Defective Retinoic Acid Regulation of the Pit-1 Gene Enhancer: A Novel Mechanism of Combined Pituitary Hormone Deficiency
Laurie E. Cohen,
Kerstin Zanger,
Thierry Brue,
Fredric E. Wondisford and
Sally Radovick
Divisions of Endocrinology Departments of Medicine
Childrens Hospital (L.E.C., K.Z., T.B., S.R.) and Beth Israel
Deaconess Medical Center (F.E.W.), Harvard Medical School
Boston, Massachusetts 02115
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ABSTRACT
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Pit-1 is a pituitary-specific transcription factor
responsible for pituitary development and hormone expression in
mammals. Pit-1 contains two protein domains, termed POU-specific and
POU-homeo, which are both necessary for DNA binding and activation of
the GH and PRL genes and regulation of the
PRL, TSH-ß subunit (TSH-ß), and
Pit-1 genes. Pit-1 is also necessary for retinoic
acid induction of its own gene during development through a
Pit-1-dependent enhancer. Combined pituitary hormone deficiency is
caused by defective transactivation of target genes in the anterior
pituitary. In the present report, we provide in vivo
evidence that retinoic acid induction of the Pit-1 gene can
be impaired by a Pit-1 gene mutation, suggesting a new
molecular mechanism for combined pituitary hormone deficiency in man.
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INTRODUCTION
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Pit-1 (whose official nomenclature is now POU1F1) is a member of a
family (POU) of transcription factors regulating mammalian development.
Pit-1 contains two protein domains, termed POU-specific and POU-homeo,
which are both necessary for high-affinity DNA binding on the
GH and PRL genes (1). When bound to DNA, Pit-1
activates GH and PRL gene expression, in part,
through an N-terminal transactivation domain rich in hydroxylated amino
acid residues (2, 3). During development, Pit-1 gene
expression precedes GH and PRL gene expression in
the somatotroph and lactotroph, respectively, and is thought to be the
major cell-specific activator of hormone expression from these cell
types (4, 5, 6, 7, 8, 9). Additional nuclear factors, however, appear to be
necessary for expression of the GH and PRL genes.
Lipkin et al. (10) cloned a zinc finger transcription
factor, termed Zn-15, which is responsible with Pit-1 for synergistic
activation of the GH gene. In addition, some investigators
suggest that Pit-1 synergistically activates the GH gene in
the presence of thyroid hormone receptors (11). Other investigators,
however, have not confirmed this finding (12). Likewise, the estrogen
receptor is required, along with Pit-1, for PRL gene distal
enhancer activation by estradiol (13, 14, 15).
Rhodes et al. (16) explored the molecular mechanism
responsible for activation of the Pit-1 gene in
vivo. They demonstrated that an enhancer element, located more
than 10 kb upstream of the transcriptional start site, was essential
for pituitary-specific expression of the Pit-1 gene in
transgenic mice. Like the GH and PRL genes, Pit-1
alone was not sufficient to direct pituitary-specific expression of its
own gene via this distal enhancer element. In addition to several Pit-1
DNA-binding sites, this enhancer contains at least two retinoic acid
response elements (RAREs). Interestingly, the retinoic acid receptor
(RAR) functionally interacts with Pit-1 on one of these latter elements
to mediate a synergistic activation of the Pit-1 gene in
response to retinoic acid (RA). It is intriguing that a developmental
factor (Pit-1) and a morphogen (RA) would interact at this element and
suggests that RA may play a critical role in pituitary development.
Point mutations in the Pit-1 gene have been found in
association with combined pituitary hormone deficiency (CPHD) of GH,
PRL, and TSH (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28), some on only one allele (19, 21, 22). Unlike the
autosomal recessive mutations in which loss of function from one allele
can not cause CPHD, the molecular mechanism of the sporadic mutations
is unclear. One mutation (R271W) dominantly inhibits activation of the
GH and PRL genes by wild-type (wt) Pit-1, and its
properties have been described elsewhere (19, 22).
In the present report, we have explored the mechanism of CPHD in a
patient with a novel Pit-1 gene mutation present on only one
allele. This mutation (K216E) does not inhibit activation of the
GH and PRL target genes. It, however, blocks RA
induction via the Pit-1 gene distal enhancer. Our results
indicate that Pit-1 gene mutations can cause CPHD by
selectively inhibiting gene activation by the superfamily of nuclear
hormone receptors.
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RESULTS AND DISCUSSION
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A Novel Pit-1 Gene Mutation in a Patient with
CPHD
A patient was evaluated at Childrens Hospital for short stature.
The patient had measurable GH levels, but insulin-induced hypoglycemia
and glucagon stimulation resulted in deficient GH release to a maximum
of 1.4 µg/liter (normal, >7 µg/liter). He also had partial PRL
deficiency with a normal basal PRL (5.4 µg/liter), but no increase in
PRL response to TRH stimulation. Although he initially had normal
thyroid function, he developed secondary hypothyroidism over the first
2 yr of life, as evidenced by a low serum T4 level of 39
nmol/liter (normal 58193) and a blunted response of TSH to TRH (<1.7
mU/liter, below the limits of detection of the assay at that time) at
28 months of age. An A-to-G mutation in codon 216 of the
Pit-1 gene has been identified in one-half of independent
clones sequenced (data not shown), resulting in an amino acid change
from a lysine to a glutamic acid in one allele (K216E). The rest of the
Pit-1 gene was sequenced, and no other mutation was
identified, indicating that this patient is not a compound
heterozygote. The parents DNA could not be obtained, but they were of
normal height, suggesting that this is most likely a sporadic mutation.
Codon 216 encodes the third amino acid in a phosphorylation consensus
sequence of the Pit-1 gene specified by the amino acid
sequence (K/R)4RT(S/T)I (29). This region is completely
conserved among the homeodomains of other POU domain proteins. The
molecular properties of the K216E Pit-1 mutation were evaluated in this
study.
Superactivation of the Mutant Pit-1 Protein on the GH
and PRL Genes
The effect of the K216E Pit-1 mutant on the expression of the
GH and PRL genes was investigated in CV-1 and
JEG-3 cells (Pit-1 deficient). The GH and PRL
genes were chosen for these studies because their responses to Pit-1
have been well characterized. A construct containing 236 bp of rat (r)
GH or 1.0 kb of bovine (b) PRL 5'-flanking DNA
was fused to the luciferase reporter gene. The proximal GH
promoter contains both GH1 and GH2 sites (Pit-1 binding sites) and the
intervening Z-box sequence (10). The human GH promoter is
similar to the rat GH promoter and is virtually identical to
the rat GH gene in the GH1-Z box-GH2 region. The rat Pit-1
cDNA was chosen because rat Pit-1 and its isoforms are much better
characterized than human Pit-1, and rat and human Pit-1 are virtually
identical at the amino acid level.
Figure 1
illustrates the effect of wt
Pit-1 and the human mutation of Pit-1, K216E, on GH and
PRL gene activation. Wild-type Pit-1 activated the
GH promoter 1.5-fold in both CV-1 and JEG-3 cells (Fig. 1A
).
Interestingly, the K216E mutant Pit-1 was a better stimulator of the
GH promoter than wt in both CV-1 and JEG-3 cells (3.7- and
2.5-fold, respectively). The same pattern of stimulation, but to a much
greater extent, was seen for the proximal PRL promoter in
JEG-3 cells. Wild-type Pit-1 stimulated the proximal PRL
promoter 25-fold, while the K216E mutant Pit-1 stimulated it 41-fold
(Fig. 1B
). Relative activation of the proximal PRL promoter
in CV-1 cells was minimal compared with empty vector (EV) (data not
shown), suggesting that other factors not found in CV-1 cells may be
important in PRL gene activation. Western blot analysis
(Fig. 1C
) confirmed equivalent expression of wt and mutant K216E Pit-1
in the transfection system. Thus, the differences in activation cannot
be explained by differential levels of protein expression. The
increased activation of pituitary genes by the mutant Pit-1 leaves the
mechanism of CPHD yet unexplained.

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Figure 1. Activation of the GH and
PRL Promoters in Pit-1-Deficient Cell Lines (CV-1 and
JEG-3)
A pSG5 wt or mutant K216E Pit-1 expression construct or pSG5 EV
(0.5 µg in CV-1, 0.3 µg in JEG-3) was cotransfected with a 236-bp
rGH luciferase reporter construct (3 µg) (panel A) or
a 1.0-kb bPRL luciferase reporter construct (3 µg)
(panel B). Each experiment was performed in triplicate. Data are the
mean ± SE stimulation of the reporter construct after
correction for transfection efficacy as monitored by pTKGH. For the
rGH promoter, P < 0.01 when EV is
compared with wt, and p<0.005 when K216E mutant is compared with wt;
for the bPRL promoter, P < 0.005
when EV is compared with wt, and P < 0.005 when
K216E mutant is compared with wt, using Students unpaired
t test. C, Western blot analysis of the cellular
extract. Equivalent amounts of wt and K216E mutant Pit-1 protein of the
expected 31- and 33-kDa size isoforms were detected with a Pit-1
monoclonal antibody.
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DNA-Binding Properties of the Mutant Pit-1 Protein
We next determined the DNA-binding properties of the K216E mutant
Pit-1 molecule, since an alteration in DNA binding might explain the
functional changes that were observed above. For these studies, wt and
K216E mutant rat Pit-1 were expressed in Escherichia coli
using a glutathione S-transferase (GST) gene fusion system.
GST is synthesized as a 29-kDa protein, while both wt and mutant Pit-1
GST fusion proteins are synthesized as approximately 60-kDa proteins in
equal amounts (Fig. 2A
).

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Figure 2. DNA Binding Properties of WT and Mutant Pit-1 on
the PRL-1P-Binding Site
A, An SDS-PAGE analysis after purification of the GST-Pit-1 fusion
proteins revealed the expected 29-kDa GST protein and approximately
equal amounts of the wt and mutant constructs. B, Gel-mobility shift
analysis of the PRL response element utilizing purified Pit-1 proteins
from E. coli. Equal amounts of the GST and Pit-1
proteins were used, the last lane containing GST protein only. DNA
fragment used: PRL-1P, -60 TTATATATATATTCATGAA -42. The longer
arrows denote specific protein-DNA complexes; the solid
arrow may represent a monomer, and the dashed
arrow may represent a dimer. The shorter arrow
denotes the supershifted complex with the addition of Pit-1 antiserum
(Pit-1 AS). The * denotes a nonspecific complex also observed with GST
protein alone.
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The DNA-binding properties of wt and K216E-purified bacterial
recombinant Pit-1 were evaluated in a gel mobility shift assay using a
high-affinity PRL binding site (PRL-1P). As shown in
Fig. 2B
, recombinant Pit-1 formed two distinct complexes with the
PRL probe, consistent with monomeric and dimeric species.
These complexes were specifically shifted with Pit-1 antisera, but not
c-Jun antisera, confirming their identity. Protein from E.
coli containing GST alone did not result in formation of a band.
The K216E mutant protein had increased binding to the PRL
site relative to wt Pit-1. An increase in DNA binding of the mutant
Pit-1 protein may explain the increased transactivation properties on
the GH and PRL promoters.
The K216E mutation is found in the N-terminal basic region of the POU
homeodomain. Crystallographic structure of the POU homeodomain
indicates that the K216E mutation lies within the spacer region between
the POU-specific and POU-homeo domains (30) and does not contact DNA
(31). Hence, the gel mobility shift assay confirms that codon 216 is
not likely to contact the Pit-1-binding site. However, this codon may
be important in interactions between Pit-1 and other proteins.
Defective RA Signaling on the Pit-1 Gene
Enhancer
These data did not clarify how the K216E mutation caused CPHD,
since K216E appeared to be a superactive stimulator of GH
and PRL gene expression. However, recent data suggest that
an important additional role for Pit-1 involves its interaction with
the nuclear hormone superfamily of receptors (13, 14, 15, 16). During
development, Pit-1 gene activation requires RA induction of
a Pit-1-dependent Pit-1-autoregulatory feedback loop (16). Since Pit-1
functionally interacts with the RAR on the Pit-1 gene distal
enhancer, defective interaction with the K216E mutant and this receptor
could result in abnormal regulation of the Pit-1 gene. A
lack of Pit-1 induction would then result in a cascade of events
leading to disruption of pituitary gene expression. We tested,
therefore, whether this mutation would alter RA signaling on the
Pit-1 gene distal enhancer.
Figure 3
is the functional assessment of
this enhancer in CV-1 cells (Pit-1 deficient). pSG5 expression vectors
containing either RAR-
, and wt rat Pit-1 or the K216E mutation in
rat Pit-1 were cotransfected with two copies of the Pit-1
gene distal enhancer (-10.7 to -10.2 kb) fused upstream of TK109Luc.
Cotransfection of the RAR with an EV expression vector yielded a
minimal RA response of 1.9-fold (seventh set of bars). In
contrast, wt Pit-1 and RAR cotransfection mediated a 36.0-fold response
by RA (first set of bars), which confirmed a previous report
showing a Pit-1 dependence for the RA response on this element (16).
Cotransfection of RAR and the K216E mutant, however, resulted in a
minimal RA response of 3.4-fold (sixth set of bars).

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Figure 3. Effect of a Pit-1 Mutation on RA Regulation of the
Pit-1 Gene Distal Enhancer
Two copies of the mouse Pit-1 enhancer were inserted
upstream of TK109Luc. This construct (0.5 µg) was cotransfected with
a pSG5 RAR- expression vector (0.1 µg) in CV-1 cells and varying
amounts of pSG5 wt and/or mutant K216E Pit-1 expression construct or
pSG5 wt Pit-1 and/or EV expression construct to a total of 0.5 µg.
Stimulation was performed with 10-6 M
9-cis-RA for 24 h. Each experiment was performed in
triplicate. Data are expressed as the mean ± SE fold
activation of the Pit-1 distal enhancer reporter
construct relative to EV, after correction for transfection efficacy as
monitored by pTKGH. After RA stimulation, P < 0.01
when comparing 0.25 µg K216E mutant plus 0.25 µg EV with 0.5 µg
wt, and P < 0.005 when comparing 0.1 µg K216E
mutant plus 0.4 µg EV or 0.5 µg K216E mutant with 0.5 µg wt,
using the unpaired Student t test. However,
P = NS when comparing all concentrations of
wt plus EV with 0.5 µg wt.
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To determine whether cotransfection of increasing amounts of K216E
mutant vs. wt Pit-1 resulted in a progressive decrease in RA
induction, varying amounts of mutant Pit-1 were cotransfected with wt
Pit-1 and RAR expression vectors, and the degree of RA induction was
determined (Fig. 3
). When total Pit-1 was kept constant at both a 1:1
and 4:1 mutant to wt plasmid DNA ratio (second and fourth set of
bars, respectively), there was decreased RA induction of the
Pit-1 gene distal enhancer element relative to wt Pit-1, to
a level of 19.8- and 11.6-fold, respectively. To control for the
decreasing amount of wt Pit-1 used, total DNA was also kept constant by
the addition of EV (third and fifth set of bars). There was
not a significant change in RA induction of the Pit-1 gene
distal enhancer when the mutant K216E Pit-1 was not present, indicating
that the decreased responsiveness was not due to decreased amount of
functional wt Pit-1. These data indicate that this Pit-1 mutation is
functionally defective and blocks wt Pit-1 in mediating RA induction of
the Pit-1 gene through its distal enhancer element, with a
1:1 wt to mutant Pit-1 ratio resulting in a decrease in activation of
the Pit-1 distal enhancer by approximately 50%.
Aberrant Physical Interaction between RAR and the Pit-1 Mutant
on the Pit-1 Gene Distal Enhancer
To test for structural interactions between Pit-1 and RAR, gel
mobility shift analysis with a radiolabeled fragment of the
Pit-1-dependent RARE (PRARE) was performed. Figure 4
is the gel shift assay with the PRARE
element. RAR-
alone did not form a specific protein-DNA complex
either alone or in combination with RA. wt And K216E mutant Pit-1
protein alone formed a protein-DNA complex with the probe, which was
further shifted in mobility by Pit-1 antisera (Pit-1 AS), indicating
that Pit-1 was present in the complex. However, the supershifted wt and
mutant bands differ, suggesting that the conformation of the K216E
mutant on this element could differ from wt Pit-1. When both wt Pit-1
and RAR were present in the binding reaction, a slightly larger complex
was formed (long arrow), which was shifted in mobility
(short arrow) by RAR-
antisera (RAR AS), indicating that
wt Pit-1 and RAR bind cooperatively on the Pit-1 gene distal
enhancer. The light intensity of the band is consistent with the weak
cooperative binding interactions between RAR and Pit-1 on the PRARE
(16). This effect was not seen with the K216E mutant Pit-1, indicating
that there is decreased binding cooperativity between the mutant Pit-1
and RAR on the Pit-1 gene distal enhancer.
Effect of the Mutant Pit-1 Molecule on Pit-1 mRNA in
Vivo
To determine whether the defect of the mutant Pit-1 in mediating
RA induction of the Pit-1 gene was significant in
vivo, Northern hybridization analysis was performed utilizing mRNA
obtained from a Pit-1-expressing cell line (GH3). Wild-type
or mutant K216E Pit-1 or EV was transfected into the GH3
cell line, and Northern hybridization was performed with a rat Pit-1
cDNA probe. Figure 5a
shows the detection
of two mRNA transcripts of the expected 2.6-kb and 1.2-kb sizes (32).
Figure 5b
is the densitometric analysis of this Northern blot after
correction for ß-actin levels. After transfection of wt Pit-1, RA
stimulation led to an increase in Pit-1 mRNA, comparable to the level
seen after RA stimulation after transfection of EV. This result was
expected, since the GH3 cell line expresses endogenous
Pit-1 at high levels. However, there was no increase in Pit-1 mRNA
after RA stimulation when the mutant Pit-1 was transfected. Thus, the
mutant K216E Pit-1 interferes with RA stimulation of the endogenous
Pit-1 gene in GH3 cells.

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Figure 5. Northern Hybridization Analysis of Pit-1 mRNA
Expression in GH3 Cells (Pit-1 Sufficient) after
Transfection with a pSG5 wt or Mutant K216E Pit-1 Expression Construct
or pSG5 EV (8 µg)
Stimulation was performed with 10-6 M
all-trans-RA for 24 h. mRNA (0.5 and 1.5 µg) was
used for each sample. A, mRNA transcripts of 2.6 and 1.2 kb were
detected. B, Densitometric analysis of the Pit-1 mRNA. Data are
expressed as Pit-1 mRNA, divided by the densitometric analysis of
ß-actin in each lane to correct for loading, and expressed relative
to wt Pit-1.
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Impaired RA Signaling Leads to Defective Activation of Pit-1
Target Genes
To ascertain whether the defect in RA induction
of the Pit-1 gene affected pituitary hormone gene
expression, a construct containing two copies of the Pit-1
gene distal enhancer, fused upstream of the human Pit-1
promoter, linked to either wt or mutant K216E rat Pit-1 cDNA was made
(Fig. 6a
). A third vector containing no
Pit-1 cDNA was also constructed. These vectors were cotransfected with
a pSG5 expression vector containing the RAR-
receptor and a
luciferase reporter construct containing 236 bp of the rGH
promoter in CV-1 cells. Figure 6b
shows that relative to EV, the wt
construct activated the GH promoter after RA stimulation,
while the K216E mutant construct was completely defective. Since the
K216E mutation is found on only one allele, the wt and mutant
constructs were cotransfected in a 1:1 ratio, keeping total amount
constant (Fig. 6c
). RA induction of the GH promoter was
blocked, indicating that the K216E mutant acted as a dominant inhibitor
of wt Pit-1 in this transfection system. To confirm that this decrease
was not due to using half the amount of wt construct, the wt construct
and EV construct were also cotransfected in a 1:1 ratio, keeping the
total amount constant. There was RA induction of the GH
promoter similar to that seen with the wt construct alone. Thus,
RA induction of the Pit-1 gene distal enhancer leads to
defective GH promoter activation when the K216E mutation is
present, and wt Pit-1 function is inhibited as well.

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Figure 6. Activation of the GH Promoter after
RA Regulation Stimulation of the Pit-1 Gene Distal
Enhancer in CV-1 Cells
A, Two copies of the mouse Pit-1 gene enhancer were
fused to 991 bp of the human Pit-1 promoter placed
upstream of wt or mutant K216E rat Pit-1 cDNA and an SV40 polyA site in
the PGEM9z vector (PRARE-Pit-1). B, This construct (1.5 µg) was
cotransfected with a pSG5RAR- expression vector (0.1 µg) in CV-1
cells and 236 bp of the rGH promoter fused to luciferase
(3 µg). Stimulation was performed with 10-6
M all-trans-RA for 24 h. Each
experiment was performed in triplicate. Data are standardized to
EV and are the mean ± SE stimulation of the
GH reporter construct after correction for transfection
efficacy as monitored by pTKGH. After RA stimulation,
P < 0.01 when EV is compared with wt, and
P < 0.005 when K216E mutant is compared with wt
using the unpaired Student t test; or using varying
amounts of the constructs to a total of 1.5 µg (panel C). After RA
stimulation, P = NS when comparing 0.75 µg
PRARE-wt plus 0.75 µg PRARE-EV with 1.5 µg wt-PRARE using
Students unpaired t test. However,
P < 0.005 when comparing 0.75 µg PRARE-K216E
mutant plus 0.75 µg PRARE-EV with 1.5 µg wt-PRARE.
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Molecular Mechanisms Causing CPHD
Pit-1 gene mutations are found sporadically or
are inherited as either autosomal-recessive or dominant traits
(18, 19, 20, 21, 22, 23, 24, 25). Autosomal-recessive mutations usually disrupt Pit-1 DNA
binding leading to a lack of gene activation in the pituitary. Other
mutations, present on only one allele, are thought to dominantly
inhibit the function of wt Pit-1 and also lead to CPHD. We have
previously described one mutation (R271W) with these properties in two
different patients (19, 22). In the present report, we have
characterized the mechanism of transrepression by a new mutation,
K216E. This mutation is found in the 5'-region of the POU homeodomain
in an area that does not appear to be important for DNA binding to
target pituitary genes (31). Indeed, structural analysis indicated that
the mutant Pit-1 does not appear to decrease DNA binding. Since the
mutant Pit-1 does not function on certain elements such as the
Pit-1 distal enhancer, this region may be important for
protein-protein interactions between Pit-1 and other transcription
factors.
Initial studies indicated that the K216E mutant acted as a
superactivator of GH and PRL gene expression,
demonstrating that the K216E mutant must cause CPHD through yet another
mechanism. Pit-1 is known to interact with the steroid/thyroid hormone
superfamily of nuclear receptors and modify hormonal regulation of
certain target genes. Pit-1 is required for estrogen induction of the
PRL gene via the distal PRL enhancer and RA
induction of the Pit-1 gene during development via the
Pit-1 gene distal enhancer. We determined that the K216E
mutation was unable to support RA induction of the Pit-1
gene distal enhancer either alone or in combination with wt Pit-1. The
mutant Pit-1 was unable to interact with the RAR, and RA was unable to
stimulate Pit-1 expression in vivo when the mutant K216E
Pit-1 was present. Furthermore, this mutant Pit-1 resulted in defective
Pit-1-dependent RA induction of the GH promoter. Since the
patient has one normal and one mutant allele, the net result may be a
significant reduction in RA activation of the Pit-1 gene
during a crucial period of development. Our data indicate that in the
patient, reduction in Pit-1 gene activation leads to
decreased Pit-1 mRNA levels and presumably decreased Pit-1 protein
levels. The final result is decreased activation of target genes, such
as GH (model, Fig. 7
). We
suggest that the ability to selectively impair interaction with the
superfamily of nuclear hormone receptors is a new molecular mechanism
responsible for CPHD caused by certain Pit-1 gene
mutations.

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Figure 7. Model of Selective Impairment of the Interaction of
Pit-1 with the RAR as a Molecular Mechanism Responsible for CPHD
Pit-1 is required for RA induction of the Pit-1 gene
during development via the Pit-1 gene distal enhancer.
The K216E mutant is unable to support RA induction of the
Pit-1 gene distal enhancer, either alone or in
combination with wt Pit-1, resulting in decreased Pit-1 levels. The
lack of Pit-1 leads to decreased activation of target genes.
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MATERIALS AND METHODS
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Plasmids
Wild-type and mutant K216E rat Pit-1 cDNA, confirmed by DNA
sequencing, were cloned into the EcoRI/BamHI
sites of the SV40 viral expression vector, pSG5. Rat GH
promoter (236 bp) or bovine PRL promoter (1.0 kb) was
inserted upstream of the luciferase reporter gene in pSVOAL
5'. A
500-bp mouse Pit-1 gene distal enhancer (-10.7 to -10.2
kb, Pit-en) was obtained from a PCR of mouse genomic DNA and subjected
to DNA sequencing. Two copies of this fragment were cloned as an Asp
718 fragment upstream of TK109 luciferase. Two copies of the mouse
Pit-1 gene enhancer were fused to 991 bp of the human
Pit-1 promoter placed upstream of wt or mutant K216E
rat Pit-1 cDNA and an SV40 polyA site in the pGEM9z vector
(PRARE-Pit-1). The human RAR-
was also inserted into the pSG5
expression vector (kind gift of B. Neel, Beth Israel Hospital, Boston,
MA).
Transfections
Various concentrations of pSG5 Pit-1 expression constructs
(total 0.5 µg per well in CV-1 cells, 0.3 µg per well in JEG-3
cells) were cotransfected with a rGH promoter (3 µg per
well), bPRL promoter (3 µg per well), or Pit-1
distal enhancer (0.5 µg per well) luciferase reporter construct into
CV-1 or JEG-3 cells (both are Pit-1 deficient) in triplicate using a
calcium-phosphate precipitation technique (Specialty Media, Inc.,
Lavallette, NJ) in six-well tissue culture plates, and luciferase
activity in relative light units was measured at 36 h. Various
concentrations of PRARE-Pit-1 expression constructs (total 1.5 µg per
well) were cotransfected with a rGH promoter luciferase
reporter construct (3 µg per well) in CV-1 cells. Transfection
efficiency was monitored by cotransfecting 0.4 µg per well of a human
GH expression construct (pTKGH) and measuring GH secretion in the media
by chemiluminescent assay (Nichols Institute, San Juan Capistrano, CA).
The ability of mutant Pit-1 to interfere with the function of wt Pit-1
was assessed by cotransfecting either EV or K216E mutant Pit-1 with wt
Pit-1 expression vector. In the Pit-1 enhancer experiments, 0.1 µg of
pSG5 RAR-
was cotransfected per well; and 10-6
M 9-cis-RA (Hoffman-Roche, Nutley, NJ) or
10-6 M all-trans-RA (Sigma
Pharmaceutical Co., St. Louis, MO) was added to phenol-free Dulbeccos
modified essential media with glutamine for 24 h before assay.
Western Blot Analysis
After transient transfection in CV-1 cells as above, proteins
were extracted and Western blot analysis was performed according to
published protocols (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
and Amersham Life Science, Arlington Heights, IL, respectively).
A Pit-1 monoclonal antibody (Pit-1 AS) (Transduction Laboratories,
Lexington, KY) was used for detection.
Gel-Shift Analysis
Gel-shift analysis was performed with recombinant Pit-1 proteins
and 32P-labeled DNA fragments. For testing Pit-1 binding, a
high-affinity Pit-1 DNA-binding site from the bovine PRL
gene (-60 to -42) was used: -60 TTATATATATATTCATGAA -42. For
testing of Pit-1 and RAR binding to the Pit-1 gene distal
enhancer, the composite Pit-1-RARE was used:
5'-TAATATGTGCTCAAAGTTCAGGTATGAATATAA-ATG-3'
(RAR half-sites underlined, a half-site for Pit-1 binding
site in bold). For these studies, RAR-
was synthesized in
a coupled transcription/translation reaction (Promega Biotech, Madison
WI), and 1 µg of bacterial recombinant purified Pit-1 was used.
9-cis RA (10-6 µM)
(Hoffman-Roche) was added to reactions containing RAR.
Recombinant wt and mutant K216E Pit-1 were synthesized in bacteria by
inserting the corresponding cDNAs into the pGEX-4T-2 vector. The
pGEX-4T-2 vector, which contains GST, is induced to express Pit-1 in an
E. coli strain (JM109) by 0.5 M
isopropyl-ß-D-thiogalactopyranoside treatment.
Recombinant Pit-1 fused to GST was purified by affinity chromatography
using glutathione Sepharose 4B (Pharmacia Biotechnology,
Uppsala, Sweden) using manufacturers instructions. As a negative
control in these experiments, GST protein was used. Protein
concentrations were measured in a protein assay (Bio-Rad, Hercules,
CA).
The Pit-1 complexes were supershifted with a Pit-1 monoclonal antibody
(Pit-1 AS) (Transduction Laboratories). As a control, c-Jun polyclonal
antibody (C-Jun AS) (Santa Cruz Biotechnology, Inc.) was used. The RAR
complexes were supershifted with an RAR-
polyclonal antibody (Santa
Cruz Biotechnology, Inc.).
Northern Hybridization Analysis
pSG5 wt or mutant Pit-1 or EV expression construct (8 µg) was
transfected into GH3 cells (Pit-1 deficient) using
Lipofectamine Plus (GIBCO BRL, Gaithersburg, MD) in 175-cm2
tissue culture flasks. mRNA was isolated using a Guanidine Direct mRNA
purification kit (CPG, Inc., Lincoln Park, NJ) using
manufacturers instructions. mRNA (0.5 µg and 1.5 µg) was run on a
1.4% gel, and mRNA was transferred to Gene Screen Plus nylon membrane
(NEN Life Science Products, Boston, MA). The membrane was hybridized
with a wt rat Pit-1 cDNA probe prepared by random primed DNA labeling
(Boehringer Mannheim, Indianapolis, IN) and then washed and probed with
ß-actin (provided by M. Zakaria, Childrens Hospital, Boston, MA).
Densitometry was performed using Adobe Photoshop 4.0 (Adobe Systems
Corp., San Jose, CA) and NIH Image 1.6 (Scion Corp., Frederick,
MD).
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Yukiko Hashimoto, Rebecca Marier, and
Diane Stafford for technical assistance. The patient was identified
using a computerized database at Childrens Hospital, Boston (33).
RAR-
was the kind gift of B. Neel (Beth Israel Hospital, Boston,
MA), and ß-actin was the kind gift of M. Zakaria (Childrens
Hospital, Boston, MA).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Laurie E. Cohen, M.D., Childrens Hospital, Division of Endocrinology, Enders 4, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail:cohen l{at}a1.tch.harvard.edu
This work was supported by grants from the NIH (L.E.C., S.R.) the
Charles H. Hood Foundation (L.E.C.), and the Genentech Foundation
for Growth and Development (S.R.).
Received for publication March 17, 1998.
Revision received October 5, 1998.
Accepted for publication November 30, 1998.
 |
REFERENCES
|
---|
-
Rosenfeld MG 1991 POU-domain transcription factors:
pou-er-ful developmental regulators. Genes Dev 5:897907[CrossRef][Medline]
-
Theill LE, Castrillo J-L, Wu D, Karin M 1989 Dissection of
functional domains of the pituitary-specific transcription factor
GHF-1. Nature 342:945948[CrossRef][Medline]
-
Ingraham HA, Flynn SE, Voss JW, Albert V, Kapiloff MS, Wilson
L, Rosenfeld MG 1990 The POU-specific domain of Pit-1 is essential for
sequence-specific, high affinity DNA binding and DNA-dependent
Pit-1-Pit-1 interactions. Cell 61:10211033[Medline]
-
Nelson C, Albert VR, Elsholtz HP, Lu LW, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and
prolactin genes by a common transcription factor. Science 239:14001405[Medline]
-
Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, Wilson L,
Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein,
Pit-1, activates both growth hormone and prolactin promoters
transcriptionally. Genes Dev 3:946958[Abstract]
-
Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS,
Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes involve
cell-specific Pit-1 mRNA translation and synergistic interactions with
other classes of transcription factors. Genes Dev 4:695711[Abstract]
-
Schaufele F, West BL, Reudelhuber TL 1990 Overlapping Pit-1
and Sp1 binding sites are both essential to full rat growth hormone
gene promoter activity despite mutually exclusive Pit-1 and Sp1
binding. J Biol Chem 265:1718917196[Abstract/Free Full Text]
-
Fox SR, Jong MT, Casanova J, Ye ZS, Stanley F, Samuels HH 1990 The homeodomain protein, Pit-1/GHF-1, is capable of binding to and
activating cell-specific elements of both the growth hormone and
prolactin gene promoters. Mol Endocrinol 4:10691080[Abstract]
-
Supowit SC, Ramsey T, Thompson EB 1992 Extinction of
prolactin gene expression in somatic cell hybrids is correlated with
the repression of the pituitary-specific trans-activator GHF-1/Pit-1.
Mol Endocrinol 6:786792[Abstract]
-
Lipkin SM, Naar AM, Kalla KA, Sack RA, Rosenfeld MG 1993 Identification of a novel zinc finger protein binding a conserved
element critical for Pit-1-dependent growth hormone gene expression.
Genes Dev 7:16741687[Abstract]
-
Schaufele F, West BL, Baxter JD 1992 Synergistic activation of
the rat growth hormone promoter by Pit-1 and the thyroid hormone
receptor. Mol Endocrinol 6:656665[Abstract]
-
Suen C-S, Chin WW 1993 Ligand-dependent, Pit-1/GHF-1
independent transcriptional stimulation of rat growth hormone gene
expression by thyroid hormone receptors in vitro. Mol Cell
Biol 13:17191727[Abstract]
-
Day RN, Maurer RA 1989 The distal enhancer region of the rat
prolactin gene contains elements conferring response to multiple
hormones. Mol Endocrinol 3:39[Abstract]
-
Nowakowski BE, Maurer RA 1994 Multiple Pit-1-binding sites
facilitate estrogen responsiveness of the prolactin gene. Mol
Endocrinol 8:17421749[Abstract]
-
Day RN, Koike S, Sakai M, Muramatsu M, Maurer RA 1990 Both
Pit-1 and the estrogen receptor are required for estrogen
responsiveness of the rat prolactin gene. Mol Endocrinol 4:19641971[Abstract]
-
Rhodes SJ, Chen R, DiMattia GE, Scully KM, Kalla KA, Lin S, Yu
C, Rosenfeld MG 1993 A tissue-specific enhancer confers Pit-1-dependent
morphogen inducibility and autoregulation on the Pit-1 gene. Genes Dev 7:913932[Abstract]
-
Li S, Crenshaw EB, Rawson EJ, Simmons DM, Swanson LW,
Rosenfeld MG 1990 Dwarf locus mutants three pituitary cell types result
from mutations in the POU-domain gene Pit-1. Nature 347:528533[CrossRef][Medline]
-
Tatsumi K, Miyai K, Notomi T, Kaibe K, Amino N, Mizuno Y,
Kohno H 1992 Cretinism with combined hormone deficiency caused by a
mutation in the PIT1 gene. Nat Genet 1:5658[Medline]
-
Radovick S, Nations M, Du Y, Berg LA, Weintraub BD, Wondisford
FE 1992 A mutation in the POU-homeodomain of Pit-1 responsible for
combined pituitary hormone deficiency. Science 257:1115[Medline]
-
Pfäffle RW, DiMattia GE, Parks JS, Brown MR, Wit JM,
Jansen M, Van der Nat H, Van den Brande JL, Rosenfeld MG, Ingraham HA 1992 Mutation of the POU-specific domain of Pit-1 and hypopituitarism
without pituitary hypoplasia. Science 257:11181121[Medline]
-
Ohta K, Nobukuni Y, Mitsubuchi H, Fujimoto S, Matsuo N,
Inagaki H, Endo F, Matsuda K 1992 Mutations in the Pit-1 gene in
children with combined pituitary hormone deficiency. Biochem Biophys
Res Commun 189:851855[Medline]
-
Cohen LE, Wondisford FE, Salvatoni S, Maghnie M, Brucker-Davis
F, Weintraub BD, Radovick S 1995 A "hot spot" in the Pit-1 gene
responsible for combined pituitary hormone deficiency: clinical and
molecular correlates. J Clin Endocrinol Metab 80:679684[Abstract]
-
Irie Y, Tatsumi K-I, Ogawa M, Kamijo T, Preeyasombat C,
Suprasongsin C, Amino N 1995 A Novel E250X Mutation of the PIT1 Gene in
a Patient with Combined Pituitary Hormone Deficiency. Endocrine J 42:351
-
Okamoto N, Wada Y, Ida S, Koga R, Ozono K, Chiyo K-A, Hayashi
A, Tatsumi K-I 1994 Monoallelic expression of normal mRNA in the PIT1
mutation heterozygotes with normal phenotype and biallelic expression
in the abnormal phenotype. Hum Mol Genet 3:1565[Abstract]
-
de Zegher F, Pernasetti F, Vanhole C, Devlieger H, Van den
Berghe G, Martial JA 1995 Fetal immaturity and maternal alactogenesis
in fetomaternal Pit-1 deficiency. J Clin Endocrinol Metab 80:3127[Abstract]
-
Pellegrini-Bouiller I, Belicar P, Barlier A, Gunz G, Charvet
J-P, Jaquet P, Brue T, Vialettes B, Enjalbert A 1996 A new mutation of
the gene encoding the transcription factor Pit-1 is responsible for
combined pituitary hormone deficiency. J Clin Endocrinol Metab 81:2790[Abstract]
-
Brown MR, Parks JS, Adess ME, Rich BH, Rosenthal IM, Voss TC,
VanderHeyden TC, Hurley DL 1998 Central hypothyroidism reveals compound
heterozygous mutations in the Pit-1 gene. Horm Res 49:98102[CrossRef][Medline]
-
Pernasetti F, Milner RDG, Ashwal AAZ, de Zegher F, Chavez VM,
Muller M, Martial JA 1998 Pro239Ser: a novel recessive mutation of the
Pit-1 gene in seven Middle Eastern children with growth hormone,
prolactin, and thyrotropin deficiency. J Clin Endocrinol Metab 83:20792083[Abstract/Free Full Text]
-
Kapiloff MS, Farkash Y, Wegner M, Rosenfeld MG 1991 Variable
effects of phosphorylation of Pit-1 dictated by the DNA response
elements. Science 253:786789[Medline]
-
Andersen B, Rosenfeld MG 1994 Pit-1 determines cell types
during development of the anterior pituitary gland. J Biol Chem 269:2933529338[Free Full Text]
-
Jacobson EM, Li P, Leon-del-Rio A, Rosenfeld MG, Aggarwal AK 1997 Structure of Pit-1 POU domain bound to DNA as a dimer: unexpected
arrangement and flexibility. Genes Dev 11:198212[Abstract]
-
Zhang K, Kulig E, Jin L, Lloyd RV 1993 Effects of Estrogen and
Epidermal Growth Factor on Prolactin and Pit-1 mRNA in GH3
Cells. Proc Soc Exp Biol Med 202:193200[Abstract]
-
Kohane IS, McCallie, A dynamically reconfigurable clinicians
workstation with transparent access to remote and local databases.
Program of the First Annual American Medical Informatics Conference,
Snowbird, UT, 1990, vol 1:70 (Abstract)