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INTRODUCTION |
Thyroid transcription factor-1
(TTF-1)1 is a
homeodomain-containing protein implicated in the transcriptional
activation of genes expressed exclusively in thyroid (1, 2) and lung
(3). In addition, TTF-1 plays an important role in thyroid, lung, and brain morphogenesis before the onset of cell type-specific
transcription (4). The ability of TTF-1 to activate transcription of
different genes in distinct cell types and to play diverse roles during development suggests that the activity of this transcription factor is
highly regulated.
Mechanisms involved in modulating the transcriptional potential of a
transcription factor, without changes in its intracellular concentration, are post-translational modification, such as
phosphorylation (5), and/or modulation of the redox state (6).
Cofactors have also been shown to be important modulators of
transcription factors activity and, in some cases, to be able to assign
to a given transcription factor distinct promoter specificity in
defined cell types (7-10). Cofactors are important for transcriptional activation through different mechanisms; they can act as a bridge between a transcription factor and the basal transcription apparatus (11, 12), they can be involved in chromatin reorganization (13), or
they can modify the DNA binding properties of a transcription factor
(14-16). Other proteins act as cofactors by activating the conformation of transcription factors and modulating in this way their
DNA binding capability (17).
The DNA binding activity of TTF-1 has been proposed to be regulated by
redox (18, 19) and phosphorylation (20-23), even though for the latter
modification no effect has been observed in heterologous cells (24).
However, no TTF-1 cofactor has been identified yet.
We searched for TTF-1 cofactors using a modified yeast one-hybrid
system and found that TTF-1 can interact with calreticulin, a
60-kDa protein and a major Ca2+-binding component in
non-muscle cells. Calreticulin plays a role in Ca2+
storage, its expression is modulated during cell differentiation, and
the amount of this protein varies among different cell types (25).
Calreticulin is also an important chaperone involved in glycoprotein maturation (26) as it promotes the efficient folding and assembly of class I histocompatibility molecules (27).
Moreover, it has been demonstrated that calreticulin modulates gene
expression by interaction with steroid hormone nuclear receptors (15,
16).
We show in this report that TTF-1 and calreticulin interact both
in vitro and in vivo. Overexpression of
calreticulin in HeLa cells results in an increased activity of TTF-1,
suggesting that the interaction between these two proteins may have
functional significance. Calreticulin binds to the TTF-1 homeodomain
and may act by promoting its folding because the DNA binding activity of heat-denatured TTF-1 HD synthesized in Escherichia coli
is improved significantly by calreticulin. These findings suggest that
calreticulin is able to modulate TTF-1 activity by regulating the
folding state of TTF-1 HD.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The reporter plasmid Tg-
Gal has been described
(28). The TTF-1 deletion mutant
36 (29), containing the TTF-1 coding sequence from amino acid 96 to 296, was inserted downstream of the ADH1
promoter in the plasmid pGAD424 (),
replacing the GAL4 activation domain. From this plasmid we obtained, by cleavage with SphI, a fragment containing the ADH1 promoter,
36, and ADH1 terminator sequence. Cloning this fragment in pGBT9
plasmid () by replacing the fragment
between the SphI sites yielded the plasmid ADH1
36. The
plasmids Tg-
Gal and ADH1
36 were used for the yeast one-hybrid
system. The complete sequence of the calreticulin open reading frame
was cloned by reverse transcriptase-polymerase chain reaction using RNA
extracted from the rat thyroid cell FRTL-5 (29). The plasmid
CMVcalreticulin was generated by inserting the entire calreticulin
cistron in the expression vector pKW10 (30). The plasmids CMVTTF-1,
G7,
G13, G5E1b, C5E1b, CMV-Luc, and pTACAT3, used in transient
transfection of HeLa cells, have been described previously (29,
31).
Yeast Strains and Methods--
The yeast strain used was INVSC1
(MATa, his3-
1, leu2, trp1-289, ura3-52) (Invitrogen). Yeast were
grown in YEPD or selective minimal medium (32). Transformations were
made by the method of Schiestl and Gietz (33).
-Galactosidase
activity was assayed in liquid as described previously (34).
Yeast One-hybrid System--
A yeast strain containing both the
reporter plasmid Tg-
Gal and the ADH1
36 bait was constructed in
INVSC1 yeast cells and maintained by selection on
Ura
Trp
medium. These cells were used for
transformation with an FRTL-5 cDNA expression library, constructed
in the GAL4 activation domain plasmid pGAD10
(). After transformation, cells were plated on Ura
Trp
Leu
agar medium. The
screening was performed by
-galactosidase activity using a colony
color assay as described (, Matchmaker two-hybrid system protocol).
In Vitro Transcription and Translation--
1 µg of
CMVcalreticulin was transcribed and translated using the
TnT®-coupled reticulocyte lysate system (Promega)
according to the manufacturer's instructions.
In Vitro Protein Binding--
Both full-length TTF-1 as well the
NH2-terminal coding sequence were expressed in E. coli as fusion proteins with a 6-histidine tail and purified as
described (28, 35). Expression and purification of TTF-1 HD were done
as reported (36). For the in vitro protein binding, 80 µg
of purified full-length TTF-1 was incubated with 0.2 ml of
Ni+-nitrilotriacetic acid-agarose resin (Qiagen) for 30 min
at 4 °C in a final volume of 2 ml of buffer B250 (25 mM
Tris/Cl, pH 7.5, 15 mM MgCl2, 0.15 mM EGTA, 0.3% Triton X-100, 1 mM
dithiothreitol, 250 mM NaCl, and the protease inhibitors
leupeptin, pepstatin, and phenylmethylsulfonyl fluoride). After
incubation the resin was washed twice with 2 ml of B250 and resuspended
in 2 ml of B250. Each in vitro translated
[35S]methionine-labeled protein was incubated with 0.1 ml
of resin containing either TTF-1 protein or BSA. After incubation, the resins were washed using different salt concentration (B250, B500, B700). Elution was carried out with 40 µl of 2 × SDS loading
buffer. The eluted proteins were run on a 12% denaturing
SDS-polyacrylamide reducing gel. After fixation for 20 min with 10%
acetic acid and 10% methanol, the gel was enhanced with an Enlightning
solution (NEN Life Science Products), dried, and exposed for autoradiography.
An in vitro protein binding assay with deletion mutants of
TTF-1 was performed using both the TTF-1 NH2-terminal
domain and TTF-1 HD linked to CNBr-activated Sepharose 4B resin
(Amersham Pharmacia Biotech Inc.) at a final concentration of 1 mg of
protein/ml of resin. As control, BSA was linked to CNBr-activated
Sepharose 4B resin at the same final concentration. 0.2 ml of packed
resin containing the immobilized proteins was preincubated with 2 ml of
B250 for 30 min at 4 °C. The resin was washed twice and resuspended in 2 ml of B250. 0.1 ml of B250 resin was incubated with in
vitro translated [35S]methionine-labeled
calreticulin. The in vitro protein binding assay was carried
out as described for full-length TTF-1 protein.
Cell Culture and Transfection--
HeLa cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. For transient expression assay, cells were plated at 5 × 105 cells/60-mm tissue culture dish 4-6 h before
transfection. Transfections and measurement of luciferase activity on
cell extracts were determined as described (29). CAT expression was
detected using a CAT enzyme-linked immunosorbent assay (Boehringer
Mannheim) according to the manufacturer's instructions. The thyroid
cell line PC was maintained as described in a medium supplemented with
thyroid-stimulating hormone (TSH) and insulin (37), except when
otherwise stated.
In Vitro Folding and Band Shift Assay--
Thermal denaturing of
TTF-1 HD was performed in 40 mM Hepes, pH 7.9, and 1 mg/ml
BSA for 10 min at 60 °C followed by fast cooling in ice for 3 min. Native and heat-treated TTF-1 HD were incubated with increasing
amounts of in vitro translated calreticulin for 15 min at
room temperature. Incubation with reticulocyte lysate alone was used as
control. The DNA binding activities of 2 ng of native and denatured
TTF-1 HD, before and after incubation with calreticulin, were measured
by band shift assay using oligonucleotide C (38).
Northern Blot Analysis--
15 µg of total RNA, prepared from
PC cells maintained under different conditions (37), was
electrophoresed in 1% agarose gel containing 2.2 M
formaldehyde and transferred to Hybond N-plus membrane (Amersham
Pharmacia Biotech Inc.). The blot was hybridized with a
32P-labeled HindIII/BamHI fragment of
the plasmid CMVcalreticulin, encoding calreticulin from amino acid 1 to
193. To normalize for RNA loading, hybridization with
32P-labeled TTF-1 (38) and
-actin (39) probes was
carried out.
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RESULTS |
Isolation of cDNA Clones Encoding Proteins That Interact with
TTF-1--
We used a modification of the yeast one-hybrid system to
identify TTF-1-interacting proteins. The indicator yeast strain carries a chimeric gene where transcription of a lacZ reporter is
under the control of a segment of the thyroglobulin (Tg) promoter fused to the minimal yeast CYC-1 promoter (Fig.
1A). The portion of the Tg
promoter used contains three TTF-1 binding sites and has been shown to
function as an enhancer (31). Furthermore, we have demonstrated
previously that this Tg promoter segment can be activated efficiently
by TTF-1 in mammalian non-thyroid cells (29). This reporter showed
little background activity in yeast, but it was transactivated
efficiently by full-length TTF-1 (Fig. 1B).

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Fig. 1.
Calreticulin interacts with TTF-1 in
yeast. Panel A, the structure of the constructs used in
the modified yeast one-hybrid system is shown. Tg-lacZ is the
thyroglobulin promoter upstream region, in which the TTF-1 binding
sites are indicated, fused to the CYC minimal promoter upstream of the
lacZ cistron; TTF-1 is schematic structure of the TTF-1
protein, with HD indicating the TTF-1 homeodomain; 36 is the TTF-1
deletion used for the modified one-hybrid system; cl 11.3 is the clone
encoding calreticulin fused to the GAL4 activation domain (GAL4
AD) isolated in the modified one-hybrid screen. Panel
B, INVSC 1 yeast cells were transformed with the reporter plasmid
Tg-lacZ. Single colonies were then transformed with expression vectors
encoding the indicated proteins. -Galactosidase activity was
measured in triplicate by quantitative liquid culture assay.
Bars indicate the S.D.
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As bait, we expressed in the indicator strain a deletion mutant of
TTF-1 (
36, amino acids 96-296) which is not able to transactivate the reporter gene (Fig. 1, A and B).
36 is
also unable to activate transcription from the Tg promoter in mammalian
cells (29) but can compete with the full-length protein. Because
3,
another TTF-1 deletion that includes the homeodomain, is unable to show the same competing activity (29), we hypothesize that
36 does not
compete with TTF-1 for the DNA binding site but for a common coactivator.2 The indicator cells
were then transformed with a FRTL-5 cDNA library constructed in a
pGAD10 vector, in which each cDNA clone is fused to the
NH2-terminal end of the GAL4 transcriptional activation domain. GAL4AD-cDNA fusion proteins, which can interact with
36 or bind directly to the Tg promoter, will activate transcription of
lacZ and will show a blue phenotype by colony color assay. A
screen of 1 × 106 primary transformants yielded nine
potential positive clones. All but three of these could activate the
reporter gene upon transformation into yeast lacking
36, and they
were discarded. In contrast, three plasmids could only activate the
reporter gene in the presence of
36. Sequence analysis showed that
one of the plasmids (clone 11.3) contained a cDNA insert encoding calreticulin.
As shown in Fig. 1B, coexpression of GAL4AD-calreticulin
fusion protein encoded by clone 11.3, together with
36, resulted in
increased reporter gene activity. This increased activity was dependent
upon the presence of the bait, as it was not observed when the
GAL4-calreticulin hybrid was expressed alone.
In Vitro Association of TTF-1 and Calreticulin--
To confirm the
specific interaction between calreticulin and TTF-1 observed in yeast
cells, we first examined the interaction between TTF-1 and calreticulin
in vitro. Full-length TTF-1 was expressed in E. coli as a fusion protein with a 6-hystidine residue tag (35). The
fusion protein was immobilized on a Ni+-nitrilotriacetic
acid-agarose resin and incubated with in vitro translated,
[35S]methionine-labeled calreticulin. To analyze the
strength of interaction, we measured the amount of calreticulin bound
on TTF-1 after washing with a buffer of increasing ionic strength. As
shown in Fig. 2A, calreticulin
binds to TTF-1 but fails to associate with a control resin, and it is
not able to bind the unrelated protein TTF-2 (data not shown). The
interaction between calreticulin and TTF-1 seems to be strong, as shown
by the resistance of the complex to a 700 mM NaCl wash.

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Fig. 2.
In vitro binding between
calreticulin and TTF-1 or TTF-1 deletion mutants. In
vitro translated, [35S]methionine-labeled
calreticulin was incubated with immobilized full-length TTF-1
(panel A), TTF-1 HD (panel B), or the TTF-1
NH2-terminal domain (panel C). After incubation
the resins were washed using 250, 500, and 700 mM NaCl.
Bound proteins were eluted with SDS loading buffer and resolved by
SDS-polyacrylamide gel electrophoresis. Full-length TTF-1 is a 6 histidine-tagged protein, immobilized on a nitrilotriacetic acid resin.
Control is the resin without protein. The two TTF-1 deletions (HD) and
NH2 terminal domain (N-ter) were immobilized on
CNBr-activated Sepharose. The same resin coupled to BSA was used as
control.
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To establish which region of TTF-1 interacts with calreticulin, we
immobilized TTF-1 HD (36), the TTF-1 NH2 terminus (28), and
BSA as a negative control on CNBr-activated Sepharose 4B resin. The
different resins were incubated with in vitro translated, [35S]methionine-labeled calreticulin. Incubation and
washing conditions were the same as described above for the binding
assay with full-length TTF-1. As shown in Fig. 2B,
calreticulin is able to bind TTF-1 HD, although it fails to interact
with the TTF-1 NH2 terminus (Fig. 2C) and with a
control resin containing immobilized BSA (Fig. 2B). The
binding of calreticulin to TTF-1 HD in vitro is consistent
with the data obtained in the yeast one-hybrid system because the bait
36 used in yeast contains the entire TTF-1 HD and 63 amino acids of
the NH2-terminal domain.
Calreticulin Increases TTF-1 Activity in HeLa Cells--
To assess
the in vivo significance of the TTF-1-calreticulin
interaction shown in vitro and in yeast, we determined
whether overexpression of calreticulin in HeLa cells would modulate
TTF-1 transcriptional activity. We transfected HeLa cells with a
plasmid containing Tg minimal promoter fused to a CAT coding
sequence (pTACAT3) (31). This promoter showed very little activity
in HeLa cells, as demonstrated previously (29), but it was
transactivated efficiently upon cotransfection with a TTF-1 expression
vector (CMVTTF-1) containing the TTF-1 cistron under the control of the human cytomegalovirus promoter (29). As shown in Fig.
3A (lane 4),
cotransfection of a calreticulin expression vector resulted in an
approximately 3-fold increase of TTF-1-induced CAT expression. This
increased activity was dependent upon the presence of TTF-1 because
there was no effect of calreticulin alone on Tg promoter transcription
(Fig. 3A, lane 2). To provide further support for the direct role of calreticulin on TTF-1 activity, we cotransfected in
HeLa cells the CAT reporter plasmid under the control of the C5E1b
promoter, which contains five TTF-1 binding sites upstream of the E1b
TATA box (29). This promoter is transcribed efficiently only upon
cotransfection of the TTF-1 expression vector (Fig. 3B,
lane 3), and CAT expression is still increased 3-fold when calreticulin is overexpressed (Fig. 3B, lane
4).

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Fig. 3.
Effect of overexpression of calreticulin in
HeLa cells on TTF-1 activity. Panel A, HeLa cells were
transiently transfected with the reporter plasmid pTACAT3 (3 µg) and
expression vectors encoding TTF-1 (0.25 µg) and calreticulin (2.5 µg) as indicated. CMV-Luc was cotransfected to normalize for
transfection efficiency. Values are expressed as the fold of activation
of the reporter gene above that observed with the reporter alone and
represent the average of three independent experiments. The error
bars show the S.D. for the mean. Panel B, HeLa cells
were transiently transfected with the reporter plasmid C5E1b (3 µg)
and expression vectors encoding TTF-1 (0.25 µg) and calreticulin (2.5 µg) as indicated. Values are presented as in panel A and
represent the average of three independent experiments. The error
bars show the S.D. for the mean. The structure of the reporters
used is illustrated in the bottom part of the figure.
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Because calreticulin binds to the TTF-1 HD, we asked whether this
interaction is necessary for the observed calreticulin-mediated increase of TTF-1 activity. To this end we tested the effect of calreticulin overexpression on the activity of TTF-1 deletion mutants
fused to the DNA binding domain of GAL4. These TTF-1-GAL4 fusions were
tested on the G5E1b reporter plasmid (40) containing five GAL4 binding
sites upstream of the E1b TATA box. It has been demonstrated previously
that TTF-1 has two distinct activation domains and that either the
entire region NH2-terminal to the homeodomain (mutant
G7) or a segment of TTF-1 from the COOH-terminal (mutant
G13)
fused with the DNA binding domain of GAL4 is able to activate the
expression of the G5E1b reporter, containing five DNA binding sites for
GAL4 (29). We tested the role of calreticulin overexpression on both
G7 and
G13 fusions. The experiment revealed that both fusions are
insensitive to calreticulin overexpression (Fig.
4B), suggesting that the target of
calreticulin function is the TTF-1 HD.

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Fig. 4.
Effect of calreticulin overexpression on
TTF-1 activation domains. Panel A, structure of the
reporter construct and of the activators containing the DNA binding
domain of GAL4 fused either to the amino- ( G7) or the carboxyl-
( G13) activation domains of TTF-1. Numbers refers to the
amino acid residue in the TTF-1 protein. Panel B, HeLa cells
were transiently transfected with the reporter plasmid G5E1b (3 mg) and
expression vectors encoding G7 (0.1 µg), G13 (0.25 µg), and
calreticulin (2.5 µg), as indicated. CMV-Luc was cotransfected to
normalized for transfection efficiency. Values are expressed as the
fold of activation of the reporter gene above that observed with the
reporter alone and represent the average of three independent
experiments. The error bars show the S.D. of the mean.
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Calreticulin Is Able to Refold HD in Vitro--
Because the
calreticulin-TTF-1 interaction resulted in an increased activity of
TTF-1 and calreticulin binds TTF-1 HD in vitro, we asked
whether calreticulin can exert its effect by modulating the DNA binding
activity of TTF-1 HD. It has been demonstrated previously that the
helical content of TTF-1 HD is very sensitive to temperature variation
and that this thermal denaturing resulted in a greatly decreased DNA
binding activity of TTF-1 HD (36). Thus, we tested the capability of
calreticulin to improve the refolding of TTF-1 HD after heat
denaturing. To this end, TTF-1 HD was first heat treated at 60 °C
and then incubated at room temperature either with or without an
increasing amount of in vitro translated calreticulin. The
folding state of TTF-1 HD was detected by band shift assay using the
oligonucleotide C specifically recognized by TTF-1 (38). As shown in
Fig. 5, the refolding of TTF-1 HD increases
after the addition of an increasing amount of calreticulin, whereas
incubation with the control reticulocyte lysate has no effect on the
heat-denatured TTF-1 HD. To define better the role of calreticulin on
TTF-1 HD, we analyzed by band shift assay the effect of calreticulin on
TTF-1 HD not denatured. Native TTF-1 HD was incubated for 10 min with
an increasing amount of in vitro translated calreticulin,
then its affinity for the labeled oligonucleotide C was detected by
band shift assay. Fig. 5 shows that the specific binding of TTF-1 HD on
DNA does not change after incubation with calreticulin. These data
strongly suggest that calreticulin is able to stimulate refolding of
denatured TTF-1 HD but is not able to increase the DNA binding activity of the native TTF-1 HD. Calreticulin alone does not bind to
oligonucleotide C (Fig. 5, lane 16), and the addition of
calreticulin to heat-treated TTF-1 HD produces a band comigrating with
the untreated native TTF-1 HD (Fig. 5, lanes 10-12),
suggesting that calreticulin does not remain in the TTF-1 HD·DNA
complex. Together, these results indicate that calreticulin is able to
affect the folding state of TTF-1 HD but does not bind stably to either
the DNA or protein component of the final complex.

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Fig. 5.
Calreticulin refolds TTF-1 HD in
vitro. TTF-1 HD, either native (lanes 2-8)
or heat-denatured (lanes 9-15), was incubated with
32P-labeled oligonucleotide C in the presence or absence of
in vitro translated calreticulin, as indicated. As a
control, the same amount of reticulocyte lysate, without calreticulin,
was used. Lane 1 is the free probe. Lane 16 is
calreticulin-containing reticulocyte lysate alone.
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Calreticulin Expression in Thyroid Cells Is Regulated by TSH and
Insulin--
The DNA binding activity of TTF-1 in thyroid cells has
been reported to be under TSH control because of redox regulation (19) even though TTF-1 mRNA appears to be down-regulated by the hormone (41, 42). To investigate whether calreticulin can contribute to
hormonal regulation of the DNA binding activity of TTF-1, we measured
calreticulin mRNA levels in PC thyroid cells cultured in medium
with or without TSH and insulin for different times. As shown in Fig.
6, calreticulin expression is reduced
strongly after just 1 day of depletion of hormones, and it is
reexpressed 12 h after the addition of TSH and insulin. The
variation of calreticulin mRNA levels is specific, as demonstrated
by the different effects of the hormonal starvation on
-actin and
TTF-1 mRNA. These data suggest that calreticulin could be involved
in the hormonal regulation of DNA binding activity of TTF-1.

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Fig. 6.
Expression of calreticulin mRNA in
thyroid cells. PC cells, grown in the presence of TSH and insulin
(CTRL), were depleted of both hormones for 24 h,
48 h, 72 h, and 5 days. After 5 days of starvation, the
hormones were added again, and cells were cultured for 12 and 24 h. Total RNA was extracted at all time points, electrophoresed on
formaldehyde-agarose gel, and transferred to nylon membrane.
Hybridization was carried out with a probe encoding the
NH2-terminal-specific domain of calreticulin. To control
for RNA, loading hybridizations with probes recognizing -actin and
TTF-1 mRNAs were carried out.
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DISCUSSION |
We show in the present paper that calreticulin binds to the
homeodomain of TTF-1 and is capable of stimulating its refolding. We suggest that the ability of calreticulin to promote renaturing of
the TTF-1 HD may be responsible for the observed stimulation by
calreticulin of TTF-1 transcriptional activity in cotransfection experiments, perhaps by increasing the steady-state concentration of
properly folded homeodomain. The hypothesis that the stimulation of
TTF-1 activity by calreticulin depends on its interaction with the
homeodomain is supported by experiments showing no effect of
calreticulin overexpression on the activity of the two transactivation domains of TTF-1 fused to the heterologous DNA binding domain of GAL4.
Calreticulin functions as a Ca2+-binding protein in the
endoplasmic reticulum (43), but it has also been found in the nucleus
(44), suggesting a role for this protein in regulation of gene
expression. In keeping with this hypothesis, calreticulin has been
shown to interact with the KXFFKR amino acid sequence
present in the DNA binding domain of nuclear hormone receptors and to
inhibit their DNA binding activity (15, 16). Furthermore,
overexpression of calreticulin in cultured cells interferes with
transcriptional activation mediated by several nuclear hormone
receptors, thus indicating that calreticulin is an important negative
coregulator of gene expression (15, 16, 45-48). At variance, however,
from the negative interference exerted by calreticulin on
transcriptional activation by nuclear hormone receptors, coexpression
of TTF-1 and calreticulin results in transcriptional stimulation,
both in yeast and in mammalian cells, suggesting that in the case of
TTF-1 calreticulin operates with a different mechanism.
Furthermore, even though calreticulin interacts also in the case of
TTF-1 with its DNA binding domain, the TTF-1 HD lacks the sequence
KXFFKR, suggesting that in this case calreticulin recognizes
a different structural motif. The positive effect of calreticulin on
the transcriptional activity of TTF-1 parallels the stimulation of DNA
binding which was observed only on the thermally denatured homeodomain.
In this respect it could be of relevance the observation that the TTF-1
HD appears to be more sensitive than other homeodomains to thermal
denaturing, a property that has been related to the peculiar DNA
binding specificity of this homeodomain (36, 49).. Thus, it
is conceivable that the DNA binding activity of the TTF-1 HD, and
perhaps other HDs of the same NK-2 class which show similar properties
(50, 51), could be regulated by mechanism that impinges on their
peculiar flexibility. The conformational activation of TTF-1 HD seems
to result from a transient interaction between calreticulin and
unfolded TTF-1 HD. In fact, in an in vitro assay,
calreticulin is not found associated with the TTF-1 HD·DNA complex.
These data are similar to those demonstrating that HSP90 is able to
activate the DNA binding potential of MyoD1 by a transient interaction
with it (17). However, the in vivo assay in yeast cells
showing TTF-1-dependent stimulation of transcription by the
GAL4AD-calreticulin fusion protein suggests that this interaction
could be stable enough to activate transcription in vivo but
not to survive the conditions of the DNA binding assay. Alternatively,
the interaction between calreticulin and TTF-1 could be stabilized
in vivo by additional factors that are absent in our assay.
Homeodomains have their primary function in the recognition of specific
DNA sequences (52). However, this function can be highly regulated,
mostly as consequence of protein-protein interactions. For example, the
specific DNA sequence that is recognized by a given homeodomain can be
changed by the interaction with other homeodomain-containing proteins
(53, 54). Furthermore, homeodomains have been shown to interact with
non-homeodomain proteins (55, 56), thus expanding the protein-protein
interaction functions of this highly regulated protein domain. The
interaction with calreticulin which we report in this study could be of
a general relevance, and it could be instrumental in regulating the
amount of functional homeodomain present in the cell by regulation of its folding. In this respect it is of interest that calreticulin expression in thyroid cells is highly and rapidly regulated by TSH and
insulin. A regulation of calreticulin gene expression by the cAMP
pathway has already been reported in mouse melanoma cells (46). In
thyroid cells TSH and insulin have been demonstrated to stimulate the
expression of the thyroglobulin gene (39), whose promoter is activated
by TTF-1 (31). Previous studies have shown that TTF-1 is
redox-regulated and that the redox state of TTF-1 is
TSH-dependent (18, 19). The phosphorylation of TTF-1 also
seems to be regulated by hormones (21, 23). The regulation of the
folding state of TTF-1 HD by calreticulin could be an additional
mechanism involved in hormonal control of gene expression in thyroid cells.