(Received for publication, September 9, 1994)
From the
We have examined how retinoic acid receptors (RARs) and retinoid
X receptors (RXRs) at physiological concentrations regulate distinct
retinoid-responsive elements, hRAR2/
RARE (DR5) and
rCRBPII/RXRE (DR1), in keratinocytes from human skin, a major retinoid
target. In vitro, endogenous RAR
and RXRs bound to these
elements as heterodimers (RAR
RXR) but not homodimers
(RAR
RAR or RXR
RXR). In cultured keratinocytes,
all-trans retinoic acid, 9-cis retinoic acid, and
CD367 activated
RARE but not RXRE via endogenous RAR
RXR
(ED
= 2.3, 3.8, and 0.3 nM, respectively)
whereas SR11237 showed no significant effect. All-trans retinoic acid, 9-cis retinoic acid, and SR11237 activated
RXRE via overexpressed RXR
RXR (ED
= 110, 120,
and 11 nM, respectively), indicating interconversion between
retinoic acid isomers, whereas co-overexpression of RAR
or
RAR
suppressed this activation. Unlike 9cRA, CD367 neither induced
formation of nor activated RXR
RXR. Overexpression of RAR or RXR
mutated in transactivation domain AF-2 suppressed endogenous receptor
activity over
RARE. Our data suggest that 1) in keratinocytes,
RAR
RXR-mediated pathway dominates over that mediated by
RXR
RXR; 2) RAR-selective CD367 and RXR-selective SR11237 can be
used to identify these two distinct pathways, respectively; 3)
RARE is mainly regulated by RAR
RXR, in which RAR alone
confers ligand inducibility whereas AF-2 of unliganded RXR is required
for transactivation by liganded RAR AF-2; 4) lack of RXRE activity in
keratinocytes is due to low endogenous levels of RXR
RXR and
inhibition by RAR
RXR; and 5) interaction among RXRs is much lower
than that between RAR and RXR.
Retinoic acid (RA) ()is an important regulator of
normal epidermal cell homeostasis(1) . For many years, RA and
synthetic retinoids have been used to treat cystic acne, psoriasis, and
certain epithelial malignancies. Recent studies in mice and humans have
demonstrated that topical RA improves the wrinkled appearance of
sun-damaged skin(2, 3, 4) , as well as
post-inflammatory hyperpigmentation (5) . In vitro, RA
inhibits differentiation of keratinocytes
(KCs)(6, 7) . The biological effects produced by RA
are believed to be mediated all or in part by two families of nuclear
receptors, retinoic acid (RARs) (8, 9, 10, 11) and retinoid X (RXRs)
receptors(12, 13, 14) .
RARs and RXRs are
ligand-dependent transcription factors that bind to cis-acting
DNA sequences called RAREs, which are composed of directly repeated
hexameric half-sites with consensus sequences (5`-PuG(G/T)TCA-3`),
within the transcriptional regulatory regions of target genes and
thereby regulate the rate of transcription of these genes (for review
see (15) and (16) and references therein). RARs and
RXRs are members of the steroid/thyroid hormone receptor superfamily,
each of which consists of three major functional domains, an N-terminal
transactivation domain (AF-1), a central DNA binding/dimerization
domain, and a C-terminal multi-functional domain, which is involved in
ligand binding, dimerization, and transactivation function
(AF-2)(13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
The RAR family is composed of RAR,
, and
and their
isoforms(27, 28, 29) , which recognize two
natural stereoisomers of RA, all-trans RA (tRA) and 9-cis RA (9cRA)(8, 30, 31) . The RXR family
consists of three members,
,
and
(13, 14) , which are activated exclusively by
9cRA(30, 31) . Recently, synthetic retinoids such as
SR11237 and SR11217 have also been identified as RXR-selective
ligands(32) .
In cell-free systems, RARs form heterodimers
with RXRs(13, 33, 34) . On the other hand,
RXRs are able to form not only homodimers but also heterodimers with
other members of the steroid/thyroid hormone receptor superfamily
including peroxisome proliferator-activated receptor, thyroid hormone
receptor, vitamin D receptor, apoAI regulatory protein, and
chicken ovalbumin upstream transcription
factor(15, 18, 19, 35, 36, 37, 38) .
Interestingly, RAREs with different spacing between half-sites have
been found to be selectively activated by heterodimers and/or
homodimers formed among overexpressed RARs and RXRs. For example, in vitro, RARE present in both human and mouse RAR
2
gene promoters(39, 40) , which is a DR5 core motif
composed of two half-sites separated by 5 base pairs, binds
RAR
RXR heterodimers efficiently in a ligand-independent manner (13, 33, 34, 41) and in vitro translation-derived and 9cRA-activated RXR homodimers to a certain
degree(35) . In contrast, retinoid X response element (RXRE) in
the rCRBPII gene promoter(42) , which is a DR1 core motif
consisting of five consensus half-sites spaced from each other by one
nucleotide, interacts efficiently with not only RAR
RXR
heterodimers in a ligand-independent way(35, 38) but
also in vitro translation-derived and 9cRA-activated RXR
homodimers(26, 35) . A synthetic reporter gene tk-CAT
containing
RARE was activated by overexpressed RARs and/or RXRs in
various cell lines(34, 41) , whereas the same reporter
gene containing RXRE was transactivated by only RXRs but not RARs in
certain cell types(34, 38, 42, 43) .
However, in living tissues, endogenous levels of the RAR and RXR members and their isoforms are tightly controlled(14, 27, 28, 29) . Recently, Dolléet al.(44, 45, 46, 47) and others (14, 48, 49) have shown that developing mouse embryos display distinct spatio-temporal expression patterns for the transcripts of each RAR and RXR member. The transactivation properties and differential expression pattern of these receptors suggest that the pleiotropic effects of retinoids may be in part due to combinatorial dimerization of various RAR and RXR members whose expression is ultimately controlled in a cell type-specific way(50) . In addition, receptor activity may be further regulated by cell-specific levels of natural retinoids such as tRA and 9cRA, which have been shown recently to be interconverted in mammalian cell lines(30, 31, 51) .
In adult human
epidermis and cultured KCs, mRNAs for RAR and
and RXR
and
were found, with RAR
and RXR
being the predominant
species(14, 52, 53, 54, 55) .
To date, very little is known about the DNA binding and transactivation
properties of these endogenous RAR and RXR proteins in adult human
epidermal KCs, although induction of
RARE activity by retinoids
was observed in human foreskin KCs (56) and transformed
epidermal KCs(57) . In this study, we took advantage of the
special features of two distinct RAREs mentioned above,
RARE (DR5)
and RXRE (DR1), to determine how at physiological concentrations RARs
and RXRs act to regulate transcription in cultured normal KCs from
adult human skin in response to various natural and synthetic
retinoids.
Figure 1:
Ligand-dependent transactivation of
retinoid-responsive reporter genes, RARE
-tk-CAT and
RARE-tk-CAT, but not RXRE-tk-CAT, by endogenous retinoid receptors
in KCs. The y axis represents relative CAT activity, which is
the average value from four independent experiments (n = 4), and the x axis shows types of ligands used
to treat cells and reporter genes transfected. Standard errors are
shown as verticalbars. Cultured KCs were transfected
with reporter gene DNA, 3 µg for
RARE
-tk-CAT and 2
µg for
RARE-tk-CAT and RXRE-tk-CAT. After transfection, cells
were treated with vehicle (0.1% ethanol) or 0.1 µM tRA or
0.1 µM 9cRA for 48 h.
Figure 2:
Gel electrophoretic mobility shift
analysis of endogenous RAR and RXRs from human epidermis and
cultured KCs and of RAR
and RXR
overexpressed in KCs using
RARE. Resolved complexes are indicated by labeledtriangles along both sides of the gels.
Antibodies used (1.2 µl for RAR
Ab and 0.8 µl for RXR Ab
unless indicated differently) in postincubation are shown immediately above the gels. 1 µM 9cRA was included in all
binding reactions unless indicated differently immediately below gels. A, competition for formation of specific
RARE-bound A complexes by wild type
RARE but not that
containing mutations in half-sites. Types of
P-labeled
probes are shown below the gel, and types and amounts of
unlabeled competitor DNA (equivalent to 2.5-, 10-, 50-, and 100-fold
excess) are shown on the top. In vitro binding
reactions were performed with 8 µg of nuclear extracts from
cultured KCs. Similar or identical results were obtained in the absence
of 9cRA (data not shown). B, endogenous RAR
and RXRs in
nuclear extracts from human epidermis and cultured KCs bind to
RARE as heterodimers but not RAR nor RXR homodimers. Types of
P-labeled probes are indicated at the top, and
those of nuclear extracts are indicated at the bottom. Amount
of nuclear extracts used in binding reactions was 8 µg for human
epidermis (lanes1-5) and cultured KCs (lanes6-10) and 2 µg for KCs containing
co-overexpressed RAR
and RXR
(lanes 11-15).
Similar or identical results were obtained in the presence of vehicle
(10% ethanol) or 1 µM tRA (data not shown). Photographs
containing lanes1-5, 6-10, and 11-15 are derived from autoradiography of the same gel
for 90, 180, and 30 min, respectively. The lowerpanel (lanes 1`-15`) shows autoradiography of the same
gel for 30 min. C, antibody titration shows that the A1
complexes are derived from two types of RXR-containing complexes
interacting with
RARE. Types of
P-labeled probes are
indicated at the top. 8 µg of nuclear extracts from human
epidermis were used. D, RAR
overexpressed in KCs binds to
RARE preferentially as RAR
RXR heterodimers but not RAR
homodimers, whereas RXR
overexpressed alone binds to
RARE as
the RAR
RXR heterodimers and RXR homodimers with the later
being the minor species (E). Types of
P-labeled
probes are indicated at the top, and those of nuclear extracts
from transfected KCs (2 µg) are indicated at the
bottom.
The protein content of the A complexes was further
examined by immunological gel mobility shift assays (Fig. 2B). Postincubation of binding reaction mixtures
with mouse monoclonal antibodies specific to either RAR (RAR
Ab) or that recognize all three members of the RXR family (
,
, and
) (RXR Ab) resulted in formation of supershifted
complexes B (lane8) and C (lane9), respectively, and reduction of fast migrating
complexes A1 but not complexes A2 in the A complexes (in comparison
with lane7), indicating that the A1 complexes
contain RAR
and/or RXR. The A1 and A2 complexes are too close to
be completely separated from each other under our experimental
conditions. Complexes B and C were also obtained with KC nuclear
extracts (2 µg) containing co-overexpressed RAR
and RXR
,
respectively (lanes13 and 14), using the
same antibodies. The nature of the A2 complexes, which also
specifically recognize
RARE, is not known. Note that under the
assay conditions (lower amounts of extracts and shorter exposure time)
the A2 complexes were absent or less visible with nuclear extracts from
KCs transfected with RAR
and/or RXR
. Interestingly, addition
of RAR
Ab to the RXR Ab-containing reaction mixture (lane9) further supershifted a portion of the C complexes
while resulting in double-supershifted D complexes (lane10) whose amount is approximately equal to that of B in lane8. Thus, formation of the D complexes identifies
RAR
RXR heterodimers, which were also observed with
co-overexpressed RAR
and RXR
(lane15).
Interestingly, the amount of the B complexes (lane8)
is less than that of C (lane9). Furthermore, the sum
of the amount of double-supershifted D complexes and that of
single-supershifted C complexes migrating faster than D in lane10 almost equals that of the C complexes in lane9. These observations suggest that the C complexes in lane10 may possibly be heterodimers formed between
RXRs and unidentified dimerization partners, which were supershifted by
RXR Ab but not RAR
Ab. Similar supershift patterns were obtained
with nuclear extracts (8 µg) prepared from human epidermis (lanes1-5), although levels of RAR
and
RXRs bound to
RARE in epidermis (lanes 1`-5`) are
relatively higher than those in cultured KCs (lanes
5`-10`). Complexes analogous to A2 were not detectable in
epidermis (lanes1-5). No significant
differences were observed among experiments performed in the presence
of vehicle (ethanol) or 1 µM 9cRA or tRA (data not shown).
To exclude the possibility that both A1 (lane3)
and C (lane5) complexes not supershifted by RAR
Ab result from insufficient amounts of this antibody used, an antibody
titration experiment was performed with nuclear extracts from epidermis
that contain higher levels of the endogenous receptors than those from
cultured KCs (Fig. 2C). As little as 0.4 µl of RXR
Ab completely supershifted the A1 complexes (lane4).
On the other hand, increasing amounts of RAR
Ab above and beyond
1.2 µl did not significantly increase the amount of the B complexes
nor reduce that of A1 (compare lane9 with lane12). The simultaneous decreases of the C and D complexes
in lanes10 and 14, respectively, were most
likely caused by an excessive quantity of protein contained within the
combined RAR
and RXR antibody fluids. Similar titration data were
obtained with nuclear extracts from cultured KCs.
RAR and
were not detected in nuclear extracts from cultured KCs by the same
assays using appropriate antibodies (data not shown), although under
the same conditions RAR
present at very low levels but not
RAR
was readily detectable in extracts from
epidermis(68) . No complexes corresponding to endogenous
RAR
or RXR homodimers were observed with both extracts, which
would have been double-supershifted by monoclonal RAR
Ab or RXR Ab
alone (Fig. 2B, lanes3, 4, 8, and 9).
To know whether RAR at higher
concentrations is able to bind to
RARE as RAR
homodimers,
nuclear extracts from KCs transfected with RAR
and/or RXR
were analyzed by gel mobility shift assays (Fig. 2D).
In comparison with KCs transfected with parental expression vector pSG5 (lanes1-3), transfection of KCs with RAR
alone increased levels of
RARE-bound A1 complexes in the absence (lane4) or presence of tRA (lane5). Mutations affecting both half-sites in
RARE
abolished formation of these complexes (lane6). The
increased A1 complexes apparently correspond to RAR
RXR
heterodimers formed between overexpressed RAR
and endogenous RXRs,
since these complexes were completely supershifted by RAR
Ab
and/or RXR Ab (lanes7-9). Similar binding took
place with KCs transfected with RXR
alone (lanes
10-12). No complexes corresponding to RAR
homodimers
were observed, which would have been double-supershifted by RAR
Ab
alone. Combining the extracts containing overexpressed RAR
(lanes4 and 5) with those containing
overexpressed RXR
(lanes10 and 11)
synergistically increased the amount of the A1 complexes (lanes13 and 14), similar to the case with extracts
containing co-overexpressed RAR
and RXR
(lanes16 and 17), indicating that KCs transfected with
single receptors did contain high levels of RAR
or RXR
,
respectively. Thus, RXR is required for RAR
to efficiently bind to
RARE, confirming the previous
finding(13, 33, 34) .
Taken together, our
experiments demonstrate that endogenous RAR and RXR proteins in
human epidermis and cultured KCs bind to
RARE in vitro almost exclusively as RAR
RXR heterodimers but not
RAR
homodimers nor RXR homodimers. Similar binding can occur when
RAR
is overexpressed alone or co-overexpressed with RXR
. In
addition, more RXR proteins versus RAR
most likely bind
to
RARE as heterodimers formed between RXR and as yet unidentified
dimerization partners. In the case with the extracts from epidermis,
RAR
proteins most likely contribute in part to the formation of
such heterodimers(68) .
Figure 3:
Differential regulation of reporter gene
RXRE-tk-CAT by overexpressed RXR, RAR
, and RAR
in KCs.
The y axis shows average CAT activity expressed as % maximal
induction, and the x axis shows types of expression vectors
cotransfected. Data are presented as average values derived from three
independent experiments (n = 3). The open and filledboxes represent cells treated with
vehicle (0.1% ethanol) or 0.1 µM 9cRA, respectively. KCs
were transfected with 2 µg of reporter RXRE-tk-CAT alone or
together with parental expression vector pSG5 or expression vectors for
RXR
, RAR
, and RAR
.
Figure 4:
Gel electrophoretic mobility shift
analysis of endogenous RAR and RXRs from human epidermis and
cultured KCs and of RAR
and RXR
overexpressed in KCs using
RXRE. Resolved complexes are indicated by labeledtriangles along both sides of the gels. N,
nonspecific complexes. Antibodies used (1.2 µl for RAR
Ab and
0.8 µl for RXR Ab unless indicated differently) in postincubation
are shown immediately above the gels. 1 µM 9cRA
was included in all binding reactions unless indicated differently
immediately below gels. A, competition for formation
of specific RXRE-bound A1 complexes in KC nuclear extracts by wild type
RXRE and
RARE but not those containing mutations in half-sites
(RXREm and
RAREm2). Types of
P-labeled probes are
shown below the gel, and types and amounts in pmoles of
unlabeled competitor DNA (equivalent to 10- and 50-fold excess) are
shown on the top. In vitro binding reactions were
performed with 8 µg of nuclear extracts from cultured KCs. B, endogenous and co-overexpressed RAR
and RXR in KCs
bind to RXRE as heterodimers but not RAR homodimers nor RXR homodimers.
Types of
P-labeled probes are indicated at the top, and those of nuclear extracts are indicated at the
bottom. Amounts of nuclear extracts used in binding reactions were 4
µg for human epidermis (lanes1-5), 8
µg for cultured KCs (lanes 6-10), and 2 µg for
mock-transfected KCs (lane 11) and transfected KCs containing
overexpressed RXR
(lanes 12-18) or co-overexpressed
RAR
and RXR
(lanes 19-23). Gels including lanes1-10 were subjected to autoradiography
for a duration at least four times longer than those comprising lanes 11-23, due to relatively lower levels of
endogenous RAR
and RXRs versus overexpressed receptors. C, competition and antibody titration analysis of
overexpressed RXR
bound to RXRE. Types of
P-labeled
probes are indicated at the bottom, and those of nuclear
extracts and competitor DNA are indicated at the top. Amounts
of nuclear extracts used in binding reactions were 2 µg for both
mock-transfected KCs (lanes1 and 2) and
transfected KCs containing overexpressed RXR
(lanes3-12). Lanes1` and 2` on the left were obtained from autoradiography of lanes1 and 2 for a longer
duration.
The protein content of the
A1 and A3 complexes bound to RXREwt was further analyzed by
immunological gel mobility shift assays. As shown in Fig. 4B, the A1 complexes (lane7)
found with KC nuclear extracts were also formed with co-overexpressed
RAR and RXR
(lane20). Note that the A3
complexes were present at very low levels in nuclear extracts from
human epidermis or KCs transfected with RAR
and/or RXR
under
the assay conditions (lower amount of extracts and/or shorter exposure
time). Postincubation of binding reaction mixtures with antibodies
specific to either RAR
or RXRs significantly supershifted the A1
but not the A3 complexes, resulting in complexes B (lane8) and C (lane9), respectively. Note
that the amount of the C complexes is higher than that of B, similar to
the case with
RARE. Formation of the B complexes identifies
RAR-containing heterodimers and that of the C complexes, RXR-containing
heterodimers. Thus, the A1 complexes apparently correspond to
RXREwt-bound endogenous RAR
and/or RXRs. Complexes B and C were
also obtained with co-overexpressed RAR
and RXR
using the
same antibodies (lanes21 and 22). During
postincubation, addition of RAR
Ab into binding reaction together
with RXR Ab further supershifted a portion of the C complexes, causing
formation of D complexes (lane10), which were also
obtained with co-overexpressed RAR
and RXR
(lane23). Formation of the D complexes identifies
RAR
RXR heterodimers. The remaining C complexes (lane10) not double-supershifted by RAR
Ab may possibly
correspond to RXR Ab-associated heterodimers formed by RXRs and
unidentified dimerization partners other than RAR
. Binding similar
to that with cultured KCs occurred with nuclear extracts prepared from
epidermis (lanes1-5) although unlike cultured
KCs, the C complexes in human epidermis were almost completely
double-supershifted by RAR
Ab (lane5). In these
three types of extracts, no complexes corresponding to the RAR
and
RXR homodimers were found, which would have been double-supershifted by
RAR
Ab or RXR Ab alone. Similar results were obtained in the
presence of vehicle (ethanol) or 1 µM 9cRA. The nature of
the A3 complexes is not known. Whether the A3 complexes are formed by
nuclear proteins involved in formation of the A2 complexes over
RARE remains to be determined.
To know whether, at relatively
higher levels versus RARs and other dimerization partners,
RXRs are able to form RXR homodimers, RXR overexpressed alone in
KCs was analyzed (Fig. 4B, lanes11-18). In the presence of vehicle, incubation of
nuclear extracts containing overexpressed RXR
with RXREwt gave
rise to E complexes (lane12), which is absent in a
control reaction performed with the same amount of nuclear extracts
from mock-transfected KCs (lane11). Addition of 9cRA
into the binding reaction resulted in formation of F complexes (lane15) whose mobility is lower than that of the
RAR
RXR heterodimers (A1 in lane20).
Mutations in the half-sites of RXRE (RXREm) abolished both E and F
complexes (lane14). The specificity of these two
complexes were confirmed by the competition experiment shown in Fig. 4C, since RXREwt (lane6) but
not RXREm (lane7) specifically competed both
complexes. As shown in Fig. 4B, the F complexes
apparently correspond to the RXR homodimers bound to RXRE, since
RXR
Ab (lane17) but not RAR
Ab (lane16) completely supershifted the F complexes while
resulting in formation of G complexes. The mobility of the G complexes
is close to that of the double-supershifted RAR
RXR
heterodimers (D in lane23). The observation that the
RXR
proteins overexpressed in KCs form homodimers in a
9cRA-dependent way is consistent with the previous finding with in
vitro transcribed-translated RXR
(35) . The nature of
the E complexes is yet unknown. These complexes found only in KCs
transfected with RXR
alone are not related to RAR
because
they were not supershifted by RAR
Ab (lane16).
However, as shown in Fig. 4C, although RXR Ab reduced
the intensity of E to a certain extent, adding excessive amounts of RXR
Ab did not completely supershift the E complexes (lanes10-12) while the F complexes were completely
double-supershifted by the lowest amount (0.2 µl) of the antibody (lane10).
To determine whether RXRs overexpressed
alone in KCs are also capable of binding to RARE as homodimers in
addition to RAR
RXR
heterodimers, nuclear extracts from
KCs transfected with RXR
alone were analyzed. As shown in Fig. 2D, overexpression of RXR
increased the
amount of the A1 complexes (compare lanes10 and 11 with lanes1 and 2) and resulted
in E complexes (lane10 in Fig. 2D and lane1 in Fig. 2E) when
RAREwt was used as a probe. The presence of tRA (Fig. 2D, lane11) or 9cRA (Fig. 2E, lane2) did not
significantly alter the binding patterns. As shown in Fig. 2E, mutations in both half-sites of
RARE
(
RAREm2) abolished formation of both A1 and E complexes (lane3). RAR
Ab supershifted only a portion of the A1
complexes resulting in B complexes (lane4), while
RXR Ab almost completely supershifted the A1 complexes to give C
complexes (lane5). In addition, G complexes
corresponding to RXR homodimers double-supershifted by RXR Ab were also
detected with
RARE (lane5), as in the case with
RXRE. The fact that the quantity of the G complexes is very low
suggests that complexes analogous to F (RXR
RXR) free of RXR Ab
may be masked by the E complexes and/or background signals (lane2). Addition of RAR
Ab together with RXR Ab into
binding reaction further supershifted a portion of the C complexes to
give D complexes (RAR
RXR) (lane6), which
co-migrate with G (RXR
RXR) (lane5). In this
case, C complexes not double-supershifted by RAR
Ab were found
again as expected (lane6). These data indicate that
RXRs bind to
RARE as homodimers only when its levels are much
higher than other dimerization partners and that RAR
RXR and other
RXR-containing heterodimers have higher affinity for this element than
the RXR homodimers do. The idea that the RXR homodimers have lower
affinity for
RARE than for RXRE was further confirmed by the
competition experiment shown in Fig. 4C.
RAREwt (lane8) but not
RAREm2 (lane9) competed specifically but less efficiently for
formation of the F complexes than did RXRE (lane6).
Thus, results from these experiments clearly indicate that 1)
endogenous RXRs bind to RXRE as RARRXR heterodimers but not
homodimers due to the presence of RAR
and other unidentified
dimerization partners (at least in the case of cultured KCs), 2) RXRs
are able to form RXRE-bound homodimers only when their concentrations
are much higher than other dimerization partners and in the presence of
9cRA, and 3)
RARE has much lower affinity for RXR homodimers than
RXRE does.
Figure 5:
Selective and dose-dependent activation of
RA-responsive reporter genes, RXRE-tk-CAT via overexpressed RXR
homodimers (A) and
RARE
-tk-CAT via endogenous
RAR
RXR heterodimers (B), by tRA, 9cRA, CD367, and
SR11237 in cultured KCs. The y axis represents average CAT
activity expressed as % maximal response, and the x axis
represents concentrations of ligand in a log scale. Labels
corresponding to tRA, 9cRA, CD367, and SR11237 are indicated on the top. n refers to number of human subjects from whom
KCs were prepared and analyzed independently. Standard errors are shown
as verticalbars. KCs were transfected with 2 µg
of RXRE-tk-CAT together with 400 ng of the RXR
expression vector (A) or 3 µg of
RARE
-tk-CAT alone (B).
Figure 6:
Suppression of endogenous RARRXR
heterodimer-mediated transactivation of
RARE in KCs by the RAR and
RXR dominant negative mutants. The y axis shows average CAT
activity in % maximal induction, and the x axis shows types of
expression vectors cotransfected. dnRAR and dnRXR refer to expression
vectors for the RAR and RXR dominant negative mutants, respectively.
KCs were transfected with 2 µg of
RARE
-tk-CAT
alone or together with 400 ng of expression vectors. Results were
expressed as average values derived from three independent experiments (n = 3). The open and different filledboxes represent cells treated with vehicle (0.1% ethanol)
or 0.1 µM tRA or 0.1 µM 9cRA, respectively.
The standard errors are represented by verticalbars.
In this study, we demonstrated that natural retinoids
including tRA and 9cRA strongly induced RARE (DR5) activity but
not that of RXRE (DR1) in cultured KCs. This result suggests that KCs
contain functional endogenous retinoid receptors that specifically
activate
RARE but not RXRE. Availability of monoclonal antibodies
and synthetic ligands specific to RAR or RXR allowed us to identify
roles of endogenous RARs and RXRs in this restricted regulation.
Several lines of evidence indicate that RAR
RXR heterodimers
but not RXR homodimers nor RAR
homodimers are the major regulators
of
RARE and RXRE in cultured KCs.
Our in vitro binding
studies clearly showed that endogenous RAR and RXR proteins in
nuclear extracts from human epidermis and cultured KCs bind to
RARE as RAR
RXR heterodimers but not RAR
homodimers
nor RXR homodimers. In fact, retinoid receptor-related binding activity
observed in epidermis was mainly contributed by RAR
and RXR
,
which represent 90% of total RAR and RXR proteins,
respectively(68) , in good correlation with their mRNA levels
previously
reported(14, 52, 53, 54) . In
cultured KCs, RAR
was not detected by specific antibodies in gel
mobility shift assays due to both its low levels and limitation on
amounts of nuclear extracts that could be loaded on gels (data not
shown). However, in human epidermis, low levels of RAR
proteins
were readily detectable by the same assays(68) . RAR
was
not detected in human epidermis (68) nor in cultured KCs (data
not shown), consistent with the absence of the corresponding mRNA in
this tissue(52) . On the other hand, RAR
overexpressed in
KCs also binds to
RARE exclusively as RAR
RXR heterodimers,
and this binding is quantitatively regulated by the levels of RXRs
available in KCs. These data further support the notion that RXRs, as
cofactors for RARs, are required for RARs to efficiently bind to
targets such as
RARE(13, 33, 34, 41) .
In
both epidermis and cultured KCs, endogenous RXRs, which are present in
relative excess versus RARs, appear to form heterodimers with
unknown dimerization partners besides RARs. In addition to endogenous
RARRXR heterodimers, overexpressed RAR
proteins bound to
RARE exclusively as RAR
RXR heterodimers but not RAR
RAR
homodimers through heterodimerizing with endogenous RXRs (Fig. 2D), indicating that the unknown dimerization
partners can be dissociated from RXRs by RARs. Recently, Baes et
al.(71) identified a new orphan receptor, called MB67,
which shares high sequence homology with the retinoid receptor
families. It binds as MB67
RXR heterodimers to consensus
half-sites present in only
RARE (DR5) but not any other direct
repeats, and its activity is not regulated by retinoic acid. Their
finding suggests that receptors capable of binding to
RARE (DR5)
are not limited to receptor dimers formed among the RAR and RXR family
members. Whether the endogenous RXR-containing heterodimers other than
RAR
RXR observed in this study correspond to dimers formed between
RXR and unidentified orphan receptors present in human epidermis and
cultured KCs remains to be further characterized.
The ED values of tRA, 9cRA, and CD367 in induction of
RARE activity
via endogenous retinoid receptors correlate well with the affinity of
these ligands for RARs but not RXRs, with CD367 being the most
potent(51, 58, 70, 72) . The fact
that RXR-specific ligand SR11237 did not significantly activate
RARE through endogenous RXRs excludes the possibility that
RAR-unrelated RXR-containing heterodimers or undetectable endogenous
RXR homodimers also contribute to transactivation of
RARE. This
result also indicates that binding of ligands to RXR in RAR
RXR
heterodimers does not confer ligand-dependent transactivation of
RARE. In vitro ligand binding assays have showed that
CD367 does not interact with RXRs(68) . In this study, we found
that this ligand neither induces formation of RXR homodimers in
vitro nor activates RXRs in vivo. Therefore, binding of
ligands such as CD367 to only RAR in RAR
RXR heterodimers seems to
be sufficient for conferring the ligand inducibility to transactivation
of
RARE. In other words, occupation of the E domain of RXR by
ligands is most likely not required.
Overexpression of dominant
negative mutants, dnRAR and dnRXR, drastically repressed endogenous
receptor-mediated induction of RARE in cultured KCs. These two
mutants have been previously shown to be functional in dimerization and
DNA binding but not in ligand-dependent transactivation(62) .
The repression we observed here most likely resulted from formation of
transactivation-deficient receptor dimers dnRAR
RXR or
RAR
dnRXR involving endogenous wild type RXRs and RARs. These
mutant dimers, which contain only one AF-2 domain with transactivation
function preserved, most probably competed the remaining endogenous
RAR
RXR from binding to
RARE, but their own binding failed to
activate this element. Thus, based on the results from both
transactivation in cultured KCs and in vitro binding studies,
we conclude that transactivation of
RARE in KCs is mainly mediated
by endogenous RAR
RXR heterodimers, in which RXR is required for
RAR to efficiently bind to
RARE, and the RAR ligand binding domain
confers ligand inducibility whereas the RXR AF-2 domain without the
need for bound ligands cooperates with the AF-2 domain of liganded RAR
in transactivation.
Transactivation of either isolated RXRE or the
natural RXRE-containing rCRBPII gene promoter was initially observed
only under experimental conditions where RXRs were overexpressed
alone(34, 42, 43) . Recently, Nakshatri and
Chambon (38) have further demonstrated that whether RXRE is
transactivated by overexpressed RXR homodimers or RARRXR
heterodimers depends on cell type. In this study, we found that in
contrast to
RARE (DR5), RXRE (DR1) was not activated by endogenous
retinoid receptors in KCs. Endogenous RAR
RXR heterodimers bound
to but failed to transactivate this element in the presence of
appropriate ligands. In vitro, when RXRE was used as a probe,
9cRA-dependent RXR homodimers were obtained with RXR
overexpressed
alone in KCs but not with RXR expressed at endogenous levels in both
human epidermis and cultured KCs, similar to the case with in vitro transcribed-translated RXRs(26, 35) . Under the
same conditions, RXR
interacts with
RARE mainly as
heterodimers and barely as RXR homodimers. Previous in vitro binding studies have shown that RXR homodimers produced by in
vitro transcription-translation were able to bind to a synthetic
RARE, called DR1G, but not RXRE in the absence of 9cRA(38) .
All of these observations suggest that organization and/or sequences of
half-sites in RAREs may also play important roles in ligand-dependent
formation of RXR homodimers.
In addition, we found that when RARs
were co-overexpressed, high levels of overexpressed RXRs bound to RXRE
preferentially as RARRXR heterodimers but not RXR homodimers even
in the presence of 9cRA. Thus, ligand-independent interaction between
RARs and RXRs appears to be much stronger than ligand-dependent
interaction among RXRs themselves. In cultured KCs, RARs overexpressed
alone did not significantly activate RXRE. Furthermore, overexpressed
RARs were able to suppress transactivation of RXRE mediated by
overexpressed RXRs, similar to the case with monkey kidney epithelial
cells, CV1(42) . These observations together with those from in vitro binding studies suggest that in KCs formation of
RAR
RXR heterodimers dominate over that of RXR homodimers, and the
resulting dominant RAR
RXR heterodimers, which are poor activators
for RXRE, may suppress RXR-mediated activation of this element by
competing the remaining RXR homodimers from binding to this element,
while their own binding does not produce effective transactivation.
Recently, a number of groups (36, 38) have shown that
in addition to RARs, chicken ovalbumin upstream transcription factor
and apoAI regulatory protein are able to efficiently form
heterodimers with RXRs and bind to RXRE. In this study, we also
observed that in cultured KCs, unidentified proteins bound to RXRE as
RXR-containing heterodimers. Taken together, we predict that in KCs,
RXRs would form 9cRA-induced homodimers over RXRE only when their
concentrations are much higher than those of RARs and of other
dimerization partners.
Finally, our finding that in human epidermal
KCs the RARRXR heterodimer-mediated nuclear retinoid signal
transduction pathway dominates over that mediated by RXR homodimers in
regulating transcription may be physiologically relevant to the recent
finding that in contrast to tRA, SR11237 produced no detectable changes
when applied on rhino mouse skin(73) . On the other hand, the
fact that tRA and 9cRA showed similar potencies in activating RXR
homodimers overexpressed in KCs suggests that there is efficient
interconversion between these two natural ligands in these cells,
similar to that observed with other continuous mammalian cell
lines(30, 31, 51) . This idea was further
supported by results from HPLC analysis of retinoids extracted from KCs
treated with 0.1 µM tRA or 9cRA for 48 h, which revealed
the presence of both ligands in either case. (
)Unlike
natural retinoids tRA and 9cRA, which are subjected to interconversion
in living tissues such as human epidermis, synthetic retinoids SR11237
and CD367 are able to independently trigger the RXR homodimer- and
RAR
RXR heterodimer-mediated nuclear signal pathways,
respectively. Advantages of these synthetic retinoids over natural
retinoids may open an avenue leading to not only tissue targeting for
therapeutic purposes but can also be used to address the question of
whether an RXR homodimer-mediated signal transduction pathway exists in
other human tissues.