(Received for publication, May 3, 1995; and in revised form, September 11, 1995)
From the
Activin is a polypeptide growth factor which exerts endocrine,
paracrine, and autocrine effects in a variety of tissues. In the
pituitary somatotrope, activin represses proliferation and growth
hormone (GH) biosynthesis and secretion. We previously demonstrated
that decreases in GH biosynthesis in MtTW15 somatotrope cells are due
at least in part to decreased binding of the tissue-specific
transcription factor, Pit-1, to the GH promoter, resulting in decreased
transcription of the GH gene. The objective of the current study was to
determine the extent to which activin-mediated decreases in GH
transcription were the result of decreased Pit-1 activity and/or
decreased Pit-1 protein content in MtTW15 cells. Activin caused rapid
increases in Pit-1 phosphorylation, which were temporally correlated
with decreases in GH DNA binding. Pit-1 phosphorylation preceded marked
decreases in steady-state levels of Pit-1 protein. The rate of Pit-1
synthesis was only moderately decreased by activin, with a time-course
similar to that observed for decreases in GH biosynthesis. However,
Pit-1 stability was markedly decreased after more than 4 h of activin
treatment. These data demonstrate that activin decreases GH expression
in MtTW15 cells through multilevel regulation of Pit-1, which may
represent a more general mechanism whereby activin and other
transforming growth factor family members modulate gene
expression through regulation of transcription factor activity as well
as content.
Activins and inhibins are members of the TGF- (
)superfamily of growth and differentiation factors which
exert endocrine, paracrine, and autocrine effects in a variety of
tissues. Other members of this growing superfamily include
Müllerian inhibiting substance(1) ,
glial-derived neurotrophic factor(2) , several bone
morphogenetic proteins(3) , dorsalin(4) ,
nodal(5) , the Drosophila decapentaplegic (6) and Vgr60A (7) gene products, and Xenopus Vg-1(8) . Activins are dimeric proteins, composed of two
subunits (homo- or heterodimers of
A
A,
A
B, or
B
B) whereas the related inhibins
are heterodimers composed of one
subunit and one of the
subunits. Although first purified from gonadal sources, activins are
also found in brain, uterus, placenta, adrenals, bone marrow, and the
gonadotropes of the anterior pituitary gland(9) . Activins
exert effects on the secretion and production of a variety of endocrine
products, as well as altered growth, differentiation, and function of a
variety of cells. These include ovarian theca and granulosa cells,
testicular Leydig cells, erythroid progenitor cells,
gonadotropin-releasing hormone-producing hypothalamic cells, and
pituitary gonadotropes, somatotropes, and corticotropes(10) ,
as well as oxytocin-producing hypothalamic paraventricular
neurons(11) . In addition, within the last few years, a number
of investigators have demonstrated a major role for activin in the
developing embryo as a dorsalizing mesoderm-inducing signal (12, 13, 14) .
The molecular mechanisms by
which activin exerts these multiple actions in a variety of cell types
are incompletely understood. Activin forms a heteromeric receptor
complex with a high affinity ligand binding component (type II,
75-kDa protein) and a second component (type I,
55-kDa
protein) whose activin binding is dependent upon the presence of type
II receptor and whose role is thought to be that of propagating the
activin signal to downstream targets. Multiple cDNAs of both types of
activin receptors have been cloned (type II (15, 16, 17) and type
I(18, 19, 20, 21) ) and were found
to be members of the same gene superfamily, along with receptors for
related ligands such as TGF-
and Müllerian
inhibiting substance. These receptor molecules are serine/threonine
kinases, each with a single transmembrane domain. The type II receptor
proteins (II and IIB) have been shown to be autophosphorylated on
serine and threonine residues when expressed in mammalian cells, as
well as in in vitro kinase assays (22, 23, 24) similar to the TGF-
system(25) . The type I receptors (ActRI and ActRIB) show
limited autophosphorylation, but are highly phosphorylated on serine
and threonine residues (26) in the presence of type II receptor
kinase activity. Evidence in both the activin (26) and the
TGF-
systems (27, 28) indicates that the kinase
domains and/or activities of both type I and type II receptors are
required for downstream signaling. Thus, the effects of activin (as
well as TGF-
) are thought to be mediated by a signaling pathway
initiated by phosphorylation of both types of receptors.
Activin has
been demonstrated to be a negative regulator of the pituitary
somatotrope, in which activin represses cellular proliferation and
growth hormone (GH) biosynthesis and
secretion(29, 30) . The somatotrope has been the
subject of extensive studies to characterize the regulation of GH gene
transcription (reviewed in (31) ) and has provided a useful
system for analyzing the regulatory mechanisms controlling cell
commitment, proliferation, and differentiation in
vertebrates(32, 33) . Using a transplantable rat
somatotrope tumor model, MtTW15, which is responsive to both growth
hormone releasing factor (34) and activin, we have previously
demonstrated that activin decreases GH biosynthesis at least in part
through decreased transcription of the GH gene in primary cultures of
MtTW15 cells(35) . Electrophoretic mobility shift analyses
indicated that decreased transcription was the result of decreased
interaction of the tissue transcription factor Pit-1, also called
GHF-1, to its proximal and distal cis-acting elements on the
GH promoter. Pit-1 is a member of the POU-domain family of
transcriptional and cell-specific regulators which constitute a
subclass of the homeobox genes(32, 33, 36) .
Expression of Pit-1 protein is restricted to pituitary somatotropes,
lactotropes, and thyrotropes, where it transactivates the GH,
prolactin, and thyroid stimulating hormone (TSH-
) genes,
respectively, in addition to autoregulating its own
synthesis(31) . Pit-1 also plays a critical role in the
specification and proliferation of these cell types during development
( (31) and references therein). Three isoforms of Pit-1 have
been identified which transactivate the GH promoter. Two translation
initiation sites generate 33- and 31-kDa isoforms of
Pit-1(37) , and a third 35-kDa variant is generated by a
26-amino acid insertion into the activation domain as a result of
alternative splicing(38, 39) .
The purpose of the
current study was to determine the causes of activin repression of
Pit-1GH promoter interactions. We report that activin exerts
multilevel regulation of Pit-1 involving early decreases in Pit-1 DNA
binding activity associated with increased Pit-1 phosphorylation, later
decreases in the overall Pit-1 content as a result of decreased
stability, and, to a lesser extent, synthesis of Pit-1.
For metabolic labeling with
[P]orthophosphate, MtTW15 cells were plated in
60-mm dishes at a density of 2
10
cells/dish and
allowed to incubate in 10% FCS-
-Pit Julep for 48-72 h. Cells
were then washed with phosphate-free Dulbecco's modified
Eagle's medium three times and incubated in the same medium for
30 min, followed by the addition of 0.5 mCi/ml
[
P]orthophosphate (ICN) in 2 ml of medium. Cells
were then incubated with labeled phosphate for 4-12 h. Activin
stimulation was performed such that all samples were exposed to labeled
phosphate for the same time prior to harvest for immunoprecipitation as
described below.
Figure 1:
Activin decreased binding of Pit-1 to
the GH promoter in mobility shift assays of MtTW15 extracts. Mobility
shift assays were performed as described under ``Experimental
Procedures.'' A, nuclear extracts were prepared from
MtTW15 cells grown under control conditions or stimulated with 20 ng/ml
activin for the indicated times. Extracts (1 µg) were incubated
with 0.9 ng of labeled wild-type Pit-1 oligonucleotide probe
(-97/-66) in the absence or presence of a 50-fold molar
excess of Pit-1 oligonucleotide competitor (DNA). Other control
extracts were incubated with a mutant Pit-1 oligonucleotide
probe(36) . Specific proteinDNA complexes are indicated
on the left (I, II, and III). Complexes were
shifted to higher molecular weight species when preincubated for 15 min
with a 1:200 dilution of
132-Pit antiserum (Ab). B, increasing amounts of control or nuclear extracts (0.05,
0.15, 0.5, and 1 µg) prepared from MtTW15 cells stimulated with 20
ng/ml activin for the indicated times were incubated with 0.8 ng of
labeled GH promoter fragment (183/+6). Specific protein
DNA
complexes were visualized by gel electrophoresis. The figures shown are
representative of at least 3 separate
experiments.
Because Pit-1 is highly abundant in nuclear extracts of somatotrope
cells, it was possible that a subset of Pit-1 molecules were affected
by the activin treatment, which would not be reflected in measurements
of microgram quantities of nuclear extracts. Therefore, additional
mobility shift analyses were performed to determine the minimum amount
of Pit-1-containing nuclear extract required to form a complex with an
intact GH promoter fragment in the presence or absence of activin. An
intact P-labeled GH promoter fragment (-183/+6)
containing the proximal and distal binding sites for Pit-1 binding (36, 44) was incubated with increasing amounts of
nuclear extract from control or activin-treated MtTW15 cells. As
previously reported, gel shifts with control MtTW15 nuclear extracts
demonstrated the formation of a major protein
DNA complex,
visualized as a doublet (Fig. 1B and (35) ). By
contrast to Fig. 1A, in which microgram quantities of
extract were capable of forming strong complexes after short exposure
to activin, use of smaller amounts of nuclear extract in Fig. 1B facilitated the earlier detection of an
activin-dependent decrease in Pit-1 binding to DNA. Progressive
decreases in Pit-1
DNA binding were first observed within 15 min,
and DNA binding was abolished within 24 h of activin treatment (Fig. 1B). Similar titrations using the (-97 to
-66) proximal Pit-1 oligonucleotide probe were also observed
(data not shown).
Figure 2:
Activin decreased Pit-1 protein content in
MtTW15 cells as detected by immunoblot analysis. Nuclear extracts were
prepared from MtTW15 cells following activin stimulation for the times
indicated, and immunoblot analysis was performed as described under
``Experimental Procedures'' using 150 µg of protein and 1
µg/ml Pit-1 antiserum from Santa Cruz Biotechnology. A
representative blot from 3 experiments is shown. Migration of molecular
mass markers (1000) are indicated on the right, and
Pit-1 protein (33-kDa species) is indicated on the left.
Figure 3:
Activin repression of GH synthesis was not
mediated by decreases in Pit-1 synthesis. MtTW15 cells were
metabolically labeled with [S]methionine,
-cysteine as described under ``Experimental Procedures,'' and
the rates of Pit-1 (A) and GH (B) synthesis following
an activin time course were compared by immunoprecipitation of
equivalent trichloroacetic acid-precipitable counts and SDS-PAGE.
Molecular mass markers (
1000) are indicated on the right; Pit-1 protein (31-kDa and 33-kDa species) and GH
(22-kDa) are indicated on the left. Autoradiogram exposure
times were 16 h for A and 1 h for B. In this
representative gel from at least 3 experiments, scanning densitometry
demonstrated that GH synthesis diminished 39% after 4 h of activin,
compared to a 22% decrease in Pit-1 synthesis. After 48 h of activin
treatment, biosynthesis of GH and Pit-1 decreased by 83% and 46%,
respectively.
The analyses of Pit-1 content and rate of synthesis
indicated that decreased Pit-1 synthesis was not solely sufficient to
account for the low levels of Pit-1 detected in the immunoblot analysis
after 24-48 h of activin stimulation. Therefore, the rate of
Pit-1 degradation was analyzed by pulse-chase labeling of MtTW15 cells
with S-labeled amino acids. Analyses of four experiments
indicated that control MtTW15 cells demonstrated a Pit-1 half-life of
4 h (Fig. 4, upper panel). The half-life for Pit-1
was unchanged in MtTW15 cells treated with activin for up to 4 h.
However, within 12 h of activin exposure, Pit-1 stability decreased,
leading to a much shorter half-life (less than 2 h). The half-lives of
both Pit-1 species were similarly affected by activin treatment. These
data suggest that Pit-1 stability is unaffected by short exposure to
activin (4 h or less), but that within 12 h of treatment, Pit-1 becomes
more sensitive to degradation. This activin-destabilization effect was
specific to Pit-1, since the stability of ActRII (used as a control
protein) in these same samples was not decreased by activin treatment (Fig. 4, lower panel). Taken together, these data
indicate that the rapid activin-mediated decreases in Pit-1 binding to
GH promoter DNA (Fig. 1B) could not be accounted for by
rapid decreases in Pit-1 synthesis or stability.
Figure 4:
Activin increased the degradation of Pit-1
protein in MtTW15 cells as analyzed by S pulse-chase
analysis. MtTW15 cells were stimulated with activin for the indicated
times and incubated with
S-labeled amino acids for 1 h
followed by the indicated chase periods with complete unlabeled medium
as described under ``Experimental Procedures.'' Lysates were
prepared and equivalent protein contents were sequentially
immunoprecipitated with
132Pit antiserum (upper panel)
followed by antibody 199D to ActRII (lower panel) as described
under ``Experimental Procedures'' and analyzed by SDS-PAGE. A
representative gel from 3 experiments is shown. Autoradiogram exposure
times were 5 days for both panels. Estimates of Pit-1 half-life (t
) were determined by scanning densitometry to
be
4 h in unstimulated and in 4-h activin-treated cells. After 12
h of activin treatment, Pit-1 t
was reduced to
less than 2 h, whereas the t
for ActRII was
unchanged.
Figure 5:
Activin increased Pit-1 phosphorylation in
MtTW15 cells. A, MtTW15 cells were metabolically labeled with
[P]orthophosphate and stimulated with 20 ng/ml
activin or a 100 nM concentration of the protein kinase C
activator 12-O-tetradecanoylphorbol-13-acetate. Lysates of
equivalent protein content were immunoprecipitated with 1:1000
132-Pit antiserum in the presence (+) or absence(-) of
blocking 33-kDa recombinant Pit-1 protein followed by Protein
A-Sepharose and analyzed by SDS-PAGE. A representative gel from 5
experiments is shown. Pit-1 phosphoproteins are indicated (31-kDa and
33-kDa species) on the left, and molecular mass markers
(
1000) are indicated on the right.
To determine if the increases in Pit-1 phosphorylation
were responsible for the observed decreases in GH promoter binding
following short-term activin treatment, mobility shift assays were
performed following dephosphorylation of nuclear extracts. Nuclear
extracts from untreated or 15-min activin-stimulated MtTW15 cells were
dephosphorylated with potato acid phosphatase prior to incubation with
the P-labeled proximal Pit-1 oligonucleotide probe. The
results in Fig. 6using smaller amounts of extract (250 ng) are
representative of four separate experiments. Phosphorylated and
dephosphorylated Pit-1 from control extracts exhibited comparable
protein-DNA complex formation. Addition of the potato acid phosphatase
inhibitor 4-nitrophenyl phosphate also had little effect. Nuclear
extracts from 15-min activin-treated cells exhibited decreased complex
formation compared to that observed using control extracts.
Importantly, dephosphorylation of nuclear extracts by potato acid
phosphatase resulted in the restoration of Pit-1 binding to DNA to a
level comparable to control extracts. Potato acid phosphatase activity
was partially blocked by co-incubation with 4-nitrophenyl phosphate,
indicating that restored DNA binding in the potato acid
phosphatase-treated extracts was dependent upon protein
dephosphorylation within the complex. Similar restoration of complex
formation was also observed using 30-min and 1-h activin-stimulated
nuclear extracts (data not shown).
Figure 6: Activin-induced Pit-1 phosphorylation resulted in decreased Pit-1 binding to the GH promoter. Untreated (Control) or 15-min activin-treated (Activin) MtTW15 nuclear extracts (250 ng) were incubated for 20 min in the absence(-) or presence of 0.02 or 0.04 unit of potato acid phosphatase (PAP) in a constant potato acid phosphatase buffer volume of 4 µl per 20-µl reaction as described under ``Experimental Procedures.'' Inhibition of potato acid phosphatase and/or endogenous phosphatases was achieved by co-incubation with 10 mM 4-nitrophenyl phosphate (Inh) in the reactions. Extracts were subsequently incubated for 20 min with 0.5 ng of labeled wild-type Pit-1 oligonucleotide probe (-97/-66) in the absence or presence of a 50-fold molar excess of Pit-1 oligonucleotide competitor (DNA). A representative gel from 4 experiments is shown.
Taken together, these data indicate that short-term stimulation of MtTW15 cells with activin increases Pit-1 phosphorylation, which causes coordinate decreases in Pit-1-containing complex formation with GH promoter DNA. Dephosphorylation of activin-treated extracts restores Pit-1 binding to control levels, consistent with phosphorylation reducing the affinity of Pit-1 for DNA. In addition, long-term activin stimulation causes decreases in Pit-1 stability and synthesis, resulting in decreased Pit-1 content, which contributes to less Pit-1 available for binding and transactivation of the GH promoter.
Our previous observations demonstrated long-term (6-day) effects of activin on decreasing GH mRNA and GH transcription in MtTW15 somatotrope cells(35) . These effects were associated with decreased binding of the tissue-specific transcription factor Pit-1 to the GH promoter. The current studies indicate that decreased Pit-1 binding, and thus decreased GH biosynthesis in MtTW15 cells, is the result of multilevel effects of activin on Pit-1 activity as well as on total Pit-1 content, which may involve alterations in the autoregulatory loop maintaining Pit-1 transactivation. The first measurable effects of activin were temporally correlated increases in Pit-1 phosphorylation (Fig. 5) and decreases in GH promoter binding after 15 min of activin treatment (Fig. 1B and Fig. 6). These effects were followed by decreases in Pit-1 stability and synthesis, and finally decreased Pit-1 content and continued diminished rates of synthesis (Fig. 2, Fig. 3, and Fig. 4).
Activin treatment of MtTW15 cells caused rapid
decreases in Pit-1GH complex formation in the absence of altered
Pit-1 protein content. Since transcription factor phosphorylation is a
well utilized mechanism for regulation of DNA binding and
transactivation (reviewed in (46, 47, 48) ),
these data suggested that altered phosphorylation of Pit-1 might be
involved in decreased GH promoter binding. Indeed, earlier
phosphorylation studies of Pit-1 in vitro and in metabolic
labeling of GC cells demonstrated increased protein kinase A- or
protein kinase C-dependent phosphorylation of Pit-1 within 7 min of
cAMP or 12-O-tetradecanoylphorbol-13-acetate treatment,
respectively(45) . Associated with this increased Pit-1
phosphorylation was decreased binding affinity for the proximal GH
element, slightly lower affinity for the prolactin-1P site, and no
change in affinity for the positive Pit-1 autoregulatory site, PB-1 (45) . Conversely, phosphorylation by protein kinase A (or
protein kinase C) enhanced Pit-1 binding to sites in the TSH-
gene, which appeared to be functionally correlated with protein kinase
A-dependent increases in TSH-
transcription(49) . These
differences have been attributed to variations within the consensus
sequences for Pit-1 binding in the TSH-
, GH, prolactin, and Pit-1
genes(49) . It has also been suggested that additional factors
interacting with Pit-1 are the true protein kinase A and protein kinase
C phosphorylation targets which are involved in regulating GH and
prolactin promoter
activity(50, 51, 52, 53) .
Although the intracellular intermediates of activin signaling have
not been identified, protein kinase A and protein kinase C do not
appear to play major roles in mediating a variety of activin effects.
The current studies demonstrated that activin increased Pit-1
phosphorylation, which was temporally correlated with significant
decreases in Pit-1 binding to the GH promoter (15 min-4 h).
Phosphatase treatment of short-term activin-treated nuclear extracts
restored binding to control levels. This result highlights the
importance of phosphorylation in mediating the early decreases in Pit-1
binding to DNA and suggests that activin-dependent Pit-1
phosphorylation decreases its affinity for binding to the GH promoter.
Activin may also target the phosphorylation of a Pit-1 co-factor, such
as Zn-15(54) , which subsequently decreases the ability of the
Pit-1-cofactor complex to bind to the GH promoter. Although previous
activin-mediated decreases in Pit-1DNA complex formation have
been correlated with repression of GH gene transcription(35) ,
the requirement of Pit-1 phosphorylation for activin-dependent
repression of GH promoter transactivation has yet to be confirmed.
Increased Pit-1 phosphorylation upon activin treatment was
temporally associated with decreases in Pit-1 binding to GH DNA. Since
not all of the nuclear Pit-1 was inhibited from binding to DNA after
short-term activin treatment, this may indicate that phosphorylation of
Pit-1 is incomplete after 15 min, or that sites are also rapidly
dephosphorylated. In addition, increased Pit-1 phosphorylation on some
sites may alter Pit-1 sensitivity to proteases. Several examples of
phosphorylation-dependent degradation have been reported. The stability
of c-Fos is decreased when the protein dimerizes with phosphorylated
c-Jun(55) . Induced phosphorylation of cytoplasmic IB when
complexed with NF
B targets I
B for degradation, resulting in
NF
B activation and translocation to the
nucleus(56, 57) . Phosphorylation of the tumor
suppressor protein, p53, by p34
is also associated with
rapid protein degradation (58) . However, in MtTW15 cells, the
time delay between the increased Pit-1 phosphorylation, observed within
15 min of activin treatment, and the increased degradation of Pit-1,
observed within 9-12 h of activin treatment, suggests that
inducible degradation of Pit-1 may be controlled by different
mechanisms.
Activin decreases not only the stability of Pit-1, but also its rate of synthesis. While this effect on Pit-1 synthesis may reflect an activin action directly on Pit-1 transcription or translation, other mechanisms are also possible. Because Pit-1 positively autoregulates its synthesis, activin-mediated degradation of Pit-1 would disrupt this loop because less Pit-1 is available, further decreasing the level of Pit-1 in the cell. In addition, activin may exert a direct inhibitory effect on regulatory sequences in the Pit-1 gene to down-regulate Pit-1 transcription.
In conclusion, activin
exerts multilevel regulation of the tissue-specific transcription
factor, Pit-1, involving increased phosphorylation and decreased
stability of the protein, resulting in decreased Pit-1 available for
transactivation of the GH gene in the somatotrope. Activin-mediated
phosphorylation of tissue-specific transcription factors may provide a
more general mechanism whereby other tissue-specific genes, such as
-inhibin in the gonad (59) and globin genes during
erythrocyte differentiation(60, 61) , are regulated by
activin through phosphorylation-induced modulation of transcription
factor activity and/or content.