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).
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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.
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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.
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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.
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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).
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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.
Molecular Cancer Research 627
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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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RARh2 Target Genes630
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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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