From the Institut Fédératif de Recherche Claude de Préval, IFR 30, Université Paul Sabatier, and Centre Hospitalo-Universitaire de Toulouse, INSERM Unité 563, Department of Lipoproteins and Lipid Mediators, Hôpital Purpan, F31059 Toulouse Cedex, France
Received for publication, October 28, 2002
, and in revised form, February 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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The regulation of epithelial cell growth and functional differentiation is susceptible to various influences along the crypt-villus axis, including growth factor-derived signals. The overall importance of growth factors in intestinal epithelial cell renewal is underscored by the fact that the normal intestinal development is severely perturbed in EGFR1 knock-out mice (4). Interestingly, EGF binding in situ along the crypt-villus axis is higher in crypt than in villus enterocytes (5). These data correlated with studies performed with colon adenocarcinoma Caco-2 cells indicating that the expression of cell surface EGFR is dramatically decreased in well differentiated Caco-2 cells (6). Even if the cell growth arrest is well known to be associated with the differentiation process, the link between these two phenomena remains a confused point in the study of epithelial cell differentiation.
We have recently described enterophilins as a new family of intestinal proteins, with a carboxyl-terminal B30.2 domain and an extended leucine zipper in their amino-terminal part. Three members were identified: enterophilin-1 (Ent-1), enterophilin-2 (Ent-2), and a short form of Enterophilin-2 (Ent-2S). Ent-1 contains up to 45 regular heptad repeats and corresponds to a 65-kDa protein. Interestingly, Ent-1 was mostly expressed in the mid-crypt-villus axis, when cells stop their proliferation to start the differentiation process. In human intestinal epithelial carcinoma Caco-2 cells, Ent-1 ortholog expression pattern was positively correlated to growth arrest and terminal differentiation program. In addition, transfection of HT-29 cells with Ent-1 full-length cDNA inhibited cell growth and promoted an increase in alkaline phosphatase activity, an intestinal differentiation marker. Taken together, these results suggested a close relationship between Ent-1 expression and the enterocyte differentiation program (7).
To investigate the role of Ent-1 in growth arrest associated with enterocytic differentiation, we performed a yeast two-hybrid screen to identify proteins interacting with Ent-1. We report herein that Ent-1 interacts with sorting nexin 1 (SNX1). The sorting nexins are an emerging family of proteins (for a review see Ref. 8), characterized by the presence of a phox homology domain (9, 10). They are involved in the intracellular trafficking of several membrane receptors (11, 12, 13). SNX1, the best studied member of this family, recognizes the lysosomal targeting sequence code in EGFR. Moreover, SNX1 overexpression decreases the amount of EGFR on the cell surface as a result of enhancing trafficking in the endosome-to-lysosome pathway (11). Furthermore, recent data demonstrate that mice lacking both SNX1 and SNX2 display alterations in proper cellular trafficking (14). We confirm the association of Ent-1 with SNX1 by biochemical and immunofluorescence experiments in mammalian cells. Furthermore, Ent-1 causes a significant diminution of cell surface EGFR. In this context, the identification of SNX1 as an Ent-1 partner and their cooperative role in cell surface EGFR decrease provide more evidence about the involvement of Ent-1 in the inhibition of cell proliferation associated with the enterocytic differentiation process.
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EXPERIMENTAL PROCEDURES |
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PlasmidsEnt-1 full-length cDNA was cloned in pcDNA3.1/Myc-His vector (Invitrogen®) as previously described (7). We thus obtained the pcDNA3/Myc-His-Ent-1 vector, encoding a c-Myc-tagged protein in the COOH-terminal region. Full-length Ent-1 was cut off from pcDNA3/Myc-His vector using KpnI/ApaI sites and was inserted in pEGFP-C2 plasmid (Clontech Laboratories) to encode green fluorescent protein (GFP)-tagged protein in the NH2-terminal region (pEGFP-C2-Ent-1). Ent-1 full-length cDNA was also cloned in pDsRed2-N1 vector (Clontech Laboratories) to generate pDsRed2-N1-Ent-1 construct, encoding COOH-terminal DsRed-tagged Ent-1 protein. The NH2-terminal GFP-tagged SNX1 (pEGFP-C1-SNX1) and the NH2-terminal FLAG-tagged SNX1 (pcDNA3/FLAG-SNX1) constructs were generously provided by Dr. Gordon N. Gill (University of California at San Diego, La Jolla).
Cell Culture and TransfectionSeveral cell lines (ATCC) were used: COS-7 (African green monkey kidney fibroblasts), Madin-Darby canine kidney cells, HeLa (human cervix epitheloid carcinoma) cells, and Caco-2 (human colon carcinoma) cells.
The cells were grown in Dulbecco's modified Eagle's medium (Invitrogen®) supplemented with 10% fetal bovine serum (Invitrogen®) and 100 µg/ml penicillin/streptomycin (Invitrogen®) in a humidified atmosphere containing 5% CO2. Nonessential amino acids (0.1 mM; Invitrogen®) were added to the culture medium of Caco-2 cells. All cells were transfected with the different plasmids using FuGENETM6 (Roche Applied Science), except Caco-2 cells, which were transfected with a cationic lipid (LipofectAMINE; Invitrogen®) according to the manufacturer's protocols.
Immunoprecipitation ExperimentsCaco-2 cells were co-transfected with both pcDNA3/Myc-His-Ent-1 and pcDNA3/FLAG-SNX1. The experiments were performed 48 h after transfection, and all of the procedures were done at 4 °C. The cells were washed three times with phosphate-buffered saline (PBS) and lysed for 15 min with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol) containing protease and phosphatase inhibitors (1 mM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 20 mM NaF, 2 mM Na3VO4). Insoluble debris were removed by centrifugation at 14,000 x g for 15 min, and the supernatant was subjected to a preclearing with 30 µl of protein G-Sepharose for 30 min. Ent-1 was immunoprecipitated for 2 h with 5 µg of monoclonal anti-Myc antibody, followed by 50 µl of protein G-Sepharose for 1 h. After washing three times in buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 10% glycerol) containing protease and phosphatase inhibitors, the bound proteins were eluted by boiling the beads in Laemmli buffer (15). The precipitated proteins were then submitted to SDS-polyacrylamide gel electrophoresis and detected by immunoblotting.
Confluence-induced Differentiation of Caco-2 CellsCaco-2 cells were seeded at 18,000 cells/cm2 and grown in 60-mm dishes in the complete medium. The medium was changed every 2 days. The cells were washed twice in PBS and scraped at 4 °C in PBS containing protease and phosphatase inhibitors (1 mM phenylmethanesulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 20 mM NaF, 2 mM Na3VO4) at various times up to 23 days after plating. The samples were sonicated, and the protein concentration was determined according to the method of Bradford (16). Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis. Then Ent-1 and EGFR expression were analyzed by immunoblotting.
ImmunoblottingThe protein samples were submitted to SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and subjected to immunoblotting according to standard protocols (17). Rabbit polyclonal anti-Ent-1 peptide antibody was obtained from Eurogentec as previously described (7). Monoclonal antibody against c-Myc epitope (9E10) and polyclonal antibody against epidermal growth factor receptor (1005) were from Santa Cruz Biotechnology. Monoclonal antibody against flag (M2) was purchased from Sigma, and monoclonal antibody against GFP was from Roche Applied Bioscience. Revelation was done with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody (Promega) and ECL (Amersham Biosciences) detection.
Immunofluorescence MicroscopyCOS-7 cells were grown on sterile glass coverslips and transfected with pEGFP-C2-Ent-1 or pEGFP-C1-SNX1 or co-transfected with both pcDNA3/Myc-His-Ent-1 and pEGFP-C1-SNX1. For EGF stimulation, the cells were serum-starved for 24 h. The cells were incubated with 100 ng/ml EGF for 1 h at 4 °C, rinsed twice in PBS, and finally incubated with warmed medium at 37 °C for 10, 15, or 30 min. The cells were washed three times with ice-cold PBS, fixed for 15 min with 3% paraformaldehyde, permeabilized for 2 min with 0.2% Triton X-100, and saturated for 30 min with 0.2% gelatin. The cells were then incubated 60 min with the primary antibody (anti-EEA1 from Transduction Laboratories or anti-c-Myc (9E10) from Santa Cruz Biotechnology), and an immunostaining was performed with TRITC-conjugated anti-mouse IgG secondary antibody (Southern Biotechnology Associates). The coverslips were examined with a Zeiss Axioskop microscope or with a confocal microscope (Zeiss, LSM 510, Axiovert 100).
Size Exclusion ChromatographyCOS-7 cells were
co-transfected with pEGFP-C2-Ent-1 and pcDNA3/FLAG-SNX1, grown for 24 h, and
then serum-starved for an additional 24 h. The cells were incubated with 100
ng/ml EGF for 1 h at 4 °C, rinsed twice in PBS, and finally incubated with
warmed medium at 37 °C for 10 min to synchronize EGFR endocytosis. As
previously described by Chin et al.
(18), COS-7 cells were lysed
for 30 min at 4 °C in lysis buffer (50 mM Tris-HCl, pH 7.6, 50
mM NaCl, 0.1% Triton X-100, 1% Nonidet P-40) containing protease
inhibitor mixture tablet (Roche Applied Science). After centrifugation at
16,000 x g for 15 min, the supernatant was concentrated on
Microcon (Millipore; cut-off, 10,000 Da), and the extract was loaded onto a
Superose 6 HR 10/30 column. The column was eluted with 40 mM HEPES,
pH 7.8, 50 mM KCl, 2 mM EDTA, 1 mM
dithiothreitol, 0.2 mM phenylmethanesulfonyl fluoride, and 10%
glycerol, using a flow rate of 0.5 ml/min, and 0.25-ml fractions were
collected after a delay of 10 min. The column was calibrated with protein
standards (Bio-Rad), including thyroglobulin (670 kDa), globulin (158
kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B-12 (1.35 kDa).
Equal volumes of fractions were subjected to immunoblotting analysis.
Analysis of Cell Surface EGFR Density by Flow CytometryCOS-7 cells were transfected with pEGFP-C2-Ent-1, pEGFP-C1-SNX1, or empty vector and grown for 72 h. Then cells were collected by treatment with 2 mM EDTA in PBS and fixed in 1% formaldehyde for 30 min, and nonspecific sites were saturated with 1% bovine serum albumin for 30 min. Cell surface EGFR was detected by incubation with the primary antibody (clone LA1; Euromedex) for 60 min, followed by incubation with a R-phycoerythrin (RPE)-conjugated goat anti-mouse IgG secondary antibody (DAKO) for 60 min. The cells were then analyzed by flow cytometry (Beckman Coulter XL 4C). Expression of the GFP-fused proteins was monitored by fluorescence measurement, and the surface binding of the anti-EGFR antibody was measured specifically on the transfected cell population. To evaluate the effect of both Ent-1 and SNX1 on plasma membrane EGFR, we co-transfected COS-7 cells with pDsRed2-N1-Ent-1 and pEGFP-C1-SNX1 or both empty vectors. Cell surface EGFR was similarly immunolabeled, except for the secondary antibody: RPE-Cy5-conjugated rabbit anti-mouse IgG antibody (DAKO). The percentage of cell surface EGFR was measured by RPE (red) or RPE-Cy5 (deep red) fluorescence mean. The results were analyzed by paired t test and were considered significantly different (*, p < 0.005; **, p < 0.001) from controls (GFP-transfected cells for single transfection or GFP- and DsRed-co-transfected cells for double transfection).
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RESULTS |
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To pinpoint the binding domains of Ent-1 with its identified partners, we used the two-hybrid one to one interaction assay with the leucine zipper part or the B30.2 domain as a bait. The results showed that the B30.2 domain was unable to bind SNX1 and SNX2. Unfortunately, the leucine zipper part of Ent-1 was toxic for the two yeast strains. Accordingly, we suggested that the leucine zipper domain and/or full-length Ent-1 was necessary to mediate the binding with the sorting nexin proteins.
We previously demonstrated that Ent-1 overexpression inhibited cell proliferation. Thus, we decided to focus our investigations on SNX1 because it is the best characterized members of SNX family, and it has clearly been involved in the decrease of cell surface EGFR by enhancing trafficking in the endosome-to-lysosome pathway. To further confirm the association of Ent-1 with SNX1, we performed co-immunoprecipitation experiments in intestinal mammalian cells. Myc-tagged Ent-1 and FLAG-tagged SNX1 were co-expressed in Caco-2 cells, and Ent-1 was subjected to immunoprecipitation with an anti-Myc antibody. Immunoprecipitated complexes were analyzed by immunoblotting, and data revealed the presence of both Ent-1 and SNX1, confirming the interaction between these two proteins in intestinal cells (Fig. 2).
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Enterophilin-1 Co-localizes with SNX1 on Vesicular and Tubulovesicular StructuresIdentification of SNXs as Ent-1 partners led us to focus on the cytoplasmic localization of Ent-1 by fluorescence microscopy. COS-7 cells were co-transfected with COOH-terminally tagged Ent-1 (pcDNA3/Myc-His-Ent-1) and NH2-terminally tagged SNX1 (pEGFP-C1-SNX1). Both Ent-1 (Fig. 3, top panel) and SNX1 (Fig. 3, middle panel) displayed a vesicular and tubulovesicular signal. We observed that part of both staining patterns perfectly overlapped (Fig. 3, bottom panel, inset), indicating that within the cell, Ent-1 and SNX1 co-localized in similar structures. However, we noticed that Ent-1 was also located in cellular areas lacking SNX1 staining. Similarly, SNX1 was found in cytoplasmic pools that did not contain Ent-1. Thus, Ent-1 and SNX1 showed substantial but not complete co-localization.
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Furthermore, we confirmed that NH2-terminally tagged Ent-1 displayed a cytoplasmic distribution similar to COOH-terminally tagged Ent-1 in different epithelial cell lines. Indeed, we also observed vesicular and tubulovesicular structures with pEGFP-C2-Ent-1 in COS-7 cells, as well as in Madin-Darby canine kidney, HeLa, and Caco-2 cells (data not shown).
Ent-1-containing Structures Are Different from EEA1-positive Early EndosomesBecause SNX1 has been reported to be substantially associated to early endosomal membranes (19, 20), we investigated whether Ent-1-containing vesicles corresponded to early endosomes. COS-7 cells were transfected with pEGFP-C2-Ent-1 and the early endosomal specific marker EEA1 was labeled with a specific antibody after various times of EGF stimulation (100 ng/ml) as indicated under "Experimental Procedures". Confocal microscopy analysis revealed that Ent-1 was still present on vesicular and tubulovesicular structures in cells stimulated for 10, 15, or 30 min with EGF (Fig. 4). However, no merge was observed with EEA1 staining at any time of stimulation, suggesting that Ent-1-containing vesicles were different from the previously defined EEA1-positive early endosomes (Fig. 4). By contrast, SNX1 partially overlapped with EEA1 signal under the same conditions of EGF stimulation. Nevertheless, as previously documented (19, 21), a large proportion of SNX1 vesicles was clearly EEA1-negative (Fig. 5) and could correspond to Ent-1 merging area. Our immunofluorescence data demonstrated that Ent-1 and SNX1 co-localized in tubular and vesicular structures that were different from EEA1-positive early endosomes.
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Enterophilin-1 and SNX1 Coexist in Macromolecular Complexes Containing
EGFR in the CellSorting nexins are supposed to act as a multimeric
protein complex named the retromer complex by analogy with their yeast
orthologs (22). Thus, we
checked for the presence of Ent-1 in such macromolecular complexes by gel
filtration chromatography. COS-7 cells were co-transfected with pEGFP-C2-Ent-1
and pcDNA3/FLAG-SNX1. After stimulation with 100 ng/ml EGF for 10 min to
stimulate EGFR endocytosis, the cytosolic fractions were prepared and loaded
onto the column. The different fractions were analyzed for the presence of
GFP-Ent-1, FLAG-SNX1, and endogenous EGFR by Western blotting. An important
signal for Ent-1 was obtained from fractions 2630, corresponding to
large complexes of 670310 kDa, with a major elution in 435 kDa
(Fig. 6). Conversely, no signal
was detected in the fractions corresponding to the size of monomeric Ent-1 (92
kDa for the GFP-tagged Ent-1), suggesting that Ent-1 does not exist as
monomers in the cell. Interestingly, SNX1 displayed an elution peak in
fractions 2630 of
310670 kDa that perfectly matched with
the Ent-1-enriched complexes. However, as already reported
(18,
22), SNX1 also presented a
wide elution profile because of its property to self-assembly or to associate
with other proteins. Thus, SNX1 was recovered in a second peak of higher size
macromolecular structures (fractions 2022,
1700 kDa). Furthermore,
SNX1 did not appear in the monomeric form (
60 kDa for the FLAG-tagged
SNX1). Endogenous EGFR presented an elution profile in fractions 2228,
overlapping the Ent-1 and SNX1 peaks in fraction 26 corresponding to
670-kDa heteromeric protein complexes. As with Ent-1 and SNX1, EGFR was
not found as monomeric form. We can notice that a part of the three proteins
was observed in the void volume (fractions 115) that could correspond
to very large molecular mass complexes excluded from the column.
Alternatively, this could be the result of nonspecific aggregation.
Nevertheless, gel filtration data demonstrated a convincing overlap between
Ent-1 and SNX1 elution profiles and strongly suggested the existence of
Ent-1/SNX1/EGFR multimeric complexes involved in EGF-induced EGFR
endocytosis.
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Enterophilin-1 Enhances the Decrease of Cell Surface EGF ReceptorsGel filtration data led us to check for the effects of Ent-1 on EGFR cell surface pool. COS-7 cells were transfected with pEGFP-C2-Ent-1, pEGFP-C1-SNX1, or empty vector for 72 h, and cell surface EGFR was immunolabeled. By flow cytometry, cell surface EGFR expression was specifically monitored in the GFP- or GFP-Ent-1-transfected cells as indicated under "Experimental Procedures." Our results showed that Ent-1 induced a significant decrease of 35% of plasma membrane EGFR, compared with the GFP-transfected cell population used as control (Fig. 7A). A similar decrease was obtained with cells overexpressing SNX1 in our experimental conditions (Fig. 7A), in agreement with previously published data (11). These results indicated that overexpressed Ent-1 was able to promote the down-regulation of cell surface EGFR.
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To evaluate the cooperation between Ent-1 and SNX1 in the regulation of EGFR endocytosis, we co-transfected COS-7 cells with pDsRed2-N1-Ent-1 and pEGFP-C1-SNX1. Cell surface EGFR was immunolabeled and identically analyzed on double-transfected cell population. We demonstrated that Ent-1 and SNX1 displayed a synergetic effect on cell surface EGFR removal because a 65% decrease of plasma membrane EGFR was quantified in double-transfected cells (Fig. 7B). Taken together, these data suggested a cooperative effect of Ent-1 and SNX1 on cell surface EGFR removal during endocytosis.
Enterophilin-1 Expression Correlates with the Decrease of EGFR during the Differentiation of Caco-2 CellsTo investigate the physiological relevance of the effects of Ent-1 overexpression on cell surface EGFR, we analyzed the expression patterns of both proteins in relation to enterocyte differentiation. Human colon carcinoma Caco-2 cell line is a well characterized model for the study of intestinal differentiation. We thus performed the confluence-induced differentiation of Caco-2 cells. As previously shown (7), the cells reached confluence after 7 days, and the human ortholog of Ent-1, identified as a 65-kDa protein, displayed an increased expression beginning at day 9, sustained up to 23 days in culture. Interestingly, Western blot analysis showed a marked decrease of EGFR expression beginning at day 14, when the human ortholog of Ent-1 was highly expressed (Fig. 8). At day 2, we observed a weak expression of EGFR that could be expected knowing that Caco-2 cells start to proliferate after a lag time of 2 days after plating (7, 23). The functional differentiation was followed by appearance of alkaline phosphatase activity, typically used as intestinal differentiation marker (data not shown). These results demonstrated a correlation between the Ent-1 expression pattern and the decrease of EGFR expression during intestinal epithelial differentiation.
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DISCUSSION |
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SNX1 and SNX2 are the mammalian orthologs of the yeast vacuolar protein
sorting Vps5p (25). Vps5p is a
subunit of a large multimeric complex, termed the retromer complex, involved
in retrograde transport of proteins from endosomes to the trans-Golgi network
(26). In mammalian cells,
homodimerization and heterodimerization of SNXs have been reported
(22,
24), and it has been proposed
that complex formation between SNXs may be necessary for organizing functional
units for receptor sorting and degradation. We showed a perfect co-elution of
Ent-1 and SNX1 in 310670-kDa macromolecular complexes. Part of EGFR was
also detected in the same complexes. We also demonstrated that neither Ent-1
and SNX1 nor EGFR eluted in their respective monomeric form. These data
suggested that the three proteins interacted with each other or with other
proteins in 435670-kDa macromolecular complexes. This was
consistent with the work of Chin et al.
(18), which demonstrated the
elution of SNX1 in similar size complexes and the lack of monomeric-SNX1 form.
Additionally, SNX1, but not Ent-1, was detected in higher molecular mass
complexes (above 1700 kDa). Because a certain proportion of SNX1 and Ent-1
co-localized by immunofluorescence, these results emphasized that both
proteins could interact with each other in the same multimeric complexes.
Ent-1 could be a component of macromolecular complexes involved in endocytosis
of EGFR. However, EGFR endocytosis is a very dynamic process, involving
continuously remodeled complexes and structures. Thus, the presence of EGFR in
Ent-1/SNX1-enriched complexes could be transient, strictly depending on well
defined macromolecular complex formation along the EGFR endosome-to-lysosome
pathway.
SNXs have been shown to modulate endocytosis of a variety of receptors (for a review, see Ref. 8), including EGFR (11), protease-activated receptor-1 (13), or low density lipoprotein receptors (27). Overexpressed SNX1 was clearly involved in the decrease of EGFR on the cell surface as a result of enhancing the rate of constitutive and ligand-induced degradation (11). We then investigated the role of Ent-1 on EGFR degradation. Our results showed that overexpression of Ent-1 significantly decreased the cell surface EGFR. Interestingly, EGFR degradation was strongly extended when Ent-1 and SNX1 were co-expressed. These results highlighted the synergetic effect of both proteins and were in favor of a role of Ent-1 in cell surface EGFR removal by endocytosis through its interaction with SNX1. Ent-1 could thus be considered as a new partner of SNX1, such as ACK2 (activated Cdc42-associated kinase 2), which promotes EGFR degradation through its interaction with SNX9 (28) or Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) that inhibits EGFR endocytosis through its binding to SNX1 (18). It will be interesting now to define whether Ent-1 could regulate other types of cell surface receptors as described for SNXs.
EGFR is an important mediator of intestinal epithelial cell proliferation. In fact, undifferentiated crypt cells display a higher EGF binding activity than differentiated villus enterocytes (5), which is correlated with the decrease of EGFR surface expression along the crypt-villus axis (6). Our data indicate that Ent-1 expression during spontaneous differentiation of Caco-2 cells correlates with the decrease of EGFR expression. Among several intestinal epithelial cell lines, Caco-2 cells have proven to be the most useful in vitro models. Indeed, Caco-2 cells are unique in their ability to initiate spontaneous differentiation on reaching confluence under normal culture conditions and undergo a maturation process closely resembling the functional differentiation found in normal intestine (23). Furthermore, we recently published that the onset of Ent-1 expression corresponded to cell growth arrest, preceding functional differentiation. Additionally, we reported a decrease of proliferation in Ent-1-transfected HT-29 cells (7). In this context, Ent-1-mediated cell surface EGFR removal adds to our understanding of the regulation of growth arrest in intestinal epithelium. This is directly related with the reported decrease of EGFR at the mid-villus axis (5).
To summarize, we demonstrated that Ent-1 was a new partner of SNX1. According to our two-hybrid results, we hypothesized an interaction mediated by the coiled-coil structures present in both the COOH-terminal region of SNXs and the NH2-terminal leucine zipper part of Ent-1. Works in progress in our laboratory presently aim at defining the exact SNX1-binding regions on Ent-1 by generating Ent-1 mutants defective in binding SNX1. Additionally, Ent-1 was localized with SNX1 on vesicular and tubulovesicular structures, which were different from EEA1-positive early endosomes. Interestingly, Ent-1 co-eluted with SNX1 in macromolecular complexes containing part of EGFR and induced cell surface EGFR removal. Moreover, Ent-1 and SNX1 displayed a cooperative effect on EGFR degradation, strongly increasing plasma membrane EGFR removal. Further studies will bring new insights to precise exact molecular mechanisms by which Ent-1 and SNX1 regulate receptor vesicular trafficking. These data provide new evidence about Ent-1 involvement in down-regulation of mitogenic signal leading to cell growth arrest and enterocyte differentiation.
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FOOTNOTES |
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Supported by fellowships from Ministère de l'Education Nationale, de
la Recherche et de la Technologie and from La Ligue Nationale contre le
Cancer.
To whom correspondence should be addressed: INSERM Unité 563, Bat C,
Department of Lipoproteins and Lipid Mediators, Hôpital Purpan, 31059
Toulouse Cedex, France. Tel.: 33-5-61-77-94-00; Fax: 33-5-61-77-94-01; E-mail:
Ama.Gassama{at}toulouse.inserm.fr.
1 The abbreviations used are: EGFR, EGF receptor; EGF, epidermal growth
factor; EEA1, early endosome antigen 1; Ent-1, enterophilin-1; GFP, green
fluorescent protein; SNXs, sorting nexins; TRITC, tetramethylrhodamine
isothiocyanate; PBS, phosphate-buffered saline; RPE, R-phycoerythrin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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