&p.1:Abstract The aim of this work was to show in serum-free medium a convergent effect of physiological factorsand extracellular matrix proteins on the differentiationprocess of enterocytes by taking as a model the HT29-D4 clone that has the feature of differentiating whensubcultured in fetal bovine serum glucose-free medium.We show that triiodothyronine (T3) as well as insulinpromotes limited cell growth and differentiation, where-as fibronectin or bovine serum albumin (BSA) inducescell growth and a low level of differentiation. However,insulin, T3, fibronectin, and BSA together with epider-mal growth factor and transferrin promoted satisfactorygrowth and enterocyte morphology with epithelial elec-trophysiological properties in HT29-D4 cells. Withthese factors adequate protein targeting was achievedsince cells apically expressed the carcinoembryonic an-tigen, and basolaterally transferrin and insulin receptors,β1 and αvβ6 integrins, talin, vinculin, and focal adhe-sion kinase (FAK). Talin, vinculin, FAK, and αvβ6 inte-grin, the fibronectin receptor, were clustered in focalcontacts, which agrees with a possible role of fibronec-tin in final cell growth, the latter process mediating thefinal phase of differentiation. This level of differentia-tion can be maintained for a long time. Thus HT29-D4cells appear to be a suitable model to study the implica-tion of integrins in the differentiation process of humanenterocytes.&bdy:

Introduction

We sought to determine the convergent effect of growthfactors, hormones, and extracellular matrix (ECM) proteinsthat without serum would sustain growth and differentiationof clone D4 [12] from a colon adenocarcinoma cell line(HT29; [17]). It has been shown by Pinto et al. [44] and byZweibaum et al. [58], respectively, that in glucose-free me-dium supplemented with galactose and in glucose-free me-dium only, this cell line grows in a monolayer and express-es intestinal hydrolases at the apical cell brush border. Likethe HT29 cell line [44, 58], HT29-D4 cells display an en-terocyte morphology when subcultured in glucose-free me-dium supplemented with galactose and serum [12].

Different cell culture models have been developed inserum-free medium to achieve enterocyte differentiationin long term studies [26, 29]. Caco-2 cell, a colic adeno-carcinoma cell line that spontaneously differentiates in15% FBS and in a high glucose medium, can grow anddifferentiate in Dulbecco’s modified Eagle’s medium(DMEM) supplemented only with insulin, transferrin, andselenium [29]. Moreover, it was shown that a clone froma rectal adenocarcinoma (HRA-19) could be differentiat-ed with insulin, transferrin, and ascorbic acid into absorp-tive, endocrine, and mucous phenotypes [26]. Tumor cells[45, 54] show a high rate of glycolysis parallel with theslowing down of mitochondrial function. Moreover, sub-stituting a high glucose concentration for a low galactoseconcentration concomitantly induces the differentiationprocess and a decrease in lactate production [15, 44],which is compatible with an activation of the mitochon-drial pathway. Thyroid hormone can directly activate mi-tochondrial metabolism [25, 53] or act by gene activationvia c-erbA [55]. Accordingly, triiodothyronine (T3) hasbeen shown to play a preponderant role in enterocyte dif-ferentiation of the suckling mouse small intestine [37]and Caco-2 cell line in serum-free medium [29–31] andto be a direct stimulator of mRNA transcription, expres-sion, and enzymatic activity of sucrase-isomaltase [31].

J.-B. Rognoni (✉) · V. Pichard · S. Honore · V. RigotM. Lehmann · M. Roccabianca · G. Carles · J. Luis · J. MarvaldiC. BriandUPRES-A CNRS 6032, Faculté de Pharmacie,Université Aix-Marseille I et II, 27,Bd Jean Moulin 13385 Marseille Cedex 05, FranceTel.: +33-4-91-835625Fax: +33-4-91-782024&/fn-block:

Differentiation (1998) 63:305–317 © Springer-Verlag 1998

O R I G I N A L A RT I C L E

&roles:Jean-Baptiste Rognoni · Véronique PichardStéphane Honore · Véronique RigotMaxime Lehmann · Monique RoccabiancaGérard Carles · José Luis · Jacques MarvaldiClaudette Briand

Convergent effects of growth factors, hormones, and fibronectin arenecessary for the enterocyte differentiation of a colon adenocarcinomacell line (HT29-D4)

&misc:Accepted in revised form: 16 June 1998

The role of the ECM on the enterocyte mitogenic anddifferentiation processes [32, 51], via transmembraneglycoproteins (integrins), has been reconsidered in thelast few years. Possible differentiation [27, 43] and mito-genic [2, 48] signaling pathways triggered from integrinshave been described, after their clustering on the basalpart of the basolateral plasma membrane [57].

Preliminary studies showed that the HT29-D4 clone isnot able to differentiate in high glucose serum-free medi-um whatever the factors assayed (hormones, growth fac-tors, ECM constituents) although it does differentiatewith suramin (an anti-trypanosomiasis; [16]). The firststep of this work (short term study) was thus to seek bywhich pathways predifferentiated HT29-D4 cells couldgrow and differentiate and thus to identify the growthand differentiating factors. In the second step (long termstudy) we associated some of these factors to obtain ahigh level of epithelial differentiation.

The epithelial phenotype has been characterized bymany approaches previously used for colon adenocarci-noma cell lines: morphological and ultrastructural [14,44, 58], specific apical [10, 14, 44, 58] and basolateral[20] antigen expression, and electrophysiological [16].

Methods

Materials

For cell culture, 75-cm2 and 25-cm2 polystyrene flasks and 9-cm2polystyrene Petri dishes were purchased from Falcon, Becton-Dickinson and Co. (Oxnard, Calif., USA). Transwell porous cellculture inserts (Nuclepore filter, 3-µm pores) were purchased fromCostar (Cambridge, Mass., USA). Millicell-ERS resistance systemfrom Millipore Corporation (Bedford, Mass., USA) was used forelectrophysiological studies. DMEM, FBS, and trypsin were pur-chased from GIBCO (Cergy-Pontoise, France) and DMEM with-out glucose was from EUROBIO (Les Ullis, France). Soybeantrypsin inhibitor, retinoic acid, epithelial growth factor (EGF), in-sulin, hydrocortisone, T3, linoleic acid, oleic acid, bovine serumalbumin (BSA), and HEPES were from Sigma (St Louis, Mo.,USA). Human fibronectin was purchased from CRTS (Lyon-Bey-nost, France) and Dulbecco’s phosphate-buffered saline (PBS) wasfrom Oxoid (Basingstoke, UK). Selenous acid was obtained fromCollaborative Biomedical Products (Bedford, Mass., USA) andCollagen type I was from Seromed (Berlin, Germany). Mousemonoclonal antibodies of anti-human carcinoembryonic antigen(CEA) and anti-human insulin receptor were purchased from Bios-ys (Compiegne, France) and Seralab (Paris, France), respectively;anti-transferrin receptor, anti β1, α2β1, α3β1 integrins, and anti-vinculin and talin were obtained from Immunotech-Coulter (Mar-seille, France) and Sigma (St Louis, Mo., USA), respectively; anti-focal adhesion kinase (FAK) was obtained from Transduction Lab-oratories (Lexington, Ky., USA). Rat monoclonal antibody (mab)anti-human αvβ6 integrin was purified from clone 69-6-5 in ourlaboratory [34]. Phalloidin-TRITC abd FITC-antibodies werefrom Sigma (St. Louis, Mo., USA).

Cell culture

Cell cultures were incubated in a humidified incubator at 37°C inan atmosphere of 5% CO2 in air. Distilled water from Milli Q ap-paratus (Millipore) and pyrogen-free water were used for mediawith serum and for serum-free media, respectively.

For cell culture in standard medium (undifferentiating medi-um), HT29-D4 cells were cultured as usual in DMEM (glutamine

4 mM; pyruvate 1 mM) containing glucose (25 mM), and 10%FBS. At confluence, cells were harvested with 0.05% tryp-sin/0.53 mM EDTA in PBS (pH 7.3) for 5 min at 37°C. For cellcultures in glucose-free medium supplemented with galactose (dif-ferentiating media), glucose-free DMEM (glutamine 4 mM; pyru-vate 1 mM) was supplemented with 10% dialyzed FBS, galactose(5 mM), and selenous acid (10–2 µg/ml). Trypsinization with tryp-sin 0.25%/0.53 mM EDTA in PBS (pH 7.3) for 10 min was neces-sary to detach cells at late confluence [12].

For characterization of factors inducing cell growth and differ-entiation, HT29-D4 cells were adapted to grow in decreasing se-rum concentrations. Cells grown in 1% FBS Gal-medium werethereafter cultured in this medium supplemented with differentfactors during 2 days. Cells were then harvested in PBS-EDTA(0.53 mM) with trypsin (0.25%). The trypsinization was stoppedby Gal-medium containing BSA (2%) and soybean trypsin inhibi-tor (240 µg/ml). Cells were rinsed once in the same medium andplated at 8×104 cells/cm2 on an ECM component-coated substrate[incubation of collagen I (50 µg/ml) or fibronectin (30 µg/ml) for1 h at 37°C in a 25-cm2 flask].

All factors were tested in differentiating media without serumeither in glucose-free DMEM (glutamine 4 mM; pyruvate 1 mM)or, for IT3FB2, (see below) in glucose-free DMEM/Ham’s F12,1/1, (glutamine 4 mM; pyruvate 1 mM). DMEM or DMEM/F12was buffered with HEPES (15 mM) and contained selenous acid(10–2 µg/ml) and galactose (5 mM). It had been shown that EGF(5×10–3 µg/ml) and transferrin (5 µg/ml) were necessary and suffi-cient for the growth of undifferentiated cells in high glucose con-centration containing DMEM/F12 medium [13]. Two kinds ofBSA, either with lipids (BSA L+), or fatty acid-free (BSA L–),were used. The factors and hormones tested were called like themain component acting independently of EGF (E) and transferrin(T) and associated as follows: ET (EGF, transferrin), hydrocorti-sone-ET (hydrocortisone 10 nM), T3-ET (T3 1.5 nM), insulin-ET(insulin 5 µg/ml), fibronectin-ET (fibronectin 10 µg/ml), BSAL+-ET (BSAL+ 2 mg/ml), IT3FB1 [EGF, transferrin, insulin (5 µg/ml),T3 (1.5 nM), fibronectin (10 µg/ml), and BSAL+ (2 mg/ml)] andIT3FB2 [EGF, transferrin, insulin (5 µg/ml), T3 (1.5 nM), fibro-nectin (10 µg/ml), BSAL– (2 mg/ml), linoleic acid (5 µg/ml), andoleic acid (5 µg/ml)].

Cell growth curves

Cells were seeded in multidish plates at a density of 3×104 cells/cm2 and then trypsinized and counted every 2 days until theyreached confluence. For each time, cells were counted in fourwells. A maximum of 5% of trypan blue permeable cells was ac-cepted. The generation times were calculated by the method ofManford and Patterson [38].

Histological techniques

Cell cultures were monitored daily by light microscopy. For trans-mission electron microscopy, samples were prepared as described[12, 14] and observed on a Geol 100 C electron microscope. Forthe observation of extracellular antigens, anti-CEA and anti-inte-grins (αvβ6, β1), antibodies were fixed to antigens at 4°C for 1 h.For integrin labeling, cell monolayers were first incubated at 37°Cin a Ca2+-free medium for 2 h to break tight junctions [23]. For in-tracellular antigens, i.e. actin, vinculin, talin, and FAK, HT29-D4cells were first fixed in 3.7% formaldehyde and then treated with70% methylic alcohol and acetone at –20°C. The cells were thenincubated with the first antibody at room temperature for 1 h. Allthe cell cultures were then incubated for 45 min at room tempera-ture with an FITC antibody. Control experiments were performedwithout primary antibody to check the non-specific staining. Foractin labeling, direct phalloidin-TRITC binding was done in PBSfor 1 h. An Olympus microscope BH-2 was used for fluorescenceobservations with a 100× lens for basolateral domains and a 20×lens for apical antigen (CEA).

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Flow cytometry

Cell cultures were treated with 0.53 mM EDTA/PBS, and cellswere harvested and rinsed on ice in DMEM (NaHCO3 14 mM)supplemented with 20% serum, 2% BSA and 15 mM HEPES. Af-ter detachment, cells were incubated for 1 h at 4°C with a slightagitation in presence of monoclonal antibodies (mabs) anti-α2β1,anti-α3β1, and anti-αvβ6 integrins, and incubated thereafter withthe second FITC antibody (rat or mouse anti-IgG) for 1 h at 4°C.After being washed with DMEM (20% FBS, 2% BSA), cells werefixed with 3% paraformaldehyde in PBS for 10 min at room tem-perature. Flow cytometry measurements were performed on aFACScan flow cytometer (Becton Dickinson).

Improvement of the tightness of filter-grownmonolayers and electrophysiological studies

Standard medium, Gal-medium, and IT3FB1 factors in glucose-,serum-free medium were assayed for HT29-D4 cells growth anddifferentiation on Nuclepore filters (3-µm pores, transwell cell cul-ture inserts). After confluence, basal chambers were provided withthe respective media, whereas monolayers were apically incubatedfor 48 h in their corresponding media deprived of FBS or BSA.Filter-attached cell layers were assayed for electrophysiologicalparameters with a Millicell-ERS (Millipore).

Cell surface radioiodination and immunoprecipitation

Cell monolayers were radioiodinated with 125I as described byGoding [21]. CEA, transferrin and insulin receptors were immuno-precipitated as described [14] by anti-CEA mab, and anti-transferrin and anti-insulin receptor mabs, respectively. Antigenswere thereafter detected by SDS-PAGE followed by autoradiogra-phy. To reach and radioiodinate the antigens of the membrane ba-solateral domain of differentiated cells, we incubated cell culturesfor 2 h in Ca2+-free medium at 37°C before membrane radioiodina-tion.

Results

Cell growth studies

For all Gal-media, HT29-D4 cell growth (Fig. 1) wasclearly different from that for standard medium. Cellgeneration time did not differ for the Gal-media (58 h,56 h, 59 h, 56 h, and 60 h for 10%, 5%, 2.5%, 1% FBS,and for IT3FB1 conditions, respectively), but was differ-ent from standard medium (48 h). The total cell numberafter exponential growth was significantly lower in Gal-media and for IT3FB1 conditions than in standard medi-um; this feature is accounted for by the cell multilayersobserved in standard medium cell cultures (Fig. 4a),which contrasts with the monolayer observed in Gal-me-dium (Fig. 4b–e).

Microscopic observations

Effects of differentiating medium (Gal-medium)supplemented with FBS on HT29-D4 cells

At confluence, the cell culture appeared disorganized instandard medium (Fig. 2a), as compared to cells cultured

in 10% FBS Gal-medium (Fig. 2b), and was undifferen-tiated and grew in multilayers (Fig. 4a). Whatever theFBS concentrations (Fig. 2b–d), cells cultured in differ-entiating medium displayed flat foci that have been de-

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Fig. 1 Growth curves of HT29-D4 cells. HT29-D4 cells wereseeded in 24× multidish plates (3×104 cells/cm2). Cells weregrown in standard medium (black square), and in differentiatingmedium supplemented with 10% fetal bovine serum (FBS; emptycircle) and 1% FBS (empty square), and with epithelial growthfactor (EGF), transferrin, insulin, triiodothyronine (T3), fibronec-tin and BSAL+ (IT3FB1; black circle). For each time, cells werecounted in four wells. Experiments were conducted in triplicate.For more details, see legend of Fig. 5&/fig.c:

Fig. 2a–d Phase contrast micrographs of HT29-D4 cells at con-fluence (early differentiation phase). HT29-D4 cells were seeded 8days before micrography at a density of 4×104 cells/cm2 and cul-tured in standard medium (a). Cells were seeded 10 days beforemicrography at a density of 8×104 cells/cm2 and cultured in differ-entiating medium supplemented with 10% FBS (b), 1% FBS (c),and 0.5% FBS+T3 (1.5 nM; d). For 1% FBS (c) and 0.5%FBS+T3 (1.5 nM; d) cultures, cells were plated on a collagen-coated substrate (50 µg/ml). Note that the cell cultures were in“flat foci state” (early differentiation state). Experiments wereconducted in triplicate (bar 10 µm)&/fig.c:

scribed [4]. Flat foci correspond to cell cultures or partof cell cultures in which cells become flattened, thus re-quiring a different microscopic focus. Also their plasmaand intracellular membranes appear very thin. These as-pects correspond to the early phase of cell differentiation(approximately 10 days after seeding). At late conflu-ence (Fig. 3), cells became polygonal and their plasmamembranes refringent. This refringency is due to the di-lated intercellular spaces, and this cell appearance inlight microscopy corresponds to the late phase of celldifferentiation (high level of ultrastructure differentia-tion). This cell organization was conserved with0.5%+T3 (Fig. 3c). Whatever the FBS concentrations,cells formed domes (Fig. 3c). The ultrastructure ofHT29-D4 cells subcultured in Gal-medium displayed anenterocyte morphology (Fig. 4b–d) except for 0.1%FBS+T3 conditions.

Effects of differentiating medium supplemented withinsulin, T3, fibronectin, and BSAL+ on HT29-D4 cells(Table 1)

Whatever the conditions, attempts to culture HT29-D4cells in serum-free medium without collagen coatingwere unproductive in differentiating medium, whereas

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Fig. 4a–e Transmission electron microscopy micrographs ofHT29-D4 cells cultured in standard medium and with decreasingserum concentrations in differentiating medium. HT29-D4 cellswere plated at a density of 4×104 cells/cm2 8 days before microg-raphy in standard medium (a) or plated at a density of 8×104

cells/cm2 20 days before micrography in differentiating mediumsupplemented with 10% FBS (b), 1% FBS (c), 0.5% FBS+T3 (d)and 0.1% FBS+T3 (e). The ultrastructure of HT29-D4 cells sub-cultured in Gal-medium displays an enterocyte morphology exceptfor cells subcultured in 0.1% FBS+T3 for which the ratioheight/width was low. However, a more differentiated pattern thanwith the other FBS concentrations was observed for cells culturedwith 0.5% FBS+T3. Note that the cells were at late confluence(late differentiation phase). Experiments were conducted threetimes. Data summarize the observations relative to two to threehistological preparations originating from different sites of a T25flask cell culture (bb brush border, arrow tight junction, arrow-headmicrovilli, is intercellular space. Bar 3 µm)&/fig.c:

Fig. 3a–d Phase contrast micrographs of HT29-D4 cells at lateconfluence (late differentiation phase). HT29-D4 cells were cul-tured in differentiating medium supplemented with 10% FBS (a),1% FBS (b), 0.5% FBS+T3 (c), and 0.1% FBS+T3 (d). T3 wasused at a concentration of 1.5 nM. Cells were seeded at a densityof 8×104 cells/cm2, 20 days before micrography. For 1% FBS (b),0.5% FBS+T3 (c), and 0.1% FBS+T3 (d) cell cultures, cells wereplated on a collagen-coated substrate (50 µg/ml). Note that thecells were polyhedric or displayed fibroblastic morphology andthat the plasma membranes were refringent. Domes (arrows) wereobserved (c); focus was set on the adherent cell culture and not onthe dome. These features correspond to the late differentiationstate. Experiments were conducted in triplicate (bar 10 µm)&/fig.c:

cells adhered onto fibronectin and thereafter quicklydied. Two days after plating, cells cultured in differenti-ating medium with ET or hydrocortisone-ET had poorlyattached to collagen-coated substrate and then died,whereas T3-ET, insulin-ET, fibronectin-ET, BSAL+-ET,and IT3FB1 minus fibronectin stimulated cells to attachand spread out moderately onto collagen-coated sub-strate. During the same time IT3FB1 and IT3FB2 strong-ly induced cell adhesion and spreading.

When cultured 20 days (long term study) with T3-ETor insulin-ET (Fig. 5a,b), HT29-D4 cells showed the dif-ferentiated aspect “flat foci” (early phase of differentia-tion) but never reached the full differentiation state andcomplete confluence. Moreover, T3-ET and insulin-ET(Fig. 6a,b) induced tight junctions and disorganizedbrush border and caused cells to grow as monolayers.

Fibronectin-ET and BSAL+-ET promoted partial cellgrowth (Fig. 5c,d), and fibronectin-ET led to a slight dif-ferentiation (Fig. 6c). Similar results were obtained forcells stimulated by BSAL+-ET. IT3FB1 without fibronec-tin (Fig. 5e) did not allow optimal cell growth but in-duced the flat foci pattern observed in the predifferentia-tion state (early phase of differentiation). Note that inthis condition (Fig. 5e), the differentiation state, similarto that for FBS Gal-media (Fig. 2b–d), was higher thanas described above (Fig. 5a–d).

On the other hand, when fibronectin was present as inthe IT3FB1 condition (Fig. 5f), HT29-D4 cells achieved apattern characterized by refringent plasma membranes,numerous polyhedric cells, and presence of domes. At theultrastructural level, IT3FB1 factors, either on collagen-coated substrate (Fig. 7a,c,e–h) or on collagen-coated Nu-clepore filter (Fig. 7b,d), caused cells to strongly differen-tiate. These characteristics, considered together with lightmicroscopy observations, show that IT3FB1 factors sus-tained a high level of epithelial differentiation and thatthey correspond to those in typical absorptive intestinalcells [42]. Finally, IT3FB2 factors in Gal+DMEM/Ham’sF12 medium (Figs. 5g, 6d) caused HT29-D4 cells togrow in multilayers and undifferentiated.

Indirect immunofluorescence studies

Undifferentiated HT29-D4 cells grown in standard medi-um displayed minute anti-CEA immunofluorescencestaining distributed on the whole plasma membrane(Fig. 8a). The preponderant CEA labeling of apicalmembrane in HT29-D4 cells cultured in FBS differenti-ating medium (Fig. 8b,c) or IT3FB1 factors (Fig. 8d)proves there was a selective CEA targeting to this mem-brane domain. IT3FB1 factors as well as dialyzed FBStriggered the expression of different surface antigens onplasma membrane. β1 integrins appeared clustered assmall patches of fluorescence, called point contacts andconsidered as functional adhesion sites in neuronal cells([3]; Fig. 9c,d), whereas αvβ6 integrins heterogeneouslyclustered as large patches, called focal contacts, andpoint contacts (Fig. 9a,b). β1 subunit staining likely cor-

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Fig. 5a–h Phase contrast micrographs of HT29-D4 cells stimu-lated with insulin, T3, fibronectin, and BSAL+. HT29-D4 cellswere plated in differentiating medium supplemented with EGF(5.10–3 µg/ml) and transferrin (5 µg/ml) at a density of 8×104

cells/cm2, 20 days before micrography on a collagen-coatedsubstrate and stimulated with T3 (1.5 nM, T3-ET; a), insulin(5 µg/ml, insulin-ET; b), fibronectin (10 µg/ml, fibronectin-ET; c), BSAL+ (2 mg/ml, BSAL+-ET; d), T3+insulin+BSAL+(IT3FB1 minus fibronectin, same concentrations as above; e),insulin+T3+fibronectin+BSAL+ (same concentrations as above:IT3FB1; f), insulin+T3+fibronectin+BSAL– (same concentra-tions as above)+ linoleic acid (5 µg/ml)+oleic acid (5 µg/ml;IT3FB2; g), and 1% FBS supplemented with IT3FB1 factors (h) atthe same concentrations as in f for 2 days. The latter cell cultureconditions corresponded to the starting point of the IT3FB1 cellculture. Note that flat foci (early differentiation phase) were ob-served (a, b, e) and that complete differentiation state was ob-served (late phase of differentiation; f, h). Micrographs a–e arerelated to cell colonies (105 to 3×105 cells). The experiments wereconducted three times (bar 10 µm)&/fig.c:

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Fig. 6a–d Transmission electronmicroscopy micrographs ofHT29-D4 cells stimulated withinsulin, T3, fibronectin, orBSAL+. HT29-D4 cells wereplated in differentiating mediumon a collagen-coated substrate ata density of 8×104 cells/cm2 20days before micrography, in thepresence of T3-ET (a), insulin-ET (b), fibronectin-ET (c), andIT3FB2 (d). Note that the celllayer in contact with the mediumshowed some differentiation fea-tures (c), i.e., disorganized brushborders and tight junctions, andthat the cells grew in multilayers;numerous intracellular cystswere observed (d) with a densebrush border facing the lumina,showing an imperfect state ofdifferentiation, the enterocytedifferentiation state being revert-ed in cells cultured with IT3FB2factors. Thus Gal+DMEM/Ham’sF12 medium in the presence ofIT3FB2 factors made it possibleto induce cell adhesion, spread-ing, and growth but was not ap-propriate to differentiate HT29-D4 cells. Data summarize theobservations relative to two tothree histological preparationsoriginating from different sites ofa T25 flask cell culture (bbbrushborder, arrow tight junction, ar-rowheadmicrovilli, star intracel-lular cysts, is intercellular space).For more details see legend ofFig. 5 (bar 3 µm)&/fig.c:

Table 1 Potentialities of HT29-D4 cells to grow and differentiatein different media. These data represent qualitative and semi-quan-titative evaluation of the different potentialities of HT29-D4 cellsto grow and differentiate. All factors were assayed on collagen-coated substrate except for ET-collagen-. E : EGF ; T : transferrin ;I : insulin ; T3 : triiodothyronine ; F : fibronectin ; B : BSAL+.Whatever the experimental conditions, 2×104 cells per well wereseeded in 24 x multidish plates. Cell adhesion, initial growth andgrowth were respectively investigated after 5 h, 3 days and 8 daysby cell counting. Differentiation state was assessed by light andelectron microscopy observations [(i) presence or not of flat foci or

refringent plasma membranes, (ii) cell monolayer, (iii) tight junc-tions, (iv) microvilli], immunofluorescence, electrophysiologicaland biochemical analysis. Cell countings were done in four fieldsof light microscopy (x 200) on an inverted Olympus microscopeIMT2 and expressed in cell number per 10–2 cm2. For cell adhesion: +, ++, +++ correspond respectively to 20 to 50, 50 to 80, and 80to 100 cells per 10–2 cm2 ; for initial cell growth : ++, +++, ++++correspond respectively to 100 to 150, 150 to 200, and 200 to 400cells per 10–2 cm2 ; for cell growth : ++, +++, ++++ correspond re-spectively to 200 to 400, 400 to 600, and 2000 to 2500 cells per10–2 cm2. For more details, see legend of figure 5&/tbl.c:&tbl.b:

Cell adhesion Initial growth Growth Differentiation

ET collagen– no no no noET collagen+ + no no noHydrocortisone-ET + no no noBSAL+-ET ++ ++ ++ +Fibronectin-ETT3-ET, ++ ++ ++ ++Insulin-ETIT3FB1 fibronectin–

IT3FB2 +++ +++ +++ +IT3FB1 +++ +++ +++ +++FBS Gal- +++ +++ +++ +++mediumStandard +++ ++++ ++++ nomedium

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Fig. 7a–h Transmission electron microscopy micrographs ofHT29-D4 cells stimulated with insulin, T3, fibronectin, andBSAL+. HT29-D4 cells were plated in differentiating mediumwith EGF and transferrin 20 days before micrography in the pres-ence of insulin, T3, fibronectin, and BSAL+ (IT3FB1) at a densityof 8×104 cells/cm2 on a collagen-coated substrate (a, c, e–h) or ona collagen-coated Nuclepore filter in Transwell Costar culturechamber (b, d). Brush borders were developed as in cells subcul-tured in FBS Gal-media, and tight junctions were present. Mito-chondria and lysosomes were normally developed. Desmosomesand intercellular spaces were present. Moreover, coated vesiclesand pits were observed at the basolateral membrane (e–h). Notethat complete differentiation state was reached (late differentiationphase). Data summarize the observations relative to two to threehistological preparations originating from different sites of a T25flask cell culture details see legend of Fig. 5 (c, d bb brush border,arrowhead microvilli, fine arrow desmosome, thick arrow tightjunctions, is intercellular space, e–h arrow coated pit, star coatedvesicle. Bars a, b 6 µm, c, d 2 µm, e–h 0.5 µm). For more detailssee legend of Fig. 5&/fig.c:

responds to α2β1 and α3β1 integrins, considering thatHT29-D4 cells express these integrins (see below presentdata), and do not express α1β1, α5β1, and α6β1 (unpub-lished results). Finally, when HT29-D4 cells were stimu-lated by IT3FB1 factors or dialyzed FBS, FAK(Fig. 9e,f), vinculin, and talin (Fig. 10a–d) were ob-served as typical spear-shaped focal contacts [9]. Actinlabeling (Fig. 10e,f) showed F-actin organization instress fibers converging in focal contacts where αvβ6,

FAK, vinculin, and talin were localized. For the threetypes of integrins and other cytoskeleton-related anti-gens, the staining (not shown here) in T3-ET, insulin-ETand IT3FB1 minus fibronectin was similar to that ofIT3FB1 conditions although less strong. In contrast, in fi-bronectin-ET and BSAL+-ET conditions, α2β1, α3β1,and αvβ6 staining was observed on the whole plasmamembrane. In the latter conditions, CEA was expressedmostly peripherally and slightly apically, whereas it wasthe inverse situation for the other conditions. These dis-crepancies are likely caused by the different level of celldifferentiation.

Flow cytometry studies

Flow cytometry analysis of HT29-D4 cells with mabsanti-α2β1, α3β1, and αvβ6 integrins showed no statisti-cal differences of integrin expression on the plasmamembrane of cells subcultured in FBS Gal-medium andin serum-free medium (Fig. 11).

Leak-proof monolayers and electrophysiological studies

Cell culture tightness was first assessed 8 days afterseeding on Nuclepore membranes. Serum BSA and se-rum-free medium BSA were detected before confluence

in the apical medium for cells grown, respectively, instandard, and in FBS Gal-medium and IT3FB1 condi-tions. BSA was no longer detected in apical chambers ofcells cultured in Gal-media later after confluence (20days after seeding) but it was detected in standard medi-um. These results proved the tightness of HT29-D4 cellsgrown in Gal-medium and with IT3FB1 factors. This fea-ture matched the presence of cell domes in confluent cellcultures and the morphological differentiation exhibitingapical and basolateral membrane domains delimited bytight junctions (Fig. 7d).

Moreover, the transepithelial resistance values forGal-medium (166±21 ohms/cm2, SEM, n=5) and withIT3FB1 factors (155±25 ohms/cm2, SEM, n=5) were sig-nificantly higher than the values for cells cultured instandard medium (27±2 ohms/cm2, SEM, n=5), thus in-dicating that the occluding junctions were functional.Hence IT3FB1 factors were able to induce electrophysio-logical characteristics of an enterocyte.

Cell surface radioiodinationand immunoprecipitation studies

The pattern of radioactive polypeptides (Fig. 12a, lanes1–3) showed a shift toward the high molecular weightsrelated to the apical membrane proteins of cells grown inGal-medium (lane 2) and with IT3FB1 factors (lane 3).The 180 kDa polypeptide (Fig. 12b) corresponding to theCEA [22] molecule was immunoprecipitated from cellsgrown in standard medium (lane 1), in Gal-medium (lane

312

Fig. 8a–d Indirect immunofluorescence staining of the carcino-embryonic antigen (CEA) in HT29-D4 cells. HT29-D4 cells wereseeded at a density of 4×104 cells/cm2 to be cultured in standardmedium and investigated 8 days after seeding (a). Cells were seed-ed 20 days before analysis at a density of 8×104 cells/cm2 in dif-ferentiating medium supplemented with 10% FBS (b), with 1%FBS (c) and with insulin, T3, fibronectin, and BSAL+ (IT3FB1)(d). Substrate was coated with collagen I (c, d). For more detailssee legend of Fig. 5. Note that some cell clusters did not expressCEA in Gal-medium whatever the cell culture conditions. Nostaining was observed in control experiments performed withoutprimary antibody. (a arrowsperipheral staining, b–d arrowsapicalstaining. Bar 10 µm)&/fig.c:

Fig. 9a–f Indirect immunofluorescence staining of β1, αvβ6, andfocal adhesion kinase (FAK) in HT29-D4 cells. Cells were seededat a density of 8×104 cells/cm2 in differentiating medium supple-mented with 10% FBS (a, c, e) or with insulin, T3, fibronectin,and BSAL+ (IT3FB1) on collagen-coated coverslips (b, d, f) andinvestigated 15 days after seeding (late differentiation phase). Forintegrin labeling, differentiated cell cultures were preincubated 2 hat 37°C in Ca2+-free medium. Anti-human αvβ6 monoclonal anti-body (mab; a, b), anti-human β1 mab (c, d), and anti-human FAKmab (e, f) were used in these experiments to investigate the ex-pression and localization of integrins and FAK. No staining wasobserved in control experiments performed without primary anti-body. Note that for the αvβ6 and β1 labeling, focal and point con-tacts, respectively, are observed. The microscope focal point wasset on substrate surface, proving the basal localization of the anti-gens observed here. For more details, see Fig. 5 (arrows fluores-cence patches corresponding to the different antigens. Bar 5 µm)&/fig.c:

2) and with IT3FB1 factors (lane 3). Transferrin [49] andinsulin [50] receptors were evidenced in HT29-D4 cellsgrown in standard medium (Fig. 13, lanes 1). These re-ceptors were expressed at the basolateral membrane incells subcultured in FBS Gal-medium (Fig. 13, lanes 3)and with IT3FB1 factors (Fig. 13, lanes 5). Togetherthese data indicate that IT3FB1, like dialyzed serum, in-duced protein cell sorting, which entails selective segre-gation of proteins known to be apical and basolateral.

The electrophysiological results together with immuno-precipitation and immunofluorescence findings showedthat the morphological differentiation of IT3FB1-stimulat-ed HT29-D4 cell agrees with a marked change in the pro-tein targeting to the two plasma membrane domains andthus proved the true functionality of these monolayers.

Discussion

Growth and differentiating factors for HT29-D4 clone

Cell culture strategy

Our goal was to determine which physiological factorsand ECM proteins used individually or in associationcould induce highly differentiated HT29-D4 cells. Pre-liminary studies showed that the HT29-D4 clone doesnot differentiate in high glucose serum-free medium

whatever the physiological factors assayed (hormones,growth factors, ECM constituents) although it does dif-ferentiate with suramin (an anti-trypanosomiasis; [16]).The strategy was thus to assay factors on potentially dif-ferentiated cells, i.e. pretreated by differentiating medi-um (glucose-free DMEM supplemented with galactoseand FBS; [12]). Thus, first, we determined the minimalserum requirement that allows HT29-D4 cell differentia-tion. In fact, with very low FBS concentrations on colla-gen-coated substrate, the differentiation state wasreached, as shown by light and electron microscopyanalysis and by CEA apical expression, which is knownto be associated with a more differentiated phenotype[10, 14].

313

Fig. 10a–f Indirect immunofluorescence staining of vinculin andtalin, and phalloidin-TRITC actin-labeling in HT29-D4 cells.Cells were seeded at a density of 8×104 cells/cm2 in differentiatingmedium supplemented with 10% FBS (a, c, e) or with insulin, T3,fibronectin, and BSAL+ (IT3FB1) on collagen-coated coverslips(b, d, f) and investigated 15 days after analysis (late differentiationphase). Anti-human vinculin mab (a, b), anti-human talin mab (c,d), and phalloidin-TRITC (e, f) were used in these experiments toinvestigate some proteins constituting focal contacts. No stainingwas observed in control experiments performed without primaryantibody. (a–d thin arrows fluorescence patches where vinculinand talin are localized (focal contacts), e, f thick arrows actinstress fibers, e, f small arrowsfocus-points of stress fibers con-verging in focal contacts). The Microscope focal point was set onsubstrate surface. For more details, see legend of Fig. 5 (bar 5 µm)&/fig.c:

Fig. 11a–f Flow cytometry analysis of HT29-D4 integrins. Cellswere seeded 20 days before analysis at a density of 8×104

cells/cm2 in differentiating medium supplemented with 10% FBS(a, c, e) or with insulin, T3, fibronectin, and BSAL+ (IT3FB1) oncollagen-coated substrate (b, d, f). After being stained with mabsanti-α2β1, anti-α3β1, and anti-αvβ6 and then with second FITCantibodies, cells were fixed with 3% paraformaldehyde. Flow cy-tometry measurements were performed on a FACScan flow cytom-eter. The logarithmic x axis represents fluorescence in arbitraryunits reflecting antigen density on the cell surface. The fluores-cence corresponding to a peak was computed as the mean value ofthe fluorescence of 10 000 cells. α2β1, α3β1, and αvβ6 integrinswere observed in a, b, in c, d, and in e, f, respectively. No statisti-cal differences (Student’s t-test) was observed in the three experi-ments, between the cells subcultured with 10% FBS Gal-mediumand IT3FB1 factors. The peak on the left in different parts indi-cates the non-specific cell labeling by the second FITC-antibody.For more details, see legend of Fig. 5&/fig.c:

Effects of factors assayed individuallyon predifferentiated HT29-D4 cells

EGF and insulin, both potent mitogenic but also differen-tiating polypeptides, are very efficient in numerous celllines [6–8, 13, 26, 29]. Moreover, brush border enzymat-ic maturation and enterocyte proliferation have beenshown to be in vivo dependent on insulin [37], EGF [36],thyroxine [37, 41], and cortisone [37, 41]. Insulin [18]and EGF [35] receptors have been evidenced in HT29cell line and in HT29-D4 cells. Moreover, the clonegrowth depends on EGF and transferrin in serum-freemedium containing a high glucose concentration [13].The two factors with or without hydrocortisone induceonly initial cell adhesion in serum-free differentiatingmedium on a collagen-coated substrate (Table 1). T3 aswell as insulin together with EGF and transferrin inducesthe early phase of differentiation process and they each

sustain limited cell growth. Moreover, T3 plays a pre-ponderant role for the same processes with FBS concen-trations as low as 0.5% and 0.1%. Our data parallel thoseof Jumarie and Malo [29, 30] and Jumarie et al. [31]showing that T3 increases the alkaline phosphatase andpeptidase activities, and the sucrase-isomaltase expres-sion of Caco-2 cells subcultured in serum-free medium.T3 could be a direct activator of mitochondrial metabo-lism [25, 53] or an indirect activator via nuclear T3 re-ceptors [55].

Fibronectin or BSAL+ together with EGF and trans-ferrin induces cell adhesion and growth, and poor entero-cytic differentiation of HT29-D4 cells in differentiatingmedium. BSAL+ is a potent lipid supplier, thus a possi-ble mitogenic factor [7], and considered a detoxifyingagent that could act in the differentiation process [7]. Weused fibronectin as a soluble factor rather than as an ad-hesion protein; it stimulated cell growth and it could in-duce a low level of differentiation. Finally, insulin, T3,fibronectin, and BSAL+ (IT3FB1) together with EGF andtransferrin are potent factors for full cell growth and dif-ferentiation of the HT29-D4 clone.

314

Fig. 12a, b Membrane protein radioiodination and CEA immuno-precipitation studies of HT29-D4 cells. HT29-D4 cells were seed-ed 8 days before analysis in 25-cm2 flasks at a density of 4×104

cells/cm2 in standard medium (a, lanes 1, 4, b, lane 1), 20 daysbefore analysis at a density of 8×104 cells/cm2 in differentiatingmedium supplemented with 10% FBS (a, lanes 2, 5, b, lane 2) andwith IT3FB1 factors on collagen-coated substrate (a, lanes 3, 6, b,lane 3). With the technique used here, the plasma membranes ac-cessible to lactoperoxydase were radioiodinated; thus when cellshave matured and not destructured tight junctions, only the apicalmembrane proteins are labeled. Only apical membrane proteinswere thus 125I labeled in lysates corresponding to lanes 2, 3, 5, 6(a) and lanes 2, 3(b), whereas all the plasma membrane proteinswere radioiodinated in lysates corresponding to lanes 1, 4(a) andto lane 1(b). a After membrane solubilization with Triton X 100,an aliquot of the 125I iodinated lysate was analyzed by SDS-PAGE(12.5%) and the dried gels were autoradiographed. CoomassieBlue staining of the corresponding lysates (lanes 4–6). b Aftermembrane solubilization with Triton X 100, lysates were incubat-ed with the mab anti-CEA and immunoprecipitated. The immuno-precipitated material was analyzed by SDS-PAGE in reducingconditions and autoradiographed, revealing a 180-kDa polypeptidecorresponding to CEA. The level of cell differentiation waschecked by light microscopy. For more details, see legend ofFig. 5&/fig.c:

Fig. 13a, b Transferrin and insulin receptor immunoprecipitationstudies. HT29-D4 cells were seeded 8 days before analysis in 25-cm2 flasks at a density of 4×104 cells/cm2 in standard medium (a,b, lanes 1), 20 days before analysis at a density of 8×104 cells/cm2

in differentiating medium supplemented with 10% FBS (a, b,lanes 2, 3) and with IT3FB1 factors on collagen-coated substrate(a, b, lanes 4, 5). Cells were incubated 2 h at 37°C in Ca2+-con-taining medium (a, b, lanes 2, 4) or in Ca2+-free medium (to de-structure tight junctions) before 125I radioiodination (a, b, lanes 3,5). After membrane solubilization with Triton X 100, 125I iodina-ted lysates were incubated either with the anti-transferrin receptormab (a) or with the anti-insulin receptor mab (b). The immunopre-cipitated material was analyzed by SDS-PAGE in reducing condi-tions and autoradiographed. See monomer and dimer (90 kDa and180 kDa, respectively; a) of transferrin receptor and α and β sub-units (135 kDa and 95 kDa, respectively; b) of insulin receptor.Level of cell differentiation was checked by light microscopy. Formore details, see legend of Fig. 5&/fig.c:&/tbl.b:

HT29-D4 cell growth:cell adhesion proteins and growth factors

HT29-D4 cell adhesion and focal contacts

The epithelial cell differentiation of intestine could beviewed in terms of embryonic differentiation or cell dif-ferentiation from the crypt to the villous tip. Induction ofenterocyte differentiation can thus be first initiated in vi-vo by either embryonic mesenchyme or underlying fibro-blasts [32, 51]. One process can act via hormones- (cor-ticoids) stimulated mesenchyme-derived cells [33] ormore directly by the basement membrane [51, 56]. Ratfetal endodermal cells display enterocyte-like differentia-tion when cultured on ECM extracted from EHS cellsbut not when cultured solely on collagen I [24]; this pro-cess being mediated by integrins directly implicated inthe recognition of specific sequences of ECM compo-nents [27]. In this view a specific pattern of integrins hasbeen characterized in normal human gut: α2β1, α3β1,α6β4, and αvβ6 [1].

Differentiated HT29-D4 cells in the presence of serumdo not need ECM component coating to adhere. Serum fi-bronectin cannot be implicated in this process since afterinitial cell adhesion onto fibronectin the cells detachedand died. One can hypothesize that serum factors stimu-late cells to synthesize and secrete some ECM compo-nent. In this view, little collagen IV mRNA was observedin rat enterocytes [56]. It must also be noted that HT29-D4 cells and Caco-2 cells [29] fully differentiate in thepresence of serum, whereas serum inhibits the differentia-tion process of HRA-19 cells [26]. PredifferentiatedHT29-D4 cells in serum-free medium require collagenbut not fibronectin for their initial adhesion; these resultsparallel those of Jumarie and Malo [29], who showedthat, in serum-free medium, Caco-2 cells need ECM con-stituents to adhere. This agrees with the fact that cloneD4 expresses on its plasma membrane α2β1 and α3β1integrins, the collagen receptors [27] and thus they can beexpected to mediate initial cell adhesion.

Moreover, in serum-free medium, HT29-D4 cellsseeded on collagen also require fibronectin for their ini-tial and late growth (late differentiation phase) since,when fibronectin was added to IT3B1 factors, cell differ-entiation and growth were completely achieved, whereasthis protein as an ECM component was inefficacious forinitial cell adhesion. These results show that fibronectin,as a soluble factor, could act as an early effector on cellgrowth and, when present with other factors, as a late ef-fector on final cell growth, which leads to complete cellconfluence. This process arises in predifferentiated cells,allowing the late phase of differentiation. Consideringthat (1) the cells express integrin αvβ6, (2) this integrinis the fibronectin receptor [2, 27, 34] and promotes cellgrowth [2], and (3) it was highly clustered (focal con-tacts) at the basolateral membrane, one can hypothesizethat integrin αvβ6 could mediate, at least partly, the fi-bronectin effect. However, only αvβ6 blockade by a spe-cific antibody or antisense RNA could prove this effect.

The fibronectin receptor acts by a signaling pathway(GRB2/SOS-RAS-MAPK; [48]) via activation of FAK,which is expressed in HT29-D4 cells when stimulated byIT3FB1 factors. These processes linked to highly clus-tered integrins [57] occur in cytoplasmic microdomains,called focal contacts [9], which were evidenced in thiswork by immunolocalizing αvβ6, vinculin, talin, andFAK, and toward which actin stress fibers converged.

EGF, insulin, and glucose metabolism in HT29-D4 cells

Tumor cells [54] or transfected cells [45, 52] have a highrate of glycolysis. This feature and glycogen accumula-tion occur simultaneously in colon cancerous cells [44,46, 47, 58]. A high rate of glycolysis has also been foundin undifferentiated HT29-D4 cells cultured with a highglucose concentration [15]. Glucose uptake, glycolysis,and glycogen synthase activity are known to be regulatedby insulin and EGF [11, 28] and are associated withgrowth factor mitogenic effects. For HT29-D4 cells sub-cultured in differentiating medium, glucose was replacedby galactose, ruling out that the mitogenic effect could belinked to a high glycolysis rate although basal lactate pro-duction has been evidenced in HT29 cell line subculturedin glucose-free medium supplemented with galactose[19]. However, attempts to culture HT29-D4 cells withIT3FB1 factors without EGF and/or insulin in glucose-free medium were unproductive. It remains likely that en-terocyte differentiation of HT29-D4 cells, which parallelsthe apical hydrolases maturation [44, 58], is tightly linkedto a shift in sugar metabolism, i.e., a decrease in glycoly-sis, as observed by Zweibaum et al. [58]. Indeed theyshowed that when glucose is added again to a long termglucose-free HT29 cell culture, glycolysis is decreasedand concomitantly the cell glycogen amount is increased,which can be explained by a glucose 6-phosphate alloste-ric activation of glycogen synthase [47].

The more recent concept of the EGF- or insulin-acti-vated tyrosine kinase receptors linked to the GRB2/SOS-RAS-MAP kinase pathway [39] could explain why thegrowth factor mitogenic effect seems independent ofsugar metabolism in HT29-D4 cells subcultured in se-rum-free medium. Moreover, through a specific pathway,EGF (by kinase C activation via PLC γ1; [40]) and insu-lin (by PI3K activation via tyrosine phosphorylation ofIRS-1; [5]), can each be differently implicated in the mi-togenic signal.

For the HT29-D4 clone, the control of the cell adhe-sion, growth, and differentiation processes in serum-freemedium was multifactorial, i.e., the ECM component(collagen) promoted initial cell adhesion; together withEGF and transferrin, T3 as well as insulin induced limit-ed cell growth and the early phase of differentiation; fi-bronectin seems to be involved as a soluble factor in thecell growth during the late phase of differentiation. Infact, we found that it is only when EGF, transferrin, insu-lin, T3, and fibronectin act together that full differentia-tion is reached. We conclude that for enterocyte differen-

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tiation with respect to HT29-D4 cells, a convergent sig-nal from growth factors, hormones, and ECM proteinsfunctions with a necessary shift in glucose metabolism.This multifactorial control promotes (1) satisfactory cellgrowth, (2) an enterocyte morphological pattern withwell targeted apical (CEA) and basolateral (insulin andtransferrin receptors and integrins) antigens, (3) thetranslocation of cytoskeletal proteins (vinculin, talin,FAK) with the convergence of stress fibers to adhesionplaques, and (4) epithelial electrophysiological featuresproving the functional integrity of tight junctions. Ourdata in serum-free medium indicate that HT29-D4 cloneis a suitable model to study the implication of integrinsin the differentiation process of human enterocytes with-out the multiple factors present in serum.

&p.2:Acknowledgements We thank F. Gianellini and J. Amic for ex-cellent technical assistance and J.J. Roccabianca and H. Bouteillefor micrographs. We acknowledge Dr. J. Fantini for his helpfulcriticism. This work was supported by grants from the Associationpour la Recherche sur le Cancer contract 6534 and contract 1323.

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