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The Department of Pharmacology, The University of Missouri School of Medicine, Columbia, Missouri 65212
Address all correspondence and requests for reprints to: Judith A. Cole, Ph.D., M517B Medical Sciences Building, The University of Missouri School of Medicine, Columbia, Missouri 65212. E-mail: ColeJ{at}health.missouri.edu
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Abstract |
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2 h) and
dose-dependent (EC50 3 x 10-10
M) activation of MAPK. Immunoblot analysis with an
activation- specific MAPK antibody indicated that PTH activated both
p42 and p44 MAPK. Epidermal growth factor (EGF) also activated p42 and
p44MAPK in a time- (peak at 5 min, return to basal within 2 h) and
dose-dependent (EC50 3 ng/ml) fashion. PTH-dependent
MAPK activation was mimicked by the protein kinase C activator (PKC)
phorbol myristate acetate (PMA), and the protein kinase A activators 8
bromo-cAMP (8-Br-cAMP) and forskolin but was not affected by pertussis
toxin pretreatment. PMA or 8-Br-cAMP pretreatment blocked MAPK
activation by reexposure to each kinase activator but caused no
significant reduction in MAPK activation by PTH. MAPK activation by
PTH, EGF, and 8-Br-cAMP was inhibited by the MAPK kinase
inhibitor PD98059 and an EGF receptor (EGFR)-selective inhibitor
tyrphostin AG1478. AG1478 also blocked MAPK activation by insulin-like
growth factor-1 and platelet-derived growth factor. EGF and PTH caused
time- and AG1478-sensitive phosphorylation of the EGFR, but EGFR
desensitization did not affect MAPK activation by PTH. EGF, PMA, and
low doses of PTH (1012 to 10-9
M) stimulated while 8-Br-cAMP and high doses of PTH
(10-8 to 10-6 M) inhibited
[3H]thymidine uptake. These data demonstrate that PTH
activates MAPK and suggest that PKC, protein kinase A, and the EGFR
play roles in PTH signaling. The biphasic effect of PTH on DNA
synthesis suggests that MAPK activation by the hormone leads to
distinct cellular responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to regulating cellular functions by activating effectors
such as adenylyl cyclase and phospholipase C, many G protein-coupled
receptors (GPCRs) regulate proliferative or differentiative signals via
MAPK. Gi-coupled agonists activate MAPK in a pertussis toxin
(PTX)-sensitive and Gß
- and Ras-dependent fashion that does not
require protein kinase C (PKC) activation (2, 3, 4, 5, 6). In contrast, MAPK
activation by Gq-coupled agonists is PKC dependent but PTX insensitive
and may or may not involve Ras (4, 6, 7, 8, 9). In addition, Gi- and
Gq-coupled MAPK activation converges at the level of phospholipase C
and proceeds through a common Ras-dependent pathway involving
Ca2+/calmodulin, c-src, and the tyrosine
phosphorylation of FAK-related kinase Pyk2 or the EGFR (5, 6, 7, 9, 10, 11, 12, 13).
In this model, the tyrosine-phosphorylated Pyk2 or EGFR serves as a
"scaffolding" molecule where the Ras mitogenic complex assembles
after GPCR activation. In some circumstances, non-RTKs such as
c-src lie upstream of EGFR phosphorylation (5, 7, 12), while
in others, ligand-independent (transactivation) of the EGFR tyrosine
kinase leads to EGFR autophosphorylation (4, 10, 11, 14, 15, 16). Unlike
Gi- and Gq-coupled MAPK activation, Gs-dependent regulation of MAPK
activity seems more cell specific. In some cells, cAMP attenuates
growth factor-mediated MAPK activation (17, 18) while activating MAPK
in others (3, 19, 20, 21). This may reflect a balance Gs
-dependent
inhibition and Gsß-mediated stimulation of MAPK activity (22) or
the differential expression of cAMP-activated B-Raf and cAMP-inhibited
Raf-1 (19, 21, 23). Involvement of c-src, Pyk2, or the EGFR
in Gs-coupled MAPK activation has not been described, but protein
kinase A (PKA) phosphorylates and inactivates the c-src
regulatory protein Csk, which could lead to c-src-dependent
MAPK activation (24).
In cells expressing PTH/PTH-related peptide receptors (PTHR), PTH leads to activation of Gs/adenylyl cyclase and Gq/phospholipase C (25) and in the proximal tubule-like OK cell line, PTH causes PKC- and PKA-dependent inhibition of Na-dependent phosphate (Na/Pi) transport (26, 27, 28). In addition to its well described regulation of renal transport function, several findings suggest that PTH may regulate MAPK in target tissues. First, the PTHR couples to signaling pathways that regulate MAPK in many systems. Second, PTH has proliferative effects in kidney and bone (29, 30). Finally, PTH causes compensatory renal growth when administered to uninephrectomized rats (31). In fact, PTH activates MAPK in parietal yolk sac carcinoma (PYS-2) cells and in CHO-R15 cells transiently expressing high levels of the PTHR (3). However, the effects of PTH on both cell proliferation and MAPK activity have not been assessed together. In this study we show that PTH causes time- and concentration-dependent increase in OK cell MAPK activity. PTH regulation of MAPK activity appears to involve PKC and PKA, as well as phosphorylation of the EGFR, and leads to biphasic regulation of DNA synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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32P]ATP (6000 Ci/mmol) and
[methyl-3H]thymidine (6.7 Ci/mmol) were obtained from
NEN Life Science Products (Boston, MA).
Immunoprecipitation/immunoblotting reagents were purchased from the
following vendors: Amersham Pharmacia Biotech (Arlington
Heights, IL), enhanced chemiluminescence (ECL) reagents; Bio-Rad Laboratories, Inc. (Richmond, CA), goat antimouse
IgG-horseradish peroxidase; New England Biolabs, Inc., polyclonal phospho-specific (tyr 204) and monoclonal
phospho-specific (thr202/tyr204) MAPK antibodies; Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), protein A-agarose;
Transduction Laboratories (Lexington, KY), monoclonal anti-EGFR and
antiphosphotyrosine antibodies; and Upstate Biotechnology, Inc. (Lake Placid, NY), rabbit antimouse IgG.
Cell culture
OK cells were grown in DMEM/Ham’s F12 (DMEM/F12) containing 5%
FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained
at 37 C in a humidified atmosphere of 95% air/5% CO2..
Subculturing was performed weekly using Ca/Mg-free HBSS and 0.025%
trypsin/0.02% EDTA. Cells were plated at 60,000 cells/ml, were
confluent in 3 days, and were used between 3–4 days in culture. In all
experiments, cells were serum deprived 24 h in DMEM/F12 containing
0.1% BSA, 100 U penicillin/ml, and 100 µg streptomycin/ml
(DMEM/F12-BSA). Drugs were added to this medium, and cells were
incubated in CO2 incubators for the times indicated.
Measurement of MAPK activity
An immune complex assay measuring the phosphorylation of myelin
basic protein was used to assess MAPK activity. Briefly, cells grown in
60-mm dishes were treated as described in each experiment. Dishes were
then washed three times with ice-cold PBS and lysed with 0.5 ml of
ice-cold lysis buffer (20 mM Tris, pH 7.5, 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%
Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
ß-glycerol phosphate, 1 mM
Na3VO4, and 0.2 mM PMSF). Dishes
were incubated 5 min at 4 C and then scraped and the lysates were
collected in microfuge tubes. The lysates were vortexed at high speed
for 20 sec and then microfuged 10 min at 4 C to remove insoluble
material. Equal amounts of protein (200 µg) were incubated with a
polyclonal phospho (activation)-specific (tyr 204) MAPK antibody
(1:100) overnight at 4 C. This antibody detects phosphorylated tyrosine
204 of p42 and p44MAPK but does not cross-react with p38
MAPK or JNK/SAPK homologs. Immunocomplexes were precipitated with
protein A-agarose and washed twice with 0.5 ml of lysis buffer and
twice with 0.5 ml kinase buffer (25 mM Tris, pH 7.5, 2
mM dithiothreitol, 5 mM ß-glycerol phosphate,
10 mM MgCl2, and 0.1 mM
Na3VO4). The pellets were resuspended in 50
µl of kinase buffer containing 100 µM ATP (10 µCi
[32P]ATP/ml) and 500 µg/ml myelin basic protein and
were incubated 30 min at 30 C. Reactions were terminated by spotting 20
µl of the reaction mix on Whatman (Clifton, NJ) P81
filter paper. Filters were washed three times in 75 mM
phosphoric acid and once in 95% ethanol and dried; 32P was
determined by liquid scintillation counting. Kinase activity is
reported in nanomoles/200 µg/30 min or as the fold increase over
basal activity. In some experiments, increases in threonine 203 and
tyrosine 204 phosphorylation indicative of p42/p44 MAPK activation were
assessed by immunoblot analysis with a monoclonal phospho-specific
(thr202/tyr204) anti-MAPK antibody (anti-pMAPK). Equal amounts of
protein from kinase assay lysates were separated by 12% SDS-PAGE,
equilibrated in transfer buffer (25 mM Tris, pH 8.3, 250
mM glycine, and 20% methanol), and then transferred to
nitrocellulose. Blots were blocked overnight in 5% nonfat dry milk
followed by a 2-h incubation in a 1:1,000 dilution of the anti-pMAPK.
Blots were then incubated with goat antirabbit IgG-horseradish
peroxidase and developed using ECL.
Analysis of EGFR phosphorylation
OK cells grown in 60-mm dishes were treated as described
in each experiment. Dishes were washed three times with PBS, lysed in
0.5 ml of lysis buffer (10 mM Tris, pH7.4, 1% Triton
X-100, 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 0.2 mM Na3VO4, 0.2
mM PMSF, and 0.5% NP-40) and rocked 30 min at 4 C. Dishes
were scraped and lysates were collected in microfuge tubes and vortexed
20 sec at high speed; insoluble materials were removed by microfuging
10 min at 4 C. Equal amounts of protein (1–2 mg) from each sample were
incubated overnight at 4 C with 5 µg of a monoclonal anti-EGFR
(anti-EGFR) or a monoclonal antiphosphotyrosine (anti-PY20) antibody.
Samples were then incubated 2 h at 4 C with rabbit antimouse IgG
and protein A-agarose, and the antibody-antigen complexes were
collected by centrifugation. Pellets were washed three times with lysis
buffer, resuspended in 2x SDS-PAGE sample buffer, and resolved on
7.5% SDS-PAGE gels. After transfer to nitrocellulose, samples were
immunoblotted with anti-PY to assess EGFR phosphotyrosine content or
with anti-EGFR to identify the EGFR and assess equivalency of
immunoprecipitation.
Cell proliferation
Cells grown in 48-well dishes were changed to serum-free medium
upon reaching confluency. Twenty-four hours later, cells were changed
to serum-free medium containing [3H]thymidine (2
µCi/ml) and increasing concentrations of PTH, EGF, PMA, or 8-Br-cAMP.
After 24 h, [3H]thymidine uptake was terminated by
washing each well three times with ice-cold PBS followed by extraction
with 5% TCA for 20 min at 4 C. TCA-insoluble material was dissolved in
0.5 ml of 0.2 N NaOH, and [3H]thymidine
content was determined by liquid scintillation counting. DNA synthesis
is expressed as the fold increase over control
[3H]thymidine uptake.
Statistical analyses
Data are means ± SEM of at least four
experiments assayed in duplicate and performed over several passages of
OK cells. Statistical significance was determined by ANOVA using
Dunnetts or Bonferroni’s multiple comparison test as indicated in the
figure legends. Differences are considered significant at
P 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
in the phosphothreonine and phosphotyrosine content of both p42
and p44 MAPK (Fig. 1A, inset). Activation was maximal
between 15–30 min, and was approximately 5-fold greater than basal at
30 min (from 1.4 ± 0.4 to 7.2± 2.3 nmol/200 µg/30 min). MAPK
activity began to decline at 60 min and returned to basal levels at
2 h. EGF also activated MAPK, with approximately 5-fold increases
occurring within 5 min (from 2.2 ± 0.8 to 10.0 ± 3.3
nmol/200 µg/30 min). EGF stimulation declined at 30 min, returning to
basal levels by 2 h. EGF also enhanced the phosphorylation of p42
and p44 MAPK (Fig. 1A, inset). A 15-min incubation with
increasing concentrations of PTH stimulated MAPK activity with an
EC50 of approximately 3 x 10-11
M (Fig. 1C). Significant increases (P
0.05) were first observed at 10-11 M, and
maximally effective concentrations of PTH increased MAPK activity
approximately 4-fold (from 2.0 ± 0.8 to 8.6 ± 1.2 nmol/200
µg/30 min). MAPK activation by EGF was detectable at 0.1 ng/ml, but
significant increases were not observed until reaching 100 ng/ml where
activity increased approximately 3-fold (from 4.0 ± 1.7 to
11.0 ± 2.6 nmol/200 µg/30 min) (Fig. 1C).
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-subunits, Gq/PKC,
Ca2+/calmodulin, and Gs/PKA (1, 6). To characterize the
mechanisms involved in PTH-dependent MAPK activation, we first assessed
the involvement of Gi by pretreating cells for 24 h with 100 ng/ml
PTX. Cells were then incubated 15 min with PTH (10-7
M) or the Gi-coupled 2A adrenergic receptor agonist
UK-14,304 (10-5 M), which activates MAPK in OK
cells (2). As expected, PTX pretreatment significantly reduced MAPK
activation by UK14,304 but had no effect on PTH-stimulated MAPK
activity (Fig. 2). These data indicate
that PTX-sensitive G proteins do not mediate PTH-dependent MAPK
activation in OK cells.
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). PMA
caused a rapid (within 5 min) 4-fold increase in MAPK activity (from
1.2 ± 0.1 to 4.6 ± 1.5 nmol/200 µg/30 min), which
returned to basal levels by 2 h. 8-Br-cAMP caused a slower (peak
within 60 min) 4-fold increase in MAPK activity (from 1.4 ± 0.2
to 6.0 ± 0.9 nmol/200 µg/30 min), which was still elevated at
2 h. 8-Br-cAMP and PMA enhanced the phosphorylation of both p42
and p44MAPK (Fig. 3A, inset). The intensity of MAPK
activation by PMA makes it difficult to resolve p42 and p44
phosphorylation at high doses of the PKC activator. Fifteen-minute
incubations with increasing concentrations of PMA enhanced MAPK
activity with an EC50 of approximately 3 x
10-9 M. Significant increases
(P 0.05) were observed at 10-8
M, and maximally effective concentrations enhanced activity
approximately 4-fold (from 4.2 ± 1.5 to 17.6 ± 2.3 nmol/200
µg/30 min) (Fig. 3B). 8-Br-cAMP and the adenylyl cyclase activator
forskolin (FSK) also caused dose-dependent (EC50 values of
10-5 M) and statistically significant
activation of MAPK (Fig. 3C).
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). However, PMA pretreatment did not
significantly reduce MAPK activation by PTH (7.5 ± 1.4
vs. 5.4 ± 0.6 nmol/200 µg/30 min; PTH vs.
PTH + PMA pretreatment, respectively). 8-Br-cAMP pretreatment had
no effect on basal MAPK activity and blunted MAPK activation by acute
reexposure to 8-Br-cAMP (6.5 ± 1.9 vs. 2.8 ± 0.9
nmol/200 µg/30 min; 8-Br-cAMP vs. 8-Br-cAMP + 8-Br-cAMP
pretreatment). Although 8-Br-cAMP pretreatment caused a 50% reduction
in PTH-dependent MAPK activation (9.0 ± 3.4 vs.
4.5 ± 1.0 nmol/200 µg/30 min, PTH vs. PTH +
8-Br-cAMP pretreatment, respectively) this change was not statistically
significant (Fig. 4B). These data suggest that either PTH activation of
MAPK does not require costimulation of PKC and PKA or that other
signaling pathways mediate the PTH response.
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(top panel) illustrates PTH
(10-7 M)-stimulated and EGF (100
ng/ml)-stimulated MAPK activity in cells pretreated with the broad
spectrum tyrosine kinase inhibitor genistein (IC50 =
3–25 µM for EGFR and c-src, >100
µM for PKA and PKC), the c-src-selective
inhibitor herbimycin A (IC50 = 0.9 µM
for c-src; no significant effect on PKA or PKC), the
EGF-RTK-selective inhibitor tyrphostin AG1478 (AG1478)
[IC50 = 3 nM; > 100 µM for
Erb-2/neu and the platelet-derived growth factor (PDGF)
receptor], and the MEK-specific inhibitor PD98059
(IC50 = 4 µM for MEK1 and 50
µM for MEK2 with no effect on any related dual
specificity kinases). Genistein (10-4 M),
which competitively interferes with ATP binding and noncompetitively
with the protein substrate, reduced agonist-stimulated MAPK activity.
These changes were significant in PTH + genistein-treated cells
(P 0.05), but not in EGF + genistein-treated cells.
Herbimycin A (10-6 M), which irreversibly
binds to c-src thiol groups and targets the protein for
degradation, had no significant effect on MAPK activation by either EGF
or PTH. The quinazoline AG1478 (10-7 M), which
competitively inhibits ATP binding and protein substrate binding in an
unknown fashion, significantly (P 0.01) reduced
EGF-stimulated MAPK activity from 9.1 ± 1.0 to 1.1± 0.4 nmol/200
µg/30 min. Surprisingly, AG1478 also blocked MAPK activation by PTH
(P 0.01), reducing activation from 7.8 ± 1.7
to 1.2 ± 0.5 nmol/200 µg/30 min. These data suggest that
transactivation of the EGFR contributes to MAPK activation by PTH. The
MEK1/2-specific inhibitor PD98059 (5 x 10-4
M), which binds to inactive MEK and prevents its activation
by Raf-1, blocked MAPK activation by PTH (P 0.01),
indicating the hormone activates MAPK in a MEK-dependent fashion.
Although MAPK activation by PMA (10-7 M) was
modestly reduced by all four inhibitors, these changes were not
statistically significant (Fig. 5, lower panel). In
contrast, MAPK activation by 8-Br-cAMP was blocked (P
0.01) by AG1478 and PD98059, suggesting MAPK activation by PTH is
MEK-dependent and may involve PKA-dependent EGFR activation.
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), which was identified as the EGFR by
reprobing with anti-EGFR (Fig. 6B). In addition, anti-PY20 and
anti-EGFR immunoprecipitates from EGF- and PTH-treated cells contained
four additional tyrosine phosphorylated proteins (61, 73, 85, and 110
kDa; Fig. 6C). EGFR phosphorylation was not detected in anti-PY20 or
anti-EGFR immunoprecipitates from unstimulated control cells, and the
presence of the tyrosine-phosphorylated proteins in anti-EGFR
immunoprecipitates indicated these proteins were associated with the
EGFR.
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). To be certain that AG1478 was
specific for the EGF-dependent MAPK activation, we evaluated its effect
on MAPK activation by IGF-1 and PDGF. PTH, EGF, IGF-1, and PDGF all
caused dose-dependent increases in the phosphorylation of p42 and p44
MAPK. Surprisingly, a 30-min pretreatment with AG178 blocked MAPK
activation by all four receptor agonists (Fig. 7B). While AG1478
clearly blocks EGFR phosphorylation, it is not selective for
EGF-dependent signaling in OK cells. Whether this reflects nonselective
effects on other growth factor RTKs or downstream signaling molecules,
it is clear that AG1478 cannot be used to evaluate EGFR transactivation
in OK cells. Therefore, we assessed the effects of EGFR desensitization
on MAPK activation. Pretreating the cells for 6 h with EGF (100
ng/ml) blocked MAPK activation by reexposure to the growth factor, but
had no effect on MAPK activation by PTH (Fig. 7C). Taken together, the
lack of AG1478 selectivity and the inability of EGFR desensitization to
block MAPK activation by PTH suggest that the PTH-induced tyrosine
phosphorylation of the EGFR results from the activation of a
non-RTK.
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).
Although 8-Br-cAMP caused marked increases in MAPK activity (Fig. 2
),
it inhibited DNA synthesis in OK cells (Fig. 8B). Interestingly, a 24-h
incubation with low doses of PTH (10-12 to
10-9 M) significantly increased DNA while
doses of PTH 
10-8 M appeared to reduce
DNA synthesis (Fig 8B). Thus, PTH produced EGF- and PMA-like effects on
DNA synthesis at low doses and cAMP-like inhibition of proliferation at
high doses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PTH caused time- and concentration-dependent increases in MAPK activity and in the phosphotyrosine content of p42 and p44MAPK. Although EGF produced a more rapid increase in MAPK activity, PTH and EGF were equally effective activators as the enzyme. While PTH-dependent activation of MAPK has been observed in CHO-R15 and Psy2 cells (3), the hormone caused cAMP-dependent inhibition of MAPK in ROS17/2.8 and UMR106 osteosarcoma cells (18). These conflicting results in PTHR-expressing cells reflect the heterogeneity of responses produced by GPCR agonists in different cell types. Gq-coupled receptor activation leads to PKC-dependent and Ras-independent MAPK activation in CHO cells (36), Ca2+/calmodulin- and Ras-dependent activation in vascular smooth muscle cells (8, 10, 15), and Ca2+- and Pyk2-dependent activation in PC12 cells (6, 11). Similarly, cAMP inhibits MAPK in many cells (17, 18) yet activates the enzyme in others (3, 20, 21, 34). Since GPCR-dependent regulation of MAPK activity depends, to a large extent, on the cellular complement of receptors, G proteins, and effectors, these data underscore the importance of identifying the signaling molecules leading to GPCR-induced MAPK activation by a specific agonist in a given cell type.
In OK cells, activation of PKA or PKC leads to PTH-like inhibition of Na/Pi transport, and the loss of signaling in either pathway prevents PTH-dependent regulation of the transport process (26, 28). This suggests that PKA and PKC signals must be integrated in order for PTH to inhibit Na/Pi transport. In the present study, PMA and cAMP mimic PTH-dependent MAPK activation, yet PKC down-regulation and PKA desensitization did not significantly reduce the hormone response. The MEK inhibitor PD98059 blocked MAPK activation by PTH and 8-Br-cAMP, but had no effect on the response to PMA. The inability to block PMA-induced MAPK activation most likely reflects the relatively weak inhibitory effect of PD98059 in the face of the substantial MAPK activation produced by PMA and, in fact, the EGF response was similarly unaffected by PD98059, even though MAPK activation by this growth factor is MEK dependent (1). Similarly, AG1478 eliminated MAPK activation by EGF, PTH, and 8-Br-cAMP as well as IGF-1 and PDGF but had no effect on the response to PMA. These data indicate that AG1478 is not selective for the EGFR in OK cells and may also inhibit the activity of a downstream signaling molecule used by growth factors and GPCRs to activate MAPK. Even with this lack of selectivity, the fact that AG1478 does not inhibit PMA-induced MAPK activation suggests the inhibitor’s target is not present in the pathway leading to PKC-dependent activation of MAPK. The preceding data may be interpreted in several ways. First, they may indicate that the integration of PKA and PKC signaling is not required for PTH regulation of MAPK activity. If this is true, then PKC down-regulation or PKA desensitization would not affect PTH-dependent MAPK activation through the remaining pathway. For example, MAPK regulation by PKA may be Ras dependent (2, 3, 22, 37) while activation by PKC may be Ras independent (4, 6, 8, 9). On the other hand, PKA may cause Rap1- and B-Raf-dependent MAPK activation (19, 21, 38) while PKC may directly phosphorylate and activate Raf-1 (35). Second, the observation that AG1478 and PD98059 produced comparable effects on PTH- and 8-Br-cAMP-dependent activation of MAPK may indicate that PTH-dependent regulation of MAPK activity is mediated solely by PKA. In CHO-R15 cells, PMA and increases in intracellular cAMP both activate MAPK, but MAPK stimulation by PTH was mediated solely by cAMP (3). Third, Gq/phospholipase C-induced Ca2+ mobilization rather than PKC activation may mediate the PTH response. Recent studies have shown that Gi- and Gq-coupled receptor agonists cause Ca2+/calmodulin-dependent and PKC-independent MAPK activation (6, 7, 10, 11, 15). In these studies, PKC down-regulation prevents MAPK activation by PMA, but does not affect the response to Gi- or Gq-coupled agonists. Finally, these data may indicate that PTH regulates MAPK activity through signaling pathways other than PKA or PKC. PTH does activate phospholipase A2 in rat proximal tubule cells (39) and phospholipase D in rat osteosarcoma cells (40). Identifying signaling molecules upstream from MAPK is required to determine the common or unique targets for PTH/PKA and/or PTH/PKC-dependent regulation of MAPK activity.
PTH, like a number of GPCR agonists, causes an AG1478-sensitive tyrosine phosphorylation of the EGFR with a time course preceding that for MAPK activation. This observation is not unique to PTH since the inhibition of EGFR catalytic activity with AG1478 or dominant negative EGFRs prevents GPCR-induced MAPK activation in a variety of cell types (4, 6, 10, 11, 14). In this model, transactivation of the EGFR leads to receptor phosphorylation and its use as a "scaffold" for the assembly of the Ras mitogenic complex. Although the AG1478 sensitivity of PTH-induced EGFR phosphorylation suggests that PTH transactivates the EGFR, EGFR desensitization has no effect on MAPK activation by PTH. These data, coupled with AG1478’s lack of EGFR selectivity, suggest the catalytic activity of EGFR is not required for PTH-dependent MAPK activation. This does not mean that the EGFR is not involved in PTH signaling, since non-RTKs such as c-src phosphorylate the EGFR and lead to Ras-dependent MAPK activation. Recent studies have shown that dominant negative c-src, overexpression of the c-src regulatory protein Csk, and the c-src inhibitor herbimycin A impair GCPR-induced EGFR phosphorylation and MAPK activation (5, 6, 7, 10, 12, 15). c-src phosphorylation of the EGFR can occur even when EGFR’s catalytic activity is inhibited (10). Thus, our data are consistent with PTH phosphorylating the EGFR by activating a non-RTK rather than transactivating the EGFR. Regardless of the mechanisms involved in EGFR phosphorylation, the results are consistent from study to study: GPCR-induced EGFR phosphorylation allows the receptor to serve as a scaffold for assembling the Ras mitogenic complex and leads to MAPK activation. Thus, it seems likely that PTH-induced EGFR phosphorylation is involved in PTH-dependent MAPK activation. Additional studies will be required to define the mechanism involved in PTH-dependent EGFR phosphorylation and determine how this leads to MAPK activation.
Renal tubular proliferation occurs during the recovery phase
after renal ischemia, and a variety of growth factors including EGF
contribute to the recovery process (41, 42). In rabbit proximal tubule
cells, concentrations of PTH and PTH-related peptide ranging
from 10-11 M to 10-8
M enhanced DNA synthesis (29). These responses were
mimicked by PMA and 8-Br-cAMP, suggesting that PKA and PKC mediate
these effects. Likewise, comparable doses of PTH and PMA stimulated
[3H]thymidine uptake in OK cells. However, at higher
doses of PTH ( 10-8 M), DNA synthesis began
to decline. Interestingly, the decrease in DNA synthesis occurred at
10-8 M PTH, which is the EC50 for
activation of adenylyl cyclase reported in many studies (26, 28). This,
coupled with the fact that 8-Br-cAMP inhibited DNA synthesis, suggests
that high doses of PTH cause cAMP-dependent inhibition of cellular
proliferation. These data are consistent with the responses in
LLC-PK1 cells, where increases in intracellular cAMP
suppress DNA synthesis and accelerate the appearance of Na-dependent
glucose transport (i.e. induce differentiation) while PMA
and the expression of constitutively active Gq stimulates proliferation
and suppresses the appearance of Na-dependent glucose transport (43, 44). If the biphasic effect of PTH on DNA synthesis indicates that PTH
stimulates both proliferation and differentiation, how might this
occur? In many systems the duration of MAPK activation is an important
determinant of cellular responses. In PC12 cells, transient activation
of MAPK by EGF stimulates proliferation, while sustained activation by
nerve growth factor induces differentiation (21, 34). Nerve
growth factor-induced PC12 differentiation appears to require a
sustained increase in MAPK activity, which is mediated by
cAMP-dependent activation of Rap1 (38). In OK cells, EGF and PMA caused
rapid (within 5 min) and shorter-lived increases in MAPK
activity than PTH and 8-Br-cAMP. If the duration of MAPK activation
determines the cellular response to PTH, then PTH-dependent PKC
activation or EGFR phosphorylation-associated signaling may induce
proliferation while PTH-induced PKA (and Rap1?) activation may lead to
growth arrest and the initiation of cellular differentiation.
In summary, PTH causes time- and concentration-dependent activation of MAPK that is mimicked by PMA and 8-Br-cAMP. MAPK activation is preceded by PTH-induced EGFR phosphorylation, suggesting that the EGFR is involved in the hormone response. This has important implications in understanding how PTH regulates cellular function and is particularly compelling in renal failure where reductions in functioning renal mass lead to up-regulation of EGFRs and secondary hyperparathyroidism (41, 45). The biphasic changes in DNA synthesis caused by PTH suggest that the hormone may cause both MAPK-dependent proliferation and differentiation depending upon the signal generated by the activated PTHR. Further characterization of the signaling pathways used by PTH will be required to define the mechanisms involved in EGFR phosphorylation, the roles of PKA and PKC in MAPK stimulation, and the cellular responses to increases in MAPK activity.
Received May 7, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-adenoceptors in opossum
kidney cells couple to the stimulation of mitogen-activated protein
kinase independently of adenylyl cyclase inhibition. Arch Pharm
(Weinheim) 356:225–232[CrossRef]
-dependent activation and a
Gs-cAMP-mediated inhibition. J Biol Chem 270:25259–25265This article has been cited by other articles:
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  W. B. Sneddon, Y. Yang, J. Ba, L. M. Harinstein, and P. A. Friedman Extracellular signal-regulated kinase activation by parathyroid hormone in distal tubule cells Am J Physiol Renal Physiol, March 1, 2007; 292(3): F1028 - F1034. [Abstract] [Full Text] [PDF]
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 D. Gesty-Palmer, M. Chen, E. Reiter, S. Ahn, C. D. Nelson, S. Wang, A. E. Eckhardt, C. L. Cowan, R. F. Spurney, L. M. Luttrell, et al. Distinct beta-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation J. Biol. Chem., April 21, 2006; 281(16): 10856 - 10864. [Abstract] [Full Text] [PDF]
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 M. J. Mahon, T. M. Bonacci, P. Divieti, and A. V. Smrcka A Docking Site for G Protein {beta}{gamma} Subunits on the Parathyroid Hormone 1 Receptor Supports Signaling through Multiple Pathways Mol. Endocrinol., January 1, 2006; 20(1): 136 - 146. [Abstract] [Full Text] [PDF]
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R. Cunningham, X. E, D. Steplock, S. Shenolikar, and E. J. Weinman Defective PTH regulation of sodium-dependent phosphate transport in NHERF-1-/- renal proximal tubule cells and wild-type cells adapted to low-phosphate media Am J Physiol Renal Physiol, October 1, 2005; 289(4): F933 - F938. [Abstract] [Full Text] [PDF]
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C. A. Syme, P. A. Friedman, and A. Bisello Parathyroid Hormone Receptor Trafficking Contributes to the Activation of Extracellular Signal-regulated Kinases but Is Not Required for Regulation of cAMP Signaling J. Biol. Chem., March 25, 2005; 280(12): 11281 - 11288. [Abstract] [Full Text] [PDF]
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 T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
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 S. M. Khan, R. H. Oliver, and J. Yeh Epidermal Growth Factor Receptor Inhibition by Tyrphostin 51 Induces Apoptosis in Luteinized Granulosa Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 469 - 473. [Abstract] [Full Text] [PDF]
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 A. Shih, S. Zhang, H. J. Cao, H.-Y. Tang, F. B. Davis, P. J. Davis, and H.-Y. Lin Disparate Effects of Thyroid Hormone on Actions of Epidermal Growth Factor and Transforming Growth Factor-{alpha} Are Mediated by 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase II Endocrinology, April 1, 2004; 145(4): 1708 - 1717. [Abstract] [Full Text] [PDF]
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M. J. Mahon, J. A. Cole, E. D. Lederer, and G. V. Segre Na+/H+ Exchanger-Regulatory Factor 1 Mediates Inhibition of Phosphate Transport by Parathyroid Hormone and Second Messengers by Acting at Multiple Sites in Opossum Kidney Cells Mol. Endocrinol., November 1, 2003; 17(11): 2355 - 2364. [Abstract] [Full Text] [PDF]
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L. Qin, P. Qiu, L. Wang, X. Li, J. T. Swarthout, P. Soteropoulos, P. Tolias, and N. C. Partridge Gene Expression Profiles and Transcription Factors Involved in Parathyroid Hormone Signaling in Osteoblasts Revealed by Microarray and Bioinformatics J. Biol. Chem., May 23, 2003; 278(22): 19723 - 19731. [Abstract] [Full Text] [PDF]
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S. Chauvin, M. Bencsik, T. Bambino, and R. A. Nissenson Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and {beta}-Arrestin Mol. Endocrinol., December 1, 2002; 16(12): 2720 - 2732. [Abstract] [Full Text] [PDF]
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T. A. Doggett, J. T. Swarthout, S. C. Jefcoat Jr., D. Wilhelm, A. Dieckmann, P. Angel, and N. C. Partridge Parathyroid Hormone Inhibits c-Jun N-Terminal Kinase Activity in Rat Osteoblastic Cells by a Protein Kinase A-Dependent Pathway Endocrinology, May 1, 2002; 143(5): 1880 - 1888. [Abstract] [Full Text] [PDF]
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N. Miyakoshi, Y. Kasukawa, T. A. Linkhart, D. J. Baylink, and S. Mohan Evidence That Anabolic Effects of PTH on Bone Require IGF-I in Growing Mice Endocrinology, October 1, 2001; 142(10): 4349 - 4356. [Abstract] [Full Text] [PDF]
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 H. A. W. Tawfeek, J. Che, F. Qian, and A. B. Abou-Samra Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E545 - E557. [Abstract] [Full Text] [PDF]
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O. Kifor, R. J. MacLeod, R. Diaz, M. Bai, T. Yamaguchi, T. Yao, I. Kifor, and E. M. Brown Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells Am J Physiol Renal Physiol, February 1, 2001; 280(2): F291 - F302. [Abstract] [Full Text] [PDF]
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W. B. Sneddon, F. Liu, F. A. Gesek, and P. A. Friedman Obligate Mitogen-Activated Protein Kinase Activation in Parathyroid Hormone Stimulation of Calcium Transport But Not Calcium Signaling Endocrinology, November 1, 2000; 141(11): 4185 - 4193. [Abstract] [Full Text] [PDF]
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W. Tian, Z. Zhang, and D. M. Cohen MAPK signaling and the kidney Am J Physiol Renal Physiol, October 1, 2000; 279(4): F593 - F604. [Abstract] [Full Text] [PDF]
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J. T. Swarthout, T. A. Doggett, J. L. Lemker, and N. C. Partridge Stimulation of Extracellular Signal-regulated Kinases and Proliferation in Rat Osteoblastic Cells by Parathyroid Hormone Is Protein Kinase C-dependent J. Biol. Chem., March 2, 2001; 276(10): 7586 - 7592. [Abstract] [Full Text] [PDF]
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D. Miao, X.-K. Tong, G. K. Chan, D. Panda, P. S. McPherson, and D. Goltzman Parathyroid Hormone-related Peptide Stimulates Osteogenic Cell Proliferation through Protein Kinase C Activation of the Ras/Mitogen-activated Protein Kinase Signaling Pathway J. Biol. Chem., August 17, 2001; 276(34): 32204 - 32213. [Abstract] [Full Text] [PDF]
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D. Cussac, S. Schaak, C. Gales, C. Flordellis, C. Denis, and H. Paris alpha 2B-Adrenergic receptors activate MAPK and modulate proliferation of primary cultured proximal tubule cells Am J Physiol Renal Physiol, May 1, 2002; 282(5): F943 - F952. [Abstract] [Full Text] [PDF]
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