|
|
|
|||||||
|
|||||||||
(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
Hormones and vitamins, such as thyroid hormone (T3) ( 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
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
In the absence of RA, the RARs are without effect (Fig. 1).
In the presence of RA, all three retinoic acid receptors (hRAR
Figure 1:
Regulation of keratin gene expression
by RAR and T3R. Regulation of K5, K14, and K17 keratin promoters by T3R
and RAR
To test whether T3 also regulates keratin gene
expression, we co-transfected HeLa cells with the keratin-promoter CAT
constructs and a cT3R 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
Figure 2:
Unliganded T3R blocks suppression of
keratin gene expression by RA and RAR
Figure 3:
cT3R
cT3R Similarly, hRAR 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
Figure 4:
Effects of ligands on binding and
dimerization of cT3R
The presence of T3 or RA did
not change the pattern of binding of cT3R 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
Figure 5:
The DNA binding domain is essential for
the regulation by T3R. A, the NH
Figure 6:
The DBD mutant specifically interferes
with the regulation by T3R and not by RAR. A, the
cT3R
In view of the fact that
cT3R
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
Figure 8:
Regulation of keratin gene expression by
cT3R
cT3R 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 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
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1416-1423
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
)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) . -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.
Plasmids and Their Growth and
Purification
Plasmids pK#14CAT, pK#5CAT pK#17CAT, and pRSVZ have
been described previously (13, 17) . The plasmids
containing human RAR, RAR
, and RAR nuclear receptors
were gifts from Dr. P. Chambon. Plasmids cT3R
(51-408),
NH-terminal deletion mutant of T3R,
cT3R(120-408) DBD mutant, heptad mutants
cT3R
(L365R) and cT3R(L372R), and v-ErbA were also described
previously(23, 24, 26) . ()Plasmids were grown in JM101 Escherichia coli host to saturation density in LB medium. DNA was extracted and
purified using the Magic Mega Prep Kit from Promega.Cell Growth
HeLa cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10%
calf serum at 37 °C in a 5% CO atmosphere in media
containing penicillin and streptomycin as
described(20, 27) . The day before transfection, cells
were plated onto 60-mm dishes. Four hours before transfection the
medium was changed to DMEM supplemented with 10% calf serum depleted of
RA and T3 as described(20) .Transfection Using
Ca
We
have generally followed published procedure for cells that were at 80%
confluence(27) . At the time of transfection into each dish
were added 3 µg of the CAT plasmid, 1 µg of the nuclear
receptor expression vector plasmid, 1 µg of pRSVZ reference plasmid
and a sufficient amount of carrier to bring the total to 10 µg of
DNA. The cells were harvested 48 h after transfection by scraping into
5 ml of phosphate-buffered saline, washed once more in
phosphate-buffered saline, and resuspended in 150 µl of 0.25 M Tris buffer, pH 7.8. All transfections were performed in duplicate
plates, and each transfection experiment was repeated two to five
times. CAT and 
(PO
)-galactosidase assay were performed as
described(20) .Electrophoretic Gel Mobility Shift Assays
E.
coli-expressed hRAR and cT3R were purified as described
previously(28) . Oligonucleotides were synthesized on a
Pharmacia Gene Assembler Plus Synthesizer. The sequence of
oligonucleotides flanked by HindIII overhangs (5`-AGCTT-3`)
are as follows: TREp, AGGTCATGACCT; mTRE, ACGTCATGACGT; K14RE,
GCTAGCCTGTGGGTGATGAAAGCCAAGGGGAATGT. Double-stranded oligonucleotides
corresponding to the K14RE and TRE palindrome were labeled with
[-P]dATP, using the Klenow fragment of E. coli DNA polymerase I. 30,000 cpm of the resulting probe
was mixed with 2.5 fmol of purified receptor proteins and incubated
first for 30 min at room temperature then for 10 min at +4 °C.
The incubation was done in a 30-µl volume in 25 mM Tris,
pH 7.8, 500 µM EDTA, 88 mM KCl, 10 mM 2-
-mercaptoethanol, 0.1 µg of aprotinin, 0.1 µg of
poly(dI-dC), 0.05% Triton X-100 (v/v), 10% glycerol (v/v). Samples were
loaded on 4% polyacrylamide gel and separated by electrophoresis
(20-25 mA) at +4 °C for 2 h with a buffer containing 10
mM Tris, 7.5 mM acetic acid, and 40 µM EDTA, pH 7.8. Gels were dried and analyzed by autoradiography. (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) .
Novel Regulation of the Keratin Promoters by Retinoic
Acid and Thyroid Hormone Receptors
To analyze regulation of
keratin gene expression by RA and T3 we used the promoters of the K14,
K5, and K17 keratin genes linked to a CAT reporter gene. HeLa cells
were co-transfected with the keratin promoter-CAT constructs along with
vectors expressing wild type and mutants of chicken T3R and human
RARs in the presence or absence of the respective ligands. We chose
HeLa cells because we have shown previously that transfected keratin
gene promoters behave identically in HeLa cells and in human epidermal
keratinocytes(20, 21) . However, the endogenous
receptors are expressed at much lower levels in HeLa cells, which
facilitates the interpretation of results with transfected receptors. ,
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.
, 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.
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. 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.
. Note, however, that the
unliganded RAR does not block the induction by
T3R.
hRAR
To study the interaction of T3R and RAR
with receptor-responsive sequences, we focused on the
-96/-51 region of the K14 gene promoter in which we
previously identified a TRE/RARE using site-specific
mutagenesis(21) . Gel mobility shift DNA binding assays were
performed using hRAR and cT3R Bind to a Functional Element in
the K14 Gene Promoter and cT3R expressed and purified from E. coli (Fig. 3).
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.
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). 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. 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) .
and hRAR. Autoradiograms of the gel
mobility shift assays are presented with K14RE (A) and TREpal probe (B).
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) ., 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.The Amino-terminal Region of the T3R Is Not Essential for
Keratin Gene Regulation
To study the mechanism of keratin
regulation by cT3R, we used variants of the receptor that have
specific deletions and mutations in the NH-terminal A/B
region, the DNA binding domain, or the ligand and heterodimerization
domains, as well as v-ErbA(22, 23, 24) . We
first analyzed the role of the 50-amino acid NH-terminal
region of cT3R, because this region has been reported to be
important for hormone-independent activation of a sequence in the Rous
sarcoma virus LTR (RSV-LTR)(22, 32) . The receptor
mutant cT3R(51-408) has a complete deletion of the 50-amino
acid NH-terminal A/B domain but has normal DNA binding and
ligand binding properties(22) . Both in the absence and in the
presence of T3, cT3R(51-408) functions essentially identical
to the wild type cT3R (compare Fig. 5A with Fig. 1). Thus, the NH-terminal A/B region of
cT3R is not essential for either constitutive activation or
ligand-dependent inhibition of keratin promoter activity.
-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.
The DNA Binding Domain (DBD) of cT3R
In contrast with the
cT3R Is Essential
for Keratin Gene Regulation(51-408), a mutant lacking both the DNA binding domain
and the NH-terminal A/B region (cT3R(120-408);
also referred to as DBD) (24) , did not
influence keratin promoter activity (Fig. 5B).
cT3R
(120-408) has been shown to act as a dominant negative
inhibitor of wild type T3Rs and RARs(26, 28) , indeed
it blocked the RA-dependent stimulation of TREpCAT (Fig. 6A). The inhibitory effect was enhanced by the
addition of T3 to the medium. Unexpectedly, the suppression of keratin
genes by hRAR was not affected by addition of the
cT3R(120-408) in the presence or absence of T3 (Fig. 5A).
(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.
(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) .v-ErbA Constitutively Activates Keratin Gene
Promoters
v-ErbA is an oncogenic variant of cT3R that binds
T3 with very low affinity and constitutively represses promoters that
contain a number of positive regulatory elements, including the TREp in
TREpCAT (Fig. 7A)(24, 33, 34) . In
contrast with the repression seen with other elements, we find that
v-ErbA constitutively activates the K5, K14, and K17 promoters about
2-3-fold, which is similar to the activation found for unliganded
wild type cT3R (Fig. 7A). v-ErbA has also been
found to act as a weak dominant negative inhibitor of wild type T3Rs
and RARs(24) . This effect is thought to result from direct
competition for the DNA binding site rather than from interference with
heterodimerization with RXR(24) . In contrast to
cT3R(120-408), which did not affect inhibition by
hRAR-RA, we found that v-ErbA efficiently blocked the effect of
hRAR-RA (Fig. 7B). v-ErbA was equally efficient in
blocking T3-dependent suppression of keratin promoter activity by
cT3R (Fig. 7C). Thus, v-ErbA is not only a
constitutive activator of keratin gene expression, but also an
inhibitor of the suppression of keratin genes mediated by RA and T3.
. C, v-ErbA blocks T3-dependent suppression of the K14
gene expression and induction of TREpCAT by
T3R.
Constitutive Activation of Keratin Gene Expression by T3R
Does Not Require Heterodimerization with RXR
To study the role
of homo- and heterodimerization in the constitutive activation of
keratin genes by cT3R, we used mRXR and cT3R receptors
in our gel mobility shift experiments (Fig. 8A).
cT3R can bind as three complexes with TREpal in the presence of
mRXR. These can be identified by size as T3R monomer, T3R
homodimer, and RXR-T3R heterodimer. Addition of T3 did not change the
binding pattern. In contrast RXR-T3R heterodimers are not formed with
K14RE (Fig. 8A). The two complexes identified are the
monomer and the homodimer of T3R. The addition of hormone promotes
monomer binding at the expense of the homodimer. These results suggest
that the regulation of keratin gene expression does not require
heterodimer formation with RXR. To investigate this possibility
further, we used two cT3R mutants in the ninth heptad of the
ligand binding domain, cT3R(L365R) and cT3R(L372R), which
have been shown to be critical for heterodimerization with
RXR(23) . These mutants bind to response elements as homodimers
as efficiently as the wild type cT3R, but do not bind as
heterodimers with RXR in the absence of T3(23) . With
cT3R(L365R), but not with cT3R(L372R), T3 mediates a
conformational change that results in the formation of cT3R/RXR
heterodimers(23) . The binding pattern at cT3R(L372R)
mutant was identical to that of the wild type receptor; it formed two
complexes with K14RE: the monomer and the homodimer (Fig. 8B). The addition of mRXR did not change the
binding pattern, as expected, because this mutant is not capable of
forming heterodimers with RXR receptors.
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.
(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.
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) .
))
hspace=3 SRC="/icons/back.GIF">
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. L. Varley, J. Stahlschmidt, B. Smith, M. Stower, and J. Southgate Activation of Peroxisome Proliferator-Activated Receptor-{gamma} Reverses Squamous Metaplasia and Induces Transitional Differentiation in Normal Human Urothelial Cells Am. J. Pathol., May 1, 2004; 164(5): 1789 - 1798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Jho, N. Radoja, M. J. Im, and M. Tomic-Canic Negative Response Elements in Keratin Genes Mediate Transcriptional Repression and the Cross-talk among Nuclear Receptors J. Biol. Chem., November 30, 2001; 276(49): 45914 - 45920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Radoja, M. Komine, S. H. Jho, M. Blumenberg, and M. Tomic-Canic Novel Mechanism of Steroid Action in Skin through Glucocorticoid Receptor Monomers Mol. Cell. Biol., June 15, 2000; 20(12): 4328 - 4339. [Abstract] [Full Text]
|
|
|
|
|
|
C. Oberste-Berghaus, K. Zanger, K. Hashimoto, R. N. Cohen, A. N. Hollenberg, and F. E. Wondisford Thyroid Hormone-independent Interaction between the Thyroid Hormone Receptor beta 2 Amino Terminus and Coactivators J. Biol. Chem., January 21, 2000; 275(3): 1787 - 1792. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Presland and B. A. Dale Epithelial Structural Proteins of the Skin and Oral Cavity: Function in Health and Disease Critical Reviews in Oral Biology & Medicine, January 1, 2000; 11(4): 383 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Satoh, T. Ishizuka, T. Monden, N. Shibusawa, T. Hashida, M. Kishi, M. Yamada, and M. Mori Regulation of the Mouse Preprothyrotropin-Releasing Hormone Gene by Retinoic Acid Receptor Endocrinology, November 1, 1999; 140(11): 5004 - 5013. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. Wan, N. Oridate, D. Lotan, W. K. Hong, and R. Lotan Overexpression of Retinoic Acid Receptor {beta} in Head and Neck Squamous Cell Carcinoma Cells Increases Their Sensitivity to Retinoid-induced Suppression of Squamous Differentiation by Retinoids Cancer Res., July 1, 1999; 59(14): 3518 - 3526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Belandia, M. J. Latasa, A. Villa, and A. Pascual Thyroid Hormone Negatively Regulates the Transcriptional Activity of the beta -Amyloid Precursor Protein Gene J. Biol. Chem., November 13, 1998; 273(46): 30366 - 30371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Palomino, D. Barettino, and A. Aranda Role of GHF-1 in the Regulation of the Rat Growth Hormone Gene Promoter by Thyroid Hormone and Retinoic Acid Receptors J. Biol. Chem., October 16, 1998; 273(42): 27541 - 27547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-F. Langlois, K. Zanger, T. Monden, J. D. Safer, A. N. Hollenberg, and F. E. Wondisford A Unique Role of the beta -2 Thyroid Hormone Receptor Isoform in Negative Regulation by Thyroid Hormone. MAPPING OF A NOVEL AMINO-TERMINAL DOMAIN IMPORTANT FOR LIGAND-INDEPENDENT ACTIVATION J. Biol. Chem., October 3, 1997; 272(40): 24927 - 24933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Olson and R. J. Koenig 5'-Flanking Sequences in Thyroid Hormone Response Element Half-sites Determine the Requirement of Retinoid X Receptor for Receptor-mediated Gene Expression J. Biol. Chem., April 11, 1997; 272(15): 9907 - 9914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Hollenberg, T. Monden, J. P. Madura, K. Lee, and F. E. Wondisford Function of Nuclear Co-repressor Protein on Thyroid Hormone Response Elements Is Regulated by the Receptor A/B Domain J. Biol. Chem., November 8, 1996; 271(45): 28516 - 28520. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |