Identification and Characterization of Retinoic Acid ...Identification and Characterization of Retinoic Acid Receptor B 2 Target Genes in F9 Teratocarcinoma Cells Yong Zhuang,1 Teresa

Identification and Characterization of Retinoic Acid Receptor B2

Target Genes in F9 Teratocarcinoma Cells

Yong Zhuang,1 Teresa N. Faria,1,* Pierre Chambon,2 and Lorraine J. Gudas1

1Pharmacology Department, Weill Medical College of Cornell University, New York, NY, and 2Institut de Genetique et deBiologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, College de France, CU de Strasbourg, France

AbstractRetinoids, a group of natural and synthetic analogues of

vitamin A (retinol), modulate the differentiation of many

cell types. Retinoids are also used for the prevention and

treatment of cancer. The actions of retinoids are

generally mediated by the retinoic acid receptors (RARs

A, B, and ;) and the retinoid X receptors (RXRs A, B,

and ;). One of the RARs, RARB, is expressed at reduced

levels in many human carcinomas, and F9 RARB2�/� cells

do not growth arrest in response to RA. To determine if

RARB2 regulates the expression of a unique set of genes,

through the use of subtractive hybridization and DNA

array analysis we have identified and characterized

genes that are differentially expressed in F9 RARB2�/�

teratocarcinoma cells. These genes, which encode

transcription factors, cell surface signal transduction

molecules, and metabolic enzymes, include c-myc,

FOG1, GATA6 , glutamate dehydrogenase, glutathione

S-transferase homologue (p28), Foxq1 , Hic5 , Meis1a ,

Dab2, midkine , and the PDGF-a receptor. These genes

are regulated specifically by RARB2 in F9 wild-type (Wt)

cells as indicated by their expression profiles in F9

RARB2�/� cells as compared to F9 Wt, RARA

�/�, or

RAR;�/� cells, and their responsiveness to specific

retinoid receptor agonists. The basal expression levels

of some of these genes, such as c-myc , are higher in the

F9 RARB2�/� cells than in F9 Wt in the absence of

exogenous retinoids, suggesting that RARB2 can inhibit

gene expression in the absence of a ligand. The RARB2

target genes are transcriptionally activated by retinol, as

well as RA, in F9 Wt cells. Because the lack of RARB2

alters both the control of proliferation and differentiation

in F9 cells, the genes that we have characterized may

mediate key effects of RA, via RARB2, on these

processes.

IntroductionRetinoids, a group of natural and synthetic analogues of

vitamin A, exert profound effects on many biological

processes, such as vertebrate embryonic development (1)

and cell growth and differentiation (2). Vitamin A insuffi-

ciency during pregnancy results in the death of the fetus, as

well as congenital malformations affecting the eyes and ocular

tissues, myocardium, respiratory, urogenital, and circulatory

systems (1). Vitamin A deficiency in experimental animals is

also associated with a higher incidence of some types of

cancer, and with increased susceptibility to chemical carci-

nogens (3). Experimental models of carcinogenesis have

demonstrated the efficacy of pharmacological administration

of retinoids in preventing the development of cancers of the

skin, oral cavity, lung, mammary gland, prostate, bladder,

liver, and pancreas in animals exposed to carcinogenic agents

(3). Clinical trials have indicated that retinoids may be useful

for prevention of cancers of the upper aerodigestive tract, skin,

breast, and ovaries (4). In addition, retinoids have been

successfully used in the treatment of acute promyelocytic

leukemia (APL) (5, 6), and other cancers (for review, see

Refs. 7 and 8).

The biological effects of retinoids are primarily mediated

by two classes of nuclear retinoid receptors: retinoic acid

receptors (RARs) and retinoid X receptors (RXRs) (9, 10).

RARs and RXRs are members of the nuclear receptor

superfamily that also include estrogen, androgen, thyroid

hormone, vitamin D, PPAR, and orphan receptors. The

retinoid receptors are encoded by six distinct genes: RARa ,RARb , RARc , RXRa , RXRb , and RXRc . Each of these

receptors includes several isoforms formed by different

splicing and usage of alternative promoters (9, 10). All-trans

retinoic acid (RA) binds and activates RARs, and 9-cis-

retinoic acid (9-cis RA) binds and activates both RARs and

RXRs (11). Retinoid receptors activate transcription in a

ligand-dependent manner by binding to retinoic acid response

elements (RAREs) located in the promoter regions or

enhancers of target genes. RAREs generally consist of two

directly repeated half-sites of the consensus sequence

AGGTCA spaced by 2 or 5 bp (DR2 and DR5 elements)

(12). Recently, various coactivators for the nuclear receptors

have been identified, including CBP/p300, SRC-1, TIF-2/

Grip1, and ACTR/RAC3/p/CIP. They are recruited by ligand-

activated receptors and enhance transcription (13, 14). Many

of these coactivators possess acetyltransferase activity that can

modify histones and may increase promoter access to DNA-

binding proteins and the transcriptional machinery (15–18).

The strong conservation of each RAR across vertebrates

suggests that each receptor performs unique functions (19). In

Received 2/18/03; revised 5/9/03; accepted 5/9/03.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: NIH grant R01 CA43796 to L.J.G.; a research grant from theAmerican Institute for Cancer Research; and in part by a Cancer PharmacologyTraining Grant (CA62948-08) (Y.Z.).Requests for reprints: Lorraine J. Gudas, Room E409, Pharmacology Depart-ment, Weill Medical College of Cornell University, 1300 York Avenue, NewYork, NY 10021. Phone: (212) 746-6250; Fax: (212) 746-8858. E-mail:[email protected]*Current address: Bristol-Myers Squibb Company, New Brunswick, NJ 08903.Copyright D 2003 American Association for Cancer Research.

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our laboratory, we generated F9 teratocarcinoma cells with

mutations in both alleles of RARa, RARb2, or RARc by

homologous recombination and showed that different RARs

have different functions (20–23). Mice homozygous for

RARa, RARh, or RARg have also been generated to

understand the function of each receptor. Mutant mice lacking

RARa, RARh2, RARh, or RARg are viable but exhibit

abnormalities in several tissues (24–27). For example,

RARh�/� mice exhibit greatly reduced hippocampal CA1

long-term potentiation (LTP) and long-term depression (LTD)

(28), activities which are likely to play a role in learning

and memory.

The RARb gene has four isoforms: h1, h2, h3, and h4. The h2

isoform is the most abundant RARh isoform and the major RA

inducible form (29). An RARE that mediates RA-induced

RARh2 gene expression in many different cell types was

identified in the promoter region (30, 31). The RARh2 RARE

consists of two direct repeats of the core motif sequence

AGGTCA separated by five nucleotides (31, 32). Activation of

the RARh2 promoter is mediated by RAR/RXR heterodimers

(33). A thyroid hormone receptor/RXR heterodimer can also

bind strongly to the hRARE and activates the RARh2 promoter

in response to RXR ligands (34). RARh exhibits a pattern of

expression during development and in the mature organism (35)

which is different from those of the other RARs. This suggests

that RARh performs specific functions.

RARh is not expressed, or is expressed at low levels, in a

number of malignant tumors, including lung carcinoma,

squamous cell carcinoma of the head and neck, breast cancer

cell lines, and esophageal carcinoma (36–40). RARh is

encoded by a gene located on the short arm of chromosome

3 (p24) (41). In breast cancer, loss of heterozygosity (LOH)

has frequently been detected at chromosome 3p22–25 (42).

All of these findings support the concept that the specific

loss of RARh expression may be an important event in

tumorigenesis.

Recent studies have indicated that a decrease in RARhexpression results in resistance to the growth inhibitory

actions of retinoids. Indeed, transfection of RARh into

RARh-negative cervical, breast, and lung cancer cells

increased responsiveness to growth inhibition and induction

of apoptosis by retinoids (43–47), and the restoration of

RARh expression in RARh-negative lung cancer cell lines

inhibited tumorigenicity in nude mice (48, 49). Adminis-

tration of 13-cis RA to patients with premalignant oral

lesions can restore the expression of RARh, as well as

reverse the lesions (50). Conversely, the loss of both alleles

of the RARb2 in an F9 teratocarcinoma line, generated in this

laboratory by homologous recombination, resulted in the loss

of growth inhibition by RA (21).

We hypothesized that each RAR regulates a specific subset

of target genes. Thus, to understand the functions of the

transcription factor RARh2, the target genes specifically

regulated by RARh2 must be identified and analyzed. Very

few RARh target genes have been identified to date. By

comparing RA-treated F9 wild-type (Wt) teratocarcinoma cells

and F9 RARh2�/� cells, we identified some of the RARh2

target genes through the use of subtractive hybridization and

DNA expression microarray techniques. The target genes

identified include transcription factors, protein tyrosine

kinases, homeobox proteins, and oncoproteins. We have

initiated the further characterization of eleven target genes in

this study.

ResultsIdentification of RARb2 Target Genes in F9 Cells

We hypothesized that RARh2, which is induced by RA in

many cell types, mediated the growth inhibitory actions of RA

in F9 cells. To test this hypothesis, an F9 teratocarcinoma cell

line that contained targeted disruptions in both alleles of the

RARh2 gene was generated by homologous recombination. We

showed that the F9 Wt and RARh2+/� heterozygous lines could

undergo RA-induced growth arrest, but that RA did not cause

growth arrest in the F9 RARh2�/� cells (21). While these data

confirmed our hypothesis, more information about the target

genes regulated by RARh2 was then required to understand the

downstream effects of RARh2. We anticipated that RARh2

positively regulated the transcription of some of its target genes,

and negatively regulated other target genes.

To isolate RARh2 target genes, F9 Wt cells and F9

RARh2�/� cells were treated with 1 AM all-trans RA for 24 h.

Subtractive hybridization and microarray analysis were then

employed. By comparing the gene expression patterns between

F9 Wt cells and the F9 RARh2�/� cells, we identified

approximately 80 genes from subtractive hybridization and

300 target genes from the microarray analyses (see ‘‘Materials

and Methods’’). Approximately one-half of the putative target

genes were expressed at higher levels in RA-treated F9 Wt cells

as compared to F9 RARh2�/� cells, while the remaining genes

were expressed at higher levels in RA-treated RARh2�/� cells

than in F9 Wt.

Target Genes Identified by Subtractive HybridizationWe examined 20 of the genes isolated by subtractive

hybridization by Northern analysis of F9 Wt versus F9

RARh2�/� cells and found that 7 out of 20 target genes were

reproducibly altered in the Northern blot analysis. We

sequenced these seven target genes and identified them using

NCBI Blast Programs. Among them, six are known genes

(Table 1) and one is an uncharacterized gene. The six known

genes are Dab2 (p96 form), glutamate dehydrogenase , Foxq1 ,

Meis1a , midkine , and glutathione S-transferase homologue

(p28). To determine whether or not these genes were specific

RARh2 targets, the regulation of these genes by RA was also

examined by Northern blot analysis in F9 RARa�/� and

RARg�/� lines, generated by homologous recombination in

our laboratory (22, 23). We also examined RARh2 mRNA

expression. As expected, RARh2 mRNA was not expressed in

F9 RARh2�/� cells and its expression was stimulated by RA in

F9 Wt, RARa�/�, and RARg�/� cell lines (Fig. 1).

Dab2 (disabled-2), one of the two mammalian orthologues

of the Drosophila Disabled gene, is thought to be a tumor

suppressor in ovarian cancer since its expression is lost or

greatly reduced in 85% of breast and ovarian cancers (51). We

show here that Dab2 mRNA expression was up-regulated by

RA in F9 Wt cells, while Dab2 mRNA levels were not affected

by RA in F9 RARh2�/� cells (Table 1 and Fig. 1, C and D).

RARh2 Target Genes620



Furthermore, Dab2 basal mRNA levels were about 10-fold

lower in the F9 RARh2�/� cells as compared to Wt. This

indicates that in the absence of exogenous RA, RARh2 can

positively regulate the Dab2 gene. Dab2 mRNA expression in

F9 RARg�/� cells was also low, both before and after RA

treatment, as compared to Wt cells (Fig. 1, C and D), indicating

that RARg may also modulate its expression.

Foxq1 (formerly Hfh-1L) belongs to the family of the FOX

(Forkhead box) transcription factors (previously called HNF-3/

forkhead transcription factors) (52). RA strongly induced Foxq1

expression in F9 Wt cells, but not in F9 RARh2�/� cells. For

instance, Foxq1 mRNA was induced by 5.6- and 7.8-fold after

48 and 72 h RA treatment in F9 Wt cells. By contrast, in F9

RARh2�/� cells, after 48 and 72 h, Foxq1 mRNA was only

slightly induced (Fig. 1, A and B; Table 1). However, Foxq1

appeared not to be a specific RARh2 target. Foxq1 mRNA

expression in F9 RARg�/� cells was also low, both before and

after RA treatment, as compared to Wt cells (Fig. 1, A and B),

indicating that RARg may also modulate its expression.

Meis1a , a member of a family of genes encoding the TALE

(three-amino-acid loop extension) subset of homeodomain-

containing proteins (53), acts as a cofactor of certain Hox

proteins and may play a major role in normal hematopoiesis

(54). Meis1a mRNA was expressed in F9 Wt, RARa�/�, and

RARg�/� cell lines in the absence of RA. In contrast, Meis1a

mRNA is barely detectable in the untreated F9 RARh2�/� cells,

indicating that the basal expression level ofMeis1a mRNA in F9

Wt, RARa�/�, and RARg�/� cells may be regulated by RARh2

in the absence of exogenously added ligand. After 24 h of RA

treatment, the Meis1a mRNA level was increased by 3-, 3-, and

2.5-fold in the F9 Wt, RARa�/�, and RARg�/� cell lines,

respectively, but it was not changed in the F9 RARh2�/� cells

(Fig. 1, A and B).Meis1a mRNAwas only slightly induced in the

F9 RARh2�/� cells, even after 48 or 72 h RA treatment, indicating

that Meis1a is a specific RARh2 target gene in these cells.

Midkine is a member of a family of heparin-binding growth

factors and was originally isolated as a retinoic acid-responsive

gene (55). midkine mRNA was increased by RA in F9 Wt,

RARa�/�, and RARg�/� cells, and after 48 h, RA increased

midkine mRNA levels by 5.5-, 4-, and 5-fold, respectively. In

contrast, RA led to a slight reduction in the midkine mRNA

level in F9 RARh2�/� cells (Fig. 1, C and D). Thus, midkine

displays the features of a RARh2 specific target gene.

Glutamate dehydrogenase (GDH) is a mitochondrial enzyme

that catalyzes the oxidative deamination of glutamate to a-

ketoglutarate using NAD+ or NADP+ as a cofactor (56). In the

brain, besides its function in metabolism, GDH influences

glutamate neurotransmitter availability (57). The basal GDH

mRNA level was lower in F9 RARh2�/� cells than in F9 Wt

cells (data not shown). RA treatment caused an induction of

glutamate dehydrogenase mRNA expression in F9 Wt cells, but

not in F9 RARh2�/� cells (Table 1).

Finally, glutathione S-transferase homologue (p28) is a small

stress response protein which belongs to a family of GST-like

(glutathione S-transferase) proteins (58). p28 expression was not

affected byRA at either 24 or 48 h inWt, RARa�/�, or RARg�/�

cells, and its expression was 4-fold lower in F9 RARh2�/� cells

compared to the other three cell lines (Fig. 1, C and D). Thus, p28

exhibits the properties of an RARh2 specific target gene.

Target Genes Identified by Microarray AnalysesWe used gene microarray analyses to identify RARh2 target

genes, and obtained EST clones for the genes showing the

largest fold changes between F9 Wt cells and F9 RARh2�/�

cells. Seven out of the seven target genes from microarray

analysis were confirmed by semi-quantitative RT-PCR or

Northern analysis, indicating that the microarray analysis was

generally reliable. These genes are listed in Table 1. Dab2 and

Foxq1 were also identified by subtractive hybridization and

were discussed above.

c-myc is a proto-oncogene that is activated in various animal

and human tumors. It is a transcription factor that plays a

pivotal role in many biological functions including cell growth

and differentiation (59, 60). c-myc mRNA in untreated

RARh2�/� cells was expressed at a 3.4-fold higher level than

in untreated F9 Wt, RARa�/�, or RARg�/� cells. Furthermore,

c-myc mRNA expression in F9 RARh2�/� cells is 4.5-fold

higher than in F9 Wt cells after 6 h of RA treatment (Fig. 1, A

and B). After 24 h of RA treatment, the expression of c-myc

mRNA is reduced by 3.7-, 4-, and 4.9-fold in F9 Wt cells,

RARa�/�, and RARg�/� cells, respectively, while in the

RARh2�/� cells, the c-myc mRNA level is still higher than in

Wt cells. These data suggest that RARh2 suppresses c-myc

mRNA expression in F9 Wt cells even in the absence of

exogenously added RA.

Table 1. Summary of Genes Differentially Expressed in RA-Treated F9 Wt and F9 RARB2�/� Cells

GenBank Identity GenBankAccession No.

Method of Identification Regulation by RAin Wild-Type F9 Cells

RARh2

Specific

Myelocytomatosis oncogene (c-myc ) L00039 Microarray # YFriend of GATA-1 (FOG ) AF006492 Microarray " YGATA-binding protein 6 U51335 Microarray " YGlutamate dehydrogenase (GLUD ) X57024 Subtractive hybridization " YGlutathione S -transferase homologue (p28) MMU80819 Subtractive hybridization NC YFoxq1 (Hfh-1L) AF010405 Microarray and subtractive hybridization " NHic-5 L22485 Microarray NC YMeis1a U33629 Subtractive hybridization " YMitogen-responsive phosphoprotein p96 (Dab2 ) U18869 Microarray and subtractive hybridization " Nmidkine M35833 Subtractive hybridization " YPDGF-a receptor M57683 Microarray " N

Note: NC, no change; Y, yes; N, no.

Molecular Cancer Research 621






FOG1 is a member of the FOG (for Friend of GATA) family

of multitype zinc finger proteins that interact with GATA and

play essential roles in development (61). FOG1 is expressed in

erythroid cells and megakaryocytes, liver, and testis (61).

GATA6 is a member of the GATA family of transcription

factors which regulate gene expression during development

(62). GATA6, together with GATA4 and GATA5, is involved in

the formation of the extra-embryonic and embryonic endoderm,

as well as the cardiogenic mesoderm (63). FOG1 and GATA6

mRNAs were increased by RA in the F9 Wt, RARa�/�, and

RARg�/� cell lines, but were not increased by RA or increased

to a lesser extent in the F9 RARh2�/� cells (Fig. 1A). Thus,

FOG1 and GATA6 are RARh2 specific target genes.

Hic5 is a focal adhesion protein that is very similar to

paxillin (64). Studies have suggested that Hic5 is involved in

the negative regulation of cell growth, since overexpression of

Hic5 results in cell growth inhibition (65). Like p28 , the

expression of Hic5 is not affected by RA treatment, but basal

Hic5 mRNA expression is much higher in F9 Wt cells than in

F9 RARh2�/� cells (Table 1).

PDGF-a receptor (PDGFaR) is one of the two receptors for

platelet-derived growth factor (PDGF); it has been suggested

that the PDGFaR plays a role in embryonic development (66).

It was previously shown that PDGFaR expression is up-

regulated during RA-induced F9 cell differentiation (67). Our

results show that the PDGFaR mRNA level is increased by RA

in F9 Wt cells, but not in F9 RARh2�/� cells (Table 1).

PDGFaR mRNA levels are also lower in F9 RARa�/� and F9

RARg�/� cells, indicating that multiple RARs can regulate the

expression of this gene.

Target Genes are Transcriptionally Regulated by RARb2

To determine if the target genes were transcriptionally

regulated by RARh2, we inhibited transcription by using

actinomycin D, an inhibitor of RNA polymerase II (68). F9 Wt

cells and F9 RARh2�/� cells were first treated for 12 h with or

without 1 AM RA. At 12 h, RNAwas isolated from some dishes

of cells, while actinomycin D (2 Ag/ml) (69) was added to other

dishes of cells to inhibit RNA transcription. RNA was isolated

after an additional 6 h. The RNA samples were analyzed by

Northern blot analysis (Fig. 2). Consistent with results shown in

Fig. 1, RA induced target gene expression in F9 Wt cells, but

not in the F9 RARh2�/� cells. It is also worth noting that the

stimulatory effect of RA can be seen as early at 12 h. Treatment

of Wt cells with actinomycin D alone resulted in a decrease in

target gene mRNA. Furthermore, RA induction of target gene

mRNA was completely blocked by treatment with actinomycin

D, indicating that ongoing transcription is required for RA-

induced expression of target genes. The c-myc mRNA level

seen after 18 h of RA treatment in F9 RARh2�/� cells was

greatly reduced after 6 h of actinomycin D treatment, which is

consistent with the previous finding that the half-life of c-myc

transcripts in F9 cells is about 40 min (70).

RARb Agonists, in the Presence of a RXR Agonist,Stimulate the Expression of Target Genes in F9 Wt butnot in F9 RARb2

�/� CellsTo study further the function of RARh2 in the regulation

of its target genes, F9 Wt and RARh2�/� cells were treated

for 48 h with 1 AM RA or RARh selective agonists (100 nM

BMS185411 or 1 AM CD2314), and/or a pan-RXR agonist

(1 AM BMS188649 [BMS649]) (Fig. 3). Neither BMS185411

nor CD2314, the RARh selective agonists, alone could

induce the expression of any target genes in F9 Wt cells.

Similarly, BMS649 (pan-RXR) alone had no effect on the

expression of target genes. However, BMS649 (pan-RXR)

and the RARh selective agonists (BMS185411 or CD2314)

synergistically induced the expression of all RARh2 target

genes in F9 Wt cells, but not in RARh2�/� F9 cells. Thus,

RXR-RARh2 heterodimers appear to play a major role in

FIGURE 2. Northern blot analysis of the effects of actinomycin D onRARh2 target gene expression. F9 Wt cells and F9 RARh2

�/� cells weregrown for 12 h in the absence (�) or presence (+) of 1 AM RA. At 12 h, RNAwas isolated or 2 Ag/ml transcriptional inhibitor actinomycin D (ACT D )was added to the medium and RNA was isolated after 6 h. After sizefractionation on the gel, the RNA was transferred to nylon and hybrid-ization to different cDNAs for the target genes was performed. Thisexperiment was performed twice with similar results; one experimentis shown.

FIGURE 1. Northern blot analysis of the clones obtained from microarray analysis and subtractive hybridization analysis. A. F9 Wt cells, RARh2�/�, a�/�,

and g�/� F9 cells were treated with 1 AM RA for different times and total RNA was extracted. After size fractionation on the gel, the RNA was transferred to

nylon and hybridized to different cDNAs for the target genes. h-Actin was used as an RNA loading control. This experiment was repeated three to five timeswith different RNA preparations and very similar results were obtained. A representative experiment is shown. B. Quantitative analysis of the effect of 1 AM

RA on the mRNA levels of some target genes. C. Northern blot analysis of three clones obtained from subtractive hybridization analysis. This experiment wasrepeated three to five times with different RNA preparations and very similar results were obtained. One representative experiment is shown. D. Quantitativeanalysis of the effect of 1 AM RA on the mRNA levels of the target genes. Quantitative analyses in panels B and D were performed using NIH Image 1.62 orImage Quant 1.2. The mRNA levels are normalized to h-actin mRNA and are expressed relative to the value derived from Wt cells not treated with RA.Columns, means from three to five experiments; bars, SE. Y-axes , arbitrary density units.




mediating the induction of the expression of RARh2 target

genes. This is consistent with the conclusion that RXR-RAR

heterodimers are the functional units transducing the retinoid

signal (9).

Expression of RARb2 Target Genes Is Induced in F9 WtCells Treated With RACT, but not in F9 RARb2

�/� CellsRACT (retinoic acid, dibutyryl cAMP, and theophylline)

treatment causes F9 Wt cells to differentiate into parietal

endoderm, while RA alone results in primitive endoderm (71).

We examined the regulation of the expression of some of the

RARh2 target genes in F9 cells treated with RACT. F9 Wt or

F9 RARh2�/� cells were treated with RACT, or different

combinations of RARh agonist and/or RXR agonist in the

presence of CT (dibutyryl cAMP and theophylline) for 48 h.

The RNA samples were analyzed by Northern blot analysis

(Fig. 4). As expected, CT alone did not have an effect on

expression of target genes (72). Like RA, RACT induced the

expression of target genes in F9 Wt cells. Addition of the pan-

RXR agonist BMS649 decreased the Foxq1 , FOG1 , and

Meis1a expression induced by RACT in F9 Wt cells, but

increased the GATA6 expression induced by RACT. In the

presence of CT, BMS649 (pan-RXR) and the RARh selective

agonists (BMS185411 or CD2314) induced the expression of

FIGURE 3. Expression of RARh2 target genes is increased by a RARh-specific agonist and pan-RXR agonist. A. F9 Wt cells and RARh2�/� cells were

treated for 48 h with 1 AM RA, 1 AM or 100 nM RARh-specific agonist (BMS185411 or CD2314 ), 1 AM pan-RXR agonist (BMS188649), or differentcombinations of drugs. Total RNA was then extracted and size fractionated on the gel. The RNA was transferred onto nylon and hybridized to different cDNAsfor the target genes. The experiments were repeated with different RNA preparations. h-Actin is used as an RNA loading control. One representativeexperiment is shown here. B. Quantitation of the effects of different agonists on the mRNA levels of the target genes in F9 Wt cells. The mRNA levels arenormalized to h-actin mRNA and are expressed relative to the value derived from Wt cells not treated with RA. Columns, means from three experiments;bars, SE. Y-axes , arbitrary units.

FIGURE 4. Expression of RARh2 target genes in F9 Wt cells and RARh2�/� cells treated with RACT. F9 Wt cells and F9 RARh2

�/� cells were treated for 48h with 1 AM RA, 100 nM (CD2314 ) or 1 AM (BMS185411 ) RARh-specific agonist, 1 AM pan-RXR agonist, or different combinations in the presence of CT[dibutyryl cAMP (250 AM) and theophylline (250 AM)]. Total RNA (10 Ag) was then extracted and size fractionated on the gel. The RNA was transferred ontonylon and hybridized to different cDNAs for the target genes. The experiments were performed two times with different RNA preparations. h-Actin is used asan RNA loading control. Quantitation of some of the mRNAs is shown below the figure.







all RARh2 target genes in F9 Wt cells. In addition, retinol (Rol;

vitamin A) induced the expression of all of the RARh2 target

genes in F9 Wt cells. In contrast, in F9 RARh2�/� cells,

expression of FOG1 , Foxq1 , GATA6 , and Meis1a mRNA was

very low with various treatments (Fig. 4).

We also studied the regulation of expression of the Hoxa1

gene, a gene we have studied previously (23, 69, 73, 74).

Unlike the RARh2 target genes, a similar pattern of Hoxa1

mRNA expression was observed in F9 Wt and F9 RARh2�/�

cells (Fig. 4). Hoxa1 mRNA expression was induced by RACT

in both F9 Wt and the RARh2�/� cells. Furthermore, Hoxa1

mRNA expression was not significantly increased by the RXR

agonist (BMS649) and RARh selective agonists (BMS185411

or CD2314) in the presence of CT (Fig. 4). Rol only slightly

induced Hoxa1 mRNA expression in F9 Wt cells; thus, Rol

treatment was more effective in inducing several of the RARh2

target genes, especially GATA6 and Foxq1 , than in inducing

Hoxa1 mRNA, consistent with our prior genetic data that the

Hoxa1 gene is primarily transcriptionally regulated by RARg

and not RARh2 in F9 cells (23).

DiscussionIn this study, using subtractive hybridization and microarray

analyses, we identified some of the RARh2 targets in F9 cells.

To our knowledge, this is the first time that specific RARh2

target genes have been identified and confirmed by a

comparison of Wt, RARh2�/�, RARa�/�, and RARg�/� cell

lines. Most of the target genes are regulated by RA, though our

data suggest that some genes, such as Dab2 , Hic5 , and p28 , are

also regulated by RARh2 in the absence of exogenously added

RA. The RARh2 target genes are also induced by Rol (Fig. 4)

to a greater extent than a primary RA target gene such as

Hoxa1 , which is not an RARh target (23). As we have shown

previously that Rol is not metabolized to RA in F9 cells (75), a

Rol metabolite, 4-oxoRol, is likely to be involved in the

regulation of these target genes in F9 cells. We have not yet

determined the number of primary versus secondary RARh2

target genes, as this requires much more extensive character-

ization of the promoters of the genes. The target genes

identified in this study encode a wide range of proteins that

regulate a variety of biological processes. This suggests that, in

addition to the regulation of cell proliferation, the transcription

factor RARh2 participates in the regulation of many biological

processes via the transcription of multiple target genes.

We found that some target genes are RARh2 specific

(Table 1) in that the F9 RARh2�/� cells exhibited reduced or no

expression of these target genes, but the F9 RARa�/� and F9

RARg�/� cell lines exhibited expression levels similar to those

in F9 Wt cells. These data provide evidence that each RAR

isoform has distinct functions.

Target Genes and Cell Growth and CancerConsistent with our data showing that the induction of

RARh2 is necessary for the growth arrest of F9 cells, two of the

target genes we identified, c-myc and Dab2 , from this study

have been shown to play major roles in cell growth and

proliferation. The c-myc proto-oncogene, the cellular homologue

of the retroviral v-myc oncogene, is activated in various animal

and human tumors (76). c-Myc is a transcription factor which

regulates many biological processes, including the cell cycle and

cell differentiation (77). The c-Myc protein can regulate some

cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors,

which are cell cycle regulators (78). Deregulated c-myc

expression often results in increased cyclin E and cyclin A

expression (79, 80). In addition, expression of c-myc can

decrease p27 levels and interfere with its function (81).

Our data indicate that expression of c-myc mRNA is repressed

by RARh2 in F9Wt cells, even in the absence of exogenous RA,

and that c-myc mRNA is expressed at a much higher level in

untreated and 24 h RA treated F9 RARh2�/� than F9 Wt cells

(Fig. 1). We conclude that in F9 Wt cells, a low level of RARh2

expression (without exogenous RA addition but in the presence

of low levels of Rol from serum) inhibits c-myc mRNA

expression. The reduction of c-myc mRNA by RA in F9 Wt

cells has been observed in several studies, and down-regulation

of c-myc mRNA by RA is associated with RA-induced growth

arrest of F9 cells (82). The increased level of c-myc mRNA in

RARh2�/� cells may play a role in preventing these cells from

undergoing growth arrest on RA treatment. Since changes in the

c-myc gene or its expression are associated with approximately

one-seventh of U.S. cancer deaths (76), experiments will be

carried out to determine if the loss of RARh2 expression in

tumors causes the overexpression of c-myc transcripts and

concomitant abnormal regulation of tumor cell growth.

RA has been shown to regulate Dab2 expression during

embryonic development (83). Moreover, Dab2 has been

suggested to be a major mediator of RA cell growth inhibition.

Transient transfection of Dab2 was sufficient to uncouple

MAPK activation and c-fos expression, and was associated

with growth inhibition in F9 cells (84). Since we found Dab2 to

be a RARh2 target in this study, RARh2 could mediate the

growth inhibitory effects of RA by up-regulating Dab2

expression. Studies of GATA6-deficient mouse embryos

suggest that Dab2 expression is regulated by GATA6 (85).

Since we have shown that GATA6 is also a RARh2 specific

target, Dab2 may be an indirect target of RARh2 and may be

regulated by RARh2 through GATA6. The lack of a known

RARE in the Dab2 promoter (86) supports this suggestion. The

observation that the expression of Dab2 mRNA is eliminated in

85–95% of breast and ovarian tumors suggests that it is a tumor

suppressor gene (51). Indeed, overexpression of Dab2 reduced

the tumorigenicity of carcinoma cells (87). Conversely, the

reduction of RARh2 levels in human breast tumors (88) could

result in the down-regulation of Dab2 , which in turn could alter

the control of cell proliferation.

Target Genes and DevelopmentSeveral targets identified in this study provide insight into

the roles of RARh2 in development. Meis1a is a member of the

homeobox gene family of transcription factors and has been

suggested to play a role in peripheral neural system develop-

ment. Correct expression of Meis1a is essential for correct

patterning in the embryonic peripheral nervous system (89).

Our finding that Meis1a is a target of RARh2 is consistent with

the finding that RARh is expressed and plays a role in the

developing nervous system (90).




FOG1 is the founding member of the FOG family of

proteins that interact with GATA transcription factors and

modulate their activity (61). FOG1 knockout mice die at

embryonic day 11.5 of gestation because of arrested erythroid

cell maturation and complete failure of megakaryopoiesis (91).

As FOG1 was shown to be a target of RARh2 in these F9 cells,

RARh2 may play a role in megakaryopoiesis.

The GATA6 transcription factor is involved in the formation

of the extra-embryonic and embryonic endoderm, as well as in

cardiogenic mesoderm (92). GATA6 is essential for lung

epithelial differentiation in the mouse embryo (63). Our result

that GATA6 is a RARh2 specific target suggests that RARh has

a role in the lung epithelial cell differentiation. Consistent with

this, RARh is expressed in the epithelia of the lungs (93).

In the midgestation period, midkine is expressed in the

endoderm-derived lung epithelium and the adjacent mesen-

chyme, which suggests a key role for midkine in the regulation

of lung development (94). Interestingly, RARh is also

expressed in the endoderm-derived lung epithelium and the

adjacent mesenchyme at the same stage of embryogenesis. In

the embryo, PDGF-A is expressed primarily by epithelial cells,

whereas the PDGF-a receptor is expressed primarily in

mesenchymal cells. This ligand-receptor pair is thought to be

important to epithelial-mesenchymal signaling in the lung

during development (95). These findings, together with our

results that midkine and the PDGF-a receptor are RARh2

target genes, suggest that RARh plays a role in epithelial-

mesenchymal signaling during lung development.

Foxq1 is expressed throughout embryonic development of

the mouse (52). Although its function remains unclear, it is

likely to play a crucial role during embryogenesis, given its

similarity to other family members. Another family member,

Foxp2 , is involved in the development of speech (96).

Further characterization of the RARh2 target genes identi-

fied in this study will be helpful in increasing our understanding

of the functions of RARh2 in a variety of biological processes.

Future studies should also provide important insights into the

molecular mechanisms by which the silencing of the RARh2

gene leads to cancer.

Materials and MethodsMaterials

The RARh agonist BMS185411 and the pan-RXR agonist

BMS188649 are from Bristol-Myers Squibb (Buffalo, NY). The

RARh agonist CD2314 is from CIRD Galderma (Sophia

Antipolis, Valbonne, France). Actinomycin D was purchased

from Sigma Chemical Co. (St. Louis, MO) and used at a

concentration (2 Ag/ml) shown to inhibit RNA synthesis (69).

Cell Culture, RNA Extraction, and Northern Blot AnalysisF9 Wt, RARh2

�/�, RARa�/�, and RARg�/� cells were

maintained in DME supplemented with 10% bovine calf serum

and 2 mM glutamine. Total cellular RNA was extracted using

RNA-STAT 60 (Tel-Test, Friendswood, TX) following the

manufacturer’s protocol. Northern blot analysis was performed

as described previously (97). Briefly, total RNA (10 Ag) wasresolved on 1% agarose gels containing 5% formaldehyde and

RNA was transferred to nylon filters. Nylon filters were

prehybridized, and hybridized at 42jC in 50% (v/v) formamide,

5� SSC, 50 mM NaH2PO4-Na2HPO4 (pH 7.4), 5 mM EDTA,

and 0.1 mg/ml salmon sperm DNA. cDNA probes of target

genes were labeled with [32P]dCTP using a random primer

labeling kit (Roche, Indianapolis, IN). cDNA inserts for target

genes were either from the F9 cDNA library described below or

were commercially available EST clones. EST clones for

GATA6 (AI894084), PDGF-a receptor (BE630572), and FOG1

(AW763708) were obtained from Incyte (Palo Alto, CA). EST

clones for c-myc (BG243402) and Hic5 (BF536993) were from

ATCC. Northern blots were quantitated by phosphorimager

analysis, and the software NIH Image 1.62 or Image Quant 1.2.

Subtractive HybridizationA PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto,

CA) was used to carry out subtractive hybridization analysis. F9

Wt teratocarcinoma cells and the F9 RARh2�/� line were treated

with 1 AM all-trans RA for 24 h and total RNA was extracted.

After reverse transcription, cDNAs from the two cell lines were

subjected to subtractive hybridization according to the manu-

facturer’s protocol. Products from the secondary PCR reactions

were radiolabeled with [a-32P]dCTP and hybridized to duplicate

filters containing phage clones from a cDNA library in a Lambda

ZAP vector; this cDNA library was synthesized from F9Wt cells

treated with RA for 72 h.1 After autoradiography, the filter

patterns of F9 Wt cells and of F9 RARh2�/� cells were compared

for each spot. Differentially hybridized clones were picked and

the inserts were subjected to Northern blot analysis.

DNA Microarray AnalysisTotal cellular RNA was extracted from F9 Wt teratocarci-

noma cells and the F9 RARh2�/� cells, both of which were

treated with 1 AM all-trans RA for 24 h. Microarray analysis

was carried out according to the Affymetrix Genechip

expression analysis technical manual. Briefly, total RNA from

both cell lines was reverse transcribed into cDNA. After

second-strand synthesis, cDNAs were then in vitro transcribed

into cRNAwith biotinylated ribonucleotides (Enzo Diagnostics,

Farmingdale, NY). cRNA (20 Ag) was fragmented by heating at

94jC for 35 min. A cocktail containing fragmented cRNA,

control oligonucleotide B2, control cRNA (Biotin B, Biotin C,

Biotin D, and Cre), and herring sperm DNA was hybridized to

microarray chips (MG-U74Av2) for 16 h at 45jC. After

washing and staining, the distribution of fluorescent material on

the array was measured using a laser scanner. The resultant

image was processed with MAS (Microarray Suite) software

(Affymetrix, Santa Clara, CA). The microarray experiments

were repeated three times with different RNA preparations.

Only genes which exhibited different expression patterns

between the F9 Wt and F9 RARh2�/� cells in three independent

array experiments were further analyzed.

Microarray Data AnalysisThe Microarray Core of the Weill Medical College of

Cornell performed data preparation and data analysis.

1Thompson and Gudas, unpublished data.




The expression level of each gene and the fold change

between genes from F9 Wt cells and F9 RARh2�/� cells were

calculated using a computer program (GeneSpring, Redwood,

CA). This computer program was also used for functional

gene grouping analyses.

AcknowledgmentsWe thank members of the Gudas laboratory for scientific discussions.

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2003;1:619-630. Mol Cancer Res Yong Zhuang, Teresa N. Faria, Pierre Chambon, et al. Pharmacology Training Grant (CA62948-08) (Y.Z.).Institute for Cancer Research; and in part by a CancerR01 CA43796 to L.J.G.; a research grant from the American

NIH grant 1 1 Target Genes in F9 Teratocarcinoma Cells2βIdentification and Characterization of Retinoic Acid Receptor

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Identification and Characterization of Retinoic Acid ...Identification and Characterization of Retinoic Acid Receptor B 2 Target Genes in F9 Teratocarcinoma Cells Yong Zhuang,1 Teresa