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J. Biol. Chem., Vol. 277, Issue 47, 45141-45148, November 22, 2002
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From the Division of Endocrinology/Metabolism, Neurology and
Hematology/Oncology,
Received for publication, March 27, 2002, and in revised form, August 19, 2002
Pit-1 stimulates the expression of growth
hormone, prolactin, and thyrotropin A pituitary-specific transcription factor, Pit-1, also known as
GHF-1, is a member of the homeobox POU family of DNA-binding proteins
(1, 2). It is involved in the development of the three pituitary cell
types, somatotrophs, lactotrophs, and thyrotrophs, as well as the gene
expression of growth hormone
(GH),1 prolactin (PRL),
GH-releasing hormone receptor (GHRH-R), thyrotropin To date, at least 16 different mutations of the human Pit-1
gene related to combined pituitary hormone deficiency (CPHD) have been
reported (11-24). Most mutations are seen in the POU domain of Pit-1,
which is important for its ability to dimerize and bind to DNA
(25).
However, two mutations located in the transactivation domain have also
been reported in patients with CPHD. One is a C to T mutation in codon
14 of the Pit-1 gene, resulting in an amino acid change from
proline (P) to leucine (L) (P14L) (19) and the other is a Cys to
Thr mutation in codon 24 leading to a corresponding change from Pro to
Leu (P24L) (20). Because the patients with these mutations were both
heterozygous for the mutation, it is believed that P14L and P24L may
dominantly inhibit the transcriptional activity of the wild type Pit-1
(21-23). However, the precise mechanism causing CPHD in patients with
these mutations has not been clarified.
In the study presented here, we showed that P14L exerts its
transcriptional effect to a similar extent to the wild type. This suggests that P14L may not be responsible for CPHD. On the other hand,
P24L showed reduced transcriptional activity and could be a cause of
CPHD. In addition, P24L could not recruit its coactivator, CREB-binding
protein (CBP) into the complex containing Pit-1 in the cultured cells.
Furthermore, although P24L did not lose its transcriptional activity
completely, it did not respond to stimulation by cAMP. Although it
remains possible that non-CBP proteins are responsible for this
phenomenon, CBP as well as Pit-1 appears to be involved in
cAMP-mediated enhancement of the gene expression via Pit-1- binding
DNA elements.
Materials and Cell Culture--
Fetal calf serum and
Dulbecco's modified Eagle's medium were obtained from
Invitrogen (Tokyo, Japan). COS7 cells were maintained in
Dulbecco's modified Eagle's medium with 10% (v/v) calf serum. All
culture medium contained penicillin G (100 units/ml) and
kanamycin (100 µg/ml).
Plasmid Construction--
Human Pit-1 cDNA was subcloned
into an expression vector (pcDNA3.1), which was named
pcDNA3.1-wild type-Pit-1. The mutant forms of Pit-1 (P14L, P24L,
and E250X) expression vectors were constructed with a site-directed
mutagenesis kit (Stratagene, La Jolla, CA) according to the
manufacturer's instructions. Briefly, Pfu Turbo DNA polymerase was
used to react 50 ng of template DNA (pcDNA3.1-wild
type-Pit-1) with mutant sense primer (5'-GATACCTTTATACTTCTGAATTCTG-3') and mutant antisense primer (5'-CAGAATTCAGAAGTATAAAGGTATC-3') for P14L,
with mutant sense primer (5'-CTGCAACTCTGCTTCTGATAATGC-3') and mutant
antisense primer (5'-GCATTATCAGAAGCAGAGTTGCAG-3') for P24L, and
with mutant sense primer (5'-GGATGGCTGAATAACTGAATCTGG-3') and mutant
antisense primer (5'-CCAGATTCAGTTATTCAGCCATCC-3') for E250X. This
reaction involved 30 s of denaturation at 95 °C and 15 cycles
consisting of 30 s of denaturation at 95 °C, 1 min of annealing
at 55 °C, and 5 min of extension at 68 °C. After digestion of the
nonmutated parental DNA template with DpnI, the mutant forms
of Pit-1 expression vectors (named pcDNA3.1-P14L,
pcDNA3.1-P24L, and pcDNA3.1-E250X) were successfully
transformed. The correct sequence was confirmed by DNA sequencing.
E250X is a previously reported mutant form of Pit-1 that has completely
lost its transcriptional activity because of the defect in the binding
activity to DNA (16). The 1.8-kb rat GH and 0.6-kb rat
PRL 5'-flanking regions were inserted upstream of the
luciferase reporter gene and these plasmids were named GH-Luc and
PRL-Luc, respectively. cDNAs for wild type Pit-1, P14L, P24L, the
transactivation domain containing the codons from the first to the
128th, and the POU domain containing the codons from the 129th to the
terminal codon of the wild type Pit-1 were inserted in-frame into the
XhoI-KpnI sites in the multiple cloning site of
pEGFPC3 (Clontech Laboratories, Inc. Palo Alto, CA)
and named wild type pPit-1-EGFP, pP14L-EGFP, pP24L-EGFP, pTRANS-EGFP, and pPOU-EGFP, respectively. Wild type Pit-1, P14L, and P24L cDNA were inserted in-frame into BglII-KpnI sites in
the multiple cloning site of the pFLAG-CMVTM-6a expression
vector (Sigma) and these plasmids were named wild type pPit-1-FLAG,
pP14L-FLAG, and pP24L-FLAG, respectively. DNA fragments encoding the
transactivation domain and the POU domain were inserted in-frame into
the BglII-KpnI sites in the multiple cloning site
of the pFLAG-CMVTM-6a expression vector to produce
pTRANS-FLAG and pPOU-FLAG, respectively. Expression vectors for
adenovirus E1a, which binds with CBP and abrogates its function
(pcDNA3.1-E1a), and the mutant form of E1a, which cannot bind with
CBP (pcDNA3.1- Transient Expression Analysis Using Luciferase Assay--
In all
the transient expression experiments using luciferase assay, plasmid
was transfected to cells in 35-mm dishes, unless otherwise indicated,
using LipofectAce (Invitrogen). First, 2 µg of GH-Luc or PRL-Luc were
transfected into COS7 cells with 0.3 µg of pcDNA3.1,
pcDNA3.1-wild type-Pit-1, pcDNA3.1-P14L, pcDNA3.1-P24L, or
pcDNA3.1-E250X to evaluate the transcriptional activity of P14L and
P24L. To clarify whether P14L and P24L have a dominant negative effect,
0.3 µg of pcDNA3.1-wild type-Pit-1, pcDNA3.1-P14L, or
pcDNA3.1-P24L were transfected with 0.3 µg of pcDNA3.1-wild type-Pit-1 and 2 µg of PRL-Luc. In the experiment for dose dependent activation of PRL-Luc by Pit-1, varying amounts (0-0.3 µg) of wild
type Pit-1 or mutant forms of Pit-1 (P14L and P24L) were used.
To compare the function of P24L to activate Pit-1-targeted genes in
response to cAMP with that of wild type Pit-1, 2 µg of PRL-Luc or
1P-Luc, which contains seven Pit-1-responsive elements derived from a
Pit-1-binding DNA element, 1P, of the rat PRL gene, was
transfected into the COS7 cells with 0.3 µg of empty expression vector (pcDNA3.1), pcDNA3.1-wild type-Pit-1, or
pcDNA3.1-P24L. Twenty-four hours after transfection, 1.0 mM CPT-cAMP was added to the medium of the transfected cells.
To assess the effect of exogenous CBP on Pit-1-targeted gene
expression, 2 µg of 1P-Luc with or without 0.5 µg of CBP expression vector was transfected into the COS7 cells with 20 ng of empty expression vector (pcDNA3.1), pcDNA3.1-wild type-Pit-1, or
pcDNA3.1-P24L. We used a minimal dose of Pit-1 expression vectors
to assess the effect of CBP more clearly. Twenty-four hours after
transfection, 1.0 mM CPT-cAMP was added to the medium of
the transfected cells.
To clarify the effect of endogenous CBP on the Pit-1-targeted gene
expression, 2 µg of 1P-Luc was transfected with 1 µg of pcDNA3.1, pcDNA3.1-E1a, or pcDNA3.1-
In all the experiments, 20 ng of pRL-CMV containing the cDNA
encoding Renilla luciferase (Promega, Tokyo, Japan) was co-transfected in each transfection to normalize the luciferase activity. In each of
the experiments, cells were harvested 48 h after transfection, and
luciferase activities were measured with a Turner design luminometer TD-20/20 using the dual luciferase assay system (Promega). Values are
expressed as multiples of induction relative to the basic luciferase
activity when only an empty expression vector was co-transfected and
they represent the mean ± S.E. of at least three determinations.
Expression of Wild Type and Mutant Forms (P14L and P24L) of
Pit-1--
Three µg of pcDNA3.1-wild type-Pit-1,
pcDNA3.1-P14L, or pcDNA3.1-P24L were introduced into COS7 cells
in 100-mm dishes using LipofectAce with or without 3 µg of
pcDNA3.1-wild type-Pit-1. An empty expression vector (pcDNA3.1)
was also co-transfected to fix the total amount of these plasmids at 6 µg. Forty-eight hours after transfection, proteins were extracted
from the cells and immunoprecipitated with anti-Pit-1 polyclonal
antibody, Pit-1 (X-7) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
These samples were resolved by SDS-PAGE and anti-Pit-1 antibody was used for Western blotting. As a negative control, 6 µg of
pcDNA3.1 alone was transfected and analyzed.
Expression of Fusion Protein of Wild Type, Mutant Forms of Pit-1
(P14L and P24L), Transactivation Domain Alone, or POU Domain Alone of
the Wild Type Pit-1 with Green Fluorescent Protein (GFP) in the
Cultured Cells--
One µg of pEGFPC3, pPit-1-EGFP, pP14L-EGFP,
pP24L-EGFP, pTRANS-EGFP, or pPOU-EGFP was introduced into COS7 cells in
35-mm dishes with the aid of LipofectAce. Forty-eight hours after
transfection, fluorescence images were examined.
Co-immunoprecipitation Analysis--
Ten µg of
pcDNA3.1-wild type-Pit-1 or pcDNA3.1-P24L were introduced into
COS7 cells in 100-mm dishes with 20 µg of PRL-Luc and 10 µg of
RSV-HA-CBP (hemagglutinin (HA)-tagged CBP expression vector).
Forty-eight hours after transfection, proteins were extracted from the
cells, and divided into two equal parts, one part for immunoprecipitation with anti-HA antibody (Santa Cruz Biotechnology) and the other part for immunoprecipitation with anti-Pit-1 antibody. These samples were resolved by SDS-PAGE and anti-Pit-1 antibody was
used for Western blotting.
Furthermore, whether the transactivation domain alone or the POU domain
alone can recruit CBP was examined. Ten µg of PRL-Luc and 5 µg of
RSV-HA-CBP were introduced into COS7 cells in 100-mm dishes with 5 µg
of wild type pPit-1-FLAG, pP14L-FLAG, pP24L-FLAG, pTRANS-FLAG, or
pPOU-FLAG using LipofectAce. In the experiment to assess whether P24L
blocks the interaction of wild type Pit-1 with CBP, 10 µg of PRL-Luc
and 5 µg of RSV-HA-CBP were introduced into COS7 cells in 100-mm
dishes with 5 µg of wild type pPit-1-FLAG, 5 µg of pP24L-FLAG, 5 µg of pPit-1-FLAG + 5 µg of pP24L-FLAG, or 10 µg of pPit-1-FLAG.
pFLAG-CMVTM-6a was used to fix the total amounts of
plasmids transfected. In both experiments, proteins were extracted from
the cells 48 h after transfection and each sample was divided into
two equal parts, one for immunoprecipitation with anti-HA antibody and
the other for immunoprecipitation with anti-FLAG M2 monoclonal antibody (Sigma). These samples were then resolved by SDS-PAGE and anti-FLAG M2
monoclonal antibody was used for Western blotting.
Mammalian Two-hybrid Assay--
Expression vectors for fusion
protein of Pit-1 or Pit-1 domains with the GAL4 DNA binding domain,
pGAL4-Pit-1, pGAL4-TRANS, pGAL4-POU, and pGAL4-P24L were constructed by
inserting wild type Pit-1, the transactivation domain of wild type
Pit-1, the POU domain of wild type Pit-1, and P24L cDNA in-frame
into BamHI-XbaI sites in the multiple cloning
site of the pCMV-BD vector (Stratagene), respectively. Expression
vectors for the fusion protein of CBP with the NF- Reduced Ability of P24L, but Not P14L, to Activate GH and PRL
Genes--
Transcriptional activity of P14L and P24L for the
expression of GH and PRL genes was
investigated in COS7 cells (Pit-1 deficient). P24L activated GH-Luc
containing the 1.8-kb rat GH 5'-flanking region and PRL-Luc
containing the 0.6-kb rat PRL 5'-flanking region up to only
69 and 56%, respectively, of activation by wild type Pit-1. On the
other hand, P14L activated GH-Luc and PRL-Luc up to a similar level to
wild type Pit-1 (Fig. 1, A and
B). Furthermore, whereas P14L showed a similar
dose-dependent enhancement of its power as a transcription
factor to the wild type Pit-1, the dose-dependent enhancement of that of P24L was obviously weak (Fig. 1C).
However, whereas one mutant form of Pit-1, E250X, which cannot bind to DNA, completely lost its transcriptional activity (16), P24L did not
completely lose it (Fig. 1, A-C).
Neither P14L nor P24L Dominantly Inhibited the Transcriptional
Activity of Wild Type Pit-1--
Because both of the probands with
P14L or P24L were found to be heterozygous for these mutations (19,
20), it has been hypothesized that P14L and P24L might inhibit the
activity of the wild type Pit-1 (21-23). To test the ability of these
mutants to interfere with the transactivation of Pit-1-targeted gene
expression by wild type Pit-1, the expression vector for P14L or P24L
was transfected into COS7 cells together with the PRL-Luc and wild type
Pit-1 expression vector. Whereas P14L, like wild type Pit-1, enhanced
the transcriptional effect of the wild type in an additive manner, the
effect of P24L on the activity of the wild type Pit-1 was very weak.
However, neither P14L nor P24L dominantly inhibited the activity of the
wild type Pit-1 (Fig. 2A).
Mutant Forms of Pit-1 (P14L and P24L) Protein Showed Similar
Expression Levels to Wild Type Pit-1 Protein--
To rule out the
possibility that the differences in expression levels of wild type
Pit-1 and mutant forms of Pit-1 (P14L and P24L) could explain the minor
effect of P24L on basal transcription rates and the absence of a
dominant negative effect of P14L and P24L on wild type Pit-1, we
examined protein levels of wild type Pit-1 and mutant forms of Pit-1
using Western blot analysis. The Pit-1 protein levels were similar to
each other whether 3 µg of wild type Pit-1, P14L, or P24L expression
vector was transfected (Fig. 2B, lanes 1-3).
Furthermore, the levels of Pit-1 protein were similar to each other
whether 3 µg of wild type Pit-1, P14L, or P24L expression vector was
co-transfected with 3 µg of wild type Pit-1 expression vector (Fig.
2B, lanes 4-6). These results suggested that the
reduced activity of P24L and the absence of a dominant negative effect
of P14L and P24L were not explained by the difference in the levels of
wild type and mutant forms of Pit-1 protein.
Nuclear Accumulation of Wild Type Pit-1 and Also Mutant Forms of
Pit-1 (P14L and P24L)--
Chimera constructs of GFP with wild type
Pit-1, P14L, P24L, the transactivation domain alone, or the POU domain
alone of the wild type Pit-1 (pPit-1-EGFP, pP14L-EGFP, pP24L-EGFP,
pTRANS-EGFP, and pPOU-EGFP) were produced. These constructs were
transiently transfected into COS7 cells, and the fluorescence image of
the expressed fusion proteins were analyzed. Cells transfected with only the GFP expression vector were also analyzed as a control. In the
control cells, GFP was distributed homogeneously in both the cytoplasm
and the nucleus (Fig. 3A). On
the other hand, nuclear accumulation of wild type-Pit-1-GFP, P14L-GFP,
and P24L-GFP was observed in all the cells analyzed (Fig. 3,
B-D). Furthermore, nuclear accumulation of POU-GFP was
observed in all the cells analyzed (Fig. 3F). On the other
hand, TRANS-GFP was distributed homogeneously in both the cytoplasm and
nucleus (Fig. 3E), suggesting that the POU domain but not
the transactivation domain is involved in nuclear localization. A basic
cluster RKRKRR is present in the POU homodomain, one of the subdomains
of the POU domain. Because the basic cluster RKRKRR, which is highly
conserved in POU proteins, has been identified as NLS in Tst-1/Oct6, a
member of POU protein family (25), it appears to work as NLS in Pit-1
as well as in Tst-1/Oct6.
In Cultured Cells, Wild Type Pit-1, but Not P24L Could Recruit
Coactivator CBP into the Complex Which Contains Pit-1--
Because
P24L showed normal nuclear translocation and contained normal POU
domain that is important for dimerization and DNA binding (26), we
assumed that some transcriptional cofactors may be related with the
reduced activity of P24L. Because CBP is involved in the activities of
many transcription factors including Pit-1 (28-33), we investigated
whether the impaired function of P24L is linked to the inability to
recruit CBP. The whole cell extract prepared from COS7 cells, which had
been transfected with the expression vector for wild type Pit-1 or P24L
in addition to the expression vector for HA-tagged CBP (RSV-HA-CBP) and
PRL-Luc, was immunoprecipitated with anti-HA antibody. Whether or not
CBP is present in the complex containing wild type Pit-1 or P24L was analyzed by Western blotting with anti-Pit-1 antibody (Fig.
4A, lanes 1 and
2). Although wild type Pit-1 was co-immunoprecipitated with
CBP, P24L was not co-immunoprecipitated with CBP, suggesting that the
ability to recruit CBP was decreased by the substitution of Leu for Pro
at the 24th amino acid of Pit-1. Furthermore, whether or not the
transactivation domain alone or the POU domain alone could recruit CBP
was tested. The whole cell extract prepared from COS7 cells, which had
been transfected with RSV-HA-CBP and PRL-Luc in addition to the
FLAG-tagged expression vectors, pPit-1-FLAG, pP14L-FLAG, pP24L-FLAG,
pTRANS-FLAG, or pPOU-FLAG, was divided into two equal parts. One part
was used for immunoprecipitation with anti-HA antibody and the other
for immunoprecipitation with anti-FLAG antibody and both were analyzed
by Western blotting with anti-FLAG antibody (Fig. 4B).
pPit-1-FLAG, pP14L-FLAG, pP24L-FLAG, pTRANS-FLAG, or pPOU-FLAG appeared
to be equally transfected (Fig. 4B, lanes 6-10;
immunoprecipitated with anti-FLAG antibody and Western blotted with
anti-FLAG antibody). However, neither P24L, the transactivation domain,
nor the POU domain were detected in the complex containing CBP (Fig.
4B, lanes 1-5; immunoprecipitated with anti-HA
antibody and Western blotted with anti-FLAG antibody). Therefore, the
transactivation domain, especially proline at codon 24 of Pit-1 seemed
to be important to make a complex containing CBP in the cultured cells
(Fig. 4B, lanes 3 and 8). Furthermore, in
contrast to the result of a previous study (28), the POU domain was not
able to recruit CBP (Fig. 4B, lanes 5 and
10). It was surprising because a previous report (28)
suggested that the POU domain was sufficient to make a complex with the
CBP fragment, although the binding activity appeared to be weak. Our
result suggested that the transactivation domain alone or the POU
domain alone are insufficient to make a high affinity complex that
contains CBP in vivo cell culture. In addition, whether or
not P24L blocks the interaction of wild type Pit-1 with CBP was also
examined (Fig. 4C). The whole cell extract prepared from
COS7 cells, which had been transfected with RSV-HA-CBP and PRL-Luc in
addition to expression vectors for FLAG-tagged wild type Pit-1,
FLAG-tagged P24L, or both of them, was divided into two equal parts.
One part was used for immunoprecipitation with anti-HA antibody and the other for immunoprecipitation with anti-FLAG antibody and both were
analyzed by Western blotting with anti-FLAG antibody (Fig. 4C). Although P24L could not make a complex containing CBP
in the cultured cells, it did not block the interaction of wild type Pit-1 with CBP (Fig. 4C, lanes 2 and
3, immunoprecipitated with anti-HA antibody and Western
blotted with anti-FLAG antibody).
Mammalian Two-hybrid Assay Also Revealed That P24L, Transactivation
Domain Alone, or POU Domain Alone Could not Interact with CBP--
It
is possible that loss of the nuclear localization is responsible for
the disturbed interaction of the transactivation domain and CBP in the
co-immunoprecipitation experiment. Therefore, we performed mammalian
two-hybrid assays. When pGAL4-Pit-1-producing fusion protein of the
GAL4 DNA binding domain with Pit-1 and the pAD-CBP coding NF- Loss of Ability of Pit-1 to Make a Complex Containing CBP Appears
to be Associated with the Absence of cAMP-induced Expression of
Pit-1-targeted Genes--
CBP has been reported to be involved in
cAMP-regulated gene expression via Pit-1-binding DNA elements (28, 34,
35). Therefore, we examined whether wild type Pit-1 and the mutant form
(P24L) Pit-1 differ in their activation of the target genes after cAMP
stimulation. Whereas cAMP stimulated the transcription of the
PRL gene by wild type Pit-1, it did not affect the
transcription by P24L (Fig.
6A). In addition, 1P-Luc
containing seven Pit-1-responsive elements derived from a Pit-1 binding
DNA element, 1P, of the rat PRL gene was also used because
there were reports that the proximal region of the PRL gene
contains a cAMP responsive element-like DNA element as well as Pit-1
binding sites (36, 37). cAMP stimulated the transcription of 1P-Luc by
wild type Pit-1, but it did not affect the transcription by P24L (Fig.
6B). These results suggest that for Pit-1 making a complex
containing CBP is associated with the cAMP-mediated signal pathway that
stimulates the expression of the targeted genes. Besides, co-expression
study revealed that CBP and cAMP enhanced the gene transcription by
wild type Pit-1, but did not enhance the transcription by P24L (Fig.
7A). Furthermore, in the
pituitary-derived GH3 cells, co-expression study revealed that
adenovirus E1a, which binds with CBP and abrogates its function, suppressed the basal and cAMP-induced expression of 1P-Luc, whereas This is the first functional analysis of Pit-1 with mutations in
the transactivation domain found in patients with CPHD (19, 20). The
case reported by Fofanova et al. (19) was a 3-year-old girl
who showed total GH/PRL and partial TSH deficiency without mental
retardation. All six exons of the Pit-1 gene and its
promoter region were directly sequenced by the authors and it was found that the patient was heterozygous for a mutation at codon 14 of exon 1 (CCT to CTT) resulting in the substitution of
Leu for Pro. However, her mother, her maternal aunt, and grandmother
were phenotypically normal, although they had the same heterozygous
mutation. The need to thoroughly assess genomic imprinting was
emphasized by the authors. The patient presented by Ohta et
al. (20) was a 5-year-old boy of short stature without mental
retardation. Both of the plasma GH and PRL levels were undetectable
either before or after the provocative stimulation tests. The basal TSH
level was also low. TRH caused a weak TSH response. DNA sequence of the
Pit-1 gene showed that the patient had a heterozygous mutation at codon
24 in exon 1 (CCT to CTT) resulting in
substitution of Leu for Pro. Neither phenotype nor genotype of the
family was described.
As mentioned above, because both of the patients had only one mutant
allele, it had been predicted that P14L and P24L were likely to possess
the dominant negative effect (21-23). However, neither of these
mutations is likely to show dominant negative activity judging from our
data. Especially, P14L showed normal transcriptional activity and
therefore P14L did not seem to be responsible for CPHD, although it
remains possible that this mutant lead to CPHD through an unknown
mechanism in vivo. On the other hand, P24L could be a cause
of CPHD in view of a previous report of monoallelic expression of the
Pit-1 gene (38). It was possible that only the mutant
allele was expressed in this patient through a genomic imprinting
mechanism rather than some other genomic or nongenomic etiology.
Because CBP is associated with so many different transcription factors,
mutations in the gene encoding CBP cause developmental disorder (known
as the Rubinstein-Taybi syndrome) comprised of multiple abnormalities,
broad thumbs and halluces, mental retardation, growth retardation,
developmental delay, microcephaly, and craniofacial abnormalities (39,
40). Rubinstein-Taybi syndrome is believed to be a haploinsufficient
disorder (41) and homozygous pathological mutation of CBP is lethal
(42). On the other hand, CBP appears to be haplosufficient for GH, PRL,
and TSH gene expression, because there have been no reports of
Rubinstein-Taybi syndrome with deficiencies of these hormones (43).
However, it is possible that if only the mutant form of Pit-1, which
cannot recruit CBP, is expressed, not only the expression of the
Pit-1-targeted genes, but also the development of somatotrophs,
lactotrophs, and thyrotrophs might be impaired.
Previously, we showed that Pit-1-binding DNA elements mediate the
transcriptional response to cAMP through a mechanism that does not
require inducible phosphorylation of Pit-1 (44). Although major
phosphorylation sites of Pit-1 are serine 115 and threonine 220 (45),
the mutations of those amino acids did not affect the transcriptional
function in response to cAMP (44). This finding suggested the cAMP
effects on the Pit-1- dependent gene expression might involve
other proteins that associate with Pit-1 (44).
Because the present study demonstrated that mutation of Pro at codon 24 of Pit-1 completely disrupted the transcriptional response to cAMP, and
that adenovirus E1a that binds to CBP and abrogates its function
blocked the stimulatory effect of cAMP on Pit-1-dependent
gene transcription in the pituitary-derived GH3 cells, we speculated
that the inability of the mutant form of Pit-1 (P24L) to recruit CBP is
involved in the phenomenon (Fig. 8).
However, how CBP is associated with the cAMP-mediated activation of
Pit-1-targeted genes still remains to be clarified (28, 35, 41). It was
very interesting that the basal activity of P24L as a transcription
factor was not disrupted completely. This suggests that other
coactivators for Pit-1 that function independently of CBP may exist.
This functional analysis of the naturally occurring mutation in the
transactivation domain of Pit-1 provided an interesting insight into
the mechanism by which Pit-1 stimulates gene expression. However, the
structure and function of the transactivation domain of Pit-1 remains
to be clarified, although those of the POU domain have been extensively
studied using NMR, crystallographic methods, and mutational analysis
(26, 46, 47).
In conclusion, it was shown first that the transactivation domain,
especially Pro at codon 24 of Pit-1 is important to recruit CBP in the
cultured cells. Second, CBP is a mediator in the cAMP signal
transduction pathway to activate Pit-1-targeted gene transcription. And
third, CBP does not appear to be an essential coactivator for Pit-1 to
maintain basal transcription, suggesting that some other unknown
coactivators functioning independently of CBP may exist.
We thank Dr. Fukamizu for the gift of
RSV-HA-CBP, Dr. Yang Shi for the gift of E1a expression vector, and
Chika Ogata for excellent technical assistance.
*
This work was supported in part by a grant-in-aid for
Scientific Research from the Japanese Ministry of Education, Science, Sports, and Culture, and grants from the Japanese Ministry of Health,
Labor and Welfare and from Growth Science Research Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Basic
Allied Medicine, Kobe University School of Medicine, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0142, Japan. Tel.: 81-78-796-4540; Fax:
81-78-796-4540; E-mail: okimura@ams.kobe-u.ac.jp.
Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M202991200
The abbreviations used are:
GH, growth
hormone;
GHRH-R, growth hormone-releasing hormone receptor;
TSH
Novel Function of the Transactivation Domain of a
Pituitary-specific Transcription Factor, Pit-1*
,
,,,,, and
Department of Clinical Molecular
Medicine, and § Department of Basic Allied Medicine,
Department of Brain Sciences, Kobe University Graduate
School of Medicine, Kobe University School of Medicine, 7-10-2, Tomogaoka, Suma-ku, Kobe 654-0142, Japan, and the ** College
of Nursing Art and Culture, Hyogo 655-0048, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit genes.
Consequently, abnormality of the Pit-1 gene results
in combined pituitary hormone deficiency (CPHD). In this study, we
analyzed the function of Pit-1 with a mutation (proline to leucine at
codon 24) in the transactivation domain, P24L, which has a normal POU
domain important for binding to DNA, because this mutation had been
reported in a patient with CPHD. We found that codon 24 proline in the
transactivation domain as well as the POU domain of Pit-1 was crucial
to recruit coactivator CREB-binding protein (CBP) in the cultured
cells. P24L completely lost the responsiveness to cAMP to stimulate the
expression of the Pit-1-targeted genes. Furthermore, CBP and Pit-1, but
not P24L, markedly enhanced the expression of the Pit-1-targeted gene to cAMP, and adenovirus E1a that binds to CBP and abrogates its function blocked the induction by cAMP of Pit-1-stimulated gene transcription in the pituitary-derived GH3 cells. These results suggest
that CBP and proline at codon 24 in the transactivation domain of Pit-1
are important for the cAMP-induced activation of Pit-1-targeted genes.
However, P24L maintained basal transcriptional activity, suggesting
that CBP is unlikely to be an essential coactivator for
Pit-1.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit (TSH
), and Pit-1 itself by binding to the specific DNA elements of these
genes (3-10). Therefore, abnormalities of the Pit-1 gene
result in GH, PRL, and TSH deficiencies.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-E1a), were constructed by inserting DNA
fragments of E1a and 2-36 E1a to pcDNA.3.1, respectively.
-E1a into the GH3
cells in 35-mm dishes. Twenty-four hours after transfection, 1.0 mM CPT-cAMP was added to the medium of the transfected cells.
B transactivation
domain, pAD-CBP, pAD-CBP-(1-319), pAD-CBP-(320-420),
pAD-CBP-(421-1457), pAD-CBP-(1458-1891), and pAD-CBP-(1892-2441),
were constructed by inserting cDNAs coding full-length CBP,
CBP-(1-319), CBP-(320-420), CBP-(421-1457), CBP-(1458-1891), and
CBP-(1892-2441) in-frame into BamHI-HindIII
sites in the multiple cloning site of the pCMV-AD vector (Stratagene),
respectively. Two µg of reporter plasmid, pFR-Luc (Stratagene), which
contains GAL4-responsive elements and 1 µg of pGAL4-Pit-1 were
transfected into the COS7 cells in 35-mm dishes with 1 µg of pCMV-AD,
pAD-CBP, pAD-CBP-(1-319), pAD-CBP-(320-420), pAD-CBP-(421-1457),
pAD-CBP-(1458-1891), or pAD-CBP-(1892-2441). Furthermore, 2 µg of
pFR-Luc and 1 µg of pAD-CBP were transfected into the COS7 cells in
35-mm dishes with 1 µg of pGAL4-TRANS, pGAL4-POU, or pGAL4-P24L. In
all the experiments, 20 ng of pRL-CMV was co-transfected to normalize the luciferase activity. Cells were harvested 48 h after
transfection and values are expressed as multiples of induction
relative to the basic luciferase activity when pFR-Luc was
transfected with pGAL4-Pit-1 and pCMV-AD.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Functional analysis of mutant forms of Pit-1
(P14L and P24L). A, 2 µg of reporter plasmid,
PRL-Luc, which contains the 0.6-kb rat PRL 5'-flanking
region, was transfected into COS7 cells in 35-mm dishes with 0.3 µg
of empty expression vector, wild type Pit-1, P14L, P24L, or E250X
expression vectors (named pcDNA3.1.-wild type-Pit-1,
pcDNA3.1.-P14L, pcDNA3.1-P24L, and pcDNA3.1-E250X,
respectively). E250X is a previously reported nonfunctioning mutant
form of Pit-1 that cannot bind to DNA. Although P14L had a similar
effect on the PRL promoter to wild type Pit-1, the ability
of P24L to activate the promoter was obviously reduced. B,
similar experiments were also performed with the reporter plasmid
GH-Luc that contains the 1.8-kb rat GH 5'-flanking region.
Although P14L again showed a comparable transcriptional activity to the
wild type Pit-1, the ability of P24L to activate the GH
promoter was remarkably reduced. C, 2 µg of PRL-Luc
was transfected into the COS7 cells in 35-mm dishes with
pcDNA3.1-wild type-Pit-1, pcDNA3.1-P14L,
pcDNA3.1-P24L, or pcDNA3.1-E250X. Transcriptional activity was
evaluated under experimental conditions using 0.1, 0.2, or 0.3 µg of
these Pit-1 expression vectors. An empty expression vector
(pcDNA3.1) was also co-transfected to fix the total amount of
transfected plasmids. Although P14L showed similar dose-dependent enhancement of its
transcriptional activity to wild type, the activity of P24L was
obviously weak. A-C, 20 ng of pRL-CMV containing the
cDNA encoding Renilla luciferase was co-transfected into each
transfection to normalize the luciferase activity. Experiments were
performed in triplicate, and values (mean ± S.E.) are expressed
as multiples of induction relative to luciferase activity when the
empty expression vector was transfected.
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Fig. 2.
No dominant negative effect of mutant forms
of Pit-1 (P14L and P24L) on wild type Pit-1. Because both of the
probands with P14L or P24L showed heterozygous for these mutations (19,
20), we examined whether P14L and P24L function dominant
negatively. A, 0.3 µg of pcDNA3.1-wild type-Pit-1,
pcDNA3.1-P14L, or pcDNA3.1-P24L were transfected with 0.3 µg
of pcDNA3.1-wild type-Pit-1 and 2 µg of PRL-Luc. When
co-transfected with the pcDNA3.1-wild type-Pit-1, P14L showed
a similar additive effect to the wild type. On the other hand, the
additive effect of P24L was minimal. Twenty ng of pRL-CMV was
co-transfected into each transfection to normalize the luciferase
activity. Values are expressed as multiples of induction relative to
the basic luciferase activity when only an empty expression vector was
transfected. The experiment was performed in triplicate and data show
the mean ± S.E. stimulation of the reporter construct.
B, to evaluate the wild type and mutant forms of Pit-1
expression levels, COS7 cells were transfected with 3 µg of
pcDNA3.1-wild type-Pit-1, pcDNA3.1-P14L, or pcDNA3.1-P24L,
with or without 3 µg of pcDNA3.1-wild type-Pit-1 (lanes
1-6). Forty-eight hours after transfection, the extract of the
transfected cells was immunoprecipitated with Pit-1 antibody. These
samples were resolved by SDS-PAGE and anti-Pit-1 antibody was used for
Western blotting. In addition, 6 µg of pcDNA3.1 without Pit-1
expression vector was introduced and analyzed as a negative control
(lane 7).
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Fig. 3.
Nuclear accumulation of wild type Pit-1 and
also mutant forms of Pit-1 (P14L and P24L). COS7 cells,
cultured in 35-mm dishes, were transfected with the expression vector
containing chimera constructs of GFP with wild type Pit-1, P14L, or
P24L, and 48 h after transfection their fluorescence image was
analyzed. Cells transfected with only the GFP expression vector as a
control were also analyzed to demonstrate that GFP was distributed
homogeneously in both the cytoplasm and the nucleus (A).
Nuclear accumulation of GFP-wild type-Pit-1, GFP-P14L, and GFP-P24L was
observed in all the cells analyzed (B-D). Although nuclear
accumulation of the GFP-POU domain was observed in all the cells
analyzed (F), the GFP-transactivation domain was distributed
homogeneously in both the cytoplasm and the nucleus
(E).
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Fig. 4.
Wild type Pit-1, but not P24L could recruit
CBP in the cultured cells. A, 20 µg of PRL-Luc and 10 µg of RSV-HA-CBP (HA-tagged CBP expression vector) were
co-transfected with 10 µg of pcDNA3.1-wild type-Pit-1 or
pcDNA3.1-P24L into COS7 cells in 100-mm dishes with the aid of
LipofectAce. Forty-eight hours after transfection, proteins were
extracted from the cells and each sample was equally divided into two
parts, one for immunoprecipitation with anti-HA antibody (lane
1, wild type Pit-1; lane 2, P24L) and the other for
immunoprecipitation with Pit-1 antibody (lane 3, wild type
Pit-1; lane 4, P24L). These samples were resolved by
SDS-PAGE and anti-Pit-1 antibody was used for Western blotting.
Although wild type Pit-1 was co-immunoprecipitated with CBP, P24L was
not co-immunoprecipitated with CBP (lanes 1 and
2). B, to test whether the transactivation domain
alone or the POU domain alone are co-immunoprecipitated with CBP or
not, 5 µg of pPit-1-FLAG, pP14L-FLAG, pP24L-FLAG, pTRANS-FLAG, or
pPOU-FLAG were transfected into COS7 cells in 100-mm dishes with 10 µg of PRL-Luc and 5 µg of RSV-HA-CBP. Forty-eight hours after
transfection, each cell extraction was divided into two equal parts,
one for immunoprecipitation with anti-HA antibody (lane 1,
wild type Pit-1; lane 2, P14L; lane 3, P24L;
lane 4, transactivation domain (TRA); lane 5, POU
domain (POU)) and the other for immunoprecipitation with anti-FLAG
antibody (lane 6, wild type Pit-1; lane 7, P14L;
lane 8, P24L; lane 9, TRA; lane 10,
POU). These samples were resolved by SDS-PAGE and anti-FLAG antibody
was used for Western blotting. pPit-1-FLAG, pP14L-FLAG, pP24L-FLAG,
pTRANS-FLAG, and pPOU-FLAG appeared to be transfected well (Fig.
4B, lanes 6-10; immunoprecipitated with
anti-FLAG antibody and Western blotted with anti-FLAG antibody).
However, neither P24L, the transactivation domain, nor the POU domain
were detected in the complex containing CBP (Fig. 4B,
lanes 3-5; immunoprecipitated with anti-HA antibody and
Western blotted with anti-FLAG antibody). C, to test whether
P24L blocks the interaction of wild type Pit-1 with CBP, pPit-1-FLAG (5 µg), pP24L-FLAG (5 µg), pPit-1-FLAG (5 µg) + pP24L-FLAG (5 µg),
or pPit-1-FLAG (10 µg) were transfected into COS7 cells in 100-mm
dishes with 10 µg of PRL-Luc and 5 µg of RSV-HA-CBP. Forty-eight
hours after transfection, each cell extraction was divided into two
equal parts, one for immunoprecipitation with anti-HA antibody
(lane 1, wild type Pit-1 (5 µg); lane 2, P24L
(5 µg); lane 3, wild type Pit-1 (5 µg) + P24L (5 µg);
lane 4, wild type Pit-1 (10 µg)) and the other for
immunoprecipitation with anti-FLAG antibody (lane 5, wild
type Pit-1 (5 µg); lane 6, P24L (5 µg); lane
7, wild type Pit-1 (5 µg) + P24L (5 µg); lane 8,
wild type Pit-1 (10 µg)). These samples were resolved by SDS-PAGE and
anti-FLAG antibody was used for Western blotting. Although P24L could
not make a complex containing CBP in the cultured cells, it did not
block the interaction of wild type Pit-1 with CBP.
B
activation domain and CBP were co-transfected into the COS7 cells, the
pFR-Luc that contains the GAL4-binding elements was activated (Fig.
5). However, when pAD-CBP was
co-transfected with pGAL4-P24L, pGAL4-TRANS producing a fusion protein
of GAL4 and the transactivation domain, pGAL4-POU producing a fusion
protein of GAL4 and the POU domain, pFR-Luc was not activated (Fig. 5). This result indicated that the transactivation domain alone could not
interact with CBP, and was in accordance with the result from immunoprecipitation. On the other hand, pAD-CBP- (1-319),
pAD-CBP-(320-420), pAD-CBP-(421-1457), pAD-CBP-(1458-1891), or
pAD-CBP-(1892-2441) were co-transfected with pGAL4-Pit-1 and pFR-Luc
to identify domains in CBP that interact with Pit-1. In accordance with
the previous reports using the glutathione S-transferase
pull-down assay (27, 34, 35), the regions containing the CH1 or CH3
domains seemed to be important for CBP to interact with Pit-1, because
pAD-CBP-(320-420) and pAD-CBP-(1458-1891) could activate pFR-Luc
(Fig. 5).
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Fig. 5.
Mammalian two-hybrid assay also revealed that
P24L, transactivation domain alone, or POU domain alone could not
interact with CBP. A, the structure of CBP.
B, 2 µg of pFR-Luc and 1 µg of pGAL4-Pit-1 were
introduced into COS7 cells in 35-mm dishes with 1 µg of pCMV-AD,
pAD-CBP, pAD-CBP-(1-319), pAD-CBP-(320-420), pAD-CBP-(421-1457),
pAD-CBP-(1458-1891), or pAD-CBP-(1892-2441). One µg of pAD-CBP and
2 µg of pFR-Luc were also co-transfected with pGAL4-P24L,
pGAL4-TRANS, or pGAL4-POU. Twenty ng of pRL-CMV was also co-transfected
to normalize the luciferase activity. Cells were harvested 48 h
after transfection and values are expressed as multiples of induction
relative to the basic luciferase activity when pFR-Luc was transfected
with GAL4-Pit-1 and pCMV-AD. Co-transfection of pGAL4-Pit-1 with
pAD-CBP-(320-420) or pAD-CBP-(1458-1891) could activate pFR-Luc. When
pGAL4-Pit-1 and pAD-CBP were co-transfected into the COS7 cells, the
pFR-Luc containing GAL4-binding elements was activated. However, when
pAD-CBP was co-transfected with pGAL4-P24L, pGAL4-TRANS, or pGAL4-POU,
pFR-Luc was not activated.
-E1a that cannot bind to CBP did not (Fig. 7B).
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Fig. 6.
Loss of ability of Pit-1 to make a complex
containing CBP appears to be associated with the defect in
cAMP-mediated activation of the Pit-1-targeted genes. Two µg of
PRL-Luc or 1P-Luc containing seven Pit-1-binding elements was
transfected into the COS7 cells in 35-mm dishes with 0.3 µg of empty
expression vector (pcDNA3.1), pcDNA3.1-wild type-Pit-1, or
pcDNA3.1-P24L. Twenty-four hours after transfection, 1.0 mM CPT-cAMP was added to the medium of the transfected
cells. cAMP strongly enhanced the luciferase activity of PRL-Luc
(A) and 1P-Luc (B) in the presence of wild type
Pit-1, but not P24L. Experiments were performed in triplicate and data
show the mean ± S.E. stimulation of the reporter construct.
Twenty ng of pRL-CMV was also co-transfected to normalize the
luciferase activity.
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Fig. 7.
CBP is a mediator in the cAMP signal
transduction pathway to activate Pit-1-targeted gene
transcription. A, 2 µg of 1P-Luc containing seven
Pit-1-binding elements was transfected into the COS7 cells in 35-mm
dishes with or without 0.5 µg of CBP expression vector, in addition
to 20 ng of empty expression vector (pcDNA3.1), pcDNA3.1-wild
type-Pit-1, or pcDNA3.1-P24L. A minimal dose of Pit-1 expression
vector was used to assess the effect of CBP more clearly. Empty
expression vectors were used to fix the total amount of plasmids
transfected. Twenty-four hours after transfection, 1.0 mM
CPT-cAMP was added to the medium of the transfected cells. CBP and cAMP
enhanced the gene transcription by wild type Pit-1, but did not enhance
the transcription by P24L. B, 2 µg of 1P-Luc with 1 µg
of pcDNA3.1, pcDNA3.1-E1a, or pcDNA3.1- -E1a was
transfected into the GH3 cells in 35-mm dishes. Twenty-four hours after
transfection, 1.0 mM CPT-cAMP was added to the medium of
the transfected cells. Wild type E1a, which binds to CBP and abrogates
its function, suppressed the basal and cAMP-induced expression of
1P-Luc, whereas -E1a, which is unable to bind to CBP did not.
Experiments were performed in triplicate and data show the mean ± S.E. stimulation of the reporter construct. Twenty ng of pRL-CMV was
also co-transfected to normalize the luciferase activity.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Role of coactivator CBP in cAMP-stimulated
activation of the Pit-1-targeted genes. CBP recruitment is
involved in the cAMP-mediated signal pathway that stimulates gene
expression via the Pit-1 binding site.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, thyrotropin subunit;
CPHD, combined pituitary hormone deficiency;
CBP, CREB-binding protein;
CREB, cAMP-response element-binding protein;
CMV, cytomegalovirus;
GFP, green fluorescent protein;
EGFP, epidermal
growth factor protein;
HA, hemagglutinin;
RSV, Rous sarcoma virus;
CPT, chlorophenylthio.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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