Received for publication, November 19, 1999, and in revised form, January 24, 2000
Retinoids are essential for normal
epidermal growth and differentiation and show potential for the
prevention or treatment of various epithelial neoplasms. The retinoic
acid receptors (RAR
, -
, and -
) are transducers of the retinoid
signal. The epidermis expresses RAR and RAR, both of which are
potential mediators of the effects of retinoids in the epidermis. To
further investigate the role(s) of these receptors, we derived
transformed keratinocyte lines from wild-type, RAR, RAR, and
RAR null mice and investigated their response to retinoids,
including growth inhibition, markers of growth and differentiation, and
AP-1 activity. Our results indicate that RAR is the principle
receptor contributing to all-trans-retinoic acid
(RA)-mediated growth arrest in this system. This effect partially correlated with inhibition of AP-1 activity. In the absence of RARs,
the synthetic retinoid N-(4-hydroxyphenyl)-retinamide
inhibited growth; this was not observed with RA, 9-cis RA,
or the synthetic retinoid (E)-4-[2-(5, 5, 8, 8 tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propenyl] benzoic
acid. Finally, both RAR and RAR differently affected the
expression of some genes, suggesting both specific and overlapping roles for the RARs in keratinocytes.
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INTRODUCTION |
Vitamin A derivatives (retinoids) play central roles in embryonic
development and maintenance of various tissues in the adult (1-3).
Retinoids also exhibit potent antitumorigenic properties in diverse
model systems and show potential for the treatment of a number of human
malignancies, including diverse epithelial cancers or pre-cancerous
lesions (4-9).
The retinoid signal is transduced by two families of nuclear receptors,
the retinoic acid (RA)1
receptors (RAR, -, and - and their isoforms) and the retinoid x receptors (RXR, -, and -) (10-13). RARs function as
ligand-inducible transcription regulators by binding, together with an
RXR partner, to specific cis-acting response elements
(RAREs). RARs can be activated by both RA and its stereoisomer,
9-cis RA, whereas RXRs are activated only by
9-cis RA (14). RXRs are also essential heterodimeric
partners for a number of other nuclear receptor signaling pathways,
including thyroid hormone, vitamin D, and certain orphan receptors (15,
16). Although 9-cis RA is not obligatory for transcriptional
regulation via these pathways, some results suggest that RXR-specific
ligands can elicit transcriptional activation in certain settings
(e.g. Refs. 17-20).
RARs, like several other nuclear receptors, can function in a
ligand-dependent manner to inhibit AP-1 activity, and it
has been suggested that the affect of retinoids on the growth of
transformed cells may occur through this trans-repression
mechanism (21-23). This inhibition is believed to be due, at least in
part, to competition for limiting amounts of transcriptional
co-factors, such as CBP and/or its homologue p300, common to both
pathways (24, 25). Other mechanisms, such as inhibition of the
expression of AP-1 family members or c-Jun N-terminal kinase (JNK), may
also contribute to this cross-talk (26-29).
Gene targeting of the various RARs has revealed essential and
diverse roles for these receptors (2, 30, 31). However, because of
perinatal or embryonic lethality inherent to many of these RAR null
backgrounds, there is a void in our knowledge of RAR function in a
number of contexts, such as tumorigenesis.
Exogenous retinoids can attenuate the effects of tumor promoters in the
two stage skin carcinogenesis protocol (9, 32). Among the retinoid
receptors, normal epidermis expresses RAR and RAR as well as
RXR and RXR, with RAR and RXR as the predominant
heterodimer (33, 34). This pattern of expression prompted us to
investigate the roles of RAR and RAR in mediating the
antitumorigenic effects of retinoids in epithelial keratinocytes. To
this end, we established RAR, RAR, and RAR null
keratinocyte lines by transformation with a dominant-negative p53
expression vector and compared the properties of these various lines.
Our results demonstrate that RAR and RAR affect different aspects of retinoid response in these transformed cells, with RAR being the
primary mediator of RA-induced growth inhibition. However, other
synthetic ligands affected proliferation independent of the RARs.
RAR-dependent, but not -independent, growth inhibitory effects generally correlated with the attenuation of AP-1
transcriptional activity. Finally, the effects of RAR and RAR on
expression of certain keratinocyte markers suggests that each RAR may
perform a subset of specific functions, which cannot be entirely
fulfilled by other RARs in this cell type.
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EXPERIMENTAL PROCEDURES |
Primary Keratinocyte Culture and Immortilization--
The RAR
null mice used in these studies have been described previously (35,
36). RAR, RAR, and RAR mutants were generated from the
appropriate matings, whereas wild-type offspring were obtained from
RAR+/
intercrosses. Fetuses were procured by caesarean
section at 18.5 days post coitus, and genotype was determined by
polymerase chain reaction as described (37). Primary keratinocyte
cultures were established from the epidermis by standard means (38) and
cultured in S-minimal essential medium with 10% chelex-treated fetal
calf serum (calcium concentration of 0.5 mM), insulin (5 µg/ml), hydrocortisone (0.5 µM), MgCl2 (1.5 mM), cholera toxin (1.2 × 1011
M), adenine (24 µg/ml), and gentimycin (10 µg/ml). The
next day, the cells were fed with medium further supplemented with
epidermal growth factor (10 ng/ml) and expanded for several days.
Cultures were treated at 3-5 days post-plating with versene (0.5 mM EDTA in phosphate-buffered saline) to remove
contaminating fibroblasts. The cells were subcultured at a 1:3 ratio at
most 2 times prior to transformation.
A single 10-cm plate of cells (~2 × 106) of each
genotype was harvested, and cells were resuspended in 800 µl of
medium. The cells were then electroporated (250 mV, 960 microfarads in
a 0.4-cm gap cuvette) with 25 µg of a linearized expression vector
harboring a mutated p53 from the Friend erythroleukemia cell line CB7
(39). Cells were plated and routinely subcultured until past crisis. All experiments were performed using cultures between passage 16 and 26.
Growth Assays--
Transformed keratinocytes were seeded into
96-well plates at a cell density of 500 cells/well and were treated the
following day with vehicle (Me2SO) or the appropriate
retinoid (RA, 9-cis RA, 4-HPR, or TTNPB). Medium was
replenished every second day. Growth was assessed either in response to
varying concentrations of retinoid at eight days post-plating or over
time in response to 106 M ligand. DNA content
was assessed as a measure of cell growth using crystal violet staining
as described previously (40). Relative dye binding was assessed by OD
at 590 nm using a microplate reader. Results were expressed either as
A590 values or as growth relative to untreated
controls and were derived from the mean (± S.D.) of four replicate wells.
Transient Transfection and AP-1 Activity
Assay--
Transfections were performed using Lipofect ACE reagent
(Life Technologies, Inc.). Briefly, cells were plated in 6-well cluster plates at 4 × 104 cells/well. Transfections consisted
of 0.5 µg of AP-1 reporter or appropriate control (41), either alone
or with expression vectors encoding c-Fos, c-Jun, CBP, p300, or RARs.
Total DNA (5 µg; normalized with KS+) was mixed with 10 µl of lipid
and added to 100 µl of serum-free S-minimal essential medium. The
lipid/DNA mixture was then added to the cells in 1 ml of complete
medium and incubated at 37 °C overnight. Medium was changed daily,
and luciferase activity was assessed 48 h post-transfection.
Results were corrected for protein concentration and are expressed as the mean (± S.D.) from three independent transfections. All
experiments were repeated at least three times with comparable results.
Electrophoretic Mobility Shift Assays and Western Blot
Analysis--
Cells were cultured in 10-cm plates in the presence of
RA (106 M) or vehicle for 48 h prior to
harvest. Nuclear proteins were isolated from each cell line, and
protein concentration was determined using the DC protein assay kit
(Bio-Rad). Electrophoretic mobility shift assays were performed
essentially as before (42). Briefly, binding reactions containing ~2
ng of probe (50,000 cpm) and 5 µg of nuclear protein were separated
by electrophoresis through a 6% polyacrylamide gel containing 0.25 × Tris borate and EDTA. Specificity of binding was assessed by
competition with a 10-fold excess of unlabeled RARE
(5'-GGGTAGGGTTCACCGAAAGTTCACTCGCA) or AP-1 (5'-GATCCGATGAGTCAGCCA)
double-stranded oligonucleotides.
For Western blot analysis, 40 µg of nuclear protein from the various
cell lines were size fractionated on a 10% SDS-polyacrylamide gel
electrophoresis and electroblotted to Immobilon-P polyvinylidene difluoride membrane as recommended by the supplier (Millipore). Proteins of interest were detected by incubation with the desired antibodies and detection with an ECL kit (Amersham Pharmacia Biotech) as per the manufacturer's instructions. Antibodies were purchased from
Santa Cruz Biotechnology.
Northern Blot Analysis--
Fifteen micrograms of total RNA,
isolated by Trizol reagent (Life Technologies, Inc.), were size
fractionated on a 1% agarose-formaldehyde gel in MOPS buffer and
transferred to a MAGNA nylon membrane (MSI). Fragments were isolated by
restriction digestion of cDNAs followed by purification by
Geneclean and used to generate probes by labeling with
[-32P]CTP by random priming with an oligo labeling kit
(Amersham Pharmacia Biotech). Membranes were hybridized according to
the manufacturer's directions.
| |
RESULTS |
Generation of RAR Null Cell Lines--
Primary cultures of
wild-type and RAR null keratinocytes showed no major differences in
morphology, growth, or immortalization with dominant-negative p53. All
lines grew well for at least 40 passages, suggesting that they were
immortalized. None of the lines formed colonies in soft agar or were
tumorigenic in nude mice.2
Electrophoretic mobility shift assay revealed that, relative to
wild-type extracts, disruption of RAR, and to a greater extent RAR, decreased specific binding to an RARE, and association was completely abolished in extracts from RAR double null cultures (Fig. 1). Northern blot analysis
confirmed the disruption of RAR and/or RAR message in the
appropriate cell line (data not shown). RAR transcripts were
undetectable in all lines by Northern blot or polymerase chain reaction
approaches, consistent with previous studies indicating that this
receptor type is not expressed in epidermal keratinocytes (33, 43).
These data suggest that there is no compensatory up-regulation of the
remaining receptors in response to disruption of a given RAR.
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Fig. 1.
Electrophoretic mobility shift assay of an
RARE sequence by nuclear extracts from transformed keratinocyte
lines. Nuclear extracts (5 µg) were incubated with a labeled
double-stranded RARE oligonucleotide probe (50,000 cpm) and bound and
free probe separated by polyacrylamide gel electrophoresis. Binding was
competed with 10-fold excess RARE probe (C) but not by a
nonspecific probe comprised of an AP-1 recognition sequence
(NSC). Note the presence of a nonspecific complex
(N/S) migrating slightly faster than the RAR-containing
complex (RAR). WT, wild type.
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Contribution of Specific RARs to Retinoid-mediated Growth
Inhibition--
All transformed cell lines exhibited similar
morphology and growth characteristics in the absence of retinoid
treatment (Fig. 2). However, wild-type
and RAR/ cultures were growth inhibited by
106 M RA (Fig. 2). In marked contrast,
RAR/ cells were highly resistant and
RAR/ cultures were completely resistant to these
effects. The growth arrest observed in wild-type and RAR null
cultures was likely because of the inhibition of proliferation as
opposed to apoptosis, as judged by thymidine incorporation and
programmed cell death assays (data not shown).
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Fig. 2.
Growth of transformed keratinocyte
lines. The plots indicate growth of transformed keratinocyte lines
in the absence (closed circles) or presence of
106 M RA (open circles) over 11 days. Microphotographs show representative cultures after 6 days of
growth in the presence of vehicle (DMSO, Me2SO)
or 106 M RA as denoted on the top of the
columns. Genotypes of the cultures are indicated to the
left. WT, wild type.
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Dose-response experiments were performed to determine the relative
sensitivity of the various cell lines to growth arrest by RA,
9-cis RA, or the synthetic retinoids TTNPB or 4-HPR. As shown in Fig. 3, wild-type keratinocytes
exhibited a significant reduction in proliferation at 109
M RA, with the maximal affect at
107-106 M RA.
RAR/ keratinocytes exhibited a similar profile,
although their response to RA was slightly more pronounced than
wild-type cultures. Consistent with time-course analysis,
RAR/ keratinocytes were only marginally inhibited by
the highest dose of RA examined (106 M), and
RAR/ keratinocytes were not significantly
affected by RA at any dose tested.
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Fig. 3.
Analysis of keratinocyte proliferation in
response to RA, 9-cis RA, TTNPB, and 4-HPR. Cells
were grown in the presence of vehicle or retinoids (from
1013 M to 106 M)
for 8 days. Growth was assayed by DNA content as described under
"Experimental Procedures." Results are the mean ± S.D. of
quadruplicate samples and are expressed relative to untreated cultures
for each line. WT, wild type.
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9-cis RA is a ligand for both RARs and RXRs, and RXR
agonists have been shown to induce effects on growth or differentiation in several model systems. Proliferation of both wild-type and RAR/ cultures was inhibited by 9-cis RA,
although higher concentrations were required compared with RA (Fig. 3).
Interestingly, 9-cis RA had no significant outcome on the
growth of either RAR/ or RAR/
cultures. This finding suggests that RXR activation does not lead to
growth arrest in this model system, at least in the absence of RARs.
Whether RXR-specific signaling has other biological consequences remains to be investigated.
The RAR agonist TTNPB was a very potent inhibitor of growth in
wild-type or RAR/ cultures with an effect evident at
1011-1010 M (Fig. 3). However,
TTNPB affected RAR/ and RAR/
cultures only at the highest dose tested (106
M). Whether this is indicative of effects on other pathways
or is because of nonspecific cytotoxicity is unknown.
The synthetic retinoid 4-HPR has been shown to be a potent inducer of
growth arrest and/or apoptosis in several model systems (44-46). This
compound was the least efficient of all those tested in inhibiting
proliferation of wild-type and RAR/ cultures (Fig.
3). However, in marked contrast to the other retinoids, 4-HPR affected
the growth of RAR and RAR cultures at high doses, consistent
with receptor-dependent and -independent mechanisms of
action for this compound (47-50).
RAR Regulation of AP-1 Transcriptional Activity--
RARs can
repress AP-1 transcriptional activity, and this mechanism of action has
been proposed to underlie at least some of the antitumorigenic effects
of retinoids (8, 9, 43, 51). In transient transfection assays, we found
that RA (106 M) inhibited AP-1 activity
8-10-fold in wild-type and RAR/ cultures (Fig.
4A). AP-1 activity in
RAR/ cultures was more modestly affected, typically
exhibiting 10-30% reduction, whereas activity in
RAR/ cultures was not affected. The latter line
was capable of response following re-introduction of either RAR or
RAR by transient transfection (Fig. 4B). Thus,
attenuation of AP-1 activity requires the presence of at least one
functional RAR, although there does not appear to be discrimination
between receptor types for this outcome.
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Fig. 4.
Effects of RA on AP-1 activity.
A, AP-1 reporter vector (0.5 µg/well) was transfected into
transformed keratinocyte lines and luciferase activity was assessed
following treatment with vehicle (closed bars) or RA
(106 M; open bars) as described
under "Experimental Procedures." B, empty vector or
RAR2 or RAR2 expression vectors were transfected into RAR
null keratinocytes, and cells were treated as above. The closed
bar represents expression in untreated RAR cultures, and
open bars are values from cells treated with RA. Samples
transfected with RAR expression vectors (0.1 or 0.5 µg/well) are
denoted by the triangles and RAR type at the bottom of the
figure. For both A and B, results are the
mean ± S.D. of three independent transfections and are expressed
as percentage activity relative to untreated controls. WT,
wild type.
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Dose-response studies revealed a close parallel between AP-1 activity
and growth inhibition mediated by all four compounds in wild-type
cultures (Fig. 5). However, growth arrest
induced by 4-HPR in RAR and RAR mutant lines never correlated
with a reduction of AP-1 activity (data not shown). This finding
underscores a unique and unknown mechanism of action for this retinoid
in affecting proliferation.
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Fig. 5.
Effects of RA, 9-cis
RA, TTNPB, and 4-HPR on AP-1 reporter activity in wild-type
cultures. Transfection was performed as under "Experimental
Procedures." Cells were treated with carrier or with the indicated
concentrations of the retinoids, and luciferase activity was assessed
48 h post-transfection. Results are the mean value of triplicate
samples ± S.D. and are expressed as percentage values relative to
untreated control.
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We next determined the effect of RA on the expression of AP-1
members in wild-type and RAR null lines. Both the basal mRNA levels
and RA response of several of the AP-1 members varied across the
different RAR null lines. In untreated cells, c-fos
expression was comparable across all four lines, although it was
slightly reduced in RAR cells (Fig.
6). RA strongly inhibited
c-fos in both wild-type and RAR/ lines
but had no effect in RAR or RAR cultures. This pattern was
also observed at the protein level (Fig.
7). In contrast, treatment affected
c-jun expression only in RAR null cultures, although
basal mRNA levels varied across the lines. However, c-Jun protein
did not reflect its cognate mRNA levels and was reduced by RA
treatment in wild-type, RAR/, and
RAR/ cultures. Phosphorylated c-Jun (P-Jun) levels
paralleled those of c-Jun, suggesting that variations in
phosphorylation were because of alterations in total c-Jun levels,
rather than affects on JNK activity.
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Fig. 6.
Northern analysis of AP-1 family
members. A Northern blot was prepared using total RNA (15 µg)
from the different keratinocyte lines treated for 48 h with
carrier ( ) or 106 M RA (+). The blot was
probed with cDNAs encoding AP-1 family members as noted to the
right. Hybridization to ribosomal RNA (18S) was
used as loading control. WT, wild type.
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Fig. 7.
Effects of RAR ablation and RA treatment on
c-Fos, c-Jun, and P-jun protein levels. Nuclear protein
extracts (40 µg) from cells treated for 48 h with carrier ()
or 106 M RA (+) were used to prepare a
Western blot. Specific antibodies used to probe the blots are denoted
to the right, and the various cell lines are noted at the
top. WT, wild type.
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Fra-1 expression was barely detectable in wild-type,
RAR/, and RAR/ lines but was
elevated in RAR/ cultures; Western blot analysis was
inconclusive, as the signal was too weak to be distinguished (data not
shown). junB and junD expression did not vary
significantly with the exception that junB levels were
slightly reduced in the RAR/ line (Fig. 6 and
data not shown).
A number of mechanisms have been suggested to underlie retinoid
repression of AP-1 activity. These include inhibition of JNK activity,
which is unlikely given the observation that P-Jun levels appear to
change as a function of c-Jun levels. Alternatively, the observed
down-regulation of c-Fos and/or c-Jun proteins might play a role,
especially if either of them are limiting. A third mechanism involves
competition for limiting ancillary factors common to both RAR and AP-1
transcriptional complexes, such as p300/CBP (24). We addressed the
latter two possibilities by assessing the ability of exogenous CBP,
p300, c-Fos, or c-Jun to negate the effects of RA treatment on AP-1 activity.
CBP or p300 transfection in wild-type cells resulted in a
dose-dependent increase in AP-1 activity in the absence of
RA (Fig. 8A). Interestingly,
p300 appeared to be more potent in affecting AP-1 activity, suggesting
that it may be preferred over CBP in this context. Despite this
increase in activity, expressing the data as fold inhibition indicated
that both factors resulted in only a modest reversal of inhibition
(Fig. 8B).
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Fig. 8.
Rescue of AP-1 repression. Wild-type
keratinocytes were transfected with an AP-1 reporter in the absence or
presence of various amounts of expression vectors encoding CBP, p300,
c-Fos (0.1, 0.5 or 1.0 µg) or c-Jun (0.5 µg), or c-Fos plus c-Jun
(0.5 µg each). A, cells were treated with vehicle
(closed bars) or 106 M RA
(open bars), and luciferase activity was assessed 48 h
post-transfection. Results are expressed as percentage activity
relative to untreated wild-type control. B, the results from
A were expressed as fold AP-1 activity relative to untreated
transfected cultures. Results are the mean ± S.D. of three
independent transfections for both A and B.
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Overexpression of either c-Fos or c-Jun also resulted in an increase in
basal AP-1 activity, again with only a marginal reduction in fold
repression mediated by RA (Fig. 8). Although this rescue effect was
more pronounced when both c-Jun and c-Fos were co-transfected, repression was not completely abolished (Fig. 8B). These
observations suggest that several mechanisms, including titration of
limiting co-factors and inhibition of expression of AP-1 family
members, act in concert in an RAR-dependent manner to
attenuate AP-1 activity in these transformants.
Effect of RAR Ablation on Gene Expression--
Northern blot
analysis was performed to study the effect of receptor disruption on
the expression levels of several genes implicated in keratinocyte
growth and differentiation. The major integrin isoforms found in
epidermis are integrin 2, 3,
6, 1, and 4. These are
expressed in basal keratinocytes, and a decrease in integrin expression
is generally correlated with differentiation and loss of proliferative
potential (52, 53). Northern blot analysis revealed that, with the
exception of the 3 isoform, all integrins were
down-regulated by RA treatment in wild-type, RAR/,
and RAR/ cultures in a manner that correlated with
the effects of treatment on proliferation (Fig.
9). Moreover, although integrin
expression was not affected by RA treatment in RAR cultures,
basal expression of integrins 2, 3, and
4 was substantially reduced. The seemingly contradictory
observation that both RA excess and RAR loss can reduce expression of
several integrins is perhaps indicative of an altered differentiation
state in the double mutant line. Interestingly, integrin
1 expression decreased in untreated
RAR/ cells and was up-regulated in untreated RAR
mutant cultures.
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Fig. 9.
Northern blot analysis of keratinocyte gene
expression. Wild-type (WT) and RAR null keratinocytes
were treated with carrier () or 106 M RA
(+) for 48 h. 15 µg of total RNA from each cell line were used
to prepare Northern blots, which were probed with cDNAs encoding
various keratinocyte markers, as denoted to the right of the
figure. Ribosomal RNA (18S) was used as loading control. The
results are typical of at least two experiments.
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Keratin expression patterns reflect the differentiation states of the
various epithelial strata (52, 53). K5/K14 are expressed in basal
epidermal cells, whereas K1/K10 are associated with early differentiation steps and predominate in suprabasal cells. K6 and K19
are not expressed in normal epidermal keratinocytes but are often
observed in situations of aberrant proliferation, such as psoriasis,
wound healing, and propagation in tissue culture. With the exception of
RAR null cultures, RA treatment repressed expression of K10 (Fig.
9). This observation may be related to the fact that RA excess can
inhibit keratinocyte differentiation (54, 55). However, RAR disruption
did not result in up-regulation of this differentiation marker,
suggesting that K10 is not normally regulated by the RARs but responds
to pharmacological levels of RA.
RA suppressed K6 expression in wild-type, RAR/, and
(to a lesser extent) RAR/ cultures, consistent with
the effect of treatment on both AP-1 activity and proliferation. K19
expression was induced in wild-type and RAR/
cultures. Interestingly, this gene was also up-regulated in
RAR/ cells in the absence of treatment. These data
indicate that, as for the integrins, the roles of the various RARs on
keratin expression vary depending on both the receptor and the gene of interest. Moreover, most of the integrin and keratin markers were affected in both RAR and RAR null cultures. This demonstrates that both receptor types transduce effects on expression of many responsive genes.
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DISCUSSION |
We present, for the first time, the effects of RAR disruption on
the characteristics and RA response of transformed epidermal keratinocytes. These data indicate that each RAR type plays both specific as well as overlapping roles in events related to keratinocyte growth and gene expression.
RARs Are Not Necessary for Survival and Growth of Transformed
Keratinocytes in Culture--
Previous work using a dominant-negative
RAR under the control of a basal-keratinocyte-specific promoter
suggested that RA signaling is essential for normal keratinocyte
differentiation (56). We found that wild-type and RAR null
keratinocytes are comparable in regards to growth and morphology,
although expression of some markers, such as integrin 2,
did differ. It is unlikely that RAR plays any compensatory role in
these cells, as we have never observed expression of this receptor in
these cultures irrespective of RAR status. Moreover, the RAR line
was completely resistant to excess RA with respect to all outcomes
examined. Thus, these cells are likely completely devoid of functional
RARs. The difference between the relatively mild effects observed in
the present study, compared with a dominant-negative RAR (56), suggests
that transgene expression affects other pathways, perhaps by
sequestration of RXRs. Alternatively, we cannot exclude an unrecognized
compensation mechanism in the RAR null animals and derivative cells
that may mask certain roles for these receptors in skin.
RAR Is the Principle Mediator of Growth Arrest in Transformed
Keratinocytes--
Analysis of the effects of the various RARs on
growth inhibition suggests that the major player in transducing this
effect is RAR with only a negligible contribution by RAR. This
may simply be because of the prevalence of the former receptor type in
keratinocytes (57) rather than indicative of receptor-specific function. Nevertheless, irrespective of the basis for this finding, these data suggest that targeting RAR is a logical strategy to affect disorders of keratinocyte proliferation.
In the absence of the RARs, the RXR ligand 9-cis RA had no
effect on proliferation, suggesting that liganded RXRs do not impact on
keratinocyte growth, at least in the absence of RARs. This is in
contrast to previous reports, which suggests that growth inhibitory
effects can be mediated by RXR-selective agonists in several other
transformed cell types (17, 19, 58-61). This suggests either that RXR
ligands inhibit growth in a cell type-specific manner or that these
synthetic agonists have effects that cannot be mimicked by
9-cis RA. It will be of interest to determine if such
ligands exert an effect in the RAR null line.
Although 9-cis RA inhibited the growth of wild-type and
RAR null lines, it was consistently less potent than RA or TTNPB. Because both RA and 9-cis RA have comparable affinities for
the RARs (62), this observation suggests either that 9-cis
is more labile than RA or is titered away from RAR signaling. In
contrast to 9-cis RA, the synthetic RAR agonist TTNPB was a
strong inhibitor of growth. As displacement assays suggest that TTNPB
binds to the RARs with a lower affinity than RA, the greater relative
potency of this analog likely lies in its enhanced stability and/or
weaker association with cellular RA-binding proteins relative to RA
(63). Moreover, although full manifestation of growth inhibition
by TTNPB required the RARs, a slight inhibition at the highest
dose used was seen in RAR and RAR null cultures,
suggesting either nonspecific cytotoxicity or effects via
nonreceptor-mediated pathways.
Evidence for RAR-independent Mechanisms of Growth Inhibition by
4-HPR--
4-HPR can inhibit growth and induce apoptosis in a
number of model systems (44, 64, 65). Although this compound can act
directly via the RARs, some of its effects may also be mediated by
receptor-independent mechanisms (47, 66). Indeed, we found that 4-HPR
inhibited proliferation in all lines assessed but that this occurred at
lower concentrations in RAR-positive cultures. This is consistent with
4-HPR acting through both RAR-dependent and -independent
mechanisms. As 4-HPR is well tolerated at high doses (65), it should
mediate effects even in those epithelial tumors that lack retinoid
responsiveness to "pure" RAR agonists.
RARs and Inhibition of AP-1 Activity--
RA is a potent inhibitor
of AP-1, and this mechanism of action has been proposed to underlie
some of the antitumorigenic effects of retinoids (2, 67-70, 72, 73).
We found that both RAR and RAR can repress AP-1 activity in
p53-transformed keratinocytes in culture. However, attenuation of AP-1
activity in RAR null cultures (~30%) did not agree well with
growth inhibition in this cell line, which was minimal. A lack of
correlation was especially notable in the case of 4-HPR, which never
repressed AP-1 reporter activity in RAR or RAR null cultures
at concentrations that inhibited their growth. This observation
supports the existence of retinoid-mediated mechanisms of growth
inhibition unrelated to AP-1 activity, at least in this model.
Multiple Pathways for RA in Inhibition of AP-1
Activity--
Several mechanism have been proposed to underlie the
cross-talk between AP-1 and RA signaling pathways. These include
titration of common transcriptional co-regulators, such as CBP/p300
(24), effects on expression of AP-1 family members (27, 74, 75), or
inhibition of JNK activity (28). We found evidence that supports the
two former possibilities in our model.
RA inhibited c-Fos and c-Jun (and P-Jun) protein levels in a manner
that partially correlated with the observed attenuation of AP-1
activity. Although c-Jun levels paralleled the effect of treatment on
AP-1 across the various cell lines, c-Fos was not affected by RA in
RAR or RAR null cultures. These data underscore a specific
role for RAR in inhibiting c-Fos expression, whereas either RAR
or RAR affected c-Jun. In this regard, it is interesting to note
that c-Jun was also elevated in untreated RAR and RAR lines,
indicating that RAR, presumably in its unliganded state, represses
c-Jun expression. Consistent with this possibility, prior studies also
suggest that certain RARs may be antagonistic to one another in F9
cells (71, 76).
Transfection of either CBP or p300 induced basal AP-1 expression in
wild-type keratinocytes and modestly attenuated the effects of RA on
AP-1 activity. The persistent inhibition of AP-1 by RA, even following
transfection of higher levels of CBP/p300 expression vectors, suggests
that the RARs remain in excess. Alternatively, as c-Fos and/or c-Jun
also appear to contribute to this relationship, the combination of
several distinct events probably underlies retinoid antagonism of
AP-1.
RAR and RAR Play Both Overlapping and Specific Roles in
Keratinocytes--
The phenotype of RAR mutant mice clearly indicates
that these receptors are highly but not completely redundant. This is
supported by work using RAR null F9 embryocarcinoma cells
(e.g. Refs. 71 and 76). Our present findings suggest that
specificity also exists regarding RAR function in keratinocytes.
Although certain aspects of this selectivity may be explained by the
relative levels of expression, with RAR being the predominant
receptor type, this is not always the case. For example, repression of
integrin 6 and integrin 4 was more
affected by the loss of RAR than the loss of RAR.
In addition to specific functions, it is also interesting to note that
RAR may actually attenuate the effects of RAR in certain
instances. For example, growth inhibition mediated by RA,
9-cis RA, or TTNPB was greater in RAR null cells relative to wild-type cultures, suggesting that RAR is a more efficient inducer of growth arrest in the absence of RAR. Other complex interactions were also observed. RA induced keratin 19 expression in
wild-type cells, and this induction was greatly reduced in RAR null
lines, indicating that RAR positively regulates this gene. However,
in RAR null cultures, K19 basal expression was induced, and RA
induction was lost. One mechanistic explanation for this is that RAR
normally mediates repression of this gene in the absence of RA and that
RAR induces its expression in the presence of ligand. Consistent
with this, K19 expression in RAR cells returns to basal values,
and regulation is lost, supporting a model for opposing function
between RAR and RAR in regulating this gene; such antagonism has
been suggested previously (71). Although analysis of the regulatory
sequences governing this response is necessary to understand the nature
of these observations, these examples offer strong evidence that RAR
and RAR are not completely functionally equivalent in this model system.
We thank P. Chambon for the RAR null mice, M. Nemer, K. McBride, M. Karin, R.Goodman, and S. Benchimol for expression
vectors and AP-1 reporters, Jana Krosl for several of the antibodies, and members of the laboratory for support and suggestions.
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TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Inst. de
Recherches Cliniques de Montréal, 110 Ave. des Pins, Ouest,
Montréal, Québec H2W 1R7, Canada. Fax: 514-987-5767;
E-mail: lohnesd@ircm.qc.ca.
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