J Biol Chem, Vol. 275, Issue 3, 2103-2114, January 21, 2000
Janus Kinase 2-dependent Activation of p38 Mitogen-activated
Protein Kinase by Growth Hormone
RESULTANT TRANSCRIPTIONAL ACTIVATION OF ATF-2 AND CHOP,
CYTOSKELETAL RE-ORGANIZATION AND MITOGENESIS*
Tao
Zhu and
Peter E.
Lobie
From the Institute of Molecular and Cell Biology, National
University of Singapore, 30 Medical Drive, Singapore 117609, Republic of Singapore
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ABSTRACT |
We demonstrate here that p38 mitogen-activated
protein (MAP) kinase is activated in response to cellular stimulation
by human GH (hGH) in Chinese hamster ovary cells stably transfected
with GH receptor cDNA. This activation requires the proline-rich
box 1 region of the GH receptor required for JAK2 association and is
prevented by pretreatment of cells with the JAK2-specific inhibitor AG490. ATF-2 is both phosphorylated and transcriptionally activated by
hGH, and its transcriptional activation also requires the proline-rich box 1 region of the GH receptor. Expression of wild type JAK2 can
further enhance hGH-induced ATF-2-, CHOP-, and Elk-1-mediated transcriptional activation, whereas pretreatment with AG490 is inhibitory. Use of either specific pharmacological inhibitors or
transient transfection of cells with p38
MAP kinase cDNA or a
dominant negative variant demonstrated that hGH-stimulated
transcriptional activation of ATF-2 and CHOP, but not Elk-1, is
regulated by p38 MAP kinase. Both the p38 MAP kinase and p44/42 MAP
kinase are critical for hGH-stimulated mitogenesis, whereas only p38
MAP kinase is required for hGH-induced actin cytoskeletal
re-organization. p38 MAP kinase is therefore an important regulator in
coordinating the pleiotropic effects of GH.
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INTRODUCTION |
Growth hormone (GH),1 as
the major regulator of postnatal body growth, possesses diverse and
pleiotropic effects on the growth, differentiation, and metabolism of
cells (1, 2). The GH receptor is a single membrane-spanning
glycoprotein in the cytokine receptor superfamily (3) that consists of
a ligand-binding external domain and a 350-amino acid residue
cytoplasmic domain required for intracellular signal transduction (1,
2). GH receptor signaling involves ligand-induced receptor
homodimerization and activation of the tyrosine kinase JAK2 by
association with the GH receptor (1, 2, 4). The proline-rich Box1 motif of the receptor has been identified as the site of association with
JAK2. Multiple effector molecules downstream of JAK2-mediating GH
signal transduction have now been identified (5-11). We have also
recently demonstrated that GH stimulates the formation of a
multiprotein signaling complex centered around p125FAK (12)
and p130Cas-CrkII (13) leading to JNK/SAPK activation.
The mitogen-activated protein kinase (MAPK) superfamily, encompassing
p44/42 MAP kinase, c-Jun amino-terminal kinases (JNK), and p38 MAP
kinase, are proline-directed serine-threonine protein kinases that have
important functions as mediators of cellular responses to a variety of
extracellular stimuli (14, 15). p44/42 MAP kinase has been implicated
in mitogenic growth in a variety of cell contexts, and its activation
is via a well known sequential cascade involving SHC, Grb2,
son-of-sevenless, Ras, Raf, and MAP/extracellular signal-regulated
kinase (MEK) (14, 15). The p38 MAP kinases have been shown to be
activated by a series of cytokines, growth factors, and autonomic
neurotransmitters (16-19) in addition to the stress and
pro-inflammatory signals (20-22). Activation of p38 MAP kinase
involves phosphorylation on threonine and tyrosine residues present in
a TGY amino acid motif (23, 24), resulting in increased enzyme activity
(25, 26). At least four isoforms of p38 MAP kinase have been described (20-22, 26-30). Experiments with dominant-negative or active mutant proteins have demonstrated that p38 MAP kinase lies downstream of Rac,
Cdc42, GCK, PAK1, TAK1, ASK1, TPL1, and RAFTK/PYK2 (31-39) and is
directly activated by three dual-specificity MAPK kinases, MKK3, MKK4,
and MKK6 (36, 40-43). Some of the substrates for p38 MAP kinase that
may be physiologically relevant have been identified. These include the
transcription factors ATF-2 (23, 42), CHOP (44), Elk-1 (45, 46), CREB,
ATF-1 (47, 48), Sap1a (45, 46), MEF2C, and MEF-2A (49-51), which can
be phosphorylated and transcriptionally activated by p38 MAP kinase.
p38 MAP kinase also activates the following downstream protein kinases:
MAPKAP kinase-2, MAPKAP kinase-3, and p38-regulated/activated protein kinase (22, 52, 53).
We and others (54-56) have previously demonstrated GH activation of
both p44/42 MAP kinase and c-Jun amino-terminal kinase (JNK/SAPK1)
(13). We demonstrate here that hGH transiently phosphorylates and
activates p38 MAP kinase in CHO cell lines stably transfected with rat
GH receptor cDNA and that the activation of the p38 MAP kinase
pathway by hGH is JAK2-dependent. Furthermore, both ATF-2 and CHOP (GADD153) are transcriptionally activated by hGH in a JAK2-dependent manner, and p38 MAP kinase is required for
both cytoskeletal re-organization and cell proliferation stimulated by
hGH. The p38 MAP kinase pathway is therefore an important regulator of
the pleiotropic effects of GH.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human growth hormone (hGH) was a
generous gift of Novo-Nordisk (Singapore). The JAK2 inhibitor
tyrphostin AG490, MEK1 inhibitor PD98059, and the p38 MAP kinase
inhibitor SB203580 were purchased from Calbiochem. Protein A/G plus
agarose was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
The enhanced chemiluminescence (ECL) kit was purchased from Amersham
Pharmacia Biotech. The immobilized phospho-p38 MAP kinase
(Thr-180/Tyr-182) monoclonal antibody, immobilized phospho-p44/42 MAP
kinase (Thr-202/Tyr-204) monoclonal antibody, phospho-p38 MAP kinase
(Thr-180/Tyr-182) polyclonal antibody, phospho-ATF-2 (Thr-71)
polyclonal antibody, ATF-2 polyclonal antibody, phospho-Elk-1 (Ser-383)
polyclonal antibody, Elk-1 polyclonal antibody, and Elk-1 fusion
protein were purchased from New England Biolabs (Beverly, MA). The
glutathione S-transferase fusion protein of the
amino-terminal portion of activating transcription factor-2 (ATF-2)
(base pairs 1-109) was a generous gift of Dr. Shengcai Lin (Institute
of Molecular and Cell Biology, Singapore). Transfection reagent DOTAP
was purchased from Roche Molecular Biochemicals. [3H]Thymidine was purchased from Amersham Pharmacia
Biotech. All other reagents were purchased from Sigma.
The wild type JAK2 expression plasmid has been described previously
(57). The fusion trans-activator plasmids (pFA-ATF-2, pFA-CHOP, and pFA2-Elk-1) consisting of the DNA binding domain of Gal4
(residue 1-147) and the trans-activation domain of ATF-2, CHOP, or Elk-1 were purchased from Stratagene (La Jolla, CA). pFC2-dbd
plasmid is the negative control for the pFA plasmid to ensure the
observed effects are not due to the Gal4 DNA binding domain and was
also obtained from Stratagene. Wild type or p38 MAP kinase dominant
negative (TGY-AGF) mutant and wild type p42 MAP kinase in pcDNA3
expression vector were kindly provided by Dr. Jiahua Han (San Diego,
CA). SPI-GLE1-Luc plasmid has been previously described (58). All
plasmids were prepared with the plasmid megaprep kit from Qiagen
(Hilden, Germany).
CHO Cell Lines Stably Transfected with GH Receptor
cDNA--
Rat GH receptor cDNAs were cloned into an expression
plasmid containing an SV40 enhancer and a human metallothionein IIa
promoter. The cDNAs were transfected into CHO-K1 cells using
Lipofectin together with the pIPB-1 plasmid, which contains a neomycin
resistance gene fused to the thymidine kinase promoter. Stable
integrants were selected using 1000 mg/ml G418. The complete rat GH
receptor cDNA coding for amino acids 1-638 was expressed in
CHO4-638 or CHOA-638 cells and will be referred to as
CHO-GHR1-638 (54). The construction of GH receptor
cDNA expression plasmids containing a deletion of box 1 (
297-311) and the individual substitution of proline residues 300, 301, 303, and 305 in box 1 for alanine has been described previously
(59). These cDNAs were stably transfected into CHO-K1 cells; the
297-311 mutation was expressed in
CHO-GHR1-638297-311 cells, and P300A,P301A,P303A,P305A was expressed in CHO-GHR1-638P300,301,303,305A cells
(cells were a kind gift of Dr. Nils Billestrup) (59). The level of receptor expression for the individual cell clones is comparable between clones and has been described previously (54, 59).
Cell Culture and Treatment--
CHO-GHR1-638,
CHO-GHR1-638297-311, and
CHO-GHR1-638P300,301,303,305A were maintained in Ham's
F-12 medium (F-12) plus 10% v/v fetal calf serum, 100 units/ml
penicillin, and 100 mg/ml streptomycin in a humidified atmosphere
containing 5% CO2 and 95% air at 37 °C as described
previously (59). Prior to treatment, cells were deprived of serum for
12-16 h in Ham's F-12 medium. Unless otherwise indicated, the final
concentration of the MEK1 inhibitor PD98059 was 30 µM,
p38 MAP kinase inhibitor SB205380 was 10 µM, and hGH was
50 nM. This concentration of GH is within the physiological
range for circulating rodent GH (60, 61).
Immunoblotting--
Immunoblotting for p38 MAP kinase, dual
phospho-p38 MAP kinase, Elk-1, phospho-Elk-1, ATF-2, and phospho-ATF-2
were carried out as described previously (12, 13). After preincubation with inhibitors for respective time and/or incubation with hGH for the
appropriate duration, the cells were washed twice with ice-cold PBS,
and cells were lysed at 4 °C in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton-100, 150 mM
NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM
sodium orthovanadate, 0.5% Nonidet P-40, 0.1% phenylmethylsulfonyl
fluoride, 10 µg/ml each aprotinin and leupeptin) for 30 min with
regular vortices. Cell lysates were centrifuged at 14,000 × g for 15 min, and the resulting supernatants were collected,
and protein concentration was determined. Cell lysates dissolved in 1×
SDS-polyacrylamide gel electrophoresis sample buffer containing 25 mM Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, and
bromphenol blue, boiled for 10 min, and centrifuged at 14,000 × g for 2 min were analyzed on 10% SDS-polyacrylamide gels
and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline with 0.1% Tween
20 (PBST) for 1 h at 22 °C. The blots were then treated with
the primary antibody in PBST containing 1% non-fat dry milk at 4 °C
overnight. After three washes with PBST, immunolabeling was
detected by ECL according to the manufacturer's instructions. Membranes were then stripped by incubation for 30 min at 50 °C in a
solution containing 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 0.7% mercaptoethanol. Blots were then washed for 30 min with several changes of PBST at room temperature. Efficacy of stripping was determined by re-exposure of the membranes to ECL. Thereafter, blots
were reblocked and immunolabeled as described above.
Kinase Assays--
p38 MAP kinase assays were performed using
New England Biolabs assay kit specific for p38 and p38
MAP kinase
according to the manufacturer's instructions. In brief,
CHO-GHR1-638 cells were serum-deprived for 16 h,
treated with 50 nM hGH, and the cells lysed at 4 °C in 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
glycerol phosphate, 1 mM Na3VO4, 0.1% phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin) per sample. The
lysates were centrifuged at 15,000 × g for 15 min at
4 °C. The supernatant containing 300 µg of protein per sample was
incubated overnight at 4 °C with 20 µl of immobilized phospho-p38
MAP kinase (Thr-180/Tyr-182) monoclonal antibody in a final volume of
500 µl in 1× lysis buffer. The bead slurries were washed twice with 500 µl of lysis buffer containing 0.1% phenylmethylsulfonyl fluoride and twice with 500 µl of kinase buffer (25 mM Tris-HCl,
pH 7.5, 5 mM glycerol phosphate, 2 mM
dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2). 3 µg of recombinant glutathione
S-transferase-ATF2 protein containing 1-109 amino-terminal
amino acids of ATF-2 was added to each sample. The kinase reactions
were carried out in the presence of 100 mM ATP at 30 °C
for 30 min. Phosphorylation of ATF-2 at Thr-71 was determined by
Western blot using phospho-ATF-2 (Thr-71) antibody (1:1000). p44/42 MAP
kinase assays were also performed using New England Biolabs assay kit
according to the manufacturer's instructions. In brief, cells were
serum-deprived for 16 h, treated with 50 nM hGH, and
lysed at 4 °C in 1 ml of lysis buffer (as described above) per
sample. The lysates were centrifuged at 15,000 × g for
15 min at 4 °C. The supernatant containing 200 µg of protein per
sample was incubated overnight at 4 °C with 15 µl of immobilized
phospho-specific p44/42 MAP kinase (Thr-202/Tyr-204) monoclonal
antibody in a final volume of 500 µl in 1× lysis buffer. The pellets
were washed twice with 500 µl of lysis buffer containing 0.1%
phenylmethylsulfonyl fluoride and washed twice with 500 µl of kinase
buffer (25 mM Tris-HCl, pH 7.5, 5 mM glycerol
phosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, 10 mM MgCl2).
The kinase reactions were performed in the presence of 2 µg of Elk-1
fusion protein and 200 µM ATP at 30 °C for 30 min.
Elk-1 phosphorylation was selectively detected by western immunoblotting using a chemiluminescent detection system and a specific
phospho-Elk-1 (Ser-383) antibody (1:1000).
Transfection Procedure and Luciferase
Assays--
CHO-GHR1-638 cells were cultured to 60-80%
confluence for transfection experiments in 6-well plates. 1 µg of
reporter plasmid pFR-Luc was transfected together with 20 ng of the
respective fusion trans-activator plasmid (pFA-ATF-2,
pFA-CHOP, pFA-Elk-1, or pFC2-dbd). For transient transfection of MAP
kinases, either 1.5 µg of pCDNA3-p38 MAPK, dominant negative
pCDNA3-p38 MAPK, or pCDNA3-p42 MAPK were transfected in each
well. Control empty vector was used to normalize the amount of plasmids
in each well. For each well, 4 µg of DOTAP for each µg of DNA was
used as per the manufacturer's instructions. DNA and the DOTAP
reagents were diluted separately in 100 µl of serum-free medium mixed
and incubated at room temperature for 30 min. DNA-lipid complex was
diluted to a final volume of 6 ml (for triplicate samples) with
serum-free medium. Cells in each well were rinsed once with 2 ml of
serum-free medium, and 2 ml of diluted DNA-lipid complex was overlaid
in each well and incubated for 6 h. After incubation, complete
Ham's F-12 medium containing 2% FBS was added to each well so as to incubate the cells in 0.5% serum for 12-16 h. Cells were pretreated with 10 µM SB203580 or 30 µM PD98059 or
with the equivalent amount of solvent (Me2SO) as a control
for 45 min prior to stimulation with hGH. 50 nM hGH was
added for an additional 5-7 h. The cells were washed in PBS and lysed
with 300 µl of 1× lysis buffer (25 mM Tris phosphate, pH
7.8, 2 mM EDTA, 2 mM dithiothreitol, 10% glycerol, 1% Triton X-100) by a freeze-thaw cycle, and lysate was
collected by centrifugation at 14,000 rpm for 15 min. The supernatant
was used for the assay of luciferase and -galactosidase activity.
The luciferase activities were normalized on the basis of protein
content as well as on the -galactosidase activity of pCMV vector.
The -galactosidase assay was performed with 20 µl of precleared
cell lysate according to a standard protocol (62). Mean and standard
deviations of at least three independent experiments are shown in the figures.
Visualization of Filamentous Actin and Confocal Laser Scanning
Microscopy--
Cells were fixed in ice-cold 4% paraformaldehyde,
washed with PBS, permeabilized for 5 min with 0.1% Triton X-100,
blocked in 2% bovine serum albumin, and incubated with
phalloidin-TRITC (0.2 mg/ml) after the indicated treatments. Excess
phalloidin-TRITC was removed by extensive washing with PBS. Labeled
cells were visualized with a Carl Zeiss Axioplan microscope equipped
with epifluorescence optics and a Bio-Rad MRC1024 confocal laser
system. Images were converted to the tagged information file format and processed with the MacIntosh Photoshop program.
Cell Proliferation Assays--
Cell proliferation was assayed by
measuring incorporation of [3H]thymidine during DNA
synthesis (63). Subconfluent CHO-GHR1-638 cell monolayers
in 24-well plates were grown to quiescence in serum-free Ham's F-12
medium at 37 °C for 16 h. Cells were then incubated for 24 h in Ham's F-12 medium with hGH to a final concentration as indicated
in the figure legends. Each cell line was plated in triplicate for each
treatment. In case of pretreatment of cells with chemical inhibitors,
cells were pretreated with 10 µM SB203580 or 30 µM PD98059 or with the equivalent amount of solvent
(Me2SO) as a control for 45 min.
[3H]Thymidine (1 µCi per well, 1 Ci = 37 GBq,
Amersham Pharmacia Biotech) was added, and the cells were incubated at
37 °C for a further 8 h. Cells were rinsed twice with ice-cold
PBS incubated with 1 ml of ice-cold 5% trichloroacetic acid for 30 min
at 4 °C and 0.5 ml of 0.5 N NaOH, 0.5% SDS was added
subsequently at room temperature. Solubilized samples were subjected to
liquid scintillation counting in a scintillation counter.
Statistics and Presentation of Data--
All experiments were
repeated at least three times. Figures presented for Western blot
analyses are representative of multiple experiments. The text under
"Results" summarizes the results from multiple Western blot
analyses. Consequently, the text under "Results" (e.g.
description of the time course of phosphorylation and
dephosphorylation) may not exactly correspond to the actual figure
presented. All numerical data are expressed as mean ± S.D. Data
were analyzed using the two-tailed t test or analysis of variance.
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RESULTS |
Time- and Dose-dependent Activation of p38 MAP Kinase
by Cellular Stimulation with hGH--
We and others (13, 54-56) have
previously shown that GH activates both p42/44 MAP kinase and JNK. In
order to determine whether cellular stimulation with hGH also results
in the activation of p38 MAP kinase, we first examined whether hGH
stimulation of cells resulted in the phosphorylation of p38 MAP kinase.
Dual phosphorylation of p38 MAP kinase on Thr-180 and Tyr-182 is
required for its activation (14, 25, 64). We therefore treated
serum-deprived CHO-GHR1-638 cells for 0-60 min with 50 nM hGH and prepared cell extracts for Western blot
analysis. Phosphorylated p38 MAP kinase was detected by use of a
phospho-p38 MAP kinase (Thr-180/Tyr-182) antibody that detects p38 MAP
kinase after dual phosphorylation at Thr-180 and Tyr-182. hGH
stimulation of CHO-GHR1-638 cells resulted in increased
p38 MAP kinase phosphorylation detectable 5 min after stimulation.
Maximal hGH-stimulated phosphorylation of p38 MAP kinase was observed
at 15 min and followed by a subsequent decline in phosphorylation state
to 60 min (Fig. 1A). Equal
loading of samples was verified by stripping of the membrane and
subsequent reblotting with a p38 MAPK antibody (Fig. 1B). We
also examined whether the hGH-stimulated phosphorylation of p38
resulted in enhanced p38 MAP kinase activity using ATF-2 as an in
vitro substrate (26, 41). We therefore treated serum-deprived
CHO-GHR1-638 cells for 0-60 min with 50 nM
hGH and immunoprecipitated dual-phosphorylated p38 MAP kinase from cell
extracts with immobilized phospho-p38 MAP kinase (Thr-180/Tyr-182)
monoclonal antibody and measured p38 MAP kinase activity by using ATF-2
as the substrate. Phosphorylation of ATF-2 by the immunoprecipitated
p38 MAP kinase was detected by immunoblotting with an antibody that
detects ATF-2 when phosphorylated on threonine residue 71 (Thr-71).
Thus the appearance of phosphorylation of p38 MAP kinase observed in
hGH-stimulated cell extracts corresponds temporally to the increase in
p38 MAP kinase activity in hGH-stimulated cell extracts. We also
examined the dose dependence of the hGH stimulation of p38 MAP kinase
phosphorylation and activity. We therefore stimulated
CHO-GHR1-638 cells with 0, 0.005, 0.05, 0.5, 5, and 50 nM hGH for 15 min and prepared cell extracts. Determination of p38 MAP kinase phosphorylation and p38 MAP kinase activity was
performed as described above. The maximal phosphorylation (Fig.
1D) and activation (Fig. 1F) of p38 MAP kinase
was achieved at a concentration of 50 nM hGH in
CHO-GHR1-638 cells. Again, equal loading of samples was
verified by stripping of the membrane and subsequent reblotting with a
p38 MAPK antibody (Fig. 1E). Therefore, 50 nM
hGH was used for all subsequent experiments.
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Fig. 1.
Time and dose dependence of the
hGH-stimulated dual phosphorylation and activation of p38 MAP kinase in
CHO cells stably transfected with GH receptor cDNA. For time
course experiments, CHO-GHR1-638 cells were stimulated
with 50 nM hGH for the indicated times. For dose dependence
experiments, CHO-GHR1-638 cells were stimulated with the
indicated concentration of hGH for 15 min. The samples containing equal
amounts of proteins were electrophoresed and immunoblotted with the
antibodies specific for activated p38 MAP kinase
(phospho-Thr-180/Tyr-182) (A and D) and total p38
MAP kinase (B and E). Kinase assays (C
and F) were performed by immunoprecipitating activated p38
MAP kinase with immobilized phospho-p38 MAP kinase (Thr-180/Tyr-182)
monoclonal antibody, and p38 MAP kinase activity was determined as
described under "Experimental Procedures." The data presented are
representative of at least three separate experiments.
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Activation of p38 MAP Kinase by hGH Is
JAK2-dependent--
The activation of JAK2 upon cellular
stimulation with GH is thought to be required for subsequent signal
transduction events (4, 5, 12, 65). To examine the potential role of
JAK2 in the activation of p38 MAP kinase by hGH, we utilized well
characterized CHO cell clones stably expressing the wild type receptor
(CHO-GHR1-638), a receptor mutation in which the
proline-rich box 1 region had been deleted
(CHO-GHR1-638297-311), and a receptor mutation in
which the individual proline residues of box 1 had been converted to
alanine (CHO-GHR1-638P300,301,303,305A) (Fig.
2A). The proline rich box 1 region of the GH receptor is required for the association of JAK2 with
the receptor and its subsequent activation after receptor dimerization
(59). We therefore stimulated the respective serum-deprived cell line
with 50 nM hGH for 15 min and prepared cell extracts for
determination of p38 MAP kinase activity. hGH stimulation of
CHO-GHR1-638 cells resulted in activation of p38 MAP
kinase, whereas no hGH-dependent activation of p38 MAP
kinase activity was observed in either the
CHO-GHR1-638297-311 or
CHO-GHR1-638P300,301,303,305A cell lines (Fig.
2B). For control purposes we also treated
CHO-GHR1-638, CHO-GHR1-638297-311, and CHO-GHR1-638P300,301,303,305A cell lines
with sorbitol (41, 66) and demonstrated equipotent activation of p38
MAP kinase in all three cell lines. Thus it is apparent that the hGH activation of p38 MAP kinase requires the proline-rich box 1 region of
the GH receptor indicative that p38 MAP kinase is activated in a
JAK2-dependent manner.
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Fig. 2.
JAK2 dependent activation of p38 MAP kinase
by hGH in CHO cells stably transfected with wild type GH receptor or
Box1-deficient GH receptor cDNAs. A, schematic
diagram of the GH receptor and the various GH receptor
mutations/deletions used. The wild type receptor has the extracellular,
transmembrane, and intracellular regions indicated.
CHO-GHR1-638297-311 has the proline-rich region (Box1)
deleted, CHO-GHR1-638P300,301,303,305A has the individual
proline residues in box 1 mutated to alanine. CHO-GHR1-638
cells and CHO cells expressing the GH receptor box 1 mutations or
deletions were stimulated with 50 nM hGH for 15 min, cell
extracts prepared, and p38 MAP kinase activity determined
(B). CHO-GHR1-638 cells were preincubated with
100 µM tyrphostin AG490 or vehicle Me2SO for
16 h at 37 °C prior to treatment with 50 nM hGH for
0 or 15 min, cell extracts prepared, and p38 MAP kinase activity
determined (C). CHO-GHR1-638 cell
(preincubated ± AG490), CHO-GHR1-638297-311
cells, and CHO-GHR1-638P300,301,303,305A cell were treated
with sorbitol for 5 min, cell extracts prepared, and p38 MAP kinase
activity determined (D). The data presented are
representative of at least three separate experiments.
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Tyrphostin AG490, a reportedly specific inhibitor of JAK2 tyrosine
kinase activity (67-69), was also utilized to verify that hGH
stimulation of p38 MAP kinase activity was JAK2-dependent. CHO-GHR1-638 cells were therefore pretreated with 100 µM AG490 or vehicle (Me2SO) for 16 h,
followed by stimulation with 50 nM hGH for 15 min and
determination of p38 MAP kinase activity. The hGH-stimulated p38 MAP
kinase activation was abolished by the pretreatment of the cells
with AG490 (Fig. 2C). In contrast, pretreatment of the
CHO-GHR1-638 cells with AG490 as described above did not
alter the ability of sorbitol to stimulate p38 MAP kinase activity
(Fig. 2D). Thus, hGH stimulation of p38 MAP kinase activity
by hGH is JAK2-dependent.
Time- and Dose-dependent Phosphorylation of
Cellular ATF-2 by Cellular Stimulation with hGH--
Since ATF-2 has
been demonstrated to be a substrate of p38 MAP kinase (23, 26, 42), we
wished to determine if hGH stimulation of
CHO-GHR1-638 cells also resulted in phosphorylation of cellular ATF-2. CHO-GHR1-638 cells were serum-deprived and stimulated with 50 nM hGH for the indicated times. Cell
lysates were prepared, and phosphorylation of ATF-2 was detected by
immunoblotting with a specific antibody against ATF-2 phosphorylated on
Thr-71 (Fig. 3A).
Phosphorylation of ATF-2 was first observed 5 min after cellular
stimulation with hGH, and maximal hGH-dependent
phosphorylation of ATF-2 was observed at 30 min with a relative decline
in ATF-2 phosphorylation observed at 60 min. Equal loading of samples
was verified by stripping of the membrane and subsequent reblotting with an ATF-2 antibody (Fig. 3B). We also examined the dose
dependence of the hGH stimulation of ATF-2 phosphorylation. We
therefore stimulated CHO-GHR1-638 cells with 0, 0.005, 0.05, 0.5, 5, and 50 nM hGH for 15 min and prepared cell
extracts. The maximal phosphorylation of ATF-2 was achieved at a
concentration of 5-50 nM hGH in CHO-GHR1-638
cells (Fig. 3C). Equal loading of samples was again verified
by stripping of the membrane and subsequent reblotting with an ATF-2
antibody (Fig. 3D).
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Fig. 3.
Time- and dose-dependent
phosphorylation of ATF-2 by hGH in CHO cells stably transfected with
wild type GH receptor or Box1-deficient GH receptor
cDNAs. CHO-GHR1-638 cells were stimulated with 50 nM hGH for the indicated times. The samples containing
equal amounts of proteins were electrophoresed and immunoblotted with
the antibodies specific for ATF-2 phosphorylated at Thr-71
(A) and total ATF-2 antibody (B).
CHO-GHR1-638 cells were stimulated with the indicated
concentrations of hGH for 15 min. The samples containing equal amounts
of proteins were electrophoresed and immunoblotted with the antibody
specific for ATF-2 phosphorylated at Thr-71 (C) and total
ATF-2 antibody (D). The data presented are representative of
at least three separate experiments.
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Phosphorylation of the ATF-2 by hGH Is
JAK2-dependent--
We also examined the requirement of
JAK2 for hGH-stimulated phosphorylation of ATF-2. We utilized the same
well characterized CHO cell clones stably expressing either the wild
type receptor (CHO-GHR1-638), a receptor mutation in which
the proline-rich box 1 region had been deleted
(CHO-GHR1-638297-311), or a receptor mutation in which
the individual proline residues of box 1 had been converted to alanine
(CHO-GHR1-638P300,301,303,305A) (Fig. 2A) as
described above. Each serum-deprived cell line was stimulated with 50 nM hGH for 30 min, and cell extracts were prepared for
Western blot analysis of ATF-2 phosphorylation. Human
GH-dependent phosphorylation of ATF-2 was only detected in
the CHO cell line that expressed the full-length wild type GH receptor
(Fig. 4A). Equal loading of
total cellular ATF-2 from the three CHO cell clones was verified by
reblotting the membrane with an ATF-2 antibody (Fig. 4B).
Thus, it is apparent that the GH-induced ATF-2 phosphorylation requires
the proline-rich box 1 region of the GH receptor.
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Fig. 4.
JAK2-dependent phosphorylation of
ATF-2 by hGH in CHO cells stably transfected with wild type GH receptor
or Box1-deficient GH receptor cDNAs. The schematic diagram of
the GH receptor and the various GH receptor mutations/deletions is
presented in Fig. 2A. CHO-GHR1-638,
CHO-GHR1-638297-311, and
CHO-GHR1-638P300,301,303,305A cells were stimulated with
50 nM hGH for 30 min. The samples containing equal amounts
of proteins were electrophoresed and immunoblotted with the antibodies
specific for ATF-2 phosphorylated at Thr-71 (A) and total
ATF-2 (B). CHO-GHR1-638 cells were preincubated
with 100 µM tyrphostin AG490 or vehicle Me2SO
for 16 h at 37 °C prior to treatment with 50 nM
hGH. The samples containing equal amount of proteins were
electrophoresed and immunoblotted with the antibodies specific for
ATF-2 phosphorylated at Thr-71 (C) and total ATF-2
(D). The data presented are representative of at least three
separate experiments.
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To verify further the JAK2 dependence of the hGH-stimulated
phosphorylation of ATF-2, we also pretreated CHO-GHR1-638 cells with 100 µM AG490 or vehicle followed by cellular
stimulation with 50 nM hGH for 30 min. Pretreatment with
the JAK2-specific inhibitor prevented hGH stimulation of ATF-2
phosphorylation (Fig. 4C). Again equal loading of total
cellular ATF-2 for the various experimental conditions was verified by
reblotting the membrane with an ATF-2 antibody (Fig.
4D).
hGH Stimulates the Transcriptional Activation of ATF-2, CHOP, and
Elk-1--
To determine if hGH-stimulated phosphorylation of ATF-2
resulted in ATF-2-dependent transcription, we utilized a
trans-activation reporter assay specific for ATF-2. We
therefore transiently transfected CHO-GHR1-638 cells with
the fusion trans-activator plasmid pFA-ATF-2 consisting of
the DNA binding domain of GAL4 (residue 1-147) and the
trans-activation domain of ATF-2 together with the fusion
trans-activator plasmids pFA-ATF-2 consisting of the DNA
binding domain of GAL4 (residue 1-147) and the
trans-activation domain of ATF-2, together with luciferase
reporter plasmid and pCMV vector, respectively. The luciferase
activities were measured and normalized on the basis of protein content
as well as on the -galactosidase activity of pCMV vector. We
demonstrated an hGH-dependent transcriptional activation of
ATF-2 (Fig. 5A) in
CHO-GHR1-638 cells. hGH failed to stimulate ATF-2-mediated
reporter expression in cells transfected with a plasmid encoding the
GAL4 DNA binding domain (residue 1-147) lacking an activation domain,
indicative that the ATF-2 transcriptional activation domain is required
for hGH-stimulated reporter expression. Since transcriptional
activation of CHOP has also been shown to be p38 MAP
kinase-dependent, we utilized a trans-activation
reporter assay specific for CHOP to determine if hGH can also stimulate
CHOP-dependent transcription. We demonstrated a
hGH-dependent transcriptional activation of CHOP (Fig.
5B) in CHO-GHR1-638 cells. hGH failed to
stimulate CHOP-mediated reporter expression in cells transfected with a plasmid encoding the GAL4 DNA binding domain (residue 1-147) lacking an activation domain, indicative that the CHOP transcriptional activation domain is required for hGH-stimulated reporter expression. We have also incorporated an Elk-1 reporter assay for control purposes
(Fig. 5C), since it was previously shown that GH-stimulated transcriptional activation of Elk-1 was p44/42 MAP
kinase-dependent (70, 71). We demonstrated a
hGH-dependent transcriptional activation of Elk-1 in
CHO-GHR1-638 cells. hGH failed to stimulate Elk-1-mediated
reporter expression in cells transfected with a plasmid encoding the
GAL4 DNA binding domain (residue 1-147) lacking an activation domain,
indicating that the Elk-1 transcriptional activation domain is required
for hGH-stimulated reporter expression.
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Fig. 5.
hGH stimulates the transcriptional activation
of ATF-2, CHOP, and Elk-1 in CHO cells stably transfected with GH
receptor cDNA. A, CHO-GHR1-638 cells
were co-transfected with the following: 1) -galactosidase expression
vector pCMV; 2) reporter plasmid pFR-Luc; 3) fusion
trans-activator plasmid either pFA-ATF-2 (A),
pFA-CHOP (B), or pFA2-Elk-1 (C); and 4) pFC2-dbd
as indicated in the figures. Cells were treated with vehicle
(shaded bars) or 50 nM hGH (solid
bars) as indicated. The relative luciferase activities presented
were normalized by protein concentrations as well as -galactosidase
activity (mean ± S.D., n = 3). Luciferase
activity in cell lysates is normalized to the activity in untreated
control cells (control = 1). The data presented are representative
of at least three separate experiments, each measured in triplicate.
Bars represent mean ± S.D. *, p < 0.01.
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hGH-stimulated ATF-2, CHOP, and Elk-1 Transcriptional
Activation Is JAK2-dependent--
We have demonstrated
above that the activation of p38 MAP kinase by hGH is
JAK2-dependent and therefore reasoned that hGH-stimulated transcriptional activation of ATF-2 and CHOP should also be
JAK2-dependent. hGH stimulation of Elk-1 has previously
been demonstrated to be JAK2-dependent (73). We first
examined the hGH-dependent transcriptional activation of
ATF-2, CHOP, and Elk-1 in CHO cell clones stably expressing either the
wild type receptor (CHO-GHR1-638), a receptor mutation in
which the proline-rich box 1 region had been deleted
(CHO-GHR1-638297-311), or a receptor mutation in which
the individual proline residues of box 1 had been converted to alanine
(CHO-GHR1-638P300,301,303,305A) as described above.
Neither the CHO-GHR1-638297-311 nor
CHO-GHR1-638P300,301,303,305A) cell lines responded to
hGH with transcriptional activation of ATF-2, CHOP, or Elk-1 in
contrast to CHO-GHR1-638 cells that responded with the
expected magnitude of transcriptional activation (Fig.
6A). We also demonstrated that
transient transfection of a JAK2 expression plasmid enhanced the
hGH-dependent transcriptional activation of ATF-2, CHOP,
and Elk-1. Furthermore, pretreatment of the cells with 100 µM AG490 prevented both the hGH-dependent transcriptional activation of ATF-2, CHOP, and Elk-1 and the
enhancement of hGH-dependent transcriptional activation
observed upon transient transfection of the JAK2 expression plasmid
(Fig. 6B).
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Fig. 6.
hGH-stimulated transcriptional
activation of ATF-2, CHOP, and Elk-1 requires the proline rich (Box1)
domain of the GH receptor and is JAK2-dependent.
A, CHO-GHR1-638,
CHO-GHR1-638297-311, and
CHO-GHR1-638P300,301,303,305A cells were co-transfected
with the following: 1) -galactosidase expression vector pCMV; 2)
reporter plasmid pFR-Luc; and 3) fusion trans-activator
plasmid pFA-ATF-2, pFA-CHOP, or pFA2-Elk-1 as indicated. Cells were
treated with vehicle (shaded bars) or 50 nM hGH
(solid bars). B, CHO-GHR1-638 cells
were co-transfected with the following: 1) -galactosidase expression
vector pCMV; 2) reporter plasmid pFR-Luc; 3) fusion
trans-activator plasmid either pFA-ATF-2, pFA-CHOP, or
pFA2-Elk-1 as well as pRC-JAK2 as indicated. CHO-GHR1-638
cells were preincubated with 100 µM tyrphostin AG490 or
vehicle Me2SO for 16 h at 37 °C prior to treatment
with 50 nM hGH for 6-8 h, and cell extracts were prepared.
The relative luciferase activities presented were normalized by protein
concentrations as well as -galactosidase activity (mean ± S.D., n = 3). Luciferase activity in cell lysates is
normalized to the activity in untreated control cells (control = 1). The data presented are representative of at least three separate
experiments, each measured in triplicate. Bars represent
mean ± S.D. *, p < 0.01.
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Lack of Cross-inhibition between the MEK1-specific Inhibitor and
the p38 MAP Kinase-specific Inhibitor--
PD98059 selectively blocks
the activity of MEK1 thereby inhibiting the activation of the p44/42
MAP kinase and the subsequent phosphorylation of p44/42 MAP kinase
substrates both in vitro and in vivo (73-75).
SB203580 is a pyridinyl imidazole derivative that is a highly specific
inhibitor of p38 MAP kinase activity (26, 76, 77). We utilized these
pharmacologic agents to determine which pathway was utilized by hGH to
stimulate the transcriptional activation of ATF-2, CHOP, and Elk-1 and
therefore needed to establish the absence of cross-inhibition of the
respective pathways. CHO-GHR1-638 cells were pretreated
with either 30 µM PD98059 or 10 µM SB203580 prior to hGH stimulation, and the activity of both MAP kinases was
determined. 30 µM PD98059 did not inhibit the
hGH-stimulated activation of p38 MAP kinase (Fig.
7A), and 10 µM
SB203580 did not inhibit the hGH-stimulated activation of p42/44 MAP
kinase (Fig. 7B). Both of the inhibitors at the same
concentration specified above effected inhibition of the desired
respective pathway (Fig. 7, A and B). Thus,
PD98059 and SB203580 were suitable for delineation of the MAP kinase
pathway required for hGH-dependent activation of ATF-2,
CHOP, and Elk-1 in the CHO-GHR1-638 cell line.
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Fig. 7.
Inhibition of p38 MAP kinase attenuates
hGH-stimulated ATF-2 and CHOP but not Elk-1-mediated transcriptional
activation. CHO-GHR1-638 cells were preincubated with
the MEK1 inhibitor PD98059 (30 µM) or the p38 MAP kinase
inhibitor SB203580 (10 µM) for 45 min prior to treatment
with 50 nM hGH for 15 min. Cell extracts were prepared, and
p38 MAP kinase activity (A) and p44/42 MAP kinase activity
(B) were determined as described under "Experimental
Procedures." CHO-GHR1-638 cells were
co-transfected with the following: 1) -galactosidase expression
vector pCMV; 2) reporter plasmid pFR-Luc; 3) fusion
trans-activator plasmid either pFA-ATF2 (C),
pFA-CHOP (D), or pFA2-Elk1 (E).
CHO-GHR1-638 cells were preincubated with MEK1 inhibitor
PD98059 (30 µM), p38 MAP kinase inhibitor SB203580 (10 µM), or vehicle (Me2SO) as indicated for 45 min prior to treatment with 50 nM hGH for 6-8 h. The
relative luciferase activities presented were normalized by protein
concentrations as well as -galactosidase activity (mean ± S.D., n = 3). Cells were treated with vehicle
(solid bars) or hGH (solid bars) as above. The
data presented are representative of at least three separate
experiments. Bars represent mean ± S.D. *,
p < 0.01.
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Inhibition of p38 MAP Kinase Attenuates hGH-stimulated ATF-2 and
CHOP but Not Elk-1-mediated Transcriptional Activation--
To
investigate the possible role of p44/42 MAP kinase and p38 MAP kinase
in hGH-induced ATF-2- and CHOP-mediated transcriptional activation,
CHO-GHR1-638 cells transfected with the fusion transactivator plasmid pFA-ATF-2 or pFA-CHOP, and the luciferase reporter plasmids were treated with PD98059, SB203580, or
Me2SO prior to hGH stimulation. Human GH-stimulated
ATF-2-mediated transcriptional activation in CHO-GHR1-638
cells was significantly abrogated by the pretreatment with SB203580 but
was unaffected by pretreatment with PD98059, indicating that
hGH-stimulated ATF-2-dependent gene expression requires p38
MAP kinase activation but not MEK1 and subsequent p44/42 MAP kinase
activation (Fig. 7C). Similar results were demonstrated in
the pFA-CHOP-transfected CHO-GHR1-638 cells (Fig.
7D). These data indicate that hGH-induced ATF-2- and CHOP-mediated transcriptional activation are downstream of p38 MAP
kinase and are independent of p44/42 MAP kinase activation. In
contrast, both p44/42 MAP kinase and p38 MAP kinase have shown to be
upstream of Elk-1 (78). To determine whether inhibition of the p44/42
MAP kinase or the p38 MAP kinase activity affected the ability of Elk-1
to mediate transcriptional activation in response to hGH,
CHO-GHR1-638 cells transfected with pFA-Elk-1 fusion
transactivator plasmid and luciferase reporter plasmid were treated
with 30 µM PD98059, 10 µM SB203580, or
Me2SO prior to hGH stimulation. Human GH-stimulated
Elk-1-mediated transcriptional activation in CHO-GHR1-638
cells was largely abrogated by pretreatment with PD98059 but unaffected
by pretreatment with SB203580, indicating that hGH-stimulated
Elk-1-dependent gene expression requires MEK1 activation
but not p38 MAP kinase activation (Fig. 7E).
Expression of p38 MAP Kinase Enhances hGH-induced ATF-2- and
CHOP-mediated but Not Elk-1-mediated Transcriptional
Activation--
To verify further that hGH-stimulated ATF-2 and
CHOP-mediated transcriptional activation were p38 MAP
kinase-dependent, we transiently transfected either wild
type p38 MAP kinase, dominant negative p38 MAP kinase, or p42 MAP
kinase expression plasmids with the respective fusion
trans-activator plasmids (pFA-ATF-2, pFA-CHOP, and
pFA2-Elk-1) and the luciferase reporter plasmid and examined
hGH-stimulated ATF-2-, CHOP-, and Elk-1-mediated transcription. As
observed in Fig. 8, transfection of wild
type p38 MAP kinase enhanced both hGH-induced ATF-2- and CHOP- but not Elk-1-mediated transcriptional activation. Transfection of the
dominant negative p38 MAP kinase abolished both hGH-induced ATF-2-
and CHOP-mediated but not Elk-1-mediated transcription. Transfection of
p42 MAP kinase did not alter hGH-induced ATF-2- or CHOP-mediated
transcriptional activation, although hGH-induced Elk-1-mediated
transcriptional activation was remarkably enhanced upon co-transfection
of p42 MAP kinase. These data suggest that activation of p38 MAP kinase
is required for hGH-dependent ATF-2- and CHOP-mediated but
not Elk-1-mediated transcriptional activation in
CHO-GHR1-638 cells.
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Fig. 8.
Expression of p38
MAP kinase enhances hGH-induced ATF-2- and CHOP-mediated but not
Elk-1-mediated transcriptional activation. A,
CHO-GHR1-638 cells were co-transfected with the following:
1) -galactosidase expression vector pCMV; 2)reporter plasmid
pFR-Luc; and 3) fusion trans-activator plasmid either
pFA-ATF2, pFA-CHOP, or pFA2-Elk1 and the indicated p38 MAP kinase,
p38 MAP kinase dominant negative, or the p42 MAP kinase (ERK2)
expression plasmids. CHO-GHR1-638 cells were incubated
with 50 nM hGH for 6-8 h, and cell extracts were prepared.
The relative luciferase activities presented were normalized by protein
concentrations as well as -galactosidase activity (mean ± S.D., n = 3). Cells were treated with vehicle
(hatched bars) or hGH (solid bars) as above.
Luciferase activity in cell lysates is normalized to the activity in
untreated control cells (control = 1). The data presented are
representative of at least three separate experiments. Bars
represent mean ± S.D. *, p < 0.01.
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hGH-induced Actin Cytoskeletal Re-organization Is p38 MAP
Kinase-dependent--
hGH has previously been demonstrated
to stimulate re-organization of the actin cytoskeleton including stress
fiber breakdown and the formation of membrane ruffles (79). Since p38
MAP kinase has been implicated to be the required for actin
cytoskeletal reorganization stimulated by a variety of stimuli (18, 80, 81), we therefore examined whether pretreatment of cells with SB203580
affected hGH-induced actin cytoskeletal re-organization. CHO-GHR1-638 cells were grown on coverslips to 50%
confluence and pretreated with PD98059 or SB203580 or the equivalent
volume of Me2SO for 45 min followed by stimulation with 50 nM hGH for 5 min. In vehicle-treated cells, hGH initially
stimulated the depolymerization of actin stress fibers with maximal
depolymerization of stress fibers observed 5 min after addition of hGH
(Fig. 9, A and B).
Pretreatment of cells with SB203580 prevented the ability of hGH to
stimulate stress fiber breakdown in CHO-GHR1-638 cells
(Fig. 9D). In contrast, CHO-GHR1-638 cells
pretreated with PD98059 still exhibited stress fiber breakdown upon hGH
stimulation similar to that of the vehicle-treated cells (Fig.
9C). These results suggest that the stress fiber breakdown
stimulated by hGH is mediated specifically through the p38 MAP kinase
pathway.
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Fig. 9.
hGH-stimulated actin cytoskeletal
re-organization is p38 MAP kinase-dependent.
CHO-GHR1-638 cells were pretreated with Me2SO
(A and B) or 30 µM PD98059
(C) or 10 µM SB208350 (D) for 45 min before subsequent stimulation with 50 nM hGH for 5 min
and fixed in 4% paraformaldehyde as described under "Experimental
Procedures." Filamentous actin was visualized with phalloidin-TRITC
and analyzed by confocal laser scanning microscopy. Similar results
were obtained in at least three separate experiments.
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STAT5-mediated Transcriptional Activation Is Independent of p44/42
MAP Kinase and p38 MAP Kinase--
One major mechanism by which hGH
affects cellular function is by use of the JAK-STAT pathway, especially
JAK2 and STAT5 (1, 2). We have demonstrated here that the p38 MAP
kinase pathway is JAK2-dependent, and therefore, we wished
to determine if p38 MAP kinase was therefore also required for STAT5
activation. CHO-GHR1-638 cells were transfected with the
SPI-GLE1-LUC plasmid and pCMV vector and were pretreated with 30 µM PD98059, 10 µM SB203580, or an
equivalent volume of Me2SO for 45 min prior to hGH
stimulation. hGH-induced STAT5-mediated transcriptional activation was
not affected in the presence of either 30 µM PD98059 or
10 µM SB203580 or combined 30 µM PD98059,
10 µM SB203580, indicating that hGH-induced STAT5-mediated transcriptional activation is independent of both the
p44/42 MAP kinase and the p38 MAP kinase pathways in this system (data
not shown).
Roles of p44/42 MAP Kinase and p38 MAP Kinase Pathways in
hGH-induced Mitogenesis--
To characterize the roles of p38 MAP
kinase and p44/42 MAP kinase pathways in hGH-induced cell mitogenesis,
we examined the effect of the specific chemical inhibitors of the
p44/42 MAP kinase and p38 MAP kinase pathways on hGH-induced cell
proliferation in CHO-GHR1-638 cells. Cell proliferation
was estimated by the [3H]thymidine incorporation assay.
The effects of these inhibitors on DNA synthesis were first examined
over a range of inhibitor concentrations with or without the presence
of 50 nM hGH as indicated. Thus we observed that both
PD98059 and SB203580 inhibit hGH-stimulated [3H]thymidine
incorporation in a dose-dependent manner (Fig.
10, A and B). We
next examined [3H]thymidine incorporation over a range of
hGH concentrations when the concentration of inhibitors was held
constant (Fig. 10C). Inhibition of MEK1 by PD98059
attenuated hGH-induced DNA synthesis by 40-45% in
CHO-GHR1-638 cells. A similar pattern of attenuation of
hGH-induced CHO-GHR1-638 cell proliferation was observed upon inhibition of p38 MAP kinase by SB203580. A combination of both
inhibitors resulted in an additive effect on the inhibition of cell
proliferation, reducing hGH-induced DNA synthesis by 60-70% compared
with cells treated with Me2SO alone.
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Fig. 10.
Role of p44/42 MAP kinase and p38 MAP kinase
pathways in hGH-induced mitogenesis. Cell proliferation was
estimated by the [3H]thymidine incorporation assay. The
effects of the MEK1 inhibitor (A) or p38 MAP kinase
inhibitor (B) on DNA synthesis were first examined over a
range of indicated concentrations of the inhibitors in the presence of
50 nM hGH in CHO-GHR1-638 cells.
[3H]Thymidine incorporations were also examined in
CHO-GHR1-638 cells untreated ( ) or pretreated with
Me2SO ( ), 30 µM PD98059 ( ), 10 µM SB203580 ( ), or a combination of 30 µM PD98059, 10 µM SB203580 ( ) for 45 min
and stimulated with the range of indicated hGH concentrations
(C). Experiments were performed in triplicate, and the
results from three independent experiments are shown; S.E. values were
below 10%.
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DISCUSSION |
In the present study we have demonstrated that hGH induces the
rapid phosphorylation and activation of p38 MAP kinase. We (13, 54) and
others (55, 56) have previously demonstrated that other members of the
MAP kinase family, namely p44/42 MAP kinase and c-Jun amino-terminal
kinase/stress-activated protein kinase (JNK/SAPK), are also activated
by GH. In support of our demonstration of GH-dependent
activation of p38 MAP kinase, the groups of Schwartz and Carter-Su (73)
have recently reported a minimal (0.6-fold) increase in p38 MAP kinase
activity after GH stimulation of 3T3-F442A cells. The role of
activation of the MAP kinases in the cellular effects of GH requires
further delineation. It has been demonstrated that GH activation of
p44/42 MAP kinase is required for Elk-1-mediated transcription and also
for expression of c-fos, egr-1, and
junB (73). We have also previously demonstrated that p44/42
MAP is required for the hyperproliferation of mammary carcinoma cells
induced by autocrine production of GH (82). We have demonstrated here
that p38 MAP kinase is required for hGH stimulation of ATF-2- and
CHOP-mediated transcriptional activation, for hGH-stimulated
re-organization of the actin cytoskeleton, and also for hGH-stimulated
mitogenesis. Thus it is apparent that GH activation of p38 MAP kinase
is pivotal in mediation of the pleiotropic cellular effects of GH.
It is apparent that hGH stimulation of p38 MAP kinase activity is
JAK2-dependent. JAK2 has been proposed to be the initial event in GH signal transduction (1, 4, 83), and no
GH-dependent tyrosine phosphorylation of proteins is
observed in cells transfected with cDNAs encoding GH receptor
mutations lacking the ability to activate JAK2 (58). JAK2 has also been
demonstrated to be required for GH-dependent activation of
p44/42 MAP kinase and a variety of other signaling molecules including
p125FAK (12), IRS-1, and IRS-2 (6). In the case of p44/42 MAP kinase,
JAK2 is required for the phosphorylation of SHC and Grb-2 (8) and therefore subsequent coupling of Ras-Raf upstream of MEK1 (5, 58, 83).
Interestingly though, JAK2 is apparently not required for GH
stimulation of calcium ion influx through L-type calcium channels (84).
In any case this is the first demonstration that p38 MAP kinase is
activated in a JAK-dependent manner despite the fact that
p38 MAP kinase has been reported to be activated by other members of
the cytokine receptor superfamily which also utilize JAK2 such as
erythropoietin (85) and granulocyte colony-stimulating factor (19). It
is most probable that JAK2 is upstream of a previously described
pathway for the activation of p38 MAP kinase (3). We (12) and others
(86) have placed the focal adhesion protein-tyrosine kinase family
(including FAK and Pyk2) as downstream components of JAK activation
(FAK downstream of JAK2 and Pyk2 downstream of JAK3). It has recently
been reported that Pyk2 is critical for the JAK-mediated p44/42 MAP
kinase (87) and p38 MAP kinase (39) activation, and therefore a
JAK2-FAK coupling may be one mechanism for GH activation of the p38 MAP
kinase pathway. In this regard it is interesting that GH utilizes
JAK2-dependent phosphorylation of the EGF receptor to
activate p44/42 MAP kinase (89).
ATF-2 is one member of the ATF/CREB family of transcription factors and
binds to the cAMP response element as a homodimer or heterodimer with
c-Jun (90). Interestingly, GH has previously been demonstrated to
increase the cellular level of c-Jun resulting in increased binding to
AP-1 sites (91). The amino-terminal region of ATF-2 contains the
transcriptional activation domain, which is phosphorylated on Thr-69,
Thr-71, and Ser-90 by stress-activated kinases (including p38 MAP
kinase) (3, 23, 42) leading to increased ATF-2
trans-activation (42, 90). We have demonstrated here that
hGH stimulation of cells results in phosphorylation of ATF-2 and
subsequent ATF-2-mediated transcriptional activation. As could be
expected from the JAK2-dependent activation of p38 MAP
kinase, we also demonstrated that ATF- dependent. ATF-2
trans-activation stimulated by GH may also provide a
mechanism for the increase in c-Jun transcription noted after cellular
exposure to GH since ATF-2 is found in a multiprotein complex with p300
binding to the Jun2 element of the c-jun promoter (92).
ATF-2 trans-activation may also provide one mechanism for
the p38 MAP kinase-dependent portion of GH-stimulated cell
proliferation since ATF-2 has been demonstrated to cooperate with v-Jun
to promote cell proliferation (93).
We have also demonstrated here that hGH stimulation of cells results in
the transcriptional activation of CHOP. CHOP, also known as GADD153
(growth arrest and DNA damage), is a mammalian gene that encodes for a
small nuclear protein related to the CCAAT/enhancer-binding protein
(C/EBP) family of transcription factors (44). We have demonstrated here
that the hGH-stimulated transcriptional activation of CHOP is p38 MAP
kinase-dependent. This is concordant with the demonstration
that CHOP undergoes stress-induced phosphorylation on two adjacent
serine residues (78 and 81) by p38 MAP kinase in vivo with a
resultant enhancement of CHOP trans-activation (44). CHOP
was initially proposed to be involved in cell cycle arrest and
apoptosis (94-96). It is therefore interesting that we observed
hGH-stimulated proliferation, both here in CHO-GHR1-638 cells and in hGH-producing mammary carcinoma cells (82), to be p38 MAP
kinase-dependent. Autocrine production of hGH in mammary carcinoma cells results in the transcriptional up-regulation of CHOP
and increased CHOP-mediated
transcription.2
Overexpression of CHOP by stable transfection of CHOP cDNA in mammary carcinoma cells results in enhanced cell proliferation in
response to hGH,2 suggesting that CHOP is also a positive
regulator of cell number. Recent evidence derived from in
vivo models of CHOP deficiency indicates that CHOP may possess a
dual function by regulation of both apoptosis and cellular regeneration
(97). It has been observed that CHOP protein influences gene expression
as both a dominant negative regulator of C/EBP binding to one class of DNA targets and positively by directing CHOP-C/EBP heterodimers to
other sequences (98), and this observation may constitute the mechanism
of its apparent dichotomous function. hGH may therefore utilize CHOP as
a positive regulator of gene transcription with resultant mitogenesis
in the cell systems described. It is also interesting that GH itself
has also been reported to be both mitogenic and growth-inhibitory
depending on the cellular context (1, 2).
We have demonstrated here that both p38 MAP kinase and p44/42 MAP
kinase cooperate during hGH-stimulated mitogenesis in the CHO-GHR1-638 cell line. Such cooperation between p38 MAP kinase and P44/42 MAP kinase has also been observed for granulocyte colony-stimulating factor stimulated proliferation of hemopoietic cells
(19). Granulocyte colony-stimulating factor is another member of the
cytokine receptor superfamily (1-3). Interestingly, however, we did
not observe such cooperativity between p38 MAP kinase and p44/42 MAP
kinase in mammary carcinoma cells with autocrine hGH production, as the
autocrine hGH-stimulated cell proliferation was completely inhibited
with either the inhibitor for MEK1 (PD98058) or the inhibitor for p38
MAP kinase (SB203580) (82). The target molecules activated by either
p38 MAP kinase or P44/42 MAP kinase responsible for the cooperative
effect on proliferation in the CHO-GHR1-638 cell line are
not defined. Presumably, however, they must represent a class of
molecules, which are exclusively activated by GH stimulation of either
p38 MAP kinase or p44/42 MAP kinase. Such an example may be provided in
this paper whereby the GH-stimulated activation of ATF-2 and CHOP were
p38 MAP kinase-dependent, whereas GH-stimulated activation
of Elk-1 was p44/42 MAP kinase-dependent (73). GH has been
reported to utilize Elk-1 to mediate GH-induced transcription of
egr-1 (99) which may provide a mechanism for the p44/42 MAP
kinase-dependent component of GH-stimulated proliferation. GH is a comparatively weak mitogen, and many effects of GH are exerted
on differentiated cell function. GH may therefore also utilize p38 MAP
kinase for regulation of differentiation or differentiated cell
function. For example, p38 MAP kinase has been reported to be required
for erythroid differentiation (100), chondrogenesis (101), and myotube
formation (102).
We have demonstrated here that inhibition of p38 MAP kinase prevents
hGH-stimulated stress fiber breakdown in the CHO-GHR1-638 cell line, whereas inhibition of MEK1 was without effect. Previous reports (80, 103, 104) have demonstrated that modulation of actin
dynamics by p38 MAP kinase requires the phosphorylation of HSP27
downstream of MAPKAP-2 and MAPKAP-3 (80, 103-105). MAPKAP-2 has
previously been reported to be activated by GH (73). It is therefore
likely that GH also modulates the phosphorylation state of HSP27,
although this has not yet been demonstrated. GH-stimulated re-organization of the actin cytoskeleton has been demonstrated to
require phosphatidylinositol 3-kinase activity (79), suggesting that
phosphatidylinositol 3-kinase may also be upstream of the GH-dependent increase in p38 MAP kinase activity. A
previous study has reported that p38 MAP kinase activated by the
chemotactic peptide N-formyl-Met-Leu-Phe requires
phosphatidylinositol 3-kinase activity (106). Such is the case for hGH
stimulation of both p44/42 MAP kinase (73, 110) and
JNK/SAPK.3
In summary we have demonstrated here that hGH transiently
phosphorylates and activates p38 MAP kinase in CHO cell lines stably transfected with rat GH receptor cDNA and that the activation of
the p38 MAP kinase pathway by hGH is JAK2-dependent.
Furthermore, both ATF2 and CHOP are transcriptionally activated by hGH
in a JAK2-dependent manner, and p38 MAP kinase is required
for both cytoskeletal re-organization and cell proliferation stimulated by hGH. Since p38 MAP kinase has also been reported to phosphorylate and activate several other protein kinases, including MNK1, MNK2, MAPKAPK2, MAPKAPK3, MSK1, and PRAK (22, 52, 108-112), it is likely to
be central to the pleiotropic cellular effects of GH.
| |
ACKNOWLEDGEMENTS |
We thank Drs. Nils Billestrup, Shengcai Lin,
and Jiahua Han for contribution to this work.
| |
FOOTNOTES |
*
This work was supported by the National Science and
Technology Board of Singapore (to P. E. L.).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.: 65-8747847; Fax:
65-7791117; E-mail: mcbpel@mcbsgs1.imcb.nus.edu.sg.
2
H. C. Mertani, T. Zhu, G. Morel, K. O. Lee, and
P. E. Lobie, manuscript in preparation.
3
E. L. Goh, T. Zhu, and P. E. Lobie,
manuscript in preparation.
| |
ABBREVIATIONS |
The abbreviations used are:
GH, growth hormone;
hGH, human GH;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
CHO, Chinese hamster ovary;
JNK, c-Jun amino-terminal kinases;
SAPK, stress-activated protein kinase;
MEK, MAPK/extracellular
signal-regulated kinase kinase;
ATF-2, activating transcription
factor-2;
DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulfate;
PBS, phosphate-buffered saline;
TRITC, tetramethylrhodamine isothiocyanate.
| |
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