From the Departments of Cell Biology and Orthopedics
and the Yale Cancer Center, Yale University School of Medicine, New
Haven, Connecticut 06520 and the ¶ Department of Physiology and
Division of Oral Biology, Faculty of Medicine and Dentistry, The
University of Western Ontario, London, Ontario N6A 5C1, Canada
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ABSTRACT |
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While it is well established that adenylyl
cyclase and phospholipase C- are two proximal signal effectors for
the calcitonin receptor, the more distal signaling pathways are less
well characterized. G protein-coupled receptors can activate Erk1/2 by
Gs-, Gi-, or Gq-dependent signaling pathways, depending on
the specific receptor and cell type examined. Since the calcitonin
receptor can couple to all three of these G proteins, the ability
of calcitonin to activate Erk1/2 was investigated. Calcitonin induced
time- and concentration-dependent increases in Shc
tyrosine phosphorylation, Shc-Grb2 association and Erk1/2
phosphorylation and activation in a HEK 293 cell line that stably
expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin,
which inactivates Gi, and calphostin C, a protein kinase C
inhibitor, each partially inhibited calcitonin-induced Shc tyrosine
phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In
contrast, neither forskolin nor H89, a protein kinase A inhibitor, had
a significant effect on basal or calcitonin-stimulated Erk1/2
phosphorylation. Our results suggest that the calcitonin receptor
induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by
parallel Gi- and PKC-dependent mechanisms. The
calcitonin-induced elevation of cytosolic free Ca2+ was
required for Erk1/2 phosphorylation, since preventing any change in
cytosolic free Ca2+ by chelating both cytosolic and
extracellular Ca2+ abolished the response. However, the
change in Ca2+ that is induced by calcitonin is not
sufficient to account for the calcitonin-induced Erk1/2
phosphorylation, since treatment with 100 nM ionomycin or
10 µM thapsigargin, each of which induced elevations of
Ca2+ comparable to those induced by calcitonin, induced
significantly less Erk1/2 phosphorylation than that induced by
calcitonin. Erk1/2 may have important roles as downstream effectors
mediating cellular responses to calcitonin stimulation.
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INTRODUCTION |
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Calcitonin (CT)1 is a
polypeptide hormone that regulates calcium homeostasis by inhibiting
osteoclastic bone resorption and enhancing renal calcium excretion (1).
High affinity CT binding has also been demonstrated in other tissues,
including the central nervous system, pituitary gland, and
gastrointestinal tract. Because of its marked hypocalcemic effect, CT
has been widely used for treating diseases that are characterized by
elevated osteoclast activity, such as hypercalcemia of malignancy,
osteoporosis, and Paget's disease (1). The CT receptor (CTR) couples
to multiple heterotrimeric G proteins, leading to the activation of the
proximal effectors adenylyl cyclase and phospholipase C (2-4).
Recently, the CTR-dependent activation of phospholipase D
has also been reported (5). CT-induced activation of adenylyl cyclase
and phospholipase C
appear to elicit different components of a
cell's response to CT. For example, in isolated osteoclasts,
cAMP-dependent mechanisms lead to reduced cell motility
while protein kinase C-dependent events are apparently
involved in the CT-induced retraction of the cell (6, 7). In contrast
to the well documented activation of adenylyl cyclase and phospholipase
C
following stimulation of the CTR, less progress has been made in
characterizing the more distal signaling pathways elicited by CT
(8).
Mitogen-activated protein kinases (MAPKs) are a group of
serine/threonine protein kinases, including Erk1/2, JNK, and SAPK, that
lie at the end of parallel protein kinase cascades. MAPKs are activated
in response to the stimulation of several distinct classes of
cell-surface receptors, including receptor tyrosine kinases and G
protein-coupled receptors (GPCRs), and play important roles in
integrating the effects of extracellular signals on multiple cellular
functions, including differentiation, proliferation, and transformation
(9, 10). Erk1/2 (also denoted as p42/44 MAPK) were the first to be
identified and are the best characterized. Their activation by several
GPCR ligands has recently been well documented (11). Depending on the
receptors and the cell types, several distinct signal transduction
pathways leading from GPCRs to Erk1/2 have been demonstrated. The
pertussis toxin-sensitive Gi-coupled receptors
2A-adrenergic receptor (AR) and
M2A-muscarinic acetylcholine receptor activate Erk1/2 by a
mechanism that involves the sequential tyrosine phosphorylation of the
adaptor protein Shc, association of phosphorylated Shc with the
Grb2·mSOS complex, and activation of Ras (12). The same pathway is
used by the receptors for EGF and other growth factors. The
Gq-coupled receptors
1B-AR and
M1A-muscarinic acetylcholine receptor can activate Erk1/2
via a PKC-dependent but Ras-independent pathway in some cells (12) and via a Ras-dependent pathway in others (13, 14). cAMP, which antagonizes Erk1/2 activation in some cells (15-18),
activates Erk1/2 in PC12 cells (19) and in COS-7 cells (20).
Many GPCRs couple to more than one type of heterotrimeric G protein. For example, the thrombin and lysophosphatidic acid (LPA) receptors couple to Gi, Gq, and G12/13, and the thyrotropin receptor couples to Gs, Gi, Gq, and G12/13 (21-23). Interestingly, the LPA receptor-dependent activation of Erk1/2 is virtually abolished by pertussis toxin, indicating that activation is primarily via Gi with little contribution from Gq or G12/13 (24). The PTH/PTHrP receptor also couples to at least two different types of heterotrimeric G proteins, Gs and Gq (25). Recent reports indicate that the PTH/PTHrP receptor can regulate Erk1/2 activity by different pathways, with the specific mechanism and resulting effect on Erk1/2 activity depending on the cellular context (15, 26). These observations suggest that the mechanisms by which individual receptors regulate Erk1/2 activity are highly receptor- and cell type-specific.
Since the CTR is capable of coupling to Gs, Gi, and Gq, we examined the ability of CT to regulate Erk1/2 activity. We report here that calcitonin induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 activation in HEK 293 cells that stably express the rabbit osteoclast CTR C1a isoform. Additive effects of pertussis toxin and PKC inhibition suggest that both Gi-dependent and pertussis toxin-insensitive PKC-dependent mechanisms contribute to the CTR-induced activation of Erk1/2. Erk1/2 are therefore downstream effectors of the CTR which may mediate certain cellular responses to CT.
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EXPERIMENTAL PROCEDURES |
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Materials--
Salmon calcitonin was purchased from Peninsula
Laboratories, Inc. (Belmont, CA). Antibodies against total Erk1/2 and
phosphorylated Erk1/2 were purchased from New England Biolabs, Inc.
(Beverly, MA). The polyclonal antibody used for Shc
immunoprecipitation, the monoclonal anti-phosphotyrosine antibody 4G10,
the antibody against Grb2, and MAP kinase MBP(95-98) substrate peptide
were from Upstate Biotechnology (Lake Placid, NY). Fetal bovine serum, forskolin, epidermal growth factor (EGF), pertussis toxin (PTX), phorbol 12-myristate 13-acetate (PMA) and LPA were from Sigma. The
MEK-specific inhibitor PD98059 and calphostin C were purchased from
Calbiochem-Novabiochem International (San Diego, CA). Indo-1 penta(acetoxymethyl)ester (indo-1 AM), BAPTA-AM, and thapsigargin were
obtained from Molecular Probes (Eugene, OR). Enhanced chemiluminescence solutions and nitrocellulose membranes were from Amersham and Schleicher & Schuell (Keene, NH), respectively. The pRK5 vector and
pRK-ARK1ct were generous gifts from Dr. Robert J. Lefkowitz.
Stable Cell Line and Transient Transfections--
HEK 293 cell
stably expressing the rabbit CTR C1a (C1a-HEK)(4) were maintained in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
serum and 50 units/ml penicillin (Sigma) and 50 µg/ml streptomycin
(Sigma) in 10-cm plates until 80% confluent. Cells were then incubated
in DMEM with 0.5% fetal bovine serum for 18 h prior to the
treatments. For transient transfection, the HEK 293 cells were plated
in 6-well plates until 80-90% confluent and transfected with 0.4 µg/well of CTR C1a in the presence of either 1 µg/well empty vector
pRK5 or 1 µg/well ARKct using LipofectAMINE reagent (Life
Technologies). The cells were cultured in DMEM with 0.5% fetal bovine
serum for 18 h prior to stimulation with CT.
Cell Stimulation and Preparation of Lysates-- Cells were treated with agents as indicated in the figure legends. After treatment, cells were washed once with ice-cold phosphate-buffered saline and lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysates were centrifuged for 30 s at 12,000 × g to pellet insoluble material. Protein concentrations were assayed with Bradford protein assay reagent (Bio-Rad).
Detection of Shc Tyrosine Phosphorylation and Shc-Grb2 Complex Formation-- For analysis of Shc tyrosine phosphorylation and its association with Grb2, 1.5 mg of cell lysate protein were incubated with 5 µg of polyclonal anti-Shc antibody plus 50 µl of a 50% slurry of protein A-Sepharose (Sigma) with mild agitation for 1 h at 4 °C. Immune complexes were washed three times with ice-cold modified RIPA buffer and denatured in Laemmli sample buffer (27). Following resolution by SDS-polyacrylamide gel electrophoresis, proteins were transferred to nitrocellulose membranes which were then cut at the position of the 30-kDa protein marker. The upper part of the membrane was immunoblotted with anti-phosphotyrosine antibody 4G10 (1:1000 dilution) to detect the extent of tyrosine phosphorylation of Shc. The membrane was stripped and reblotted with monoclonal anti-Shc antibody (1:1000 dilution) to determine the amounts of Shc proteins. The lower part of the membrane was immunoblotted with antibody against Grb2 (1:1000 dilution) to detect the presence of co-precipitated Grb2.
Measurements of Erk1/2 Phosphorylation-- Total cell lysates (20 µg) were electrophoresed on 12% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose membranes. Phosphorylation of Erk1/2 was measured by immunoblotting with a polyclonal antibody (anti-phospho-MAPK) that recognizes only the activated Erk1/2 (13). Total Erk1/2 was detected by blotting with the antibody against a phosphotyrosine-independent Erk1/2 epitope (anti-p44/42 MAPK). Membranes were incubated with the anti-phospho-MAPK antibody (1:1000) or the anti-p44/42 MAPK antibody (1:1000) overnight at 4 °C. The next day, after three washes in 50 mM Tris, pH 7.5, containing 150 mM NaCl and 0.1% Tween 20, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-linked secondary antibody (1:20,000). After repeated washes, bound antibody was detected by the ECL method.
Erk1/2 Activity Assay--
C1a-HEK cells were grown to 80%
confluence, then the medium was replaced by DMEM with 0.5% fetal
bovine serum and penicillin/streptomycin for 18 h. The next day,
the cells were incubated with or without the MEK inhibitor PD 98059 (50 µM) for 30 min prior to the addition of 1 nM
CT or 10 ng/ml EGF. After 5-min exposure to the ligands, the cells were
harvested in 50 mM -glycerophosphate, pH 7.3, containing
1.5 mM EGTA, 1 mM EDTA, 1 mM
dithiothreitol, 1 mM sodium fluoride, 10 µg/ml aprotinin,
10 µg/ml leupeptin (28). The cells were homogenized by passing
through a 27-gauge needle five times, and the homogenates centrifuged
for 30 min at 12,000 × g. The kinase assay was
performed in 25 µl of kinase buffer containing 50 mM
HEPES, pH 7.4, 10 mM MgCl2, and 50 µM ATP (specific activity 0.8 Ci/mmol). MBP(95-98)
substrate peptide was added at a final concentration of 1 µg/µl.
The reaction was initiated by adding 5 µg of cellular extracts to the
above assay buffer, allowed to proceed for 30 min at room temperature,
and terminated by spotting the reaction mixture onto Whatman p81
phosphocellulose paper. Free [
-32P]ATP was removed by
three extensive washes in 150 mM phosphoric acid. The p81
phosphocellulose paper was rinsed briefly in acetone and the
radioactivity incorporated into MBP peptide was measured by
scintillation counting.
Measurement of Cytosolic Free Ca2+ Concentration ([Ca2+]i)-- C1a-HEK cells were loaded with indo-1 by incubation in conditioned medium with indo-1 AM (2 µM) for 30 min at 37 °C. Cells were then washed and harvested by brief exposure to nominally Ca2+- and Mg2+-free buffer containing trypsin (0.05%) and EDTA (0.5 mM). Conditioned medium was added to inactivate trypsin, following which cells were sedimented and resuspended in HEPES-buffered minimum essential medium. Aliquots of cell suspension were sedimented and resuspended in 2 ml of continuously stirred Na+-HEPES buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 20 mM HEPES, pH 7.3, 290 mOsmol/liter) in a fluorimetric cuvette maintained at 37 °C. In experiments investigating dependence on extracellular Ca2+, cells were suspended in nominally Ca2+-free Na+-HEPES buffer or in Ca2+-containing Na+-HEPES buffer to which EGTA (5 mM) was added. For most experiments, solutions in the cuvette contained 0.5% fetal bovine serum. Where indicated, cells were loaded with BAPTA-AM (50 µM) for 30 min concomitantly with indo-1 AM. Test substances were added directly to the cuvette.
[Ca2+]i was monitored using a dual wavelength fluorimeter (model RF-M2004, from Photon Technology International, London, Canada) with excitation wavelength of 355 nm and emission wavelengths of 405 and 485 nm. The system software was used to subtract background fluorescence and calculate the ratio R, which is the fluorescence intensity at 405 nm divided by the intensity at 485 nm. [Ca2+]i was determined from the relationship [Ca2+] = Kd((R ![]() |
RESULTS |
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Calcitonin Induces Transient and Concentration-dependent Activation of Erk1/2-- To examine the effect of CT on Erk1/2 phosphorylation, C1a-HEK cells were treated with 1 nM CT for increasing periods of time, then lysed and analyzed for phosphorylated Erk1/2 as described under "Experimental Procedures." CT induced rapid phosphorylation of Erk1/2, which was apparent as early as 30 s, reached a maximum at 5 min, and decreased to a low level by 60 min (Fig. 1A, upper panel). Parallel samples blotted with Erk1/2 antibody (lower panel) showed no change in the amount of Erk1/2 protein. The cells express predominantly the 42-kDa Erk2 protein, and both forms are phosphorylated at levels proportional to their expression. The effect of CT on Erk1/2 phosphorylation, measured after 5 min of treatment, was concentration-dependent. A detectable increase in Erk1/2 phosphorylation was induced by as little as 0.001 nM CT and the maximum response occurred at concentrations of 1 nM or higher. The maximum phosphorylation induced by CT was comparable to that induced by 10 ng/ml EGF (Fig. 1B).
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The Cyclic AMP-Protein Kinase A Pathway Has Little Effect on Erk1/2 Phosphorylation-- GPCR may regulate Erk1/2 activity via one or more of several different pathways involving Gs-, Gi-, and Gq-dependent mechanisms, depending on the receptors and the cellular context (11). Since the CTR is capable of coupling to Gs, Gi, and Gq, we investigated which pathways are involved in CT-induced Erk1/2 activation. Although cAMP generally attenuates growth factor-induced Erk1/2 activation (16-18), the activation of Erk1/2 by cAMP has been reported (19, 20). We therefore examined whether cAMP participated in CT-induced Erk1/2 activation (Fig. 3). Serum-starved C1a-HEK cells were treated with 200 µM forskolin for 25 min alone or followed by 1 nM CT for an additional 5 min. Forskolin induced only a slight increase in Erk1/2 phosphorylation (lane 3) and had no apparent effect on CT-induced Erk1/2 phosphorylation (lane 4). Furthermore, the protein kinase A-specific inhibitor H89 (10 µM) had little effect on CT-stimulated Erk1/2 phosphorylation (lane 6). Taken together, these data suggest that the cAMP/PKA pathway does not contribute significantly to CT-induced Erk1/2 activation in HEK-293 cells.
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Both Gi and PKC Mediate CT-induced Shc Tyrosine
Phosphorylation and Erk1/2 Phosphorylation--
In HEK 293 cells, both
PTX-sensitive Gi-coupled receptors and PTX-insensitive
Gq-coupled receptors can activate Erk1/2 via the
Shc/Grb2/Ras pathway (13) by which receptor tyrosine kinases such as
the EGF receptor activate Erk1/2 (30). To determine whether Shc also
plays a role in the mechanism of CT-induced Erk1/2 activation, the
tyrosine phosphorylation of Shc and its association with Grb2 were
analyzed. C1a-HEK cells were treated with 1 nM CT for
various times and lysed as described under "Experimental Procedures." Shc was immunoprecipitated from 1.5 mg of cell lysates and the immunoprecipitated proteins were analyzed for Shc
phosphorylation and Grb2 association as described under "Experimental
Procedures." As shown in Fig.
4A, 1 nM CT
induced the tyrosine phosphorylation of predominantly the 52-kDa Shc,
which reached a maximum level at 5-10 min and remained elevated during
the time course examined for up to 120 min. The broad band below 52-kDa
Shc was a nonspecific interaction of the immunoprecipitation antibody
with the immunoblotting antibodies, since it was present in mock
immunoprecipitations performed in the absence of cell lysate (not
shown). The amount of immunoprecipitated Shc is shown in the
middle panel of Fig. 4A. The induction of Shc
tyrosine phosphorylation was also concentration-dependent, with the maximum effect induced by CT concentrations 1 nM
(Fig. 4B). Co-precipitated Grb2 was readily detectable in
the samples in which Shc was phosphorylated. As expected, EGF also
induced Shc tyrosine phosphorylation and its association with
Grb2 (Fig. 4B), although to a much greater extent than did
CT.
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CT-induced Increase in Cytosolic Calcium Is Required for Erk1/2 Phosphorylation-- A recent report suggested that the increase in cytosolic Ca2+ is one of the key mediators of both Gi- and Gq-coupled adrenergic receptor-stimulated Erk1/2 phosphorylation in HEK 293 cells (13). On the other hand, the PTH/PTHrP receptor (structurally related to CTR) activated Erk1/2 via a Ca2+-independent mechanism in Chinese hamster ovary cells (26). Since CT increases [Ca2+]i (32), we investigated the role of [Ca2+]i in CT-induced Erk1/2 phosphorylation. [Ca2+]i levels were manipulated using EGTA and BAPTA to chelate extracellular and intracellular Ca2+, respectively, the calcium ionophore ionomycin to induce the elevation of [Ca2+]i, and thapsigargin to deplete the intracellular Ca2+ stores. Changes in [Ca2+]i were monitored by fluorescence spectroscopy as described under "Experimental Procedures."
Consistent with earlier reports (32, 33), 1 nM CT induced a biphasic elevation of [Ca2+]i, which rose within 30 s from basal levels of 134 ± 19 nM to peaks of 319 ± 37 nM above basal levels, followed by a sustained plateau of 57 ± 5 nM above basal levels (n = 7, Fig. 7B). The earlier reports showed that the initial transient results from the release of Ca2+ from intracellular stores and the sustained phase results from the influx of extracellular Ca2+ (33). No change in fluorescence was detected when C1a-HEK cells were treated with vehicle alone (Fig. 7A). When cells were loaded with BAPTA and then suspended in nominally Ca2+-free Na+ buffer, the Ca2+ response to CT was virtually abolished (Fig. 7E). Similarly, pretreatment of cells with 5 mM EGTA for 30 min markedly decreased the amplitude of both the peak and plateau phases of the [Ca2+]i elevation induced by CT (Fig. 7F). These treatments abolished the CT-induced Erk1/2 phosphorylation (Fig. 9A, lanes 5 and 6), indicating that an increase in [Ca2+]i is required for the CT-mediated response. In contrast, preincubation of the cells with EGTA for 30 min had little effect on EGF-induced Erk1/2 phosphorylation (Fig. 9A, lanes 7 and 8).
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DISCUSSION |
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This study demonstrates that CT activates Erk1/2 in HEK 293 cells that express the C1a isoform of the rabbit CTR in a time- and concentration-dependent manner. The CT-induced activation of Erk1/2 is largely independent of cAMP/PKA-dependent pathways. Rather, in these cells the CTR activates the Shc/Grb2/Erk1/2 pathway via both Gi- and PKC-coupled mechanisms. The CTR-stimulated sustained increase in [Ca2+]i is necessary to support the full CT-dependent Erk1/2 phosphorylation, but the concurrent activity of other signaling events (e.g. PKC activation) is required for the maximal CT-induced response.
Recent results from us and others have shown that signaling via the CTR
involves the activation of at least two G proteins, Gs and
Gq (2-4). In the present study, we demonstrate that the CTR can also activate Gi since the CTR-stimulated events
described here are partly PTX-sensitive. The CTR stimulates prolonged
Shc tyrosine phosphorylation, as reported for some other GPCR, such as
the thrombin and endothelin receptors, and for the insulin receptor or
growth hormone receptor (34-37). This prolonged Shc tyrosine
phosphorylation is in contrast to the transient effects observed with
the Gi-coupled 2A-AR (31) or the
Gq-coupled
1B-AR (38). On the other hand,
the CTR-induced Erk1/2 phosphorylation is transient. While CT induces
prolonged Shc phosphorylation, it is likely that activated Erk1/2
trigger a negative feedback loop in which MAP kinase phosphatase(s) are
activated and in turn dephosphorylate and inactivate Erk1/2, since
treatment with the phosphatase inhibitor sodium orthovanadate prolonged
CT-stimulated Erk1/2 phosphorylation.2 The difference in
the time course of phosphorylation of the two proteins is the subject
of further investigation.
The finding that PTX and calphostin C have additive effects on both Shc and Erk phosphorylation is important for several reasons. It suggests that the Gi-dependent and PKC-dependent mechanisms that are activated by the CTR each can activate the Shc/Grb2/Erk1/2 pathway independently of the other, and that the Gi- and PKC-dependent mechanisms converge at or above the level of Shc in HEK 293 cells. Furthermore, it demonstrates for the first time that a single GPCR, the CTR, can activate Erk1/2 through parallel independent signaling pathways in the same cells.
Our results confirm the recent report of Della Rocca and colleagues
(13) that both Gi- and Gq-coupled receptors
activate Erk1/2 through the Shc/Grb2 pathway in HEK cells. However, our results differ in some respects. First, inhibition of PKC partially blocks the CTR-induced Erk1/2 phosphorylation, but has no effect on the
responses elicited by the Gi-coupled 1B-AR
or the Gq-coupled
2A-AR. This difference may
be due to the receptors, the G protein isoforms or other intermediate
effectors that are activated by the CTR in our study and by the
1B-AR and
2A-AR in the other. Second,
these authors found that 10 µM A23187 stimulated Erk1/2 phosphorylation to the same degree as the adrenergic receptors and that
Erk phosphorylation was mostly inhibited by BAPTA. On this basis, they
proposed that the receptor-induced increase in [Ca2+]i leads to activation of tyrosine kinases
and subsequently of the Shc/Ras/Erk cascade. In contrast, we found that
treatment with 100 nM ionomycin, which induced a
[Ca2+]i increase that was comparable to the
CT-dependent increase in [Ca2+]i,
resulted in much lower Erk1/2 phosphorylation than was seen following
CT treatment. Moreover, BAPTA did not inhibit the Erk1/2
phosphorylation induced by CT. Although the absence of data on the
actual changes in [Ca2+]i induced by the
adrenergic receptors makes it impossible to fully compare these
results, our results clearly demonstrate that the maximal
phosphorylation of Shc and Erk1/2 in response to CT requires a
sustained [Ca2+]i response that is at least
40-50 nM above basal levels, and that CT can stimulate
Erk1/2 phosphorylation when the initial Ca2+ transient is
completely suppressed by BAPTA. Furthermore, the changes in
[Ca2+]i that are induced by stimulation of the
CTR are not sufficient to cause the maximal phosphorylation of Shc and
Erk 1/2 in the absence of other signaling events.
The fact that CT-stimulated Erk1/2 phosphorylation requires sustained [Ca2+]i elevation of 40-50 nM above basal levels is in keeping with the recent report on differential activation of transcription factors by a small Ca2+ plateau versus a large Ca2+ transient in B-lymphocytes (39). In many situations, particularly in cells that express voltage-sensitive Ca2+ channels, an influx of extracellular Ca2+ is both necessary and sufficient for activation of Erk1/2 (40, 41). However, in C1a-HEK cells, the increase in [Ca2+]i alone is not sufficient to account for the full CT-induced increase in Erk1/2 phosphorylation, since 100 nM ionomycin or 1 µM thapsigargin each induced changes in [Ca2+]i comparable to those induced by CT, but stimulated little Erk1/2 phosphorylation. Furthermore, although challenging the thapsigargin-treated cells with CT failed to induce any additional change in [Ca2+]i, the level of Erk1/2 activation was much higher in cells treated with both thapsigargin and CT than in cells treated only with thapsigargin. Thus, both the increase in [Ca2+]i and the activation of other signaling effectors, including PKC, are required for the full CTR-dependent Erk1/2 response.
Although the main focus of investigations of Erk1/2 function has been on their role in mediating the mitogenic effects of diverse ligands for receptor tyrosine kinases and GPCR, these proteins were originally identified as enzymes that phosphorylate microtubule-associated proteins and thereby regulate cytoskeletal stability (42-44). It is now well established that Erk1/2 are involved in far more than just mitogenic signaling (10). The ability of the CTR to activate the Shc to Erk1/2 pathway by two independent mechanisms implies that Erk1/2 may play an important role in mediating the morphological and/or biochemical effects of CT on osteoclast.
The duration of Erk activation has been proposed to determine the cell fate in PC 12 cells (45). Nerve growth factor stimulates sustained Erk activation which leads to the outgrowth of neurites and cessation of cell division, whereas EGF induces transient Erk activation which correlates with cell proliferation. If the model of PC 12 cells can be extended to cells expressing CTR, CT-induced transient Erk1/2 activation may be responsible for the proliferative effect of CT on a human prostate cancer cell line (46). The effect of CT on the proliferation of T47D and MCF-7, two other carcinoma cell lines that express the CTR, is currently under investigation.
In conclusion, we have demonstrated that CT induces Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 activation in C1a-HEK by parallel independent pathways involving Gi and PKC. Both pathways require the elevation of [Ca2+]i, but the change in [Ca2+]i that occurs in response to treatment with CT cannot by itself induce full Erk1/2 phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert J. Lefkowitz for
providing the pRK5 and pRK-ARK1ct plasmids and Dr. Hong Sun for
helpful discussion.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DE-04724 (to R. B.) and Medical Research Council of Canada (MRC) Grant MT-10854 (to S. J. D.).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.
§ Recipient of a Fulbright Award. Present address: Dept. of Biology and Anatomy, National Defense Medical Center, P. O. Box 90048-502, Taipei, Taiwan 100, Republic of China.
Recipient of a Medical Research Council Studentship Award.
** Present address: First Department of Internal Medicine, Tokushima University School of Medicine, 3-18-15 Kuramato-cho, Tokushima-shi, Tokushima 770, Japan.
Present address: Research Institute of Molecular Pathology, Dr.
Bohr-Gasse 7, A-1030, Vienna, Austria.
§§ To whom correspondence should be addressed: Dept. of Orthopedics, Yale University School of Medicine, P. O. Box 208044, New Haven, CT 06520-8044. Tel.: 203-785-2165; Fax: 203-785-2744; E-mail: william.horne{at}yale.edu.
1 The abbreviations used are: CT, calcitonin; AR, adrenergic receptor; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; [Ca2+]i, cytosolic free calcium concentration; CTR, calcitonin receptor; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PTX, pertussis toxin; PMA, phorbol 12-myristate 13-acetate; PTH, phenylthiohydantoin.
2 Y. Chen, J-F. Shyu, A. Santhanagopal, D. Inoue, J.-P. David, S. J. Dixon, W. C. Horne, and R. Baron, unpublished observations.
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