From the Institute of Molecular and Cell Biology,
117609 Singapore, Republic of Singapore and the Departments of
§ Physiology and ¶ Internal Medicine, Sahlgrenska
University Hospital, University of Göteborg,
Göteborg S-41345, Sweden
Received for publication, July 26, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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In the rat, a growth hormone-binding protein
(GHBP) exists that is derived from the growth hormone (GH) receptor
gene by an alternative mRNA splicing mechanism such that the
transmembrane and intracellular domains of the GH receptor are replaced
by a hydrophilic carboxyl terminus. In isolation, the GHBP is inactive, although it does compete with the receptor for ligand binding in the
extracellular space and therefore inhibits the cellular response to GH.
The GHBP is also located intracellularly and is translocated to the
nucleus upon ligand stimulation. We show here that endogenously
produced GHBP, in contrast to exogenous GHBP, was able to enhance the
STAT5-mediated transcriptional response to GH. Interestingly, when the
GHBP was targeted constitutively to the nucleus by the addition of the
nuclear localization sequence of the SV40 large T antigen,
greater enhancement of STAT5-mediated transcription was obtained. The
transcriptional enhancement did not require GH per se and
was not specific to the GH receptor, since similar enhancement of
STAT5-mediated transcription by nuclear localized GHBP was obtained
with specific ligand stimulation of both prolactin and erythropoietin
receptors. Thus, the GHBP exerts divergent effects on STAT5-mediated
transcription depending on its cellular location. The use of a soluble
cytokine receptor as a location-dependent transcriptional
enhancer and the ligand-independent involvement of the extracellular
domain of a polypeptide ligand receptor in intracellular signal
transduction provide additional novel mechanisms of transcriptional control.
Growth hormone (GH)1 is
the major regulator of postnatal body growth and initiates its
biological actions, including the induction of a number of RNA species
in mammalian tissues, by interaction with a specific membrane-bound
receptor (1, 2). The GH receptor was the first cloned member of the now
extensive cytokine receptor superfamily, which includes the receptors
for prolactin (PRL), erythropoietin (EPO), granulocyte
colony-stimulating factor, granulocyte-macrophage colony stimulating
factor, ciliary neutrophic factor, thrombopoietin, leptin,
cardiotrophin I, and the A soluble rat growth hormone-binding protein (GHBP) exists that is
derived from the GH receptor gene by an alternative mRNA splicing
mechanism such that the transmembrane and intracellular domains of the
GH receptor are replaced by a hydrophilic carboxyl-terminal sequence
(12). An analogous GHBP exists in other species (13), such as humans,
but is derived by proteolytic cleavage of the full-length
membrane-bound receptor (14), presumably by the action of specific
metalloproteases (15). The GHBP has been demonstrated to compete with
the receptor for ligand binding in the extracellular space and has been
shown to inhibit the cellular response to GH in vitro (16,
17). In vivo, the GHBP has been demonstrated to increase the
biological activity of GH by prolongation of the half-life of plasma GH
(18). The GHBP is also located intracellularly (19-21) and has also
been prominently localized to the nucleus (20). Other components of the
GH signal transduction pathway are also located in the nucleus or
translocate to the nucleus upon GH stimulation (21-25). Thus, the GH
receptor is subject to ligand-dependent nuclear
translocation (21), and constitutively nuclear JAK2 is phosphorylated
by exogenous GH stimulation (24). Internalization of the GH receptor
has been reported not to be necessary to achieve transcriptional
activation by GH (26), and therefore the function of the nuclear
localization of components of the GH signal transduction pathway is unknown.
We demonstrate here that nuclear localized GHBP functions as a potent
enhancer of STAT5-mediated transcription, not only for GH but also for
other members of the cytokine receptor superfamily. Thus, the GHBP
exerts opposing effects on STAT5-mediated transcription depending on
its extra-/intracellular location. The use of a soluble cytokine
receptor as a location-dependent transcriptional enhancer and the ligand-independent involvement of the extracellular domain of a
polypeptide ligand receptor in intracellular signal transduction provides additional novel mechanisms of transcriptional control.
Materials--
Human growth hormone (hGH) was a generous gift of
Novo Nordisk (Singapore). oPRL and rGH were obtained from NIDDK
(National Institutes of Health), and mEPO was purchased from Roche
Diagnostics. All cell culture medium and supplements (for culture
medium) were obtained from Sigma. The luciferase assay system was
purchased from Promega (Madison, WI). The ECL kit was obtained from
Amersham Biosciences. The GH, PRL, and EPO receptor cDNAs used here
were as described previously (27). Transfection reagent DOTAP,
poly(dI/dC) and the DNA 3'-end labeling kit were purchased from Roche
Diagnostics. Monoclonal antibody against hemagglutinin was obtained
from Clontech, monoclonal antiserum against
phospho-STAT5A/B were from Upstate Biotechnology, Inc. (Lake Placid,
NY), monoclonal antisera against green fluorescent protein (GFP) were
from Molecular Probes, Inc. (Eugene, OR), and polyclonal antibody
against STAT5B were from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA). mAb 4.3 and recombinant rat GHBP were generous gifts of Dr.
W. R. Baumbach (Monsanto Corp.). The production and
characterization of the recombinant rat GHBP has been previously
described by us (20).
Generation of Stable Cell Transfectants--
BRL cells were
stably transfected with the complete rat GH receptor cDNA inserted
into an expression vector containing the human cytomegalovirus enhancer
and promoter (pcDNA1). The characterization and use of these cells
has previously been described in detail (28). These cells will be
referred to as BRL-GHR1-638 cells.
Cell Culture--
BRL cells were grown in DMEM (supplemented
with 10% heat-inactivated fetal calf serum (FCS), 100 units/ml
penicillin, 100 µg/ml streptomycin, and 2 mM
L-glutamine) at 37 °C in 5% CO2. hGH, rGH,
oPRL, and recombinant rat GHBP were prepared as a stock solution of 1 mg/ml in distilled water. For treatment of cells, hGH, rGH, oPRL, mEPO,
and recombinant rat GHBP were diluted in fresh DMEM serum-free medium
and added to the cells after transient transfection. Cells were treated
with 100 nM hGH unless otherwise specified. oPRL was used
at 100 nM. mEPO was used at 10 units/ml.
Construction of Expression Plasmids--
The cDNA expression
plasmid encoding the wild type GHBP under the control of the
metallothionein Ia promotor was as previously described (17). In the
XS-GHBP construct, the rat GHBP was PCR-amplified without its
NH-terminal signal sequence, and an ATG was introduced in the primer
just upstream of where the mature GHBP protein is coded. The nuclear
localization sequence (NLS)-GHBP was constructed in a similar way, but
a nuclear localization signal from the SV40 large T antigen (PKKKRKV)
(29) was added upstream of where the mature GHBP is coded. For
GHBP-GFP, wild type GHBP was subcloned into a N-terminal enhanced green
fluorescent protein vector from Clontech (pEGFP-N3)
under the control of a cytomegalovirus IE promoter. For the
construction of epitope-tagged GHBP mutants, wild type GHBP and
NLS-GHBP with or without a stop codon at the position of amino acid 115 were subcloned into a pCI-neo vector with a double hemagglutinin
tag at the N terminus. The integrity of the reading frame for the GHBP
modifications was confirmed by sequence analysis. The construction of
GH receptor cDNA expression plasmids containing a deletion, a
deletion of box 1 ( Transient Transfection and Reporter Assay--
BRL and
BRL-GHR1-638 cells were cultured to confluence in six-well
plates. Transient transfection was performed in serum-free DMEM with
DOTAP according to the manufacturer's instructions. 1 µg of reporter
plasmid (SPI-GLE1-CAT) and 1 µg of pSV2-LUC were transfected per
well. The control or empty vectors served to normalize the amount of
DNA transfected. For receptor cDNA transfection into BRL cells, 1 µg of each receptor cDNA was used. Cells were incubated with
DOTAP/DNA for 12 h before the medium was changed to serum-free
DMEM containing either the respective hormones or GHBP at the indicated
concentrations. After a further 24 h, cells were washed in PBS and
scraped into lysis buffer. The protein content of the samples was
normalized, and CAT and luciferase assays were performed as previously
described (31). Results were normalized to the level of luciferase to
control for transfection efficiency and calculated as the -fold
stimulation of unstimulated (non-hormone-treated) cells.
Confocal Laser-scanning Microscopy--
BRL cells were grown on
glass coverslips in six-well plates and transiently transfected as
described above. Fixation was performed with PBS, pH 7.4, containing
4% paraformaldehyde for 10 min at room temperature. Cells were
permeabilized with PBS, 0.1% Triton X-100 for 1 min and processed for
immunofluorescence as described (32). The location of the expressed
GHBP was determined using the mAb 4.3 directed against the hydrophilic
carboxyl terminus of the GHBP (20). Noncross-reactive mAbs 50.8 and 7 at the same concentration were used as control (20). Labeled
cells were visualized with a Carl Zeiss Axioplan microscope equipped
with epifluorescence optics and a Bio-Rad MRC1024 confocal laser system.
For the GFP-GHBP translocation, BRL-GHR1-638 cells were
cultured to confluence in six-well plates on 50-µm-thick sapphire glass coverslips. Transient transfection was performed in serum-free DMEM with DOTAP according to the manufacturer's instructions. Cells
were kept for 12 h in serum-free medium and then stimulated with
50 nM hGH or transferred into DMEM containing 10% fetal
bovine serum as indicated. The cells were fixed by using an
ethane-freezing/methanol fixation (33). To enhance the GFP signal, the
fixed cells were exposed to an anti GFP-antibody.
Western Blot Analysis--
Medium from BRL cells transiently
transfected with the different GHBP constructs was collected and
concentrated. Fractions were normalized for protein content and loaded
onto a 7.5% polyacrylamide gel as described (32). Proteins were
transferred to nitrocellulose membranes using a semidry apparatus in
Laemmli electrophoresis buffer containing 15% methanol. Membranes were
blocked for 1 h with 5% skim milk powder in TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4). mAb 4.3 at
0.25 µg/ml in TTBS (TBS plus 0.1% Tween 20) with 1% skim milk
powder was used for GHBP detection. Membranes were further processed
and developed using the ECL system as previously described (32).
Gel Electrophoretic Mobility Shift Assay--
The gel
electrophoretic mobility shift assay was performed according to
standard protocols. The binding reactions were performed by
preincubating 10 µg of nuclear extracts or cytosolic extracts of both
hGH-treated and -untreated control vector and NLS-GHBP-transfected cells with 3 µg of poly(dI-dC) acid in 15 ml of buffer containing 20% Ficoll, 60 mM HEPES, pH 7.9, 20 mM Tris,
pH 7.9, 0.5 mM EDTA, and 5 mM dithiothreitol
for 15 min on ice. For supershift analysis, the extracts were incubated
with the antibodies against STAT5B (Santa Cruz Biotechnology) or
control antibodies for another 10 min on ice.
32P-3'-end-labeled double-stranded SPI-GLE1 probe was added
(5'-TGTTCTGAGAAATA-3'), and the mixture was incubated for 5 min on ice
followed by another 10 min at room temperature. The samples were
electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.25×
TBE (22.5 mM Tris borate, pH 8.0, 0.5 mM EDTA)
at 250 V at 4 °C for 2 h. The gel was dried, and the
radioactive pattern was then visualized by autoradiography.
Statistics and Presentation of Data--
All experiments were
repeated at least three times. Figures presented for Western blot
analyses are representative of multiple experiments. All numerical data
are expressed as mean ± S.E., and the data were analyzed using
Instat 3.0 from GraphPad Software Inc.
Effect of Exogenous GHBP on GH-stimulated STAT5-mediated
Transcription--
We have used a BRL (Buffalo rat liver) cell
co-transfection assay (28) to study the role of the GHBP in the signal
transduction pathway of GH. The characterization and use of BRL cells
stably transfected with GH receptor cDNA has previously been
described in detail (28), and these cells will be referred to as
BRL-GHR1-638 cells. We first used the
BRL-GHR1-638 cells to demonstrate the effect of
exogenously added recombinant rat GHBP on GH-stimulated STAT5-mediated
transcription utilizing a reporter plasmid containing the STAT5 binding
element of the serine protease inhibitor 2.1 gene promoter
(SPI-GLE1-CAT) (31). Exogenously added recombinant rat GHBP decreased
in a dose-dependent manner the GH stimulation of
STAT5-mediated transcription (Fig. 1).
With the GH concentration fixed at 1 nM, a
dose-dependent inhibition of GH-stimulated STAT5-mediated transcription was observed over the range of 1-100 nM GHBP
with an ED50 of 10 nM. The inhibition of
GH-stimulated STAT5-mediated transcription is in accord with previous
demonstrations that exogenous GHBP inhibits GH-stimulated function
(16).
Effect of Endogenous GHBP on GH-induced STAT5-mediated
Transcription--
The GHBP has also previously been localized
intracellularly both attached to intracellular membranes (19, 20) and
soluble in the cytoplasm and nucleoplasm (20). To determine the effect of endogenously produced GHBP on the GH-stimulated STAT5-mediated transcriptional response, we transiently transfected BRL cells with
both GH receptor cDNA and WT-GHBP cDNA and examined the
STAT5-mediated transcriptional response to GH. The transfected WT-GHBP
cDNA resulted in GHBP protein expression in a predominantly
perinuclear location and also secretion of the GHBP to the
extracellular space (see Fig. 2). In
contrast to exogenously added GHBP, 1 µg of transiently transfected
WT-GHBP cDNA resulted in a significant increase in the
STAT5-mediated transcriptional response to GH (Fig.
3A). This effect was observed
at concentrations up to 100 nM GH, and the subsequent
decrease in GH-stimulated STAT5-mediated transcription at GH
concentrations higher than 100 nM is consistent with
antagonism of the GH effect at high ligand concentrations (34).
Transfection of increasing amounts of WT-GHBP cDNA resulted in less
enhancement of the GH-stimulated STAT5 transcriptional response
presumably due to increased secretion of GHBP to the medium with
the consequent inhibition of GH function. Transient transfection of
WT-GHBP cDNA exerted no significant effect on STAT5-mediated
transcription in the absence of concomitant transfection of GH receptor
cDNA (Fig. 3B). Transient transfection of WT-GHBP
cDNA into BRL-GHR1-638 cells produced similar
results as transient transfection of both WT-GHBP and GH
receptor cDNA (data not shown). These results indicated that
extracellular and intracellular GHBP exerted opposing effects on
GH-stimulated STAT5-mediated transcription.
Cytoplasmic GHBP Enhances GH-stimulated STAT5-mediated
Transcription--
To determine whether nonsecreted cytoplasmically
localized GHBP would enhance GH-stimulated STAT5-mediated
transcription, we removed the secretion sequence from the GHBP cDNA
(XS-GHBP) (12). In the XS-GHBP construct, the rat GHBP was
PCR-amplified without its signaling peptide, and an ATG was introduced
in the primer just upstream of where the mature GHBP protein is coded. XS-GHBP is expressed throughout the cytoplasm of the cell as observed by confocal laser-scanning microscopy and is not secreted to the extracellular space (Fig. 2). Transient transfection of XS-GHBP cDNA also increased the STAT5-mediated transcriptional response to
GH. Transfection of increasing amounts of XS-GHBP cDNA further enhanced GH-stimulated STAT5-mediated transcription. As observed with
WT-GHBP, transient transfection of XS-GHBP cDNA exerted no significant effect on STAT5-mediated transcription in the absence of
concomitant transfection of GH receptor cDNA (Fig.
4). Thus, the intracellular GHBP enhances
GH-stimulated STAT5-mediated transcription independent of its secretion
to the extracellular space.
Ligand-dependent Nuclear Translocation of the
GHBP--
It has been previously reported that the GHBP is located
intracellularly in both the cytoplasm and the nucleus in both an insoluble form attached to membranes and a soluble form in the cytoplasm or nucleoplasm. To determine whether the GHBP was subject to
ligand-dependent nuclear translocation, we subcloned the
WT-GHBP into a N-terminal enhanced fluorescent protein vector. The
WT-GHBP was therefore expressed as a fusion protein to the N terminus of the enhanced GFP. The integrity of the reading frame was
confirmed by sequence analysis, and protein expression was examined by
Western blot analysis and confocal laser-scanning microscopy (Fig.
5A). The WT-GHBP-GFP was
expressed as a protein with a molecular mass of 67 kDa in contrast to
the native GFP, which was expressed as a protein of 27 kDa. Examination
of the cellular distribution of the WT-GHBP-GFP demonstrated distinct
perinuclear localization in contrast to the native GFP, which exhibited
a diffuse cytoplasmic distribution. We transfected GHBP-GFP in
BRL-GHR1-638 cells to demonstrate the effect of exogenous
GH stimulation on the cellular distribution of GHBP. Stimulation of
cells with 50 nM hGH first resulted in a contraction of the
perinuclear distribution of the WT-GHBP-GFP and subsequent nuclear
translocation, which intensified to 30 min after stimulation with GH.
In contrast, the native GFP was not translocated to the nucleus upon
cellular stimulation with GH. Cellular stimulation with fetal bovine
serum also resulted in nuclear translocation of the WT-GHBP-GFP (Fig.
5) but not the native GFP. Thus, the GHBP is subject to
ligand-dependent nuclear translocation indicative of an
intracellular/intranuclear function. We therefore focused our attention
on the nuclear GHBP.
Effect of Nuclear Localized GHBP on GH-stimulated STAT5-mediated
Transcription--
To examine the function of the nuclear localized
GHBP, we introduced the NLS of the SV40 large T antigen at the N
terminus of the GHBP (NLS-GHBP). The NLS-GHBP was constructed by
replacement of the secretion sequence of the WT-GHBP with the nuclear
localization signal from the SV40 large T antigen (PKKKRKV) (29). The
integrity of the reading frame for the GHBP modification was confirmed
by sequence analysis, and translation and location of the protein product was determined by confocal laser-scanning microscopy (Fig. 2).
The NLS-GHBP was predominantly localized to the nucleus and was not
secreted from the cell and therefore could be utilized to study the
functional effect of nuclear localized GHBP on GH-stimulated STAT5-mediated transcription. Transient transfection of BRL cells with
both GH receptor cDNA and NLS-GHBP cDNA resulted in a dramatic enhancement of GH-stimulated STAT5-mediated transcription (Fig. 6A). Transient transfection of
BRL-GHR1-638 cells with NLS-GHBP cDNA also resulted in
dramatic enhancement of GH-stimulated STAT5-mediated transcription
(data not shown). The transcriptional enhancing effect of NLS-GHBP on
GH-stimulated STAT5-mediated transcription was increased with the
transfection of increasing amounts of NLS-GHBP cDNA (Fig.
6A). No STAT5-mediated transcriptional response to GH was
obtained upon transfection of NLS-GHBP cDNA without concomitant transfection of GH receptor cDNA (Fig. 6A). The ability
of NLS-GHBP to enhance GH-stimulated STAT5-mediated transcription was
observed over a wide concentration range of both the homologous rat GH (Fig. 6B) and human GH (Fig. 6C). The ability of
the nuclear localized GHBP to enhance GH-stimulated transcription was
not observed when a STAT1/3-responsive reporter plasmid (c-fos-SIE-CAT)
(35) was utilized instead of the STAT5 reporter (SPI-GLE1-CAT) (data
not shown). Thus, the transcriptional enhancing effect of the nuclear localized GHBP has some specificity for GH-stimulated STAT5-mediated transcription. Similarly transient transfection of NLS-GHBP cDNA does not alter the activity of reporter plasmids that constitutively express either CAT (pCMV-CAT) or luciferase (pSV2-LUC).
Regions in the Intracellular Domain of the GH Receptor
Required for NLS-GHBP to Enhance GH-stimulated STAT5-mediated
Transcription--
As described above, no STAT5-mediated
transcriptional response to GH was obtained upon transfection of
NLS-GHBP cDNA without concomitant transfection of GH receptor
cDNA. We therefore next examined the regions in the intracellular
domain of the GH receptor required for NLS-GHBP to enhance
GH-stimulated STAT5-mediated transcription. NLS-GHBP cDNA was
co-transfected into BRL cells with the cDNA encoding various
receptor mutations or deletions as indicated in Fig.
7. No GH stimulation of STAT5-mediated
transcription was observed with GH receptor mutations that lacked the
proline-rich box 1 region of the GH receptor (GHR1-294,
GHR1-638 NLS-GHBP Increases the Rate of GH-stimulated STAT5-mediated
Transcription--
To determine the potential molecular mechanisms by
which nuclear localized GHBP enhanced GH-stimulated STAT5-mediated
transcription, we first examined the effect of the NLS-GHBP on the rate
of GH-stimulated STAT5-mediated transcription. As is observed in Fig.
8, the rate of GH-stimulated
STAT5-mediated transcription was dramatically increased in the presence
of NLS-GHBP. The differential rate of GH-stimulated STAT5-mediated
transcription was limited to the first 6 h after cellular
stimulation with GH. Thus, nuclear localized GHBP enhances the rate of
GH-stimulated STAT5-mediated transcription.
Effect of NLS-GHBP on Tyrosine Phosphorylation and DNA Binding
Activity of STAT5--
We subsequently examined whether the
GHBP-enhanced rate of GH-stimulated STAT5-mediated transcription was
due to alteration in the phosphorylation state of STAT5. Tyrosine
phosphorylation of STAT molecules is requisite for their dimerization,
nuclear translocation, and DNA binding (40). We examined both the
nuclear translocation of STAT5 and the appearance of
tyrosine-phosphorylated STAT5 in the nucleus. Cellular stimulation with
GH resulted in the nuclear translocation of STAT5 concordant with the
concomitant appearance of tyrosine-phosphorylated STAT5 in the nucleus.
The presence of NLS-GHBP did not alter the ability of GH to stimulate either the tyrosine phosphorylation or nuclear translocation of STAT5
(Fig. 9A). Furthermore, the
presence of the NLS-GHBP did not alter the rate of removal of
tyrosine-phosphorylated STAT5 from the nucleus as observed by the equal
reduced amount of tyrosine-phosphorylated STAT5 180 min after
stimulation with GH (Fig. 9A) nor after a prolonged period
to 8 h (data not shown). We next examined the effect of nuclear
localized GHBP on the ability of GH to stimulate binding of STAT5 to
its DNA response element. Electrophoretic mobility shift assay with use
of the GAS-like element from the SPI 2.1 gene promoter used in reporter
assays demonstrated two distinct binding species in response to
cellular stimulation with GH. These two SPI-GLE1 binding species have
previously been identified as STAT5 (slower migrating) and STAT1
(faster migrating) (41) in BRL-GHR1-638 cells stimulated
with GH. By supershift analysis, we have also demonstrated that the
slower migrating DNA binding species does indeed contain STAT5 (Fig.
9B). GH-stimulated STAT5 DNA binding activity was evident 5 min after GH stimulation and declined thereafter to 3 h when DNA
binding of STAT5 was minimal (Fig. 9B). NLS-GHBP did not
alter the ability of GH activated STAT5 to bind to its DNA response
element (Fig. 9B). The GHBP was not present in the
STAT5-containing DNA binding complex as indicated by failure of
supershift of the complex with monoclonal antibody to the GHBP (data
not shown). Thus, NLS-GHBP does not enhance GH-stimulated
STAT5-mediated transcription by alteration of STAT5 tyrosine
phosphorylation and subsequent DNA binding.
A Truncated Version of NLS-GHBP Also Enhances GH-stimulated
STAT5-mediated Transcription--
To determine whether GH was required
to bind the GHBP to observe the NLS-GHBP enhancement of GH-stimulated
STAT5-mediated transcription, we simply truncated the NLS-GHBP at amino
acid number 115. We introduced a stop codon at the amino acid position 115 in the NLS-GHBP construct, thereby destroying the ability of the
GHBP mutant to bind GH (42). Truncation of the GHBP would also result
in loss of the 17-amino acid hydrophilic tail, which contains the
epitope for the monoclonal antibody (mAb 4.3) used so far to examine
the expression of the transfected GHBP constructs. We therefore
introduced an epitope tag (hemagglutinin) at the amino terminus of
either the wild type GHBP, the nuclear localized GHBP, or the truncated
nuclear localized GHBP (NLS-GHBP1-115). The integrity of
the reading frame was determined by sequence analysis. Transient
transfection and Western blot analyses of these constructs demonstrated
expression of the WT-GHBP, NLS-GHBP, and NLS-GHBP1-115 at
the appropriate molecular weights (Fig. 10A). Subsequent analysis of
STAT5-mediated transcription demonstrated that
NLS-GHBP1-115 was able to enhance GH-stimulated
STAT5-mediated transcription to a similar extent as the full-length
NLS-GHBP (Fig. 10B). Thus, the nuclear localized GHBP is
able to function independently of GHBP-bound ligand to enhance
GH-stimulated STAT5-mediated transcription.
NLS-GHBP Enhances STAT5-mediated Transcription Stimulated by Other
Members of the Cytokine Receptor Superfamily--
Since the GHBP could
enhance STAT5-mediated transcription without bound ligand, we examined
whether NLS-GHBP could function as a transcriptional enhancer for other
cytokine receptor superfamily members that also utilize STAT5 for
transcriptional activation (43). We therefore transiently transfected
either the PRL or EPO receptors into BRL cells (27) and determined the
STAT5-mediated transcriptional response in the presence of NLS-GHBP
(Fig. 11). Human GH is also a ligand
for the PRL receptor (44), and therefore an activation of the PRL
receptor and a STAT5-mediated transcriptional response to hGH via the
PRL receptor can be expected (27). STAT5-mediated transcription,
stimulated specifically through the PRL receptor either with hGH or
with ovine PRL, was also enhanced in the presence of NLS-GHBP to a
similar extent as the enhancement of STAT5-mediated transcription by
NLS-GHBP through the GH receptor. Enhancement of STAT5-mediated
transcription by NLS-GHBP was also observed upon activation of the EPO
receptor with erythropoietin, whereas GH stimulation of cells
transfected with the EPO receptor failed to activate STAT5 with or
without the presence of the NLS-GHBP (Fig. 11). An expression plasmid
encoding for nuclear localized hGH (NLS-hGH) also did not result in
enhancement of EPO-stimulated STAT5-mediated transcription, indicating
that the NLS-GHBP-enhanced STAT5-mediated transcription is not due to
any effect of the NLS of the SV40 large T antigen (data not shown).
Thus, the nuclear localized GHBP functions as an enhancer of
STAT5-mediated transcription for members of the cytokine receptor
superfamily.
We have described here a new functional and ligand-independent
role for the soluble extracellular domain of the growth hormone receptor otherwise known as the GHBP. Exogenously applied GHBP behaves
as expected (16) and inhibits the cellular response to GH in
vitro. In contrast, endogenously produced GHBP functions as an
enhancer of cytokine receptor-stimulated STAT5-mediated transcription.
Such enhancement of cytokine receptor-stimulated STAT5-mediated
transcription is mediated predominantly in the nucleus and does not
require the presence of the ligand per se. The use of a
soluble cytokine receptor as a location-dependent transcriptional enhancer and the ligand-independent involvement of the
extracellular domain of a polypeptide ligand receptor in intracellular
signal transduction provides additional novel mechanisms of
transcriptional control.
The intracellular (19-21) and nuclear localization of the GHBP (19,
21) have been reported previously. The localization of the GHBP to the
nucleus has been observed both in vivo (19, 21) and in
vitro in experimental systems (21). The localization of the GHBP
to the nucleus was heterogenous (19), suggesting that the nuclear
localization of the GHBP was dynamic rather than constitutive. We have
now demonstrated here that a GHBP-GFP fusion protein translocates to
the nucleus upon cellular stimulation with GH or serum. Thus, the GHBP,
in addition to secretion to the extracellular space, is also
specifically localized to the nucleus. This localization is not
constitutive but requires exposure of the cells to a stimulus and is
therefore presumably an active process. We had also previously reported
that both GH (23) and the GH receptor (21) are subject to a rapid and
transient nuclear translocation. At least one function of the nuclear
translocation of the growth hormone and its receptor appears to be the
phosphorylation of nuclear localized JAK2 (24, 25) (45). Interestingly, however, the nuclear translocation of both GH and the GH receptor are
independent of JAK2,2
suggesting that nuclear translocation of the ligand-receptor/binding protein complexes are distinct from classical JAK-STAT pathways. Whether the GHBP requires ligand for nuclear translocation remains to
be determined, as does the mechanism of the nuclear translocation. The
characterization of the GHBP-GFP reported here should greatly facilitate delineation of the mechanism of secretion of the GHBP and
also translocation of the GHBP to the nucleus.
We have demonstrated here that nuclear localized GHBP functions as an
enhancer of STAT5-mediated transcription not only for GH but also for
other members of the cytokine receptor superfamily, which utilize STAT5
for transcriptional responses. STAT5 has been demonstrated to be
utilized by a variety of hormones and cytokines including
interleukin-2, -3, -5, -7, and -15, erythropoietin, granulocyte-macrophage colony stimulating factor, thrombopoietin, and
epidermal growth factor (43, 46, 47). Thus, there must exist a complex
interaction between different cytokines at the cellular level for
regulation of STAT5-mediated transcription. Ligands that do not bind to
the GHBP, such as EPO, would not be subject to the extracellular
binding and subsequent inhibition by GHBP as would GH. It is plausible,
however, that EPO or other factors may modulate the production and/or
secretion of the GHBP such that the response of the cell to GH and
subsequent STAT5-mediated transcription is altered. The EPO receptor
and the GH receptor and their respective ligands, in addition to the
endocrine distribution of the ligands, are widely co-expressed in
various tissues such as placenta, mammary gland, the central nervous
system, and smooth muscle (48-52). Thus, the final hormonal response
of the cell would depend on a complex interplay of the ratio of
extracellular to intracellular (nuclear) GHBP and the identity of the
stimulating ligand. Thus, physiological factors promoting the nuclear
localization of the GHBP would enhance the otherwise limited
transcriptional response of the cell to various ligands. Other
physiological factors, which could up-regulate GHBP production and
secretion, would presumably increase the STAT5-mediated transcriptional
responses to other ligands such as EPO. Many analogous soluble isoforms
of various cytokine and growth factor receptors have also been reported
(4-7). Presumably, the complexity of the cellular STAT5-mediated
transcriptional response would be further increased if other soluble
cytokine receptors/binding proteins (such as the PRL-binding protein
(53)) function as transcriptional enhancers similar to GHBP. This
regulatory strategy may also be one mechanism by which the cell can
filter multiple redundant signals initiated by cytokine molecules
sharing the same signal transduction pathway (9, 47, 54). Such regulatory mechanisms would presumably play an important role during
physiological states such as puberty, pregnancy, and lactation.
The molecular mechanism by which the NLS-GHBP enhances cytokine
receptor-stimulated STAT5-mediated transcription remains to be
determined. We have observed that the nuclear localized GHBP does not
alter GH-stimulated tyrosine phosphorylation, nuclear translocation, or
DNA binding of STAT5. We are also unable to detect an association
between the GHBP and STAT5.3
Tyrosine phosphorylation, nuclear translocation, and even DNA binding
of STAT5 is not sufficient for STAT5 to induce transcriptional activity
(55),3 suggesting that additional factors are involved in
the activation of STAT5. Indeed, multiple co-activators and repressors
that interact with STAT5 have been identified (56-58). One possibility
for the observed GHBP-enhanced STAT5-mediated transcription could be
that GHBP binds to a repressor of STAT5-mediated transcription, thereby preventing an inhibitory association between the repressor and STAT5,
releasing STAT5 to function as an activator of transcription. A family
of STAT transcriptional repressors has been identified (59). The
protein inhibitor of activated STAT (PIAS) family consists of five
members including PIAS1, PIAS3, PIASx In conclusion, we demonstrate here that nuclear localized GHBP
functions as a potent enhancer of STAT5-mediated transcription, not
only for GH but also for other members of the cytokine receptor superfamily. Thus, the GHBP exerts opposing effects on STAT5-mediated transcription depending on its extra-/intracellular location. The use
of a soluble cytokine receptor as a location-dependent transcriptional enhancer and the ligand-independent involvement of the
extracellular domain of a polypeptide ligand receptor in intracellular
signal transduction provide additional novel mechanisms of
transcriptional control. What remains to be determined is the mechanism
by which the nuclear localized GHBP functions as a transcriptional enhancer. Identification of proteins interacting with the GHBP should
be useful in this regard, and such experiments are in progress.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain of interleukin (IL)-2 through IL-7,
IL-9, and IL-11 to IL-13 (3). Most receptors of the cytokine receptor
superfamily exist in a soluble and transmembrane form (4-7). The
function of the transmembrane forms is well documented and includes
signal transduction predominantly but not exclusively through the
JAK-STAT pathway, resulting in gene transcription (8, 9). The role of
the soluble cytokine receptors, with the notable exception of the
soluble forms of the IL-6 and ciliary neurotropitir factor
(CNTF) receptors (10, 11), appears confined to ligand sequestration in
the extracellular space with a consequent impairment of the cellular
response to exogenous ligand (4).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
297-311), or the individual substitution of
proline residues 300, 301, 303, and 305 in box 1 for alanine has been
described previously (30).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of exogenous recombinant rat GHBP on
GH stimulation of STAT5-mediated transcription in
BRL-GHR1-638 cells. BRL-GHR1-638 cells
were cultured to confluence and transiently transfected with
SPI-GLE1-CAT as described under "Experimental Procedures." Cells
were treated for 24 h with 1 nM hGH in the presence of
the indicated concentrations of recombinant rat GHBP. Results are
presented as the mean ± S.E. of triplicate determinations of the
-fold stimulation above non-hormone-stimulated cells.
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Fig. 2.
Expression in BRL cells of transiently
transfected wild type GHBP (WT-GHBP), a GHBP with the amino-terminal
secretion sequence removed (XS-GHBP), and a GHBP with the
amino-terminal secretion sequence replaced by the nuclear localization
sequence of SV40 large T antigen (NLS-GHBP). A,
schematic diagram of the WT-GHBP, XS-GHBP, and NLS-GHBP proteins
encoded by their respective cDNAs. B-E, localization of
the expressed proteins in BRL cells by immunofluorescence with
the empty vector (B) as a control. WT-GHBP is expressed in
the perinuclear region of the cell (C), XS-GHBP is expressed
in the cytoplasm (D), and NLS-GHBP is expressed in the
nucleus. mAb 4.3 directed against the hydrophilic C terminus of the
GHBP was used for detection. F, Western blot analysis of
medium from BRL cells transiently transfected with WT-GHBP,
XS-GHBP, and NLS-GHBP cDNAs.
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Fig. 3.
A, effect of transient transfection of
WT-GHBP cDNA on the STAT5-mediated transcriptional response to GH
in BRL cells transiently transfected with GHR cDNA. BRL cells were
cultured to confluence and transiently transfected with GHR cDNA,
SPI-GLE1-CAT, and the indicated amounts of GHBP cDNA. Cells were
treated for 24 h with 50 nM GH. C, control.
Raw data for the cells transfected with 1 µg of either vector or
WT-GHBP cDNA are as follows: nonstimulated, 585 ± 42 and
554 ± 57, respectively; stimulated, 1778 ± 351 and
2836 ± 193, respectively. B, effect of increasing
concentrations of GH on the STAT5-mediated transcriptional response to
GH in the presence of transiently transfected vector and WT-GHBP
cDNA. BRL cells were cultured to confluence and transiently
transfected with GHR cDNA, SPI-GLE1-CAT, and 1 µg of WT-GHBP
cDNA. Cells were treated for 24 h with the indicated
concentrations of GH. Results are presented as the mean ± S.E. of
triplicate determinations of the -fold stimulation above
non-hormone-stimulated cells.
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[in a new window]
Fig. 4.
Effect of transient transfection of XS-GHBP
cDNA on the STAT5-mediated transcriptional response to GH in BRL
cells transiently transfected with GHR cDNA. BRL cells were
cultured to confluence and transiently transfected with GHR cDNA,
SPI-GLE1-CAT, and the indicated amounts of XS-GHBP cDNA. Cells were
treated for 24 h with 50 nM GH. Results are presented
as the mean ± S.E. of triplicate determinations of the -fold
stimulation above non-hormone-stimulated cells. C,
control.
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[in a new window]
Fig. 5.
Expression of GHBP-GFP in
BRL-GHR1-638 cells and its cellular distribution after
stimulation with exogenous GH and fetal bovine serum. Cells were
transiently transfected with GHBP-GFP and serum-starved for 12 h
before they were treated as indicated with either 50 nM hGH
or 10% fetal bovine serum. A, Western blot analysis of cell
extract from BRL-GHR1-638 cells transiently transfected
with GFP-vector or GHBP-GFP. B, localization of the green
fluorescent protein or GHBP-GFP expressed in BRL-GHR1-638
cells in minutes after stimulation with GH or fetal bovine serum. Cells
were fixed using an ethane-freezing/methanol fixation, and the GFP
signal was further enhanced by using an anti-GFP antibody.
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Fig. 6.
Effect of transient transfection of NLS-GHBP
cDNA on the STAT5-mediated transcriptional response to GH in BRL
cells transiently transfected with GHR cDNA. BRL cells were
cultured to confluence and transiently transfected with GHR cDNA,
SPI-GLE1-CAT, and the indicated amounts of NLS-GHBP cDNA. Cells
were treated for 24 h with 50 nM GH. C,
control (panel A). Effect of increasing concentrations of
hGH (panel B) and rat GH (panel C) on the
STAT5-mediated transcriptional response in the presence of transiently
transfected vector and NS-GHBP. BRL cells were cultured to confluence
and transiently transfected with GHR cDNA, SPI-GLE1-CAT, and 5 µg
of NLS-GHBP cDNA. Cells were treated for 24 h with the
indicated concentrations of GH. Results are presented as the mean ± S.E. of triplicate determinations of the -fold stimulation above
non-hormone-stimulated cells.
297-311, and GHR1-638
P300A, P301A,P303A,P305A) required for association and
activation of JAK2 (30, 36) either in the presence or absence of
cotransfected NLS-GHBP cDNA. Truncation of the distal intracellular
domain of the GH receptor at amino acid residue 454 or 540 diminished
the GH stimulation of STAT5-mediated transcription as expected (37) and
completely prevented the ability of NLS-GHBP to enhance GH-stimulated
STAT5-mediated transcription. Thus, the ability of the nuclear
localized GHBP to enhance GH-stimulated STAT5-mediated transcription
requires the activation of JAK2 and residues of the intracellular
domain of the GH receptor located between amino acids 541 and 638 that
are required for binding and full activation of STAT5 (38, 39).
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Fig. 7.
A, regions of the GH receptor required
for the STAT5-mediated transcriptional enhancing effect of NLS-GHBP.
BRL cells were cultured to confluence and transiently transfected with
the cDNA for the respective GHR mutation. Cells were treated for
24 h with 50 nM GH. Results are presented as the
mean ± S.E. of triplicate determinations of the -fold stimulation
above non-hormone-stimulated cells. B, schematic diagram of
the GH receptor mutations encoded by their respective cDNAs.
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Fig. 8.
Effect of transient transfection of NLS-GHBP
cDNA on the rate of the STAT5-mediated transcriptional response to
GH in BRL cells transiently transfected with GHR cDNA. BRL
cells were cultured to confluence and transiently transfected with GHR
cDNA, SPI-GLE1-CAT. Cells were treated for various times with 50 nM GH. Results are presented as the mean ± S.E. of
triplicate determinations of the -fold stimulation above
non-hormone-stimulated cells.
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[in a new window]
Fig. 9.
Effect of transient transfection of NLS-GHBP
cDNA on the tyrosine phosphorylation and nuclear translocation of
STAT5 (A) and on STAT5 DNA binding activity after GH
stimulation (B). BRL-GHR1-638 cells
were cultured to confluence and transiently transfected with NLS-GHBP
or the control vector and treated with 50 nM GH for the
indicated times. A, nuclear extracts were prepared and
analyzed for phosphorylated STAT5 and total STAT5 by Western blotting.
B, nuclear extracts were prepared, and DNA binding activity
to the SPI-GLE1 probe was analyzed by electrophoretic mobility shift
assay.
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[in a new window]
Fig. 10.
Effect of transient transfection of NLS-GHBP
cDNA and the mutant NLS-GHBP1-115 on the
STAT5-mediated transcriptional response to GH in BRL cells.
A, Western blot analysis of cell extract from BRL-GHR cells
transiently transfected with a plasmid containing the
hemagglutinin-tagged cDNA for either the WT-GHBP, NLS-GHBP, or
NLS-GHBP truncated at amino acid 115. B, transcriptional
response to GH in BRL cells transiently transfected with GHR cDNA
and either the WT-GHBP, NLS-GHBP, or NLS-GHBP1-115. BRL
cells were cultured to confluence and transiently transfected with GH
receptor cDNA and SPI-GLE1-CAT. Cells were treated for 24 h
with 50 nM GH. Results are presented as the mean ± S.E. of triplicate determinations of the -fold stimulation above
non-hormone-stimulated cells.
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Fig. 11.
Effect of transient transfection of NLS-GHBP
cDNA on the STAT5-mediated transcriptional response to hGH and rGH,
oPRL, and mEPO in BRL cells transiently transfected with the GH
receptor, PRL receptor, or EPO receptor cDNA, respectively.
BRL cells were cultured to confluence and transiently transfected with
GH receptor, PRL receptor, or EPO receptor cDNAs, SPI-GLE1-CAT, and
5 µg of NLS-GHBP cDNA. Cells were treated for 24 h with 50 nM hGH or rGH, 100 nM oPRL, or 10 units/ml
mEPO, respectively. Results are presented as the mean ± S.E. of
triplicate determinations of the -fold stimulation above
non-hormone-stimulated cells.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, PIASx
, and PIASy (59).
Members of the PIAS family bind to activated STAT molecules and prevent
STAT binding to DNA and subsequent transcription (59, 60). Indeed,
nuclear localized PRL has just been demonstrated to bind to PIAS3
independent of the PRL receptor, thereby allowing STAT5 to bind
preferentially to its DNA-responsive element instead of the PIAS
protein (61). Alternatively, the GHBP may participate in the formation
of the transcriptional complex required for STAT5-mediated
transcription. Several enhancers of STAT5-mediated transcription have
been identified, including the p300-CBP complex (62), the Nmi
(N-Myc-interacting protein) (63),
and the glucocorticoid receptor (64). PRL has been demonstrated to
stimulate the association between STAT5 and the histone
acteyltransferase p300-CBP, enhancing STAT-mediated transcription by
linkage with the transcriptional complex (62). Nmi has been
demonstrated to enhance STAT5-mediated transcription by increased
formation of STAT5-p300-CBP complexes (63). STAT5 cooperates with the glucocorticoid receptor for transcriptional activation without a need
for the DNA or ligand binding domains of the nuclear receptor (65),
supporting the idea that even minimal promoter sites are enough to
attract complex transcriptosomes without DNA binding of all
components. Another transcription factor interacting with STAT5, YY1,
requires additional DNA recognition sites on its promoter region to
cooperate with STAT5 in transcriptional regulation (56). The SPI-GLE1 sequence used here is alone sufficient for formation of
the GH-induced STAT5 DNA binding complexes and does not contain other
consensus sequences used by other transcription factors (28). Since we
observed no interaction of the GHBP with STAT5, it is therefore not
surprising that the GHBP was not contained in the DNA binding complex.
Further studies to delineate proteins, which interact with the GHBP,
should allow identification of the mechanism by which the GHBP enhances
cytokine receptor-stimulated STAT5-mediated transcription.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. W. R. Baumbach for the generous gift of recombinant GHBP and mAb 4.3.
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FOOTNOTES |
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* 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: Institute of
Molecular and Cell Biology, National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore. Tel.:
65-68747847; Fax: 65-67791117; E-mail: mcbpel@imcb.nus.edu.sg.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M207546200
2 Mertani, H., Raccurt, M., Abatte, A., Nilsson, J., Törnell, J., Billestrup, N., Usson, Y., Morel, G., and Lobie, P. E. (2003) Endocrinology, in press.
3 R. Graichen and P. E. Lobie, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: GH, growth hormone; PRL, prolactin; EPO, erythropoietin; IL, interleukin; GHBP, growth hormone-binding protein; hGH, human GH; rGH, rat GH; oPRL, ovine PRL; mEPO, murine EPO; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; WT-GHBP, wild type GHBP; CAT, chloramphenicol acetyltransferase; STAT, signal transducers and activators of transcription; PIAS, protein inhibitor of activated STAT; NLS, nuclear localization signal; DOTAP, N-[1-(2,3-dioleoylloxy)propyl]-N,N,N-trimethyl ammonium methyl sulfate.
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