Regulation of MAP kinase by calcium-sensing receptor in bovine parathyroid and CaR-transfected HEK293 cells

Olga Kifor1, R. John MacLeod1, Ruben Diaz2, Mei Bai1, Toru Yamaguchi1, Tham Yao1, Imre Kifor1, and Edward M. Brown1

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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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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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-beta gamma -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-beta gamma -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. 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 beta -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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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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Fig. 1.   High ionized Ca2+ concentration (Cao2+) stimulates extracellular signal-related kinase (ERK)1/2 phosphorylation in parathyroid cells in a time- and dose-dependent manner. Parathyroid cells were incubated with 0.5, 2.0, or 3.0 mM Cao2+ in "standard medium" at 37°C for the times indicated. A: ERK1/2 phosphorylation (P-ERK1/2) was measured using Western blotting with a phospho-ERK1/2-specific antiserum and an anti-ERK2 antiserum recognizing both phosphorylated and nonphosphorylated forms of the enzyme. B: integrated optical densities were determined for phosphorylated ERK1/2. Results in (B) show the data in (A) normalized to the values observed at 0.5 mM Cao2+ (denoted as 1). The sums of P-ERK1 and P-ERK2 are shown. There was statistically significant stimulation at 2, 10, and 30 min (P < 0.05). Results are expressed as means ± SE of the results from 4-5 separate experiments, each performed in duplicate.

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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Fig. 2.   High Cao2+ stimulates ERK1/2 phosphorylation in human embryonic kidney Cao2+-sensing receptor (HEKCaR) cells in a dose- and time-dependent fashion. Serum-deprived HEKCaR and HEK293 cells were incubated for the times indicated in the presence of 0.5, 2.0, or 3.0 mM Cao2+. A: ERK1/2 phosphorylation (P-ERK1/2) was measured using Western blot analysis with the phospho-ERK1/2-specific antiserum as in Fig. 1. B: results in (A) normalized to those at 0.5 mM Cao2+, with the control level set at 1. The sums of P-ERK1 and P-ERK2 are shown. Data represent means ± SE of the results from 3 independent experiments, each performed in duplicate. There was statistically significant stimulation by 2.0 and 3.0 mM Cao2+ (P < 0.05) at 2, 10, and 30 min in HEKCaR cells.

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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Fig. 3.   NPS R-467 increases ERK1/2 phosphorylation in parathyroid and HEKCaR cells in a dose-dependent manner. HEKCaR and bovine parathyroid cells (Pt., A) were incubated in the presence of 1.0 mM Cao2+ with 0.3, 1.0, or 3.0 µM NPS R-467 or the same concentrations of NPS S-467 for 10 min. B: averaged data quantified by densitometry from A (parathyroid and HEKCaR cells). C: HEKCaR cells were incubated in the presence of 0.5, 1.0, or 3.0 mM Cao2+ with 3.0 µM NPS R-467. ERK1/2 phosphorylation was measured using Western blot analysis with the same phospho-ERK1/2-specific antiserum as in Fig. 1. D: results obtained in C, expressed as fold increases in activation relative to that observed with 1.0 mM Cao2+ + vehicle, without any added NPS R-467, which was defined as 1.0. Results indicate means ± SE of the results from 3 (A, parathyroid cells), 5 (A, HEKCaR cells), and 4 (C) experiments, each performed in duplicate. There was statistically significant stimulation only by NPS R-467 (P < 0.05) in both cell types.

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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Fig. 4.   Phorbol 12-myristate 13-acetate (PMA) and sodium o-vanadate stimulate, whereas pertussis toxin (PTX), protein kinase C (PKC), and protein tyrosine kinase inhibitors reduce the high Cao2+-induced ERK1/2 activation in parathyroid and HEKCaR cells. Dispersed parathyroid cells and serum-starved HEKCaR cells were treated for 15 min before stimulation with DMSO (0.1%, control), 1 µM PMA, 1 µM bisindolylmaleimide (GFX), or 0.1 mM sodium orthovanadate (OV), or for 20 min with 5 µM genistein (Gen) or 1 µM herbimycin A (HB) or were preincubated overnight with or without 100 ng/ml PTX. Cells were then incubated with 0.5 or 3.0 mM Cao2+ for 10 min, and ERK1/2 phosphorylation was determined as described in the legend for Fig. 1. Values shown represent means ± SE of the results from 2-4 separate experiments, each performed in duplicate.

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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Fig. 5.   Combined inhibitory effects of PTX, GFX, and/or genistein (Gen) on high-Cao2+-stimulated ERK1/2 phosphorylation in HEKCaR cells. Serum-starved cells were preincubated overnight with or without 100 ng/ml PTX. Before stimulation, cells were treated for 15 min with vehicle or with GFX or were treated for 20 min with genistein or GFX plus genistein and then incubated with 0.5 and 3.0 mM Cao2+. ERK1/2 phosphorylation was determined as described in Fig. 1. Values shown represent means ± SE of the results from 3 separate experiments, each performed in duplicate.

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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Fig. 6.   Combined inhibitory effects of PTX and the phosphotidylinositol-specific phospholipase C (PI-PLC) inhibitor and GFX on high Cao2+-stimulated ERK1/2 phosphorylation in HEKCaR cells. A: serum-starved cells were preincubated overnight with or without 100 ng/ml PTX. Before stimulation, cells were treated for 15 min with vehicle, 10 µM PI-PLC inhibitor (U73122) (A and B) or with 1.0 µM GFX (A and C) and then stimulated with 3.0 mM Cao2+. ERK1/2 phosphorylation was determined as described in Fig. 1. Values shown represent means ± SE of the results from 3 separate experiments, each performed in duplicate.

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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Fig. 7.   Effects of mitogen-activated protein kinase kinase inhibitor PD98059 (PD) on high-Cao2+- and NPS R-467-stimulated ERK1/2 and cPLA2 phosphorylation. Dispersed bovine parathyroid cells (A) and serum-starved HEKCaR cells (B) were treated for 15 min with vehicle or with 50 µM PD98059 before being incubated with 0.5, 1.0, or 3.0 mM Cao2+ or in the presence of 1.0 mM Cao2+ with 3.0 µM NPS R-467 for 10 min. ERK1/2 phosphorylation was determined as described in Fig. 1. For analysis of cytosolic phospholipase A2 (cPLA2), parathyroid and HEKCaR cells were lysed in ice-cold lysis buffer, and after isolation of cell debris by centrifugation, the postnuclear supernatant was immunoprecipitated with monoclonal anti-cPLA2 antibody followed by immunoblotting with anti-cPLA2 and rabbit anti-phosphoserine antiserum, as described in EXPERIMENTAL PROCEDURES. Integrated optical densities were determined for phosphorylated ERK1/2 (C) and for phosphorylated cPLA2 (D). Results are expressed as means ± SE of the results from 3 separate experiments, each performed in duplicate.

The specific MEK inhibitor PD98059 blocks the phosphorylation of ERK1/2 and several downstream cellular responses that are mediated via the MAPK cascade. This compound has been shown to inhibit specifically the upstream MAPK kinases (MEK1 and MEK2) that phosphorylate p42/p44 MAPK in several cell lines (10, 12, 28). To study the role of ERKs in mediating the phosphorylation of cPLA2 by high Cao2+ or by NPS R-467, we examined ERK1/2 and cPLA2 phosphorylations in parathyroid and HEKCaR cells pretreated for 20 min with or without this compound. As shown in Fig. 7, preincubation of parathyroid and HEKCaR cells with PD98059 for 20 min inhibited the phosphorylation of ERK1/2 and cPLA2 induced by 3.0 mM Cao2+ or by NPS R-467. We next compared the role of PKC in the phosphorylation of ERK1/2 and cPLA2. In our previous study (24), we showed that pretreatment of parathyroid cells with PMA stimulated release of arachidonic acid at low and high Cao2+. Figure 8 shows that, in HEKCaR cells preincubated for 10 min with PMA (1 µM), there was an immediate, statistically significant increase in the levels of phosphorylation of ERK1/2 and cPLA2 at both low and high Cao2+, showing that PKC mediates the activation of both.


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Fig. 8.   Effects of PMA on ERK1/2 and cPLA2 phosphorylation in HEKCaR cells. Serum-deprived HEKCaR cells were treated for 15 min before stimulation with vehicle or with 1 µM PMA. Cells were stimulated with 0.5, 2.0, or 3.0 mM Cao2+ for 10 min, and ERK1/2 and cPLA2 (A) phosphorylation was determined as in Figs. 1 and 7. Integrated optical densities are shown for phosphorylated ERK1/2 (B) and cPLA2 (C). Rresults are expressed as means ± SE of the results from 3 experiments, each performed in duplicate.

Figure 9 summarizes the mechanisms by which the CaR likely activates various signaling pathways and, in turn, MAPK and cPLA2 in bovine parathyroid and HeKCaR cells. The subsequent metabolism of free arachidonic acid liberated by cPLA2 generates a variety of active mediators, such as hydroxyperoxyeicosatetranoic acid and hydroxyeicosatetranoic acid.


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Fig. 9.   Proposed model for mechanisms underlying CaR-induced activation of ERK1/2 and cPLA2 in bovine parathyroid and HEKCaR cells. Activation of the 7-membrane-spanning CaR by Cao2+ or the selective, calcimimetic CaR activator NPS R-467 results in a PTX-insensitive, presumably Gq/11-mediated activation of PI-PLC, leading to intracellular calcium (Cai2+) mobilization, PKC activation, and resultant PKC-mediated stimulation of the mitogen-activating protein kinase (MAPK) cascade. The CaR also activates MAPK via a PTX-sensitive G protein, probably an isoform of Gi, and subsequent downstream activation of a tyrosine kinase-dependent process, probably involving a Ras- and Raf-dependent series of steps. Activated MAPK then phosphorylates and activates cPLA2, which releases free arachidonic acid (AA) that can be metabolized to biologically active mediators such as hydroxyperoxyeicosatetranoic acid (HPETE) or hydroxyeicosatetranoic acid (HETE). *Data presented in this study.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta gamma -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-beta gamma -subunit-mediated MAPK activation suggest a role for a PTK in the G-beta gamma -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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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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