Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

doi: 10.1152/ajpgi.00295.2007293:G1178-G1189, 2007. First published 4 October 2007;Am J Physiol Gastrointest Liver Physiol

Edgard Delvin, Alain Sane, Eric Tremblay, Carole Garofalo and Emile LevyEmilie Grenier, Françoise Schwalm Maupas, Jean-François Beaulieu, Ernest Seidman,secretion, and apolipoprotein biogenesisdifferentiation as well as on lipid synthesis, lipoprotein Effect of retinoic acid on cell proliferation and

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Effect of retinoic acid on cell proliferation and differentiation as well ason lipid synthesis, lipoprotein secretion, and apolipoprotein biogenesis

Emilie Grenier,1 Francoise Schwalm Maupas,1 Jean-Francois Beaulieu,2 Ernest Seidman,2,3

Edgard Delvin,4 Alain Sane,1 Eric Tremblay,2 Carole Garofalo,1 and Emile Levy1,2

Departments of 1Nutrition and 4Biochemistry, CHU Sainte-Justine, Universite de Montreal, Montreal; 2Group on theIntestinal Epithelium, Canadian Institute of Health Research and Department of Cellular Biology, Faculty of Medicine,Universite de Sherbrooke, Sherbrooke; and 3Research Institute, McGill University, Montreal, Quebec, Canada

Submitted 28 June 2007; accepted in final form 2 October 2007

Grenier E, Maupas FS, Beaulieu J-F, Seidman E, Delvin E,Sane A, Tremblay E, Garofalo C, Levy E. Effect of retinoic acid oncell proliferation and differentiation as well as on lipid synthesis,lipoprotein secretion, and apolipoprotein biogenesis. Am J PhysiolGastrointest Liver Physiol 293: G1178–G1189, 2007. First publishedOctober 4, 2007; doi:10.1152/ajpgi.00295.2007.—Dietary vitamin Aand its active metabolites are essential nutrients for many functions aswell as potent regulators of gene transcription and growth. Althoughthe epithelium of the small intestine is characterized by rapid andconstant renewal and enterocytes play a central role in the absorptionand metabolism of alimentary retinol, very little is known about thefunction of retinoids in the human gastrointestinal epithelium andmechanisms by which programs engage the cell cycle are poorlyunderstood. We have assessed the effects of 10 �M 9- and 13-cis-retinoic acid (RA) on proliferation and differentiation processes, lipidesterification, apolipoprotein (apo) biogenesis and lipoprotein secre-tion along with nuclear factor gene transcription. Treatment of Caco-2cells with RA at different concentrations and incubation periodsrevealed the reduction of thymidine incorporation in 60% preconflu-ent or 100% confluent cells. Concomitantly, RA 1) modulated D-typecyclins by reducing the mitogen-sensitive cyclin D1 and upregulatingcyclin D3 expressions and 2) caused a trend of increase in p38 MAPK,which triggers CDX2, a central protein in cell differentiation. RAremained without effect on lipoprotein output and apo synthesis, evenfor apo A-I that possesses RARE in its promoter. RA, in combinationwith 22-hydroxycholesterol, could induce apo A-I gene expressionwithout any impact on apo A-I mass. Only the gene expression ofperoxisome proliferator-activated receptor (PPAR)�, retinoic receptor(RAR)�, and RAR� was augmented and no alteration was noted inPPAR�, PPAR�, liver X receptor (LXR)�, LXR�, and retinoid Xreceptors. Taken together, these data highlight RA-induced cell dif-ferentiation via specific signaling without a significant impact on apoA-I synthesis.

retinoids; apolipoproteins; lipoproteins; nuclear factors; lipids

RETINOIC ACID (RA), a low-molecular-weight lipophilic metab-olite of vitamin A or retinol, is the most potent natural retinoidable to influence biological processes, such as cell growth anddifferentiation of various epithelia in the body (17, 51). RAacts by binding to retinoic receptors (RAR)�, -�, -�, whichform heterodimers with retinoid X receptors (RXR-�, -�, -�),thereby inducing transcription of a wide array of specific targetgenes (41). Synthesis and degradation of RAR and RXR byproteasomes and phosphorylations control amount and timingof the retinoid responses. During development, absence of

vitamin A can lead to an uncontrolled proliferation of epithelialstem cells that fail to differentiate into the normal phenotype inmany lining epithelia (59). Although the small intestine is thefirst gateway for contact with dietary retinol as well as theunique organ for its absorption and metabolism, limited infor-mation is available as to the effects of RA on the mechanismsgoverning functional maturation.

Earlier studies established that mammalian apolipoprotein(apo) A-I is synthesized principally in the small intestine inaddition to liver (15, 76). Apo A-I represents the major pro-tein component of high-density lipoprotein (HDL) particlesand has been attributed antiatherogenic properties in view of itspotential to initiate the removal of excess cholesterol fromperipheral tissues by enhancing cholesterol efflux and deliver-ing to the liver through the reverse cholesterol transport path-way (16, 33). Apo A-I exhibits supplementary functions, whichinclude the protection against oxidative stress, inflammation,endothelium vasoconstriction, and thrombosis (3, 52, 55, 58).Therefore, multiple efforts have been exerted to find outnutrients and chemical agents that raise the levels of apo A-I,which may provide new therapeutic options for the preventionof atherosclerotic cardiovascular disease. There have beenseveral tantalizing clues that revealed diverse influences on apoA-I expression in liver vs. intestine in response to develop-mental, dietary, hormonal, toxic, and pharmacological stimuli(23, 50, 54, 64, 67). Retinoid status seems to regulate hepaticapo A-I gene expression. The administration of vitamin A torats lowered hepatic apo A-I mRNA (78), and treatment of rathepatocytes with RA also resulted in a decrease in apo A-Igene expression (5). These observations indicate that RAsuppressed the expression of apo A-I in rats. In contrast, astimulatory effect on apo A-I synthesis was noted in hepatomacell lines (6) and in primary cultures from cynomolgus monkey(30). The reason for the discrepancy among studies is not fullyclear. Inconsistent findings were also reported in intestinal cells(22, 71), which prompted us to investigate the regulation of apoA-I by RA in Caco-2 cell line, an interesting model frequentlyused to study lipid transport and lipoprotein assembly (37). Aconsiderable body of evidence supports the concept that intes-tinal cell differentiation is an obligatory prerequisite for apobiogenesis. Studies using pulse-labeling experiments in iso-lated rat enterocytes (11) and dot-blot hybridization analysis ofrat mucosal scrapings (13) showed that apo synthesis wasprimarily localized in villus-associated enterocytes. Further-

Address for reprint requests and other correspondence: E. Levy, ResearchCentre, Gastroenterology, Hepatology and Nutrition Unit, CHU-Sainte-Jus-tine, 3175 Cote Ste-Catherine, Montreal, Quebec, Canada H3T 1C5 (e-mail:[email protected]).

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

Am J Physiol Gastrointest Liver Physiol 293: G1178–G1189, 2007.First published October 4, 2007; doi:10.1152/ajpgi.00295.2007.

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more, rat intestinal cell line IEC-6, which retains the charac-teristics of normal rat crypt jejunal cells (57), was unable tosynthesize apos and lipoproteins (13, 45). We have, therefore,surmised that RA-mediated cell differentiation might promoteapo synthesis. An additional aim of the present work was todetermine the expression profile of various nuclear factorsrepresenting transacting proteins known to bind to the RAREthat has been identified in the promoter region of the apo A-Igene promoter (60).

MATERIALS AND METHODS

Cell culture. Caco-2 cells (American Type Culture Collection,Rockville, MD) were grown at 37°C with 5% CO2 in minimalessential medium (MEM) (GIBCO-BRL, Grand Island, NY) contain-ing 1% penicillin-streptomycin and 1% MEM nonessential aminoacids (GIBCO-BRL) and supplemented with 10% decomplementedfetal bovine serum (FBS) (Flow, McLean, VA). Caco-2 cells (pas-sages 30–40) were maintained in T-75-cm2 flasks (Corning GlassWorks, Corning, NY). Cultures were split (1:6) when they reached70–90% confluence, by use of 0.05% trypsin-0.5 mM EDTA(GIBCO-BRL). For individual experiments, cells were plated at adensity of 1 � 106 cells/well on 24.5-mm polycarbonate Transwellfilter inserts with 0.4-�m pores (Costar, Cambridge, MA), in MEM(as described above) supplemented with 5% FBS. The inserts wereplaced into six-well culture plates, permitting separate access to theupper and lower compartments of the monolayers. Cells were culturedfor various periods, including 21 days, at which the Caco-2 cells arehighly differentiated and appropriate for lipid synthesis (44, 46). Themedium was refreshed every second day.

9-cis RA was then added to cells at different concentrations for24 h. Sucrase activity was measured as a marker of cell differentiationand transepithelial resistance as a marker of monolayer integrity(Millipore, Bedford, MA) (44, 46). Cell viability was assessed byTrypan blue exclusion (46). Furthermore, DNA and protein contentwas evaluated as previously described (46). All these parameters weretested in the presence or absence of RA. Since no differences werenoted between different RA isoforms, we have decided to use only9-cis RA at a concentration of 10 �M that was found optimal forvarious biological effects (data not shown).

Cell proliferation analysis by [3H]thymidine incorporation. Theeffect of RA on Caco-2 cell proliferative activity was determined byomitting FBS for 18 h of incubation. Thereafter, Caco-2 cells wereincubated with or without RA, in the presence of unlabeled oleic acid(as above) for 4 h. [3H]thymidine (10 �Ci; sp act, 20 �Ci/�mol; DuPont, Markham, ON, Canada) was added to each well. Cells wereincubated for 4 h, washed with MEM (serum free), and then harvestedwith a rubber policeman. Cells were sonicated and the incorporationof [3H]thymidine into DNA was measured in homogenates(counts �min�1 �DNA�1). DNA was quantified by a microfluorometricmethod.

Measurement of lipid synthesis and secretion. Lipid synthesis andsecretion were assayed as previously described (38, 46, 65). Briefly,radiolabeled [14C]oleic acid (sp act, 53 mCi/mmol; Amersham,Oakville, ON, Canada) was added to unlabeled oleic acid and thensolubilized in fatty acid-free bovine serum albumin (BSA) [BSA/oleicacid, 1:5 (mol:mol)]. The final oleic acid concentration was 0.7 mM(0.45 �Ci)/well. Cells were first washed with phosphate-bufferedsaline (PBS) (GIBCO), and the [14C]oleic acid-containing mediumwas added to the upper compartment. RA was added to the upperchamber in serum-free MEM. At the end of a 24-h incubation period,cells were washed and then scraped with a rubber policeman in a PBSsolution containing antiproteases (phenylmethylsulfonyl fluoride,pepstatin, EDTA, aminocaproic acid, chloramphenicol, leupeptin,glutathione, benzamidine, dithiothreitol, sodium azide, and Trasylol,all at a final concentration of 1 mM). An aliquot was taken for lipid

extraction by standard methods (38, 46, 65) in the presence ofunlabeled carrier [phospholipids (PL), monoglycerides, diglycerides,triglycerides (TG), free fatty acids, free cholesterol, and cholesterylester (CE)].

The various lipid classes synthesized from [14C]oleic acid werethen separated by thin-layer chromatography (TLC) using the solventmixture of hexane, ether, and acetic acid (80:20:3, vol/vol/vol), aspreviously described (38, 46, 65). The area corresponding to eachlipid was scratched off the TLC plates, and the silica powder wasplaced in a scintillation vial with Ready Safe counting fluid (Beck-man, Fullerton, CA). Radioactivity was then measured by scintillationcounting (LS 5000 TD, Beckman). Cell protein was quantified by theBradford method and results were expressed as disintegrations perminute per milligram of cell protein. Lipid secreted in the basolateralcompartment was analyzed and quantified, as described above, aftercentrifugation (2,000 rpm for 30 min at 4°C) to remove cell debris.

Lipid carrier. Blood was drawn 2 h after the oral intake of a fatmeal by human volunteers, and postprandial plasma was prepared toserve as a carrier for the lipoproteins synthesized by Caco-2 cells asdescribed previously (38, 39).

Isolation of lipoproteins. For the determination of secreted lipopro-teins, Caco-2 cells were incubated with the lipid substrate as describedabove, in the presence or absence of RA. The medium supplementedwith antiproteases (as described above) was first mixed with a plasmalipid carrier (4:1, vol/vol) to efficiently isolate de novo lipoproteinssynthesized. The lipoproteins were then isolated by sequential ultra-centrifugation using a TL-100 ultracentrifuge (Beckman), as de-scribed previously (30). Briefly, chylomicrons (CM) were isolatedafter ultracentrifugation (20,000 rpm for 20 min). Very-low-densitylipoprotein (VLDL) (1.006 g/ml) and low-density lipoprotein (LDL)(1.063 g/ml) were separated by spinning at 100,000 g for 2.26 h witha tabletop ultracentrifuge 100.4 rotor at 4°C. The HDL fraction wasobtained by adjusting the LDL infranatant to density at 1.21 g/ml andcentrifuging for 6.5 h at 100,000 g. Each lipoprotein fraction wasexhaustively dialyzed against 0.15 M NaCl and 0.001 M EDTA, pH7.0, at 4°C for 24 h.

De novo apo synthesis. The effect of RA on newly synthesized andsecreted apos (A-I, A-IV, B-48, B-100, and E) was assessed asdescribed previously (38, 44, 46, 65). To first induce apo synthesis,cells were incubated apically with unlabeled oleic acid bound toalbumin in serum-free medium, 24 h before [35S]methionine incuba-tion. The concentration of the unlabeled lipid was equivalent to thelabeled substrate described above. During this time, RA was againadded to the apical chamber. After 24-h incubation, cells as well as theouter chambers were rinsed twice with PBS (GIBCO). The apicalcompartment was replaced with 1.5 ml of methionine-free mediumcontaining the unlabeled substrate and 100 �Ci/ml [35S]methionine(50 mCi/mmol, Amersham Life Sciences). After incubation for 4 h at37°C with 5% CO2, the medium from the basolateral compartmentwas collected. Cells were scraped off the inserts in the cell lysisbuffer, as described above. The medium and cell lysates were sup-plemented with an antiprotease cocktail. To assay a considerableamount of de novo apo synthesis, the material from two wells waspooled.

Immunoprecipitation of apo. The medium and cell lysates were firstsupplemented with unlabeled methionine to act as a carrier (finalconcentration, 0.1 mM). Immunoprecipitation was performed in thepresence of excess polyclonal antibodies to human apos (BoehringerMannheim) at 4°C overnight (38, 46). Samples were then washed withNonidet P-40 (0.05%). They were subsequently centrifuged andresuspended in sample buffer (1.2% SDS, 12% glycerol, 60 mM Tris,pH 7.3, 1.2% �-mercaptoethanol, and 0.003% bromophenol blue) andanalyzed by a linear 4–15% polyacrylamide gradient preceded by a3% stacking gel, as described previously. Radioactive molecularweight standards (Amersham Life Sciences) were run in the sameconditions. Gels were sectioned into 2-mm slices and counted after anovernight incubation with 1 ml of Beckman tissue solubilizer (0.5 N

G1179RETINOIC ACID, CELL GROWTH, AND LIPID TRANSPORT

AJP-Gastrointest Liver Physiol • VOL 293 • DECEMBER 2007 • www.ajpgi.org


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quaternary ammonium hydroxide in toluene) and 10 ml of liquidscintillation fluid (Ready Organic, Beckman). Results for each apostudied were expressed as percent disintegrations per minute per milli-gram protein to assess the specific effect of RA on apo synthesis andsecretion.

RT-PCR. PCR experiments for the various genes as well as �-actin(as a control gene) were performed by using the GeneAmp PCRSystem 9700 (Applied Biosystems) as described previously (61).Approximately 30–40 cycles of amplification were used at 95°C for30 s, 58°C for 30 s, and 72°C for 30 s. Amplicons were visualized onstandard ethidium bromide-stained agarose gels. Importantly, we haveestablished the experimental conditions relative to RT-PCR, whichcorrespond to the linear portion of the exponential phase for everygene expression.

Statistical analysis. All values were expressed as means � SE.Data were analyzed by two-tailed Student’s t-test.

RESULTS

Effect of RA on Caco-2 cell proliferation and integrity. Cellproliferation was assessed by [3H]thymidine incorporation atvarious periods of confluence. RA caused a decrease in Caco-2cell proliferation as measured by [3H]thymidine incorporation(Fig. 1, A and B). On the other hand, RA remained withoutimpact when the differentiation process was engaged, i.e., 10and 21 days postconfluence (Fig. 1, C and Fig. 1D).

Following treatment with RA, Caco-2 cell integrity wasassessed by the determination of cell monolayer transepithelialresistance and cell viability by Trypan blue exclusion. Nocytotoxic effect of RA was observed (results not shown).

Effect of RA on Caco-2 cell differentiation. Since D-typecyclins are believed to be cell cycle-promoting agents, we firststudied their protein expression in proliferative and differenti-ated states. The treatment of Caco-2 cells with RA loweredcyclin D1 and raised cyclin D3 (data not shown). Conse-quently, the cyclin D3/cyclin D1 ratio increased at 0, 10, and21 days postconfluence (Fig. 2).

Then, we examined the protein expression of phosphatidyl-inositol 3-kinase (PI3K), an enzyme required for morphologi-cal and differentiation of enterocytes. The addition of RAresulted in little effect on PI3K protein expression at 60% aswell as at 0 and 10 days postconfluence. However, RA was

able to reduce PI3K protein expression at 21 days postconflu-ence (results not shown).

p38 Mitogen-activated protein kinase (p38 MAPK) has re-cently emerged as a key modulator of various vertebrate celldifferentiation processes (26, 77). We therefore investigatedwhether it is modulated by RA. A significant raise was noted inits protein expression at 60% confluence, whereas only a trendwas noted at confluence or at 10 and 21 days postconfluence(results not shown). Additionally, we tested the influence ofRA on activated p38 MAPK by exposure of Caco-2 cells toenvironmental stress and found a fall at 10 days postconfluence(results not shown).

Finally, we sought to address the effect of RA on caudal-related homeobox 2 (CDX2), a protein capable of suppressingproliferation and promoting differentiation. Interestingly, RAsignificantly increased CDX2 protein expression at 60%(Fig. 3A) confluence and lowered it at 10 and 21 days post-confluence (Fig. 3, C and D).

Effect of RA on lipid esterification and apo synthesis inCaco-2 cells. We explored the modulation of lipid esterifica-tion and de novo apo synthesis by RA in the presence andabsence of 22-hydroxycholesterol (22-OH), a natural ligandagonist for liver X receptor (LXR), solubilized in ethanol andused at a concentration of 4 �g/ml during 6- and 24-h incuba-tion culture. No changes were observed in the esterification ofTG, PL, and CE (results not shown). For the short incubationperiod, RA was unable to influence the biogenesis of apo A-Iand apo A-IV (Fig. 4) as well as that of apo B-48, apo B-100,and apo E (Fig. 5). In contrast, 22-OH could increase thesynthesis of most of the apos. However, the addition of RA to22-OH suppressed the 22-OH-induced biogenesis.

During the long incubation period, RA only enhanced theapo A-IV synthesis whereas 22-OH was effective in activatingthe production of apos (A-I, A-IV, B-100, and E). The com-bination of RA and 22-OH did not induce any change in thelevel of synthesis of all the apos.

Effect of RA on lipoprotein secretion by Caco-2 cells. Nosubstantial variations were observed in lipoprotein output fol-lowing the addition of RA either at 4 or 24 h of incubation(results not shown).

Fig. 1. Effects of retinoic acid (RA) on Caco-2 cellline proliferation. The incorporation of [3H]thymi-dine was studied in Caco-2 cells at various condi-tions: 60% confluence (A), 0-day confluence (B), 10days postconfluence (C), 21 days postconfluence (D).Experiments were performed in duplicate and valuesare expressed as means � SE for n � 4 in each groupof experiments. *P 0.05 vs. controls.

G1180 RETINOIC ACID, CELL GROWTH, AND LIPID TRANSPORT



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Effect of RA on nuclear factors in Caco-2 cells. To approachthe mechanisms triggered by RA, we assessed the gene expres-sion of peroxisome proliferator-activated receptors (PPAR)s,LXR, RXR, and RAR in Caco-2 cells at 4 and 24 h ofincubation. RA and 22-OH did not exhibit an influence onPPAR� at the short incubation period (Fig. 6A). A significantlowering effect was recorded with 22-OH and 22-OHRA(Fig. 6B). RA elicited an enhancement in the gene expressionof PPAR� at 4 and 24 h of incubation (Fig. 6, C and D). The

combination of RA and 22-OH significantly lowered PPAR�gene expression (Fig. 6D). RA and 22-OH alone or in combi-nation reduced the PPAR� gene expression (Fig. 6, E and F).

When we tested the influence on RA and 22-OH on geneexpression of LXR� and �, we could notice only a trend ofincrease (results not shown).

We also explored the impact of RA treatment on RXRs inCaco-2 cells. We mostly noted an increase in RXR� (Fig. 7B)and RXR� (Fig. 7D) following the administration of RA to

Fig. 2. Effects of RA on cyclins D3 and D1 inCaco-2 cells. The proteins of cell homogenateswere fractionated by SDS-PAGE and electro-transferred onto nitrocellulose membranes. Theblots were then incubated with the polyclonalantibody overnight at 4°C. Immunocomplexeswere revealed by means horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin andan enhanced chemiluminescence’s kit. D1 andD3 mass were quantitated by using an HP Scanjetscanner equipped with a transparency adapterand software. Thereafter, D3-to-D1 ratio wascalculated in 4 experimental conditions: 60%confluence (A), 0-day confluence (B), 10 dayspostconfluence (C), 21 days postconfluence (D).Values are expressed as means � SE. *P 0.05vs. controls; **P 0.01 vs. controls.

Fig. 3. Effects of RA on the protein expres-sion of CDX2, a caudal-related homeoboxgene encoding an intestine-specific transcrip-tion factor in Caco-2 cells. CDX2 profile wasexamined by SDS-PAGE and Western blot-ting at various conditions: 60% confluence(A), 0-day confluence (B), 10 days postcon-fluence (C), 21 days postconfluence (D). Val-ues represent means � SE for n � 4 in eachgroup of experiments. *P 0.05 vs. respec-tive controls; **P 0.01 vs. respective con-trols.




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Caco-2 cells at 24 h of incubation. 22-OH alone or in combi-nation with RA seemed to cause a decline in their geneexpression (Fig. 7, B and D) without altering RXR� (data notshown).

We finally tackled the RAR regulation by RA and 22-OH(Fig. 8). In general, a raise of RAR�, RAR�, and RAR�resulted from the addition of RA. A trend of decrease wasapparent in the gene expression of these nuclear factors fol-

Fig. 4. Effects of RA and 22-hydroxycho-lesterol (22-OH) on the synthesis of apoli-poproteins (apo) A-I and A-IV in Caco-2cells. These epithelial cells at 21 days post-confluence were incubated with [35S]methi-onine in the presence of unlabeled oleic acidfor 4 or 24 h to stimulate the biogenesis ofapos. At the end of the labeling period, cellswere washed, homogenized, and centrifuged.Supernatants from the cell homogenateswere then reacted with excess antibodies for18 h at 4°C to precipitate specific apos.Immune complexes were washed and ana-lyzed by linear 4–20% SDS-PAGE. Afterelectrophoresis, gels were sliced and countedfor radioactivity. Data represent means � SEof 4 experiments in each group: apo A-I (4-hincubation) (A), apo A-I (24-h incubation)(B), apo A-IV (4-h incubation) (C), apo A-IV(24-h incubation) (D). Values representmeans � SE for n � 4 in each group ofexperiments and are reported as percent dif-ference relative to control values represent-ing 100%. *P 0.05 vs. controls.

Fig. 5. Effects of RA and 22-OH on the synthe-sis of apos (B-48, B-100, and E) in Caco-2 cells.These epithelial cells at 21 days postconfluencewere incubated with [35S]methionine in thepresence of unlabeled oleic acid for 4 or 24 h tostimulate the biogenesis of apos. At the end ofthe labeling period, cells were washed, homog-enized, and centrifuged. Supernatants from thecell homogenates were then reacted with excessantibodies for 18 h at 4°C to precipitate specificapos. Immune complexes were washed and an-alyzed by linear 4–20% SDS-PAGE. After elec-trophoresis, gels were sliced and counted forradioactivity. Data represent means � SE of 4experiments in each group: apo B-48 (4-h incu-bation) (A), apo B-48 (24-h incubation) (B), apoB-100 (4-h incubation) (C), apo B-100 (24-hincubation) (D), apo E (4-h incubation) (E), apoE (24-h incubation) (F). Values representmeans � SE for n � 4 in each group of exper-iments and are reported as percent differencerelative to control values representing 100%.*P 0.05 vs. controls.




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lowing the addition of 22-OH, and an enhancement wasrecorded in RAR� when RA and 22-OH were combined(Fig. 8C).

DISCUSSION

RA is known to exert pleiotropic effects given its ability tomodulate gene expression of various genes involved in growth,adaptation, and function. We have, therefore, undertaken sys-tematic studies to examine the impact of RA treatment on

intestinal epithelial cells to examine its regulatory role incellular proliferation and differentiation, as well as on lipidtransport processes and transcriptional programs elicited bynuclear factors. Our data indicate that RA acted as a differen-tiation factor since, at 60% preconfluence and at 0-day conflu-ence, it 1) lowered labeled thymidine incorporation; 2) modu-lated D-type cyclins (cell cycle regulatory proteins) by down-regulating the mitogen-sensitive cyclin D1 expression (therebyblocking DNA synthesis and cell proliferation) and upregulat-

Fig. 6. Effects of RA and 22-OH on peroxi-some proliferator-activated receptor (PPAR)gene expression in Caco-2 cells. PCR analysiswas performed on Caco-2 cells at 21 dayspostconfluence to analyze mRNA of PPAR�(4-h incubation) (A), PPAR� (24-h incubation)(B), PPAR� (4-h incubation) (C), PPAR�(24-h incubation) (D), PPAR� (4-h incuba-tion) (E), PPAR� (24-h incubation) (F). Val-ues represent means � SE for n � 4 in eachgroup of experiments and are reported as per-cent difference relative to control values rep-resenting 100%. *P 0.05 vs. control; **P 0.01 vs. control.




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ing cyclin D3 expression (thereby inducing cell differentia-tion); and 3) showed a trend of increase in p38 MAPK, whichtriggers CDX2, a central protein in cell differentiation. Despiteits capacity to diminish cell proliferation, RA was unable toincrease intestinal apo A-I gene expression, apo A-I synthesis,and HDL production. Furthermore, it remains without effect onthe esterification of TG, CE, and PL; the production of CM,VLDL, and LDL; and the biogenesis of apos (A-IV, B-48,B-100, and E). Moreover, RA augmented the gene expressionof various nuclear receptors, such as PPAR�, RAR�, andRAR�. Finally, when combined with 22-OH, RA could induceapo A-I gene expression without any impact on apo A-I mass.

Caco-2 cells, an intestinal cell line derived from a humancolorectal carcinoma that spontaneously differentiates understandard culture conditions, lends itself to the in vitro study ofhuman gut in view of its efficient intestinal transport processes(37). Their distinct cellular polarity is characterized by apicalmicrovilli with associated brush border hydrolases and baso-laterally positioned nuclei, features comparable to those ofnormal, mature intestinal absorptive cells. Caco-2 cells havethe ability to form a high transepithelial electrical resistance,indicating a functional tight junction barrier. They are amongthe few intestinal cell lines capable of synthesizing sucrase-

isomaltase, a well-characterized marker for functional differ-entiation of enterocytes. Among their multiple biological func-tions are those related to the absorption, transport, and metab-olism of lipids and lipoproteins (37). The present work hasdescribed the regulation of cell differentiation, apo andlipoprotein synthesis, and nuclear factor expression by RA.However, these findings obtained in Caco-2 cells should beinterpreted with caution since there are still different shortcom-ings intrinsic to the model, which have to be taken intoconsideration when using the Caco-2 model as a screeningtool, including the absence of mucus, the lack of cytochromeP450 enzymes, and the inability to study regional intestinaldifferences in oral absorption. Further studies are necessarybefore extrapolating the results to human intestine in vivo.

In our studies with Caco-2 cells, RA diminished cell prolif-eration and raised cell differentiation in conditions of precon-fluence and postconfluence. Similarly, a number of in vivo andin vitro studies using various experimental models have re-vealed that RA plays a key role in embryonic development, theprevention and treatment of tumors, as well as cell proliferationand differentiation (10, 12). In particular, our data are in linewith many reports suggesting that RA is more active ininhibiting proliferation and inducing differentiation in rela-

Fig. 7. Effects of RA and 22-OH on RXRgene expression in Caco-2 cells. PCR analy-sis was performed on Caco-2 cells at 21 dayspostconfluence to analyze mRNA of RXR�(4-h incubation) (A), RXR� (24-h incuba-tion) (B), RXR� (4-h incubation) (C), andRXR� (24-h incubation) (D). Values repre-sent means � SE for n � 4 in each group ofexperiments and are reported as percent dif-ference relative to control values represent-ing 100%. *P 0.05 vs. control.




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tively undifferentiated systems, i.e., murine F9 teratocarcinomacells, promiolocytic HL-60 cells, L-6 melanoma cells, andneuroblastoma and embryonal stem cells P19 (2, 20, 49). Incontrast, in other cell lines, such as keratinocytes (66), embry-onal lung cells (63), and chick embryo hepatocytes (70),proliferation is stimulated. Differentiation is also blocked inchondrocytes, osteocytes, and adipocytes (49). The reportedcontrasting effects of RA can be explained by the existence ofmany types of RA-nuclear receptors, regulated both temporallyand in a tissue-specific fashion. Moreover, the divergent effects

of RA in various tissues may be explained by the differentlevels of cellular retinol binding protein (RBP) necessary forretinol binding and transfer across the plasma membrane (72).

In proliferating eukaryotic cells, the progression from G1phase to S phase of the cell cycle is controlled by the expres-sion of a subset of the cell cycle control genes. These includethe D-type cyclin genes, which consist of three family mem-bers, cyclins D1, D2, and D3. The regulatory function of theD-type cyclins is mediated by their interactions with cyclin-dependent kinases (CDK)2, CDK4, and CDK6 (43). Abnormal

Fig. 8. Effects of RA and 22-OH on RARgene expression in Caco-2 cells. PCR analy-sis was performed on Caco-2 cells at 21 dayspostconfluence to analyze mRNA of RAR�(4-h incubation) (A), RAR� (24-h incuba-tion) (B), RAR� (4-h incubation) (C), RAR�(24-h incubation) (D), RAR� (4-h incuba-tion) (E), and RAR� (24-h incubation) (F).Values represent means � SE for n � 4 ineach group of experiments and are reportedas percent difference relative to control val-ues representing 100%. *P 0.05 vs. con-trol; **P 0.01 vs. control.




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or untimely expression has the potential to disrupt the cellcycle and therefore assign them as proliferation-promotinggenes or oncogenes (4). Indeed, overexpression of the D-typecyclins has been reported in a large number of tumors (14, 18,28, 73). However, not all three D-type cyclins are overex-pressed in the same tumor and the three cyclins are differen-tially linked to poor prognosis (68, 74). In our study, theadministration of RA to Caco-2 cells decreased cyclin D1 andincreased cyclin D3, resulting in a significant D3/D1 ratio at0-day confluence, and 10 and 21 days postconfluence. Thesedata are congruent with the observation that overexpression ofcyclin D1, but not cyclin D3, is linked to cancer carcinogenesis(47). At present, the mechanism by which RA enhanced cyclinD3 and lowered D1 is not clear.

CDX2 is selectively localized in the nuclei of fetal and adultmucosal epithelial cells in the small and large intestines in bothhumans and mice (48). In our study, CDX2 protein expressionwas increased by RA at 60% confluence and decreased by RAat 10 and 21 days postconfluence. Although the reasons for thevariable CDX2 modulation by RA during the processes of cellproliferation and differentiation remain unclear, it is possible tosuggest a few explanations to enlighten this situation. The lossof CDX2 expression in the differentiation state may result from1) defects in trans-acting pathways known to regulate CDX2transcription in the 5�-flanking gene region (24); 2) CDX2 genesilencing by hypermethylation of the CpG island associatedwith the promoter region (75); and 3) the inhibition p38 MAPKpathways leading to the downregulation of CDX2 (25, 34), asexemplified by our findings showing the parallel trend of p38MAPK and CDX2.

PI3K belongs to a family of signal transducer heterodimericenzymes composed of a 85-kDa regulatory subunit (p85 � or�) containing SH2 and SH3 domains and a 110-kDa catalyticsubunit (p110 � or �) (9, 35) that phosphorylates the D3hydroxyl in the inositol ring of phosphatidylinositol. PI3K isinvolved in the regulation of normal cellular processes such ascellular growth, vesicular trafficking, insulin-regulated glucoseuptake, and apoptosis. Recently, PI3K has been shown to be animportant regulator of differentiation in certain cell types,including myogenic, intestinal, and adipocyte differentiation(53). However, PI3K may behave as a negative regulator ofcellular differentiation (8, 56). In our studies, PI3K did notchange following RA treatment at 60% preconfluence or at 0confluence. On the other hand, p38 MAPK was augmented,which is congruent with its important role in various mamma-lian cell-differentiating processes (53). Consistent with thenotion that CDX2 has a critical function in intestinal celldifferentiation within a regulatory network that establishes thedifferentiated phenotype of intestinal epithelial cells (25), wefound a RA-induced enhancement in Caco-2 cells at 60%confluence.

Expression of the APO A-I gene is regulated at both thetranscriptional and posttranscriptional level. The APO A-I genepromoter contains a TATA-like motif close to the transcrip-tional start site and several cis-elements that regulate expres-sion of the gene in either a positive or negative manner inresponse to various hormonal or metabolic signals. Varioustranscription factors assemble into multiprotein complexes onkey regulatory regions of the gene (40). General transcriptionfactors (TFs) associated with polymerase II (Pol II), such asTFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, are recog-

nized by core promoter sequences and are responsible for thebasal expression of the gene. The multistep assembly of thesefactors is involved in APO AI gene modulation. Data of ourstudy clearly demonstrated that RA does not regulate theexpression of apo A-I in the Caco-2 cells, although Hargroveet al. (23) have reported that retinoids induce APO A-I pro-moter activity and gene expression through proximal promoterelements located between nucleotides �235 and �144 (rela-tive to the transcriptional start site, 1). It seems thereforepossible that RA might act by simultaneously blocking aliver-specific enhancer element such as that described by Sas-try et al. (62). In fact, several transcriptional repressors havebeen shown to suppress APO A-I gene transcription (23),although the physiological relevance of each of these transcrip-tion elements is not always clear. For example, binding of athyroid hormone receptor monomer to a negative thyroidhormone response element located 3� of the APO A-I geneTATA-box may suppress APO A-I gene transcription onlywhen removed from the full-length promoter (69). Neverthe-less, additional studies are needed to clarify whether RAmodifies the 3 apos (A-I, C-III, and A-IV), which are groupedtogether in a cluster on 17 kb of human chromosome 11 (31).Apparently, apo A-I transcription rates are strongly influencedby an APO C-III gene enhancer element, part of the APOS (A-I,C-III, A-IV) gene cluster (29). Thus one cannot exclude thepossibility that RA may affect the distal regulatory region ofthe APO C-III promoter that acts as a common enhancer for thethree closely linked genes (32). For example, as a scenario,prevention of the binding of SP1 to the enhancer by RA coulddiminish the promoter/enhancer activity (32).

The retinoid signal is transduced by two families of nuclearreceptors, the RARs and RXRs, which work as RXR/RARheterodimers. Each family consists of three isotypes (�, �, and�) encoded by separate genes (41). RARs are activated byall-trans RA and its 9-cis isomer, whereas RXRs are onlyactivated by 9-cis RA. Our data showed that RA was able toinduce RXR� and RXR� only at the 24-h incubation, whereasRXR� gene expression was not altered by the presence of RA.On the other hand, all the RAR isoforms responded to RAeither at 4-h incubation or 24-h incubation except RAR� at 4-hincubation. Our results imply that a long period of incubationis necessary to enhance the mRNA levels of RXR� and RXR�in Caco-2 cells. Moreover, RXR� seems resistant to regulationby RA, suggesting a low dissociation constant for the boundretinoids or limited role in Caco-2 cells.

Importantly, posttranslational modification of transcriptionfactors by phosphorylation provides a rapid cellular response toenvironmental changes. Phosphorylation processes are criticalfor the transcriptional activity of RAR and RXR because anabnormal phosphorylation might result in the dysregulation ofRA target genes (7). Moreover, a link between the phosphor-ylation status of the receptor and its ubiquitination/degradationhas recently been proposed, possibly implicating this mecha-nism in the proliferative and carcinogenetic processes (1, 42).The precise mechanisms underlying the effects of retinoids inour study are still to be elucidated since RAR and RXR wereassessed only at the molecular level and the specific role of thenuclear receptors phosphorylation and degradation throughthe ubiquitin-proteasome pathway should be addressed in fu-ture investigation. Evidently, confirmation of the positive mod-ulation of the RAR and RXR factors at the protein levels by




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RA may uncover changes in their properties, including alter-ations in DNA binding capacity, ligand binding, dimerization,coactivator recruitment, and transcriptional activation.

PPAR� forms a heterodimer with RXR�, which enhancesits binding to DNA sequence elements termed peroxisomeproliferator response elements (19, 21, 36). In our studies, aparallel decline in the gene expression of PPAR� and RXR�was noticed with the administration of RA22-OH comparedwith RA alone. At present, the mechanism by which RA andRA22-OH differently impact on the gene expression ofPPARs is not clear. In view of the repression of PPAR� andRXR� by LXR� reported previously (27), one may onlysuggest that the RA22-OH-mediated trend of increase ofLXR� might behind the reduction of PPAR�.

Although much is known about the modulation of apo A-Iby diet, hormones, and development, there remains a dearth ofinformation regarding the intestinal regulation of apo A-Isynthesis and HDL production by natural and synthetic retinoidmembers of the vitamin A family. Our studies have revealedthe potential of RA to induce cell differentiation withoutmodulating apo A-I synthesis. A better understanding of theregulatory mechanisms in the future may offer us usefulinformation to manipulate apo A-I expression and to benefitpatients with coronary events.

ACKNOWLEDGMENTS

The authors thank Schohraya Spahis for expert technical assistance.

GRANTS

This study was supported by the Dairy Farmers of Canada.

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