dietary soy protein isolate modifies hepatic retinoic acid receptor- beta proteins and inhibits...

8/13/2019 Dietary Soy Protein Isolate Modifies Hepatic Retinoic Acid Receptor- Beta Proteins and Inhibits Their DNA Bindinng …

http://slidepdf.com/reader/full/dietary-soy-protein-isolate-modifies-hepatic-retinoic-acid-receptor-beta-proteins 1/6

The Journal of Nutrition

Biochemical, Molecular, and Genetic Mechanisms

Dietary Soy Protein Isolate Modifies Hepatic

Retinoic Acid Receptor- b Proteins and Inhibits

Their DNA Binding Activity in Rats

1,2

Chao Wu Xiao,3,5* Jie Mei,3 Wenxin Huang,3 Carla Wood,3 Mary R. L’Abbe ´ ,3 G. Sarwar Gilani,3

Gerard M. Cooke,4,5 and Ivan H. Curran4

3Nutrition Research Division, 4Toxicology Research Division, Food Directorate, Health Products and Food Branch, Health Canada,

2203C Banting Research Centre, Ottawa, ON, Canada K1A 0L2 and 5Department of Cellular and Molecular Medicine, Faculty of

Medicine, University of Ottawa, Ottawa, ON, Canada

Abstract

Retinoic acid receptors (RAR) belong to the same nuclear receptor superfamily as thyroid hormone receptors (TR) that

were previously shown to be modulated by dietary soy protein isolate (SPI). This study has examined the effect of dietary

SPI and isoflavones (ISF) on hepatic RAR gene expression and DNA binding activity. In Expt. 1, Sprague-Dawley rats were

fed diets containing 20% casein or 20% alcohol-washed SPI in the absence or presence of increasing amounts of ISF (5–

1250 mg/kg diet) for 70, 190, or 310 d. In Expt. 2, weanling Sprague-Dawley rats were fed diets containing 20% casein

with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10, and 20%) for 90 d.

Intake of soy proteins significantly elevated hepatic RAR b2 protein content dose-dependently compared with a casein

diet, whereas supplemental ISF had no consistent effect. Neither RAR b protein in the other tissues measured nor the

other RAR (RARa and RARg ) in the liver were affected by dietary SPI, indicating a tissue and isoform-specific effect of SPI.

RAR b2 mRNA abundances were not different between dietary groups except that its expression was markedly

suppressed in male rats fed SPI for 310 d. DNA binding activity of nuclear RAR b was significantly attenuated and the

isoelectric points of RAR b2 were shifted by dietary SPI. Overall, these results show for the first time, to our knowledge,

that dietary soy proteins affect hepatic RAR b2 protein content and RAR b DNA binding activity, which may contribute to

the suppression of retinoid-induced hypertriglyceridemia by SPI as reported. J. Nutr. 137: 1–6, 2007.

Introduction

Soy intake has been shown to be hypotriglyceridemic in bothanimals (1–3) and hyperlipidemic human subjects (4). This ac-tion of soy components has been further confirmed in our recentrat study showing dietary alcohol-washed soy protein isolate(SPI)6 containing minimal isoflavones (ISF) remarkably de-creased plasma triacylglycerol levels (5). Furthermore, we pre-viously reported that diets containing SPI significantly increasedthe hepatic thyroid hormone receptor (TR) b1 protein content

and suppressed the DNA binding activity of nuclear TR proteins(6,7). Retinoic acid receptors (RAR) are also members of thenuclear receptor superfamily and can form heterodimers withTR to interact with the target genes (8). However, whether soyintake affects RAR gene expression and function is unknown.

RA is a metabolite of vitamin A and plays important roles incontrolling immune function, reproduction, cell growth, differ-

entiation (9), and lipid metabolism (10,11). RA is important inthe prevention and treatment of various cancers (12,13). Forinstance, loss of hepatic RA function leads to development of steatohepatitis and liver tumors (14). Most of the physiologicalfunctions of RA are mediated through RAR and altered RARactivity or RAR-mediated pathway is associated with manytypes of carcinogenesis (15–18). Three types of RAR (RARa,RAR b, and RARg ) have been characterized (19,20) and areencoded by independent genes. Several isoforms are generated

from each gene by alternative splicing or usage of distinct pro-moters (21–23). For example, RAR b has 4 isoforms (RAR b1,RAR b2, RAR b3, and RAR b4) that are generated by alternativegene splicing of primary transcripts initiated from 2 promoters,P1 and P2 (23,24).

Retinoids have been extensively used in the treatment of var-ious cancers (25–27). However, one of the major adverse effectsof retinoid treatment is the induction of hypertriglyceridemia,which has been observedin both rats (28) and humans (12,29–31).Interestingly, replacement of dietary casein with SPI markedlyreduced the severity of RA-induced hypertriglyceridemia in rats(10,32). Nevertheless, the underlying mechanism(s) is not wellunderstood. It has been shown that dietary SPI had no effect on

serum retinoid level in RA-treated rats compared with the casein

1 Supported by Health Canada.2 This is publication no. 610 of the Bureau of Nutritional Sciences.6 Abbreviations used: HRP, horseradish peroxidase; ISF, isoflavone; pI, isoelec-

tric point; RAR, retinoic acid receptor; SPI, soy protein isolate; TR, thyroid

hormone receptor; 2D, 2-dimensional.

* To whom correspondence should be addressed. E-mail: chaowu_xiao@hc-sc.

gc.ca.

0022-3166/07 $8.00 ª 2007 American Society for Nutrition. 1Manuscript received 1 September 2006. Initial review completed 26 September 2006. Revision accepted 13 October 2006.

b y onF

e b r u ar y 1 1 ,2 0 0 8

j n.n u t r i t i on. or g

D ownl o a d e d f r om

http://jn.nutrition.org/





















control (32) and that retinoid-induced hypertriglyceridemia ismediated through retinoid receptors (10,33). This suggests thatSPI may exert its suppressive actions on the retinoid-inducedhypertriglyceridemia via modulation of RAR or RAR-regulatedgene expression. The objective of this study was to examine if dietary SPI and soy-derived ISF affect hepatic RAR gene expres-sion and DNA binding ability in rats.

Materials and Methods

Chemicals and reagents. Alcohol-washed SPI (Pro Fam 930 contain-

ing 90% protein) and Novasoy (soy ISF concentrate) were purchased

from Archer Daniels Midland Company. Casein protein (90% totalprotein) was from ICN and Harlan Teklad. Acrylamide, N,N’-methyl-

ene-bis-acrylamide, ammonium persulfate, dithiothreitol, Tris, phenyl-

methylsulfonyl fluoride, maleic acid, boric acid, Nonidet P-40, and

EDTA were from Sigma Chemical. Western blotting detection kits and

Hybond-N1 Membrane were obtained from Amersham. Goat anti-

rabbit and anti-mouse immunoglobulin G (H1L)- horseradish peroxidase

(HRP) conjugated antibodies, and Bio-Rad protein assay kits were pur-chased from Bio-Rad Laboratories. X-ray film was from MJS Biolynx.

Affinity purified rabbit polyclonal antibodies against human RARa,

RAR b, and mouse monoclonal antibody against human RARg werefrom Santa Cruz Biotechnology and all tested to cross-react with rat anti-

gens. RAR b antibody was shown to detectboth RAR b1 and RAR b2 iso-forms. Biotin 3# End DNA Labeling kit and LightShift Chemiluminescent

electrophoretic mobility shift assay kit were from Pierce Biotechnology.

Animals, diets, and tissue samples. The animal experimental pro-

tocol was approved by the Health Canada Animal Care Committee and

all animal handling and care followed the guidelines of the CanadianCouncil for Animal Care. Expt. 1 was designed to examine the effect of

alcohol-washed SPI (containing minimal amount of ISF) and increasing

amounts of added soy ISF using casein as a control. Briefly, Sprague-Dawley male and female rats (Charles River) at age of 50 d were ran-

domly divided into 6 groups and fed one of the 6 diets as previously

described (6) (Diet 1: 20% casein; Diet 2: 20% alcohol-washed SPI;

Diets 3–6: 20% SPI supplemented with 5, 50, 250, or 1250 mg ISF/kg

diet) for 70, 190, or 310 d. At the end of each feeding period, 10 male

and 10 female rats per dietary group were killed for collection of tissues.Expt. 2 was to determine the effect of increasing amounts of alcohol-

washed SPI and the residual ISF contained in the alcohol-washed SPI.Weanling Sprague-Dawley rats were randomly divided into 5 groups

(8 males and 8 females per group) and fed 1 of the 5 diets for 90 d. All

5 diets were formulated according to the specifications for the AIN93Gdiet (34) except that L-cystine was replaced by L-methionine and in Diets

3–5, casein by equal amounts of alcohol-washed SPI (5, 10, and 20%).

To determine the potential effect of remaining ISF in alcohol-washed SPI

(31.7 mg/kg diet of 20% SPI), Diet 2 was supplemented with 50 mg/kg

diet of ISF from Novasoy. The detaileddietary composition was reportedelsewhere (7). At theend of feeding periods, rats were necropsied and the

plasma and tissue samples were collected and stored at 280C until

analysis. The actual ISF content in each diet was determined by Waters

HPLC linear gradient with UV detection monitored at 254 nm (35) andreported previously (6,7).

Protein extraction and western-blot analysis. Total protein extrac-tion and western-blot analysis were carried out as previously described

(6) withminor modifications. Briefly, total proteins (80mg) were resolved

by 12% SDS-PAGE and electrotransferred (30 V, 4C, overnight) onto

nitrocellulose membranes. After blocking, membranes were incubatedovernight at 4C with primary antibodies and subsequently with

HRP-conjugated secondary antibodies (1:5000) at room temperature

for 45 min. Immunoreactivity was detected by chemiluminescence

autoradiography in accordance with the manufacturer’s instructions and

the images were scanned. The intensities of the protein bands of interest

and the Ponceau-stained proteins were determined densitometrically

using Scion Image software. The intensities of the target proteins were

normalized by the respective Ponceau-stained total protein (36).

RAR b mRNA quantification. Total RNA was isolated from rat liver

samples with TRIzol reagent (Life Technologies). Five hundred nano-

grams of total RNA were reverse transcribed for cDNA synthesis usingrandom primer oligonucleotides. One-tenth of the cDNA synthesized

was then amplified with the following primers: rat RAR b2 [forward:

5#-CTCTCAAAGCCTGCCTCAGT-3# (292–311), reverse: 5#-CTGTG-

CACTCCTGCTTTGAA-3# (702–683)] (GenBank accession number

AJ002942) and universal 18S rRNA primers and competimers (Ambion)

in a ratio of 2:8. PCR cycle conditions were 94 C for 5 min, 94C for30 s, 60C for 30 s, and 72C for 1 min for 30 cycles, 72C for 10 min.

Samples were resolved on 2% agarose gels and visualized with ethidium

bromide. The images were taken using BioDoc-It Imaging System (UVPInc.) and analyzed with Scion Image software. RAR b2 mRNA levels

were normalized against their respective 18S rRNA content.

Two-dimensional western-blot analysis of RAR b. Two-dimensional

(2D) gel electrophoresis was carried out according to the method of

O’Farrell (37). Briefly, liver total proteins (200 mg) pooled from 3 rats fed

thesame dietswere subjected to isoelectric focusing in 2% glass tube gels

of pH 3.5–10 (Amersham Pharmacia Biotech). After equilibration, thetube gels were sealed to the top of stacking gels on top of 10% acryl-

amide slab gels and run for ;4 h. The gel was transblotted onto poly-

vinylidene difluoride membrane overnight and stained with Coomassie

Blue. The membranes were immunostained with rabbit anti-human

RAR b polyclonal antibody (1:500 dilution) and detected using an emis-sion of chemiluminescence kit.

Nuclear protein preparation and electrophoretic mobility shift

assay. Hepatic nuclear protein extracts were prepared as previously de-

scribed (7). Double-stranded DNA oligonucleotides containing consen-

sus sequences (5#-TCGAGGGTAGGGTTCACCGAAAGTTCACTCG-3#)for RAR-specific binding was labeled at the 3#-end with biotin-N4-CTP

and terminal deoxynucleotidyl transferase. Nuclear protein extracts

(2 mg) were incubated with biotin-labeled DNA probes in the binding

buffer for 15 min at room temperature. DNA-protein complexes were

resolved on a native 6% polyacrylamide gel in Tris-buffered EDTA (pH

8.0) and electrotransferred (80 V for 1.5 h) onto positively charged nylonmembranes. The biotin-labeled DNA probes bound to the nuclear

proteins were immunostained with streptavidin-HRP conjugate and

detected by chemiluminescence autoradiography. The binding specificity

of RAR b to DNA probe was determined by cold probe (1003) com-petition and supershifting with 1 mg rabbit anti-human RAR b antibody.

The intensity of the specific nuclear protein band was densitometrically

determined and normalized by the amount of total probe (bound1 free).

Statistical analyses. Results are expressed as means 6 SEM. Effects of

treatment on hepatic RAR b2 protein content and mRNA steady-state

abundances were analyzed by 2-way ANOVA, which included the main

effects of diet and feeding duration as well as interaction of diet 3feeding duration. All the other data presented were analyzed by 1-way

ANOVA. The males and females were analyzed separately. Differencesbetween individual means were determined by Fisher’s least significant

difference test. A probability of P , 0.05 was considered significant.

Data were analyzed using STATISTICA Version 7.1 (StatSoft).

Results

Hepatic total and nuclear RAR b protein content. Consump-tion of 20% alcohol-washed SPI in the absence of supplementalISF for 70 d markedly increased hepatic RAR b protein contentin both female and male rats compared with a casein-based diet(Fig. 1A,B; P , 0.01) . Supplementation of increasing amountsof ISF to 20% alcohol-washed SPI-based diet had no additionaleffect on RAR b content compared with SPI alone (P. 0.05; Fig.1A,B) except that addition of 5 or 1250 mg ISF/kg diet slightlyelevated RAR b levels in female rats (Fig. 1A). Dietary SPIsignificantly increased hepatic RAR b protein content at all 3

time points (for 70, 190, and 310 d) examined in both female

2 Xiao et al.

b y onF e b r u ar y 1 1 ,2 0 0 8
























and male rats compared with a casein-based diet (Fig. 1C ,D).However, neither SPI nor ISF had any effect on RARa or RARg

(data not shown).Intake of increasing amounts of alcohol-washed SPI (5, 10,

and 20%) for 90 d markedly elevated hepatic nuclear RAR b pro-tein content in a dose-dependent manner in both sexes (Fig. 2A,B).Addition of ISF (50 mg ISF/kg diet) to the casein-based diet hadno effect on hepatic nuclear RAR b protein content.

Hepatic RAR b mRNA abundance. Hepatic RAR b mRNAsteady-state levels measured by relative semiquantitative reversetranscription-PCR did not differ between dietary groups infemale rats (data not shown). Consumption of 20% alcohol-

washed SPI decreased RAR b mRNA abundance in the male ratsfed for 310 d (P, 0.01; Fig. 3B) but had no effect at earlier timepoints measured (70 and 190 d).

Isoelectric points of the hepatic RAR b proteins. Intensitiesof most protein spots in the 3 Coomassie Blue-stained 2D images(Fig. 4A,C ,E) were similar, indicating even loading and transfer.Two spots with a molecular weight of 51 kDa were immuno-stained with RAR b antibody in the liver proteins of the casein-fed

male rats. Their isoelectric points (pIs) were 7.20 and 7.48,respectively (Fig. 4B). Interestingly, in addition to these spots, 2more spots with the same molecular weight but different pIs(6.59 and 6.88) were detected in the liver samples from SPI-fedrats (Fig. 4D,F ). Similar results were obtained in the female rats(data not shown).

Hepatic RAR b DNA binding activity. Three bands weredetected in the liver samples, which may represent differentforms of RAR complexes (i.e. homodimers and heterodimers).Addition of a 100-fold excess of unlabeled DNA oligonucleotideprobe (cold probe) eliminated all the binding activity of the 2lower bands and reduced the abundance of the top band (lanes 6

and 12 vs. lanes 2 and 8, respectively; Fig. 5), indicating the 2lower bands and part of the top band are RAR-specific bindingcomplex. Supershifting analysis showed that the 2 lower bandsbut not the top one were shifted by RAR b antibody (lanes 5 and11). Nuclear RAR b DNA binding abilities in both male andfemale rats fed diets containing either 5% (lanes 3 and 9) or 20%(lanes 4 and 10) SPI were lower than in those fed the casein diet(lanes 2 and 8; P , 0.05).

Tissue distribution of RAR b proteins. Distinct RAR b iso-forms were expressed in different tissues of both female ( Fig. 6)and male (data not shown) rats. The 51-kDa RAR b2 proteinwas the predominant isoform in the liver and remarkably in-creased by dietary SPI compared with casein. In kidney and

heart, the predominant form was RAR b4 (48 kDa) and wasunchanged by the diets (Fig. 6A). RAR b2 protein content wasvery low in thyroid tissue and was not different between dietarygroups (Fig. 6B).

Figure 1 Hepatic total RAR b protein content in the female (A, C ) and male (B,

D ) rats fed diets containing 20% casein or 20% alcohol-washed SPI with or

without increasing amounts of supplemental ISF (5, 50, 250, or 1250 mg/kg diet)

for70 d or feddiets containing 20%casein (Ca) or20% alcohol-washedSPI (S)for

70, 190, and 310 d. The images shown are representative of 3 replicates. Values

are means6

SEM, n ¼ 3. Means without a common letter differ,

P ,0.01.

Figure 2 Hepatic nuclear RAR b protein content in the female (A) and male (B )

rats fed diets containing 20% casein in the absence or presence of supple-

mental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10,

or 20%) for 90 d. The images shown are representative of 3 replicates. Values

are means 6 SEM, n ¼ 3. Means without a common letter differ, P , 0.01.

Figure 3 Hepatic RAR b mRNA abundance in the male rats fed 20% casein

(Ca) or 20% alcohol-washed SPI (S) for 70, 190, or 310 d. The images shown are

a representative of 3 replicates. Values are means 6 SEM, n ¼ 3. Means

without a common letter differ, P , 0.01.

Soy proteins modulate retinoic acid receptors 3

b y onF e b r u ar y 1 1 ,2 0 0 8
























Discussion

In this study, we showed that consumption of alcohol-washedSPI (containing minimal amount of ISF) significantly elevatedhepatic RAR b2 protein content in both male and female rats.This effect was further demonstrated to be dose dependent.However, the protein content of the other RAR, including RARaand RARg , did not differ among dietary groups, indicating thatthe effect of SPI is RAR isoform-specific. Supplementation withincreasing amounts of soy-derived ISF to SPI-based diets had noconsistent additional effect.

Although the SPI used in this study was subjected to alcohol

extraction to remove the associated ISF, a minimal level of ISF(;31.7 mg/kg diet) remained in the diet containing 20% SPI.Soy ISF, especially the major component genistein, are capable of modulating RAR b gene expression in various cancer cells via

inhibition of DNA methyltransferase activity (38). This mayimply that the effect of SPI observed could be a result of ISFcontamination. To exclude this possibility, we added a similaramount of ISF (42.8 mg/kg diet) from Novasoy to the casein-based diet in Expt. 2. The results showed that added ISF had nosignificant effect (P. 0.05) on the nuclear RAR b2 protein in theliver, confirming that SPI rather than ISF accounts for theincrease in hepatic RAR b2 protein.

To understand the potential underlying molecular event(s) bywhich SPI elevated hepatic RAR b2 protein content, we furthermeasured the RAR b2 mRNA steady-state levels and found thatdietary SPI failed to upregulate the RAR b2 gene expressionthroughout the feeding periods. In contrast, the long-termfeeding (310 d) of the rats with the SPI-based diet decreased theRAR b2 mRNA abundances in the liver compared with thecasein diet. This indicates that modulation of hepatic RAR b2protein content by SPI might be post-transcriptional. This notionwas supported by the evidence obtained from 2D western-blotanalysis showing that the rats fed SPI-based diets have 2 uniquehepatic proteins with the same molecular weight (51 kDa) asRAR b2 but different pIs. These proteins cross reacted with

rabbit anti-human RAR b antibody, suggesting that they mightbe modified forms of RAR b2 protein.This study showed that dietary SPI markedly suppressed the

binding activity of the hepatic nuclear RAR b to the consensusDNA sequence of target genes. Binding of the nuclear receptorsto the ligand response elements of the promoter region in thetarget genes is essential for the regulation of downstream geneexpression and critical for the receptor-mediated functions.Thus, inhibition of RAR b function (i.e. DNA binding activity) isbelieved to play a pivotal role in mediating the previouslyreported suppressive actions of SPI on retinoid-induced hyper-glyceridemia that was shown to be mediated through RAR (10).

We further demonstrated that nuclear content of RAR b2protein was also consistently elevated in both male and female

rats by dietary SPI (Fig. 2). This suggests that modification of RAR b2 protein by SPI may affect only the DNA binding abilitybut not the translocation of the receptor into nucleus. RAR bprotein content in the other tissues examined were not affectedby dietary SPI in this study, indicating that the effects of soyprotein are liver specific. Liver plays important roles in theuptake, storage, and mobilization of retinol and stores up to80% of the body retinoids (39). In addition, RAR b2 is the mostabundant RAR b isoform in the body (40). Suppression of he-patic RAR b function by soy components may have impactson vitamin A function and RAR b-regulated gene expressionand activities. RAR b has been identified as a tumor suppressor(41–44) and silenced or reduced RAR b gene expression is closely

associated with tumorigenesis (13,18,45). Whether the inhibition

Figure 4 2D western-blot analysis of hepatic RAR b protein in the male rats

fed diets containing either 20% casein (A, B ) or 20% alcohol-washed SPI in the

absence (SPI; C, D ) or presence (SPI1ISF; E and F ) of supplemental ISF (250

mg/kg diet) for 70 d. The pI were calculated using a linear regression of pI and

the location (distance) of the protein on the 2D gel, which was established with

an internal standard of a known pI. The arrows in ( D ) and (F ) represent the RAR b

protein with changed pI.

Figure 5 DNA binding activity of hepatic nuclear RAR in rats fed diets

containing either 20% casein (lanes 2 and 8) or 5% (lanes 3 and 9) or 20% (lanes

4 and 10) alcohol-washed SPI for 90 d. The binding specificity was determined

by cold probe competition (1003) (lanes 6 and 12) and supershifting with rabbit

anti-human RAR b polyclonal antibody (lanes 5 and 11). The images shown are

representatives of 3 replicates of each.

Figure 6 Total RAR b protein content in liver, thyroid, kidney, and heart (A) of

the female rats fed diets containing either 20% casein or 20% alcohol-washed

SPI for 70 d. The image shown in (A) is a representative of 3 replicates. RAR b2

expression in thyroid was further analyzed separately ( B ).

4 Xiao et al.

b y onF e b r u ar y 1 1 ,2 0 0 8
























of RAR b DNA binding ability induced by soy components in theliver is related to any type of carcinogenesis warrants furtherinvestigation.

Although the exact molecular mechanism(s) by which soycomponents elevated the hepatic RAR b2 protein level andsuppressed RAR b DNA binding activity is not understood, it isbelieved that post-translational modification of RAR b2 proteinmay play a key role in this regard. Phosphorylation is one of themost common protein modifications in animal cells (46). It hasbeen shown that phosphorylation of serine and/or threonine in

proteins such as soluble CD44, the principal hyaluronic acidreceptor (47), and rat valosin-containing protein (48) resulted inacidic shift in their pIs, an effect observed in hepatic RAR b2 of the rats fed SPI in this study. Moreover, phosphorylation isknown to be an important mechanism regulating the transac-tivation and degradation of RAR (49). Particularly, the phos-phorylation of RARa has been extensively studied and severalkinases including Akt and c-Jun N-terminal kinase have beenshown to be involved in these processes. For example, Akt phos-phorylates the Ser-96 residue of RARa DNA-binding domainand inhibits its transactivation (49). c-Jun N-terminal kinasephosphorylates RARa at residues Thr-181, Ser-445, and Ser-461, resulting in RAR dysfunction and degradation through the

ubiquitin-proteasomal pathway (50). In addition, several com-ponents of soy, including soybean trypsin inhibitors, ISF (genis-tein), and their metabolite, equol, have been shown to affectphosphorylation status of proteins and enzymes (51–53).

It was suggested that a higher arginine-to-lysine ratio in soymay be responsible for its lipid-lowering actions, because theaddition of arginine to a casein-based diet reduced the severity of retinoid-induced hypertriglyceridemia, but not as effectively asreplacing casein with soy protein (54). More recent evidenceindicates that b-conglycinin, one of the major soybean storageproteins, may contain the bioactive peptide responsible for thehypolipidemic effects of soy (2,55–57).

In summary, this study demonstrates for the first time, to our

knowledge, that intake of alcohol-washed SPI markedly in-creased hepatic RAR b2 protein content and suppressed thebinding ability of the nuclear RAR b to the consensus DNAsequence of target genes in rats. The RAR b2 mRNA abundancewas not increased by dietary SPI. The pIs of the hepatic RAR b2protein demonstrated that more acidic isoforms were prevalentin rats ingesting SPI. These results suggest that dietary SPI mayexert its effect through post-translational modification such asphosphorylation on the RAR b2 protein, thereby changing itsstructure or conformation and affecting the protein degradation(or stability) and DNA binding activity of RAR b. However, thisremains to be confirmed. Our ongoing studies include determi-nation of the type of protein modifications in a variety of proteinsin response to dietary SPI. Further work is required to identify

the bioactive component(s) in soy and their effects on proteindegradation using appropriate models.

Literature Cited

1. Lin Y, Meijer GW, Vermeer MA, Trautwein EA. Soy protein enhancesthe cholesterol-lowering effect of plant sterol esters in cholesterol-fedhamsters. J Nutr. 2004;134:143–8.

2. Moriyama T, Kishimoto K, Nagai K, Urade R, Ogawa T, Utsumi S,Maruyama N, Maebuchi M. Soybean beta-conglycinin diet suppressesserum triglyceride levels in normal and genetically obese mice by induc-tion of beta-oxidation, downregulation of fatty acid synthase, and inhi-bition of triglyceride absorption. Biosci Biotechnol Biochem. 2004;68:

352–9.

3. Ascencio C, Torres N, Isoard-Acosta F, Gomez-Perez FJ, Hernandez-Pando R, Tovar AR. Soy protein affects serum insulin and hepaticSREBP-1 mRNA and reduces fatty liver in rats. J Nutr. 2004;134:522–9.

4. Wang Y, Jones PJ, Ausman LM, Lichtenstein AH. Soy protein reducestriglyceride levels and triglyceride fatty acid fractional synthesis rate inhypercholesterolemic subjects. Atherosclerosis. 2004;173:269–75.

5. Xiao CW, Wood C, Huang W, L’Abbe MR, Gilani GS, Cooke GM,Curran I. Tissue-specific regulation of acetyl-CoA carboxylase gene ex-pression by dietary soya protein isolate in rats. Br J Nutr. 2006;95:1048–54.

6. Xiao CW, L’Abbe MR, Gilani S, Cooke G, Curran I, Papademetriou SA.

Dietary soy protein isolate and isoflavones modulate hepatic thyroidhormone receptors in rats. J Nutr. 2004;134:743–9.

7. Huang W, Wood C, L’Abbe MR, Gilani S, Cockell KA, Xiao CW. Soyprotein isolate increases hepatic thyroid hormone receptor content andinhibits its binding to the target genes in rats. J Nutr. 2005;135:1631–5.

8. Lee S, Privalsky ML. Heterodimers of retinoic acid receptors and thy-roid hormone receptors display unique combinatorial regulatory prop-erties. Mol Endocrinol. 2005;19:863–78.

9. Soprano DR, Qin P, Soprano KJ. Retinoic acid receptors and cancers.Annu Rev Nutr. 2004;24:201–21.

10. Standeven AM, Beard RL, Johnson AT, Boehm MF, Escobar M,Heyman RA, Chandraratna RA. Retinoid-induced hypertriglyceridemiain rats is mediated by retinoic acid receptors. Fundam Appl Toxicol.1996;33:264–71.

11. Villarroya F, Iglesias R, Giralt M. Retinoids and retinoid receptors in the

control of energy balance: novel pharmacological strategies in obesityand diabetes. Curr Med Chem. 2004;11:795–805.

12. Toma S, Bonelli L, Sartoris A, Mira E, Antonelli A, Beatrice F, GiordanoC, Benazzo M, Caroggio A, et al. 13-cis retinoic acid in head and neckcancer chemoprevention: results of a randomized trial from the ItalianHead and Neck Chemoprevention Study Group. Oncol Rep. 2004;11:1297–305.

13. Petty WJ, Li N, Biddle A, Bounds R, Nitkin C, Ma Y, Dragnev KH,Freemantle SJ, Dmitrovsky E. A novel retinoic acid receptor fbetagisoform and retinoid resistance in lung carcinogenesis. J Natl CancerInst. 2005;97:1645–51.

14. Shiota G. Loss of function of retinoic acid in liver leads tosteatohepatitis and liver tumor: a NASH animal model. Hepatol Res.2005;33:155–60.

15. Darwiche N, Scita G, Jones C, Rutberg S, Greenwald E, Tennenbaum T,Collins SJ, De Luca LM, Yuspa SH. Loss of retinoic acid receptors inmouse skin and skin tumors is associated with activation of the ras(Ha)oncogene and high risk for premalignant progression. Cancer Res.1996;56:4942–9.

16. MarshallGM, Cheung B, Stacey KP, CamachoML, SimpsonAM, KwanE,Smith S, Haber M, Norris MD. Increased retinoic acid receptor gammaexpression suppresses the malignant phenotype and alters the differenti-ationpotential of humanneuroblastoma cells.Oncogene.1995;11:485–91.

17. de The H. Altered retinoic acid receptors. FASEB J. 1996;10:955–60.

18. Wang Y, Fang MZ, Liao J, Yang GY, Nie Y, Song Y, So C, Xu X, WangLD, et al. Hypermethylation-associated inactivation of retinoic acidreceptor fbetag in human esophageal squamous cell carcinoma. ClinCancer Res. 2003;9:5257–63.

19. Krust A, Kastner P, Petkovich M, Zelent A, Chambon P. A third humanretinoic acid receptor, hRAR-gamma. Proc Natl Acad Sci USA. 1989;86:5310–4.

20. Chambon P. A decade of molecular biology of retinoic acid receptors.FASEB J. 1996;10:940–54.

21. Kastner P, Krust A, Mendelsohn C, Garnier JM, Zelent A, Leroy P,Staub A, Chambon P. Murine isoforms of retinoic acid receptor gammawith specific patterns of expression. Proc Natl Acad Sci USA. 1990;87:2700–4.

22. Leroy P, Krust A, Zelent A, Mendelsohn C, Garnier JM, Kastner P,Dierich A, Chambon P. Multiple isoforms of the mouse retinoic acidreceptor alpha are generated by alternative splicing and differentialinduction by retinoic acid. EMBO J. 1991;10:59–69.

23. Zelent A, Mendelsohn C, Kastner P, Krust A, Garnier JM, Ruffenach F,Leroy P, Chambon P. Differentially expressed isoforms of the mouseretinoic acid receptor beta generated by usage of two promoters andalternative splicing. EMBO J. 1991;10:71–81.

24. Nagpal S, Zelent A, Chambon P. RAR-fbetag4, a retinoic acid receptor

isoform is generated from RAR-fbetag2 by alternative splicing and

Soy proteins modulate retinoic acid receptors 5

b y onF e b r u ar y 1 1 ,2 0 0 8
























usage of a CUG initiator codon. Proc Natl Acad Sci USA. 1992;89:2718–22.

25. Yamauchi T, Arai H, Taga M, Amaya N, Lee JD, Ueda T. Adams-Stokesattack due to complete atrioventricular block in a patient with acutepromyelocytic leukemia during remission induction therapy using all-trans retinoic acid. Rinsho Ketsueki. 2005;46:206–10.

26. Berg G, Andersson T, Sjodell L, Jansson S, Nystrom E. Development of severe thyroid-associated ophthalmopathy in a patient with dissemi-nated thyroid cancer treated with recombinant human thyrotropin/ radioiodine and retinoic acid. Thyroid. 2005;15:1389–94.

27. Stadler R, Kremer A. Therapeutic advances in cutaneous T-cell lym-

phoma (CTCL): from retinoids to rexinoids. Semin Oncol. 2006;33:S7–10.28. Gerber LE, Erdman JW Jr. Hyperlipidemia in rats fed retinoic acid.

Lipids. 1981;16:496–501.

29. Marsden JR. Effect of dietary fish oil on hyperlipidaemia due to isotret-inoin and etretinate. Hum Toxicol. 1987;6:219–22.

30. Marsden JR, Trinick TR, Laker MF, Shuster S. Effects of isotretinoin onserum lipids and lipoproteins, liver and thyroid function. Clin ChimActa. 1984;143:243–51.

31. Cartmel B, Dziura J, Cullen MR, Vegso S, Omenn GS, Goodman GE,Redlich CA. Changes in cholesterol and triglyceride concentrations inthe Vanguard population of the Carotene and Retinol Efficacy Trial(CARET). Eur J Clin Nutr. 2005;59:1173–80.

32. Radcliffe JD, Imrhan VL, Hsueh AM. The use of soy protein isolate toreduce the severity of 13-cis retinoic acid-induced hypertriglyceridemia.Cancer Detect Prev. 1998;22:526–32.

33. Vu-Dac N, Gervois P, Torra IP, Fruchart JC, Kosykh V, Kooistra T,Princen HM, Dallongeville J, Staels B. Retinoids increase human apoC-III expression at the transcriptional level via the retinoid X receptor.Contribution to the hypertriglyceridemic action of retinoids. J ClinInvest. 1998;102:625–32.

34. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for lab-oratory rodents: final report of the American Institute of Nutrition adhoc writing committee on the reformulation of the AIN-76A rodentdiet. J Nutr. 1993;123:1939–51.

35. Wang HJ, Murphy P. Isoflavone content in commercial soybean foods. J Agric Food Chem. 1994;42:1666–73.

36. Xiao CW, Yan X, Li Y, Reddy SA, Tsang BK. Resistance of humanovarian cancer cells to tumor necrosis factor alpha is a consequence of nuclear factor kappaB-mediated induction of Fas-associated deathdomain-like interleukin-1beta-converting enzyme-like inhibitory pro-

tein. Endocrinology. 2003;144:623–30.37. O’Farrell PH. High resolution two-dimensional electrophoresis of

proteins. J Biol Chem. 1975;250:4007–21.

38. Fang MZ, Chen D, Sun Y, Jin Z, Christman JK, Yang CS. Reversal of hypermethylation and reactivation of p16INK4a, RARbeta, andMGMT genes by genistein and other isoflavones from soy. Clin CancerRes. 2005;11:7033–41.

39. Biesalski HK, Nohr D. New aspects in vitamin A metabolism: the roleof retinyl esters as systemic and local sources for retinol in mucousepithelia. J Nutr. 2004;134:S3453–7.

40. Xu XC, Lee JJ, Wu TT, Hoque A, Ajani JA, Lippman SM. Increasedretinoic acid receptor-fbetag4 correlates in vivo with reduced retinoicacid receptor-fbetag2 in esophageal squamous cell carcinoma. CancerEpidemiol Biomarkers Prev. 2005;14:826–9.

41. Hoffman AD, Engelstein D, Bogenrieder T, Papandreou CN,

Steckelman E, Dave A, Motzer RJ, Dmitrovsky E, Albino AP, et al.Expression of retinoic acid receptor beta in human renal cell carcinomascorrelates with sensitivity to the antiproliferative effects of 13-cis-retinoic acid. Clin Cancer Res. 1996;2:1077–82.

42. Liu Y, Lee MO, Wang HG, Li Y, Hashimoto Y, Klaus M, Reed JC,Zhang X. Retinoic acid receptor beta mediates the growth-inhibitoryeffect of retinoic acid by promoting apoptosis in human breast cancercells. Mol Cell Biol. 1996;16:1138–49.

43. Kaiser A, Herbst H, Fisher G, Koenigsmann M, Berdel WE, RieckenEO, Rosewicz S. Retinoic acid receptor beta regulates growth and dif-ferentiation in human pancreatic carcinoma cells. Gastroenterology.1997;113:920–9.

44. Lefebvre B, Brand C, Flajollet S, Lefebvre P. Down-regulation of thetumor suppressor gene RARfbetag2 through the PI3K/Akt signalingpathway. Mol Endocrinol. 2006;20:2109–21.

45. Rochaix P, Monteil-Onteniente S, Rochette-Egly C, Caratero C, Voigt JJ, Jozan S. Reduced expression of retinoic acid receptor beta protein(RAR beta) in human papillary thyroid carcinoma: immunohistochem-ical and western blot study. Histopathology. 1998;33:337–43.

46. Pagliarini DJ, Dixon JE. Mitochondrial modulation: reversible phos-phorylation takes center stage? Trends Biochem Sci. 2006;31:26–34.

47. Knepper PA, Miller AM, Choi J, Wertz RD, Nolan MJ, Goossens W,Whitmer S, Yue BY, Ritch R, et al. Hypophosphorylation of aqueoushumor sCD44 and primary open-angle glaucoma. Invest OphthalmolVis Sci. 2005;46:2829–37.

48. Klein JB, Barati MT, Wu R, Gozal D, Sachleben LR Jr, Kausar H, Trent JO, Gozal E, Rane MJ. Akt-mediated valosin-containing protein 97phosphorylation regulates its association with ubiquitinated proteins.

J Biol Chem. 2005;280:31870–81.

49. Srinivas H, Xia D, Moore NL, Uray IP, Kim H, Ma L, Weigel NL,

Brown PH, Kurie JM. Akt phosphorylates and suppresses the trans-activation of retinoic acid receptor alpha. Biochem J. 2006;395:653–62.

50. Srinivas H, Juroske DM, Kalyankrishna S, Cody DD, Price RE, Xu Xc,Narayanan R, Weigel NL, Kurie JM. c-Jun N-terminal kinase contrib-utes to aberrant retinoid signaling in lung cancer cells by phosphory-lating and inducing proteasomal degradation of retinoic acid receptorfalphag. Mol Cell Biol. 2005;25:1054–69.

51. Liu D, Homan LL, Dillon JS. Genistein acutely stimulates nitric oxidesynthesis in vascular endothelial cells by a cyclic adenosine5#-monophosphate-dependent mechanism. Endocrinology. 2004;145:5532–9.

52. Inagaki K, Kobayashi H, Yoshida R, Kanada Y, Fukuda Y, Yagyu T,Kondo T, Kurita N, Kitanaka T, et al. Suppression of urokinaseexpression and invasion by a soybean Kunitz trypsin inhibitor aremediated through inhibition of Src-dependent signaling pathways. J BiolChem. 2005;280:31428–37.

53. Joy S, Siow RC, Rowlands DJ, Becker M, Wyatt AW, Aaronson PI, CoenC, Jacob R, Kallo I, et al. The isoflavone equol mediates rapid vascularrelaxation: Ca21-independent activation of eNOS/Hsp90 involvingERK1/2 and Akt phosphorylation in human endothelial cells. J BiolChem. 2006;281:27335–45.

54. Radcliffe JD, Czajka-Narins DM. Use of arginine to reduce the severityof retinoid-induced hypertriglyceridemia. Nutr Cancer. 2000;36:200–6.

55. Aoyama T, Kohno M, Saito T, Fukui K, Takamatsu K, Yamamoto T,Hashimoto Y, Hirotsuka M, Kito M. Reduction by phytate-reducedsoybean beta-conglycinin of plasma triglyceride level of young and adultrats. Biosci Biotechnol Biochem. 2001;65:1071–5.

56. Duranti M, Lovati MR, Dani V, Barbiroli A, Scarafoni A, Castiglioni S,Ponzone C, Morazzoni P. The alpha’ subunit from soybean 7S globulinlowers plasma lipids and upregulates liver beta-VLDL receptors in ratsfed a hypercholesterolemic diet. J Nutr. 2004;134:1334–9.

57. Fukui K, Kojima M, Tachibana N, Kohno M, Takamatsu K, HirotsukaM, Kito M. Effects of soybean beta-conglycinin on hepatic lipid meta-bolism and fecal lipid excretion in normal adult rats. Biosci BiotechnolBiochem. 2004;68:1153–5.

6 Xiao et al.

b y onF e b r u ar y 1 1 ,2 0 0 8






















dietary soy protein isolate modifies hepatic retinoic acid receptor- beta proteins and inhibits...

Documents