(Received for publication, October 30, 1995)
From the
Expression of keratin proteins, markers of epidermal
differentiation and pathology, is uniquely regulated by the nuclear
receptors for retinoic acid (RAR) and thyroid hormone (T3R) and their
ligands: it is constitutively activated by unliganded T3R, but it is
suppressed by ligand-occupied T3R or RAR. This regulation was studied
using gel mobility shift assays with purified receptors and transient
transfection assays with vectors expressing various receptor mutants.
Regulation of keratin gene expression by RAR and T3R occurs through
direct binding of these receptors to receptor response elements of the
keratin gene promoters. The DNA binding ``C'' domain of these
receptors is essential for both ligand-dependent and -independent
regulation. However, the NH-terminal ``A/B''
domain of T3R is not required for either mode of regulation of keratin
gene expression. Furthermore, v-ErbA, an oncogenic derivative of cT3R,
also activates keratin gene expression. In contrast to the previously
described mechanism of gene regulation by T3R, heterodimerization with
the retinoid X receptor is not essential for activation of keratin gene
expression by unliganded T3R. These findings indicate that the
mechanism of regulation of keratin genes by RAR and T3R differs
significantly from the mechanisms described for other genes modulated
by these receptors.
Hormones and vitamins, such as thyroid hormone (T3) ()and all-trans-retinoic acid (RA), are important
regulators of development and differentiation in general and of the
epidermis in particular. The effects of vitamin A, a precursor of RA,
on the skin were observed first in 1922(1) . Since that time,
the skin has been a model tissue for the study of RA action. It has
been shown that hypovitaminosis A causes epidermal hyperkeratinization,
while non-keratinizing tissues, such as conjunctiva and cornea, become
keratinized. Conversely, hypervitaminosis A causes inhibition of
keratinization, hyperplasia, and a block of terminal
differentiation(1, 2, 3, 4, 5, 6) .
Similarly, thyroid hormone deficiency results in a number of skin
changes, including
hyperkeratosis(7, 8, 9, 10) , and
the thyroid hormone excess causes increased epidermal cell
division(11) . Similar effects of RA and T3 were observed in
keratinocytes in vitro(2, 9, 12) .
Keratins are the intermediate filament network proteins in many epithelia. Their expression is precisely controlled in various physiological and pathological states of the epidermis. When the basal keratinocyte becomes detached from the basement membrane, its commitment to differentiation is announced by suppression of the basal cell-specific keratins K5/K14 and the induction of the differentiation-specific keratins K1/K10(13, 14) . In wound healing and other hyperproliferative processes, keratinocytes express the activation-specific keratin pair K6/K16(15, 16) . During inflammation, keratin K17 is expressed, whereas transformed keratinocytes express keratins K8/K18(17, 18) .
Because a fairly large number of keratin genes are suppressed by RA and T3, these genes provide a unique opportunity to study the mechanisms of negative regulation by T3R and RAR on native regulatory elements. We have reported previously that keratin gene expression is suppressed by RA or T3(19, 20, 21) . To examine this regulation in more detail, we studied the response of three different keratin promoter-CAT constructs (K5, K14, K17) to RAR or T3R, in the presence or absence of their cognate ligands using mutants of T3R in transfection and gel mobility shift experiments(22, 23, 24) . These promoters were chosen because K5 and K14 keratins are specific for the basal layer of the epidermis, the layer most proximal to the source of RA in vivo, whereas K17, although not present in healthy skin, is a marker of various inflammatory processes. Furthermore, all three promoters are expressed at high levels when transfected into cells of epithelial origin.
Our results show that T3R regulates keratin genes
in a unique manner: unliganded T3R leads to activation while the
addition of T3 results in suppression. The NH-terminal
``A/B'' domain of cT3R
is not required for keratin gene
regulation while the ligand binding and the DNA binding domains are
essential. In addition, we found that v-ErbA is a constitutive
activator of keratin genes and that it blocks ligand-dependent
suppression by T3R and RAR. Furthermore, we found that T3R does not
form heterodimers with RXR when bound to K14RE, and that addition of T3
promotes monomer binding at the expense of the homodimer. Last, mutants
which do not form heterodimers with RXR do mediate constitutive
activation of keratin genes. Taken together these results suggest that
the regulation of keratin genes may be mediated by monomers, or perhaps
homodimers, of T3R.
Competition experiments were performed as follows: a 100 M excess of the competitor DNA was incubated with protein at room temperature for 15 min prior to addition of the radioactively labeled DNA probe. Binding reactions were further incubated at room temperature for 15 min and then at +4 °C for additional 10 min.
cT3R(L372R) was obtained by in vitro translation using
TNT T7-coupled Reticulocyte Lysate System from Promega with 1.5 µg
of purified DNA. The wild type cT3R
receptor was used as a
control. The quality of both synthesized proteins was analyzed by
SDS-gel electrophoresis and autoradiography. The relative amount at
cT3R
(L372R) protein was compared with wild type cT3R
and
determined by quantitating the incorporated
S corrected
for the number of methionine residues per protein. One µl of
reticulocyte lysate translated receptor was used in the binding
reaction, in the presence of RNase A (0.5 µg) and RNase T
(1.5 units) as described(23) .
In the absence of RA, the RARs are without effect (Fig. 1).
In the presence of RA, all three retinoic acid receptors (hRAR,
hRAR
, and hRAR
) suppress expression of each of the keratin
gene promoters 5- to 6-fold (Fig. 1). In contrast, TREpCAT,
containing an optimized thyroid hormone/retinoic acid response element,
was stimulated approximately 30-fold by all three receptors in the
presence of RA.
Figure 1:
Regulation of keratin gene expression
by RAR and T3R. Regulation of K5, K14, and K17 keratin promoters by T3R
and RAR, RAR
, and RAR
. The basic, unregulated activity
of each CAT construct is designated as 1 to show -fold regulation by
RAR and T3R. Numbers on the left ordinate represent regulation
of keratin genes, and the numbers on the right represent -fold
regulation of the TREpCAT.
To test whether T3 also regulates keratin gene
expression, we co-transfected HeLa cells with the keratin-promoter CAT
constructs and a cT3R expression vector and then incubated the
cells in the presence or absence of T3. As previously found, the
control reporter TREpCAT is stimulated approximately 35-fold by T3 and
suppressed by unliganded T3R approximately
8-fold(23, 29) . In contrast, cT3R
has the
opposite effect on keratin gene expression: unliganded T3R stimulates
keratin K5, K14, and K17 gene promoters approximately 3-fold, whereas
with T3 the basal expression of the three keratin promoter constructs
is inhibited about 5-fold (Fig. 1). Comparing the results in Fig. 1we find that RAR and T3R mediate ligand-dependent
inhibition of keratin gene activity with similar efficiency.
To
analyze the combined effect of T3R and RAR on the regulation of keratin
gene promoters, both receptors were expressed using 5-fold more
cT3R expression vector. Unliganded T3R blocked both the
ligand-dependent inhibition of keratin genes by hRAR
and the
ligand-dependent stimulation of TREpCAT (Fig. 2). Conversely,
when hRAR
was expressed in a 5-fold excess over cT3R
, it did
not block the constitutive activation of keratin gene expression by
unliganded T3R. In the presence of its ligand, however, hRAR
was
epistatic and completely blocked the activation by cT3R
(Fig. 2). The effects of RA without co-transfected hRAR
are
due to the low levels of endogenous RAR
.
Figure 2:
Unliganded T3R blocks suppression of
keratin gene expression by RA and RAR. Note, however, that the
unliganded RAR
does not block the induction by
T3R.
Figure 3:
cT3R and hRAR
specifically bind
K14RE. Autoradiograms of the gel mobility shift assay with K14RE probe
are presented with cT3R
(shown on the left) and hRAR
(shown on the right). Binding of both receptors is efficiently
competed with 100 M excesses of cold K14RE (Slf) and
TREpal but not with mTRE DNA. Note significant increase in the amount
of free probe in lanes competed with K14RE and
TREpal.
cT3R formed two mobility
complexes with the K14RE probe, the monomer and the
homodimer(28) . cT3R
predominantly binds K14RE as a
homodimer. Binding is specific because it can be efficiently competed
with a 100 M excess of cold K14RE and consensus TREpal. A
mutated TREpal that does not bind cT3R
(28) does not
compete for the binding of cT3R
to K14RE (Fig. 3).
Similarly, hRAR predominantly forms a homodimer complex with
K14RE (Fig. 3). Binding is specific because it can be competed
with an excess of K14RE or TREpal but not with mTRE. These results
confirm that the -95/-51 region of the K14 promoter
contains a functional TRE/RARE that binds both cT3R
and hRAR
receptors.
Because the addition of T3 changes transcriptional
regulation from stimulation to repression, we investigated the effects
of ligand binding. Interestingly, the addition of T3 dramatically
inhibits the formation of homodimers of cT3R while increasing the
monomer binding to K14RE (Fig. 4A). In contrast,
addition of RA did not change the binding pattern of hRAR
. A small
change in mobility is due to the conformational change caused by ligand
binding to the receptor(28) .
Figure 4:
Effects of ligands on binding and
dimerization of cT3R and hRAR
. Autoradiograms of the gel
mobility shift assays are presented with K14RE (A) and TREpal probe (B).
The presence of T3 or RA did
not change the pattern of binding of cT3R or hRAR
to the
TREpal as shown in Fig. 4B. Again there is small change
in mobility of the complexes due to a conformational change. The K14RE
has a lower binding affinity when compared with the optimized TREpal
sequence, which is similar to other previously described native
TRE/RAREs(30, 31) .
We analyzed the combined
effects of the receptors using gel mobility shift assays. In the
absence of ligands three different complexes were detected: homodimers
of cT3R, heterodimers of cT3R
/RAR
, and homodimers of
hRAR
(Fig. 4A, last four lanes). Addition of T3
inhibited the binding, whereas addition of RA did not affect it.
Figure 5:
The DNA binding domain is essential for
the regulation by T3R. A, the NH-terminal mutant
cT3R
(51-408), which contains the DBD, regulates expression
of keratin gene promoters the same as the wild type T3R (compare with Fig. 1). B, the cT3R
(120-408) mutant of the
T3R, which lacks the DBD, does not regulate expression of keratin gene
promoters.
Figure 6:
The DBD mutant specifically interferes
with the regulation by T3R and not by RAR. A, the
cT3R(120-408) (DBD
) mutant of the T3R does
not block the suppression of keratin gene expression by RAR
. Note
that it blocks the induction of the TREpCAT. B, the
cT3R
(120-408) (DBD
) mutant efficiently
blocks regulation of K14 keratin gene expression by T3R, both the
suppression and the induction in the presence and absence of T3,
respectively.
In view of the fact that
cT3R(120-408) has no effect on regulation by hRAR
, we
were surprised to find that it blocks the effects of cT3R
(Fig. 6B). The cT3R
(120-408) mutant
efficiently blocked both effects of wild type cT3R
on the keratin
K14 gene promoter: constitutive activation by unliganded receptor and
the inhibition found in the presence T3. The blocking effect is not
mediated through direct competition for the DNA binding, because
cT3R
(120-408) is not a DNA-binding protein. The inhibition
most likely result from the dimeric interactions with
cT3R
(23, 24) .
Figure 7:
Regulation by v-ErbA. A, by
itself, v-ErbA constitutively stimulates keratin gene expression. B, v-ErbA blocks suppression of the keratin gene expression by
RAR. C, v-ErbA blocks T3-dependent suppression of the K14
gene expression and induction of TREpCAT by
T3R.
Figure 8:
Regulation of keratin gene expression by
cT3R does not involve heterodimerization with RXR. A, gel
mobility shift assays using TREpal (left panel) and K14RE (right panel) as probes with purified cT3R
and mRXR
receptors. B, autoradiogram of gel mobility shift assays using
K14RE probe with mutant cT3R
(L372R) receptor expressed in
reticulocyte lysate system and purified mRXR
. C,
regulation by the two ninth heptad mutants of T3R. Mutant
cT3R
(L365R) regulates keratin genes as does the wild type T3R
(compare with Fig. 1), while cT3R
(L372R) constitutively
stimulates keratin gene expression, similar to v-ErbA (Fig. 7A). Note the difference in regulation of
TREpCAT.
cT3R(L365R) stimulates
the expression of TREpCAT in the presence of T3, but does not suppress
basal expression in the absence of T3 (Fig. 8C). In
contrast, cT3R
(L365R) regulates keratin promoters similarly to
wild type cT3R
: it activates without T3, while it suppresses
keratin expression in the presence of T3 (Fig. 8C). The
mutant cT3R
(L372R), which does not form heterodimers with or
without T3, does not stimulate or repress TREpCAT, but can
constitutively activate keratin gene promoters (Fig. 8C). cT3R
(L372R) does not mediate negative
regulation by T3 because it has a very low affinity for
ligand(23) . Constitutive activation of keratin promoters by
the two mutants with the altered ninth heptad, cT3R
(L365R) and
cT3R
(L372R), together with the results from gel mobility shift
experiments support the notion that T3-independent stimulation of
keratin gene expression by T3R occurs by a mechanism that is
independent of heterodimerization with RXR.
The regulation of keratin gene expression by T3R and RAR described in this study is the inverse of the more commonly studied positive regulation of transcription. First, T3R without T3 constitutively activates keratin gene expression instead of silencing or suppressing the level of basal expression. Second, in the presence of T3, the constitutive activation of T3R is not only reversed, but the extent of transcriptional activity is further inhibited approximately 5-fold below the level of basal expression. Although RAR does not mediate constitutive activation, incubation with RA also leads to negative regulation. A number of natural promoters have been reported to be negatively regulated by either RAR or T3R and their ligands, but not by both receptors(35, 36) . However, the large family of keratin genes is negatively regulated by both T3 and RA via their cognate receptors. Furthermore, keratin genes are the first group of genes reported which are not only suppressed by T3R in the presence of its ligand, but are also activated by unliganded T3R.
We provide three new lines of evidence for a direct effect of RAR and T3R on keratin gene promoters. Previously we have identified an RARE/T3RE in the K14 promoter using site-specific mutagenesis(21) . In this paper we have shown that the identified responsive element physically binds nuclear receptors. We also show that the oncogenic derivative v-ErbA is an efficient competitor of the ligand-dependent regulation of keratin gene expression by RAR and T3R. Since it appears that v-ErbA acts by competing for DNA binding rather than by formation of nonfunctional heterodimers(24) , our data with v-ErbA receptor support a direct regulatory mechanism. Furthermore, deletion of the DBD from the T3R aborts keratin gene regulation. Taken together, our results suggest that regulation occurs via a direct interaction between RAR or T3R and keratin gene promoters.
Several different mechanisms of negative regulation by RAR and T3R have been described in literature. One mechanism involves blocking a positive transcription factor, such as AP1(35, 36, 37, 38, 39, 40) . This mechanism is difficult to reconcile with the stimulation of keratin gene expression by the unliganded T3R. Furthermore, the negative regulation does not occur through blocking an AP1 binding site, because the K14 promoter does not appear to have an AP1 site and the apparent AP1 site in the K5 promoter is not required for inhibition by RAR and T3R(41, 42) . A second mechanism of negative regulation by T3R was observed on the RSV-LTR in which T3 inhibits stimulation mediated by unliganded T3R (32) . Negative regulation of the keratin genes differs from negative regulation of the RSV-LTR because ligand not only blocks induction by the unliganded receptor, but also suppresses basal expression by 5-fold. Furthermore, regulation of the RSV-LTR(32) , but not of the keratin genes requires the A/B domains. Therefore, we conclude that regulation of keratin gene expression by RAR and T3R occurs through a distinct molecular mechanism.
To examine the molecular mechanisms through which regulation of keratin gene regulation occurs, we used several mutated T3Rs and tested their effects on regulation of keratin gene expression. These mutant T3Rs include complete deletions of the A and B domains, the DBD domain described above, as well as v-ErbA, a native variant of T3R, and point mutations in the ligand and heterodimerization domains. The results with these mutants are summarized in Fig. 9.
Figure 9: Summary of keratin gene regulation by various mutants of T3R. Asterisks represent mutations and differences in sequence of v-ErbA versus cT3R, the wild type. Other mutations and deletions are indicated in the respective amino acid numbers.
We were particularly interested in the constitutive activation mediated by unliganded T3R. In this paper, we have identified several characteristics that make this regulation novel and distinct. First, the constitutive activation elicited by unliganded T3R does not appear to be mediated through the binding of T3R/RXR heterodimers. Evidence from studies with ninth heptad mutants suggests that the activation of keratin genes is mediated by T3R homodimers. However, our gel mobility shift results suggest that the ligand-dependent inhibition may be mediated by T3R monomers.
Second,
constitutive activation by cT3R appears to involve only a subset
of the transactivation domains thought to be important for
ligand-dependent transcriptional activation by T3R. Unlike the
reduction in ligand-dependent transactivation by T3R on several other
native response elements(22) , activation of keratin genes by
unliganded cT3R
does not require the NH
-terminal A/B
domain of the receptor. This also further distinguishes activation of
keratin gene expression from the regulation of RSV-LTR by unliganded
cT3R
, which requires the NH
-terminal region for
constitutive activation(32) . In addition, v-ErbA also acts as
a constitutive activator of keratin gene expression. This indicates
that the putative transactivation domain that is deleted in v-ErbA at
the COOH-terminal end of cT3R
does not mediate constitutive
activation(25) . Thus constitutive activation may be mediated
by another, so far unidentified, region of the receptor. This finding
is consistent with the previous observation that
cT3R
(1-392), which lacks this putative activation domain,
can constitutively activate the growth hormone or prolactin gene
promoters in GH4C1 cells(26) .
This novel mechanism of gene regulation may be particularly important in those tissues in which both T3 and RA play important roles determine the cell phenotype. While in some cells regulation that involves RXR integrates the response to hormones and vitamins, in the epidermis the response to each signal may need to be clearly distinct from responses to all other signals. If so, the RXR-independent regulation described here may provide the appropriate discrimination of signals reaching the epidermis. We expect, however, that this novel regulation operates in other systems as well.
ACKNOWLEDGEMENTS
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