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*
Yuan-Chun
Zhou and
David J.
Waxman
From the Division of Cell and Molecular Biology, Department of
Biology, Boston University, Boston, Massachusetts 02215
 |
ABSTRACT |
Hepatic peroxisome proliferation induced by
structurally diverse non-genotoxic carcinogens is mediated by the
nuclear receptor peroxisome proliferator-activated receptor (PPAR
)
and can be inhibited by growth hormone (GH). GH-stimulated Janus
kinase-signal transducer and activator of transcription 5b
(JAK2/STAT5b) signaling and the PPAR activation pathway were
reconstituted in COS-1 cells to investigate the mechanism for this GH
inhibitory effect. Activation of STAT5b signaling by either GH or
prolactin inhibited, by up to 80-85%, ligand-induced,
PPAR
-dependent reporter gene transcription. GH failed to
inhibit
15-deoxy-
12,14-prostaglandin-J2-stimulated
gene transcription mediated by an endogenous COS-1 PPAR-related
receptor. GH inhibition of PPAR
activity required GH receptor and
STAT5b and was not observed using GH-activated STAT1 in place of
STAT5b. GH inhibition was not blocked by the mitogen-activated protein
kinase pathway inhibitor PD98059. STAT5b-PPAR
protein-protein
interactions could not be detected by anti-STAT5b supershift analysis
of PPAR
-DNA complexes. The GH inhibitory effect required the
tyrosine phosphorylation site (Tyr-699) of STAT5b, an intact STAT5b DNA
binding domain, and the presence of a COOH-terminal
trans-activation domain. Moreover, GH inhibition was
reversed by a COOH-terminal-truncated, dominant-negative STAT5b mutant.
STAT5b must thus be nuclear and transcriptionally active to mediate GH
inhibition of PPAR
activity, suggesting an indirect inhibition
mechanism, such as competition for an essential PPAR
coactivator or
STAT5b-dependent synthesis of a more proximal PPAR
inhibitor. The cross-talk between STAT5b and PPAR
signaling pathways
established by these findings provides new insight into the mechanisms
of hormonal and cytokine regulation of hepatic peroxisome proliferation.
 |
INTRODUCTION |
The peroxisome proliferator-activated receptors
(PPARs)1 comprise a family of
three nuclear receptors characterized by unique functions, ligand
specificities, and tissue distributions (1-3). Ligands for PPAR
include fibrate hypolipidemic drugs, specific fatty acids and
eicosanoids, and leukotriene B4 (4-7), whereas anti-diabetic thiazolidinediones (8) and
15-deoxy-
12,14-prostaglandin J2
(15d-PGJ2) (9, 10) are high affinity ligands for PPAR
.
Like many other nuclear receptors, PPARs are transcription factors that
bind to specific DNA response elements (PPREs) upstream of target genes
in heterodimeric complexes with the 9-cis-retinoid acid
receptor RXR, leading to the activation of target gene transcription (11).
PPAR
and PPAR
play a key role in lipid homeostasis, adipocyte
differentiation, and inflammatory responses (3, 12). PPAR
target
genes include liver-expressed enzymes involved in fatty acid
-oxidation and microsomal
-hydroxylation (13-15). PPAR
thus
plays a direct role in liver homeostasis by regulating lipid storage
and by modulating the metabolism of important lipid signaling
molecules, including prostaglandins and leukotrienes. PPAR
gene
knock-out mice do not exhibit hepatic (16) or renal (17) peroxisome
proliferative responses induced by fibrate drugs and other PPAR
activators, demonstrating the key role played by PPAR
in these
processes. PPAR
also mediates the carcinogenicity of foreign
peroxisome proliferators (18), which are non-genotoxic hepatocarcinogens when administered chronically to rodents (19).
PPAR
gene expression and PPAR
-stimulated transcriptional activity
are tightly controlled by a variety of hormones that act at multiple
levels and via different mechanisms. Glucocorticoids induce PPAR
protein expression at the transcriptional level (20), which may account
for the expression of PPAR
diurnally and in a stress-inducible
manner (21), whereas insulin treatment decreases PPAR
mRNA
levels (22). Peroxisome proliferative responses in rodents are
suppressed by the thyroid hormone triiodothyronine (23), whose receptor
may compete with PPAR for heterodimerization with the retinoid X
receptor RXR and for trans-activation of PPAR DNA response
elements (PPREs) (24). Hepatic peroxisome proliferation can also be
modulated by sex hormones, with female rats being less responsive than
males to clofibrate and other peroxisome proliferators (25) and
testosterone treatment abolishing this sex difference (26).
The observation that hypophysectomy enhances peroxisome proliferation
in female rats (26) suggests that a pituitary factor(s) serves as a
negative regulator of peroxisome proliferation. In rats, the continuous
plasma growth hormone (GH) profile characteristic of adult females
fully suppresses liver expression of the clofibrate-inducible P450 4A2
fatty acid
-hydroxylase (27). The same suppressive effect is
observed in primary rat hepatocyte cultures, where GH inhibits
peroxisomal
-oxidation induced by clofibrate (28, 29). GH has
diverse effects on metabolism and growth, some of which are indirectly
mediated by insulin-like growth factor-1 but many of which reflect the
direct effects that GH has on gene expression. In particular, GH, like
many cytokines and growth factors, directly activates JAK-STAT
signaling pathways. GH binds to and thereby dimerizes its plasma
membrane receptor (GHR) in a process that leads to JAK2
kinase-catalyzed tyrosine phosphorylation of STAT proteins, which are
latent, cytoplasmic transcription factors (30). The
tyrosine-phosphorylated STAT proteins dimerize and translocate into the
nucleus, where they bind to specific DNA response element and thereby
activate target gene transcription (31). Among the seven mammalian
STATs, four forms (STATs 1, 3, 5a, and 5b) can be activated by GH
(32-35).
In the present study, we investigate the mechanism that underlies the
inhibitory effect of GH on peroxisome proliferation using COS-1 cells
transfected to express both the GHR/JAK/STAT signaling pathway and the
peroxisome proliferator-activated PPAR pathway. We find that GH
inhibits PPAR
-stimulated reporter gene transcription in a process
that is mediated by STAT5b but not by STAT1. We further demonstrate
that STAT5b tyrosine phosphorylation, DNA binding, and transcriptional
activation are each essential for GH to mediate its inhibitory effects
on PPAR
activity. These findings are discussed in the context of the
implications of this cross-talk between STAT5b and PPAR
for the
regulation of PPAR
-dependent responses by hormones and cytokines.
 |
MATERIALS AND METHODS |
Plasmids--
PPRE-firefly luciferase reporter plasmid derived
from the rabbit CYP4A6 gene, pLUCA6-880, and mouse PPAR
cloned into the expression plasmid pCMV5 (pCMV-mPPAR
) were provided
by Dr. E. Johnson (Scripps Research Institute, La Jolla, CA) (13). The PPRE-firefly luciferase reporter plasmid pHD(X3)Luc, obtained from Dr.
J. Capone (McMaster University, Ontario, Canada), contains three tandem
copies of the PPRE from the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene upstream of a minimal promoter (15) cloned into the
plasmid pCPS-luc (36). The PPAR
expression plasmid pSV-Sport-mPPAR
was obtained from Dr. J. Reddy (Northwestern University, Chicago) (37). Mouse RXR
expression plasmid pCMX-mRXR
was obtained from Dr. R. Evans (Salk Institute, San Diego) (38). Rat
GHR cloned into the expression plasmid pcDNAI and mouse JAK2 cloned
into the expression plasmid pRK5 were respectively provided by Dr. N. Billestrup (Hagedorn Research Institute, Denmark) (39) and Dr. J. Ihle
(St. Jude Children's Research Hospital, Memphis, TN) (40). pME18S
expression plasmids encoding mouse STAT1, STAT3, STAT5a, and STAT5b
were obtained from Dr. A. Mui (DNAX Research Institute of Molecular and
Cellular Biology, Inc., Palo Alto, CA) (41). Mouse prolactin receptor
long form cloned into the expression plasmid pcDNAI (42), and
COOH-terminal truncated forms of mouse STAT5a and STAT5b (designated
STAT5a
749 and STAT5b
754, respectively) and cloned into the
expression plasmid pXM were provided by Dr. B. Groner (Institute for
Experimental Cancer Research, Freiburg, Germany) (43). Human STAT5a,
STAT5b, STAT5a-Y694F, and STAT5b-Y699F cloned into the expression
plasmid pSX were provided by Dr. W. Leonard (NHLBI, National Institutes
of Health) (44). pRc/CMV expression plasmids encoding rat STAT5b and
the DNA-binding region mutants STAT5b-VVVI-(466-469) and
STAT5b-EE-(437-438), where each of the indicated residues is replaced
by alanine, were provided by Dr. L. Yu-Lee (Baylor College of Medicine)
(45).
Cell Culture and Transfections--
COS-1 cells were maintained
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum. Transfection of COS-1 cells grown in 12-well tissue culture
plates was carried out by calcium phosphate precipitation (46). At
9 h after addition of the DNA-calcium phosphate precipitate, cells
were washed and incubated in Dulbecco's modified Eagle's medium
without serum for 12 h. Peroxisome proliferators were then added
to the culture medium in combination with GH at concentrations
specified in each figure. Cells were lysed 24 h later, and firefly
luciferase and
-galactosidase (pCMV-
gal; internal control)
reporter activities were measured using a Galacto-light chemiluminescent reporter kit (Tropix, Bedford, MA). In some
experiments, Renilla luciferase expression plasmid (pRL-TK;
Promega, Madison, WI) was used in place of the pCMV-
gal internal
control plasmid, as indicated in the figure legends. Firefly and
Renilla luciferase activities were measured using a
dual-luciferase assay kit (Promega, Madison, WI). Transfections were
performed using the following amounts of plasmid DNA/well (3.8 cm2) of a 12-well tissue culture plate: 0.36 µg of
reporter construct (pHD(X3)Luc or pLUCA6-880), 14 ng of pCMV-mPPAR
,
0.2 µg of GHR expression plasmid, 0.12 µg of JAK2 expression
plasmid, and 0.2 µg of STAT expression plasmid. pCMV-
gal (0.16 µg) or pRL-TK (30 ng) were included as internal controls in each cell
transfection. The total amount of DNA was adjusted to 0.96 µg/well
using salmon sperm DNA (Stratagene, La Jolla, CA). Data shown in each
figure are mean values ± S.D. (for n = 3 replicates) or mean values ± half the range (for duplicates) and
are representative of at least three such independent experiments.
EMSA Analysis--
Whole cell extracts were prepared by lysing
transfected COS-1 cells in lysis buffer (Tropix, Inc.) containing 1 mM dithiothreitol added prior to use. 10 µg of cell
extract was added to 2 µl of 5× gel-shift buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 50 mM Tris-HCl), plus 1 µl
of 2 µg/µl poly(deoxyinosinic-deoxycytidylic) acid (Boehringer
Mannhein), with water added to adjust the total volume to 15 µl.
Samples were incubated for 10 min at room temperature. 32P-Labeled double-stranded DNA probe (1 µl; 10 fmol) was
then added, and the incubation was continued for 20 min at room
temperature and then 10 min on ice. Loading dye (2 µl of 30%
glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol) was then added
before the mixture was loaded onto an acrylamide gel (5.5% acrylamide,
0.7% bisacrylamide in 0.5× TBE). The gel was electrophoresed at
4 °C for 40 min at 100 V before loading. The gel was first
electrophoresed for 20 min at 100 V at 4 °C at which point the dye
entered the gel; electrophoresis was then continued at room temperature
for 5 h. This procedure minimizes formation of nonspecific
protein-DNA complexes (47). For STAT5b supershift assays, 3 µl of
anti-STAT5b antibody (Santa Cruz Biotechnology, Santa Cruz, CA;
antibody sc-835) was added 10 min after the labeled DNA probe, followed
by a 10-min incubation at room temperature and 10-min incubation on ice
before samples were loaded on the gel. The STAT5 binding site of the rat
-casein promoter (5'-GGA-CTT-CTT-GGA-ATT-AAG-GGA-3') was used as
gel-shift probe for GH-activated STAT5a and STAT5b, and the
sis-inducible element (SIE)-binding site
(5'-gtc-gaC-ATT-TCC-CGT-AAA-TCg-tcga-3') was used as gel-shift probe
for analyzing GH-activated STAT1 and STAT3 (34).
32P-Labeled DNA probe corresponding to the Z element of the
CYP4A6 gene
(5'-g-CGC-AAA-CAC-TGA-ACT-AGG-GCA-AAG-TTG-AGG-GCA-G-3') was used as
probe for PPAR
binding (48).
Western Blotting--
Whole cell extracts from COS-1 cells (15 µg; see above) were resolved on 10% SDS-polyacrylamide gels,
electrotransferred to nitrocellulose, and then probed with anti-STAT
antibodies. STAT5b-specific antibody sc-835 is a rabbit polyclonal
antibody raised to mouse STAT5b amino acid 711-727 (Santa Cruz
Biotechnology). Mouse anti-STAT antibodies (Transduction Laboratories,
Lexington, KY) were as follows: anti-STAT5 (antibody S21520) was raised
to sheep STAT5 amino acids 451-649; anti-STAT3 (antibody S21320) was
raised to rat STAT3 amino acids 1-175; anti-STAT1 (antibody S21120)
was raised to human STAT1 amino acids 592-731.
 |
RESULTS |
GH Activation of STAT5b Inhibits PPAR
Transcriptional
Activity--
To investigate the mechanism by which GH inhibits
PPAR
-dependent liver peroxisome proliferative responses,
we reconstituted GH signaling and PPAR
-dependent
peroxisome proliferation pathways by cotransfection of key components
into COS-1 cells. GH signal transduction was reconstituted by
cotransfection of expression plasmids encoding GHR, JAK2 kinase, and
STAT5b, the major GH-responsive STAT form in liver (34). The PPAR
pathway was reconstituted by cotransfection of a mouse PPAR
expression plasmid, together with reporter plasmid containing 880 nucleotides of 5'-flanking DNA of the rabbit CYP4A6 gene
(13) fused to a firefly luciferase reporter gene. Transfected cells
were stimulated with the peroxisome proliferator Wy-14,643 at 20 µM for 24 h in the presence or absence of GH (200 ng/ml). Fig. 1A shows that
Wy-14,643 activation of the CYP4A6 promoter is significantly
decreased in the presence of GH. Wy-14,643 activation of the
CYP4A6 promoter is mediated by PPAR
, which binds to one
strong and two weaker PPREs within the 5'-flanking 880 nucleotides (13,
49). This GH inhibitory effect may be due to GH suppression of
PPAR
-dependent transcription via the CYP4A6
PPREs; alternatively, GH may interfere with other transcription factors
required for either basal or Wy-14,643-inducible CYP4A6
promoter transcription, independent of PPRE. To distinguish these
possibilities, we examined the effect of GH on PPAR
-activation of
the reporter construct pHD(X3)Luc, which contains three tandem repeats
of a PPRE from the gene that encodes enoyl-CoA
hydratase/3-hydroxyacyl-CoA dehydrogenase, cloned upstream of a minimal
promoter and luciferase reporter gene (15). Fig. 1B shows
that GH inhibits, by up to 80-85%, PPAR
induction mediated by this
isolated PPRE. Maximal GH inhibition was achieved at 25 ng/ml, well
within the physiological range of GH concentrations and consistent with
the reported Kd of GHR for GH of ~2 ng/ml (50).
The inhibitory effects of GH on PPAR
activation were also apparent
with several other PPAR
activators, including both foreign chemicals
(nafenopin, a fibrate hypolipidemic drug) and the endogenous PPAR
activators (8S)-hydroxyeicosatetraenoic acid and leukotriene
B4 (Fig. 1C).

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Fig. 1.
PPAR activation of CYP4A6 promoter
(A) and an isolated PPRE reporter
(B) is inhibited by GH-activated STAT5b when
using multiple PPAR activators (C). COS-1 cells
were cotransfected with expression plasmids encoding PPAR , GHR,
JAK2, STAT5b, and the indicated reporter plasmid. A -galactosidase
expression plasmid (pSV- gal) was included to normalize samples for
transfection efficiency. Cells were stimulated with GH together with
the indicated peroxisome proliferators 24 h before preparation of
cell extracts and measurement of luciferase activity. Data shown are
firefly luciferase reporter activities normalized for the level of
-galactosidase activity in each extract. A, GH (200 ng/ml) suppresses Wy-14,643-stimulated (Wy; 20 µM) PPAR induction of CYP4A6 promoter
activity (nucleotides 880 to 1). B, GH suppresses
PPAR activation of an isolated PPRE linked to a firefly luciferase
reporter, pHD(X3)Luc. Shown are the effects of 1-100 ng/ml GH on
Wy-14,643-stimulated (20 µM) reporter gene activity.
C, GH suppresses PPAR activation stimulated by a range of
foreign chemicals and endogenous PPAR activators, as monitored with
the reporter plasmid pHD(X3)Luc. Luciferase activity was measured after
the cells were treated for 24 h with Wy-14,643 (20 µM), (8S)HETE (8(S)H) (5 µM), leukotriene B4 (LTB)(10
µM), or nafenopin (Naf) (100 µM)
in the absence and presence of GH (100 ng/ml), as indicated. Data shown
are relative luciferase activities (firefly
luciferase/ -galactosidase) for cells treated with PPAR activators
compared with vehicle controls (fold induction).
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Requirement of GHR, JAK2, and STAT5b for GH Suppression of PPAR
Activity--
Since some, but not all GH intracellular events involve
JAK/STAT signaling pathways (30), we examined whether GHR, JAK2, and
STAT5b are each required for the inhibitory effect of GH on PPAR
activity. Fig. 2A shows that
inhibition of PPAR
activity by 25 ng/ml GH (lanes 3 versus lane 2) was not observed in the absence of
cotransfected GHR (lane 4) or STAT5b (lane 5). A
similar dependence on GHR and STAT5b was observed at higher GH
concentrations (100-500 ng/ml GH; data not shown). In the absence of
transfected JAK2, GH could still activate STAT5b and inhibit PPAR
activity using the low levels of JAK2 expressed endogenously in COS-1
cells, although a higher GH concentration (500 ng/ml) was required for maximal PPAR
inhibition (data not shown). Interestingly,
cotransfection of GHR, JAK2, and STAT5b significantly reduced the
moderate PPAR
-dependent but Wy-14,643-independent basal
luciferase reporter activity, even in the absence of GH (Fig. 2A,
lanes 6 versus 1). This
Wy-14,643-independent activity has been associated with the presence of
endogenous PPAR activators, such as fatty acids (4, 5). Inhibition of
this basal PPAR activity likely results from the constitutive
activation of STAT5b upon overexpression of JAK2, as shown in the
gel-shift studies described below (Fig. 2B). GH stimulation
further decreased the Wy-14,643-independent PPAR
activity up to
4-fold (data not shown).

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Fig. 2.
GH activation of STAT5b is required for
inhibition of PPAR activity. A, GHR and STAT5b are both
required for GH inhibition of PPAR activity. COS-1 cells were
transiently transfected with expression plasmids encoding GHR, JAK2,
and STAT5b, as indicated in each lane, in combination with PPAR
expression plasmid and the PPRE-luciferase reporter gene pHD(X3)Luc.
Cells were stimulated with Wy-14,643 (Wy) (20 µM) and GH (25 ng/ml), as indicated. Luciferase
activities relative to a -galactosidase transfection control were
measured in whole cell lysates. B, GH activates STAT5b DNA
binding activity in transfected COS-1 cells. COS-1 cells were
transfected with expression plasmids encoding GHR, JAK2, STAT5b,
PPAR , and the pHD(X3)Luc reporter gene. Cells were treated with
Wy-14,643 (20 µM) and GH (100 ng/ml) for 24 h, as
indicated, followed by preparation of whole cell extracts. EMSA assays
were carried out with a 32P-labeled -casein promoter DNA
probe, which contains a STAT5b-binding site. The specific STAT5b-DNA
complex is marked 5b. Anti-STAT5b-specific antibody was used
to supershift STAT5b-containing DNA-protein complexes (STAT5b
SShift), as indicated. Nonspecific protein binding to the
-casein probe is labeled ns.
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To confirm the reconstitution of STAT5b activity in the GH-stimulated
COS-1 cells, EMSA assays were carried out using a
-casein promoter
probe, which contains a single STAT5b-binding site. No STAT5b DNA
binding was detected upon GH stimulation of untransfected COS-1 cells
(Fig. 2B, lanes 1 and 2). This
indicates that COS-1 cells have at most low levels of GHR and/or STAT5b
and are thus suitable for use as recipient cells in these transfection
studies. In cells cotransfected with GHR, JAK2, and STAT5b, a low basal STAT5b DNA binding was observed in the absence of GH; this STAT5b activity was not affected by Wy-14,643 treatment (Fig. 2B, lanes 3 versus 5). Thus, overexpression of JAK2
and STAT5b results in some constitutive activation of STAT5b DNA
binding activity. Stimulation with GH yielded a much higher level of
STAT5b DNA binding activity (lane 7 versus
5). The
-casein DNA-binding complexes obtained in these
experiments were confirmed to contain STAT5b, as shown by supershift
analysis using anti-STAT5b antibody (lanes 4, 6, and
8).
Inhibition of PPAR
Activity by GH-activated STAT3 but Not
STAT1--
In addition to STAT5b, GH can activate three other STAT
proteins, STATs 1, 3, and 5a (32-35). Fig.
3 shows, however, that in COS-1 cells
transfected with STAT1, GH did not decrease PPAR
-induced luciferase
reporter activity, even at a high GH concentration (500 ng/ml). Partial
GH inhibition of PPAR
activity was seen in COS-1 cells transfected
with STAT3 or with the closely related (41, 51) STAT5a. In control
experiments, all four STATs were shown by Western blotting to be highly
expressed in the transfected COS-1 cells compared with untransfected
controls (Fig. 3B; lanes 2-4 versus lane 1). The
lower effectiveness of STAT5a compared with STAT5b is not because of
its inefficient activation by GH, as shown by EMSA using
32P-labeled
-casein probe (Fig. 3C, lanes 3 and 4 versus lanes 6 and 7). STAT1 and
STAT4 expressed in GH-treated cells were also shown to be functionally
active in DNA binding using an SIE (sis-inducible element)
EMSA probe (see "Materials and Methods") (data not shown).

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Fig. 3.
Comparison of the capability of GH-activated
STATs 1, 3, 5a, and 5b to inhibit PPAR activity. A,
COS-1 cells were transiently transfected with the reporter plasmid
pHD(X3)Luc and with expression plasmids encoding PPAR , GHR, JAK2
kinase and the indicated STAT protein. Cells were stimulated for
24 h with Wy-14,643 (Wy) (20 µM) and GH
at the indicated concentrations. Whole cell extracts were prepared, and
luciferase activities relative to a -galactosidase transfection
control were determined. B, expression of STAT proteins in
transiently transfected COS-1 cells. The same cell lysates assayed in
A were analyzed for STAT protein expression by Western blot
using each of the indicated STAT form-specific antibodies. Small
amounts of STAT1 and STAT5a are seen to be present in untransfected
COS-1 cell extracts (lane 1). C, GH activates
STAT5a and STAT5b DNA binding activity in transfected COS-1 cells.
COS-1 cells were transfected with expression plasmids encoding GHR,
STAT5a or STAT5b, and PPAR . Cells were treated with GH (500 ng/ml)
for 30 min, and cell extracts were assayed for EMSA activity using the
-casein probe. Supershift analysis was carried out using anti-STAT5a
(Santa Cruz, sc-1081x) or anti-STAT5b antibody (Santa Cruz sc-835), as
indicated. GH-activated STAT5a (lane 3) migrates more slowly
than STAT5b (lane 6). ns, nonspecific protein-DNA
complex.
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Prolactin Inhibition of PPAR
Activity--
Prolactin is a
GH-related pituitary polypeptide hormone that also signals via its
cell-surface receptor through a JAK2/STAT5 pathway (42). STAT5a,
originally identified as a prolactin-activated mammary gland factor,
and STAT5b can both confer a prolactin response to the mammary
-casein gene promoter (42, 51). Fig. 4
shows that in the presence of prolactin, STAT5b, but not STAT5a,
inhibits PPAR
activation in cells cotransfected with prolactin
receptor and JAK2. This inhibition by prolactin is less extensive
(~40%) than seen for GH-activated STAT5b in the same system
(~80%; cf., Fig. 3A) and also required a
higher concentration of hormone to achieve its maximum effect (100 ng/ml prolactin versus 25 ng/ml GH). STAT5b activation by
prolactin was confirmed by gel-shift assay using a
-casein probe
(data not shown).

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Fig. 4.
Prolactin inhibition of PPAR activity
mediated by STAT5b. COS-1 cells were cotransfected with expression
plasmids encoding prolactin receptor, JAK2 kinase, STAT5a or STAT5b,
PPAR , and the reporter plasmid pHD(X3)Luc. Cells were treated with
Wy-14,643 (Wy) (20 µM) and prolactin
(PRL) (0, 10 or 100 ng/ml, as indicated) for 24 h. Cell
extracts were then prepared and assayed for luciferase activity
relative to a -galactosidase transfection control. In some
experiments, prolactin-activated STAT5a inhibited PPAR activity by
~20%.
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GH-activated STAT5b Fails to Inhibit PPRE-dependent
Transcription Induced by
15-Deoxy-
12,14,-PGJ2
(15d-PGJ2)--
15d-PGJ2 is a naturally
occurring PPAR ligand that activates PPAR
and other PPARs (52).
Treatment of COS-1 cells with 5 µM 15d-PGJ2
stimulated PPRE-dependent luciferase reporter activity by
~20-fold, even in the absence of PPAR
transfection (Fig.
5). This induction was not further
enhanced by co-transfection of a PPAR
expression plasmid (data not
shown), suggesting that the 15d-PGJ2-stimulated PPRE
response observed in this experiment involves an endogenous COS-1 PPAR
that is distinct from PPAR
or PPAR
. GH did not, however, inhibit
15d-PGJ2 -induced, PPRE-dependent reporter gene
transcription (Fig. 5), demonstrating that the GH suppressive response
has specificity for the PPAR
activation pathway.

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Fig. 5.
GH activated STAT5b fails to inhibit
activation of endogenous COS-1 cell PPAR activity stimulated by
15d-PGJ2. COS-1 cells were cotransfected with
expression plasmids encoding GHR, JAK2, STAT5b, and the reporter
plasmid pHD(X3)Luc. Transfected cells were stimulated with
15d-PGJ2 (5 µM) with or without GH (100 ng/ml). Cell extracts were prepared 24 h later and assayed for
luciferase activities relative to -galactosidase transfection
controls.
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STAT5b Inhibition of PPAR
Activity Is Not Mediated by the MAP
Kinases ERK1 and ERK2--
We next investigated whether MAP kinase
plays a role in the GH-induced down-regulation of PPAR
activity. GH
can activate the MAP kinase kinase MEK1, which phosphorylates and
thereby activates the MAP kinases ERK1 and ERK2 (53). MAP kinases are
actively involved in growth factor and cytokine receptor signaling (54, 55) and catalyze phosphorylation on serine and/or threonine of several
transcription factors, including PPAR
(56). By analogy to the MAP
kinase regulation of PPAR
, PPAR
, which is also a phosphoprotein
(57), might be regulated by MAP kinase-catalyzed serine
phosphorylation. To investigate whether MAP kinase activity is required
for GH inhibition of PPAR
activity, we used the MEK1 and MEK2
inhibitor PD98059 (58) to block MAP kinase activation. Stimulation of
transfected COS-1 cells with Wy-14,643 in combination with PD98059
increased PPRE-dependent luciferase activity compared with
Wy-14,643 treatment alone (Fig. 6). The
enhancement by PD98059 of PPRE luciferase activity varied from ~1.5-
to 4-fold in different experiments. However, GH treatment inhibited
Wy-14,643-induced reporter activity to a similar extent in the presence
and in the absence of PD98059 (Fig. 6). Since PD98059 did not block the
GH inhibitory effect, the MAP kinases ERK1 and ERK2 are not likely to
mediate GH inhibition of PPAR
activity. PD98059 treatment also
enhanced Wy-14,643-induced PPAR
activity in the absence of GHR,
JAK2, and STAT5b (data not shown). This suggests that basal MAP kinase
activity in these cells exerts a negative effect on PPAR
activity
but in a manner that is independent of GHR or STAT5b expression or GH
treatment.

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Fig. 6.
Effect of MAP kinase pathway inhibitor
PD98059 on GH inhibition of PPAR activity. COS-1 cells were
transfected with expression plasmids encoding GHR, JAK2, STAT5b,
PPAR , and the reporter plasmid pHD(X3)Luc. Transfected cells were
treated with Wy-14,643 (Wy) (20 µM) and GH
(100 ng/ml) in the presence or absence of PD98059 (PD) (20 µM). Luciferase activities relative to -galactosidase
transfection controls were measured in whole cell extracts prepared
24 h after Wy-14,643 and GH treatment. In the experiment shown, GH
inhibited PPAR activity by ~90% in the absence of PD98059 and by
~70% in its presence. Similar results were obtained in an experiment
carried out at 1 µM Wy-14,643 (73 and 84% inhibition by
GH in the absence and presence of 10 µM PD98059,
respectively), except that PD98059 induced an ~4-fold increase in
PPAR activity in Wy-14,643-treated cells (data not shown).
|
|
Tyr-699 Phosphorylation of STAT5b Is Required for GH Inhibition of
PPAR
Activity--
STAT5b is activated by JAK2
kinase-dependent phosphorylation on tyrosine 699. Mutation
of this tyrosine to phenylalanine (STAT5b-Y699F) results in a loss of
STAT5b dimerization, DNA binding, and transcriptional activation (44).
Fig. 7 shows that, when activated by GH,
wild-type human STAT5b inhibited PPAR
activity to a similar extent
as did mouse STAT5b. By contrast, the Y699F substitution abrogated GH inhibition of PPAR
activity. Accordingly, STAT5b tyrosine
phosphorylation and/or its downstream activities (STAT5b dimerization,
nuclear translocation, DNA binding, or transcriptional activation) are obligatory for STAT5b to mediate GH inhibition of PPAR
. When equal
amounts of human STAT5a expression plasmid were transfected, it failed
to inhibit PPAR
activity (Fig. 7). Partial inhibition (~40%) was
observed, however, at 4-fold higher human STAT5a plasmid levels (data
not shown).

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Fig. 7.
Mutation of STAT5b Tyr-699 abolishes GH
inhibition of PPAR activity. Expression plasmids encoding human
STAT5a, human STAT5b and their site-specific mutants (STAT5b-Y699F and
STAT5a-Y694F) were transiently transfected into COS-1 cells together
with GHR, JAK2, PPAR expression plasmids and pHD(X3)Luc reporter
plasmid. Transfected cells were treated with Wy-14,643 (Wy)
(20 µM) and GH (0, 25 or 100 ng/ml) for 24 h. Cell
extracts were then prepared and assayed for luciferase activity
relative to a -galactosidase transfection control.
|
|
Requirement of STAT5b COOH-terminal trans-Activation Domain and
Effects of Dominant-negative STAT5b Mutant--
Deletions of the
COOH-terminal trans-activation domain of mouse STAT5a and
STAT5b (constructs STAT5a
749 and STAT5b
754, respectively) result
in a loss of transcriptional activity and yield truncated STAT5
proteins that exert dominant-negative effects on wild-type STAT5-induced gene transcription (43). These COOH-terminal truncated STAT5 proteins undergo hormone-induced tyrosine phosphorylation and
retain DNA binding activity but show delayed tyrosine dephosphorylation (43). These mutants were used to examine whether the transcriptional activity of STAT5b is necessary for GH inhibition of PPAR
activity. Fig. 8A shows that, in
contrast to wild-type STAT5b, STAT5b
754 cannot mediate GH-induced
PPAR
inhibition, suggesting that the trans-activation
domain of STAT5b is required for GH inhibitory effects. In contrast,
STAT5a
749 inhibited PPAR
activity by ~40% following GH
stimulation.

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Fig. 8.
Dominant-negative STAT5b blocks GH inhibition
of PPAR activity by wild-type STAT5b. A, effects of
COOH-terminal truncated STAT5b and STAT5a on PPAR activity. COS-1
cells were cotransfected with expression plasmids encoding GHR, JAK2,
and the STAT5b COOH-terminal deletion constructs STAT5b 754
( STAT5b) or STAT5a 749 ( STAT5a).
Transfected cells were treated with Wy-14,643 (Wy) (20 µM) alone or in the presence of GH (100 ng/ml) for
24 h. Cell extracts were then prepared and assayed for firefly
luciferase activity relative to a Renilla luciferase
transfection control. STAT5a, but not STAT5b, partially inhibited
Wy-14,643-induced reporter gene activity in GH-treated cells.
B, STAT5b 754 blocks wild-type
STAT5b-dependent inhibition of PPAR activity. COS-1
cells were transfected with pHD(X3)Luc reporter and PPAR , GHR, JAK2,
and mouse STAT5b expression plasmids together with increasing amounts
of STAT5b or STAT5a expression plasmids (0-, 0.5-, 1-, and 5-fold
relative to the amount of cotransfected wild-type STAT5b, calculated on
a per microgram plasmid DNA basis). Cells were stimulated with
Wy-14,643 (20 µM) and GH (100 ng/ml) for 24 h.
Luciferase activities relative to a -galactosidase transfection
control were then assayed in whole cell extracts. C,
expression of STAT5a, STAT5b, and their COOH-terminal truncated
mutants. The same cell lysates analyzed in B were analyzed
for STAT5 protein expression by anti-STAT5 Western blotting
(Transduction Laboratories, antibody S21520). The COOH-terminal
truncated STAT5b and STAT5a bands were not detectable by a carboxyl-terminal targeted STAT5b antibody (Santa Cruz
antibody sc-835; data not shown). D, EMSA analysis
of STAT5b, STAT5a, and STAT5b DNA binding activity. The
same cell lysates shown in B and C were
analyzed for STAT5 DNA binding activity using the -casein
promoter probe, as described under "Materials and
Methods." STAT5b COOH-terminal targeted antibody (Santa Cruz
antibody sc-835) was used for supershift (SS) where
indicated (anti-5b).
|
|
To test for dominant-negative activity, COS-1 cells were transfected
with wild-type STAT5b in the presence of increasing amounts of
STAT5a
749 or STAT5b
754. Fig. 8B shows that
STAT5b
754 could fully block the STAT5b-dependent GH
inhibition of PPAR
activity in a dose-dependent manner
(lane 2 versus lanes 3 and
4). Interestingly, while STAT5a
749 has dominant-negative
activity toward wild-type STAT5b and can block its transcriptional
activity (43), this mutant had only a modest effect on
STAT5b-dependent PPAR
inhibition (lane 2 versus lanes 5 and 6). However,
interpretation of this result is complicated by the fact that
STAT5a
749 itself confers partial inhibition of PPAR
in response
to GH stimulation (Fig. 8A). Fig. 8C confirms the
expression of STAT5a
749, STAT5b
754, and wild-type STAT5 proteins
in the transfected COS-1 cells, detected with an antibody against the
STAT5 SH2 and SH3 domains, which are upstream of the deleted sequences.
As expected, the STAT5b
754 and STAT5a
749 bands seen on this blot
were of lower molecular weight (lanes 4 and 7)
and could not be detected using an antibody that specifically
recognizes the extreme COOH-terminal region of STAT5b (Santa Cruz
antibody sc-835; data not shown). When equal amounts of expression
plasmid were transfected, STAT5a
749 protein was expressed at a level
similar to wild-type STAT5b (lane 7 versus lanes
2 and 3). The level of STAT5b
754 protein (lane
4) was much lower (Fig. 8C), highlighting the potency
of its dominant-negative effects (Fig. 8B). EMSA analysis
showed that STAT5a
749 and STAT5b
754 both bind to a
-casein DNA
probe much more efficiently than wild-type STAT5b (Fig. 8D; lanes
2, 3, 8, 9, 11, and 12 versus lanes 5 and 6), in agreement with earlier studies (43). This suggests
that these mutants achieve their dominant-negative effect, at least in
part, by interfering with wild-type STAT5b DNA binding activity. As
expected, antibody specific for the STAT5b COOH-terminal peptide can
completely supershift wild-type STAT5b but not the much more intense
STAT5b
754 binding to the
-casein probe (Fig. 8D, lanes 7 versus 13).
STAT5b DNA Binding Activity Is Required for Inhibition of PPAR
Activity--
STAT5a interacts with glucocorticoid receptor through
formation of a protein-protein complex that inhibits
glucocorticoid-induced gene expression (59, 60). STAT5b inhibition of
the stimulatory effects of prolactin at the IRF-1 promoter is also
proposed to involve direct protein-protein interactions (45). We
therefore considered whether a direct interaction between GH-activated
STAT5b and PPAR
can be detected using an EMSA assay for PPAR
-PPRE
protein-DNA complexes. Fig. 9 shows that
anti-STAT5b antibody does not alter the mobility of a
PPAR
-containing DNA complex formed on a PPRE probe from the
CYP4A6 gene (lanes 3 versus 2) under
conditions where the antibody fully supershifts STAT5b-containing DNA
complexes (Figs. 2B and 3C). These data indicate
that STAT5b does not bind directly to a PPAR
-DNA complex. In control
samples, the PPAR-PPRE complex seen in lane 2 was either
disrupted or supershifted by various anti-PPAR
antibodies
(lanes 4 and 5), competed by an excess of
unlabeled PPRE probe (lane 6) and was fully dependent on
transfected PPAR
for its formation (lane 7).

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Fig. 9.
STAT5B does not bind directly to PPAR -DNA
complex. COS-1 cells were transfected with expression plasmids
encoding GHR, JAK2, STAT5b and RXR in combination with (lanes
2-6) or in the absence of PPAR expression plasmid (lane
7). Cell were stimulated with Wy-14,643 (20 µM) and
GH (100 ng/ml) for 24 h before cell extracts were prepared. EMSA
assays were carried out with a 32P-labeled
CYP4A6 Z-element EMSA probe, which contains a single PPAR
binding site. Lane 1, untransfected COS-1 cell extract.
Anti-STAT5b-specific antibody (2 µl; Santa Cruz, sc-835) was included
in lane 3. Anti-PPAR antibodies were used to verify the
presence of PPAR in the DNA-binding complex: lane 4,
`a', 4 µl of anti-PPAR antibody which specifically inhibits
complex formation (Affinity Bioreagents, Inc., cat# PA1-822);
lane 5, `b', 2 µl of a supershifting anti-PPAR
antibody provided by Dr. E. Johnson (Scripps Research Institute, La
Jolla, CA).
|
|
We next investigated whether STAT5b DNA binding activity is required to
inhibit PPAR
activity. The effects of two rat STAT5b mutants,
STAT5b-EE and STAT5b-VVVI (45), were compared with that of wild-type
rat STAT5b. In these mutants, amino acid residues EE (437-438) and
VVVI (466-469) within the VTEE and SLPVVVI sequences of the DNA
binding domain of STAT5b are replaced by alanine. The STAT5b-VVVI
mutant is devoid of DNA binding activity and is transcriptionally inactive following prolactin stimulation, whereas the
STAT5b-EE mutation does not abolish STAT5b DNA binding activity or
prolactin-induced transcription from the
-casein promoter (45).
STAT5b-EE and STAT5b-VVVI were therefore tested for their ability to
inhibit PPAR
activity in GH-treated COS-1 cells. Fig.
10 shows that wild-type rat STAT5b and
rat STAT5b-EE both inhibit PPAR
activity in GH-treated cells in a
manner similar to the effects of mouse and human STAT5b shown above. By
contrast, much less inhibition is seen with STAT5b-VVVI. Thus, the VVVI
residues in the DNA binding domain of STAT5b are critical for
GH-activated STAT5b to efficiently inhibit PPAR
activity, strongly
suggesting that this inhibition requires STAT5b DNA binding and
transcriptional activation activity.

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Fig. 10.
DNA-binding activity is required for rat
STAT5b to mediate GH inhibition of PPAR activity. COS-1 cells
were transiently transfected with pHD(X3)Luc reporter and expression
plasmids encoding PPAR , GHR, JAK2 and rat STAT5b or either of two
rat STAT5b site-specific mutants localized to STAT5b's DNA binding
domain. STAT5b-EE retains DNA-binding activity, while STAT5b-VVVI is
devoid of DNA-binding activity (45). Transfected cells were treated
with Wy-14,643 (20 µM) and GH (25 and 100 ng/ml) for
24 h. Cell extracts were assayed for firefly luciferase activity
relative to renilla luciferase activity.
|
|
 |
DISCUSSION |
GH and several other hormones, including thyroid hormone
(triiodothyronine) and glucocorticoids, modulate the pleiotropic responses of rodent liver to structurally diverse peroxisome
proliferators mediated by the nuclear receptor PPAR
. The present
studies demonstrate that the GH-activated transcription factor STAT5b,
and to a lesser extent STAT3, can mediate the inhibitory effects of GH
on PPAR
transcriptional activity and that these effects occur at a
physiological GH level. This specific inhibitory effect on PPAR
activity was seen both with rodent and human STAT5b proteins and
occurred when STAT5b was activated by either GH or prolactin. Given
that STAT5b can also be activated by a large number of cytokines and
growth factors, and is widely expressed in mammalian tissues (41, 51), the potential for inhibitory interactions between PPAR
and
JAK-STAT5b signaling pathways is widespread. Although STAT5a (mammary
gland factor) and STAT5b show ~90% amino acid sequence identity, and both bind to and trans-activate target genes via STAT5
response elements such as that found in the
-casein promoter, they
have distinct tissue-specific functions that cannot be compensated by
each other in vivo, as shown in the case of mouse
STAT5 gene knock-out studies (61, 62). The present study
provides further evidence for differences between the two STAT5 forms,
insofar as STAT5a was less effective at inhibiting PPAR
activity,
particularly when activated by prolactin (Fig. 4).
Functional interactions involving the binding of STAT factors and
another nuclear receptor family member, glucocorticoid receptor, have
been described (59, 60, 63). STAT5a and glucocorticoid receptor form a
molecular complex that enhances STAT5a-induced transcription from the
-casein promoter and inhibits glucocorticoid-stimulated transcription from a glucocorticoid response element. Specific DNA
binding by STAT5a is required for cooperation with glucocorticoid receptor on the
-casein promoter, and although the receptor does not
bind the STAT5a DNA-binding element directly, it associates with the
STAT5a-DNA complex. A potential glucocorticoid receptor-binding site is
present within the
-casein promoter, and this site is required for
the synergism between STAT5a and glucocorticoid receptor (64). These
studies establish a model in which STAT proteins cross-talk with
nuclear receptors by direct protein-protein interactions that modulate
gene expression. By contrast, we were unable to detect molecular
complexes involving both STAT5b and PPAR
in the present study, as
judged by EMSA supershift analysis. Transcriptional inhibitory effects
of prolactin-activated STAT5b have also been observed in studies of the
full-length IRF-1 (interferon regulatory factor-1) promoter but not
with an isolated IRF-1 STAT response element (45). In contrast to our
findings with PPAR
, the prolactin inhibitory effects seen on the
IRF-1 promoter are conferred equally well by STAT5a or STAT5b.
Moreover, unlike our findings in the present study, the effects of
STAT5b on the IRF-1 promoter do not require the DNA binding activity of
STAT and are proposed to involve STAT5b in protein-protein interactions
with other transcription factors (45).
GH activates the MAP kinase pathway in many cell types, including liver
cells (32, 53, 65). MAP kinase can, in turn, phosphorylate and thereby
modulate the activity of a variety of transcription factors, including
STAT proteins, which may undergo MAP kinase-catalyzed serine
phosphorylation required for full transcriptional activity (66, 67).
Growth factor-activated MAP kinase phosphorylates PPAR
, resulting in
an inhibition of the transcriptional activity of that nuclear receptor
(56, 68). In our experiments, the MAP kinase kinase inhibitor PD98059
increased the responsiveness of COS-1 cells to PPAR
activators,
suggesting that MAP kinase phosphorylates and thereby inhibits PPAR
by a mechanism similar to that described for PPAR
(56, 68). However, PD98059 did not alter the extent to which GH-activated STAT5b inhibited
PPAR
activity.
Several mechanisms may account for the inhibition of PPAR
by
GH-activated STAT5b described in the present study. First, activated STAT5b could inhibit PPAR
protein expression and thereby block PPAR
activation of PPRE. However, this is considered unlikely, since
GH-activated STAT5b had no effect on expression of the internal control
-galactosidase expression plasmid, which utilizes the same
cytomegalovirus promoter as does the PPAR
expression plasmid used in our experiments. Second, STAT5b might compete with PPAR
for
binding to the PPAR reporter PPRE elements of plasmid. This is also
unlikely, since the pHD(X3)Luc reporter used in this study does not
contain STAT5b-binding sites. Moreover, gel-shift assays revealed that
STAT5b does not bind to an isolated PPRE (Fig. 9, lane 7).
Third, by analogy to the interaction of STAT5a with glucocorticoid receptor discussed above, STAT5b may form a protein-protein complex with PPAR
and thereby prevent PPAR
from
trans-activating its target genes. However, the inability of
a STAT5b-specific antibody to supershift activated PPAR
when bound
on a PPRE element (Fig. 9) argues against a direct association of
STAT5b with PPAR
, indicating a mechanism distinct from the
previously described STAT5a-glucocorticoid receptor association. We
cannot rule out the possibility, however, that STAT5b might form a
complex with PPAR
that is independent of its binding to PPRE and
thus not detected in our experiments.
An alternative possibility is that STAT5b inhibits PPAR
activity by
an indirect mechanism. For example, when present in the nucleus in a
transcriptionally active state, STAT5b may compete for an essential
coactivator of PPAR, such as SRC-1, CBP/p300, or PBP (69-71), or
perhaps modulate the binding of PPAR to other interacting proteins,
such as RXR
or RIP140 (72), leading to inhibition of transcriptional
activity of PPAR
. Indeed, CBP and/or p300 have been implicated in
the antagonism between STAT and AP-1 signaling pathways (73).
Alternatively, given the requirement for intact, functional STAT5b DNA
binding and transcriptional activation domains, STAT5b may activate
transcription of a second gene, leading to production of a distinct
protein factor that serves as a more proximal inhibitor of PPAR
activity. Both of these possibilities are consistent with our
observation that a dominant-negative STAT5b mutant (STAT5b
754 (43))
blocks wild-type PPAR
inhibitory activity of STAT5b (Fig. 8). Of
note, some caution about the interpretation of the effects of the
STAT5b mutants employed in this study is required. For example, if
mutation of the STAT5b DNA binding domain residues VVVI (45) were to
interfere with STAT5b tyrosine phosphorylation and/or nuclear
translocation, then a failure to translocate into the nucleus rather
than the loss of DNA binding activity per se could account
for the observed lack of PPAR
inhibition (Fig. 10). The precise
mechanism underlying the dominant-negative effect of the COOH-terminal
truncated STAT5b also needs to be elucidated. Further study will be
required to test and evaluate these and other possible inhibitory mechanisms.
The inhibition of PPAR
activity by activated STAT5b described in
this study may have diverse physiological consequences. PPAR
is an
important intracellular messenger that transmits pharmacological as
well as nutritional and immunological stimuli to cells. PPAR
target
genes are involved in fatty acid oxidation, transport, and synthesis in
liver and other tissues, and PPAR
activators include certain
steroids, fatty acids, and metabolites of arachidonic acid (12). STAT5b
can be activated by many cytokines and growth factors in addition to GH
and prolactin, including erythropoietin and interleukins 2, 3, and 5 (41, 44). If, as seems likely, STAT5b inhibits PPAR
activity when
activated by these other cytokines and growth factors, additional
cytokine and hormonal effects on the regulation of lipid metabolism and
the degradation of lipid signaling molecules are also possible.
Leukotriene B4, a mediator of certain inflammatory and
immunological reactions, has been identified as an endogenous ligand for PPAR
(7). Peroxisome proliferators and PPAR
activators, such
as clofibrate, are reported to have both inductive (74, 75) and
suppressive effects (76) on the enzymes involved in leukotriene
B4
-hydroxylation, a reaction that deactivates this potent chemotactic agent and thereby shortens the duration of an
inflammatory response. The inhibition of PPAR
activity by STAT5b
activators may therefore provide a mechanism through which STAT5b-activating cytokines modulate leukotriene B4-induced
inflammatory responses. The pituitary hormones GH and prolactin can
have a marked influence on immune cell types (77, 78). Leukocytes express receptors for GH and prolactin (77) and have an intact JAK/STAT
pathway (79), indicating that these polypeptide hormones have the
potential to modulate inflammatory responses by suppression of
PPAR
-regulated leukotriene B4 degradation. Inflammatory
cells also have the capacity to synthesize and secrete GH (80), which could provide for fine adjustment of the leukotriene B4
response in immune cells.
The present demonstration that GH-activated STAT5b can inhibit
PPAR
-dependent transcriptional responses provides a
mechanistic basis for the observed inhibitory effects of GH on
peroxisomal enzyme induction (28, 29). Additional mechanisms may also be operative, however, as suggested by the longer term down-regulation of PPAR
mRNA levels seen in GH-treated liver cells (81). Foreign chemical peroxisome proliferators, including chlorinated hydrocarbons and their metabolites, induce hepatocarcinogenesis via
PPAR
-dependent non-genotoxic mechanisms (18, 19).
Increased oxidative stress and proto-oncogene induction (82) are both
postulated to contribute to the carcinogenicity of these peroxisome
proliferators; however, the precise mechanism remains unclear. PPAR
plays a critical role in tumor development in mice in response to the
foreign peroxisome proliferator Wy-14,643, and targeted disruption of
the PPAR
gene results in loss of this carcinogenic response (18). GH
suppression of PPAR
activity may therefore inhibit tumor
development, suggesting a novel mechanism whereby endogenous hormones
beneficially modulate cellular responses to non-genotoxic chemical carcinogens.
 |
ACKNOWLEDGEMENTS |
We thank Drs. E. Johnson, J. Capone, J. Reddy, R. Evans, N. Billestrup, J. Ihle, A. Mui, B. Groner, W. Leonard,
and L. Yu-Lee for provision of plasmid DNAs used in this study.
 |
FOOTNOTES |
*
This work was supported in part by the Environmental
Protection Agency via National Institutes of Health Grant ES07381 (to D. J. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biology,
Boston University, 5 Cummington St., Boston, MA 02215. Tel.: 617-353-7401; Fax: 617-353-7404; E-mail:
djw{at}bio.bu.edu.
The abbreviations used are:
PPAR, peroxisome
proliferator-activated receptor; GH, growth hormone; RXR, retinoid X
receptor; PPRE, peroxisome proliferator response element; GHR, GH
receptor; JAK2, Janus kinase 2; STAT, signal transducer and activator
of transcription; CYP, cytochrome P450; EMSA, electrophoretic mobility
shift assay; 15d-PGJ2, 15-deoxy-
12,14
prostaglandin J2; MAP kinase, mitogen-activated protein
kinase; MEK, MAP kinase kinase; IRF-1, interferon regulatory
factor-1.
 |
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