknown. In the present study, we investigated the cellular events following the binding of CT to its membrane receptors in human endometrial epithelial cell line Ishikawa. We observed that CT treatment triggers a transient rise in intracellular calcium in these cells. Most interestingly, CT treatment also led to the disappearance of E-cadherin, a critical cell adhesion molecule, from cell-cell contact sites. Blockade of intracellular calcium release by BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl) prevented the CT-induced disappearance of E-cadherin. Our studies further revealed that CT treatment markedly down-regulates the level of E-cadherin mRNA in Ishikawa cells. We also examined whether CT influences the expression of E-cadherin mRNA in intact rat uterine tissue during implantation. In pregnant rats, high levels of E-cadherin mRNA were expressed during the first 3 days of gestation when the CT mRNA in uterine epithelial cells is undetectable. Concomitant with a transient burst of CT expression during days 4–5 of pregnancy, the level of E-cadherin mRNA declined sharply. Furthermore, administration of exogenous CT to animals on day 2 of pregnancy led to a premature suppression of E-cadherin mRNA level on day 3, indicating a direct link between elevated levels of uterine CT and the down-regulation of E-cadherin expression in the surface epithelium. Collectively, our results are consistent with the hypothesis that CT-induced reduction in E-cadherin expression may remodel the adherens junctions between epithelial cells, and this change in epithelial cell phenotype might be a critical event during the implantation of the blastocyst. Implantation involves complex and progressively intimate interactions between the blastocyst and the uterine epithelium (1Psychoyos A. Greep R.O. Astwood E.G. Handbook of Physiology. American Physiological Society, Washington, D. C.1973: 187-215Google Scholar, 2Carson D.D. Bagchi I. Dey S.K. Enders A.C. Fazleabas A.T. Lessey B.A. Yoshinaga K. Dev. Biol. 2000; 223: 217-237Crossref PubMed Scopus (659) Google Scholar). In humans and rodents, the blastocyst initially adheres to and penetrates the uterine epithelium and subsequently invades the uterine stroma (1Psychoyos A. Greep R.O. Astwood E.G. Handbook of Physiology. American Physiological Society, Washington, D. C.1973: 187-215Google Scholar, 2Carson D.D. Bagchi I. Dey S.K. Enders A.C. Fazleabas A.T. Lessey B.A. Yoshinaga K. Dev. Biol. 2000; 223: 217-237Crossref PubMed Scopus (659) Google Scholar). At the initial stages of implantation, the uterine epithelium undergoes pronounced changes in cell proliferation and remodeling, which prepares it to be “receptive” to invasion by the embryo (1Psychoyos A. Greep R.O. Astwood E.G. Handbook of Physiology. American Physiological Society, Washington, D. C.1973: 187-215Google Scholar, 2Carson D.D. Bagchi I. Dey S.K. Enders A.C. Fazleabas A.T. Lessey B.A. Yoshinaga K. Dev. Biol. 2000; 223: 217-237Crossref PubMed Scopus (659) Google Scholar, 3Schlafke S. Enders A.C. Biol. Reprod. 1975; 12: 41-65Crossref PubMed Scopus (362) Google Scholar, 4Yoshinaga K. Ann. N. Y. Acad. Sci. 1988; 541: 424-431Crossref PubMed Scopus (127) Google Scholar, 5Parr M.B. Parr E.L. Wynn R.M. Jollie W.P. Biology of the Uterus. Plenum Publishing Corp., New York1989: 233-277Crossref Google Scholar, 6Glasser S.R. Denker H.W. Aplin J.D. Trophoblast Invasion and Endometrial Receptivity: Novel Aspects of the Cell Biology of Embryo Implantation. Plenum Publishing Corp., New York1990: 377-416Crossref Google Scholar). The specific modifications leading to the acquisition of the receptive state of the uterine epithelium are regulated by a complex interplay of a variety of effectors including steroid hormones, growth factors, and cytokines (2Carson D.D. Bagchi I. Dey S.K. Enders A.C. Fazleabas A.T. Lessey B.A. Yoshinaga K. Dev. Biol. 2000; 223: 217-237Crossref PubMed Scopus (659) Google Scholar, 7Psychoyos A.J. Reprod. Fertil. Suppl. 1976; 25: 17-28Google Scholar, 8Psychoyos A. Ann. N. Y. Acad. Sci. 1986; 476: 36-42Crossref PubMed Scopus (331) Google Scholar, 9Sharkey A. J. Reprod. Fertil. 1998; 3: 52-61Google Scholar). However, the precise nature of the molecular mechanisms through which these effectors promote uterine receptivity remains unclear. Our previous studies identified calcitonin (CT), 1The abbreviations used for: CT, calcitonin; BAPTA-AM 1, 2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; CGRP, calcitonin gene-related peptide; CTR(s), calcitonin receptor(s); DIG, digoxigenin; fluo-3 AM, fluo-3 acetoxymethyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; sCT, salmon calcitonin; ODN, oligodeoxynucleotide. a peptide hormone known to regulate calcium homeostasis in bone and kidney cells, as a potential regulator of implantation (10Ding Y.Q. Zhu L.J. Bagchi M.K. Bagchi I.C. Endocrinology. 1994; 135: 2265-2274Crossref PubMed Scopus (98) Google Scholar, 11Zhu L.-Z. Cullinan-Bove K. Polihronis M. Bagchi M.K. Bagchi I.C. Endocrinology. 1998; 139: 3923-3934Crossref PubMed Scopus (89) Google Scholar). The expression of CT was transiently induced in rat endometrium epithelium precisely at the onset of implantation (10Ding Y.Q. Zhu L.J. Bagchi M.K. Bagchi I.C. Endocrinology. 1994; 135: 2265-2274Crossref PubMed Scopus (98) Google Scholar, 11Zhu L.-Z. Cullinan-Bove K. Polihronis M. Bagchi M.K. Bagchi I.C. Endocrinology. 1998; 139: 3923-3934Crossref PubMed Scopus (89) Google Scholar). Our studies also showed that the expression of CT is induced in the human endometrial epithelium specifically during the mid-secretory phase (days 19–24) of the menstrual cycle, which closely overlaps with the putative window of implantation (12Kumar S. Zhu L.Z. Polihronis M. Cameron S.T. Baird D.T. Schatz F. Dua A. Ying Y.K. Bagchi M.K. Bagchi I.C. J. Clin. Endocrinol. Metab. 1998; 83: 4443-4450Crossref PubMed Scopus (110) Google Scholar). Most importantly, suppression of the steady-state level of the CT mRNAs in the preimplantation rat uterus by antisense ODNs resulted in a dramatic reduction in the number of implanted embryos (13Zhu L.J. Bagchi M.K. Bagchi I.C. Endocrinology. 1998; 139: 330-339Crossref PubMed Google Scholar). These results suggested strongly that a transient surge of CT expression in the preimplantation rat uterus is crucial for blastocyst implantation. The precise function of CT in rat and human endometrium during implantation, however, remains unknown. To gain an understanding of the function of CT during implantation, we investigated the regulation of cellular functions by this hormone in a human endometrial epithelial cell line Ishikawa. We observed that binding of CT to its cell surface receptor in Ishikawa cells leads to a transient rise in intracellular calcium, which in turn suppresses the expression of calcium-dependent cell adhesion glycoprotein, E-cadherin, at cell-cell contact sites. Consistent with this in vitro observation, we found that administration of exogenous CT down-regulates E-cadherin expression in the endometrial epithelium of pregnant rats without altering the expression of ZO-1, a marker of tight junctions. Based on these results, we favor the hypothesis that uterine CT plays a critical role during implantation by modulating adherens junctions. We postulate that the hormone-induced down-regulation of E-cadherin expression results in the reorganization of the adherens junctions between epithelial cells, thereby facilitating implantation of the blastocyst. Salmon CT (sCT), calcitonin gene-related peptide (CGRP), and CGRP antagonist 8-37 were purchased from Peninsula Laboratories (Belmont, CA). Antibodies against E-cadherin (human) and ZO-1 (rat) were purchased from Zymed Laboratories Inc. (Burlingame, CA). An antibody against E-cadherin (rat) was purchased from BD Transduction Company (Lexington, KY). All experiments involving animals were conducted according to NIH standards for the care and use of experimental animals. Virgin female rats (Sprague-Dawley, from Charles River, Wilmington, MA; 60–75 days of age), in proestrus, were mated with adult males. The different stages of the cycle in the nonpregnant rats were ascertained by examining vaginal smears. The presence of a vaginal plug after mating was designated as day 1 of pregnancy. The animals were killed at various stages of gestation and the uteri collected. In some experiments, animals were injected intravenously or intramuscularly with 100 μg of sCT as described under “Results.” The rats were killed 24 h after the final injection. Previous studies have shown that T47D cells harbor abundant CT receptors. Two forms of human CT receptors have been cloned from expression cDNA library generated from these cells (14Kuestner R.E. Elrod R.D. Grant F.J. Hagen F.S. Kuijper J.L. Matthewes S.L. O'Hara P.J. Sheppard P.O. Stroop S.D. Thompson D.L. Whitmore T.E. Findlay D.M. Houssami S. Sexton P.M. Moore E.E. Mol. Pharmacol. 1994; 46: 246-255PubMed Google Scholar). We have therefore used T47D as control cells. Ishikawa and T47D cells were grown in calcium containing Dulbecco's modified Eagle's medium (Invitrogen) and 10% fetal bovine serum. 0.5 ml of culture medium containing Ishikawa or T47D cells was placed in test tubes. Each tube received 50,000 cpm of125I-sCT (Peninsula Laboratories). Unlabeled sCT was added to the tubes to produce a cold displacement curve. The tubes were incubated overnight with gentle agitation at 4 °C, washed twice with 2 ml of culture medium, and counted in a γ-counter. The results were subjected to Scatchard analysis to determine the dissociation constant (Kd) and number of binding sites/cell. Intracellular calcium was monitored in Ishikawa cells by fluorescence microscopy. 1,000 Ishikawa cells/well were cultured in 96-well plates overnight and loaded with 10 μm fluo-3 acetoxymethyl ester (fluo-3 AM) at 37 °C for 1 h. The attached cells were rinsed three times with medium and cultured in 100 μl of fresh medium. Some of the cells were preloaded for 1 h with 10 μm BAPTA-AM in addition to fluo-3 AM. Fluorescent images were generated by excitation of fluo-3 at 450–490 nm with a mercury lamp and detecting the light emitted at 525 nm. A GenIISys image intensifier (DAGE-MTI, Inc., Michigan City, IN) was used to enhance the signal. All images were video-taped every 5 s using a Panasonic AG-7350 video recorder. The video camera (CCD 72, DAGE-MTI, Inc.) was set to reverse the image so that a computer-based image analysis system (MCID M4, Image Research, St. Catherine's, Ontario, Canada) could be utilized to determine the fluorescence intensity. [Ca2+]i was estimated using Equation 1, [Ca2+]i=Kd(F−Fmin)/(Fmax−F)Equation 1 where Kd is the dissociation constant for [Ca2+]i (316 nm),F is the fluorescence intensity, F minis the background fluorescence, and F max is the maximal fluorescence obtained by equilibrating cytoplasmic and extracellular Ca2+ using 5 μm ionomycin. 10 cells in each well were monitored to determine the average fluorescence intensity for calculation of [Ca2+]i. Ishikawa cells were grown in calcium-containing Dulbecco's modified Eagle's medium to 60–70% confluence. Cells were then treated with either 10 nm/100 nm CT or vehicle. 24 h after treatment with CT, cell surface distribution of E-cadherin was examined by immunofluorescence using a monoclonal antibody that specifically recognizes E-cadherin. For certain experiments cells were also treated with 100 nmCGRP or CGRP antagonist 8-37 for 24 h. In other experiments, cells were pretreated with 33.3 μm BAPTA-AM for 1 h before the addition of CT. The experiments were repeated at least three times. For Northern analysis 20 μg of total RNA was separated by formaldehyde-agarose gel electrophoresis and transferred to Duralon membrane (Stratagene). After transfer, the membranes were baked at 80 °C for 2 h. Blots were prehybridized in 50 mm NaPO4, pH 6.5, 5× SSC, 5× Denhardt's, 50% formamide, 0.1% SDS, and 100 μg/ml salmon sperm DNA for 4 h at 42 °C. Hybridization was carried out overnight in the same buffer containing 106 cpm/ml 32P-labeled E-cadherin cDNA fragment. The filters were washed twice for 15 min in 1× SSC, 0.1% SDS at room temperature, then twice for 20 min in 0.2× SSC, 0.1% SDS at 55 °C, and the filters were exposed to x-ray films for 24–72 h. The intensities of signals on the autoradiogram were estimated by densitometric scanning. To correct for RNA loading, the obtained signals were normalized with respect to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) signal in the same blot. For this the filters were stripped of the radioactive probe by washing for 10 min in 0.5% SDS at 95 °C. The blots were then reprobed with a32P-labeled GAPDH probe as described above. Uterine tissues from pregnant animals were collected and frozen. Tissues were fixed in 4% paraformaldehyde at 4 °C. Cryostat sections were cut at 8 μm and attached to 3-aminopropyltriethoxysilane (Sigma)-coated slides. In situhybridization was performed with digoxigenin (DIG)-labeled sense or antisense RNA probes complementary to E-cadherin gene. DIG-labeled RNA probes were synthesized from E-cadherin cDNA using T3 or T7 RNA polymerase and DIG-labeled nucleotides according to manufacturer's specifications (Roche Molecular Biochemicals). Prehybridization was carried out in a damp chamber at 37 °C for 60 min in hybridization buffer (50% formamide, 5× SSC, 2% blocking reagent, 0.02% SDS, 0.1% N-laurylsarcosine). Hybridization was carried out at 42 °C overnight in a damp humidified chamber. To develop the substrate, sections were washed sequentially in 2× SSC, 1× SSC, and 0.1× SSC for 15 min in each buffer at 37 °C. Sections were then incubated with anti-DIG alkaline phosphatase-conjugated antibody. Excess antibody was washed away, and the color substrate nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indoyl phosphate was added. Slides were allowed to develop in the dark, and the color was visualized under light microscopy until maximum levels of staining were achieved. The reaction was stopped, and the slides were counterstained in Nuclear Fast Red for 5 min. The slides were washed in water, dehydrated, and coverslipped. Control incubations utilized a DIG-labeled RNA sense strand and were performed under identical conditions. Polyclonal antibodies against rat E-cadherin and ZO-1 were diluted 1:1,000 for immunohistochemistry. Frozen uteri were sectioned at 7 μm, mounted on slides, and then fixed in 5% formaldehyde in phosphate-buffered saline. Sections were washed in phosphate-buffered saline for 20 min and then incubated in a blocking solution containing 10% normal rabbit serum for 10 min before incubation in primary antibody overnight at 4 °C. Immunostaining was performed using a streptavidin-biotin kit for rabbit primary antibody (Zymed Laboratories Inc.). Red deposits indicate the sites of immunostaining. Total RNA was subjected to RT-PCR using a Stratascript kit. Briefly, the RNA samples were mixed with oligo(dT) primer, incubated at 65 °C for 5 min, and annealed at room temperature. First strand cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase at 37 °C, and the reaction was stopped by heating the tubes at 95 °C for 5 min. PCR was then performed in a 100-μl total volume using 35 ng of CT; E-cadherin; and GAPDH-specific primers; 200 μm each dATP, dGTP, dCTP, and dTTP; 1.5 mm Mg2+; and 0.5 μl of Taq polymerase (PerkinElmer Life Sciences). The conditions for PCR were: 94 °C for 30 s for 1 cycle followed by 94 °C for 30 s, 65 °C for 30 s, and 68 °C for 2 min for 25 cycles. PCR products were electrophoresed on agarose gels and processed for Southern blot analysis. Total RNA was prepared from Ishikawa cells as well as from T47D breast cancer cells. The T47D cells are known to express abundant CTRs and served as a positive control (14Kuestner R.E. Elrod R.D. Grant F.J. Hagen F.S. Kuijper J.L. Matthewes S.L. O'Hara P.J. Sheppard P.O. Stroop S.D. Thompson D.L. Whitmore T.E. Findlay D.M. Houssami S. Sexton P.M. Moore E.E. Mol. Pharmacol. 1994; 46: 246-255PubMed Google Scholar). The RNA samples were reverse transcribed and amplified by PCR using CTR-specific primers that flank the region of the insert containing 16 amino acids. The PCR products were then subjected to Southern blot analysis employing a radiolabeled CTR cDNA fragment as a probe. PCR products (2 μl each) were run on 1% agarose gel. After electrophoresis, the gel was transferred to Duralon membrane (Stratagene). The membrane was prehybridized in 6× SSC, 5× Denhardt's, 0.5% SDS, and 100 μg/ml salmon sperm DNA for 2 h at 68 °C. Hybridization was performed in the same buffer containing 106 cpm/ml 32P-labeled cDNA fragments of CT, E-cadherin, and GAPDH overnight at 68 °C. The membrane was washed with 2× SSC and 0.1% SDS for 15 min at room temperature, in 0.1× SSC containing 0.5% SDS at 68 °C for 45 min and exposed to x-ray film for 8 h. Uteri were collected from rats on days 1, 4, and 6 of pregnancy, homogenized, and extracted in chilled buffer containing 0.5% Triton X-100, 25 mm KCl, 120 mm NaCl, 2 mm EDTA, 2 mm EGTA, 0.1 mm dithiothreitol, 0.015 mm pepstatin A, 0.085 mm bestatin, and 0.011 mm E-64 in 10 mm Tris-HCl, pH 7.5. The extract was centrifuged, and the supernatant was loaded (50–80 μg of total protein in each lane) onto a 7.5% SDS-polyacrylamide gel. The gel was then blotted onto a polyvinylidene difluoride membrane, which was transferred to a solution of 5% dried nonfat milk in TBS (0.1m Tris-buffered saline, pH 7.5) and incubated overnight with primary antibody (rabbit anti-E-cadherin polyclonal antibody or rabbit anti-ZO-1 polyclonal antibody). After washes, the membrane was probed with secondary antibody coupled to horseradish peroxidase, washed extensively before incubation in enhanced chemiluminescence reagent, and exposed to autoradiographic film. Pregnant rats were treated with sense and antisense ODNs targeted to CT mRNA as described previously (13Zhu L.J. Bagchi M.K. Bagchi I.C. Endocrinology. 1998; 139: 330-339Crossref PubMed Google Scholar). Briefly, rats were deeply anesthetized, and an incision was made in the lower abdomen. The sense or antisense ODNs were mixed with DOTAP (Roche Molecular Biochemicals) and 20% F127 pluronic gel (Sigma). The solution was maintained in liquid form at 4 °C before injection. 20 μg of this ODN solution was taken in prechilled syringes and injected into each uterine horn on day 2 of pregnancy. After surgery, the animals were returned to their cages. 48 h after the surgery, the animals were killed, uteri were collected, and mRNAs were isolated for Northern blot analysis. The blot was hybridized with 32P-labeled probes containing CT, E-cadherin, and GAPDH cDNA sequences. To investigate the regulation of cellular functions by CT and its receptor, human endometrial adenocarcinoma (glandular origin) Ishikawa cells present a convenient model system. These cells, like normal preimplantation uterine epithelial cells, harbor estrogen and progesterone receptors and retain a response to these steroid hormones. Because the responsiveness of a target cell to CT is dependent upon the presence of the cognate receptors on the cell surface, we first ascertained that CTRs are indeed expressed in Ishikawa cells. The human CTR is expressed in two molecular forms, which differ slightly in size (14Kuestner R.E. Elrod R.D. Grant F.J. Hagen F.S. Kuijper J.L. Matthewes S.L. O'Hara P.J. Sheppard P.O. Stroop S.D. Thompson D.L. Whitmore T.E. Findlay D.M. Houssami S. Sexton P.M. Moore E.E. Mol. Pharmacol. 1994; 46: 246-255PubMed Google Scholar). The larger isoform contains a 16-amino acid insert that the shorter form lacks. We initially analyzed the expression of CTR mRNAs in Ishikawa cells by RT-PCR. Because previous reports indicated that T47D cells harbor abundant functional CTRs (14Kuestner R.E. Elrod R.D. Grant F.J. Hagen F.S. Kuijper J.L. Matthewes S.L. O'Hara P.J. Sheppard P.O. Stroop S.D. Thompson D.L. Whitmore T.E. Findlay D.M. Houssami S. Sexton P.M. Moore E.E. Mol. Pharmacol. 1994; 46: 246-255PubMed Google Scholar), these cells were used as positive control cells. As shown in Fig.1, significant amounts of the two isoforms of CTR mRNAs were detected in Ishikawa cells (lane 2), and the levels of these mRNAs were comparable with those in T47D cells (lane 1). We also determined the number of CT binding sites in Ishikawa cells by using radiolabeled CT. The results of the Scatchard analysis are shown in Table I. The dissociation constants for 125I-CT binding in T47D and Ishikawa cells were statistically equivalent. The number of CT binding sites in T47D and Ishikawa cells were also similar. T47D cells exhibited 3.76 × 104 CT binding sites/cell, whereas the Ishikawa cells harbored 2.78 × 104 CT binding sites/cell. These results indicated that Ishikawa cells are likely to be responsive to CT.Table I125I-sCT binding to Ishikawa cellsCellsK dBinding sites/cellnmIshikawa1.942.78 × 104T47D1.273.76 × 104 Open table in a new tab Binding of CT to CTRs is known to elevate intracellular calcium levels in a variety of cell types, including bone and kidney cells (16Chabre O. Conkin B.R. Lin H.Y. Lodish H.F. Wilson E. Ives H.E. Catanzariti L. Hemmings B.A. Bourne H.R. Mol. Endocrinol. 1992; 6: 551-556Crossref PubMed Google Scholar, 17Force T. Bonventre I.V. Flannery M.R. Gorn A.H. Yamin M. Goldring S.R. Am. J. Physiol. 1992; 262: F1110-F1115PubMed Google Scholar). We therefore investigated the effect of CT on intracellular calcium levels in Ishikawa cells using fluorescence microscopy. In this experiment, the cells were plated at a density of 104 cells/well in a 96-well plate and loaded with the fluorescent calcium indicator fluo-3 AM. Some of the cells were preloaded with BAPTA-AM, an intracellular calcium chelator. 10 cells in each well were monitored to determine the average fluorescence intensity for calculation of [Ca2+]i. As shown in Fig.2 (top panel), no intracellular calcium elevation was observed when cells were treated with vehicle alone. Treatment of the cells with 3 or 5 nmCT led to a marginal rise in intracellular calcium (Fig. 2,middle panel, left and center images, respectively). Addition of 10 nm CT, however, elicited a sharp rise in intracellular calcium level within 5 s (Fig. 2,middle panel, right image). This rise in intracellular calcium level was blocked when cells were preloaded with BAPTA-AM (Fig. 2, bottom panel), indicating that the increased fluorescence of fluo-3 was specific for calcium. Taken together, these results demonstrated that Ishikawa cells respond to CT through receptor-mediated calcium signaling. An alteration in cellular calcium is known to control adhesiveness and polarity of epithelial cells (18Gumbiner B.M. Stevenson B. Grimaldi A. J. Cell Biol. 1988; 107: 1575-1587Crossref PubMed Scopus (655) Google Scholar). Our observation that CT alters the level of intracellular calcium in Ishikawa cells raised the possibility that such alterations may influence cellular adhesiveness by changing the expression or triggering redistribution of critical cell adhesion molecules or junctional complexes. E-cadherin is a cell surface glycoprotein that mediates calcium-dependent cell-cell adhesion and is believed to be critical for the establishment and maintenance of adherens junctions in epithelial cells (19Gumbiner B.M. Cell. 1996; 84: 345-357Abstract Full Text Full Text PDF PubMed Scopus (2948) Google Scholar, 20Huber O. Bierkamp C. Kemler R. Curr. Opin. Cell Biol. 1996; 8: 685-691Crossref PubMed Scopus (307) Google Scholar). We therefore investigated whether treatment with CT altered expression or distribution of E-cadherin in Ishikawa cells. Ishikawa cells grown in a calcium-containing medium to 60–70% confluence were treated with either 100 nm CT or vehicle. 24 h after treatment with CT, the cell surface distribution of E-cadherin was examined by immunofluorescence using a monoclonal antibody that specifically recognizes E-cadherin. E-cadherin was clearly localized at points of cell-cell contact in the untreated Ishikawa cells (Fig. 3 A,panels B and C, and Fig. 3 B,panel A). Upon treatment with CT, E-cadherin immunostaining at the cell surface was reduced dramatically after 24 h (Fig.3 A, panels b and c, and Fig.3 B, panel B). We also performed a time course to monitor the decline in E-cadherin after 2, 6, 12, and 24 h of CT treatment. Our studies detected a marked decline in the level of E-cadherin in response to CT only after 24 h of hormone treatment (data not shown). We next examined the role of CTRs in the CT-induced disappearance of E-cadherin from contact sites of Ishikawa cells. We observed that when the cells were treated with another peptide hormone, CGRP, which does not bind to CTRs, E-cadherin expression at the contact sites of epithelial cells remained unaffected (Fig. 3 B, panel C). Interestingly, when cells were treated with a modified CGRP (8-37), which binds to CTRs and functions as an agonist (40Chiba T. Yamaguchi A. Yamatani T. Nakamura A. Morishita T. Inui T. Fukase M. Noda T. Fujita T. Am. J. Physiol. 1989; 256: E331-E335PubMed Google Scholar), E-cadherin expression at the points of cell-cell contact declined (Fig.3 B, panel D), indicating that the effect of CT is dependent on CTR-mediated signaling. We next tested the hypothesis that the CT-induced rise in intracellular calcium is a critical upstream event for the observed loss of E-cadherin from the cell-cell junctions. We therefore treated the cells with BAPTA-AM, which effectively chelates intracellular calcium. We found that in the presence of BAPTA-AM, CT failed to induce a loss of E-cadherin from the cell-cell contact sites (Fig.3 B, panel E). These results suggested that this effect is indeed triggered by an intracellular calcium spike induced by CT. Collectively, these data are consistent with the view that CT acts through its cell surface receptor to initiate calcium signaling, which, through a cascade of events, eventually alters the expression of E-cadherin in epithelial cells. We next investigated whether the CT-induced change in expression of E-cadherin in Ishikawa cells is caused by a down-regulation of E-cadherin mRNA. Ishikawa cells were grown to 60–70% confluence and treated with CT or vehicle. 24 h after treatment, cells were harvested to isolate mRNA for Northern blot analysis. Our results showed that significant amounts of E-cadherin mRNA were present in Ishikawa cells treated with vehicle alone (Fig.4, lanes 1 and 2). The addition of CT to Ishikawa cells led to a marked decline in the level of E-cadherin mRNA (Fig. 4, lanes 3 and4). The relative levels of expression of E-cadherin mRNA in Ishikawa cells treated with or without CT were estimated by densitometric scanning followed by normalization with respect to the control GAPDH mRNA signal. By our estimate, the magnitude of E-cadherin mRNA reduction in Ishikawa cells upon CT administration was greater than 60%. The calcium signaling induced by CT is therefore transduced by an unknown second messenger pathway(s) to down-regulate expression of E-cadherin gene in Ishikawa cells. Our previous studies showed that CT is transiently induced in rat endometrial epithelium within the implantation window (10Ding Y.Q. Zhu L.J. Bagchi M.K. Bagchi I.C.