1Endocrine-Hypertension Division, Department of Medicine and Membrane Biology Program, Brigham and Women's Hospital and Harvard Medical School, and 2Division of Experimental Medicine, Beth Israel Deaconess Medical Center and Harvard Institutes of Medicine, Boston, Massachusetts 02115; and 3Osteoporosis and Bone Metabolic Unit, Department of Clinical Biochemistry and Endocrinology, Copenhagen University Hospital, Hvidovre, DK-2650, Denmark
Submitted 11 November 2002 ; accepted in final form 11 April 2003
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ABSTRACT |
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G protein-coupled receptor; Leydig cells; humoral hypercalcemia of malignancy; dominant negative; stress-activated protein kinase activator 1; osteolysis
Here, we have used a well-accepted HHM cell model (H-500) to investigate
the CaR-mediated intracellular signaling that underlies PTHrP release. High
[Ca2+]o, acting via the CaR, regulates PKC
activity, which in turn modulates PTH release from the bovine parathyroid
gland (22). In addition,
involvement of the PKC pathway in
[Ca2+]o-stimulated PTHrP release has been
reported in nonsmall cell lung cancer (NCI-H727); therefore, PKC is a
potential candidate for postreceptor signaling associated with CaR-induced
PTHrP release from H-500 cells
(6). On the other hand, the CaR
stimulates MAPKs in a variety of cell types, including HEK-CaR, rat-1A
fibroblasts, ovarian surface epithelial cells, and Madin-Darby canine kidney
(MDCK) cells, regulating functions such as phospholipase C activation and
proliferation (2,
16,
17). Because PKC regulates a
variety of MAPKs, including ERK1/2, in this study we sought to identify
intracellular signaling cascades that regulate CaR-mediated PTHrP release. Our
data reveal that the CaR, but not the ADP receptor [a G protein-coupled
receptor (GPCR) linked to Gq11 like the CaR], induces PTHrP
release. This effect is mediated by PKC as well as by PKC-independent
activation of ERK1/2, p38 MAPK and its downstream activating transcription
factor (ATF)-2 cascade, and the stress-activated protein kinase activator
(SEK-1). In addition, the effect of the CaR on PTHrP release is likely
transcriptional, as high [Ca2+]o upregulates
the PTHrP transcript in the absence, but not in the presence, of actinomycin
D.
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MATERIALS AND METHODS |
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Cell culture. The Rice H-500 rat Leydig cell tumor was obtained from the National Cancer Institute-Frederick Cancer Research and Development Center DCT Tumor Repository (Frederick, MD). Male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200220 g (10 wk of age) were used for all experiments. A fragment of the H-500 tumor or dispersed H-500 cells (106 per rat) were implanted or injected subcutaneously, respectively, in each rat, and the tumors were allowed to grow for 814 days. The encapsulated tumor was then excised, rinsed several times with cell culture medium (to be described), minced into small pieces, and dispersed by repeated pipetting and several passages through a 22-gauge needle. Dispersed H-500 cells were subsequently plated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-100 µg/ml streptomycin and grown at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged every 45 days with 0.05% trypsin-0.53 mM EDTA and used for experimentation within the first 10 passages. All cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY) with the exception of FBS, which was obtained from Gemini Bio-Products (Calabasas, CA). Rats were handled in accordance with local institutional guidelines.
Northern blot analysis. To study whether high [Ca2+]o exerts an effect on the expression of PTHrP mRNA, we performed Northern blot analysis as described before (7). Briefly, cellular RNA was isolated (9) using the TRIzol reagent (Invitrogen, Carlsbad, CA) by following the manufacturer's instructions. The recovered RNA was quantitated by spectrophotometry, and aliquots of 20 µg total RNA from low [Ca2+]o (0.5 mM) or high [Ca2+]o (7.5 mM) were loaded on a formaldehyde agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA so that we could document equal loading of RNA from the various experimental samples. The RNA was then blotted onto nylon membranes (Duralon; Stratagene, La Jolla, CA). Blots were hybridized with a cDNA probe for PTHrP and washed under high-stringency conditions as described previously (23). Equal loading was also confirmed by reprobing the membranes with GAPDH cDNA. Specific radioactive signals were analyzed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) with the Image-Quant program.
Gene transfer into H-500 cells with CaR constructs.
High-efficiency gene transfer into H-500 cells was accomplished using a
recombinant adeno-associated virus (rAAV)-based method. The CaR sequence with
a naturally occurring, dominant negative mutation (R185Q), as well as the same
vector containing the cDNA for the -gal (BG) protein, were under the
control of a cytomegalovirus immediate-early, or CMVIE, promoter element and
packaged as previously described
(37). The BG served as the
control for nonspecific effects of rAAV infection. Cells were seeded (1,000
cells/well) in 96-well plates in 0.1 ml of growth medium and cultured
overnight. About 1,000 virus particles/cell (as optimized by pilot studies)
were used to infect each well. Cells were washed once with serum-free
-MEM. Virus particles were then added, and the culture was incubated
for 90 min in serum-free medium at 37°C in a cell culture incubator. Equal
volumes of RPMI 1640 containing 20% serum were added to the cells to achieve a
final serum concentration of 10%. The cells were then cultured for 48 h, and
experiments with basal (low, 0.5 mM) and high calcium concentrations were
performed as described in subsequent sections.
Western blot analysis. For the determination of ERK1/2, p38 MAPK,
SEK1, or ATF-2 phosphorylation, monolayers of H-500 cells were grown on
six-well plates. 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 7.5 mM CaCl2 either alone or
with the PKC inhibitor. The cells were also preincubated with PKC inhibitor
for 0.5 h, as described in RESULTS. At the end of the incubation
period, the medium was removed, the cells were washed twice with ice-cold
phosphate-buffered saline (PBS) containing 1 mM sodium vanadate and 25 mM NaF,
and 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, 25 mM NaF, 1% Triton X-100, 10%
glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerophosphate,
and a cocktail of protease inhibitors). The protease inhibitors were
aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain
inhibitor (10 µg/ml of each), all from frozen stocks, as well as 100
µg/ml of Pefabloc. The sodium vanadate, NaF, and Pefabloc were freshly
prepared on the day of the experiment. The cells were scraped into the lysis
buffer, sonicated for 5 s, and then centrifuged at 6,000 g for 5 min
at 4°C. The supernatants were frozen at -20°C. After thawing, equal
amounts of supernatant protein (100 µg) were separated by SDS-PAGE. The
separated proteins were electro-phoretically 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. ERK1/2, p38 MAPK,
SEK1, and ATF-2 phosphorylation was detected by immunoblotting using an 18-h
incubation with 1:1,000 dilutions of rabbit polyclonal antisera specific for
phospho-ERK1/2, phospho-p38 MAPK, phospho-SEK1, or phospho-ATF-2,
respectively. Blots were washed for five 15-min periods at room temperature
(1% PBS, 1% Triton X-100, and 0.3% dry milk) and then incubated for 1 h with a
secondary goat anti-rabbit, peroxidase-linked antiserum (1: 2,000) in blocking
solution. Blots were then washed again (5 x 15 min). Bands were
visualized by chemiluminescence according to the manufacturer's protocol
(Supersignal, Pierce Chemical). The same membrane was used after stripping
(Restore Western Blot Stripping, Pierce) to measure nonphospho-ERK1/2, -p38
MAPK, and -SEK1. Protein concentrations were measured with the Micro BCA
protein kit (Pierce).
PTHrP release. The effects of [Ca2+]o, as well as MAPK and PKC inhibitors, on PTHrP release were determined by seeding cells in 96-well plates (1 x 104 cells/well) in 0.1 ml of growth medium. After 48 h, the growth medium was removed and replaced with 0.1 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. Two hours later, this medium was removed and replaced with 0.225 ml of the same medium or that supplemented with additional CaCl2 (to a final concentration of 2.5, 5.0, or 7.5 mM) for 6 h. In other experiments, the medium was supplemented either with the kinase inhibitors described in RESULTS or with 7.5 mM CaCl2 together with the same inhibitors. In the experiment with ADP, the concentrations used were 10-7, 10-8, and 10-9 M. Six hours later, the conditioned medium was removed for determination of PTHrP release. The 6-h incubation time was decided upon after a time course experiment had been carried out that examined the effects of low and high calcium on PTHrP release at 4, 6, and 24 h. The fold increase of PTHrP release at high [Ca2+]o did not vary over the first 24 h. The 6-h time point was chosen for subsequent experiments because it yielded PTHrP values falling on the linear portion of the PTHrP assay, whereas at 4 and 24 h, PTHrP was at the lower or upper portion of the curve, respectively.
PTHrP was measured in conditioned medium using a two-site immunoradiometric
assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) that detects
PTHrP-(172) with a sensitivity of 0.3 pmol/l. PTHrP assays were
initiated immediately after the conditioned medium was removed from cultures
to minimize degradation of the peptide resulting from freeze-thawing and other
manipulations. Standard curves of PTHrP concentrations were generated with the
addition of recombinant PTHrP-(186) to the treatment medium used in the
study (i.e., unconditioned Ca2+-free DMEM containing 0.5
mM CaCl2). Calcium and MAPK inhibitors alone had no effect in the
PTHrP assay.
Measurement of intracellular calcium by fluorimetry in cell populations. Coverslips with H-500 cells were loaded for 2 h at room temperature with fura 2-AM in 20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, 0.1% BSA, and 0.1% dextrose and then were washed once with a bath solution (20 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% dextrose, and 0.1% BSA) at 37°C for 20 min. The coverslips were then placed diagonally in a thermostatted quartz cuvette containing the bath solution with a modification of the technique employed previously in this laboratory (4). In the experiment with agonists for other G protein-coupled receptors, angiotensin II was used at 10-8 M, ADP at 10-8 M, thrombin peptide agonist at 10-5 M, and carbachol at 10-4 M. The angiotensin II, ADP, thrombin peptide agonist, and carbachol were added into the bath solution. Excitation monochromators were centered at 340 and 380 nm, and emission light was collected at 510 ± 40 nm through a wide-band emission filter. The 340/380-excitation ratio of emitted light was used to estimate intracellular calcium ([Ca2+]i), as described previously (4).
Statistics. The data are presented as means ± SE of the indicated number of experiments. Data were analyzed by 1) one-way ANOVA followed by Dunnett's multiple comparison test or 2) Student's t-test when appropriate. A P value of <0.05 was considered to indicate a statistically significant difference.
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RESULTS |
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Stimulation of PTHrP release is not a generalized function of GPCR activation. Because high [Ca2+]o, acting via the CaR, increased PTHrP release, we next examined whether this is a generalized effect of activation of any GPCR. We first assessed whether the agonists affected [Ca2+]i release by using cells loaded with the calcium-sensitive dye fura 2. Treatment of cells with ADP (10-8 M) produced a rapid and transient increase in [Ca2+]i in the H-500 cells, whereas [Ca2+]o, angiotensin II, thrombin agonist, and carbachol had no such effect (data not shown; n = 3). This proves that the H-500 cells express a functional ADP receptor linked to the phosphoinositol (PI)-PLC system, thereby elevating [Ca2+]i in an agonist-dependent manner. We then incubated H-500 cells with increasing ADP concentrations from 10-9 to 10-7 M in medium containing basal calcium (0.5 mM). We observed that, whereas high [Ca2+]o increased PTHrP release to 492 ± 117% of basal PTHrP release (P < 0.05), ADP had no effect (Fig. 2).
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Effect of [Ca2+]o on PTHrP transcripts. We next determined whether high [Ca2+]o-stimulated PTHrP release occurred at the level of transcription. We first pretreated the cells with actinomycin D (50 µg/ml) for 1 or 2 h and stimulated them with high [Ca2+]o for another 6 h, as described in MATERIALS AND METHODS. The inhibition of [Ca2+]o-stimulated PTHrP release in response to actinomycin D was dependent on the pretreatment time (Fig. 3A); i.e., the reduction in PTHrP release was more robust when cells were pretreated for 2 h than for 1 h. Expressed quantitatively, [Ca2+]o-stimulated PTHrP release was 129 ± 7 and 103 ± 2% of basal release with pretreatment for 1 and 2 h compared with 225 ± 13% in cells incubated at high [Ca2+]o without actinomycin D (P < 0.05). We then performed Northern analysis of mRNA extracted from H-500 cells incubated for 6 h with high (7.5 mM) or low [Ca2+]o (0.5 mM [Ca2+]o) after a 2-h preincubation with or without actinomycin D. The PTHrP transcript was upregulated by high [Ca2+]o in the absence of actinomycin D (P < 0.05). In contrast, actinomycin D markedly downregulated the level of PTHrP mRNA, and no difference was seen between high and low calcium (Fig. 3B).
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Effects of PKC and MAPK inhibitors on high [Ca2+]o-induced PTHrP release. To identify the signal transduction pathways involved in CaR-mediated stimulation of PTHrP release, we examined the effects on both basal ([Ca2+]o = 0.5 mM) and high calcium ([Ca2+]o = 7.5 mM)-stimulated PTHrP release of a PKC inhibitor, GF-109203X (1 µM) (36), a MEK inhibitor, PD-98059 (10 µM) (11), a JNK inhibitor, SP-600125 (10 µM) (5), and a p38 MAPK inhibitor, SB-203580 (10 µM) (10). None of the four inhibitors alone had any effect on basal PTHrP release (Fig. 4). In contrast, the MEK inhibitor, PD-98059, decreased calcium-stimulated PTHrP release from 432 ± 47 to 229 ± 25% of basal release (P < 0.05). This is a reduction by 61% in the CaR-mediated component of PTHrP release. Similar results were found with GF-109203X, SP-600125, and SB-203580; PTHrP release decreased to 237 ± 29, 251 ± 14, and 185 ± 13% of basal release, respectively, all of which were significant reductions compared with high calcium alone (P < 0.05). Either PD-98059 or SP-600125, when administered with GF-109203X, decreased PTHrP release to 26 ± 11 and 42 ± 12%, respectively, of the value observed with low calcium alone (P < 0.05).
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It has been previously shown that CaR-mediated activation of ERK1/2 in HEK-CaR cells is PKC dependent (20), whereas little is known about the involvement of p38 MAPK and JNK. Here, we studied the combined effects of the PKC and p38 inhibitors on [Ca2+]o-stimulated PTHrP release from H-500 cells and found that the effects of the two inhibitors on high [Ca2+]o-induced PTHrP release were not additive (170 ± 12% of basal release). Interestingly, we observed that the JNK and PKC inhibitors together had an additive effect, because in combination, these inhibitors reduced high calcium-stimulated PTHrP release to 157 ± 23% compared with SP-600125 (251 ± 14%) or GF-109203X (300 ± 30%) alone (P < 0.05). When administered together in all possible combinations of pairs, the MAPK inhibitors did not show any additive effect on PTHrP secretion (data no shown).
High [Ca2+]o stimulates phosphorylation of MAPKs. Because the MEK inhibitor PD-98059 inhibited high [Ca2+]o-stimulated PTHrP release in H-500 cells, we determined whether [Ca2+]o activated the MEKERK1/2 MAPK cascade by using a specific antibody to phosphorylated ERK1/2, a MEK substrate. High [Ca2+]o promoted the phosphorylation of ERK1/2 (Fig. 5A). [Ca2+]o-induced ERK1/2 phosphorylation was delayed, reaching a maximum level between 30 and 60 min that was sustained for 2 h before starting to decline. To determine whether PKC is capable of stimulating ERK1/2, we added PMA (100 nM) to H-500 cells and measured ERK1/2 phosphorylation; as expected, PMA stimulated ERK1/2 phosphorylation at 5 min compared with high calcium. Surprisingly, however, we found that the PKC inhibitor did not block high calcium-stimulated ERK1/2 phosphorylation at 30 and 60 min, suggesting that the CaR stimulation of the ERK pathway is PKC independent.
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Next, we studied the activation of p38 MAPK by using an antibody against
the phosphorylated form of this kinase. A delayed and sustained
phosphorylation of p38 MAPK was observed in H-500 cells in response to high
[Ca2+]o
(Fig. 5B); i.e., the
phosphorylation reached a maximum intensity at 1 h and decreased to the basal
level by 2 h. The PKC inhibitor did not reduce the calcium-induced
phosphorylation of p38 MAPK at 30 and 60 min, suggesting that PKC is not
upstream of p38 MAPK. Upon phosphorylation at its Thr-Gly-Tyr motif, p38 MAPK
is translocated into the nucleus and activates various transcription factors.
Two major substrates for phospho-p38 MAPK are ATF-2 and Elk-1. Phosphorylation
of ATF-2 occurred as early as 5 min and peaked at 2 h
(Fig. 5B). We
therefore observed an early response and a much-delayed activation for ATF-2
(2 h compared with 1 h, as observed in the case of p38 MAPK). No
phosphorylation of Elk-1 was observed after 2-h incubation with high
[Ca2+]o (data not shown).
Because SEK1 is upstream of JNK, and we observed that the JNK inhibitor diminishes calcium-induced PTHrP release, we next investigated whether high calcium promoted phosphorylation of SEK1. Indeed, we observed that SEK1 is phosphorylated by high [Ca2+]o within 5 min in H-500 cells and remains elevated for 60 min before starting to decline. Furthermore, the PKC inhibitor GF-109203X had no effect on SEK1 phosphorylation (Fig. 5C).
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DISCUSSION |
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Because the CaR is a member of the GPCR family that uses
Gq/11 for coupling to its major signaling pathway, PI-PLC,
we studied whether PTHrP release could be induced by activation of other GPCRs
coupled to G
q/11. Our data showed that ADP did not induce
PTHrP release despite mobilizing [Ca2+]i,
thereby suggesting that stimulation of PTHrP release is not a generalized
effect of GPCR-mediated activation of PIPLC. This specific induction of PTHrP
release by the CaR has important pathophysiological implications in HHM, in
which the major mediator, PTHrP, is under the control of the CaR, culminating
in a vicious cycle (12).
Interestingly, [Ca2+]o did not activate
[Ca2+]i store release. This might be due to
the low receptor density on these cells compared with parathyroid chief cells
or because the CaR uses a PLC-independent signaling pathway to regulate PTHrP
release. We have observed this phenomenon in other cells that are uninvolved
in extracellular calcium homeostasis, such as PC-3, MDA-MB 231, and U87 cells
(S. Quinn, unpublished observations).
Expression and release of PTHrP have been shown to be regulated by a variety of nuclear and membrane-bound receptor ligands. These various forms of regulation are mostly at the transcriptional level and are often exerted in a cell type-specific fashion. Much work has been carried out to identify agents that regulate PTHrP expression/release; however, relatively little is known about the mechanisms underlying these effects, particularly in relation to GPCRs. Our data show that high [Ca2+]o, acting via the CaR, augments the synthesis of PTHrP, likely acting at the transcriptional level.
The postreceptor mechanisms by which the CaR stimulates PTHrP release involve activation of PKC, MEK, JNK, and p38 MAPK pathways. Regulation of PTHrP release by PKC has been reported in the NCI-H727 nonsmall lung cancer cell and in alveolar epithelial cells (6, 13). We observed that CaR-mediated induction of PTHrP release is potentiated by a PKC activator and blunted by a PKC inhibitor. In some cell systems, activation of PKC participates in the activation of the ERK1/2 cascade (15). A Ras/MEK/ERK pathway has recently been implicated in the production of PTHrP from cardiac myocytes likely acting at the transcriptional level (15). Because the Ras/PKC/MEK pathway represents a classical signaling cascade, it is therefore conceivable that the CaR acts via this pathway to induce PTHrP production at the transcriptional level. However, surprisingly, we did not detect any effects of the PKC inhibitor on [Ca2+]o-induced ERK activation. Taken together with our data on PTHrP release, where no additive effect was seen, this result suggests that the pathways are parallel and converge distal to ERK. In contrast to the rapid and transient kinetics of ERK activation observed in other cells in response to high [Ca2+]o, in H-500 cells the ERK1/2 response was both delayed and sustained. Although delayed and sustained activation of ERK1/2 has been observed in response to a variety of stimuli in many cell systems, its role(s) is conjectural (21, 29, 33). Certainly, the most important determinant of the duration of ERK activation is the balance between activating kinases and inactivating phosphatases. However, the occurrence of nuclear translocation of ERK1/2 in response to prolonged activation suggests that high [Ca2+]o-induced activation of ERK acts at the transcriptional level to increase PTHrP production. The effect of sustained activation of ERK1/2 has been demonstrated in Swiss 3T3 fibroblasts, where it led to translocation of activated ERK and was associated with proliferation (25).
p38 MAPK is a stress-activated kinase that was originally identified as the
target of pyridinylimidazole compounds found to inhibit inflammatory cytokine
production and cell death following cellular stress
(26). Here we observed that
high [Ca2+]o-induced PTHrP release was
attenuated in the presence of SB-203580, a specific inhibitor of p38
and -
subtypes. We also observed that high
[Ca2+]o induced the phosphorylation of p38
MAPK. Phosphorylation activates p38 MAPK, which in turn activates ATF-2, which
then acts as a transcription factor for various target genes. The rapid
activation of ATF-2 observed in our studies in H-500 cells in response to high
[Ca2+]o may be due to other intracellular
signaling pathways that also activate ATF-2. Recently, p38 MAPK was shown to
be involved in the Smad-independent induction of PTHrP release from MDA-MB-231
breast cancer cells by transforming growth factor-
(19). However, our results
represent the first demonstration that a GPCR can regulate PTHrP production
via the p38 MAPK pathway in a pathophysiological setting. Similar to the
temporal pattern observed with ERK1/2 activation, we observed a delayed
activation of p38 MAPK and its downstream substrate, ATF-2, by high
[Ca2+]o in these cells. The delayed and
sustained nature of p38 MAPK activation such as that found in our system has
been reported in reactive murine astrocytes following the induction of
seizures with kainic acid and in arterial smooth muscle cells after balloon
injury (8,
18). In the field of GPCR
agonists, the thromboxane analog U-46619 has been reported to activate p38
MAPK in platelets, an activation blocked by SB-203580
(28). However, in another
study (34), the GPCR agonist
adenosine stimulated IL-6 in the intestinal epithelial cell line T84.
Adenosine-induced IL-6 production was mediated through cAMP-mediated
activation of nuclear cAMP-responsive element-binding, CREB, protein and
ATF-2. ATF-2 was activated between 1 and 3 h, which was similar to the maximal
activation at 2 h in our study.
CaR activation of the JNK pathway has been shown in MDCK cells (2). Here we show that the JNK inhibitor SP-600125 reduces CaR-mediated PTHrP release and that this action is additive with the PKC inhibitor. This suggests that the PKC and JNK pathways do not converge, as seems to be the case for MEK and p38 MAPK cascades. The fact that activation of the three MAPKs is independent of PKC might be due to the interaction of the CaR with filamin A (14). Filamin A has also been shown to interact with MEK, p38, and SEK1 (24). The CaR's potential interactions with these three MAPKs via filamin A could provide a PI-PLC-PKC-independent mode of activation of these enzymes in H-500 cells.
In conclusion, our data demonstrate that the CaR stimulates transcription of the PTHrP gene, thereby increasing release of PTHrP in H-500 cells. CaR-mediated PTHrP release occurs via PKC-, ERK1/2-, and p38 MAPK-dependent pathways that are activated in parallel but likely converge downstream of these protein kinases to regulate PTHrP synthesis and release. The JNK pathway, in contrast, appears to function independently of and parallel to the PKC pathway. Surprisingly, the activations of ERK1/2, SEK1, and p38 are independent of PKC.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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