Endocrine-Hypertension Division and Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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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
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INTRODUCTION |
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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)-
(29), tumor necrosis factor-
(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.
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MATERIALS AND METHODS |
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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.
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RESULTS |
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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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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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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
protein1 · 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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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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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
protein1 · 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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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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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
protein1 · 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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DISCUSSION |
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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-1-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 Gq and G
subunits utilizing
parallel signaling pathways. G
q stimulates MKK3 in a
Rac- and Cdc42-dependent manner and MKK6 in a Rho-dependent
manner, whereas G
-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 - and
-isoforms of p38 (11); however, the isoforms of p38
activated by the CaR have not been studied. Increases in Ca
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-, activins, and bone-morphogenetic proteins activate p38 MAP
kinase in several cell lines (27, 43). Previously, we
reported that TGF-
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-
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-
1. Distinguishing the differences in
signaling of CaR-mediated PTHrP secretion from TGF-
-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.
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ACKNOWLEDGEMENTS |
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We thank NPS Pharmaceuticals for a gift of calcimimetics and HEK-CaR cells.
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FOOTNOTES |
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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.
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