1 Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital; and 2 Endocrine Division, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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Regulation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway by the extracellular calcium (Cao2+)-sensing receptor (CaR) was investigated in bovine parathyroid and CaR-transfected human embryonic kidney (HEKCaR) cells. Elevating Cao2+ or adding the selective CaR activator NPS R-467 elicited rapid, dose-dependent phosphorylation of ERK1/2. These phosphorylations were attenuated by pretreatment with pertussis toxin (PTX) or by treatment with the phosphotyrosine kinase (PTK) inhibitors genistein and herbimycin, the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor GF109203X and were enhanced by the PKC activator phorbol 12-myristate 13-acetate. Combined treatment with PTX and inhibitors of both PKC and PTK nearly abolished high Cao2+-evoked ERK1/2 activation in HEKCaR cells, demonstrating CaR-mediated coupling via both Gq and Gi. High Cao2+ increased serine phosphorylation of the 85-kDa cytosolic phospholipase A2 (cPLA2) in both parathyroid and HEKCaR cells. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished high-Cao2+-induced ERK1/2 activation and reduced cPLA2 phosphorylation in both cell types, documenting MAPK's role in cPLA2 activation. Thus our data suggest that the CaR activates MAPK through PKC, presumably through Gq/11-mediated activation of PI-PLC, as well as through Gi- and PTK-dependent pathway(s) in bovine parathyroid and HEKCaR cells and indicate the importance of MAPK in cPLA2 activation.
calcium-sensing receptor; mitogen-activated protein kinase; signal transduction; phospholipase A2; kidney
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INTRODUCTION |
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THE PARATHYROID GLANDS play an essential role in mineral ion homeostasis by virtue of their capacity to recognize and respond to (i.e., sense) small changes in the extracellular ionized calcium concentration (Cao2+) (4, 5). Parathyroid hormone (PTH) secretion is reduced, and renal Ca2+ excretion is increased, at high Cao2+, processes mediated by a recently characterized cell surface Cao2+-sensing receptor (CaR) (6; for reviews, see Refs. 7, 8). The CaR is a member of the superfamily of G protein-coupled receptors (GPCRs) and has a large extracellular domain that binds Cao2+ and other polycationic CaR agonists such as magnesium, gadolinium, and neomycin. The CaR controls multiple signaling pathways (6, 8, 24, 25, 35). Ligand binding results in G protein-dependent activation of phosphatidylinositol-specific phospholipase C (PI-PLC), causing accumulation of inositol 1,4,5-trisphosphate (IP3) and 1,2-sn-diacylglycerol and promoting rapid release of Ca2+ from its intracellular stores (8, 15, 26, 46). CaR-mediated activation of PI-PLC in parathyroid and human embryonic kidney cells (HEK293) stably transfected with the human CaR (HEKCaR) appears to be a direct, G protein-mediated process, probably involving Gq/11, since this effect is not blocked by pertussis toxin (PTX) (8). Activation of phospholipases A2 (PLA2) and D by high Cao2+ are probably indirect, utilizing CaR-mediated, PLC-dependent activation of protein kinase C (PKC) (8, 24). High Cao2+-elicited, CaR-induced activation of these intracellular signaling pathways is thought to cause the associated decrease in the rate of PTH secretion from the parathyroid cell.
Studies of inherited disorders of Cao2+ sensing have established the CaR's central role in Cao2+ homeostasis. Inactivating CaR mutations cause an increase in the set point for Cao2+-regulated PTH secretion and promote excessive renal tubular Cao2+ reabsorption in the hypercalcemic disorders, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism (1, 7, 8). Conversely, activating mutations produce a form of autosomal dominant hypocalcemia with inappropriately normal or low PTH levels and excessive renal Ca2+ excretion at any given level of Cao2+ (1, 7, 8). Abnormal Cao2+ sensing in hyperparathyroidism (i.e., due to parathyroid adenoma or severe uremic hyperparathyroidism) is associated with reduced expression of the CaR protein (27). The selective calcimimetic CaR activator NPS R-568 effectively inhibits PTH secretion and concomitantly reduces serum calcium concentration in hyperparathyroid patients (38).
The mitogen-activated protein kinase (MAPK) pathways are found ubiquitously in eukaryotic organisms (16). The MAPK are serine/threonine-specific protein kinases and include the extracellular signal-regulated kinases ERK1 and ERK2, the cJun NH2-terminal kinases, and the p38-MAP kinases (16, 20, 21). MAPKs are regulated and activated by dual tyrosine and threonine phosphorylation (20, 28). Phosphorylated, activated ERK1/2 can phosphorylate cytosolic protein substrates [e.g., cytosolic phospholipase A2 (cPLA2)] (22, 29, 30) and also translocate to the nucleus, where they phosphorylate and activate nuclear transcription factors (16, 20, 29).
Early studies of the biological roles of MAPK emphasized their capacities to regulate cell growth and differentiation. However, more recent work has revealed that they regulate a wide variety of processes, including not only gene expression (e.g., by phosphorylating nuclear transcription factors) but also the activity of various enzymes [e.g., cPLA2 (22, 29, 30) and cAMP phosphodiesterase (34)] and ion channels [i.e., K+ channels (33) and chloride currents (49)]. Therefore, the MAPKs are pleiotropic regulators of numerous cellular activities.
In addition to being activated by various growth factors and cytokines
such as epidermal growth factor (39) and interleukin-1 (37), the MAPKs are also activated by many G
protein-coupled receptors (GPCRs) (16, 20). There are
several mechanisms by which GPCRs stimulate ERK. In COS-7 cells,
lysophosphatidic acid (LPA) potently stimulates MAPK through
PTX-sensitive, Gi-linked LPA receptors (17,
32). Activation of other Gi-linked receptors, such
as the M2 muscarinic receptor, is believed to stimulate MAP kinase through Gi--subunits acting on a Ras- and
Raf-dependent pathway (21, 31, 48). In Chinese hamster
ovary cells, inhibitors of phosphatidylcholine-specific phospholipase C
(PC-PLC) and phosphatidylinositol 3-kinase (PI 3K) diminish activation
of ERK2 through PTX-sensitive, Gi-linked receptors
(13). Furthermore, G-
-subunits stimulate MAPK
activity by activating Src-family tyrosine kinases, promoting the
phosphorylation of Shc and recruiting Grb2 and Sos complexes to the
plasma membrane (31, 32). cAMP inhibits MAPK in many cells
(48) yet activates the enzyme in others (11,
48). In contrast, receptors coupled to PTX-insensitive G
proteins stimulate ERK predominantly via a tyrosine kinase-independent
pathway mediated by PKC (16, 17, 20).
Little information is available on the regulation of the MAPK pathway by the CaR, although McNeil et al. (36) have recently shown that this receptor stimulates ERK1 kinase activity and cellular proliferation in rat-1 fibroblasts. High Cao2+ is known to regulate PLA2 in both kidney and parathyroid (2, 3, 24, 43, 44). In view of the fact that ERK1/2 phosphorylates and activates cPLA2, we have investigated the role of the CaR and of the MAPK pathway in regulating this enzyme in CaR-transfected human embryonic kidney (HEKCaR) cells and in bovine parathyroid cells. Our results show that high Cao2+ causes rapid protein tyrosine phosphorylation and activation of ERK1/2 in both HEKCaR and parathyroid cells but not in nontransfected HEK293 cells, which do not express an endogenous CaR. Studies of the time course and concentration-response relationships for the effects of high Cao2+ and the "calcimimetic" CaR activator NPS R-467 and its less active stereoisomer NPS S-467 on MAPK activity in HEKCaR and bovine parathyroid cells demonstrate the mediatory role of the CaR in the stimulation of ERK1/2 activity and the accompanying phosphorylation of cPLA2.
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MATERIALS AND METHODS |
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Materials. A polyclonal antiserum against phosphorylated ERK1/2 [phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody] was purchased from New England Biolabs (Beverly, MA), and that against phosphoserine was obtained from Zymed (San Francisco, CA). A monoclonal antibody against ERK2 was from Transduction Laboratories (Lexington, KY), and an anti-cPLA2 antibody was from Santa Cruz Biochemicals (San Francisco, CA). The R- and S-enantiomers of NPS 467 were generous gifts of Dr. Edward F. Nemeth, NPS Pharmaceuticals (Salt Lake City, UT). A selective inhibitor of MAPK kinase (also termed MEK), designated PD98059 (2'-amino-3'-methoxyflavone), and the PKC inhibitor GF109203X were obtained from Calbiochem-Novabiochem (San Diego, CA); U73122 and genistein were purchased from Biomol Research Laboratory (Plymouth Meeting, PA). The Renaissance enhanced chemiluminescence kit was purchased from DuPont-NEN. Cell culture media [Eagle's minimal essential medium (EMEM) and Dulbecco's modified Eagle's-F12 medium (DMEM-F12)] were obtained from GIBCO-BRL (Grand Island, NY). Protease inhibitors were from Boehringer Mannheim, and other reagents were from Sigma Chemical (St. Louis, MO)
Preparation and incubation of dispersed bovine parathyroid cells. Dispersed parathyroid cells were prepared by collagenase and DNase digestion as previously described (24). Cells were used immediately as acutely dispersed cells or were cultured overnight in DMEM-F12 medium with penicillin and streptomycin. Similar results were obtained with acutely dispersed cells or with cells cultured for 1 day. The cells were washed with EMEM containing 0.5 mM Cao2+, 0.5 mM Mgo2+, and 0.2% bovine serum albumin (BSA; "standard medium"). Subsequent incubations with various reagents and with varying levels of Cao2+ were performed in standard medium at 37°C as described below.
Culture and maintenance of CaR-transfected and nontransfected HEK293 cells. A clonal HEK293 cell line stably transfected with the cDNA for the human parathyroid CaR (hPCaR4.0) (1, 24) was kindly provided by Dr. Kimberly Rogers of NPS Pharmaceuticals and was selected by hygromycin resistance. We have previously shown (1) that nontransfected HEK293 cells do not express an endogenous CaR, whereas transfected HEK293 cells (HEKCaR) express the CaR protein on the cell surface at high levels and are responsive to addition of CaR agonists to the ambient medium. Cells were grown in DMEM with 10% fetal bovine serum, without sodium pyruvate, either without (wild-type HEK293 cells) or with 200 µg/ml hygromycin (HEKCaR cells). Before stimulation, subconfluent cell monolayers were serum starved in DMEM supplemented with 0.2% BSA (cell culture tested; Sigma) for 24 h. After aspiration of the culture media, the cells were incubated with the various reagents detailed in RESULTS.
Western blot analysis.
For the determination of ERK1/2 phosphorylation, parathyroid cells and
monolayers of serum-starved HEKCaR and HEK293 cells were incubated at
37°C in serum-free medium containing 0.2% BSA, with varying
concentrations of Cao2+ or of NPS R-467 or NPS S-467 at
1.0 mM Cao2+, after preincubation with inhibitors of
various signal transduction pathways, as described in
RESULTS. At the end of the incubation period, the medium
was removed, the cells were washed with ice-cold phosphate-buffered
saline (PBS) containing 1 mM sodium vanadate, and 100 µl of ice-cold
lysis buffer were added [20 mM Tris · HCl (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol,
1 mM dithiothreitol, 1 mM freshly prepared sodium vanadate, 50 mM
-glycerophosphate, and protease inhibitors (10 µg/ml each of
aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain
inhibitor, as well as 100 µg/ml Pefabloc)] and were frozen
immediately on dry ice (25, 27). After thawing and
homogenization, lysates were centrifuged at 10,000 g for 5 min at 4°C. Equal amounts of supernatant proteins (10-15 µg)
were separated by SDS-PAGE, and Western blot analysis was performed as
described previously (25, 27) and in more detail below. Alternatively, cells were lysed directly with 100 µl/well of Laemmli sample buffer, cell lysates were sonicated briefly, and proteins were
resolved by SDS-PAGE. The separated proteins were transferred electrophoretically onto nitrocellulose membranes (Schleicher and
Schuell) and incubated with blocking solution (10 mM
Tris · HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween-20)
containing 5% dry milk for 1 h at room temperature. ERK1/2
phosphorylation was detected by immunoblotting using a 3-h incubation
with a 1:1,000 dilution of a rabbit polyclonal phospho-ERK1/2-specific
antiserum and a subsequent incubation with a second, goat anti-rabbit,
peroxidase-linked antiserum diluted in blocking solution. The bands
were visualized by chemiluminescence (Renaissance enhanced
chemiluminescence system). Quantitative comparisons of the
phosphorylation of ERK1/2 under various experimental conditions were
performed using an ImageQuant and a Personal Densitometer (Molecular
Dynamics). Nitrocellulose membranes were stripped of antibodies and
reprobed using an antiserum to ERK2 that detects this protein
independently of its state of phosphorylation to confirm equal loading
of ERK proteins. Protein concentrations were measured using the Micro
BCA protein kit (Pierce).
Immunoprecipitation. Immunoprecipitation was performed to detect phospholipase A2 as before (25). Cells were washed with ice-cold PBS containing 1.0 mM sodium vanadate, lysed with lysis buffer (as described above), and centrifuged at 10,000 g for 10 min. Equal amounts of supernatant proteins (150 µg of total cell lysate) were incubated with 5 µg of anti-cPLA2 monoclonal antibody (see RESULTS) for 1 h at 4°C, followed by addition of protein A-Sepharose beads for 1 h. Bound immune complexes were washed three times with lysis buffer, and the pellet was eluted by boiling for 5 min with 2× Laemmli sample buffer (25, 27). Supernatant proteins were separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-cPLA2 and anti-phosphoserine antibodies.
Statistics. The data are presented as means ± SE of the indicated number of experiments. Statistical analyses were carried out using the unpaired Student's t-test when two groups were compared or with ANOVA, Bonferroni's method, or Dunn's one-way ANOVA on ranks when three or more groups were compared or multiple comparisons were carried out with a single control. A P value of <0.05 was considered to indicate a statistically significant difference.
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RESULTS |
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Phosphorylation of ERK1/2 in response to elevated
Cao2+ in bovine parathyroid cells.
Figure 1 shows that high
Cao2+ increased the phosphorylation of ERK1/2 in a
time- and dose-dependent manner, as assessed by use of a
phospho-ERK1/2-specific polyclonal antiserum. The phosphorylation of
ERK1/2 peaked at 10 min after raising the level of
Cao2+, decreased to the basal value after 1 h, and
remained at that level for as long as 24 h. Figure 1B
shows a densitometric analysis of data obtained from four separate
experiments. Stimulation of parathyroid cells with 3.0 mM
Cao2+ resulted in a maximal, sevenfold increase in
combined ERK1 and ERK2 phosphorylation after a 10-min incubation.
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Phosphorylation of ERK1/2 is mediated by the
CaR in HEKCaR cells.
To prove the mediatory role of the CaR in activating MAPK, we compared
the effects of elevated Cao2+ on MAPK activity in
CaR-transfected and nontransfected HEK293 cells. As shown in Fig.
2, A and B,
addition of high Cao2+ to HEKCaR cells stimulated a
rapid (within 2 min), dose-dependent phosphorylation of ERK1/2, which
then began to decline after 30-60 min but persisted at a level
above baseline for as long as 24 h. The high
Cao2+-stimulated phosphorylation of ERK1/2 in HEKCaR
cells was markedly higher than that observed in nontransfected HEK293
cells (Fig. 2, A and B). Acute stimulation of
HEKCaR cells with 3.0 mM Cao2+ produced a 12-fold
increase in combined ERK1 and ERK2 phosphorylation at 2 min and a
17-fold increase at 10 min as assessed by densitometry. In
nontransfected HEK293 cells there was only a slight (2.1-fold) stimulation of ERK1/2 phosphorylation by increased
Cao2+.
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NPS R-467 stimulates
ERK1/2 phosphorylation in parathyroid and
HEKCaR cells.
The calcimimetic CaR activators (NPS R-568 and NPS R-467)
potentiate the stimulatory effects of Cao2+ on the CaR
(38). To confirm that the CaR mediates the high Cao2+-elicited phosphorylation of ERK1/2 in parathyroid
cells, we compared the effects of NPS R-467 with those of its less
active enantiomer, NPS S-467, in parathyroid, HEKCaR, and HEK293 cells.
NPS R-467 caused concentration-dependent increases in ERK1/2
phosphorylation in both HEKCaR and
parathyroid cells (Fig. 3, A
and B). The R-enantiomer of NPS-467 was ~10- and 7-fold
more potent, respectively, in HEKCaR and in parathyroid cells, than the
S-enantiomer (Fig. 3, A-B). To confirm the specificity of
this compound for the CaR, we compared the effects of NPS R-467 in
HEKCaR and HEK293 cells. NPS R-467 (3.0 µM) increased ERK1/2
phosphorylation only in HEKCaR cells (not shown). Figure 3,
C and D shows that NPS R-467 increased the
sensitivity of the CaR to activation by Cao2+, typical
of a CaR-mediated response. That is, incubation of HEKCaR cells with
NPS R-467 shifted the calcium concentration curve to the left, and the
maximal effect was observed at 1.0 mM Cao2+,
substantially lower than the value observed without NPS R-467 (e.g.,
Fig. 2). These results support, therefore, the CaR's role in mediating
the high-Cao2+-evoked activation of ERK1/2 in
both parathyroid and HEKCaR cells.
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The role of PKC in CaR-mediated
MAPK activation.
To determine whether PKC mediates the activation of MAPK by the CaR, as
is the case for several other GPCRs (17, 20-22), we
determined the effects of either activating PKC [by brief exposure to
phorbol ester (phorbol 12-myristate 13-acetate, PMA)] or of inhibiting
the enzyme with a PKC inhibitor. Figure 4
shows that, in cells preincubated for 10 min with PMA (1 µM), there
was a significant increase in high-Cao2+-induced ERK1/2
phosphorylation in both cell types. Moreover, acute stimulation of PKC
with PMA was sufficient to induce ERK1/2 phosphorylation at low
Cao2+ (Fig. 4). The MEK inhibitor PD98059 also
abolished PMA-induced ERK1/2 activation in both cell types (not shown).
Pretreatment of cells with the specific PKC inhibitor GF109203X (GFX;
0.1 µM for 20 min), significantly inhibited 3.0 mM
Cao2+-stimulated phosphorylation of ERK1/2 in
parathyroid (by 47%) and in HEKCaR cells (by 61%; Fig. 4), suggesting
that PKC plays an important role in high Cao2+-induced,
CaR-mediated stimulation of ERK1/2 in these two cell types. Because
inhibition of PKC only partially blocked
high-Cao2+-evoked activation of MAPK, however, it
appeared likely that other intracellular signaling pathways also
participated in this process.
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Effect of protein tyrosine kinase and phosphatase inhibitors on high-Cao2+-stimulated MAPK activation. The Src family of tyrosine kinases have been implicated in activation of MAPK by several GPCRs (17-21, 32). To examine whether Src or related tyrosine kinases might be linked to the high-Cao2+-induced increases in ERK1/2 activities in parathyroid and HEKCaR cells, we tested the effects of herbimycin, a selective inhibitor of cytoplasmic tyrosine kinases (17, 21). Herbimycin pretreatment for 20 min reduced high- Cao2+-stimulated phosphorylation of ERK1/2 in both cell types, suggesting that c-Src, or a related cytoplasmic tyrosine kinase, may be a mediator in this signaling pathway (Fig. 4). Genistein is another commonly used tyrosine kinase inhibitor (21, 41). Pretreatment of parathyroid and HEKCaR cells with 1 µg/ml genistein for 20 min also substantially inhibited high-Cao2+-induced phosphorylation of ERK1/2 (Fig. 4), whereas pretreatment with the phosphotyrosine phosphatase inhibitor, o-vanadate (100 µM), enhanced this response in both cell types (Fig. 4), suggesting the presence in these cells of a common signaling pathway involving tyrosine phosphorylation that participates in activation of MAPK (11, 50).
MAPK activation is partially sensitive to
PTX in parathyroid and HEKCaR cells.
In several cell types, activation of ERK1/2 via G protein-coupled
receptors has been reported to be sensitive to PTX (20, 41). We therefore examined the effects of PTX on high
Cao2+ -induced ERK1/2 phosphorylation in parathyroid
and HEKCaR cells. PTX inhibited 3.0 mM Cao2+-stimulated
ERK1/2 phosphorylation by 65 and 60%, respectively, in parathyroid and
HEKCaR cells (Fig. 4). PTX preincubation was without effect on
PMA-induced phosphorylation in HEKCaR cells (not shown), confirming
that PKC activation of MAPK is Gi independent. Because
pretreatment of HEKCaR cells with PTX, or with PKC or protein tyrosine
kinase (PTK) inhibitors individually, only partially reduced
high-Cao2+-induced ERK1/2 phosphorylation, we next
examined the effects of various combinations of these inhibitors. As
depicted in Fig. 5,
high-Cao2+-stimulated ERK1/2 phosphorylation was almost
completely inhibited by addition of combinations of two, and especially
three, inhibitors.
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The role of PI-PLC in CaR-mediated MAPK activation.
Phosphoinositide (PI) hydrolysis produces IP3 and
diacylglycerol, leading to activation of PKC, which can stimulate MAPK
activity. Figure 6 shows that a PI-PLC
inhibitor (U73122) markedly diminished MAPK activation, and PTX
enhanced this inhibitory effect on ERK1/2 phosphorylation, supporting
the involvement of both Gq/11 and Gi in the
CaR-activated MAPK cascade in HEKCaR cells.
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Role of ERK1/2 in CaR-mediated
stimulation of cPLA2 phosphorylation in
parathyroid and HEKCaR cells.
In a previous study (24), we showed that high
Cao2+ (3.0 mM) stimulated cPLA2 activity in
parathyroid and HEKCaR cells, which is responsible for high-
Cao2+-evoked arachidonic acid release. Phosphorylation
of cPLA2 by ERK has been shown to be essential for its
activation in several different systems (22, 29, 30).
Therefore, we investigated whether the 85-kDa PLA2 was
phosphorylated in response to activation of the CaR in parathyroid and
HEKCaR cells by immunoprecipitating cPLA2 after incubation
of these cells with varying levels of Cao2+ or with NPS
R-467 and then determining the extent of serine phosphorylation of
cPLA2. As shown in Fig. 7,
the intensity of the band comigrating with cPLA2 that is
immunostained with an anti-phosphoserine antibody was greater in cells
incubated with 3.0 mM Cao2+ or with NPS R-467.
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DISCUSSION |
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The MAPKs integrate multiple intracellular signals after activation by a variety of extracellular signals (16-20, 41). Significant heterogeneity exists between cell types with regard to the mechanisms underlying MAPK activation. In Rat-1 fibroblasts, for example, Gi-coupled receptors mediate tyrosine kinase-dependent ERK1/2 activation (17). In Chinese hamster ovary cells, in contrast, stimulation of Gq/11-coupled receptors leads to ERK1/2 activation (23). In PC12 pheochromocytoma cells, both Gi- and Gq/11-coupled receptors activate ERK1/2 (17, 19-21). It is becoming increasingly clear that the pathways involved in MAP kinase activation are cell specific as well as agonist specific. We investigated, therefore, how high Cao2+ and the CaR regulate the MAPK cascade and, in turn, whether activation of MAPK is coupled to regulation of cPLA2 in a CaR-dependent manner. Indeed, in the present study, we have demonstrated that high Cao2+ activates MAPK and promotes phosphorylation of cPLA2 in both CaR-expressing bovine parathyroid and HEKCaR cells in a time- and dose-dependent manner. These results confirm and extend those of McNeil et al. (36), who demonstrated high Cao2+-evoked, CaR-mediated stimulation of ERK1 in rat-1 fibroblasts.
The CaR is activated by both polyvalent cations and polycationic molecules that interact with the extracellular domain of the receptor (6, 8, 9). NPS R-467 and NPS R-568 are potent and selective activators of the Cao2+-sensing receptor and inhibit both PTH secretion (38) and parathyroid cellular proliferation (42). These compounds act as positive allosteric modulators to increase the sensitivity of the CaR to activation by Cao2+ and its other polycationic agonists and are thought to bind to the CaR's transmembrane domains (38). The addition of NPS R-467 to parathyroid and HEKCaR cells produced a concentration-dependent increase in MAPK activity in the presence of 1.0 mM Cao2+. The concentration-response curve for Cao2+ was also significantly left shifted in HEKCaR cells by the addition of NPS R-467, as has been shown previously for the regulation of PTH secretion and the cytosolic Ca2+ concentration by Cao2+ (38). Therefore, these results strongly support the mediatory role of the CaR in the activation of MAPK by high Cao2+ in bovine parathyroid cells.
Studies with other receptors have demonstrated two signaling pathways
from GPCRs to the activation of MAPK, a PTX-sensitive, Ras-dependent
pathway that requires G protein -subunits and a PTX-insensitive,
Ras-independent pathway that involves PKC (17-23, 41). In HEK293 cells, ERK1/2 phosphorylation via endogenous lysophosphatidic acid (LPA) and thrombin receptors is mediated via both
PTX-sensitive and -insensitive G proteins (17, 31, 32).
Chang et al. (9) demonstrated, in HEK293 cells stably expressing the CaR cDNA, that there were significant reductions in cAMP
upon addition of high Cao2+, Mg2+,
Gd3+, or the CaR activator NPS R-467. Preincubation of
parathyroid cells with PTX blocked the inhibitory effects of high
Cao2+ on cAMP accumulation as well as on PTH release in
some, but not all, studies (for review, see Refs. 4 and
8). To determine which pathway is responsible for CaR stimulation of
ERK1/2 in the cell types studied here, we used specific inhibitors of
various signaling pathways. PD98059, which selectively inhibits
activation of MEK, almost entirely inhibited the activation of ERK1/2
by high Cao2+ and NPS R-467, consistent with activation
via the well characterized ERK cascade. When parathyroid and HEKCaR
cells were treated overnight with PTX, phosphorylation of ERK1/2 by
high Cao2+ was partially suppressed. Thus the CaR can
activate the MAPK pathway, in part, through PTX-sensitive G proteins.
In rat-1 fibroblasts, in contrast, which express an endogenous CaR,
elevated Cao2+ stimulated cellular proliferation and
produced PTX-insensitive increases of c-Src and ERK1 activity
(36). Thus ERK activation is complex and dependent not
only on the nature of the receptor but also on the cell type.
Many factors stimulating the activity of phospholipases (e.g., PI-PLC, PC-PLC, PLA2, and/or PLD) also activate the phospholipid-dependent isoforms of PKC (6, 15, 24, 43). In this study, we analyzed in detail the roles of PI-PLC and PKC in high-Cao2+-induced MAPK activation in parathyroid and HEKCaR cells. Receptors that couple via Gq/11 to phosphoinositide hydrolysis (such as the CaR) stimulate the formation of diacylglycerol and inositol phosphates and elevate the level of cytosolic Ca2+, leading to activation of PKC. In several cell types, the resultant activation of PKC plays an important role in MAPK stimulation (22, 41). In parathyroid and HEKCaR cells, ERK1/2 activation by elevated Cao2+ also appears to be partially PKC dependent, because it was attenuated by the selective PKC inhibitor GF109203X. Moreover, acute stimulation of PKC with phorbol ester was sufficient to induce ERK1/2 phosphorylation in the presence of low Cao2+. Elevated Cao2+ potentiated this effect of PMA, suggesting that multiple pathways are involved in ERK phosphorylation induced by the CaR. To characterize further the role of PI-PLC in MAPK activation, we studied the effect of U-73122, an aminosteroid inhibitor of PI-PLC activity (43, 45), on high-Cao2+-activated ERK1/2 phosphorylation. U-73122 inhibited both high- Cao2+- and NPS R-467-induced activation of ERK1/2, documenting the role of PI-PLC in CaR-induced MAPK activation (most probably through a rise in intracellular calcium and/or activation of PKC).
The effects of PTK inhibitors on Gi-coupled receptor- and
G--subunit-mediated MAPK activation suggest a role for a PTK in the G-
-mediated signaling pathway (31, 32).
Both genistein and herbimycin A partially inhibited
high-Cao2+-stimulated ERK1/2 phosphorylation in
parathyroid and HEKCaR cells. Therefore, the partial inhibition of
high-Cao2+-elicited phosphorylation of ERK1/2 by PI-PLC
and PKC inhibitors as well as by PTX and PTK inhibitors suggests that
members of the Gq/11 family and a member of an additional
family, most likely Gi, are both involved in this process
(20, 21, 48). Accordingly, we tested the effect of
combinations of different inhibitors. In HEKCaR cells, the high
Cao2+-stimulated ERK1/2 phosphorylations were
essentially completely inhibited by a combination of PTX, a PTK
inhibitor (genistein), and a PKC inhibitor (GF109203X), supporting the
involvement of multiple pathways in the activation of the MAPK cascade.
Further studies are needed, however, to elucidate the mechanism
underlying the CaR-mediated activation of MAPK occurring via the
Gi pathway, as well as the possible roles of other
signaling cascades, including PI 3K, sphingomyelinase, LPA, PC-PLC,
etc. (10, 13, 40).
Bourdeau and colleagues (2, 3) showed that high Cao2+ increases the release of free arachidonic acid from parathyroid cells and that the addition of exogenous arachidonic acid or the products of its further metabolism, e.g., hydroxyperoxyeicosatetranoic acids, suppress PTH secretion, suggesting a role for PLA2 activity in Cao2+-regulated PTH release. Furthermore, Wang et al. (44) demonstrated that raising of Cao2+ or addition of neomycin, which also stimulates the CaR, reduces the activity of apical K+ channels in the thick ascending limb of the kidney. cPLA2 is involved in the CaR-induced inhibition of K+ channel activity, since inhibition of cPLA2 but not PLC abolishes this effect (43). The mechanism by which the CaR activates cPLA2 in the thick ascending limb, however, has not been investigated.
In many cell types, G protein-dependent signaling systems activate cPLA2 via MAPK (20, 22, 29, 30). Full activation of cPLA2 requires an increase in the cytosolic Ca2+ concentration and phosphorylation on Ser505 by MAPK (29, 30). Treatment of E5 Chinese hamster ovary cells with 12-O-tetradecanoylphorbol 13-acetate causes phosphorylation of cPLA2 at the MAPK phosphorylation site (30). We showed previously that high Cao2+ stimulates cPLA2 activity in parathyroid and HEKCaR cells (24). In both cell types, maximal high-Cao2+-stimulated phosphorylation of ERK1/2 and arachidonic acid release occurred at the same concentration of Cao2+ (3.0 mM) and followed similar time courses. These data are consistent with the sequential activation of ERK1/2 and cPLA2. In the present studies, therefore, we investigated the possibility that cPLA2 is phosphorylated and activated via MAPK. Using antibodies to the 85-kDa cytosolic form of PLA2 and to phosphoserine, we found that cPLA2 is phosphorylated on serine in parathyroid and HEKCaR cells and that the level of phosphorylation increases in high Cao2+-stimulated cells and in the presence of PMA. Moreover, the inhibition of the ERK1/2 and cPLA2 phosphorylation by the MEK inhibitor PD98059 indicates that ERK mediates this CaR-elicited activation of cPLA2 in both cell types. Finally, activation of ERK1/2 and cPLA2 is evident only in CaR-transfected HEK293 cells and not in nontransfected cells, providing strong evidence that the CaR directly or indirectly mediates these effects.
Cellular responses to external signals require coordinated control of protein kinases and phosphatases. In the case of ERK, the mechanisms underlying not only its activation but also its dephosphorylation and inactivation are of considerable interest, because phosphorylation occurs on both threonine and tyrosine residues during activation and because sustained ERK activation is important for proliferative signaling (12, 21, 28, 47). To obtain evidence that dephosphorylation of ERK1/2 takes place in parathyroid and HEKCaR cells, we examined the effect of sodium orthovanadate, a potent inhibitor of protein tyrosine phosphatase that can serve in this fashion as a powerful activator of the Ras-Raf-MAPK cascade (28, 50). Pretreatment of parathyroid and HEKCaR cells with sodium orthovanadate before addition of high Cao2+ potentiated the sustained phase of ERK1/2 phosphorylation in both cell types, suggesting that orthovanadate-sensitive phosphotyrosine phosphatase was the basis for ERK1/2 dephosphorylation in these cells. Further studies are needed to investigate the role of this phosphorylation in the control by MAPK of various aspects of the function of parathyroid, HeKCaR, and other CaR expressing cells.
We conclude that 1) elevated Cao2+ elicits rapid, dose-dependent phosphorylation and activation of ERK1/2 in parathyroid and HEKCaR cells, but not in nontransfected HEK293 cells; 2) the CaR mediates activation of the MAPK cascade in parathyroid cells, because the selective CaR activator NPS R-467 mimics the effect of high Cao2+; 3) stimulation of the CaR by high Cao2+ and by NPS R-467 activates MAPK through multiple signaling pathways in both parathyroid and HEKCaR cells, including Gq/11-mediated activation of PI-PLC and PKC as well as a PTX-sensitive pathway likely involving Gi, as has been described in various other cell types; and 4) the CaR mediates activation of cPLA2 in HEKCaR and bovine parathyroid cells through the MAPK cascade.
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ACKNOWLEDGEMENTS |
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This work was supported by generous grants from the US Public Health Service (DK-44588 to O. Kifor and E. M. Brown; DK-41415, DK-48330, and DK-52005 to E. M. Brown; and DK-54934 to M. Bai), The St. Giles Foundation (to E. M. Brown), NPS Pharmaceuticals (to E. M. Brown), The National Dairy Council (to E. M. Brown), a Pediatric Endocrinology Fellowship from Genentech through the Lawson Wilkins Pediatric Endocrinology Society (to R. Diaz), a Postdoctoral Fellowship from the Medical Research Council, Canada (to R. J. MacLeod), a Mochida Memorial Foundation Grant for Medical and Pharmaceutical Research (to T. Yamaguchi) and a Yamanouchi Foundation Grant for Research on Metabolic Disorders (to T. Yamaguchi).
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FOOTNOTES |
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Address for reprint requests and other correspondence: O. Kifor, Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115.
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.
Received 9 June 2000; accepted in final form 24 October 2000.
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