Prolactin Enhances CCAAT Enhancer-Binding Protein-ß (C/EBPß) and Peroxisome Proliferator-Activated Receptor
(PPAR
) Messenger RNA Expression and Stimulates Adipogenic Conversion of NIH-3T3 Cells
Rika Nanbu-Wakao,
Yoshio Fujitani,
Yasuhiko Masuho,
Masa-aki Muramatu and
Hiroshi Wakao
Helix Research Institute (R.N.-W., Y.M., M.-a.M., H.W.) Chiba,
292-0812, Japan
The First Department of Internal Medicine
(Y.F.) Osaka University Medical School Osaka, 565 Japan
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ABSTRACT
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Extracellular stimuli trigger adipocyte
differentiation by inducing the complex cascades of transcription.
Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and
peroxisome proliferator-activated receptor
(PPAR
) play crucial
roles in this process. Although ectopic expression of these factors in
NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in
adipogenic conversion, little is known as to hormonal factors that
regulate adipogenesis in these cells. In this report we demonstrate
that PRL, a lactogenic hormone, enhances C/EBPß and PPAR
mRNA
expression and augments adipogenic conversion of NIH-3T3 cells.
Moreover, we show that ectopic expression of the PRL receptor in
NIH-3T3 cells results in efficient adipocyte conversion when stimulated
with PRL and a PPAR
ligand, as evidenced by expression of the
adipocyte differentiation-specific genes as well as the presence of
fat-laden cells. We further demonstrate that signal transducer and
activator of transcription 5 (Stat5), a PRL signal transducer,
activates aP2 promoter in a PRL-dependent manner. These results suggest
that PRL acts as an adipogenesis-enhancing hormone in NIH-3T3 cells.
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INTRODUCTION
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Adipogenic differentiation is accompanied by a cessation of
mitotic proliferation, morphological changes, and an alteration in gene
expression (1, 2). Preadipocyte cell lines such as 3T3-L1 and 3T3-F422A
have served as in vitro models to elucidate complex cascades
of transcriptional events during the differentiation process.
Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and the
nuclear hormone receptor peroxisome proliferator-activated receptor
(PPAR
) are potential regulators of this process, relaying external
signals to gene expressions that ultimately lead to adipocyte
differentiation.
C/EBP are characterized by a common structure, the presence of a
C-terminal leucine zipper for dimerization and basic residues
responsible for DNA binding. Among the family of C/EBPs, C/EBP
,
-ß, -
, and CHOP (Gadd153) have been shown to be involved in
adipogenesis (3, 4, 5, 6, 7). Chronologically, the expression of C/EBPß and
C/EBP
precedes that of C/EBP
during differentiation of 3T3-L1
cells (8). Evidence that these C/EBPs are crucial regulators of
adipogenesis stems in part from the observation that ectopic expression
of C/EBPß and, to a lesser extent, C/EBP
results in the conversion
of preadipocytes or multipotential mesenchymal stem cells into
adipocytes (6). As well, overexpression of a dominant-negative form of
C/EBPß inhibits 3T3-L1 cell differentiation (6). C/EBP
has also
been shown, from both antisense and overexpression studies, to play a
crucial role in adipogenesis (3, 4, 5). Recent studies using gene-targeted
mice support these findings. For example, mice ablated of C/EBP
suffer significant decreases in both brown adipose tissue (BAT) and in
white adipose tissue (WAT) (9). Also, while depletion of either the
C/EBPß or
gene results in only a mild perturbation of adipogenic
differentiation of primary embryonic fibroblasts and a slight volume
loss in epidydimal WAT, the C/EBPß and -
double knockout mice
display an almost complete abrogation of adipocyte differentiation as
well as severely reduced WAT weight due to a greatly diminished number
of adipocytes (10).
PPAR
is a member of the ligand-stimulated nuclear hormone
receptor superfamily (11, 12). PPAR
plays a central role in
adipocyte differentiation. Enforced expression of PPAR
in
multipotential mesenchymal stem cells results in adipogenic conversion
in the presence of its ligands/agonists, thiazolidinedione, or
prostaglandin (13, 14, 15, 16). In adipocytes, PPAR
is responsible for the
expression of adipose differentiation-related genes such as 422/aP2,
phosphoenol pyruvate carboxykinase, and lipoprotein lipase. Indeed,
promoters of these genes contain PPAR
binding sites (17, 18, 19).
Although these families of transcription factors have been shown to
play important roles in adipogenesis, little is known about which
extracellular stimuli drive the mesenchymal cell to commit toward the
adipogenic lineage. In vitro studies have established that
hormones such as dexamethasone (DEX), methylisobutylxanthine (MIX),
insulin, and those present in FBS trigger the adipogenic conversion of
3T3-L1 cells as well as NIH-3T3 cells ectopically expressing C/EBPs or
PPAR
. MIX and DEX are direct inducers of C/EBPß and
,
respectively (8). This induction, in turn, promotes PPAR
expression
and ultimately stimulates adipogenesis (20, 21). In contrast, simple
treatment of NIH-3T3 cells with the above mentioned adipogenic hormones
does not lead to adipogenic conversion. These observations indicate
that some signal transducers are absent or present at low levels in
NIH-3T3 cells, and the addition of some hormones or factors that
up-regulate the expression of C/EBPs or PPAR
may have a potential to
convert NIH-3T3 cells into adipocytes. Recently, it has been shown that
the PRL receptor mRNA is up-regulated during preadipocyte
differentiation (22). PRL is best known as a lactogenic hormone
responsible for the development of the mammary gland, and it plays a
crucial role in reproduction, although many other functions are also
reported (23). With regards to diseases, PRL-secreting pituitary
adenoma is occasionally associated with obesity (24). In some cases of
prolactinoma and obesity, normalization of serum PRL level results in
reduction of body weight (25). These observations prompted us to
investigate a possible role of PRL and its cognate receptor in
adipogenesis. We herein demonstrate that PRL enhances C/EBPß and
PPAR
mRNA production in conjunction with MIX and DEX in NIH-3T3
cells and provide evidence that ectopic expression of the PRL receptor
results in adipogenic conversion of NIH-3T3 cells in the presence of a
PPAR
stimulator. We also show that PRL contributes to the activity
of aP2 gene promoter via signal transducer and activator of
transcription 5 (Stat5).
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RESULTS
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PRL Augments C/EBPß Transcription in Multipotential Mesenchymal
Stem Cells (NIH-3T3 Cells)
Since C/EBPß and
are the initial transcription factors
required for adipogenesis and their mRNAs are induced by a variety of
stimuli, including MIX, DEX, insulin, lipopolysaccharide,
interleukin (IL)-1, IL-6, and GH (8, 26, 27), we first asked whether
PRL enhances C/EBPß mRNA expression in multipotential mesenchymal
stem cells (NIH-3T3) and in 3T3-L1 preadipocyte cells. Confluent
NIH-3T3 or 3T3-L1 cells were treated with or without increasing
concentrations of PRL or with various effectors, and Northern blot
analysis was performed (Fig. 1
). While
nontreated NIH-3T3 cells expressed a low level of C/EBPß mRNA, PRL
increased its transcript in a dose-dependent manner (Fig. 1A
, lanes
15). At 333 ng/ml of PRL, the induction was saturated (Fig. 1A
, lane
4). This dose of PRL was as efficacious as MIX, DEX, and insulin (Fig. 1A
, lanes 4 and 68). FBS was not as potent as PRL (Fig. 1A
, lane 9).
The effect of PRL was also examined in 3T3-L1 cells. PRL failed to
enhance C/EBPß mRNA (Fig. 1B
, lanes 15), while MIX, DEX, and
insulin increased C/EBPß mRNA expression as previously reported (Fig. 1B
, lanes 68) (8). FBS moderately enhanced its expression but less
efficiently than the other stimulators (Fig. 1B
, lane 9). No
PRL-dependent expression of C/EBP
mRNA was detected in either cell
type (data not shown). These results suggest that PRL has an inductive
effect on C/EBPß transcription, at least in NIH-3T3 cells.

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Figure 1. Induction of C/EBPß mRNA by Different Effectors
in NIH-3T3 Cells (A) and in 3T3-L1 Preadipocytes (B)
Confluent cells were exposed to different effectors in DMEM containing
10% CS (lanes 18) or to DMEM containing 10% FBS (lane 9) for 3
h. No effector (lane 1), different concentrations of PRL (P; lane 2, 37
ng/ml; lane 3, 111 ng/ml; lane 4, 333 ng/ml; lane 5, 1 µg/ml), MIX
(lane 6, M; 0.5 mM), DEX (lane 7, D; 1 µM),
and insulin (lane 8, I; 10 µg/ml). Total RNA was analyzed by Northern
blot hybridization using the DIG-labeled antisense C/EBPß RNA. An
equivalent amount of total RNA (8 µg/lane) was loaded as indicated by
staining of ribosomal RNA.
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PRL Enhances PPAR
mRNA in NIH-3T3 Cells as Well as in
Preadipocyte 3T3-L1 Cells
Since ectopic expression of C/EBPß in multipotential
mesenchymal stem cells results in activation of PPAR
expression
(20), we next asked whether PRL might influence PPAR
expression. As
optimal PPAR
mRNA production requires MIX, DEX, and insulin (21),
PRL was challenged together with these adipogenic hormones. Figure 2
shows Northern blot analysis for
NIH-3T3 (Fig. 2A
) and 3T3-L1 cells (Fig. 2B
). While PPAR
mRNA was
efficiently induced by MIX, DEX, and insulin in 3T3-L1 cells, it was
barely detected in NIH-3T3 cells in the absence of PRL (Fig. 2
, A and
B, lane 1). In both cell lines, however, addition of PRL resulted in a
dose-dependent transcriptional augmentation (Fig. 2
, A and B, lanes
25). PRL at the concentration of 333 ng/ml was as potent as FBS in
both cell lines (Fig. 2
, A and B, lanes 4 and 6). To further determine
which adipogenic effector(s) synergize with PRL for PPAR
mRNA
expression, different combinations were tested in NIH-3T3 (Fig. 2C
) and
in 3T3-L1 cells (Fig. 2D
). MIX, DEX, and insulin efficiently induced
PPAR
mRNA in 3T3-L1 cells but much less efficiently in NIH-3T3 cells
(Fig. 2
, D and C, lane 1). In NIH-3T3 cells PRL alone or in combination
with MIX, insulin, or DEX induced little mRNA (Fig. 2C
, lanes 25).
Combination of MIX and DEX, but not MIX and insulin, nor DEX and
insulin, led to strong PPAR
mRNA expression together with PRL (Fig. 2C
, lanes 68). Insulin plus MIX and DEX resulted in a maximized
induction, and FBS was as efficacious as PRL (Fig. 2C
, lanes 9 and 10).
Similar results were obtained in 3T3-L1 cells (Fig. 2D
). It is
noteworthy that DEX and PRL had an inductive effect, which was slightly
enhanced by insulin (Fig. 2D
, lanes 5 and 8). These data demonstrate
that PRL enhances MIX- and DEX-induced PPAR
expression in both cell
lines.

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Figure 2. PRL Dose-Dependent Enhancement of PPAR mRNA
Expression in NIH-3T3 Cells (A) and in 3T3-L1 Preadipocytes (B)
Confluent cells were exposed to DMEM containing 10% CS, insulin, DEX,
and MIX for 48 h with increasing concentrations of PRL (lane 1,
none; lane 2, 37 ng/ml; lane 3, 111 ng/ml; lane 4, 333 ng/ml; lane 5, 1
µg/ml). As a control, confluent cells were incubated with 10% FBS in
the presence of the same cocktail (lane 6). Total RNA (4 µg/lane) was
analyzed by Northern blot hybridization with the DIG-labeled antisense
PPAR RNA. Panels C and D, Induction of PPAR mRNA by different
adipogenic effectors in NIH-3T3 cells (C) and in 3T3-L1 preadipocytes
(D). Confluent cells were exposed to various combinations of effectors,
MIX (M; 0.5 mM), DEX (D; 1 µM), insulin (I;
10 µg/ml), and PRL (P; 1 µg/ml) in DMEM containing 10% CS (lanes
19) for 48 h. As a control, 10% FBS plus MIX, DEX, and insulin
were included (lane 10). Total RNA was extracted, and 3 µg of each
sample were subjected to Northern blot analysis.
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Ectopic Expression of the PRL Receptor Results in Enhanced
Adipogenic Conversion of NIH-3T3 Cells
The above data suggested that PRL might endow NIH-3T3 cells with
the ability to convert toward adipocytes. However, no terminal
differentiation marker gene expression or morphological changes were
observed even after 12 days of incubation with PRL under the normal
permissive conditions (data not shown). Since NIH-3T3 cells do not
undergo adipogenic conversion as readily as 3T3-L1 preadipocyte cells
in normal permissive medium, we employed strong permissive conditions
using troglitazone, a ligand of PPAR
.
Troglitazone is a member of thiazolidinediones, which has
been shown to elevate the potential of multipotential mesenchymal cells
to differentiate (14, 28). Under these conditions, we set out to study
the function of PRL in adipogenic conversion of NIH-3T3 cells. The PRL
receptor gene was stably transfected into NIH-3T3 cells along with the
neomycin resistance gene. As a control, cells harboring only the
neomycin resistance gene were also selected. G418-resistant colonies
were cultured for 2 weeks and were induced to differentiate in
situ. Before differentiation stimulation, no fat-laden
differentiated cells were observed (data not shown). However, upon
exposure to the strong permissive regimen for 10 days, some terminally
differentiated adipocyte colonies, as evidenced by Oil-Red-O staining,
appeared in the plates transfected with the PRL receptor (Fig. 3B
, PRLR). From three independent
experiments, 11% of the G418-resistant colonies cotransfected with the
PRL receptor gave rise to differentiated adipocytes. In
contrast, 2% of differentiated colonies were present in the control
cells (Fig. 3A
, Neo). The omission of PRL from the medium resulted in a
significant decrease of differentiated colony number in both cases
(data not shown). These data further reinforced our hypothesis that PRL
and the PRL receptor play an important role in adipogenic
differentiation of NIH-3T3 cells.

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Figure 3. Effect of the PRL Receptor Expression on Colonies
of NIH-3T3 Cells Exposed to the Adipogenic Hormones
NIH-3T3 cells were transfected with a neomycin resistance gene
expression vector (pSV2neo) alone (A, Neo) or together with a PRL
receptor expression vector (B, PRLR). G-418-resistant colonies (6070
per dish) were tested for differentiation in situ.
Colonies with tightly packed cells were exposed to the strong
permissive regimen in the presence of 1 µg/ml of PRL for 10 days, and
subsequently fixed and stained with Oil-Red-O. Red colonies correspond
to clones that showed substantive evidence of fat accumulation when
monitored by light microscopy.
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To further confirm these observations, stable NIH-3T3 cells ectopically
expressing the PRL receptor or the neomycin resistance gene alone were
selected by G418, and more than 20,000 individual clones were pooled.
Expression of the exogenous PRL receptor mRNA in transfectant cells was
confirmed by Northern blot hybridization (data not shown). Pooled cells
were used to avoid possible clonal variation in the ability to commit
differentiation (29). First, we examined the effect of the PRL receptor
on C/EBPß and PPAR
mRNA expression. Cells transfected only with
the neomycin resistance gene exhibited similar dose-dependent C/EBPß
mRNA expression as parental cells (Figs. 1A
and Fig. 4A
, lanes 15). In contrast, the PRL
receptor-expressing cells showed an enhanced sensitivity to a given
dose of PRL (Fig. 5A
, lanes 610). Even
at the highest concentration of PRL (1 µg/ml), the C/EBPß mRNA
level in cells expressing the neomycin resistance gene was lower than
that induced with 111 ng/ml of PRL in the PRL receptor-expressing cells
(Fig. 4A
, compare lanes 5 and 8). As for PPAR
mRNA, the same PRL
dose dependency was observed in parental and in the control cells
(Figs. 2A
and Fig. 4B
, lanes 15). Interestingly, ectopic expression
of the PRL receptor made these cells much more sensitive to PRL as in
the case for C/EBPß mRNA (Fig. 4B
, lanes 15 and 610). Addition of
111 ng/ml of PRL to the PRL receptor-expressing cells resulted in the
similar induction of PPAR
mRNA as seen with 1 µg/ml of PRL to the
control cells (Fig. 4B
, compare lanes 5 and 8). We next examined the
differentiation program of these two cells with or without PRL (Fig. 5
). PRL slightly elevated C/EBPß mRNA in the control cells after 3
days (Fig. 5A
). In the PRL receptor-overexpressing cells, further
up-regulation was detected especially at day 3 (compare lanes 4 and 9
in Fig. 5A
and those in Fig. 5B
), and thereafter PRL sustained C/EBPß
mRNA at a slightly higher level (Fig. 5
, A and B, compare lanes 911).
As for C/EBP
mRNA, addition of PRL had no effect in either cell
throughout the entire differentiation process (data not shown).
Expression of PPAR
mRNA peaked on day 2 in the control cells, and
then sharply declined in the absence of PRL (Fig. 5A
, lanes 16).
Ectopic expression of the PRL receptor resulted in enhanced expression,
even in the absence of exogenous PRL (Fig. 5B
, lane 3). Addition of PRL
kept the level of PPAR
mRNA slightly high during the later period of
the differentiation program in both control and PRL
receptor-overexpressing cells (Fig. 5
, A and B, lanes 911). Adipsin
mRNA was detected on day 5 in both control and PRL
receptor-overexpressing cells, and PRL significantly enhanced the mRNA
in the PRL receptor-overexpressing cells (Fig. 5
, A and B, lanes 5 and
10). Quite interestingly, the control cells failed to accumulate
adipsin mRNA by day 8, while the PRL receptor-overexpressing cells kept
adipsin mRNA high until day 8 (Fig. 5
, A and B, lanes 5, 6, 10, and
11). aP2 mRNA was detected as early as day 3 in both control and PRL
receptor-overexpressing cells (lanes 4 in both panels A and B).
Addition of PRL strongly augmented the expression of aP2 mRNA,
particularly in cells overexpressing the PRL receptor (lanes 911 in
both panels A and B). Glycerol-3-phosphate dehydrogenase (GPD)
mRNA was detected on day 8 in the control cells; however, the effect of
PRL on the mRNA was not clear (panel A, lanes 6 and 11). On the other
hand, PRL increased GPD mRNA in cells overexpressing the PRL receptor
(Fig. 5B
, lanes 5, 6, 10, and 11). We have repeated these experiments
four times and obtained essentially identical results.

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Figure 4. Effect of Ectopic Expression of the PRL Receptor on
C/EBPß and PPAR mRNA in NIH-3T3 Cells
A, Enhanced induction of C/EBPß mRNA by ectopic expression of the PRL
receptor. NIH-3T3 cells were transfected with pSV2neo alone (Neo) or
PRL receptor expression vector together with pSV2neo (PRLR). Resulting
G-418-resistant stable clones were pooled and grown to confluence, and
then exposed to increasing concentrations of PRL (P) (lanes 1 and 6, no
ligand; lanes 2 and 7, 37 ng/ml; lanes 3 and 8, 111 ng/ml; lanes 4 and
9, 333 ng/ml; lanes 5 and 10, 1 µg/ml) in DMEM containing 10% CS for
3 h. Total RNA was extracted, and 3 µg of each sample were
subjected to Northern blot analysis. B, Enhancement of PPAR mRNA by
ectopic expression of the PRL receptor. Confluent cells prepared as
described above were exposed to different concentrations of PRL (P) in
DMEM containing 10% CS, insulin, DEX, and MIX for 48 h. The
concentrations of PRL were the same as in panel A. Total RNA (2
µg/lane) was analyzed by Northern blot hybridization.
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Figure 5. Effect of PRL and the PRL Receptor on Adipogenic
Gene Expression in NIH-3T3 Cells
A, Expression of adipogenic genes in cells expressing only the neomycin
resistance gene (Neo). B, Expression of adipogenic genes in cells
ectopically expressing the PRL receptor gene (PRLR). G-418-resistant
stable clones were pooled and grown to confluence, and then exposed to
DMEM containing 10% FBS together with insulin, DEX, and MIX for
48 h in the absence (FBS+I+T) or presence of 1 µg/ml of PRL
(FBS+I+T+PRL). Cells were then maintained in DMEM containing 10% FBS,
2.5 µg/ml of insulin, and 5 µM of troglitazone
in the absence (FBS+I+T) or presence of 1 µg/ml of PRL (FBS+I+T+PRL)
and replenished with this medium every other day. Total RNA was
isolated at the indicated time point, and 3 µg of each sample were
analyzed by Northern blot hybridization using the indicated DIG-labeled
antisense riboprobes.
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Both cells were then stained with Oil-Red-O after 10 days of the
adipocyte-inducing regimen. While virtually no differentiation was
observed in the absence of PRL (Fig. 6A
, Neo, FBS+I+T), 4% of control cells exhibited adipogenic conversion in
the presence of PRL as judged by Oil-Red-O-positive cell number (Fig. 6A
, Neo, FBS+I+T+PRL). In contrast, 18% of the PRL
receptor-transfected cells underwent adipogenic conversion in the
presence of PRL, as assessed by the formation of fat droplets in cells
(Fig. 6B
, PRLR, FBS+I+T+PRL). On the other hand, omission of PRL
resulted in a significant decrease of differentiated cell population to
5% (Fig. 6B
, PRLR, FBS+I+T). Four independent experiments were
performed and 1323% of G-418-resistant cells overexpressing the PRL
receptor converted into lipid-laden adipocytes in the presence of PRL.
In light of these results, we concluded that PRL and its cognate
receptor up-regulate the adipogenic conversion of NIH-3T3
multipotential mesenchymal stem cells.

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Figure 6. Effect of PRL and the PRL Receptor on Adipogenic
Differentiation of NIH-3T3 Cells
A pool of NIH-3T3 cells expressing only the neomycin resistance gene
(A, Neo) or the PRL receptor gene (B, PRLR) were grown to confluence
and exposed to the strong permissive conditions as described in Fig. 5
in the absence (FBS+I+T) or presence of 1 µg/ml of PRL (FBS+I+T+PRL)
for 10 days. Cells were fixed and stained with Oil-Red-O. Original
magnification, x10.
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Stat5 Activates aP2 Promoter
Finally, we examined a possible molecular mechanism by which PRL
enhances the adipogenic conversion, particularly the induction of the
adipocyte-specific genes. To assess the role of PRL in adipogenesis, we
used the aP2 promoter as a model system. Since PRL activates Stat5
(30), we examined the effects of Stat5 on the aP2 promoter. To this
end, the aP2 promoter construct was transfected into NIH-3T3 cells
without or with murine Stat5A, PPAR
2, Stat5A plus PPAR
2, or an
ovine dominant negative form of Stat5(Y694F) (31). After transfection
cells were treated with different combinations of PRL and
troglitazone (a PPAR
ligand). When the aP2 promoter
alone was transfected, any combination of PRL/troglitazone
failed to enhance the promoter activity (Fig. 7
, lanes 14). In contrast, transfection
with Stat5A cDNA resulted in a PRL-dependent 2-fold increase in
promoter activity (Fig. 7
, lanes 7 and 8). Troglitazone
did not affect the promoter activity (Fig. 7
, lane 6). When PPAR
2
cDNA was cotransfected, troglitazone-dependent enhancement
of the promoter activity was observed. Challenge with
troglitazone resulted in 60% augmentation, while
combination with PRL gave 100% increase in promoter activity (Fig. 7
, lanes 9, 10, and 12). PRL alone had no effect in this case (Fig. 7
, lane 11). Cotransfection with both Stat5A and PPAR
2 cDNA showed
additive effects. Troglitazone or PRL challenge doubled
the promoter activity, whereas the combination of both ligands resulted
in a 2.5-fold increase (Fig. 7
, lanes 1316). These data strongly
suggested that Stat5 regulates aP2 promoter activity. To further
strengthen this hypothesis, ovine dominant negative Stat5 (Y694) was
transfected into cells together with aP2 promoter. When challenged with
PRL, no ligand-dependent promoter activity was observed (Fig. 7
, lanes
17, 18, and 19). Addition of troglitazone had no effect on
promoter activity (Fig. 7
, lanes 18 and 20). Use of Stat5B and the
corresponding dominant negative Stat5B (Y699F) gave essentially
identical results (data not shown). These data indicate that the aP2
gene is regulated by PRL, at least partially, via Stat5.

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Figure 7. PRL Contributes to the aP2 Promoter Activity via
Stat5
A luciferase reporter gene linked to the aP2 promoter pGL2-aP2 was
transfected into parental NIH-3T3 cells along with or without the
expression vectors (cDNA). After treatment with the indicated ligand(s)
for 6 h, cells were harvested and both firefly and renilla
luciferase activities were measured. Relative luciferase activity is
shown. SD is indicated by the error bar.
Lanes 14, No expression vector; lanes 58, murine Stat5A expression
vector; lanes 912, murine PPAR 2 expression vector; lanes 1316,
murine Stat5A and PPAR 2 expression vectors; lanes 1720, dominant
negative ovine Stat5(Y694F) expression vector. Lanes 1, 5, 9, 13, and
17, no ligand; lanes, 2, 6, 10, 14, and 18, troglitazone
(5 µM); lanes 3, 7, 11, 15, and 19, PRL (1 µg/ml);
lanes 4, 8, 12, 16, and 20, troglitazone+PRL.
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DISCUSSION
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In this report we provide evidence that 1) PRL augments the
expression of C/EBPß and PPAR
mRNA in conjunction with MIX and DEX
in NIH-3T3 cells. 2) PRL enhances the adipogenic conversion of NIH-3T3
cells under strong permissive conditions (i.e. in the
presence of a PPAR
ligand/agonist). 3) ectopic expression of the PRL
receptor efficiently converts NIH-3T3 cells into fat-laden adipocytes.
4) PRL regulates aP2 promoter via Stat5.
3T3-L1 cells are of preadipocyte lineage and differentiate into
adipocytes when stimulated appropriately (1). Growth factors such as
insulin, insulin-like growth factor-1, epidermal growth factor, and
platelet-derived growth factor have been shown to promote
adipogenesis in 3T3-L1 cells, and, in fact, FBS is a potent stimulus
(32, 33, 34). On the other hand, NIH-3T3 cells are considered to be a
multipotential mesenchymal cell line that does not readily
differentiate into adipocytes as 3T3-L1 cells do. Nevertheless, NIH-3T3
cells do convert into adipocytes when transcription factors such as
C/EBPs, PPAR
, and ADD-1 are overexpressed (4, 6, 13, 35). These data
imply that NIH-3T3 cells possess a potential to undergo adipogenesis.
To our knowledge, there has been no report of any growth factor or
cytokine that enhances adipogenesis in NIH-3T3 cells. Our present data
clearly show that PRL and its cognate receptor are required but not
sufficient for the activation of adipogenic program in NIH-3T3 cells.
While PRL increased C/EBPß mRNA in a concentration-dependent fashion
in NIH-3T3 cells, no such effect was observed in 3T3-L1 preadipocytes.
At present, the reason for this difference is unclear. In both NIH-3T3
and 3T3-L1 cells, PRL enhanced PPAR
mRNA expression in conjunction
with other adipogenic hormones such as MIX and DEX (Fig. 2
, AD). As
MIX and DEX induce C/EBPß and -
, respectively (8), it is
conceivable that there is a cooperation between PRL-emanated signals
and those of C/EBPß and/or -
for the maximum production of PPAR
mRNA. Further study is required to fully understand how PRL
receptor-triggered signals act together with these transcription
factors. The fact that FBS was as potent as PRL in inducing C/EBPß
and PPAR
mRNA suggests that FBS contains PRL, or that some factors
present in FBS enhance PPAR
mRNA (Figs. 1A
and 2C
and 2D). The
report that FBS is rich in PRL rather supports the former possibility
(36). The presence of PRL in FBS may explain the relatively high level
of PPAR
mRNA expression in the PRL receptor-overexpressing cells in
the absence of exogenous PRL (Fig. 5B
, lane 3). This, in turn, may
contribute the slight increase in the number of differentiated cells in
cells overexpressing PRL receptors (Fig. 6B
, PRLR, FBS+I+T).
PRL has been shown to induce calcium influx and trigger
activation of signaling molecules such as Ras, PI3 kinase, and the
STATs (23, 30, 37, 38). It remains to be determined which of these
signaling pathways are responsible for C/EBPß and PPAR
expression.
As for the transcriptional regulation of PPAR
mRNA, it has been
shown that granulocyte-colony stimulating factor (G-CSF),
12-O-tetradecanoylphorbol-13-acetate, 1,25-dihydroxyvitamin
D3, and oxidized low density lipoprotein promote
its transcription in macrophages (39, 40). Since both G-CSF and PRL
activate JAK-STAT pathway, it is tempting to speculate that the maximum
induction of PPAR
mRNA may be dependent on this pathway.
PRL has a potential in enhancing PPAR
transcript in parental NIH-3T3
cells as well as the PRL receptor-transfected cells (Fig. 5
). This
enhancement may, in part, explain why NIH-3T3 cells receiving PRL and
troglitazone exhibit morphological changes and express
adipocyte-specific marker genes such as adipsin, aP2, and GPD.
Intriguingly, PPAR
mRNA level peaked at day 2 and gradually declined
during the adipogenic conversion (Fig. 5
). This is in contrast to the
observation during the differentiation process of 3T3-L1 cells or
NIH-3T3 cells ectopically expressing C/EBPß (17, 20). In these cells
a high level of PPAR
mRNA is sustained at later steps in
differentiation. Our data suggest that transient PPAR
mRNA
production is sufficient for the induction of some terminal marker
genes, i.e. adipsin, aP2, and GPD and for the accumulation
of fat droplets (Figs. 5B
and 6
). Why do cells treated with PRL promote
adipogenic conversion to a certain extent even though the increase of
PPAR
mRNA is rather transient? A possible explanation is that
PRL-elicited signals and those emanated from the activated PPAR
might coordinate to stimulate adipogenic program. In agreement with
this hypothesis, a significant drop of adipsin, aP2, and GPD mRNAs as
well as of Oil-Red-O positive cell number was observed in the absence
of PRL, while the PPAR
mRNA expression profile remained nearly
identical in the PRL receptor-expressing cells (Figs. 5B
and 6
). Our
data from aP2 promoter analysis further indicate that aP2 gene is
controlled, at least in part, by Stat5, which is activated by PRL (Fig. 7
). These results also suggest that the effect of PPAR
and Stat5 on
the aP2 promoter is ligand dependent and additive. However, it has yet
to be elucidated how Stat5 regulates the aP2 promoter activity.
Recently, a cross-talk between PPAR
, another member of PPAR family,
and STAT5B in GH signaling has been shown in COS cells (41). It will be
interesting to examine whether there are other adipocyte-specific genes
regulated by both PPAR
and STAT5 as shown here for the aP2 gene.
It is noteworthy that GH shows opposite effects on the expression of
adipocyte-specific genes such as PPAR
, aP2, and fatty acid synthase
and on adipogenesis in primary preadipocytes (42). Whether PRL exhibits
stimulatory or inhibitory effects on primary preadipocytes remains to
be addressed. GH deficiency is often associated with obesity (43),
while some prolactinoma patients are obese (24). In both cases,
treatment with either GH or with reagents lowing serum PRL results in
improvement of obesity (25, 43). These data imply that the action of GH
and PRL is opposite in vivo. Given the fact that GH and PRL
share many signaling molecules such as Jak2 and Stat5A and B, it is
necessary to delineate molecular mechanisms underling these opposing
effects.
PRL gene knockout mice grow normally but fail to develop mammary glands
(44). PRL receptor gene ablation leads to a similar but somewhat
different phenotype, resulting in reproductive defects (45). In both
cases, gene-targeted mice grow as wild type. While mice lacking STAT5A
grow normally, STAT5B-deficient male mice manifest a significant
decrease in weight (46, 47). The STAT5A/STAT5B double-knockout
mice exhibit more severe growth impairment, and the size of the fat
pads is decreased to one-fifth of the wild type (48). These data
suggest that STAT5A and B, both of which are activated by PRL, play a
key role in the development of the fat pad. The results presented
herein appear to be in line with these observations. The reason why
mice lacking PRL or the PRL receptor do not manifest weight loss, as
observed in STAT5B or STAT5A/STAT5B knockout mice, can be explained as
follows. In the absence of PRL or its cognate receptors, other
factor(s) or receptor(s) that activate STAT5A and/or B compensate for
their functions. The redundancy in cytokine signaling may mask all of
the PRL/PRL receptor functions from gene targeting
experiments.
In summary, we have shown here that PRL enhances adipocyte
differentiation of NIH-3T3 fibroblasts. Our results may have important
implications for defining hormonal stimulation along the adipocyte
differentiation pathway. Understanding the interactions between
PRL-triggered signals and those emanated from other adipogenic inducers
should provide valuable insight into the mechanisms that control
adipogenic differentiation.
 |
MATERIALS AND METHODS
|
---|
Ovine PRL, bovine insulin, and DEX were purchased from
Sigma (St. Louis, MO), and methylisobutylxanthine was
purchased from Wako Chemical Co (Osaka, Japan).
Troglitazone was provided from Sankyo Co., Ltd. (Tokyo, Japan).
Plasmids
The cDNAs for C/EBP
, C/EBPß, C/EBP
, PPAR
, aP2, and
GPD used in this study were isolated from a 3T3-L1 preadipocyte library
constructed 9 days after induction of differentiation, using
SuperScript
system (Life Technologies, Inc.,
Gaithersburg, MD). The sequences of the cDNAs were verified by DNA
sequencing (ABI 377 DNA sequencer). aP2 promoter comprising a -1 to
-5.4 kbp fragment was inserted into the SmaI site of
pGL2-basic vector (Promega Corp., Madison, WI).
Cell Culture and Induction of Differentiation
3T3-L1 preadipocytes (NIHS cell bank, Tokyo, Japan, catalogue
number JCRB9014) and multipotential mesenchymal stem cells (NIH-3T3;
Riken cell bank, Tsukuba, Japan; catalogue no. RCB0150) were
maintained in growth medium consisting of DMEM (Nisseiken, Kyoto,
Japan) containing 10% normal calf serum (CS; Life Technologies, Inc.). To establish stable cell lines, NIH-3T3
cells were transfected with the rat PRL receptor (PRLR) in pME18S and
pSV2neo with lipofectAMINE-PLUS reagent following the protocol provided
by the supplier (Life Technologies, Inc.). Briefly, a
mixture of 8 µg of PRLR plasmids, 0.4 µg of pSV2neo plasmids, 20
µl of PLUS reagent, and 30 µl of lipofectAMINE in 1.5 ml of
OPTI-MEM I (Life Technologies, Inc.) was incubated at room
temperature for 30 min and then diluted to 8 ml with OPTI-MEM I. Cells
plated at a density of 8 x105 cells per 10-cm
dish the day before transfection were washed with OPTI-MEM I, and the
diluted DNA-lipid mixture was added. After 3 h of incubation at 37
C, cells were refed with DMEM supplemented with 10% normal calf serum
and were cultured for an additional 24 h before selection with 0.4
mg/ml of G418. The transfectants were selected for 14 days and
subjected to in situ colony differentiation assay. In some
experiments, more than 20,000 G418-resistant clones were pooled and
assayed for their ability to differentiate. To convert NIH-3T3 cells
and the transfectants into adipocytes, cells were grown to confluence
(considered as day 0). At this stage, cells were exposed to fresh DMEM
containing 10% FBS (Life Technologies, Inc.), 1
µM DEX, 0.5 mM MIX, and
10 µg/ml of insulin for 48 h. After this treatment, the medium
was replaced with DMEM containing 10% FBS, and 2.5 µg/ml of insulin,
and cells were refed every other day (normal permissive conditions).
For strong permissive conditions, 5 µM
of troglitazone was included. To distinguish the effect of
PRL, 10% CS instead of 10% FBS was used in some experiments, as
indicated in the text and figure legends. Adipocyte conversion was
assessed by the presence of accumulated fat droplets in the cytoplasts.
Cells were fixed with 2% formaldehyde, 0.2% glutaraldehyde in PBS and
stained with Oil-Red-O (49).
Northern Blot Analysis
Total RNA was isolated according to the method of Chomczynski
and Sacchi (50). The indicated amount of the total RNA was fractionated
using 1% agarose/2.2 M formaldehyde gel electrophoresis
and was transferred to a nylon membrane (51). rRNA was stained on the
filters with methylene blue to assess RNA loading and transfer
efficiency (52). The DIG-labeled RNA probes were transcribed from
EcoRI-linearized cDNA plasmid in pZL1 according to
the Roche Molecular Biochemicals protocol. Hybridization
was performed with DIG-labeled antisense RNA probes according to the
protocol provided by the supplier (Roche Molecular Biochemicals, Indianapolis, IN).
Transcriptional Activation Assay
The expression vectors for murine Stat5A and ovine Stat5(Y694F)
were constructed by ligating the EcoRI-NotI
fragment containing Stat5A or Stat5(Y694F) into the
EcoRI-NotI site of pME18S. The
SalI-NotI fragment comprising PPAR
2 was
inserted into the XhoI-NotI site of pME18S. aP2
promoter-luciferase vector (pGL2-aP2) was transiently transfected into
NIH-3T3 cells without or with expression vectors. NIH-3T3 cells were
maintained in DMEM containing 10% CS and transfected at 70%
confluency with LipofectAMINE-PLUS reagent (Life Technologies, Inc.). A renilla luciferase expression vector (pRL-CMV) was
cotransfected as a control for the transfection efficiency
(Promega Corp.). After DNA removal by washing, cells were
serum starved for 16 h in OPTI-MEM I, and then left untreated or
challenged with PRL, troglitazone, or PRL plus
troglitazone for 6 h. After cell lysis, both firefly
and renilla luciferase activities were measured. Transfections were
performed in duplicate and repeated two to four times.
 |
ACKNOWLEDGMENTS
|
---|
We thank Ms. C. Oda and Y. Kojima for technical assistance and
Prof. G. Krystal (The Terry Fox Laboratory, Vancouver, Canada) for
critical reading of this manuscript.
Helix Research Institute is supported by the Ministry of International
Trade and Industry (MITI), Chugai Pharmaceutical Co., Fujisawa Pharmaceutical Co., Hitachi Co., Mitsubishi
Chemical Co., Nippon Godou Finance Co., Kyowa Hakko Co., Sumitomo
Chemical Co., Taisho Pharmaceutical Co., Yamanouchi Pharmaceutical Co., and Yoshitomi Pharmaceutical Co.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Hiroshi Wakao, Helix Research Institute, 15323 Yana Kisarazu-shi Chiba, 292-0812, Japan.
Received for publication July 21, 1999.
Revision received September 30, 1999.
Accepted for publication November 11, 1999.
 |
REFERENCES
|
---|
-
MacDougald OA, Lane MD 1995 Transcriptional regulation of
gene expression during adipocyte differentiation. Annu Rev Biochem 64:345373[CrossRef][Medline]
-
Fajas L, Fruchart JC, Auwerx J 1998 Transcriptional control
of adipogenesis. Curr Opin Cell Biol 10:165173[CrossRef][Medline]
-
Lin FT, Lane MD 1992 Antisense CCAAT/enhancer-binding protein
RNA suppresses coordinate gene expression and triglyceride accumulation
during differentiation of 3T3L1 preadipocytes. Genes Dev 6:533544[Abstract]
-
Freytag SO, Paielli DL, Gilbert JD 1994 Ectopic expression of
the CCAAT/enhancer-binding protein alpha promotes the adipogenic
program in a variety of mouse fibroblastic cells. Genes Dev 8:16541663[Abstract]
-
Lin FT, Lane MD 1994 CCAAT/enhancer binding protein
is
sufficient to initiate the 3T3L1 adipocyte differentiation program.
Proc Natl Acad Sci USA 91:87578761[Abstract]
-
Yeh WC, Cao Z, Classon M, McKnight SL 1995 Cascade regulation
of terminal adipocyte differentiation by three members of the C/EBP
family of leucine zipper proteins. Genes Dev 9:168181[Abstract]
-
Batchvarova N, Wang XZ, Ron D 1995 Inhibition of adipogenesis
by the stress-induced protein CHOP (Gadd153). EMBO J 14:46544661[Abstract]
-
Cao, Z, Umek RM, McKnight SL 1991 Regulated expression of
three C/EBP isoforms during adipose conversion of 3T3L1 cells. Genes
Dev 5:15381552[Abstract]
-
Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde
MD, Taylor LR, Wilson DR, Darlington GJ 1995 Impaired energy
homeostasis in C/EBP alpha knockout mice. Science 269:11081112[Medline]
-
Tanaka, T, Yoshida N, Kishimoto T, Akira S 1997 Defective
adipocyte differentiation in mice lacking the C/EBPbeta and/or
C/EBPdelta gene. EMBO J 16:74327443[Abstract/Free Full Text]
-
Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U,
Mangelsdorf DJ, Umesono K, Evans RM 1994 Differential expression and
activation of a family of murine peroxisome proliferator-activated
receptors. Proc Natl Acad Sci USA 91:73557359[Abstract]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans
RM 1995 The nuclear receptor superfamily: the second
decade. Cell 83:835839[Medline]
-
Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of
adipogenesis in fibroblasts by PPAR
2, a lipid-activated
transcription factor. Cell 79:11471156[Medline]
-
Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans
RM 1995 15-Deoxy-
12,14-prostaglandin J2 is a ligand for the
adipocyte determination factor PPAR
. Cell 83:803812[Medline]
-
Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC,
Lehmann JM 1995 A prostaglandin J2 metabolite binds peroxisome
proliferator-activated receptor
and promotes adipocyte
differentiation. Cell 83:813819[Medline]
-
Hu E, Tontonoz P, Spiegelman BM 1995 Transdifferentiation of
myoblasts by the adipogenic transcription PPAR
and C/EBP
. Proc
Natl Acad Sci USA. 92:98569860
-
Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR
2: tissue-specific regulator of an adipocyte enhancer. Genes
Dev 8:12241234[Abstract]
-
Tontonoz P, Hu E, Devine J, Beale EG, Spiegelman BM 1995 PPAR
2 regulates adipose expression of the phosphoenolpyruvate
carboxykinase gene. Mol Cell Biol 15:351357[Abstract]
-
Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman RA,
Briggs M, Deeb S, Staels B, Auwerx J 1996 PPAR
and PPAR
activators direct a distinct tissue-specific transcriptional response
via a PPRE in the lipoprotein lipase gene. EMBO J 15:53365348[Abstract]
-
Wu Z, Xie Y, Bucher NL, Farmer SR 1995 Conditional ectopic
expression of C/EBPß in NIH-3T3 cells induces PPAR
and stimulates
adipogenesis. Genes Dev 9:23502363[Abstract]
-
Wu Z, Bucher NL, Farmer SR 1996 Induction of peroxisome
proliferator-activated receptor
during the conversion of 3T3
fibroblasts into adipocytes is mediated by C/EBPß, C/EBP
, and
glucocorticoids. Mol Cell Biol 16:41284136[Abstract]
-
McAveney KM, Gimble JM, Yu-Lee L 1996 Prolactin receptor
expression during adipocyte differentiation of bone marrow stroma.
Endocrinology 137:57235726[Abstract]
-
Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly PA 1998 Prolactin (PRL) and its receptor: actions, signal transduction pathways
and phenotypes observed in PRL receptor knockout mice. Endocr Rev 19:225268[Abstract/Free Full Text]
-
Creemers LB, Zelissen PM, van t Verlaat JW, Koppeschaar HP 1991 Prolactinoma and body weight: a retrospective study. Acta
Endocrinol (Copenh) 125:392396[Medline]
-
Greenman Y, Tordjman K, Stern N 1998 Increased body weight
associated with prolactin secreting pituitary adenomas: weight loss
with normalization of prolactin levels. Clin Endocrinol (Oxf) 48:547553[CrossRef][Medline]
-
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y,
Nakajima T, Hirano T, Kishimoto T 1990 A nuclear factor for IL-6
expression (NF-IL6) is a member of a C/EBP family. EMBO J 9:18971906[Abstract]
-
Clarkson RW, Chen CM, Harrison S, Wells C, Muscat GE, Waters
MJ 1995 Early responses of trans-activating factors to growth hormone
in preadipocytes: differential regulation of CCAAT enhancer-binding
protein-ß (C/EBPß) and C/EBP
. Mol Endocrinol 9:108120[Abstract]
-
Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO,
Willson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high
affinity ligand for peroxisome proliferator-activated receptor gamma
(PPAR
). J Biol Chem 270:1295312956[Abstract/Free Full Text]
-
Green H, Kehinde O 1976 Spontaneous heritable changes leading
to increased adipose conversion in 3T3 cells. Cell 7:105113[Medline]
-
Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF)
is a novel member of the cytokine regulated transcription factor gene
family and confers the prolactin response. EMBO J 13:21822191[Abstract]
-
Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces
phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA
binding and induction of transcription. EMBO J 13:43614369[Abstract]
-
Smith PJ, Wise LS, Berkowitz R, Wan C, Rubin CS 1988 Insulin-like growth factor-I is an essential regulator of the
differentiation of 3T3L1 adipocytes. J Biol Chem 263:94029408[Abstract/Free Full Text]
-
Adachi H, Kurachi H, Homma H, Adachi K, Imai T, Morishige K,
Matsuzawa Y, Miyake A 1994 Epidermal growth factor promotes
adipogenesis of 3T3L1 cell in vitro. Endocrinology 135:18241830[Abstract]
-
Bachmeier M, Loffler G 1995 Influence of growth factors on
growth and differentiation of 3T3L1preadipocytes in serum-free
conditions. Eur J Cell Biol 68:323329[Medline]
-
Kim JB, Spiegelman BM 1996 ADD1/SREBP1 promotes adipocyte
differentiation and gene expression linked to fatty acid metabolism.
Genes Dev 10:10961107[Abstract]
-
Biswas R, Vonderhaar BK 1987 Role of serum in the
prolactin responsiveness of MCF-7 human breast cancer cells in
long-term tissue culture. Cancer Res 47:35093514[Abstract]
-
Tourkine N, Schindler C, Larose M, Houdebine LM 1995 Activation of STAT factors by prolactin, interferon-
, growth
hormones, and a tyrosine phosphatase inhibitor in rabbit primary
mammary epithelial cells. J Biol Chem 270:2095220961[Abstract/Free Full Text]
-
DaSilva L, Rui H, Erwin RA, Howard OM, Kirken RA, Malabarba
MG, Hackett RH, Larner AC, Farrar WL 1996 Prolactin recruits STAT1,
STAT3 and STAT5 independent of conserved receptor tyrosines TYR402,
TYR479, TYR515 and TYR580. Mol Cell Endocrinol 117:131140[CrossRef][Medline]
-
Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK 1998 The
peroxisome proliferator-activated receptor-
is a negative regulator
of macrophage activation. Nature 391:7982[CrossRef][Medline]
-
Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM 1998 PPAR
promotes monocyte/macrophage differentiation and uptake of
oxidized LDL. Cell 93:241252[Medline]
-
Zhou YC, Waxman DJ 1999 Cross-talk between janus kinase-signal
transducer and activator of transcription (JAK-STAT) and peroxisome
proliferator-activated receptor-
(PPAR
) signaling pathways.
Growth hormone inhibition of PPAR
transcriptional activity mediated
by stat5b. J Biol Chem 274:26722681[Abstract/Free Full Text]
-
Hansen LH, Madsen B, Teisner B, Nielsen JH, Billestrup N 1998 Characterization of the inhibitory effect of growth hormone on primary
preadipocyte differentiation. Mol Endocrinol 12:11401149[Abstract/Free Full Text]
-
Wabitsch M, Hauner H, Heinze E, Teller WM 1995 The role of
growth hormone/insulin-like growth factors in adipocyte
differentiation. Metabolism 44:4549[Medline]
-
Horseman ND, Zhao W, Montecino-Rodriguez E, Tanaka M,
Nakashima K, Engle SJ, Smith F, Markoff E, Dorshkind K 1997 Defective
mammopoiesis, but normal hematopoiesis, in mice with a targeted
disruption of the prolactin gene. EMBO J 16:69266935[Abstract/Free Full Text]
-
Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H,
Edery M, Brousse N, Babinet C, Binart N, Kelly PA 1997 Null mutation of
the prolactin receptor gene produces multiple reproductive defects in
the mouse. Genes Dev 11:167178[Abstract]
-
Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A,
Hennighausen L 1997 Stat5a is mandatory for adult mammary gland
development and lactogenesis. Genes Dev 11:179186[Abstract]
-
Udy GB, Towers RP, Snell RG, Wilkins RJ, Park SH, Ram PA,
Waxman DJ, Davey HW 1997 Requirement of STAT5b for sexual dimorphism of
body growth rates and liver gene expression. Proc Natl Acad Sci USA 94:72397244[Abstract/Free Full Text]
-
Teglund S, McKay C, Schuetz E, van Deursen JM, Stravopodis D,
Wang D, Brown M, Bodner S, Grosveld G, Ihle JN 1998 Stat5a and Stat5b
proteins have essential and nonessential, or redundant, roles in
cytokine responses. Cell 93:841850[Medline]
-
Preece A 1972 Manual for Histologic Technicians. Little,
Brown, and Co., Boston, p 260
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Maniatis T, Fritsch EF, Sambrook J 1989 Molecular Cloning: A
Laboratory Manual, ed 2. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
-
Herrin DL, Schmidt GW 1988 Rapid, reversible staining of
northern blots prior to hybridization. Biotechniques 6:196197,
199200[Medline]