The Tissue-Specific Transcription Factor Pit-1/GHF-1 Binds to the c-fos Serum Response Element and Activates c-fos Transcription
C. Gaiddon1,
M. de Tapia and
J.-P. Loeffler
UMR 7519 Neurophysiologie Cellulaire et
Intégrée CNRS, Université Louis Pasteur, IPCB
Strasbourg, 67084 Cedex France
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ABSTRACT
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Pit-1, a POU domain-containing transcription
factor, is involved in two functions in the pituitary: PRL and GH
tissue-specific expression and somato-lactotroph cells expansion. To
analyze the molecular basis of the latter function, we tested whether
Pit-1 can directly transactivate expression of an early marker of cell
cycle initiation, the c-fos gene. We show that Pit-1
overexpression in PC12 cells, which do not express Pit-1, increases
c-fos expression. Moreover, cAMP-induced c-fos
promoter activity is decreased in the somato-lactotroph cell line
GH3 when Pit-1 expression is reduced by hybrid arrest with an antisense
sequence complementary to Pit-1 cDNA. In contrast to hormonal genes
regulation, where it has been shown that any Pit-1
phosphorylation site is involved, we show that the Pit-1
phosphorylation sites are required to allow increase of
c-fos promoter activity by Pit-1. We further show, by gel
shift analyses, that Pit-1 is able to specifically bind the serum
response element sequence present within the c-fos promoter
but with a lesser affinity than the Pit-1 response element.
Taken together, these results demonstrate that the tissue-specific
transcription factor Pit-1 is able to enhance expression of genes
involved in cell cycle initiation, suggesting that this mechanism
allows Pit-1 to increase somato-lactotroph cell proliferation.
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INTRODUCTION
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GH and PRL are mainly pituitary hormones produced by somatotrophs
and lactotrophs, respectively. The study of tissue-specific expression
of GH and PRL genes has led to a characterization of the transcription
factor Pit-1/GHF-1, which is required for tissue-specific expression of
these genes (1, 2, 3, 4, 5, 6, 7). Pit-1 is the first member of the POU
transcription factor family (8). The POU proteins contain two highly
conserved domains: the first, which is highly homologous to the
homeodomain (POUh), and a second, only present in the POU family
members, the POU-specific domain (POUs) (9, 10, 11). The POU homeodomain is
a minimal region required for sequence-specific DNA binding (12), but
the POUs domain contributes also to the high-affinity binding and
participates in sequence recognition (13). However, the POUh
alone is able to bind consensus homeodomain sites (13). Pit-1 binds its
site as a homodimer or a heterodimer with OCT-1 (14, 15), and each
domain, POUh and POUs, recognize one part of the consensus sequence (9, 11, 15). It has been proposed that the POUh domain binds the sequence
TAT and the POUs domain binds the sequence TACN. Within the GH
promoter, two cis-acting elements, centered around -80 and
-120 and required for somatotroph-specific expression, bind
Pit-1/GHF-1 (3, 16, 17). Similar observations have been obtained with
the rat PRL promoter, where Pit-1 binds two elements located in the
first 200 bp of the proximal promoter and one at position -1580/-1720
in the distal enhancer (1). Pit-1 itself is regulated at the
transcriptional level by different mechanisms, including a positively
acting autoregulatory mechanism (18). Pit-1 is a substrate for
different kinases: protein kinase C, protein kinase A, and a still
unknown kinase activated during the cell cycle (19, 20, 21, 22, 23). At the
molecular level, the functional relevance of these phosphorylation
events has not been determined precisely, although it appears that
phosphorylation lowers DNA binding of Pit-1 (21). In addition, at the
cellular level, the precise role of Pit-1 in the establishment of the
somatotroph, lactotroph, and thyrotroph cell types remains also poorly
understood. Several observations suggest that expression of Pit-1 could
be involved in the regulation of cell proliferation. First, in the GC
somatotroph cell line, microinjection of Pit-1 antisense sequences
block cell growth (6). Second, physiopathological evidence for such a
role comes from Pit-1-deficient dwarf mice (7). In these mice, severe
growth reduction is associated not only with PRL and GH deficiency, but
also with a marked failure in development of somato-lactotroph cells in
the pituitary. To gain some insight into the molecular mechanisms
underlying the growth-promoting effects of Pit-1, we analyzed the
transcriptional modulation by Pit-1 of the immediate early gene (IEG)
c-fos, a gene associated with cell cycle initiation. Indeed,
c-fos expression increase is an early marker of cell cycle
initiation after growth factor treatment (24, 25). The role of Fos in
cell proliferation is also suggested by its involvement in the
transcriptional complex that stimulates expression of genes that act
directly in the cell cycle, like cyclin D1 (26, 27, 28, 29, 30).
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RESULTS
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The Tissue-Specific Transcription Factor Pit-1 Stimulates
c-fos Transcription
We analyzed the effect of Pit-1 overexpression on c-fos
transcription in PC12 cell lines, which have no endogenous Pit-1. As
reporter gene we used a construct containing the human c-fos
promoter (bp -700 to +42) fused upstream of the chloramphenicol
acetyltransferase (CAT) cDNA. In a first series of experiments, this
c-fos-CAT vector is cotransfected with an expression vector
coding for the wild-type Rat Pit-1 (pRSV Pit-1; Fig. 1A
). Overexpression of Pit-1 stimulates
c-fos promoter activity more than 10-fold, as compared with
control expression vector (pRSV) (Fig. 1A
). Furthermore, activation by
forskolin treatment of the cAMP pathway enhanced c-fos
promoter activity in control cells and in cells overexpressing Pit-1.
We confirm the ability of Pit-1 to increase c-fos
transcription by measuring c-fos mRNA levels after Pit-1
overexpression into PC12 cells. In these experiments, the mRNA was
quantified only in the transfected cells using pHook expression vector
(Invitrogene, Invitrogen BV, Groningen, The Netherlands) system.
This vector allows the expression of a chimeric protein containing the
transmembrane part of the PDGF receptor fused to the variable region of
a antibody directed against the phOx hapten
(4-ethoxymethylene-2-phenyl-oxazolin-5-one). Transfected cells are
selected by using phOx hapten cross-linked to magnetic beads (Capture
Tech pHook-2 System, Invitrogene). Selected cells were lysed, and
c-fos mRNA levels were assessed by RT-PCR. After
transfection (48 h), endogenous c-fos expression increased
when Pit-1 is ectopically expressed (Fig. 1B
). Using the cyclophilin
expression as an internal standard, we can estimate that the
c-fos mRNA level is increased 2.8- fold by Pit-1 (Fig. 1B
).
It is interesting to note that Pit-1 stimulation of the
c-fos promoter activity and the c-fos mRNA levels appears
unequal. This discrepancy could be the result of the absence in the
reporter gene construct of elements that could control positively the
c-fos transcription and/or mRNA stability. To further
analyze the physiological relevance of c-fos promoter
activation by Pit-1, Pit-1 expression was inhibited in the
somato-lactotroph-derived GH3 cell line, which expresses high level of
Pit-1, by using an expression vector coding for an antisense Pit-1
sequence (pCMV Tip; Fig. 1C
). The basal c-fos promoter
activity, as detected by the c-fos-CAT reporter, was reduced
in cells transfected with the Pit-1 antisense vector (Fig. 1C
) by
3050% in three independent experiments, although this inhibition
could not be proven statistically different because of the variability
of low CAT activity of basal c-fos promoter transcription.
In contrast, inhibition of Pit-1 expression strongly reduces (70%)
forskolin induction of c-fos promoter activity,
demonstrating that Pit-1 is an essential element of transcriptional
mechanisms involved in the cAMP-mediated regulation of c-fos
promoter activity. To further validate this approach, we show that in
cells cotransfected with Pit-1 antisense and pHook vectors and further
selected, the expression of Pit-1 decreases after 48 h to only
30% of the original level. Under these conditions the protein level
for actin is not affected, showing the specificity of the Pit-1
antisense vector.

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Figure 1. Pit 1 Regulates c-fos Transcription.
Left panel, Overexpression of the Pit-1 transcription
factor stimulates c-fos transcription (A) and
c-fos mRNA levels (B) in transfected PC12 cells. A,
Pit-1 expression vector (pRSV Pit 1) was transfected into PC12 cells
along with the reporter gene containing the first 700 bp of the
c-fos promoter (-700 c-fos-CAT). A pRSV
ß-globin construct was used to keep the total amount of transfected
DNA to avoid unspecific promoter effects. Histograms represent
means ± SD (n = 3) in fold induction from one
representative experiment of three performed in triplicate. Statistical
differences (Students t test,
P 0,01) between basal level (pRSV
ß-globin/pRSV Pit-1) and forskolin-stimulated (Fk) levels (pRSV
ß-globin/pRSV Pit 1 in the presence of Fk 5 10-6
M) are indicated by an asterisk (*). B, PC12
cells were cotransfected with pRSV Pit-1 and pHook
(Invitrogen) or pRSV ß-globin and pHook. After 48
h, transfected cells were selected according to manufacturers
instruction and c-fos and cyclophilin mRNA were
quantified by RT-PCR on ethidium bromide gel electrophoresis. Numbers
below indicate means ± SD of three
independent experiments. Values are corrected by cyclophilin mRNA
levels, which served as control.
Right panel, Expression of Pit-1 antisense cDNA inhibits
c-fos transcription (C) and decreases Pit-1 protein
level (D). C, GH3 cells were cotransfected with the -700
c-fos CAT reporter construct and a Pit-1 antisense
construct (pCMV TIP-1; Pit-1 CDNA fragment used is indicated) or with
the parental expression plasmid pCMV lacking the Pit-1 insert. One
typical experiment of three is shown, and histograms are means ±
SD (n = 3) of three transfected cultures.
Asterisks indicate statistically significant differences
(Students t test, P 0.01). Fk
indicates forskolin treatment as describes in panel A. D, GH3 cells
were transfected with pHook and either pCMV or antisense expression
vector pCMV Tip-1. Selection was done after 24 h and 48 h as
described above (Fig. 1B ). Pit-1 and actin protein levels were
quantified by Western blot. Relative Pit-1 protein levels are
indicated. Actin protein was used as internal control.
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The Pit-1 Acceptor Sites for Phosphorylation Are Required for Full
c-fos Promoter Activation
Pit-1 has been shown to be phosphorylated at various
acceptor sites (19, 20). These sites have been described to be not
important for transcriptional activation of the GH and PRL genes by the
cAMP pathway (22, 23). However, it is known that the Pit-1
phosphorylation status is regulated during the cell cycle (21),
suggesting that Pit-1 phosphorylation might be required for cell
cycle regulation and particularly for c-fos activation. To
test whether the potential phosphorylation sites of Pit 1 contribute to
the control of c-fos transcription, we compared the
transactivation properties of wild-type Pit 1 and of a mutant protein
lacking phosphorylation sites (see Fig. 2A
). In preliminary control experiments,
we show that upon transfection into PC12 cells, both expression vectors
(pRSV Pit 1 and pRSV Pit-1 3A) lead to the accumulation of comparable
levels of Pit 1 proteins (Fig. 2B
). This result indicates that
differences in transcriptional activation observed with the expression
of wild-type and mutated forms of Pit-1 do not result from different
levels of expressed proteins. We next confirmed, in our cellular model,
i.e. PC12 cells, earlier data (see above) showing that the
phospho-acceptor sites are not required for PRL transcription. Using a
reporter gene containing the 250 proximal base pairs of the rat PRL
promoter, we show that wild-type and mutated Pit 1 equally stimulate
both basal and forskolin (Fk)-induced PRL transcription (Fig. 2C
). This
does not hold true for c-fos transcription, as shown in Fig. 2D
. Indeed, if wild-type Pit 1 clearly enhances basal and Fk-stimulated
c-fos transcription, mutated Pit-1 exerted only minor
effects on basal c-fos transcription and no effect at all on
Fk-induced transcription. Taken together, these results show
that, in contrast to the PRL gene, regulation of c-fos
transcription by Pit-1 is dependent on the three main phospho-acceptor
sites of Pit-1. It remains to be established whether phosphorylation of
these residues is required for transactivation or whether they are
involved in a protein-protein interaction independently of their
phosphorylation status. However, the fact that these three residues are
phosphorylated upon activation of the cAMP pathway (19, 20) and are
required for the c-fos tansactivation suggests strongly that
phosphorylation of these residues is involved in the transactivating
processes.

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Figure 2. Transcriptional Stimulation of c-fos by
Pit-1 Requires Pit-1 Phospho-Acceptor Sites
A, Schematic representation of the Pit-1 expression vectors. pRSV Pit-1
and pRSV Pit-1 3A contain, respectively, the wild-type Pit-1 cDNA or
the mutated Pit-1 3A cDNA lacking the phospho-acceptor at position
S115, T220, and T219. B, Expression analysis of the wild-type and
mutated Pit-1 proteins in PC12 cells. PC12 cells were transfected in
10-cm diameter dishes with 10 µg of the Pit-1 expression plasmids.
Thirty hours after transfection, cells were harvested and Western blot
analysis was performed with 20 µg of nuclear extract proteins. GH3
nuclear extracts (GH3) and purified Pit-1 protein (Pit-1) were used as
positive controls. NT are PC12 cells transfected with the pRSV
ß-globin expression vector. C, The PRL promoter does not discriminate
between wild-type and Pit-1 3A mutant. Experimental procedures were as
described in Fig. 1A . Cells were cotransfected with the -250 PRL-CAT
reporter gene construct along with either wild-type Pit-1 (pRSV Pit-1)
or the mutant Pit-1 3A (pRSV Pit-1 3A) expression vectors. Wild-type
and the Pit-1 3A mutant equally stimulate basal level (Ct) and
forskolin-induced (Fk; 5 x 10-6 M) PRL
transcription. Histograms are means ± SD (n =
3), and asterisks indicate significant differences
(Students t test, P 0.01) when
compared with respective controls (pRSV ß-globin with or without Fk
treatment). D, The Pit-1 phospho-acceptor sites are required for
efficient c-fos transcription. Cells were cotransfected
with the -700 c-fos-CAT reporter gene construct along
with either wild-type (pRSV Pit-1) or the mutant (pRSV Pit-1 3A) Pit-1
expression vector. In contrast to wild-type, the Pit-1 3A only modestly
stimulates basals and did not further stimulate forskolin- induced (Fk)
c-fos transcription. Histograms are means ±
SD (n = 3), and asterisks indicate
significant differences (Students t test,
P 0.01) when the efficiency of pRSV Pit-1 and
pRSV Pit-1 3A are compared.
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The Serum Response Element (SRE) of the c-fos Promoter
Is the Main Target of Pit-1
To determine which sequence(s) is the Pit-1 target within the
c-fos promoter, we performed a rough deletion analysis of
the c-fos promoter and analyzed these deletions by
transfection into PC12 cells. As shown above, the construct containing
the first 700 bp of the c-fos promoter (Fig. 3A
) can be stimulated by Pit-1 more than
10-fold (Fig. 3B
). In contrast, the activity of a construct containing
only the first 99 bp, with a cAMP response element and a
retinoblastoma response element, is only induced 3-fold. Further
deletion of these two elements (-53 c-fos-CAT) leads
to a non-Pit-1 responsive construct. These results show that the distal
part of the c-fos promoter, upstream position -99, contains
the major Pit-1-responsive element. This region contains various
elements, including SIS response element, FAP (Fos AP1-like element),
and SRE. Moreover, analysis of the region centered on SRE shows
noticeable homologies to part of the consensus Pit-1-binding site (Fig. 4A
). To analyze the responsiveness of
this region to Pit-1, we used reporter constructs containing either the
c-fos-FAP or the c-fos-SRE sequences fused
upstream to the minimal unresponsive c-fos promoter (-53
c-fos-CAT). In contrast to the FAP construct showing no
induction, the SRE construct is strongly induced by Pit-1 (Fig. 3B
). To
further strengthen these results, chimeric reporter genes containing
either the SRE or the FAP fused to the heterologous SV40 promoter were
used. Overexpression of Pit-1 is, again, only able to stimulate the
activity of the reporter gene containing the SRE element and not those
with the FAP element or without any element upstream of the SV40
promoter (Fig. 3C
). These results indicate that Pit-1 transactivates
c-fos mainly through the SRE.

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Figure 3. Delineation of a Pit-1-Responsive Element in the
c-fos Promoter
A, Schematic representation of the c-fos reporter genes.
The construct -700 c-fos-CAT contains the c-fos
promoter region from -700 bp to +42 bp upstream of the CAT cDNA. The
responsive elements present within the -700 to +42 region are
indicated: SRE; Fos AP1-like element (FAP); SIS response element
(SISRE); retinoblastoma response element (RbRE); and cAMP response
element (CRE). The -99 c-fos-CAT and -53
c-fos-CAT contain, respectively, the
c-fos -99 to +42 bp and -53 to +42 bp. The constructs
SRE c-fos-CAT and FAP c-fos-CAT contain,
respectively, the SRE or the FAP-responsive element inserted in the
-53 c-fos-CAT construct. B, Pit-1 expression vector was
transfected in PC12 cells along with the various c-fos
reporter genes. Experimental procedures were as described in Fig. 1 .
Histograms represent means ± SD in fold induction.
Asterisk indicates statistical differences between pRSV
ß-globin and pRSV Pit-1 (Students t test,
P 0.05) in fold induction as compared with their
respective controls (same reporter gene but cotransfected with pRSV
ß-globin plasmid). C, The SRE is sufficient to confer Pit-1
responsiveness to heterologous promoter. Left, Schematic
representation of the different luciferase reporter genes used. The SRE
and FAP element were cloned into the SV40-luc plasmid in front of the
minimal unresponsive SV40 promoter. Right, Same
experimental protocol and mode of representation is used as in panel
B.
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Figure 4. Gel Shift Analysis of Pit-1 Interactions with the
SRE
A, Alignment of the different sequences used in gel shift experiments.
Sequences homologies between SRE-containing sequences and Pit-1 RE are
boxed, and the region mutated in the SRE*/FAP
oligonucleotide is underlined. B, Autoradiography of
competition experiments; Pit-RE probe (104 cpm) was
incubated with purified Pit-1 protein (50 ng) either in the absence
(-) or in the presence of increasing concentrations of cold Pit-1 RE
duplex (homologous competition, left), SRE/FAP duplex
(heterologous competition, middle), and the mutated
SRE*/FAP duplex (no competition, right). A 5- and
50-fold excess was used for the Pit-1 RE homologous competition and
50-, 100-, 500-, and 1000-fold excess was used for the SRE/FAP and
SRE*/FAP competition. M and D indicate the two bands corresponding to
binding of Pit-1 as monomer (M) or dimer (D). C, Shifts were analyzed
on a phosphosimager (BAS 2000 bioimager, Fuji Photo Film Co., Ltd., Stamford, CT), and quantification was assisted by
MAC BAS software (Bio Image, Fuji Film Co., Ltd.).
Results are expressed as percent of Pit-1 shift (100%). Values are
calculated on as many as three independent experiments where each lane
is corrected with free probe input.
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Pit-1 Binds to the c-fos SRE
To determine whether Pit-1 physically interacts with the
c-fos SRE site highlighted by the deletion
experiments, we performed gel shift analysis and competition
experiments with purified Pit-1 protein and various oligonucleotide
sequences (Fig. 4A
). Purified Pit-1 protein interacts with the PRL
Pit-1 RE by generating two high molecular weight complexes, according
to previously published data (15), that are efficiently competed by a
homologous cold duplex (Fig. 4B
, left panel). To test
whether Pit-1 protein binds specifically the SRE, the Pit-1 RE/Pit-1
complex was competed by various cold duplexes spanning the SRE and FAP
sequence of the c-fos promoter (e.g. for SRE/FAP
on Fig. 4B
, middle panel). These sequences clearly compete
for Pit-1 binding although with a lesser affinity than Pit-1 RE itself
(Fig. 4C
). Moreover, mutation of the SRE (SRE1/FAP oligos) eliminates
the competition on Pit-1RE/Pit-1 shift (Fig. 4
, B and C), thus showing
the specificity of the competition obtained with the wild-type
sequence. Finally, a short sequence spanning solely the SRE is
sufficient to compete Pit-1 RE with almost equal efficiency than the
SRE/FAP sequence (Fig. 4C
). The SRE region has been described to bind
several transcription factors, such as SRF, TCF, or
SRE-binding protein, which interact with the SRE in many case as
ternary transcriptional complexes (25, 31). A requirement of such
ternary complexes including Pit-1 could explain the relatively low
affinity of Pit-1 to the SRE when present alone. To analyze how Pit-1
interacts with the SRE sequence in the context of native nuclear
proteins, we performed gel shift analysis with GH3 nuclear extracts.
Using either SRE/FAP or SRE probes, we obtain a number of bands and, to
discriminate within this pattern which one could contain Pit-1 protein,
we performed competition experiments with an excess of cold Pit-1 RE
(Fig. 5A
). In both experiments, two bands
can be competed by the Pit-1 RE sequence, suggesting the presence of
Pit-1 protein within this shift. To further verify that Pit-1 present
in nuclear extracts is able to interact with SRE/FAP probe, we
performed supershift experiments with a Pit-1-directed antibody (Ab)
(Fig. 5B
). Only two major bands are seen when Pit-1 RE probe is used
with nuclear extract, and incubation with Pit-1-directed antibodies
allowed visualization of supershifted material (Fig. 5B
, left
panel). A clear supershifted band is also visible when the SRE/FAP
probe is used. This supershifted band is further competed with cold
Pit-1 RE, indicating also the presence of Pit-1-related material. In
control experiments, antibodies unrelated to Pit-1 (e.g.
anti-cAMP response element binding protein antibody) did not
produce any supershift (data not shown). Taken together, all these
results indicate that Pit-1 present in GH3 nuclear extracts is able to
interact with the SRE sequence of the c-fos promoter.

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Figure 5. Gel Shift Analysis of Pit-1 Interactions within GH3
Nuclear Extract
A, GH3 nuclear extract (20 µg) were incubated with either SRE/FAP or
SRE probe in the absence (-) or in the presence of increasing amounts
of Pit-1 RE cross-competitor. Pit-1 RE duplex competed bands are
indicated (*). B, GH3 nuclear extract (20 µg) was incubated with
either the Pit-1 RE (left) or SRE/FAP probe
(right). A supershifted band is observed with both probe
(*) and is specifically competed by Pit-1 RE duplex (5-, 50-, and
500-fold excess were used).
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DISCUSSION
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The observation that Pit-1-deficient mice display somatotroph,
lactotroph, and thyrotroph aplasia suggests that Pit-1 could be
involved in the development of these three pituitary cell lineages (7).
However, only a limited amount of information is available about the
molecular mechanism involving Pit-1 in the establishment of the
pituitary lineages. Two mechanisms have been suggested: 1) Pit-1 could
be involved in the replication of DNA in pituitary cells, as suggested
by the observation that Pit-1 is able to increase replication of
adenovirus in vitro (32); 2) Pit-1 is required for GHRH
receptor expression (33), a stimulatory input for somatolactotroph
proliferation (34, 35), suggesting that the role of Pit-1 could be
indirect. In this paper, we show that Pit-1 could act on proliferation
of somatolactotrophs by using a third mechanism based on its ability to
increase the expression of the c-fos gene, which is involved
in cell cycle initiation. Our results show that overexpression of
Pit-1, in a cell line that does not express Pit-1 (PC12 cells), leads
to c-fos promoter activation. The physiological relevance of
this effect is demonstrated by the fact that reduction of Pit-1
expression in the GH3 somatolactotroph-derived cell line induced a
strong inhibition of c-fos promoter induced by cAMP. These
two experiments show that Pit-1 regulates c-fos promoter and
that Pit-1 may be involved in a tissue-specific regulation of
c-fos expression in the somatolactotroph lineage. The Fos
protein is involved in the cell cycle (36, 37, 38), and the AP1
transcription factor, a complex of Fos and Jun proteins, is also
required for the proliferation of fibroblasts (39). This appears to be
true for GH3 cells, since transfection of double-stranded AP1
oligonucleotides, which are expected to bind and to inactivate
endogenous AP1, strongly reduces the rate of proliferation (C. Gaiddon
and J.-P. Loeffler, unpublished observation). The AP1-binding sites are
present in the promoter regions of various genes that are directly
involved in cell cycle progression (26, 27, 29). Indeed, it has been
demonstrated that Fos regulates cyclin D1 expression (28, 30). Thus, by
increasing c-fos expression, Pit-1 could stimulate the cell
cycle by activating expression of genes from the cyclin family. The
Pit-1 protein is a substrate for different kinases, protein kinase A or
protein kinase C, and was shown to be phosphorylated under various
physiological conditions, such as activation of the cAMP pathway,
treatment with epidermal growth factor, or during the cell cycle
(19, 20, 21). However, these phosphorylation sites do not seem to play a
role in hormonal gene regulation since wild-type and mutated Pit-1
stimulate transcription through the Pit-1-binding site. In contrast,
the present study shows that regulation of c-fos
transcription by Pit-1 requires its phospho-acceptor sites. Although we
cannot formally exclude that site-specific mutation of the Pit-1
phospho-acceptor sites might also affect protein-protein intecation,
these results suggest that Pit-1 phosphorylation events are necessary
for the c-fos induction by Pit-1. To determine how Pit-1
regulates c-fos transcription and to localize the
cis-elements required for this regulation, we performed
promoter deletion analysis and gel shift assays. We showed that Pit-1
is able to enhance the transcriptional activity of the c-fos
SRE and that Pit-1 is able to interact with this sequence. The
interaction of Pit-1 with the SRE may require a ternary complex, as
suggested by the putative presence of Pit-1 in a high molecular weight
complex in GH3 nuclear extract (see Fig. 5B
) and the lower affinity of
purified Pit-1 to the SRE than to the consensus Pit-1 RE. This putative
Pit-1-interacting protein on SRE remains to be identified. One
potential partner could be SRF, since it has been shown that this
protein interacts with homeodomain transcription factors (40). As the
POU domain of Pit-1 contains an homeodomain, it is conceivable that
this domain may mediate the interaction between Pit-1 and SRF. The
effects of Pit-1 on the expression of cell cycle genes could
operate at two levels. First, Pit-1 may stimulate genes that contain
SRE sequences and second, the products of these genes, if coding for
transcription factors such as c-fos, could then stimulate
expression of a second set of genes directly involved in the control of
cell cycle (e.g. cyclin D1). These mechanisms could have
important physiological and physiopathological implications. The direct
interaction of Pit-1 with genes that are involved into cell cycle
initiation may account for the clonal expansion of the somatolactotroph
cell lineage during development. Conversely, it also provides a
molecular basis that links reduced proliferation of these cells and the
inactivation of Pit-1 in cases of murine and human dwarfism. The same
mechanism of IEG transactivation by Pit-1 may further be implicated in
the pathogenesis of human pituitary tumors after somatic mutation of
G
s proteins (41). Indeed, chronic activation of the cAMP-signaling
pathway in these tumors or in cellular models led to a permanent
stimulation of IEGs (42). These effects may not be primarily mediated
by the cAMP response element binding protein family of
transcription factors as they are rapidly shut off by rapid and potent
retrocontrol mechanisms involving phosphatases and various
transcription factors with inhibitory effects (43, 44). Taken together
with the present finding, this opens the interesting possibility that,
in human secretory tumors, Pit-1, as a target of G
s through the
cAMP/protein kinase A signaling pathway, might be the control that
stimulates both hormonal production and cell proliferation.
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MATERIALS AND METHODS
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Cell Culture and Transfections
GH3 and PC12 cells were cultured and grown as described
previously (42). Transfections were carried out by employing a
lipopolyamine-based method (Transfectam, Promega France, Charbonnieres,
France) as described previously (45). Briefly, serum-deprived
cell culture in 3.5-cm wells was transfected overnight, followed by
culture in serum-free DMEM/F12 for 20 h. In some cases, cells were
treated with Fk for the last 8 h. CAT activity was assayed and
quantified as described previously (46). Results were corrected for
transfection efficiency by using pCMV Luc, a noninducible luciferase
reporter gene, and luciferase activity was quantified following
manufacturer recommendations (Promega France).
Source of Recombinant Plasmids
The c-fos promoter reporter gene constructs (a gift
from Dr. Roeder, New York, NY), were described previously. The pRSV
Pit-1 and pRSV Pit-1 3A vectors were kindly provided by Dr. R. Maurer
(Portland, OR); and PRL-CAT was provided by Dr. C. Bancroft (New York,
NY). The pCMV Tip vector is derived from the pRC CMV
(Invitrogen, San Diego, CA) with an insertion of a
HindIII-BstXI Pit-1 fragment from the pRSV Pit-1
vector. The SV40-luc, SRE-SV40-luc, and FAP-SV40-luc were the gift of
Dr. R. Prywes (New York, NY).
mRNA Extraction and RT-PCR Analysis
Total RNA was extracted as previously described by Chomczynski
and Sacchi (47). Two micrograms of total RNA were used for reverse
transcription reactions with 100 U of MMLV-RT (Promega Corp.) in a 30-µl reaction mix containing 20 U of RNASin
(Promega Corp.) and 8 µM random hexamers.
Reaction mix was incubated at 42 C for 50 min and terminated by adding
70 µl H2O and boiling for 5 min. PCR was performed
with 3 µl of the reverse transcription products and 1 U of AmpliTaq
DNA polymerase (Perkin Elmer Corp., Branchburg, CT)
in a 50-µl reaction mix containing 15 pmol of each specific primer.
Standard cycle parameters were used for 1025 cycles followed by a
final incubation at 72 C for 10 min. Ten microliters of each PCR
reaction were electrophoresed on 2% ethidium bromide agarose gel. Gels
were subjected to photography and densitometric analysis. Control
experiments were performed to determine the range of PCR cycles over
which amplification efficiency remains constant and proportional to the
amount of input RNA. Oligonucleotides used for c-fos and
cyclophilin amplifications are, respectively: 5'-TTTCAACGCGGACTGAGG-3'
and 5'-AGGTCATTGGGGATCTTGCA-3'; 5'-GGGGAGAAAGGATTTGGC-TA-3' and
5'-ACATGCTTGCCATCCAGCCA-3'.
Nuclear Extracts and Gel Retardation Assay
GH3 cells were grown in 10-cm tissue culture dishes (Falcon,
PolyPaba, Strasbourg, France) until 7080% confluency. Cells
were serum deprived for 24 h. Nuclear extracts were isolated by
the technique of Dignam et al. (48). Final protein
concentrations were typically 23 µg/µl as determined by Bradford
assay (Bio-Rad Laboratories, Inc., Richmond CA).
Equivalent amounts of protein, 1 µg of poly(dI-dC), and one of the
different 18-bp 32P-labeled oligonucleotides were incubated
in binding buffer (20 mM HEPES, pH 7.9, 6 mM
KCl, 5 mM dithiotreitol, 5 mM spermidine, 2%
Ficoll, and 8% glycerol) to a final volume of 20 µl. The binding
reaction lasted 20 min at room temperature. In competition experiments,
an excess of cold duplex was incubated in reaction mixture with either
Pit-1 purified protein or GH3 nuclear extract for 0.5 h
before probe incubation. In experiments using anti-Pit-1 antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, ref.
SC442 X), the antibody is added in the reaction mixture at a final
concentration of 1 µg/ml for 1 h after complex formation. The
DNA-protein complexes were resolved on nondenaturating 5%
polyacrylamide gels.
Western Blot Analysis
Nuclear extracts (20 µg) were loaded on a 12% SDS-PAGE.
Transfer was performed at 4 C overnight onto nitrocellulose membrane
(0.45 µm, Bio-Rad Laboratories, Inc.). Filters were
blocked in 5% Blotto (5% nonfat dried milk, 150 mM NaCl,
50 mM Tris-HCl, pH 8.0, 0.05% Tween 20) for 5 h
before incubation with 10 µg/ml of Pit-1 polyclonal antibody (Santa
Cruz Biotechnology, Inc., ref. SC442 X) in 3% Blotto overnight. After
three washes in TBS (150 mM NaCl, 50 mM
Tris-HCl, pH 7.4, 0.05% Tween 20) a 1:500 dilution of anti-mouse Ig
horseradish peroxidase-linked whole antibody (twin sheep, NA931,
Amersham, Arlington Heights, IL) was added in 3% Blotto
for 2 h. The blots were developed using the chemiluminescence
system [ECL (Amersham) and Kodak (Eastman Kodak Co., Rochester, NY) BIOMAX MR films]).
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge the generous gifts of c-fos constructs
from Dr. Roeder (New York, NY), the expression vectors from Dr. Maurer
(Portland, OR) and Dr. C. Bancroft (New York, NY). The SV40 chimeric
reporter genes were kindly provided by Dr. R. Prywes (New York,
NY).
 |
FOOTNOTES
|
---|
Address requests for reprints to: J.-P. Loeffler, Neurophysiologie Cellulaire et Integree, Centre National de la Recherche Scientifique, Universite Louis Pasteur, Institut de Physiologie et de Chimie Biologique, 21 rue Renee Descartes, Strasbourg, 67084 Cedex France. E-mail: Loeffler{at}neurochem.u-strasbg.fr
This work was supported by funds from Association pour la Recherche
contre le Cancer (Grants 6989 and 9821).
1 Present address: Department of Biological Sciences, Columbia
University, Fairchild Building, 1212 Amsterdam Avenue, New York,
New York 10027. 
Received for publication January 6, 1998.
Revision received February 1, 1999.
Accepted for publication February 3, 1999.
 |
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