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[Cancer Research 65, 732-742, February 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

A Novel Localization of the G-Protein-Coupled CysLT1 Receptor in the Nucleus of Colorectal Adenocarcinoma Cells

Christian Kamp Nielsen1, Joan I.A. Campbell1, John F. Öhd1, Matthias Mörgelin4, Kristian Riesbeck3, Göran Landberg2 and Anita Sjölander1

Divisions of 1 Experimental Pathology, 2 Pathology, and 3 Medical Microbiology, Department of Laboratory Medicine, Malmö University Hospital and 4 Division of Molecular Pathogenesis, Department of Cell and Molecular Biology, Lund University, Malmö, Sweden

Requests for reprints: Anita Sjölander, Experimental Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital, SE-205 02 Malmö, Sweden. Phone: 464-033-7223; Fax: 464-033-7353; E-mail: anita.sjolander{at}exppat.mas.lu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Searching for a link between inflammation and colon cancer, we have found that the inflammatory mediator leukotriene D4 (LTD4), via its receptor CysLT1, induces cyclooxygenase-2 expression, survival, and proliferation in intestinal epithelial cells. In conjunction with our previous observation that CysLT1 receptor expression is increased in colorectal adenocarcinomas, we here found an increased nuclear localization of the CysLT1 receptor in colorectal adenocarcinomas. This novel discovery of CysLT1 receptors in the nucleus was further analyzed. It was found to be located in the outer nuclear membrane in colon cancer cells and in the nontransformed epithelial cell line Int 407 cells by Western blot and electron microscopy. Cancer cells displayed higher amounts of the nuclear CysLT1 receptor, but prolonged LTD4 exposure induced its nuclear translocation in nontransformed cells. Truncation of a nuclear localization sequence abrogated this translocation as well as the LTD4-induced proliferative response. In accordance, nuclear CysLT1 receptors exhibited proliferative extracellular signal-regulated kinase 1/2 signaling. The significance of these experimental findings is supported by the observed correlation between the proliferative marker Ki-67 and nuclear CysLT1 receptor localization in colorectal adenocarcinomas. The present findings indicate that LTD4 cannot only be synthesized but also signal proliferation through nuclear CysLT1 receptors, stressing the importance of leukotrienes in inflammation-induced colon carcinogenesis.

Key Words: GPCR • CysLT1 • colon cancer • epithelial cells • inflammation • Gastrointestinal cancers: colorectal • Receptors: structure and function • Cancer in minority and medically underserved populations • Antireceptors


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukotrienes (LT) constitute an important group of proinflammatory mediators that are synthesized from membrane-bound arachidonic acid via the 5-lipoxygenase (5-LO) pathway (1, 2). In general, agonist stimulation causes translocation of 5-LO from the cytosol to the nuclear membrane (3). The activity of 5-LO leads to the formation of unstable LTA4, which can be converted into either LTB4 or cysteinyl (Cys) LTs (LTC4, LTD4, and LTE4), the most potent of which is LTD4. CysLTs mediate their effects by binding to specific cell surface receptors designated CysLT1 (4) and CysLT2 (5, 6). Pharmacologic studies have revealed that CysLT1 has a 350-fold higher affinity for LTD4 than for LTC4 (7), whereas the CysLT2 receptor exhibits equivalent affinity for LTC4 and LTD4, although in both cases the attraction is significantly less than that displayed by the CysLT1 receptor (6). The receptors that bind LTs are G-protein-coupled receptors (GPCR), which constitute the largest family of cell surface receptors on living cells (8). Thus far, it has been presumed that GPCRs are almost exclusively plasma membrane receptors that are characterized by their seven transmembrane domains. GPCRs initiate a series of intracellular signaling events and wide variety of cellular responses by interacting at the plasma membrane with heterotrimeric G-proteins (9). Malfunctioning of such heterotrimeric G-proteins has been shown to result in a number of clinical symptoms, and mutations in G-proteins have been found in several types of tumors (10). Consequently, increased attention is now being focused on the involvement of GPCRs in human diseases (11), which makes GPCRs important targets in drug development.

It is well known that LTs are associated with the pathology of inflammatory bowel disease because elevated levels of these mediators have been found in the intestinal tract of patients suffering from inflammatory bowel disease (12). Furthermore, it has been shown that patients suffering from inflammatory bowel disease have an >30% higher risk of developing colon cancer (13). Therefore, it is worth mentioning that the proinflammatory mediator LTD4, but not LTC4, increases the survival of intestinal Int 407 cells by exerting an effect that is mediated by cyclooxygenase-2 (COX-2; ref. 14). Although, prostaglandin E2 is the major lipid mediator released in response to LTD4, we cannot exclude that one of the other lipid products released could also play a role in the regulation of intestinal epithelial cell survival and proliferation. LTD4 affects both the transcription (15) and the activity (14) of COX-2. The clinical relevance of these findings was strengthened in our recent study of human adenocarcinomas (16), in which we showed that CysLT1 receptor, 5-LO, COX-2, and the cell survival protein Bcl-XL are all up-regulated in colon cancer tissues. Also in that investigation, we discovered another important aspect of the up-regulation of CysLT1 receptor expression when we observed that it correlated well with poor survival in patients with Duke's B colon cancer. These findings imply that the CysLT1 receptor can contribute to the progression and outcome of colon cancer, presumably through actions occurring via COX-2 and Bcl-XL (16). Support for this assumption is provided by the numerous studies indicating that COX-2 activity and prostaglandin synthesis play a role in intestinal carcinogenesis (17). Furthermore, increased COX-2 expression in colorectal cancer cells results in a growth and survival advantage (18). It should also be mentioned that a large number of investigations have shown that elevated COX-2 expression correlates with poor prognosis, and an epidemiologic study revealed reduced mortality in a group of colon cancer patients that received long-term treatment with nonsteroidal anti-inflammatory drugs (19).

We have recently shown that LTD4 can induce cell survival and proliferation by activating two parallel signaling pathways in Int 407 cells. The one that promotes cell survival includes PKC{alpha} and the cyclic AMP response element-binding protein, whereas the one that promotes proliferation comprises PKC{varepsilon}, extracellular signal-regulated kinase (ERK)1/2, and p90RSK (20). In this study, we also found that LTD4 induced an activation of the stress kinase p38 mitogen-activated protein kinase, but not c-jun-NH2-kinase. However, we could exclude that this stress kinase participated in the regulation of intestinal cell proliferation and survival by LTD4 (20). The ability of LTs to cause cell proliferation has also been established in bone marrow cells (21) and smooth muscle cells (22). The concept that a surface receptor can induce cell proliferation rests on the assumption that the signaling pathway that it activates reaches the nucleus. Accordingly, it is interesting that over the past few years, growth factor receptors of the tyrosine kinase family, and more recently GPCRs, have been shown to reside not only in the plasma membrane, but also in the nuclear envelope (23). Moreover, a study of endothelial cells has shown that a perinuclear GPCR for prostaglandin E2 activates the transcription of eNOS (24, 25). However, little or nothing is known about the molecular mechanisms underlying the nuclear localization and physiologic function of the GPCRs found in the nuclear membrane, or whether it could be of importance in pathologic conditions such as cancer.

In light of our results indicating that the CysLT1 receptor plays a role in colorectal cancer development and the observation that this receptor can also induce proliferation in intestinal cells, we did experiments to further investigate the functional properties of the CysLT1 receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. The rabbit polyclonal anti-human CysLT1R antibody and the CysLT1R blocking peptid, used at 5 µg/mL with 1 µg/mL of the anti-CysLT1R antibody were purchased from Cayman Chemical Co. (Ann Arbor, MI). The rabbit polyclonal anti-5-LO antibody was a generous gift from Dr. A.W. Ford-Hutchinson at Merck-Frosst (Pointe-Claire-Dorual, Quebec, Canada). The monoclonal mouse anti-ki-67 (A0047) and secondary peroxidase linked goat anti-rabbit antibody were from Dako (Glostrup, Denmark). The rabbit polyclonal MEK1-NT antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The conjugated secondary antibodies Alexa 488 and 594, as well as Fluo-3, were obtained from Molecular Probes, Inc. (Leiden, the Netherlands). ZM-198,615 (ICI-198,615) was a gift from (AstraZeneca, R&D Lund, Sweden). LipofectAMINE, versene, and Trizol were from Invitrogen Co. (Carlsbad, CA). The enhanced chemiluminescence reagents and the goat polyclonal lamin B and CD71 (transferrin) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and the hyperfilm and [methyl-3H]thymidine were from Amersham Int. (Buckinghamshire, United Kingdom). The pcDNA-CysLT1 His plasmid was kindly provided by Dr. J. Evans (Merck & Co. Research Laboratories, Sunnytown Pike, PA). Unless otherwise indicated, all other reagents were of analytic grade and originated from Sigma Chemicals (St. Louis, MO) or ICN (Temecula, CA).

Immunohistochemical Staining of Patient Samples. Formalin-fixed and paraffin-embedded colon specimens from 44 patients with colorectal cancer were obtained from the archives of the Department of Pathology at Malmö University Hospital, Lund University. All specimens were taken following colectomy. Sections of normal tissue from the colectomized specimens were used as matched controls. The specimens included random variation of stages and grades of disease, which were categorized by Dukes' classification (26). Tissue arrays were prepared from the paraffin-embedded samples and were treated as previously described (16). The arrays were immunohistochemically stained with primary antibodies against the CysLT1 receptor (1:100), ki-67 (1:100), or 5-LO (1:100) and subsequently with peroxidase-linked secondary antibodies. After immunostaining, all slides were manually counterstained with Mayer's hematoxylin. The number of cells showing nuclear reactivity was calculated as a percentage of the total number of epithelial cells per microscopic field. This procedure was repeated thrice for each specimen.

Cell Culture. Cells of the nontransformed human intestinal epithelial line Int 407 (27) were cultured as a monolayer in Eagle's basal medium supplemented with 15% new born calf serum, 55 IU/mL penicillin, and 55 µg/mL streptomycin. This was carried out at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Int 407 cells display typical epithelial growth and morphology. The CaCo-2 cell line was derived from a human colon cancer, and it exhibits an adherent epithelial growth pattern. CaCo-2 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 55 µg/mL streptomycin, and 55 IU/mL penicillin. The two cell lines were used between passages 10 and 30 and grown for 5 days to ~70% to 80% confluency. The two cell lines were regularly tested to verify the absence of Mycoplasma contamination.

Reverse Transcriptase-PCR. Total RNA was isolated from Int 407 and CaCo-2 cells by using Trizol essentially according to the instructions of the manufacturer. Briefly, cells were grown in 75-cm2 flasks until they reached 80% confluence, and they were subsequently lysed in 10 mL of Trizol. The lysates were passed several times through an 18-gauge needle, after which chloroform was added and RNA was isolated in the water phase that was generated. The RNA was precipitated with isopropanol. cDNA was synthesized using reverse transcriptase and a Superscript II kit (Invitrogen). PCR analysis of the cDNA was done using specific primers coding for the whole human CysLT1 receptor (forward primer 5'-CGAGCTCAAGCTTCGAATTCTATGGATGAAACAGGAAATCTGAC-3' and reverse primer 5'-CAGGATCCTCTAGAGGTACCTATACTTTACATATTTCTTC-3'), and PCR of 5-LO was done with the primers "Exon 13 + 14A" and "Exon 14B," as previously described by Härle et al. (28).

Cell Fractionation. Cells were washed twice with buffer A [20 mmol/L NaHEPES (pH 8.0), 2 mmol/L MgCl2, 1 mmol/L EDTA, 5 mmol/L orthovanadate, 60 µg/mL phenylmethylsulfonyl fluoride, and 4 µg/mL leupeptin] and were then covered with the same buffer and placed on ice. The cells were scraped loose and subjected to nitrogen decompression at 1000 p.s.i. for 10 minutes, using a cell disruption bomb (Parr Instrument Co., Moline, IL). The intact nuclei were collected by centrifugation at 200 x g and washed twice in buffer A. The supernatant was centrifuged at 10,000 x g for 10 minutes, and the resulting supernatant was separated into cytosol and membrane fractions by centrifugation at 200,000 x g for 1 hour. The nuclear fraction was separated into an inner and an outer nuclear membrane fraction according to a modified version of the method described by Humbert (29). To achieve this, nuclei were isolated from cells by nitrogen decompression and were subsequently washed thrice in buffer 1 [0.25 mol/L sucrose, 10 mmol/L MgCl2, 1 mmol/L dithiothreit, 10 µg/mL leupeptin, 60 µg/mL phenylmethylsulfonyl fluorid, and 50 mmol/L Tris-HCl (pH 7.4)] and collected by centrifugation at 600 x g for 15 minutes. The pellet were thereafter resuspended in buffer 1 containing 1% (w/v) sodium citrate and placed on ice for 30 minutes. This suspension was centrifuged at 500 x g for 15 minutes, which resulted in a pellet containing the intact inner nuclear membranes and the disrupted outer nuclear membranes in the supernatant. The pellet was digested with DNase 1 at 4°C overnight. The inner and outer nuclear fractions were recovered by centrifugation at 200,000 x g for 30 minutes and the two pellets were resuspended in buffer A.

Gel Electrophoresis and Immunoblotting. To ensure equal protein loading, all fractions were evaluated and compensated for protein content by using the Coomassie blue protein assay. The protein content per lane varied somewhat between the different gels (25-40 µg); however, within any given gel, there was no variation. All samples were solubilized by boiling in sample buffer [62 mmol/L Tris (pH 6.8), 1% SDS, 10% glycerol, 15 mg/mL dithiothretiol, and 0.05% bromphenol blue] for 10 minutes. The solubilized proteins were loaded in the lanes of a 10% homogeneous polyacrylamide gel in the presence of SDS and subjected to electrophoresis. The separated proteins were electrically transferred to polyvinylidene difluoride membranes, which were then blocked in 5% nonfat dry milk for 1 hour at room temperature. The membranes were incubated overnight at 4°C with the indicated primary antibodies and thereafter washed extensively and incubated with a secondary antibody for 1 hour at room temperature. The membranes were then washed again, developed by enhanced chemiluminescence, and exposed to hyperfilm.

Electron Microscopy. Samples of intact cells used for electron microscopy were prepared by pelleting 5 x 106 cells at 4°C immediately after adding a fixative (4% paraformaldehyde and 0.1% glutaraldehyde). The pellets were dehydrated in ethanol for 1 hour at room temperature and then embedded in Lowicryl (30). Ultrathin sections were cut on a microtome and mounted on nickel grids. For immunostaining, the grids were floated on drops of immune reagents placed on a sheet of parafilm. Free aldehyde groups were then blocked with 50 mmol/L glycine, and the grids were subsequently incubated with 5% (v/v) donkey serum in PBS supplemented with 0.2% bovine serum albumin (BSA; pH 7.6) for 15 minutes. This blocking procedure was followed by overnight incubation with the primary antibody (dilution 1:100) at 4°C. The grids were subsequently washed by placing them successively on 10 drops of incubation buffer (5 minutes on each drop), after which the sections were incubated with the gold-conjugated secondary antibody by letting them float on drops containing the gold conjugate reagent (diluted 1:20 in incubation buffer) for 60 minutes at room temperature. After further washing on 10 drops of incubation buffer, the sections were postfixed in 2% glutaraldehyde. Finally, the sections were washed with distilled water, poststained with uranyl acetate and lead citrate, and examined in a Jeol 1200 EX transmission electron microscope operated at 60 kV accelerating voltage. Presence of the CysLT1 receptor in the plasma membrane and different nuclear membrane compartments was analyzed by negative staining and electron microscopy, as described elsewhere (31). The antibody directed against the CysLT1 receptor was labeled with 4-nm colloidal thiocyanate gold (32). Preparations of the inner and outer nuclear membranes were mixed with the labeled antibody, and 5 µL aliquots of the mixture were allowed to adsorbed onto carbon-coated grids for 1 minute. Thereafter, the grids were washed with two drops of water and stained on two drops of 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. The specimens were examined in a Jeol JEM 1230 electron microscope operated at 60 kV accelerating voltage, and images were recorded with a Gatan Multiscan 791 CCD camera.

Immunofluorescent Staining. Cells cultured on coverslips were washed thrice in PBS and then fixed for 15 minutes in 4% ice-cold paraformaldehyde in PBS and blocked for 30 minutes in 3% BSA in PBS. The fixed cells were incubated with the primary antibodies against the CysLT1 receptor and lamin B in PBS supplemented with 1% BSA for 1 hour at room temperature and then washed five times in PBS. The cells were subsequently incubated with a conjugated secondary antibody (goat anti-rabbit IgG Alexa 488 and/or goat anti-donkey IgG Alexa 594; Molecular Probes, Inc., Leiden, The Netherlands) suspended in PBS supplemented with 1% BSA for 1 hour, and then washed five times in PBS. The coverslips were mounted on glass slides using a fluorescence mounting medium (Dako) and then examined in a Nikon TE300 microscope (60 x 1.4 plan-apochromat oil immersion objective) integrated with a Bio-Rad Radiance confocal laser scanning system. The excitation wavelengths for Alexa 488 and 594 were 488 and 543 nm, respectively, and the corresponding emission filters were HQ 515/30 and HQ 600/50. Bio-Rad LaserPix software was used to process the captured images.

Flow Cytometry. Expression of the CysLT1 receptor on the surface of Int 407 cells was analyzed by flow cytometry. Untreated or LTD4-treated cells were harvested with versene and pelleted in ice-cold calcium-free medium [136 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 5 mmol/L NaHCO3, 5.5 mmol/L glucose, 1 mmol/L EDTA, and 20 mmol/L HEPES (pH 7.4)], and 4 x 105 cells were subsequently resuspended in 150 µL of PBS (supplemented with 1% BSA). These cells were then incubated with the anti-CysLT1receptor antibody (5 µg/mL) for 1 hour on ice, washed thrice with PBS (supplemented with 1% BSA), and then incubated with the fluorescin-conjugated Alexa 488 antibody. Thereafter, the cells were washed extensively and then fixed in 1% paraformaldehyde/PBS, and the cell-bound fluorescence in the different samples was quantified using a Cell Quest FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Construction of Wild-type and Nuclear Localization Sequence–Mutated EGFP-CysLT1R Fusion Proteins and Expression of these Proteins in Int 407 Cells. The wild-type human CysLT1 receptor coding sequence (Genbank accession no. NP_006630) was amplified using the pcDNA-CysLT1 His plasmid (generously provided by Dr. J. Evans, Merck Research Laboratories) as template and the CysLT1 forward and reverse primers described in the section on reverse transcriptase-PCR. The PCR product was digested with the restriction enzymes HindIII and BamHI (Promega, Madison, WI), and the resulting DNA fragment was cloned in-frame into the pEGFP-C1 vector (Genbank accession no. u55763, Clonetech, Palo Alto, CA) digested with the same enzymes. To obtain a CysLT1 receptor mutated at the predicted nuclear localization sequence (NLS), DNA coding for the eight non-NLS amino acids (GHPQKAKT) at positions 323 to 330 in the human CysLT2 receptor was exchanged for that coding for the 12 amino acids at positions 312 to 323 in the CysLT1 receptor. The hybrid molecule was generated by using the splicing-by-overlap-extension-PCR method described by Warren et al. (33). The first set of primers used to amplify the 5' or front end of the CysLT1R gene and the coding sequence for the CysLT2R non-NLS region is (a) 5'-CGAGCTCAAGCTTCGAATTCTATGGATGAAACAGGAAATCTGAC-3' and (b) 5'-CTTTGTCTTTGCCTTCTGTGGATGGCCCTTTCTAAATGTAGACAG-3'. The second pair of primers used to amplify the inserted CysLT2R non-NLS region and the 3' or rear end of the CysLT1R gene is (c) 5'-GGCCATCCACAGAAGGCAAAGACAAAGGCCTCTTTGCCAGAAAAAGG-3' and (d) 5'-CGTTGGAGTCCACGTTCTTTAATAG-3'. Briefly, the two sets of oligonucleotides were used in separate PCRs to amplify the front and rear ends of the CysLT1R gene. The expected PCR products should end and start at the inserted CysLT2R DNA coding for the non-NLS region, respectively. For both reactions, the plasmid pEGFP-CysLT1RWT served as template DNA. The resulting 984- and 520-bp DNA fragments were gel purified and mixed in a third PCR reaction where they served as template DNA and the oligonucleotides designated (a) and (d) from previous PCRs were used as primers. The resulting CysLT1-CysLT2 receptor hybrid DNA fragment was digested and ligated into the pEGFP-C1 vector as described for the wild-type CysLT1 receptor DNA fragment. All the plasmid constructs were sequenced to confirmed the integrity of the cloning. All the constructs were sequenced across the junction sites to confirm the cloning. The transfection was done on cells grown on coverslips to 60% confluence in serum-free Eagle's basal medium supplemented with 2 µg/mL plasmid DNA and 10 µL/mL Lipofectamine for 6 hours. Thereafter, the medium in which the cells were kept was changed to serum containing BME medium, and the cells were incubated in this medium overnight under previously specified culture conditions. The following day, the subcellular location of the EGFP-CysLT1 receptor in the cells was determined in a Bio-Rad Radiance confocal laser scanning system, and the recorded signals were analyzed using Bio-Rad Laserpix software.

Intracellular Calcium Measurements. Cells grown on coverslips were incubated with 4 µmol/L Fluo-3/AM for 30 minutes and subsequently washed twice in PBS. The coverslip was then mounted in a chamber containing a physiologically balanced calcium medium [136 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, 1.1 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 5 mmol/L NaHCO3, 5.5 mmol/L glucose, and 20 mmol/L HEPES (pH 7.4)], and the chamber was placed on the heated stage of the microscope (34). The cells were allowed to rest for 15 minutes in the calcium medium, and the fluorescent signal was recorded before and after stimulating the cells with 40 nmol/L LTD4. In some experiments, the cells were incubated with 40 µmol/L ZM-198,615 for 15 minutes prior to the stimulation. The fluorescent calcium signal was detected using a Bio-Rad Radiance confocal laser scanning system with an excitation wavelength of 488 nm and a HQ 500LP emission filter. The recorded signal was analyzed using Bio-Rad Laserpix software.

Thymidine Incorporation. Int 407 cells were transiently transfected with either EGFP-CysLT1RWT or EGFP-CysLT1R{Delta}NLS and then allowed to grow for 24 hours in cell culture medium supplemented with serum as described above. Thereafter, the cells were stimulated with LTD4 for 48 hours in the absence of serum. The serum-free media was changed after 24 hours and new LTD4 was added together with 0.5 µCi of 3H-labeled thymidine. After this incubation in the presence of 3H-labeled thymidine, the cells were washed twice with PBS, treated with 10% trichloroacetic acid for 30 minutes, and then lysed in 1 mol/L NaOH. The levels of radioactivity, reflecting the incorporation of 3H-thymidine into DNA, were measured using a ß-liquid scintillation counter (LKB RackBeta Wallace, Turku, Finland).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The CysLT1 Receptor and the Enzyme 5-LO Are Up-Regulated in the Nuclei of Epithelial Cells in Human Colorectal Carcinomas. Immunohistochemical staining for CysLT1 receptor in normal colon tissue (controls) showed that the receptor was located primarily in the plasma membrane of the epithelial cells (Fig. 1A, arrow), but 20% of these cells also displayed nuclear staining for the receptor (Fig. 1A, arrowhead and Fig. 1E). Immunostaining for CysLT1 receptor in colon tumor tissue indicated that the receptor was also expressed in the plasma membrane of these epithelial tumor cells (Fig. 1B, arrows), although a significantly larger number of such cells exhibited positive nuclear staining for CysLT1 receptor (Fig. 1B, arrowhead and Fig. 1E). Figure 1C (arrowheads) shows tumor specimen, with positive nuclear staining for this receptor in >70% of the epithelial cells, and six of 44 cancer specimens exhibited CysLT1 receptor immunoreactivity solely in the nuclei of epithelial cells (Fig. 1D, arrowheads). We also found that the nuclear accumulation of the CysLT1 receptor strongly correlated (using the Pearson correlation) with nuclear staining of the proliferative marker Ki-67 (Fig. 1F). The specificity of the CysLT1 antibody was shown by antigen adsorption, and by comparison with the staining obtained with a preimmune serum of the same paraffin-embedded tissues (Fig. 1G). These data are in good agreement with our previously published results (16). Because our data implied that the nuclear location of this receptor could be involved in cancer cell proliferation, it prompted us to analyze the same samples for the presence and localization of 5-LO, the enzyme responsible for initiating the synthesis of LTs. Other investigators have shown that activation of human neutrophils causes translocation of 5-LO from the cytosol to the nucleus (1). In our study, immunohistochemical staining clearly showed the presence of 5-LO in the nuclei of epithelial cells in both normal colon (Fig. 1H, arrowhead) and colon tumor tissue (Fig. 1I, arrowheads). However, it should be pointed out that, compared with normal colon specimens, the colorectal cancer tissue exhibited increased staining for 5-LO in both the cytosol (Fig. 1I, arrow) and the nuclei (Fig. 1H and I) of the epithelial cells. These results are well in line with our earlier observation that human colorectal carcinomas show increased overall expression of the CysLT1 receptor and 5-LO compared with normal colon tissue and cancer cell lines (16). Considering these findings, we further investigated the localization and function of the nuclear CysLT1 receptor, using the nontransformed intestinal epithelial cell line Int 407 and the colon cancer cell line CaCo-2.



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  		Figure 1. Immunostaining of the CysLT1 receptor and the enzyme 5-LO in normal human colon tissue and adenocarcinomas. Tissue arrays were prepared from paraffin-embedded adenocarcinomas and control colon specimens. The arrays were partitioned in 1-µm sections and immunohistochemically stained for the CysLT1 receptor or 5-LO. A, positive staining for CysLT1 receptor in the plasma membrane (arrow) and the nuclei (arrowhead) of cells in normal colon tissue. B, positive CysLT1 staining in the plasma membrane (arrow) and the nuclei (arrowhead) of cells in a colorectal carcinoma. C, cells in a colorectal carcinoma exhibiting a high degree of nuclear CysLT1 receptor staining (arrowheads). D, cells in a colorectal carcinoma showing CysLT1 receptor immunoreactivity solely in the nuclei (arrowheads). E, diagram outlining the relative number of tissue samples (11 controls and 44 colorectal carcinomas) containing nuclei stained positive for the CysLT1 receptor (CysLT1R). F, correlation between the nuclear accumulation of the CysLT1 receptor and the nuclear staining of the proliferative marker Ki-67, calculated using the Pearson correlation (inset, positive Ki-67 staining). Points, means ± SE. *, P < 0.05 (unpaired Student's t test). G, series of sections from the same paraffin block (although sections are not in order) showing 1:100 dilution of the CysLT1R antibody, antigen adsorption (CysLT1R blocking peptide 5-1 µg/mL antibody), and rabbit preimmune serum. H, normal colon cells displaying weak cytosolic but positive nuclear staining (arrowhead) for 5-LO. I, colorectal carcinoma cells showing a high degree of 5-LO immunoreactivity in the nuclei (arrowhead) and the cytosol (arrow).

 
CysLT1 and 5-LO Are Present in both Nuclear and Plasma Membrane Fractions of Int 407 and CaCo-2 Cells. Using reverse transcriptase-PCR, we detected the presence of mRNA for the CysLT1 receptor and the enzyme 5-LO in Int 407 and in CaCo-2 cells, and the discovery of 5-LO mRNA agrees well with our previous observation that both these cell lines can synthesize LTB4 (16). In cell fractionation experiments, we also found that 5-LO and CysLT1 proteins were readily detected in the nuclear fractions of both cell lines (Fig. 2B). Considering all fractions, the levels of 5-LO protein expression were higher in the CaCo-2 cells than in the Int 407 cells, and this was particularly pronounced in the cytosolic fraction (Fig. 2B). These findings suggest that, compared with the Int 407 cells, the CaCo-2 cancer cells exhibited greater intracellular formation of LTs, and they also seemed to have an increased number of CysLT1 receptors in both the membrane and the nuclear fraction (Fig. 2B). CysLT1 was not detected in the cytosolic fraction of either of the two cell lines (Fig. 2B). To determine the purity of the subcellular fractions the blots were re-probed for lamin B (the nuclear fraction), for MAP/ERK kinase (the cytosolic fraction), and for CD71 (transferrin; the membrane fraction; Fig 2B).



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  		Figure 2. Expression and location of the CysLT1 receptor and 5-LO in intestinal epithelial cell lines. A, total RNA was isolated from Int 407 and CaCo-2 cells, and reverse transcriptase-PCR was done using specific primers to study the expression of CysLT1 receptor and 5-LO mRNA. Total RNA isolated from Int 407 and CaCo-2 cells but not treated with reverse transcriptase were used as negative controls. B, Int 407 and CaCo-2 cells were disrupted by nitrogen decompression. Membrane (M), cytosolic (C), and nuclear (N), fractions were prepared by ultracentrifugation to study the protein expression of the CysLT1 receptor and 5-LO. The protein concentration of each fraction was measured to ensure that equal amounts of protein were loaded onto the SDS polyacrylamide gel. The proteins were separated by electrophoresis, transferred onto polyvinylidene difluoride membranes, and immunoblotted with antibodies against the indicated proteins. B, to determine the purity of the cellular fractions, the membranes were reprobed for lamin B (the nuclear fraction), for MEK1 (the cytosolic fraction), and for CD71 (transferring; the membrane fraction). Right, results of the densitometric analysis of Western blots probed for CysLT1 and 5-LO in three separate experiments. C, fractions of the outer nuclear (ON) and the inner nuclear (IN) membranes of Int 407 cells analyzed as in B. The fractions were prepared by homogenization of the cells, followed by centrifugation to isolate intact nuclei. The nuclei were treated with 1% sodium citrate, and the two membrane fractions were isolated by sucrose gradient centrifugation. The different nuclei fractions were then analyzed as in B. C, to characterize the nuclear fractions, the blot were reprobed with lamin B which is known to be associated with the inner nuclear membrane. Right, results of the densitometric analysis of Western blots probed for CysLT1 and 5-LO in three separate experiments. Representative of at least three independent experiments.

 
CysLT1 and 5-LO Are Located Exclusively in the Outer Nuclear Membrane in both Int 407 and CaCo-2 Cells. Isolation of a nuclear fraction of Int 407 cells and subsequent separation of the two nuclear membranes revealed that the CysLT1 receptor protein and 5-LO protein are located solely in the outer nuclear membrane (Fig. 2C). To characterize the nuclear fractions the blots were reprobed for lamin B, which is known to be associated with the inner nuclear membrane (Fig. 2C). To further explore this interesting finding, we did a more refined analysis of the subcellular localization of CysLT1 receptor in intact Int 407 and CaCo-2 cells. First, we carried out immunogold labeling of CysLT1 receptor followed by electron microscopy, which clearly revealed expression of this receptor in the plasma membrane and the nucleus of both Int 407 (Fig. 3A and B) and CaCo-2 (Fig. 3C and D) cells. Subsequently, we used nitrogen decompression to disrupt the cells and then prepared fractions of plasma membranes (Fig. 3E and H), outer nuclear membranes (Fig. 3F and I), and inner nuclear membranes (Fig. 3G and J). We investigated these subcellular fractions by negative immunogold labeling of CysLT1 receptor and electron microscopy, and the results confirmed the localization of the CysLT1 receptor in the plasma membrane and the outer nuclear membrane of both Int 407 (Fig. 3E and F) and CaCo-2 (Fig. 3H and I) cells. We did not detect any positive CysLT1 receptor staining in the inner nuclear membrane in either of the two cell lines (Fig. 3G and J).



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  		Figure 3. Visualization of the subcellular localization of the CysLT1 receptor in intestinal epithelial cells by immunogold staining and electron microscopy. Int 407 cells (A, B, and E-G) and CaCo-2 cells (C, D, and H-J) were embedded in Lowicryl, after which ultrathin sections were cut and mounted on nickel grids. The sections were then immunostained with the primary anti-CysLT1 receptor antibody overnight and subsequently with a gold-conjugated secondary antibody. Images of the immunogold staining were finally captured by electron microscopy. Location of CysLT1 receptors in intact Int 407 (A and B) and CaCo-2 (C and D) cells. White arrows, nuclear membrane; black arrowheads, plasma membrane (bar in D, 1 µm). Negative immunogold staining of the CysLT1 receptor in plasma membrane, outer nuclear membrane, and inner nuclear membrane fractions (E-G) for Int 407 cells and (H-J) for CaCo-2 cells (bar in J, 2.5 nm).

 
LTD4 Induces Translocation of the CysLT1 Receptor from the Plasma Membrane to the Nucleus in Int 407 Cells but Not CaCo-2 Cells. Int 407 cells and CaCo-2 cells were incubated in the absence or presence of 40 nmol/L LTD4, after which cell nuclei were isolated and analyzed for CysLT1 receptor expression by Western blot. These experiments clearly revealed that LTD4 statistically significantly increased the nuclear expression of the CysLT1 receptor protein in Int 407 cells, however no statistically significant effect was observed in Caco-2 cells (Fig. 4A). Confocal microscopy of antibody-labeled CysLT1 receptors and lamin B, in this case used as a nuclear marker, confirmed that the CysLT1 receptor is located in the plasma membrane and the nucleus in both Int 407 and CaCo-2 cells (Fig. 4B). The lamin B staining in the more proliferative tumor Caco-2 cells is different from that seen in the less proliferative nontumor Int 407 cells. However, this difference is in good agreement with the demonstration that lamin B is present as foci or spots in the nucleoplasm at various times during the cell cycle (35, 36) and that its expression correlate with proliferation in several cell types (37). The nuclear staining for CysLT1 receptor was more intense in the CaCo-2 cells than in the Int 407 cells (Fig. 4B). Considering that Lu et al. (38) had found that neuronal cells exposed to angiotensin II exhibited a time- and dose-dependent increase in translocation of the angiotensin type 1 receptor to the nucleus, we conducted experiments to determine whether LTD4 can influence the localization of the CysLT1 receptor in Int 407 and CaCo-2 cells. The results indicated that LTD4 (40 nmol/L) induced translocation of the CysLT1 receptor from the plasma membrane to the nucleus in Int 407 cells (Fig. 4B), but not in Caco-2 cells (Fig. 4B), and the effect in Int 407 cells could be detected within 15 minutes from initiating the stimulation (data not shown). Interestingly, the Int 407 cells exhibited fairly similar nuclear staining of the CysLT1 receptor after the exposure to LTD4 compared with CaCo-2 cells. The LTD4-induced loss of cell surface CysLT1 receptors in Int 407 cells was verified by flow cytometry. In these experiments, LTD4 caused a significant reduction in the number of CysLT1 receptors on the surface of these cells (Fig. 4C). The inability of LTD4 to induce translocation of the CysLT1 receptor in CaCo-2 cells is not due to the lack of ability of the cell surface CysLT1 receptors to respond to LTD4, because LTD4 has been found to induce a calcium signal in these CaCo-2 cells that can be blocked by the specific CysLT1 receptor antagonist ZM-198, 615 (39).



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  		Figure 4. LTD4 induces a statistically significant translocation of the CysLT1 receptor in nontransformed Int 407 cells and to less effect in CaCo-2 colon cancer cells. A, Western blot analysis of CysLT1 receptor expression in nuclear preparations from Int 407 and CaCo-2 cells that had been incubated in the absence or presence of 40 nmol/L LTD4 for 2 hours. Thereafter, the membranes were stripped and reprobed with an antibody against lamin B. Right, densitometric analysis of the Western blots probed for CysLT1 in four separate experiments. Columns, means (n = 4); bars, ±SE. *, P < 0.05 (unpaired Student's t test). B, Int 407 and CaCo-2 cells were grown on coverslips to 80% confluence and were subsequently either directly fixed in 4% PFA (Control) or were stimulated with 40 nmol/L LTD4 for the indicated periods of time and then fixed. Thereafter, the cells were immunostained for the CysLT1 receptor and lamin B (nuclear marker), and the coverslips were mounted on glass slides. Images of the immunostained cells were obtained using a Bio-Rad Radiance 2000 confocal laser scanning microscope system. C, cells were incubated in the absence (Control) or the presence of 40 nmol/L LTD4 for 2 hours at 37°C and were subsequently harvested. Cells (4 x 105) were then resuspended and incubated in 150 µL PBS supplemented with 1% BSA and either the anti-CysLT1 receptor antibody or normal rabbit IgG for 1 hour. Thereafter, the cells were washed and incubated with a secondary fluorescin-conjugated Alexa 488 antibody. After extensive washing, the cells were fixed in PBS containing 1% PFA, and the cell-bound fluorescence was quantified using a Cell Quest FACScan flow cytometer (Becton Dickinson). Representative of three independent experiments.

 
The CysLT1 Receptor Is Signaling Competent at its Nuclear Localization in Int 407 and CaCo-2 Cells. Intact Int 407 cells were loaded with the fluorescent calcium probe Fluo-3 and thereafter stimulated with 40 nmol/L LTD4. Confocal microscopy showed that this treatment resulted in a calcium signal in the cytosol and the nucleus (Fig. 5A and B). In control experiments, these LTD4-induced calcium responses could be blocked by the specific CysLT1 receptor antagonist ZM-198,615 (Fig. 5B), and addition of 40 nmol/L LTC4 induced only a very faint, if any, calcium response (Fig. 5B, bottom). Thereafter, we directly tested the signaling capacity of nuclear CysLT1 receptors in Int 407 cells by stimulating isolated nuclei from these cells with LTD4. The Western blot results indicated an activation of the proliferative ERK1/2 signal in nuclei from these cells (Fig. 5C, left). Densitometric analysis of these results, revealed a statistically significant LTD4-induced increase in the phosphorylation level of ERK1/2 (Fig. 5C, right). These data show that the nuclear receptor pool possess intracellular signaling capacity. Notably, the ERK1/2 signal from the nuclear pool of CysLT1 receptors corresponds in time with the second phase of the LTD4-induced ERK1/2 signal we have previously observed in intact intestinal cells (20).



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  		Figure 5. The nuclear CysLT1 receptor exhibits signaling capacity. A and B, Int 407 cells grown on coverslips were incubated with 4 µmol/L Fluo-3/AM for 30 minutes. The cells were washed and covered with physiologically balanced calcium medium in a special chamber placed on a heated microscope stage (37°C), and the fluorescent signal was recorded before and after stimulation with 40 nmol/L LTD4. A, images were captured with a Bio-Rad Radiance 2000 confocal laser scanning microscope system after the length of LTD4 treatment indicated (upper left corner). B, fluorescent signals from intact whole cells and nuclei were subsequently analyzed using Bio-Rad Laserpix software. LTD4-induced calcium response in the following: a whole cell ({bullet}), nuclei ({circ}), and an intact cell pretreated with 40 µmol/L ZM-198,615 (a specific CysLT1 receptor antagonist) for 15 minutes ({blacksquare}). Bottom, calcium response induced in an intact cell by exposure to 40 nmol/L LTC4 and followed by 40 nmol/L LTD4. Representative of at least four independent experiments. C, nuclear fractions were isolated as in Fig. 2 and then stimulated with 40 nmol/L LTD4 for the indicated periods of time. Thereafter, the fractions were lysed, and samples were taken for Western blot analysis, first with a phospho-specific ERK1/2 antibody and then by reprobing with a total ERK1/2 antibody and lamin B, the latter as a loading control. Right, densitometric analysis of Western blots probed for pERK1/2 and ERK expressed as the ratio between pERK1/2 and total ERK in four separate experiments. Representative of four independent experiments. Columns, means (n = 4); bars, ±SE. **, P < 0.01 (unpaired Student's t test).

 
A Nuclear Localization Sequence Is Predicted in the CysLT1 Receptor and this Sequence Is Essential for Translocation of this Receptor to the Nucleus. Proteins that are translocated from the cytoplasm or plasma membrane to the nucleus, such as the androgen steroid receptor (40), the enzyme 5-LO (41), and the angiotensin type 1 receptor (38), usually contain a NLS. A majority of the many NLSs that have been identified are found in either of two highly conserved groups of proteins: one comprising members that contain a short NLS stretch of amino acids (42), and one including proteins that have what is called a bipartite NLS, which consists of two stretches of basic amino acids separated by 10 to 12 amino acids (43). By analyzing the CysLT1 receptor primary sequence with the PSORT II Prediction and ScanProsite programs, we found that the CysLT1 protein contains a possible bipartite NLS (rkhslssvtyvprkk) in its COOH-terminal end (amino acid positions 310–324; Fig. 6A). In contrast, no NLS sequence was predicted in the CysLT2 receptor. Therefore, we exchanged the predicted CysLT1 receptor NLS sequence with the corresponding region at amino acid positions 310 to 324 from the CysLT2 receptor. The resulting CysLT1 receptor NLS mutant (CysLT1R{Delta}NLS) and wildtype (CysLT1RWT) were then fused with enhanced green fluorescence protein (EGFP), and these tagged molecules were respectively designated pEGFP-CysLT1RWT and pEGFP-CysLT1R{Delta}NLS. Transfection of these two constructs into Int 407 cells allowed us to study the role of the bipartite NLS in the LTD4-induced translocation of the CysLT1 receptor. In cells transfected with EGFP-CysLT1RWT, exposure to LTD4 (40 nmol/L) caused a time-dependent translocation of the labeled receptor, as indicated by a 50% loss of fluorescence in the plasma membrane within 15 minutes (Fig. 6B); a representative confocal image of this translocation of pEGFP-CysLT1RWT is illustrated in Fig. 6B. By comparison, cells transfected with the pEGFP-CysLT1R{Delta}NLS construct and stimulated with LTD4 did not exhibit any translocation of the fluorescent mutated receptor (Fig. 6B), which indicates that the predicted NLS domain is necessary for proper LTD4-induced translocation of the CysLT1 receptor. We have previously showed that LTD4 increases proliferation in untransfected Int 407 cells via the CysLT1 receptor (20). Here we show that cells transiently transfected with EGFP-CysLT1RWT responded to LTD4 with a 30% increased proliferation. In contrast, cells transiently transfected with the mutated EGFP-CysLT1R{Delta}NLS did not exhibit an increased LTD4-induced proliferative response (Fig. 6C). These data suggests that the NLS sequence of the CysLT1 receptor is important not only for the translocation of this receptor but also for the LTD4-induced proliferate signal in intestinal epithelial In 407 cells.



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  		Figure 6. LTD4 induces translocation of an EGFP-tagged wild-type CysLT1 receptor but not an EGFP-tagged CysLT1 receptor lacking the bipartite NLS. A, amino acid sequence comprising the bipartite NLS (gray), and amino acids flanking the NLS (bold type and underlined). B, Int 407 cells were transfected with either an EGFP-tagged wild-type CysLT1 receptor (pEGFP-CysLT1RWT) or an EGFP-tagged CysLT1 receptor with a mutated bipartite NLS (pEGFP-CysLT1R{Delta}NLS). The cells were washed twice, placed in a physiologically balanced calcium medium, and stimulated with 40 nmol/L LTD4 for 20 minutes. Time of each stimulation (upper left corner); image obtained with a Bio-Rad Radiance 2000 confocal laser scanning microscopic system. Images outlined in B: representative of pEGFP-CysLT1RWT or pEGFP-CysLT1R{Delta}NLS transfected cells that were either not stimulated (Control) or were treated with 40 nmol/L LTD4 for 10 minutes. Quantification of the fluorescent intensity in the plasma membrane region of EGFP-CysLT1RWT or EGFP-CysLT1R{Delta}NLS transfected cells during exposure to 40 nmol/L LTD4 for 20 minutes. C, thymidine uptake analysis of cells transfected with either EGFP-CysLT1RWT or EGFP-CysLT1R{Delta}NLS vectors and then incubated in the absence or presence of 40 nmol/L LTD4 for 48 hours. [methyl-3H]thymidine (0.5 µCi per well) was added during the last 24 hours of these incubations. Duplicates of lysets from each well were mixed with scintillation liquid, and their radioactivity contents were measured in a LKB Wallace, 1209 RackBeta counter. The data were obtained as described in Materials and Methods. Columns, means (n = 3); bars, ±SE. **, P < 0.01 (unpaired Student's t test).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that the LTD4 receptor CysLT1 is up-regulated in human colorectal carcinomas compared with normal tissue, and also in the colon cancer cell line CaCo-2 compared with the nontransformed epithelial cell line Int 407 (16). In the present study, we made the novel finding in human tissue samples that the expression of the CysLT1 receptor is not only in the plasma membrane, but also in the nuclei, of both normal colon epithelial cells and colon cancer cells. However, the nuclear localization of this receptor was more pronounced and more common in colon cancer cells; indeed, in some tissues CysLT1 receptor staining was detected solely in the nuclei of the cancer cells. Western blotting and electron microscopy of subcellular fractions revealed that the CysLT1 receptor resided in the outer nuclear membrane of both nontransformed Int 407 cells and colon cancer CaCo-2 cells. Interestingly, LTD4-induced significant nuclear translocation of CysLT1 receptor in the Int 407 cells but not in CaCo-2 cells. The observation that LTD4 induced the same level of nuclear staining of the CysLT1 receptor in the nontransformed cells as in the colon cancer cells agrees with our earlier finding that treatment with LTD4 up-regulated COX-2, ß-catenin, and Bcl-2 in Int 407 cells to levels equivalent to those found in colon cancer cells (14). The exact consequences of these alterations are not known, although the implicated proteins are probably directly or indirectly related to survival and/or proliferation, two key events in cancer. This is also strengthened by our finding that high CysLT1 nuclear staining correlates highly with ki-67-positive nuclei staining. The agonist-induced nuclear translocation of the CysLT1 receptor we discovered in the present study provided us with a tool to examine the role of the bipartite NLS in such transport of the receptor. Cells have developed a comprehensive machinery for directing proteins to distinct intracellular sites, including specific targeting sequences such as the NLS, as well as specific transport proteins like the importins that can bind and dock NLS-containing proteins in the nuclear pore (44). Expression of a wild-type and a mutated EGFP-CysLT1 receptor and transfection of these proteins into Int 407 cells revealed that the NLS sequence is essential for agonist-induced translocation of the CysLT1 receptor in Int 407 cells. This finding agrees well with the results of experiments conducted by Lu et al. (38), which focused on the AT1 receptor. These investigators found that the presence of an NLS sequence in the cytoplasmic tail of the AT1 receptor is essential for its nuclear targeting.

Internalization of GPCRs is an effective mechanism by which a cell can terminate the signaling activity of a ligand-receptor complex and thereby control the duration of the effects of an agonist. Repeated or prolonged agonist stimulation has also been found to reduce the number of agonist-binding receptors on the surface of a cell and in that way desensitize the cell to subsequent agonist stimulation (45). In inflammatory bowel disease, several inflammatory mediators, including LTs, are constantly present at increased levels. In our investigation, fluorescent microscopy and flow cytometry revealed that exposure to LTD4 caused translocation of CysLT1 receptors from the plasma membrane to the nucleus in nontransformed Int 407 cells, whereas such nuclear translocation had apparently already occurred in colon cancer CaCo-2 cells because they invariably exhibited positive staining for CysLT1 receptor in the plasma membrane and even stronger staining in the nucleus. The relevance of these findings is underlined by an increase in nuclear staining of CysLT1 receptors in epithelial cells in human colon cancer specimens when compared with normal colon tissue. These observations have at least two important implications. First, they indicate that exposure to LTD4 does not down-regulate the CysLT1 receptor on the surface of colon cancer cells, which suggests that there is ongoing signaling from ligand-receptor complexes located in the plasma membrane. The significance of such a situation for cancer growth is indicated by our previous observation that LTD4 can signal for cell survival and proliferation via the CysLT1 receptor (20). Indeed, GPCRs and their downstream signaling via heterotrimeric G-proteins have also in other systems been associated with cancer. For example, constitutively active G{alpha}i can lead to oncogenic transformation (46), and it has been suggested that autocrine/paracrine stimulation of GPCRs by tumor-released ligands is involved in the carcinogenesis of different types of tumors (47, 48). The second implication of the findings is that the nuclear location of CysLT1 is not characteristic of an internalized receptor that is about to be degraded or recycled to the plasma membrane, but could equally well indicate that signaling by the receptor can occur in a novel location, providing the receptor can be accessed by its ligand.

We have previously observed that, compared with normal colon tissue and the nontransformed cell line Int 407, human colorectal cancer tissue and cells of the colon cancer line CaCo-2 exhibit significantly increased expression of 5-LO, which is the key enzyme in the synthesis of LTs (16). Such a difference between normal and cancer cells was also apparent in the present study. Interestingly, we detected 5-LO in the outer nuclear membrane of cells in both normal and malignant tissue and in nontransformed and cancer cell lines, which means that this enzyme exists in the same subcellular location as LTC4 synthase (49). Cysteinyl LTs are synthesized via activation of the 5-LO pathway and LTC4 synthase, hence it is plausible that the CysLT1 receptor expressed in the nuclei of intestinal epithelial cell can be activated via an intracellular autocrine loop that is initiated by locally produced LTs. The question is whether such ligand-receptor complexes are also capable of mediating an impact on cell behavior. The most sensitive way of visualizing the signaling capacity of the CysLT1 receptor in the nuclei of intact cells is to use confocal microscopy to study the effect of LTD4 on intracellular calcium. The importance of the information that can be gained by using such methodology is emphasized by the findings by Malviya et al. (50), showing that a rise in the nuclear concentration of calcium is involved in regulation of gene transcription. We noted that LTD4 triggered an instantaneous rise in cytosolic Ca2+ that was immediately followed by an increase in calcium in the nucleus. However, it was also possible that the calcium response in the nucleus was due to an increase in cytosolic calcium caused by LTD4-induced activation of the CysLT1 receptor in the plasma membrane, which in turn triggered an influx of calcium into the nucleus. Therefore, we exposed isolated intestinal cell nuclei to LTD4 and analyzed the activation of ERK1/2, a proliferative signal that we recently found to be stimulated by LTD4 in intact intestinal cells (20). The results of these experiments clearly show that, CysLT1 located in the nucleus is involved in LTD4-induced ERK1/2 proliferative signaling. This conclusion is further supported by the present finding that transfection of cells with a defective receptor lacking the NLS domain, CysLT1R{Delta}NLS, do not exhibit a LTD4-induced proliferative response as do cells transfected with a normal, wild-type receptor, EGFP-CysLT1RWT. The observation of a delayed ERK1/2 response to LTD4 in the nucleus suggests that this response may have given rise to the second phase of ERK1/2 activation we detected in intact, LTD4-stimulated cells. The capacity for distinct nuclear signaling by the CysLT1 receptor is also indirectly supported by the finding that LTD4 can induce transcription of the COX-2 gene in both Int 407 and CaCo-2 cells (15).

Our results show a novel, agonist-regulated localization of the GPCR CysLT1 in the outer nuclear membrane of intestinal cells. We observed increased basal expression of this receptor in the nuclei of cells in human colon cancer tissue and cells of the colon cancer line CaCo-2. The presence of LT synthesizing enzymes at the same intracellular location and the capacity of nuclear located CysLT1 receptors to trigger both a proliferative ERK1/2 signal and a proliferative response suggests that this receptor plays an important role in inflammation-related carcinogenesis. Consequently, it will be necessary to use both extracellular and intracellular agonists to effectively block the effects of the inflammatory mediator LTD4 on the proliferation and possibly also the survival of intestinal cells.


   Acknowledgments
 
Grant support: Swedish Cancer Foundation (A. Sjölander), Swedish Medical Research Council (A. Sjölander), Foundations at Malmö University Hospital (A. Sjölander, J.F. Öhd, and J.I.A. Campbell), Ruth and Richard Julin Foundation (A. Sjölander), Zoega's Foundation (A. Sjölander), Magnus Bergvall's Foundation (A. Sjölander), Gunnar Nilsson's Foundation (A. Sjölander), österlund Foundation (A. Sjölander); Royal Physiographic Society in Lund (C.K. Nielsen, J.I.A. Campbell, and J.F. Öhd); Danish Research Training Council (C.K. Nielsen); and Swedish Society for Medical Research (C.K. Nielsen and J.F. Öhd).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Maria Juhas and Elise Nilsson for their invaluable technical assistance.

Received 5/13/04. Revised 11/19/04. Accepted 11/30/04.


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 Results
 Discussion
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 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
J. F. Di Mari, J. I. Saada, R. C. Mifflin, J. D. Valentich, and D. W. Powell
HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts
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B. W. Pickard, A. B. Hodsman, L. J. Fraher, and P. H. Watson
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 Hum ReprodHome page
D. Lanoix, R. Ouellette, and C. Vaillancourt
Expression of melatoninergic receptors in human placental choriocarcinoma cell lines
Hum. Reprod., August 1, 2006; 21(8): 1981 - 1989.
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M. Mezhybovska, K. Wikstrom, J. F. Ohd, and A. Sjolander
The Inflammatory Mediator Leukotriene D4 Induces beta-Catenin Signaling and Its Association with Antiapoptotic Bcl-2 in Intestinal Epithelial Cells
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L. Parhamifar, B. Jeppsson, and A. Sjolander
Activation of cPLA2 is required for leukotriene D4-induced proliferation in colon cancer cells
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