PTHrP stimulated by the calcium-sensing receptor requires MAP kinase activation

R. John MacLeod, Naibedya Chattopadhyay, and Edward M. Brown

Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increases in extracellular calcium concentration ([Ca2+]o) stimulate from normal and malignant cells secretion of parathroid hormone-related protein (PTHrP), a major mediator of humoral hypercalcemia of malignancy. Because the calcium-sensing receptor (CaR) is a determinant of calcium-regulated hormone secretion, we examined whether HEK cells stably transfected with human CaR secreted PTHrP in response to CaR stimulation. Increases in [Ca2+]o or neomycin and Gd3+ all substantially increased PTHrP secretion in CaR-HEK cells but had no effect on nontransfected cells. CaR activation likewise increased PTHrP transcripts. PD-098059 and U-0126, inhibitors of the mitogen-activated protein kinase kinase MEK1/2, abolished CaR-stimulated secretion but had no effect on basal secretion. An inhibitor of p38 MAP kinase, SB-203580, also attenuated CaR-stimulated secretion. Western analysis revealed that CaR activation caused a robust increase in MEK1/2 and p38 MAP kinase phosphorylation. A Src family kinase inhibitor, PP2, blocked both basal and CaR-stimulated secretion. We conclude that CaR specifically mediates the effect of increasing [Ca2+]o on PTHrP synthesis and secretion and that activated MEK1/2 and p38 MAP kinases are determinants of the CaR's stimulation of PTHrP secretion.

parathroid hormone-related protein secretion; calcium-sensing receptor; mitogen-activated protein kinase; p38; mitogen-activated and extracellular signal-regulated protein kinase-1 and -2


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PARATHYROID HORMONE-RELATED PEPTIDE (PTHrP) is an important paracrine/autocrine regulator of proliferation, apoptosis, and differentiation in several normal cell types (28, 34). PTHrP expression in normal and tumor cells may be regulated by glucocorticoids (20, 25), epidermal growth factor (16), transforming growth factor (TGF)-beta (29), tumor necrosis factor-alpha (35), and vitamin D (1, 29). This peptide is also associated with the endocrine neoplastic syndrome humoral hypercalcemia of malignancy (14, 39). Elevations in extracellular calcium concentration ([Ca2+]o) will stimulate PTHrP secretion from normal human keratinocytes (17) and astrocytes (7) and squamous (29), cervical (22), and breast (36) cancer cells. Recent studies have demonstrated that the [Ca2+]o-sensing receptor (CaR), originally cloned from the parathyroid gland, mediates the effect of [Ca2+]o on cell types uninvolved in systemic calcium homeostasis (3). The CaR is a G protein-coupled receptor that, when activated, stimulates mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2] using filamin-A as a scaffold (18, 21).

To unequivocally demonstrate a causal relationship between elevations of [Ca2+]o, the CaR, and PTHrP secretion, we used HEK cells stably transfected with human CaR and compared their PTHrP-secretory responses with those of nontransfected cells. Using various MAP kinase inhibitors, we report herein a distinct pharmacology of the CaR-stimulated responses.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Polyclonal antisera against phosphorylated and nonphosphorylated MEK1/2 and p38 MAP kinases were purchased from Cell Signaling (Beverly, MA). Selective inhibitors such as SB-203580 (for p38 MAP kinase), PD-98059 (for MEK1), PP2 (for Src family kinases), and GF-109203X (for pan-PKC), as well as anisomycin, were obtained from Calbiochem-Novabiochem (San Diego, CA). U-0126, the MEK1/2 inhibitor, was purchased from Biomol Research Laboratory (Plymouth Meeting, PA). The enhanced chemiluminescence kit Supersignal was purchased from Pierce (Rockford, IL). Cell culture media [Dulbecco's modified Eagle's medium (DMEM), with and without calcium] were obtained from GIBCO-BRL (Grand Island, NY). Protease inhibitors were from Boehringer Ingelheim, and other reagents were from Sigma Chemical (St. Louis, MO).

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), previously characterized by this laboratory (2), was used in the present studies. We have previously demonstrated (2) that nontransfected HEK293 cells do not express an endogenous CaR, whereas the transfected HEK293 cells (CaR-HEK) express the CaR protein on the cell surface at high levels and are responsive to the addition of CaR agonists such as calcium, neomycin sulfate, or gadolinium chloride. Cells were grown in DMEM with 10% fetal bovine serum, 4 mM L-glutamine, and 100 U/ml penicillin-100 µg/ml streptomycin either without (nontransfected HEK293 cells) or with 200 µg/ml hygromycin B (CaR-HEK cells). Before stimulation, subconfluent cell monolayers were serum starved in Ca2+-free DMEM supplemented with 4 mM L-glutamine, 0.2% BSA (fraction V, cell culture tested; Sigma), 100 U/ml penicillin-100 µg/ml streptomycin, and 0.5 mM CaCl2 for 18 h. After aspiration of this medium, the cells were incubated with CaR agonists or reagents added to this Ca2+-free DMEM medium detailed in Western blot analysis and RESULTS. Early-passage (1-5) CaR-HEK cells were used for SB-20358 experiments.

Northern analysis. RNA was extracted from CaR-HEK cells and poly(A+) RNA was prepared by oligo(dT) cellulose chromatography using previously described techniques (5, 6). Poly(A+) RNA was run on a Northern gel, transferred to nylon membranes, and probed with a full-length human PTHrP cDNA probe (1.7 kb; generously provided by Dr. E. Schipani, Massachusetts General Hospital, Boston, MA). Hybridization and washing of the blots were performed as described before (5, 6). After the final wash, the membranes were exposed to a PhosphoImager screen, and the latter was analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) with the ImageQuant program.

Western blot analysis. For the determination of MEK1/2 or p38 MAP kinase phosphorylation, monolayers of CaR-HEK cells or HEK293 cells were grown on six-well dishes. Cells were incubated for 18 h in serum-free, Ca2+-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. This medium was removed and replaced with the same medium supplemented with either CaCl2 (3 mM), neomycin sulfate (300 µM), gadolinium chloride (25 µM), or inhibitors of various MAP kinases and PKC inhibitors, as described in RESULTS. At the end of the incubation period, the medium was removed, and the cells were washed twice with ice-cold PBS containing freshly prepared 1 mM Na-vanadate and 25 mM NaF. Then, 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, 1% Triton X-100, 10% glycerol, 1 mM DTT] containing freshly prepared 1 mM Na-vanadate, 25 mM NaF, and a cocktail of protease inhibitors (10 µg/ml each of aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor, as well as 100 µg/ml freshly prepared Pefabloc). The cells were scraped in the lysis buffer, sonicated for 5 s, and then centrifuged at 10,000 g for 5 min at 4°C, and the supernatants were frozen at -20°C. After thawing, equal amounts of supernatant proteins (90 µg) were separated on 10% SDS-PAGE gels. The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell) and incubated with blocking solution (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.25% BSA) containing 5% dry milk for >= 1 h at room temperature. MEK1/2 and p38 phosphorylation was detected by use of an 18-h incubation with a 1:1,000 dilution of rabbit polyclonal antibodies against phospho-MEK1/2 or phospho-p38, respectively. Blots were washed for five 15-min periods at room temperature (1% PBS, 1% Triton X-100 and 1% dry milk) and then incubated for 1 h with a secondary goat anti-rabbit peroxidase-linked antiserum (1:1,000) in blocking solution. Blots were then washed a second time (5 × 15 min). Bands were visualized by chemiluminescence according to the manufacturer's protocol (Supersignal, Pierce Chemical). Quantitative comparisons of the phosphorylation of MEK1/2 or p38 were made using an ImageQuant and a Personal Densitometer (Molecular Dynamics). Protein concentrations were measured using the Micro BCA protein kit (Pierce).

PTHrP secretion studies. For determining the effects of [Ca2+]o, polycationic CaR agonists, or MAP kinase inhibitors on this secretion, cells were seeded in 24-well plates (5 × 104 cells/well) in 1 ml of growth medium. After 48 h, the growth medium was removed and replaced with 1 ml of Ca2+-free DMEM containing 4 mM L-glutamine, 0.2% BSA, 100 U/ml penicillin-100 µg/ml streptomycin, and 0.5 mM CaCl2. Eighteen hours later, this medium was removed and replaced with 0.3 ml of the same medium alone or supplemented with additional CaCl2 (to a final concentration of 1.0, 2.0, 3.0, 4.0, or 5.0 mM), and the polycationic agonist neomycin sulfate (300 µM) or gadolinium chloride (25 µM) was added to each well. In other experiments, this medium was supplemented either with the kinase inhibitors described in RESULTS or with 3 mM CaCl2 together with the same kinase inhibitors. Six hours later, the conditioned medium was removed for determination of PTHrP release.

PTHrP was measured in conditioned medium by use of a two-site immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA), which detects PTHrP-(1-72) with a sensitivity of ~0.3 pmol/l. PTHrP assays were initiated immediately after removal of the conditioned medium from cultures to minimize degradation of the peptide from freeze-thawing and other manipulations. Standard curves of PTHrP concentrations were generated using recombinant PTHrP-(1-86) added to treatment medium used in the study (i.e., unconditioned Ca2+-free DMEM containing 0.5 mM CaCl2). CaR agonists and MAP kinase inhibitors alone had no effect in the PTHrP assay.

Statistics. The data are presented as means ± SE of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. A P value of <0.05 was considered to indicate a statistically significant difference.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of [Ca2+]o and polyvalent ions on PTHrP secretion. To determine whether secretion of PTHrP was regulated by the activity of the CaR, we compared the response of HEK293 cells that were stably transfected with a human clone of the CaR with nontransfected HEK cells. As illustrated in Fig. 1, both increasing [Ca2+]o and polycationic agonists of CaR substantially increased the amount of PTHrP secreted from the CaR-transfected cells. These same manipulations had no effect on PTHrP secretion from the nontransfected cells. Increasing [Ca2+]o from 0.5 to 3.0 mM caused a sevenfold increase (P < 0.05) in the amount of PTHrP secreted into the medium over 24 h in the CaR-transfected cells. Well-characterized agonists of the CaR, such as neomycin (300 µM) or gadolinium (Gd3+) (25 µM), caused fourfold (P < 0.05) and threefold (P < 0.05) increases, respectively, in secretion compared with transfected cells in 0.5 mM calcium over the same time period. In stark contrast, increasing [Ca2+]o or adding neomycin or gadolinium had no effect on PTHrP secretion from the nontransfected cells. These results suggested to us that activation of the CaR was responsible for the increased secretion of PTHrP.


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Fig. 1.   Effect of elevated levels of extracellular Ca2+ concentration ([Ca2+]o) and the polycationic calcium-sensing receptor (CaR) agonists neomycin (Neo) and gadolinium (Gd3+) on secretion of parathyroid hormone-related peptide (PTHrP) over 24 h from CaR-transfected HEK and nontransfected HEK293 cells. Open bars, human (h)CaR-transfected cells; filled bars, nontransfected HEK293 cells. Cells were treated for 24 h with the indicated concentrations of [Ca2+]o, neomycin, or gadolinium (µM), and conditioned medium was removed for determination of PTHrP released during the incubation period, as described in MATERIALS AND METHODS. There was significantly more PTHrP secreted from the CaR-transfected cells at 3 mM Ca2+ as well as in the presence of neomycin or gadolinium (P < 0.05, n = 5).

Dose responsiveness of [Ca2+]o on PTHrP secretion. We then determined, by measuring PTHrP secretion after CaR stimulation with 3 mM calcium over several time intervals, that this secretion was first order (r2 = 0.958) for 24 h. We selected secretion measured at 6 h for our remaining experiments. As illustrated in Fig. 2, the stimulation of PTHrP secretion from the CaR-transfected cells was dose responsive to [Ca2+]o. The EC50 in these experiments was ~2.5 mM calcium, consistent with CaR-mediated release of intracellular calcium previously described in these cells (2). Therefore, in remaining experiments, we selected 3 mM calcium to activate the CaR compared with transfected cells treated with 0.5 mM calcium.


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Fig. 2.   Dose response of [Ca2+]o on PTHrP secretion from CaR-HEK cells. Cells were treated for 6 h with the indicated concentrations of [Ca2+]o, and conditioned medium was removed for determination of PTHrP. Amounts of PTHrP secreted in 3, 4, and 5 mM [Ca2+]o were significantly greater than in 2 mM [Ca2+]o (P < 0.05, n = 5).

Calcimimetic NPS R-467 increases PTHrP secretion from CaR-HEK cells. The CaR may be allosterically activated by the phenylalkylamine derivatives NPS R-467 and NPS S-467 (3, 31). Such activation is stereospecific, with the R-enantiomer being more potent than the S-enantiomer. As illustrated in Fig. 3, we found that addition of 10 µM NPS S-467 to CaR-transfected cells challenged with 1 mM [Ca2+]o caused no greater stimulation of PTHrP secretion than addition of 1 mM [Ca2+]o alone. However, addition of 1 mM [Ca2+]o plus 10 µM NPS R-467 caused a twofold increase in the amount of PTHrP secreted from CaR-transfected cells (2.0 ± 0.5 vs. 5.5 ± 0.5 pM · mg protein-1 · 6 h-1, P < 0.05, n = 5; Fig. 3). Together, these results suggested to us that activation of the CaR was responsible for increased secretion of PTHrP.


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Fig. 3.   Stereoselective stimulation of PTHrP secretion by calcimimetic NPS R-467 and S-467. CaR-HEK cells were treated for 6 h with different concentrations of either NPS R-467 (filled bars) or NPS S-467 (speckled bars) in the presence of 1 mM Ca2+o. Significantly more PTHrP was secreted in the presence of 3 and 10 µM NPS R-467 compared with 1 mM [Ca2+]o alone (P < 0.05, n = 5).

Effect of [Ca2+]o on PTHrP transcripts. To determine whether the CaR-stimulated PTHrP secretion occurred at the level of PTHrP transcripts, we performed Northern analyses of CaR-HEK cells that had been exposed to 3 mM Ca2+ for 6 h. As illustrated in Fig. 4, CaR activation upregulated the ~1.2-kb PTHrP transcript ~2.6-fold. These results strongly suggest that CaR-stimulated PTHrP secretion occurs at the level of mRNA expression.


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Fig. 4.   Effect of CaR activation on PTHrP mRNA expression after 6 h in CaR-HEK cells. A: cells were incubated with 0.5 mM Ca2+ (lane 1) or 3.0 mM Ca2+ (lane 2) for 6 h and probed with a 32P-labeled PTHrP cDNA probe as described in MATERIALS AND METHODS. B: equal loading of RNA shown in lanes 1 and 2, where 28S and 18S RNA bands visibly have equal intensities. A 2.6-fold increase of ~1.2-kb PTHrP transcript was observed at 3.0 mM Ca2+ compared with 0.5 mM Ca2+. Result shown here is representative of 2 independent experiments with similar data.

Effect of MAP kinase, Src family kinase, and PKC inhibitors on PTHrP secretion. To gain insight into the signal transduction cascades activated by the CaR resulting in PTHrP secretion, we assessed the effect of different MAP kinase and other protein kinase inhibitors on this secretion. The effect of each inhibitor was measured in 0.5 mM [Ca2+]o (basal secretion) as well as 3.0 mM [Ca2+]o (stimulated secretion) for 6 h. These results are illustrated in Fig. 5. Addition of 3.0 mM [Ca2+]o increased the PTHrP secretion fourfold (P < 0.05) compared with cells exposed to 0.5 mM [Ca2+]o. An inhibitor of mitogen-activated and extracellular signal-regulated kinase kinase (MEK 1), PD-098059 (10 µM), obliterated the CaR-stimulated increase but had no effect on the basal secretion of the protein in 0.5 mM calcium. [PTHrP: 10.2 ± 0.6 vs. 2.4 ± 0.3 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9; 2.3 ± 0.3 vs. 2.4 ± 0.3 pM · mg protein-1 · 6 h-1, not significant (NS), n = 9]. An inhibitor of p38 MAP kinase, SB-203580 (10 µM) reduced the CaR-stimulated secretion 63% (3.9 ± 0.4 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9). An inhibitor of most PKC isoforms, bisindanoylmaleimide (GF-109203X; 1 µM) reduced the CaR-stimulated secretion 47% (5.4 ± 0.2 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9). The PKC inhibitor alone reduced the basal secretion of the protein (1.3 ± 0.2 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9). In the presence of both the p38 and PKC inhibitors, the CaR-stimulated PTHrP secretion was reduced to basal levels (2.5 ± 0.4 vs. 10.2 ± 0.6 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9). The Src family kinase inhibitor PP2 (10 µM) abolished the CaR-stimulated secretion (1.7 ± 0.2 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9); this inhibitor also diminished the basal secretion of PTHrP (1.0 ± 0.05 pM · mg protein-1 · 6 h-1, P < 0.05, n = 9). In additional experiments, we assessed the effect of U-0126, a dual MEK1/2 inhibitor, and found that 10 µM U-0126 had no effect on basal secretion. In contrast, the CaR-stimulated PTHrP secretion was inhibited, with an IC50 of ~3 µM (data not shown). Thus inhibitors of PKC and Src family kinases inhibit both basal and CaR-stimulated secretion. Inhibitors of MEK1/2 and p38 MAP kinase block the CaR-stimulated secretion of PTHrP.


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Fig. 5.   Effect of MAP kinase/PKC inhibitor on basal and CaR-stimulated PTHrP secretion from CaR-HEK cells. Cells were incubated in either 0.5 or 3.0 mM [Ca2+]o containing PD-098059 (10 µM), SB-203580 (10 µM), GF-109203X (1 µM), PP2 (10 µM), or SB-203580 and GF-109203X together for 6 h, and PTHrP was measured in the conditioned medium. The amounts of PTHrP secreted in the presence of the inhibitors and 3 mM [Ca2+]o were all significantly reduced compared with vehicle-treated 3 mM [Ca2+]o alone (P < 0.05, n = 9). In the basal state, all inhibitors reduced secretion compared with vehicle-treated 0.5 mM [Ca2+]o alone (P < 0.05, n = 9), except that PD-098059 had no effect on basal secretion.

Effect of CaR on phosphorylated MEK1/2 and p38 MAP kinase. Because of the importance of MEK1/2 in signaling PTHrP secretion in response to CaR stimulation in these transfected cells, we established the time course of these kinases' activation. The results of Western analysis are shown in Fig. 6. Treatment of cells with 0.5 mM [Ca2+]o had no effect on the amount of immunoreactivity of phosphorylated MEK1/2 (Fig. 6A, left). Activation of the CaR by addition of 3 mM [Ca2+]o within 1 min caused an increase in the intensity of immunoreactivity, which tripled in 2 min to result in a robust signal of activation in 5 min and then declined but remained elevated 22-fold (21.7 ± 2.6/intensity at 1 min, n = 4) at 15 min (Fig. 6A, right). Western blots probed with antibody to nonphosphorylated MEK1/2 demonstrated equal loading. Thus activation of CaR is distinguished by a substantial activation of MEK1/2.


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Fig. 6.   Effect of [Ca2+]o on phosphorylated (p-)MEK1/2 and p-p38 MAP kinase in CaR-HEK cells. A: time-dependent increase in p-MEK1/2 immunoreactivity after addition of 3 mM [Ca2+]o (right) but not 0.5 mM [Ca2+]o (left). Gels show equal loading of nonphosphorylated MEK1/2 kinase. B: time-dependent increase in p-p38 MAP kinase after addition of 3 mM [Ca2+]o (right) but not 0.5 mM [Ca2+]o; (left). Gels show equal loading on nonphosphorylated p38 kinase. Results of a representative experiment are shown.

To further explore the role of p38 MAP kinase in signaling PTHrP secretion in response to CaR stimulation in these cells, we also assessed the presence of phosphorylated p38 MAP kinase (Fig. 6B). Treatment of cells with 0.5 mM [Ca2+]o did not reveal the presence of phosphorylated p38 over the course of the experiment, whereas gels probed with antibody to nonphosphorylated p38 demonstrated equivalent amounts of nonactivated kinase (Fig. 6B, left). Activation of the CaR by addition of 3 mM [Ca2+]o revealed a rapid, transient increase in the intensity of phosphorylated p38 immunoreactivity, which peaked at 5 min (16.5 ±3.5-fold/intensity at 10 min, n = 4) and then declined but remained elevated at 15 min (Fig. 6B, right). Comparable treatment of nontransfected HEK cells with 3 mM [Ca2+]o also did not generate phosphorylated p38 immunoreactivity (data not shown). Clearly, activation of the CaR in these cells also generates a rapid and sustained activation of p38 MAP kinase.

To further understand the role of p38 MAP kinase in CaR-stimulated PTHrP secretion, we examined the effect of different doses of calcium in the presence of SB-20358. For these experiments only, early passages (1-5) of CaR-transfected HEK cells were used (Fig. 7). We observed that, in contrast to the results in Fig. 4, which utilized late-passage cells, SB-20358 (10 µM) had no effect on basal PTHrP secretion. The p38 inhibitor had no effect on PTHrP secretion in 0.5 mM Ca2+ (3.0 ± 0.4 vs. 3.1 ± 0.4 pM · mg protein-1 · 6 h-1, n = 5) or in 1.0 mM Ca2+ (3.0 ± 1.1 vs. 3.1 ± 1.1 pM · mg protein-1 · 6 h-1, n = 5). However, the increases in PTHrP secretion caused by [Ca2+]o >1 mM were all inhibited a comparable amount by the p38 inhibitor. PTHrP secretion stimulated by 3 mM Ca2+ (15.3 ± 5.8 vs. 8.7 ± 2.4 pM · mg protein-1 · 6 h-1, P < 0.05, n = 5) was reduced by SB-20358. Secretion stimulated by 4 mM Ca2+ (12.4 ± 1.9 vs. 6.1 ± 1.4 pM · mg protein-1 · 6 h-1, P < 0.05, n = 5) and 5 mM Ca2+ (11.4 ± 4.3 vs 6.0 ± 0.5 pM · mg protein-1 · 6 h-1, P < 0.05, n = 5) were also reduced by SB-20358. The extent of inhibition of PTHrP secretion stimulated by 3-5 mM Ca2+ ranged from 43 to 51%. In contrast to p38 MAP kinase but consistent with the results in Fig. 4, GF-109203X and PP2 inhibited basal PTHrP secretion (data not shown).


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Fig. 7.   Effect of p38 MAP kinase inhibitor SB-20358 on dose response of Ca2+-stimulated PTHrP secretion from CaR-HEK cells. Early-passage (1-5) cells were incubated in different concentrations of Ca2+ with or without 10 µM SB-20358 for 6 h, and PTHrP was measured in the conditioned medium. In presence of SB-20358, PTHrP levels were reduced (P < 0.05, n = 5) at 2, 3, 4, and 5 mM [Ca2+]o.

Effect of ADP and anisomycin on PTHrP secretion from CaR-HEK cells. To confirm specificity of the CaR activation of PTHrP secretion in the transfected cells, we used a purinergic agonist, ADP (1 µM), which we have demonstrated to activate ERK1/2 (14) in CaR-transfected cells. Addition of ADP did not stimulate PTHrP secretion from the cells (Fig. 8). The amount of PTHrP secreted under basal conditions in 0.5 mM calcium (2.6 ± 0.5 pM · mg protein-1 · 6 h-1) was no different compared with ADP addition (2.6±0.3 pM · mg protein-1 · 6 h-1, n = 5). Addition of 3 mM [Ca2+]o caused an increase, however (9.4 ± 0.8 pM · mg protein-1 · 6 h-1, P < 0.5, n = 5). We then used the antibiotic anisomycin (7.5 ng/ml), which others have reported to selectively activate p38 MAP kinase in HEK293 cells (22, 33). As illustrated in Fig. 8, addition of anisomycin to CaR-transfected cells in 0.5 mM [Ca2+]o modestly stimulated PTHrP secretion (5.2 ± 0.3 vs. 2.6 ± 0.5 pM · mg protein-1 · 6 h-1, P < 0.05, n = 5). These results confirm the specificity of the CaR's stimulation of PTHrP secretion and suggest that p38 MAP kinase activation is an important mediator of CaR-stimulated PTHrP secretion.


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Fig. 8.   Effect of ADP or anisomycin on basal PTHrP secretion from CaR-HEK cells. Cells were incubated in 0.5 mM [Ca2+]o containing either ADP (1 µM) or anisomycin (7.5 ng/ml) for 6 h, and conditioned medium was assessed for PTHrP. Amount of PTHrP secreted after anisomycin addition was greater than vehicle control (P < 0.5, n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The activation of the CaR by [Ca2+]o, different polyvalent cation agonists, or a stereoselective calcimimetic stimulated PTHrP secretion from stably transfected HEK cells. Activation of CaR increased both MEK1/2 and p38 MAP kinase activity. CaR activation upregulated PTHrP transcripts. CaR-stimulated PTHrP secretion was prevented by MEK1/2 inhibitors and a p38 MAP kinase inhibitor. These inhibititors had no effect on the basal secretion of PTHrP from these cells. Anisomycin, a potent activator of p38 MAP kinase, also caused CaR-transfected cells to secrete PTHrP. Elevations in [Ca2+]o are known to stimulate PTHrP secretion from normal human keratinocytes (17) and from squamous epithelial cells (29), astrocytes (7), and cervical (22) and breast cancer (36) cells. These findings, considered together with the results of our present studies, for the first time provide unequivocal evidence that elevated [Ca2+]o-induced PTHrP secretion is indeed CaR mediated, involving MEK1/2 and p38 MAP kinases.

Our present studies, demonstrating that stimulation of the CaR results in a robust and sustained activation of MEK1/2, extend our previous work using CaR-transfected cells reporting transient activation of these kinases' substrate ERK1/2 following stimulation of the CaR (21). We found that well-characterized inhibitors of MEK1/2, PD-098059 (10) and U-0126 (12), blocked the CaR-stimulated secretion of PTHrP but had no effect on basal secretion from these cells. Consistent with these findings, others have shown that PD-098059/U-0126 inhibited IL-8 secretion in response to adenosine A2B receptor activation in human mast cells (32) as well as IL-1beta -stimulated PGE2 release from human bronchial epithelial cells (12). Although the present experiments did not distinguish between the effect of MAP kinase inhibitors on PTHrP that was stored vs. PTHrP that required de novo synthesis, our results suggest that pathways converging at MEK1/2 activation are an important determinant of regulated PTHrP secretion. Interestingly, although many G protein-coupled receptors activate ERK1/2 via MEK1/2 (26), clearly, additional determinants are required for regulated PTHrP secretion, since ADP, which we have shown to stimulate ERK1/2 in these cells (18), had no effect on PTHrP secretion. The stably transfected CaR-HEK cells will therefore be an ideal model to dissect and understand the components of regulated PTHrP secretion.

We found that NPS R-467, a phenylalkylamine derivative that is a positive allosteric modulator of the CaR (31), increased PTHrP secretion under conditions of low CaR activation (i.e., 1 mM [Ca2+]o). Consistent with the reported stereospecificity of these compounds, the R-enantiomer was more active than the S-enantiomer in stimulating PTHrP secretion. This stereospecificity has also been observed in the stimulation of bile secretion as well as intracellular Ca2+ mobilization from rat hepatocytes (4) but not in calcimimetic stimulation of insulin secretion (38). Although additional studies of the behavior/potencies of calcimimetics on CaR-mediated biological effects in other CaR-expressing tissues are required, our results using these agents confirm that CaR activation stimulates PTHrP secretion from these cells.

Our present findings unequivocally demonstrated that CaR activation stimulated p38 MAP kinase. This MAP kinase may be activated by several upstream activators (23, 33). For example, the m1 muscarinic acetylcholine receptor's activation of p38 MAP kinase is mediated by both Galpha q and Gbeta gamma subunits utilizing parallel signaling pathways. Galpha q stimulates MKK3 in a Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent manner, whereas Gbeta gamma -induced MKK3/6 activations were dependent on a tyrosine kinase other than c-Src (44). It is currently unknown which pathways the CaR utilizes for p38 MAP kinase activation.

The p38 MAP kinase inhibitor SB-203580 attenuated CaR-stimulated PTHrP secretion by a comparable amount at different calcium concentrations. This inhibitor has been shown to be selective against the alpha - and beta -isoforms of p38 (11); however, the isoforms of p38 activated by the CaR have not been studied. Increases in Ca<UP><SUB>o</SUB><SUP>2+</SUP></UP>, consistent with CaR activation, result in the activation of p38 MAP kinase in MC3T3-E1 osteoblasts (43), but whether the CaR truly mediated this activation was unclear. In the context of the present findings, this now seems likely. Because SB-20358 had no effect on basal PTHrP secretion (Fig. 7) and we were unable to detect phosphorylated p38 in nonstimulated CaR-HEK cells (Fig. 5B), our results suggest no role for this kinase in basal PTHrP secretion. This MAP kinase has been shown to have a role in G protein-coupled receptor-stimulated secretion of IL-8 (32) as well as LPS-stimulated TNF-alpha production (32). The effect of p38 MAP kinase may be either directly on transcription of the gene of interest or in altering the stability of the transcript (24). Which of these events p38 MAP kinase influences during CaR-regulated secretion is not known. The simultaneous presence of the p38 and PKC inhibitors reduced the CaR-stimulated PTHrP secretion to basal levels. This suggested to us that activation of p38 and PKC by the CaR occurred in parallel with the MEK/ERK cascade.

Anisomycin stimulated PTHrP secretion from CaR-transfected HEK cells in low Ca2+ medium. Anisomycin has been shown to selectively activate p38 MAP kinase in HEK and other cell types (23, 33). Because the amount of PTHrP secretion stimulated by anisomycin was ~50% of that stimulated by CaR activation, we suggest that PTHrP synthesis/secretion may be regulated in parallel by MEK/ERK and p38 MAP kinase pathways. In this regard, it is notable that TGF-beta , activins, and bone-morphogenetic proteins activate p38 MAP kinase in several cell lines (27, 43). Previously, we reported that TGF-beta 1 increased PTHrP secretion from MDA-MB231, a human breast cancer cell line (36). In those cells that expressed CaR, the combination of high [Ca2+]o (3 mM) and TGF-beta 1 produced a greater than additive stimulation of PTHrP secretion. In light of our present experiments, this may reflect that the signaling cascades of the CaR in MDA-MB231 cells are predominantly mediated through the MEK/ERK pathway, whereas p38 MAP kinase (and Smads) are activated by TGF-beta 1. Distinguishing the differences in signaling of CaR-mediated PTHrP secretion from TGF-beta -mediated stimuli will be important for designing effective therapies to block the vicious cycle of breast cancer cells metastatic to bone contributing to malignant hypercalcemia.

The Src family kinase inhibitor PP2 (15) reduced the CaR-stimulated PTHrP secretion to basal levels. When added alone to unstimulated cells, PP2 also inhibited basal secretion. It will be of interest to know which Src kinases, Lyn, Hck, or Fgr, are activated by the CaR as well as whether Src family kinases participate in tyrosine kinase receptor "transactivation" mediated by the CaR.

PTHrP can act in a paracrine/autocrine fashion to inhibit proliferation in astrocytes (37) and influence apoptosis of osteosarcoma cells (40). PTHrP is present in the differentiated villus epithelial cells but absent in the rapidly proliferating crypt epithelia (41), whereas the CaR is present on the basolateral membrane of both types of epithelial cells (6). To further understand the dynamics of CaR stimulation of PTHrP secretion in the mammalian small intestine, the stably transfected HEK cells may be a useful model to address questions about the signal transduction of PTHrP secretion stimulated by the CaR. Indeed, differentiated villus cells in the small intestine are further characterized from proliferative crypt cells by the persistent presence of phosphorylated p38 MAP kinase in their nuclei (19). Because our present experiments unequivocally demonstrate that CaR activation stimulates p38 MAP kinase, it is possible that postprandial activation of the CaR on the basal membrane of villus cells contributes to the continued p38 MAP kinase activation required for villus differentiation.

In summary, we have found that activation of the CaR in stably transfected HEK cells resulted in robust stimulation of MEK1/2 and p38 MAP kinase activity and PTHrP synthesis and secretion. MEK1/2 inhibition and p38 MAP kinase inhibition had no effect on basal secretion of PTHrP but prevented the CaR-stimulated secretion. A Src family kinase inhibitor attenuated both basal and CaR-stimulated secretion of PTHrP. Anisomycin, an activator of p38 MAP kinase, stimulated modest PTHrP secretion from cells in low-calcium medium. The stimulation caused by the CaR was specific, since a different G protein-coupled receptor agonist, ADP, had no effect on PTHrP secretion in these cells. We conclude that activation of the CaR causes PTHrP synthesis and secretion.


    ACKNOWLEDGEMENTS

We thank NPS Pharmaceuticals for a gift of calcimimetics and HEK-CaR cells.


    FOOTNOTES

Generous support was received from the following sources: National Institute of Diabetes and Digestive and Kidney Diseases (Grants DK-48330, DK-52005, and DK-41415) and the St. Giles Foundation to E. M. Brown, and a Pfizer/AFAR New Faculty Development Award and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant (AR-02215-01A2) to N. Chattopadhyay. R. J. MacLeod was a recipient of a Martin Brotman Advanced Training/Transition Award from the American Digestive Health Foundation.

Address for reprint requests and other correspondence: R. J. MacLeod, Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Ave., Boston, MA 02115 (E-mail: rmacleod{at}rics.bwh.harvard.edu).

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.

First published October 8, 2002;10.1152/ajpendo.00143.2002

Received 2 April 2002; accepted in final form 1 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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