From the
Growth hormone (GH) exerts sexually dimorphic effects on liver
gene transcription that are regulated by the temporal pattern of
pituitary GH release, which is intermittent in male rats and nearly
continuous in females. To investigate the influence of these GH
secretory patterns on intracellular hepatocyte signaling, we compared
the pattern of liver nuclear protein tyrosine phosphorylation in male
and female rats. An M
Growth hormone (GH)
GH stimulates tyrosine phosphorylation of multiple cellular
polypeptides, including GH receptor itself, as an early hormonal
response
(10, 11) . Some of these phosphorylations are
catalyzed by a GH receptor-associated kinase identified as
Jak2
(12, 13) , a member of the Janus family of tyrosine
kinases
(14, 15) , while others are catalyzed by
downstream kinases
(11) . One of these kinases, mitogen-activated
protein kinase, triggers a cascade of kinases that ultimately activate
ribosomal protein S6, which mediates the stimulatory effects of GH on
protein synthesis
(16, 17) . Studies in mouse 3T3-F442A
preadipocytes
(13, 18) and in rat liver in vivo(19) have shown that GH stimulates tyrosine phosphorylation of a
latent cytoplasmic transcription factor of M
A unique feature
of GH action is that many, though not all, of the effects of this
polypeptide hormone are dependent on the temporal pattern of hormone
release by the pituitary gland
(2) . This pattern of pituitary GH
secretion is ultimately regulated by gonadal steroids and hence is
markedly different between the sexes, particularly in rodent
models
(23, 24, 25) . The resultant sex-dependent
plasma GH profiles in turn regulate the sex-dependent effects of GH on
longitudinal bone growth and body weight
gain
(26, 27, 28) , as well as the sex-dependent
expression of a large number of liver gene products, including
polypeptide hormone and other receptors (29-31), cytochrome
P450
(32, 33) , and other liver
enzymes
(34, 35, 36) . Studies in the rat model
have established that intermittent plasma GH pulsation, a
characteristic of adult male rats, activates liver transcription of
several genes whose expression is limited to adult males, such as the
gene that encodes the steroid 2
Although some progress has been made toward the identification of
cis-acting elements that may contribute to the GH regulation of these
sex-specific liver genes
(37, 39, 40) , little is
known about the intracellular signaling events that are stimulated by
GH and the pathways that lead to the differential activation in liver
of male-expressed as compared to female-expressed genes under the
influence of pituitary GH secretory patterns. Of particular interest is
the possibility that GH may stimulate alternate signaling pathways in
liver, depending on whether hepatocytes are exposed to the hormone
intermittently or continuously. The present study examines the effects
of physiological levels of GH administered to hypophysectomized rats on
nuclear protein tyrosine phosphorylation in the liver. We report that
intermittent plasma GH, but not continuous plasma GH, stimulates a high
steady-state level of tyrosine phosphorylation and nuclear
translocation of a 93,000 dalton polypeptide, p93, which we identify as
a novel DNA binding protein related to the prolactin-activable mammary
gland factor Stat 5
(41, 42) . These findings are
discussed in the context of the cellular and molecular mechanisms
through which GH secretory patterns regulate the sexual dimorphism of
liver gene expression.
Rat GH was administered to
hypophysectomized rats by one of three protocols. Protocol A:
intraperitoneal injection of GH was at 12.5 µg/100 g body weight,
except where a lower dose (3 µg/100 g body weight) is specified.
Rats were killed at time intervals ranging from 5 min to 4 h following
a single intraperitoneal injection of GH, as specified in individual
experiments. Where indicated, a second intraperitoneal injection of GH
was administered 4 h after the first injection, and the animals were
killed 45 min later. Protocol B: repeat subcutaneous injection of GH
was at 12.5 µg/100 g body weight/injection, given at t = 0, 3, and 6 h to maintain a circulating level of GH over
time, followed by sacrifice at t = 8 h. GH administered
by the subcutaneous route produces periods of continuous circulating
plasma hormone lasting 5-6 h
(28, 44) and, when
given repeatedly, confers a female profile of liver gene
expression
(45) . Protocol C: longer term continuous GH treatment
was by hormone infusion at 2 µg/100 g body weight/h for 1, 3, or 7
days using an Alzet osmotic minipump (model 2001; Alza Corp., Palo
Alto, CA). Minipumps were implanted subcutaneously on the backs of the
animals under Ketamine anesthesia using protocols provided by the
manufacturer and approved by the Boston University Institutional Animal
Care and Use Committee. Control animals received vehicle injections or
vehicle-filled osmotic minipumps. These treatments correspond to
biologically effective GH replacement doses, which mimic the
intermittent plasma GH profile of male rats (periodic intraperitoneal
GH injection) and the continuous plasma GH profile of females (repeat
subcutaneous injection or GH infusion by osmotic minipump)
(28) .
Rat GH used in these experiments was a hormonally pure grade
obtained from the National Hormone and Pituitary Program, NIDDK
(NIDDK-rGH-B-14-SIAFP, biopotency 1.8 I.U./mg). This material was
prepared by subtractive immunoaffinity purification and was shown to be
devoid of other pituitary hormones, including prolactin, TSH, FSH, and
LH. Rat GH was dissolved in 30 mM NaHCO
The present study identifies a 93,000 dalton latent
cytoplasmic DNA binding protein, designated liver Stat 5/p93, as a
likely intracellular mediator of the stimulatory effects of
intermittent plasma GH pulses on male-specific liver gene
transcription. Pituitary GH secretory profiles, which are sexually
differentiated in many species
(23, 25, 56) ,
including humans
(57, 58) , are responsible for
establishing and for maintaining the sex-dependent patterns that
characterize liver gene expression. These effects are best understood
in the rat liver model, where in males intermittent pituitary GH
secretion, and the resultant pulsatile plasma GH pattern, activates
transcription of male-expressed genes, while in females the more
frequent occurrence of pituitary GH secretory events, and the resultant
continuous plasma GH pattern, activates an adult female-specific
pattern of gene transcription
(32, 33) . Liver Stat 5/p93
is shown to be activated by GH via tyrosine phosphorylation, which
uncovers this protein's latent DNA binding potential and
stimulates its translocation from the cytosol to the nucleus. This
phosphorylation event, which is likely catalyzed by the GH
receptor-associated tyrosine kinase Jak2
(12, 13) , is an
early, probably primary response of the hepatocyte to GH stimulation.
Liver Stat 5 thus exhibits each of the general characteristics
associated with other Stat family members, including those activated by
interferons
In contrast
to other Stats, liver Stat 5 appears to be uniquely responsive to the
temporal pattern of hormone stimulation, which in the case of GH is
crucial for discriminating between the effects of male versus female plasma hormone profiles on liver gene expression. Tyrosine
phosphorylation, nuclear translocation, and activation of liver Stat
5's DNA binding potential were supported at a high steady-state
level in an intact rat model by the intermittent plasma GH pattern
associated with adult males but not by the continuous plasma GH profile
that is characteristic of adult females. Although GH-naive
hypophysectomized female rats initially responded to GH treatment by
tyrosine phosphorylation of p93 as in hypophysectomized males,
continuous GH exposure desensitized the hepatocytes and led to a
dramatic decline in the steady-state level of tyrosine-phosphorylated
p93. The relatively long time period required for complete
desensitization of liver Stat 5/p93 with respect to GH-induced tyrosine
phosphorylation (>8-24 h) suggests that this process is
mechanistically distinct from the more rapid desensitization of Jak2
kinase with respect to further GH activation (but not to subsequent
interferon-
The GH-activated,
tyrosine-phosphorylated liver nuclear protein p93 characterized in this
study was identified as a mammary gland factor/Stat 5-related protein
on the basis of its immunochemical cross-reactivity with two individual
anti-mammary Stat 5 monoclonal antibodies but not with several
monoclonal and polyclonal antibodies reactive with Stat 1 and Stat 3.
Both of the anti-Stat 5 antibodies consistently yielded two and
sometimes three bands on Western blots of GH-stimulated rat liver
nuclear protein extracts, suggesting that multiple Stat 5-related
proteins, or perhaps a single, multiply phosphorylated Stat 5 protein,
may be expressed in rat liver. With both antibodies, the uppermost Stat
5-immunoreactive band coincided with p93 detected with
anti-phosphotyrosine antibody, suggesting that it corresponds to the
tyrosine-phosphorylated form of liver Stat 5. This identification of
p93 as a liver-expressed, mammary Stat 5-related protein is strongly
supported by our finding that the GH-activated liver nuclear factor
binds specifically to a mammary Stat 5-binding DNA response element
derived from the 5`-flank of the rat
The broad
range of physiological and metabolic effects that GH has on target
tissues
(1, 2) and in particular the occurrence of
sex-dependent effects of GH secretory profiles on long bone growth and
liver gene expression suggest that GH may activate several independent
or, perhaps, parallel signaling pathways, even within a single cell
type. This proposal is supported by the finding that GH can activate
multiple Stat proteins in hepatocytes; these include liver Stat 5, as
shown in the present study, as well as Stat 3
We thank Drs. L. Argetsinger, Z. Zhong, J. Darnell,
and D. Levy for providing anti-phosphotyrosine and anti-Stat
antibodies.
Note Added in Proof-Western blot analysis
of liver Stat 5 levels in a series of intact male rats killed at
different times of the day
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
93,000 polypeptide,
p93, was found to be tyrosine phosphorylated to a high level in male
but not female rats. GH, but not prolactin, rapidly stimulated p93
tyrosine phosphorylation in hypophysectomized rats. Intermittent plasma
GH pulses triggered repeated p93 phosphorylation, while continuous GH
exposure led to desensitization and a dramatic decline in liver nuclear
p93. p93 was cross-reactive with two monoclonal antibodies raised to
mammary Stat 5, whose tyrosine phosphorylation is stimulated by
prolactin. Intermittent GH pulsation translocated liver Stat 5/p93
protein from the cytosol to the nucleus and also activated its DNA
binding activity, as demonstrated using a mammary Stat 5-binding DNA
element derived from the
-casein gene. p93 is thus a
liver-expressed, Stat 5-related DNA binding protein that undergoes
tyrosine phosphorylation and nuclear translocation in response to
intermittent plasma GH stimulation and is proposed to be an
intracellular mediator of the stimulatory effects of GH pulses on
male-specific liver gene expression.
(
)
regulates a broad
range of physiological processes, including somatic growth and
development, carbohydrate and lipid metabolism, and liver metabolic
function
(1, 2) . Some of these effects of GH are
indirect and are mediated by insulin-like growth factor 1 produced in
the liver in response to GH stimulation, while others result from the
direct action of GH on target tissues. The effects of GH on responsive
cells are transduced by GH receptor
(3, 4) , a
transmembrane protein expressed on the surface of liver, adipose,
kidney, heart, intestine, lung, and muscle cells
(5, 6) .
GH receptor is a member of the cytokine receptor superfamily
(7) and is comprised of three domains: a 246-amino acid
extracellular domain that binds to and is dimerized by a single
molecule of GH, a short transmembrane segment, and a 350-amino acid
intracellular domain that is required for transduction of intracellular
signaling events stimulated by GH
(3, 8, 9) .
91,000, designated Stat 1. In human lymphoblastoid IM9
cells, GH can activate, via tyrosine phosphorylation, a
93,000
dalton protein that may be antigenically related to Stat
1
(13, 20) . Stat 1 belongs to a family of Stat proteins
(signal transducer/activator of transcription) that mediate the
transcriptional responses stimulated by multiple growth factors and
cytokines, including interferon-
, epidermal growth factor, and
various interleukins
(21, 22) . Tyrosine phosphorylation
of Stat proteins is associated with nuclear translocation and
activation of a latent DNA binding activity, leading to transcriptional
activation of target genes
(21, 22) .
- and 16
-hydroxylase enzyme
CYP2C11, while exposure of hepatocytes to GH continuously, which occurs
in adult female rats, stimulates the expression in liver of the steroid
sulfate 15
-hydroxylase CYP2C12 (37, 38). GH replacement studies in
hypophysectomized rats have revealed a requirement for a minimum GH
``off-time'' for effective stimulation of CYP2C11 gene
expression by intermittent GH pulsation (28). This, in turn, suggests
that hepatocytes exposed to GH continuously (as in adult female rats)
become desensitized with respect to activation of the signaling pathway
that normally induces CYP2C11 gene expression in males
(28) .
Animal Treatments
Adult male and female
Fischer 344 rats (8-10 weeks of age) were untreated or were
hypophysectomized by the supplier (Taconic, Inc., Germantown, NY), with
follow-up care provided as previously described
(43) . Animals
were maintained at the Laboratory for Animal Care Facility at Boston
University (lights on 8 a.m.-8 p.m.) for at least three weeks
following surgery to ensure completeness of hypophysectomy (absence of
body weight gain). Intact, untreated male and female rats were killed
between 7:30 and 7:45 a.m.
, pH 10.3,
containing 0.15 M NaCl, and the pH was immediately lowered to
9.5 by addition of 0.5 M sodium bicarbonate, pH 8.3. Rat
albumin (Sigma) was added to 100 µg/ml to stabilize the
hormone
(27) . In other experiments, hypophysectomized rats were
treated with human GH (NIDDK-hGH-B-1, BIO) or with rat prolactin
(hormonally pure grade, NIDDK-rPRL-B-8-SIAFP) at 12.5 µg/100 g body
weight, intraperitoneally, and the ani-mals were killed 45 min later.
Escherichia coli lipopolysaccharide (LPS) (serotype 026:B6;
Sigma L-2762) was administered to hypophysectomized rats by
intraperitoneal injection at 1 mg/150 g body weight to promote cytokine
release
(46) . Animals were killed 75 min later.
Preparation of Liver Nuclear Extract and
Cytosol
Nuclear extracts were prepared from individual,
freshly excised rat livers using established methods
(47) , with
the addition of the phosphatase inhibitors sodium fluoride (10
mM) and sodium orthovanadate (1 mM) in the
homogenization and nuclear lysis buffers and with the inclusion of the
following protease inhibitors (Sigma) in the initial homogenization
buffer: antipain, chymostatin, and pepstatin (2 µg/ml each),
aprotinin, leupeptin (5 µg/ml each), trypsin inhibitor (10
µg/ml), and phenylmethanesulfonyl fluoride (0.1 mM). The
final nuclear preparation was dialyzed against NED buffer [25
mM Hepes (pH 7.6 at 4 °C), 40 mM KCl, 0.5
mM phenylmethanesulfonyl fluoride, 0.1 mM EDTA, 1
mM dithiothreitol containing 0.5 mM sodium fluoride,
1 mM sodium orthovanadate, and 10% glycerol] and then
snap-frozen and stored in liquid nitrogen. Cytosolic fractions were
prepared by dilution of the supernatant obtained after the first
centrifugation step (pelleting of nuclei through a 2 M sucrose
cushion) with 2 volumes of NED buffer, followed by centrifugation at
100,000 g for 2 h at 4 °C to pellet a microsomal
fraction.
Antibodies
Anti-phosphotyrosine
antibodies were mouse monoclonal antibody 4G10 (Upstate Biotechnology,
Inc.), mouse monoclonal antibody PY20 (Transduction Laboratories,
Lexington, KY), and rabbit polyclonal Shafer
anti-phosphotyrosine
(11, 48) ; the latter antibody was
provided by Dr. L. Argetsinger (Univ. of Michigan). Monoclonal
anti-Stat 1 antibody, raised to a peptide fragment comprised of Stat 1
amino acids 591-731 (S21120), monoclonal anti-Stat 3, raised to
Stat 3 amino acids 1-178 (S21320), and two independent monoclonal
anti-Stat 5 antibodies, both raised to amino acids 451-649 of
sheep Stat 5 (S21520, lot 2 (clone 85) and lot 3 (clone 89)), were
purchased from Transduction Laboratories. Anti-Stat 1 and
anti-Stat 3c were provided by Drs. Z. Zhong and J. Darnell (Rockefeller
University) and were used for gel supershift experiments carried out as
described
(49) . Anti-Stat 3c was raised against Stat 3 amino
acids 688-727. Antiserum AbN, raised against amino acids
1-67 of mouse APRF (i.e. mouse Stat 3)
(50) , was
provided by Dr. D. Levy (New York University School of Medicine).
Western Blot Analysis
Rat liver nuclear
extracts (15 µg protein/lane) were electrophoresed through standard
Laemmli sodium dodecyl sulfate-polyacrylamide (10%) gels,
electrotransferred to nitrocellulose, and then probed with
anti-phosphotyrosine or anti-Stat antibodies. Nitrocellulose sheets
were blocked for 1 h at 37 °C with 4% non-fat dry milk (Blotto)
(w/v) dissolved in 10 mM potassium P, pH 7.4, 0.9%
NaCl, 0.3% Tween 20 (anti-phosphotyrosine antibody) or with 3% bovine
serum albumin dissolved in 10 mM Tris-Cl, pH 7.5, 0.1
M NaCl, 0.1% Tween 20 (anti-Stat 5). In the case of the
anti-phosphotyrosine antibody probings, a special grade of
phosphotyrosine-free non-fat dry milk (Upstate Biotechnology, Inc.,
17-1056) was used to block nonspecific binding sites.
Nitrocellulose blots were washed and then probed for 1-2 h at
20-25 °C with antibodies diluted 1/3000 in blocking solution.
Detection was with horseradish peroxidase-conjugated secondary antibody
(1/3000) followed by enhanced chemiluminescence (ECL) imaging on x-ray
film using ECL reagent (Amersham Corp.).
Phosphatase Treatment
Nuclear extract
protein samples (25 µg) were incubated for 45 min at 37 °C with
protein phosphotyrosine phosphatase 1B (20 µl containing 2 µg
of glutathione S-transferase-phosphatase 1B fusion protein
conjugated to 10 µl of agarose beads (Upstate Biotechnology,
14-109)) in a total volume of 70 µl containing 25 mM
Tris-Cl, pH 7.0, 50 µM CaCl, and 25 µg of
bovine serum albumin. Phosphatase was activated by preincubation for 15
min at 37 °C as suggested by the supplier. Control samples included
the phosphatase inhibitor sodium orthovanadate (0.1 mM).
Phosphatase-treated samples were analyzed by phosphotyrosine Western
blotting and by gel mobility shift assay using a rat
-casein
promoter probe (see below).
Immunoprecipitation Analysis
Rat liver
nuclear extracts (50 µg) were incubated with 3 µg of
anti-phosphotyrosine antibody 4G10 or 2 µl of anti-Stat 3 antibody
AbN overnight at 4 °C in IP buffer (1% Triton X-100, 150
mM NaCl, 10 mM Hepes, pH 7.4, 1 mM EDTA, 1
mM EGTA, 0.2 mM sodium orthovanadate, 0.2 mM
phenylmethanesulfonyl fluoride, 0.5% Nonidet P-40 containing 1
µg/ml each aprotinin, leupeptin, and pepstatin) in a total volume
of 100 µl. Protein A-Sepharose (25 µl of a 50% suspension) was
added to each sample followed by incubation for 2 h at 4 °C with
gentle shaking. Samples were centrifuged for 4 min at 14,000
g, and the Sepharose pellet was then washed three times with
500 µl of IP buffer. The washed beads were resuspended in 30 µl
of of 2
Laemmli sample buffer (3% sodium dodecyl sulfate, 10%
2-mercaptoethanol, 2% glycerol, 200 mM Tris-HCl, pH 6.8,
containing pyronin Y dye), boiled for 10 min, and centrifuged for 4 min
at 14,000
g; the supernatant was then analyzed on 10%
sodium dodecyl sulfate-polyacrylamide gels.
Gel Mobility Shift
Analysis
Double-stranded oligonucleotide probes,
P-end labeled on one strand using T4 kinase, were
incubated for 30 min at room temperature with 5 µg of liver nuclear
extract protein dissolved in 5 µl of NED buffer and 10 µl of 10
mM Tris-HCl buffer, pH 7.5, containing 10 fmol of DNA probe, 2
µg of poly(dI-dC), 4% glycerol, 1 mM MgCl
, 0.5
mM EDTA, 0.5 mM dithiothreitol, 50 mM NaCl
(15 µl, total volume). Samples were electrophoresed at room
temperature through non-denaturing polyacrylamide gels (4% acrylamide,
0.05% bisacrylamide) in 0.5
TBE buffer (44.5 mM Tris,
44.5 mM boric acid, 5 mM EDTA, pH 8.0) for 2.5 h at
100 volts using standard methods. DNA probes used in these studies were
as follows: (a) rat
-casein gene Stat 5/MGF response
element, nucleotides -101 to -80
(51) ,
5`-GGACTTCTTGGAATTAAGGGA-3` (sense strand, oligonucleotide ON-257) and
5`-gTCCCTTAATTCCAAGAAGTCC-3` (antisense strand, ON-258) and
(b)SIE probe, 5`-gtcgaCATTTCCCGTAAATCgtcga-3` (sense
strand, ON-242) and 5`-gacGATTTACGGGAAATGtcgac-3` (antisense strand,
ON-243)
(52) . Nucleotides used to facilitate T4 kinase
5`-labeling with [
-
P]ATP or other purposes
are shown in lower case. Unlabeled double-stranded oligonucleotides
used as competitors for the gel shift experiments were obtained from
Santa Cruz Biotechnology, Inc. and included SIE mutant oligonucleotide
(mutation of TTCCCG to CCACCG; sc-2536), and GAS/ISRE consensus
oligonucleotide (sc-2537).
Liver Nuclear Factor p93: GH-dependent Tyrosine
Phosphorylation in Male but Not Female Rats
GH stimulates
the tyrosine phosphorylation of multiple cellular proteins in a
reaction catalyzed by Jak2, a tyrosine kinase that is activated
following GH binding to the GH receptor. Because many of the effects of
GH on liver gene expression are sex dependent and are regulated by the
temporal pattern of GH stimulation of hepatocytes, we examined whether
there are any differences between male and female rats in their
patterns of liver nuclear protein tyrosine phosphorylation. We examined
nuclear proteins because the sex-dependent effects of GH on liver gene
expression are manifest at the level of transcription
initiation
(37, 38) , and tyrosine phosphorylation
stimulated by cytokines and other growth factors is associated with
protein translocation to the nucleus
(21, 22) .
Fig. 1A shows a Western blot of liver nuclear extracts
prepared from adult male and adult female rat liver and probed with
anti-phosphotyrosine antibodies. Several immunoreactive protein bands
are common to both male and female nuclear extracts. In contrast, an
anti-phosphotyrosine immunoreactive protein of 93,000 daltons,
designated p93, was prominent in male liver nuclear extracts but was
present at low or undetectable levels in female liver nuclei. This same
sex-dependent pattern of p93 tyrosine phosphorylation was observed
using three distinct anti-phosphotyrosine antibodies: Shafer polyclonal
anti-phosphotyrosine (Fig. 1A) and monoclonal antibodies
4G10 (Fig. 2, see below) and PY20 (data not shown). Treatment of
the liver nuclear extract with phosphotyrosine phosphatase resulted in
a loss of the p93 band, confirming the phosphotyrosine nature of this
protein (data not shown). p93 was not detectable with
anti-phosphotyrosine antibody in nuclear-free liver cytosol, indicating
that this tyrosine-phosphorylated protein is localized in the nucleus
(see below).
Figure 1:
Tyrosine phosphorylation of nuclear p93
in male rat liver and in GH-treated hypophysectomized rats. Panel
A, Western blot of liver nuclear extracts prepared from individual
male (M) and female (F) rats probed with Shafer
anti-phosphotyrosine antibody. Panel B, Western blot of liver
nuclear extracts probed with monoclonal anti-phosphotyrosine antibody
4G10. Shown are hypophysectomized (Hx) rats that were
sham-injected (lane2) or were treated with rat GH (3
µg/100 g body weight, intraperitoneally) and then killed 5, 15, or
45 min later (lanes3-5). Treatment of
hypophysectomized male rats with prolactin (PRL) (lane6) or with LPS (lane7) was as
described under ``Experimental Procedures.'' Lane1, untreated male control.
Figure 2:
p93 is electrophoretically distinct from
Stat 1 and Stat 3. Shown are Western blots of liver nuclear extracts
probed with anti-Stat 3 (lane1),
anti-phosphotyrosine antibody 4G10 (lanes2-6),
and anti-Stat 1 (lane7). The seven nuclear samples
were electrophoresed on a single SDS gel, which was transferred to
nitrocellulose and then cut into three strips (lanes1, 2-6, and 7) for parallel
detection with the three indicated antibodies. The three strips were
aligned prior to exposure to x-ray film for detection of the
chemiluminescent signal by ECL. Liver nuclear extracts were prepared
from individual male (M) (lanes2 and
7), female (F) (lane3), and
hypophysectomized (Hx) rats that were sham-injected (lane4) or were injected with rat GH then killed 45 min later
(lanes1, 5, and 6). *, higher
molecular weight nuclear protein whose tyrosine phosphorylation is
enhanced by GH.
The influence of GH on the tyrosine phosphorylation of
p93 was examined by hypophysectomy and GH replacement experiments. p93
was not detectable on anti-phosphotyrosine Western blots of liver
nuclear extracts prepared from hypophysectomized male rats but was
restored to a level comparable with intact male rats in nuclear
extracts prepared from livers of hypophysectomized rats given a
physiologic replacement dose of rat GH and then killed either 15 or 45
min later (Fig. 1B, lanes4 and
5). Tyrosine phosphorylation of p93 was not stimulated by
prolactin (lane6), demonstrating that this
phosphorylation is a GH-specific response. Moreover, bacterial LPS,
which promotes the release of multiple cytokines and stimulates the
phosphorylation of Stat 3 both in mouse liver
(46) and in rat
liver,(
)
did not stimulate tyrosine
phosphorylation of p93 (lane7). The rapid appearance
of tyrosine-phosphorylated p93 in the nucleus following GH treatment
in vivo suggests that GH stimulates the phosphorylation of
preexisting p93 protein.
p93 Is Distinct from Stat 1 and Stat 3
GH
can stimulate the tyrosine phosphorylation of proteins similar or
perhaps identical to the Stat proteins that can be activated by various
cytokines and growth
factors
(13, 18, 19, 20) . Therefore, we
investigated whether p93 corresponds to Stat 1 or Stat 3, two Stat
proteins that are known to be expressed in liver cells. A direct
comparison of the electrophoretic mobility of p93, detected by
phosphotyrosine Western blotting with that of Stat 1 and Stat 3, which
were detected in liver nuclear extracts using their respective Stat
form-specific antibodies, revealed that p93 migrated slower than Stat 3
(major band at M
89,000 (p89)) (Fig. 2,
lane1) and slower than both bands of the Stat
1
/Stat 1
doublet (M
91,000 (p91)
and M
84,000 (p84), respectively)
(Fig. 2, lane7). No Stat 1 or Stat 3
immunoreactivity was seen on the Western blots at a position
corresponding to the migration of p93, indicating that p93 is not a
phosphorylated form of Stat 1 or Stat 3. This conclusion was confirmed
by immunoprecipitation of p93 with anti-phosphotyrosine antibody,
followed by Western blotting with anti-Stat 1 and anti-Stat 3
antibodies (data not shown).
Intermittent Plasma GH Pulsation, but Not Continuous
Plasma GH Treatment, Triggers Repeated Phosphorylation of p93 in
Vivo
We next investigated the hormonal basis for the marked
male specificity of p93 tyrosine phosphorylation seen in intact rats.
One possibility, that p93 protein is not expressed in female rat liver,
was ruled out by our finding that GH stimulated the tyrosine
phosphorylation of p93 within 15 min of a single hormone injection in
hypophysectomized female rats (Fig. 3A). Thus, female
rats are inherently responsive to a pulse of GH.
Figure 3:
Influence of intermittent versus continuous GH treatment on p93 tyrosine phosphorylation. Shown are
Western blots of rat liver nuclear extracts probed with
anti-phosphotyrosine antibody 4G10. Panel A, liver nuclear
extracts prepared from intact female rats (F, lane2) or from hypophysectomized female rats (Hx)
that were untreated (lane3) or were treated with rat
GH (3 µg/100 g body weight) and then killed 5, 15, or 45 min later
(lanes4-6). Nuclear extract from a
hypophysectomized male rat treated with rat GH and killed 45 min later
is shown for comparison in lane1. Tyrosine
phosphorylation of the band marked * was activated by GH within 5 min.
Panel B, nuclear extracts prepared from hypophysectomized male
rats that were untreated (lane2) or were killed 45
min (lanes3 and 4) or 4 h (lanes5 and 6) following a single intraperitoneal
injection of rat GH. Lanes7 and 8, nuclear
extracts prepared from hypophysectomized male rats injected with rat GH
at t = 0 and again at t = 4 h, then
killed 45 min after the second injection (GH pulses = 2) to test
for rephosphorylation of p93. Lane1, untreated male
nuclear extract. Panel C, nuclear extracts prepared from
hypophysectomized female rats exposed to GH continuously for 8 h by
repeat subcutaneous injection at t = 0, 3, and 6 h
(lanes4 and 5) or for 1 day (lanes6 and 7), 3 days (lanes8 and
9), or 7 days (lanes10 and 11)
using an osmotic minipump. Lane1, untreated male
control; lane2, hypophysectomized female rat given
multiple subcutaneous vehicle injections (control for lanes4 and 5); lane3,
hypophysectomized female rat killed 45 min after a single
intraperitoneal injection of rat GH. Several nonspecific bands are
prominent in the blot shown in panelC.
We next considered
whether the preferential tyrosine phosphorylation of p93 seen in male
rat liver may be a consequence of the differential stimulatory effect
of intermittent GH (male plasma GH profile) versus continuous
GH (female plasma GH profile). To test this hypothesis, biologically
effective replacement doses of GH were given to hypophysectomized male
rats by intraperitoneal injection at 4 h intervals to mimic the
frequency of plasma GH pulsation that occurs naturally in intact male
rats
(2, 23) and is required to stimulate male-specific,
GH-dependent liver gene expression
(28) . As shown in
Fig. 3B, GH stimulated tyrosine phosphorylation of p93
at 45 min, as seen in our earlier experiments, and this was followed by
a significant, albeit partial decline in tyrosine-phosphorylated p93
levels after 4 h. A second injection of GH at 4 h restimulated p93
tyrosine phosphorylation to a level comparable with that achieved
following the initial GH pulse. Thus, p93 can undergo repeated tyrosine
phosphorylation in response to an intermittent, adult male plasma GH
profile. By contrast, although p93 phosphorylation was initially
induced when GH was delivered to hypophysectomized rats continuously,
sustained levels of tyrosine-phosphorylated p93 were only maintained
for a limited time period. Tyrosine-phosphorylated p93 was present at a
reduced level after 8-24 h of continuous GH exposure and was very
low or undetectable after 3 or 7 days of continuous GH treatment
(Fig. 3C). Thus, the continuous plasma GH profile in
adult female rats is ineffective in supporting high steady-state levels
of tyrosine-phosphorylated p93. This indicates that hepatocytes exposed
to GH continuously become desensitized with respect to GH-induced p93
tyrosine phosphorylation.
p93 Corresponds to a GH-regulated, Mammary Stat
5-related Liver Nuclear Protein
Mammary gland factor has
recently been identified as a Stat family member, designated Stat 5,
that can be activated by prolactin
(41, 42) . The
polypeptide hormones GH and prolactin belong to the same gene family,
bind to receptors that are structurally homologous
(7) , and both
activate the tyrosine kinase Jak2 (12, 53). We therefore investigated
whether p93 might correspond to a liver-expressed, mammary Stat 5-like
protein. Western blot analysis of adult male rat liver nuclear extracts
probed with an anti-Stat 5 monoclonal antibody (clone 85) revealed a
closely migrating protein doublet of M
92,500-93,000 (Fig. 4A). The upper band
of this doublet corresponds to p93 detected with anti-phosphotyrosine
antibody, as established by the precise overlay of the upper Stat 5
band with the p93 band when the two are visualized on separate x-ray
films prepared from a single nitrocellulose immunoblot probed
sequentially with the two antibodies. In agreement with this
conclusion, treatment of the nuclear extracts with phosphotyrosine
phosphatase resulted in an apparent conversion of the upper Stat
5-immunoreactive band to the lower band (data not shown). As was
observed for p93, the Stat 5-immunoreactive protein bands were absent
or were present at much lower levels in female liver nuclear extracts
(Fig. 4A). Moreover, the liver Stat 5 protein was
increased in the nuclear fraction following GH treatment with the same
kinetics as p93 (Fig. 4B, lanes3-6; cf.Fig. 1B). A
second, independent anti-mammary Stat 5 monoclonal antibody (clone 89)
gave the same pattern of protein bands and the same pattern of liver
Stat 5 regulation, further supporting the relatedness of liver Stat
5/p93 and mammary Stat 5. Neither anti-Stat-5 antibody was
cross-reactive with Stat 1 or Stat 3, as evidenced by the absence of
detectable bands at the migration positions of the latter two Stats.
Figure 4:
Liver Stat 5/p93 detected by anti-mammary
Stat 5 monoclonal antibody in liver nuclear extracts. Shown are Western
blots (WB) of rat liver nuclear extracts (panelsA and B) or immunoprecipitates of nuclear
extracts (panelC) probed with anti-Stat 5 monoclonal
antibody (clone 85); similar results were obtained with a second
anti-Stat 5 monoclonal antibody (clone 89; data not shown). Panel
A, male-dominant nuclear expression of liver Stat 5 protein,
evident from nuclear extracts prepared from four independent pairs of
male (M) and female (F) rats. Panel B, rapid
increase in liver Stat 5 in nucleus of hypophysectomized (Hx)
male rats treated with GH (3 µg/100 g body weight,
intraperitoneally), but not prolactin (PRL) or LPS, as
indicated. Panel C, nuclear extracts shown in panelB were immunoprecipitated with anti-phosphotyrosine
antibody 4G10 (anti-phosphotyrosine) and analyzed by sequential Western
blotting with anti-phosphotyrosine then anti-Stat 5, as indicated in
lanes1-7. Lane8, liver
nuclear extract from untreated male rat included as a
standard.
The identification of p93 as the Stat 5-immunoreactive protein seen
on these Western blots was confirmed by immunoprecipitation of p93 from
rat liver nuclear extracts with anti-phosphotyrosine antibody 4G10,
followed by SDS-gel electrophoresis and sequential Western blotting
with anti-phosphotyrosine and then anti-Stat 5 antibody
(Fig. 4C). This identification is further supported by
immunoprecipitation of the tyrosine-phosphorylated and the Stat
5-immunoreactive p93 protein by antibody AbN (data not shown). AbN is a
polyclonal antibody that was raised to a conserved region at the amino
terminus of Stat 3 and is cross-reactive with several unidentified
Stat-like proteins
(50) , including the liver Stat 5-related
protein p93. Nuclear accumulation of liver Stat 5 was
repeatedly stimulated by an intermittent pattern of GH replacement, as
revealed by Western blot analysis with anti-Stat 5 antibody
(Fig. 5A). This effect was more apparent when
tyrosine-phosphorylated liver Stat 5 (i.e. the upper band of
the Stat 5 doublet seen in Fig. 5A) was selectively
immunoprecipitated with anti-phosphotyrosine antibody
(Fig. 5B). The nuclear level of liver Stat 5 was also
suppressed by continuous GH treatment in a manner indistinguishable
from that of p93 (data not shown). Liver Stat 5/p93 apparently differs
from Stat 5 cloned from sheep mammary gland
(41) , however,
insofar as it does not respond to prolactin (Fig. 4B,
lane7, and Fig. 4C, lane6; cf. unresponsiveness of p93,
Fig. 1B).
Figure 5:
Elevation of nuclear levels of liver Stat
5 by intermittent GH stimulation. Panel A, shown is a Western
blot (WB) of liver nuclear extracts prepared from individual
hypophysectomized (Hx) male rats treated with GH by
intermittent intraperitoneal injection to mimic the pulsatile plasma GH
profile of intact males, using the protocol described under Fig.
3B, and then probed with anti-Stat 5 antibody (clone 85).
Panel B, samples from the experiment shown in panelA were immunoprecipitated with anti-phosphotyrosine
antibody 4G10 and then probed sequentially with anti-phosphotyrosine
4G10 and then anti-Stat 5 antibodies, as indicated. Samples are from
the same experiment shown in Fig. 3B. Samples shown in
panelB, lanes5 and 6,
correspond to the individual liver nuclear extracts shown in panelA, lanes6 and
7.
GH Stimulates Tyrosine Phosphorylation of Cytosolic
Liver Stat 5/p93
Liver cytosolic extracts were analyzed by
Western blotting to determine whether hepatocytes contain a cytoplasmic
pool of the mammary Stat 5-related protein that could serve as a
precursor of the tyrosine-phosphorylated p93, which accumulates in the
nucleus following GH stimulation. Western blot analysis of these
samples revealed a Stat 5-immunoreactive protein that is expressed at a
low level in liver cytosol in hypophysectomized rats
(Fig. 6A, lane4), as well as in
intact male and female rats (data not shown). The cytosolic Stat
5-immunoreactive protein seen in these samples (designated Stat 5
band b, Fig. 6A) migrated distinctly faster
than the liver Stat 5 protein seen in the nucleus (lane4versus3). Moreover, the cytosolic protein was
not tyrosine phosphorylated, as determined by immunoblotting with
anti-phosphotyrosine antibody (Fig. 6B, lane4). GH treatment led to a noticeable decrease in the
mobility of the liver Stat 5 protein detected in the cytosol (i.e. conversion of band b to band a). This mobility
decrease was first detectable at 5 min, as revealed by a longer
exposure of the blot shown in Fig. 6A, and was maximal
at 15 and 45 min after hormone injection (lanes5-7versus4). The lower mobility liver Stat 5
protein present in the cytosol (band a,
Fig. 6A) corresponds to the tyrosine-phosphorylated
form, as determined by anti-phosphotyrosine Western blotting
(Fig. 6B). Moreover, the time course for appearance of
this upper band (lanes5-7) paralleled the
appearance of the tyrosine-phosphorylated p93 in the nucleus (cf.lanes2 and 3). Thus, GH induces a
rapid tyrosine phosphorylation of liver Stat 5 present in a cytosolic
precursor pool, and this is closely followed by translocation of the
phosphorylated protein to the nucleus.
Figure 6:
GH-stimulated phosphorylation of liver
cytosolic Stat 5. Shown are Western blots of liver nuclear extracts (15
µg/lane; lanes1-3) and the corresponding
cytosols (70 µg/lane; lanes4-9) probed
sequentially with anti-Stat 5 (clone 85) (panel A) and
anti-phosphotyrosine antibody 4G10 (panel B).
Hypophysectomized (Hx) male rats were untreated (lanes1 and 4) or were treated with rat GH (3
µg/100 g body weight, intraperitoneally) and then killed 5 min
(lanes2 and 5), 15 min (lanes3 and 6), or 45 min later (lane7). Hypophysectomized rats treated with prolactin
(PRL, lane8) or LPS (lane9) are included as controls. Two bands were detected with
anti-Stat 5 in hypophysectomized liver cytosol (bands marked
a and b); bandb predominates in
the hypophysectomized rat liver sample (lane4) and
is converted to the more slowly migrating band a following
treatment with GH but not prolactin or LPS. The tyrosine-phosphorylated
p93 band seen in panelB coincides with Stat 5,
band a, as determined by direct overlay of the x-ray films of
the two ECL probings. The changes in cytosolic Stat 5 banding pattern
shown in lanes4-7 for hypophysectomized male
rats were also observed following GH treatment of hypophysectomized
female rats (data not shown).
GH Activates DNA Binding Activity of Liver Stat
5/p93
In view of the immunochemical similarity between
liver Stat 5/p93 and mammary gland Stat 5, we examined the effects of
GH treatment on liver nuclear protein binding to a mammary Stat
5-binding DNA response element found upstream of the rat -casein
gene
(51) . As shown in Fig. 7A, a discrete gel
mobility shift complex was formed upon incubation of the
-casein
promoter DNA probe with nuclear extracts prepared from male but not
female or hypophysectomized male rat liver. This nuclear DNA binding
activity was rapidly increased by a pulse of GH given to either male or
female hypophysectomized rats (Fig. 7B), with kinetics
similar to the tyrosine phosphorylation of p93 (cf.Fig. 1B and 3A). By contrast, neither
prolactin nor LPS induced this gel mobility shift complex. Continuous
GH treatment suppressed this nuclear protein binding activity in
parallel to p93 and liver Stat 5 protein (e.g.Fig. 7CversusFig. 3C). A
low level of DNA binding activity was detected in cytosolic extracts of
GH-treated hypophysectomized rats (Fig. 7D, lanes4 and 5), in agreement with the low level of
tyrosine-phosphorylated liver Stat 5/p93 protein in liver cytosol.
Little or no gel shift complex was formed by cytosolic extracts of
hypophysectomized male rat liver (Fig. 7D, lane2), despite the presence of liver Stat 5-immunoreactive
protein (albeit not tyrosine-phosphorylated protein) (cf.Fig. 6
, lane 4). This suggests that tyrosine
phosphorylation is required to activate the DNA binding activity of
liver Stat 5. This conclusion is supported by the loss of DNA binding
activity upon dephosphorylation of liver Stat 5/p93 by treatment with
phosphotyrosine-specific phosphatase (Fig. 7E). Thus,
GH-stimulated tyrosine phosphorylation and nuclear translocation of
liver Stat 5/p93 is associated with a specific activation of this
factor's latent binding activity toward a Stat 5 DNA response
element.
Figure 7:
Gel mobility shift analysis of liver Stat
5/p93 DNA binding activity using -casein promoter probe. Liver
nuclear extracts were analyzed by gel mobility shift assay using a rat
-casein gene mammary Stat 5 response element probe. Panel
A, liver nuclear extracts prepared from individual male
(M) (lanes1, 2), female
(F) (lanes3 and 4), and
hypophysectomized male rats (lane5). Panel
B, time course for activation of liver Stat 5/p93 following
intraperitoneal injection of rat GH (3 µg/100 g body weight) in
hypophysectomized (Hx) male (lanes2-5) and female rats (lanes6-9). Lane1, intact male rat
liver nuclear extract (M). Panel C, influence of
continuous GH treatment in hypophysectomized female rats for times
ranging from 45 min to 3 days. Samples analyzed are the same ones shown
in Fig. 3C. A second, lower mobility protein-DNA complex with
the same binding specificity as the major complex is evident in this
experiment. Panel D, gel shift activity detected in liver
cytosol (20 µg; lanes2-5) of male
hypophysectomized rats treated with GH. Samples are the same as those
shown in Fig. 6. Gel shift activity of liver nuclear extract from
untreated male rat (5 µg) is shown for comparison in lane1. Panel E, effect of phosphotyrosine
phosphatase (PY-PTase) treatment on gel shift complex
formation. Liver nuclear extracts from two individual untreated male
rats were incubated for 45 min at 37 °C either with or without
phosphatase (PTase) in the absence or presence of 0.1
mM sodium orthovanadate, as indicated. Panel F,
effect on gel shift complex formation of unlabeled DNA competitors
present at a 20-fold (lane2) or 50-fold (lanes3-6) molar excess over
P-labeled
-casein probe. Competitors used were
-casein probe (lanes2 and 3), SIE (lane4), SIE
mutant (lane5), and GAS/ISRE (lane6).
Competition experiments were carried out to examine the
relationship of the GH-inducible, liver Stat 5 protein/-casein DNA
gel shift complex to protein-DNA complexes formed by other Stat
proteins (Fig. 7F). Formation of the
-casein DNA
complex was fully inhibited by unlabeled
-casein DNA probe
(lanes2 and 3) but was not inhibited by an
oligonucleotide containing a consensus binding site
(21) for the
interferon-
-activated GAS/ISRE sequence of the Ly6 gene (lane6); thus, liver Stat 5/p93 does not bind to the GAS site.
The high affinity c-sis-inducible element of the human
c-fos gene (SIE site m67
(54) ), which binds tightly to
a broad range of activated Stat
proteins
(49, 50, 52, 55) , inhibited
-casein DNA complex formation only partially (lane4), suggesting that liver Stat 5/p93 binds to the
-casein promoter site with a substantially higher affinity than to
the SIE site. The partial inhibition by SIE is specific, however, since
it was not observed when using an SIE probe containing a mutated
binding site (lane5versus4). The
distinct nature of the GH-inducible liver Stat 5-
-casein gel shift
complex was further highlighted by the inability of antibodies to Stat
3 (or to Stat 1) to supershift this complex, despite the strong
supershift conferred by this antibody on a complex formed between the
SIE probe and LPS-activated rat liver nuclear extracts (data not
shown).
and
, epidermal growth factor, interleukin 6,
and various other cytokines and growth
factors
(21, 49, 59, 60) .
activation), which is seen in cultured IM-9 cells
exposed to GH continuously for 1 h
(13) . Additional studies are
required to establish the precise mechanism for the desensitization
observed in our in vivo studies. Conceivably, this
desensitization may involve a feedback inhibitory effect of continuous
GH that is manifest (a)at the level of GH receptor
activation via dimerization
(8) , (b)at the
level of Jak2 kinase recruitment and
activation
(12, 13) , or (c)by the
availability of functionally active cytoplasmic liver Stat 5 protein
for tyrosine phosphorylation. This desensitization is unlikely to
reflect plasma membrane GH receptor down-regulation in response to
prolonged GH stimulation, since GH receptors are, in fact, up-regulated
by continuous GH treatment and, consequently, are abundant on the
hepatocyte surface in female rats (61, 62).
-casein gene. The inability
of a GAS oligonucleotide or a high affinity SIE oligonucleotide, which
contain binding sites for Stat proteins 1 and 3, to significantly
compete with this DNA binding site lends further support to the
distinct nature of liver Stat 5. Although liver Stat 5 is thus related
to Stat 5 cloned from sheep mammary gland, liver Stat 5 and mammary
Stat 5 seem to be different, since the latter Stat is not detectably
expressed at the mRNA level in liver tissue
(41) . Moreover,
prolactin, which activates mammary Stat 5
(41) , did not activate
liver Stat 5 in our experiments, as judged by the lack of an increase
in p93 tyrosine phosphorylation or liver Stat 5 protein in liver nuclei
and by the absence of an effect on liver nuclear DNA binding activity
assayed with the
-casein Stat 5 binding site probe. This lack of
an effect of prolactin may reflect the absence of the
prolactin-activable mammary Stat 5 in liver, since the two other
cellular components required for prolactin-induced Stat 5 activation,
i.e. the long form of prolactin receptor
(7, 63) and Jak2 kinase
(53, 64, 65) , are
reportedly present in hepatocytes. Liver Stat 5 may not be restricted
to hepatocytes, as suggested by the presence of GH-activated,
tyrosine-phosphorylated protein(s) of similar size in other
GH-responsive cell types
(13, 20, 66) . Further
studies, including cloning of liver Stat 5 and direct study of its
responsiveness to GH and prolactin, are needed to establish the precise
relationship between liver Stat 5 and mammary Stat 5.
and, under
certain conditions, Stat 1
(19) .
It seems likely
that these three GH-responsive Stat proteins will contribute to the
activation of different subsets of GH-responsive genes and conceivably
may each respond in a distinct manner to the temporal pattern of
circulating plasma GH levels. This would help explain the broad
diversity of the effects of GH, as well as the restriction of sex- and
plasma hormone profile-dependent effects of GH to a subset of
GH-activable genes. For instance, the rapid activation of the c-fos gene by GH
(67, 68) could in part be mediated by
the binding of GH-activated Stat 3 and/or Stat 1 to the SIE sequence
found within a regulatory element upstream of that gene
(54) ,
while activation of the liver cytochrome P450 gene CYP2C11 by
intermittent GH pulsation
(37, 38) could be mediated by
the binding of GH-activated liver Stat 5 to cognate regulatory elements
within or adjacent to CYP2C11. However, since the stimulatory effects
of GH pulses on CYP2C11 gene expression take at least 1-2 days to
be manifest
(69) , as compared with the activation of liver Stat
5/p93 within 15 min of GH treatment (this study), the induction of
CYP2C11 gene transcription by GH pulsation may be an indirect response
to liver Stat 5 activation. Further studies will be required to
determine the molecular mechanisms through which liver Stat 5 activates
target genes in response to GH pulses, as well as the cellular
mechanism through which hepatocytes exposed to continuous GH become
desensitized with respect to liver Stat 5 activation.
revealed a striking individual
variability in liver nuclear Stat 5 levels, with a strong positive
correlation between the occurrence of liver Stat 5 protein in the
nucleus and the presence of GH in the plasma at the time of death. This
finding provides strong additional support for our conclusion that
liver Stat 5 undergoes repeated cycles of tyrosine phosphorylation and
nuclear translocation in response to naturally occurring plasma GH
pulses in intact adult male rats.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.