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Institute of Endocrine Science (S.C., A.L., M.P., E.B., A.S.) and Endocrine Surgery (L.V.), Ospedale Maggiore IRCCS, Milan, Italy 20122
Address all correspondence and requests for reprints to: Anna Spada, M.D., Institute of Endocrine Science, Via F. Sforza, 35, 20122 Milan, Italy. E-mail . anna.spada{at}unimi.it
Abstract
The calcium-sensing receptor (CaR) activation has recently been shown to modulate the ERK1 and ERK2 cascade in different cell lines. The present study investigated this pathway in human normal and tumoral parathyroid cells. In cells from normal parathyroids and almost all hyperplasia increasing extracellular calcium concentrations (Cao2+) induced a significant activation of ERK1 and -2, the percent stimulation over basal activity (at 0.5 mM Cao2+) being 545 ± 140 and 800 ± 205 in normal cells and 290 ± 71 and 350 ± 73 in hyperplasia at 1 and 2 mM Cao2+, respectively. This effect was mediated by CaR because it was mimicked by the receptor agonist gadolinium and neomycin. Basal and Cao2+-stimulated ERK1 and -2 activity was nearly abolished by the PKC inhibitor calphostin C, and PKA changes did not affect ERK1 and -2 activity. PI3K blockade by wortmannin, known to prevent G protein ß
subunit effect on ERK1 and -2, induced a 30% reduction of the Cao2+-stimulated ERK1 and -2 activity. Adenomatous cells showed high PKC-dependent ERK1 and -2 activity in resting conditions that was unresponsive to high Cao2+. A role of MAPK on PTH secretion was suggested by the finding that PD98059, a specific MEK inhibitor, abolished the inhibitory effect of 1.5 mM Cao2+ on PTH release from normal parathyroid cells. In conclusion, these data first demonstrate that CaR activation, through the PKC pathway and, to a lesser extent, PI3K, increases ERK1 and -2 activity in normal parathyroid cells and this cascade seems to be involved in the modulation of PTH secretion by Cao2+. Interestingly, this signaling pathway is disrupted in parathyroid tumors.
THE CALCIUM-SENSING receptor (CaR) plays a central role in extracellular calcium (Cao2+) homeostasis (1, 2). This receptor belongs to the superfamily of G protein-coupled receptors. Ligand binding results in Gq/11-dependent activation of phosphatidylinositol-specific PLC, which causes accumulation of IP3 and diacylglycerol, rapid release of calcium from intracellular stores, and activation of PKC (3, 4). Despite the large number of studies concerning intracellular signaling triggered by CaR activation, the precise nature of the cellular effectors involved in the inhibition of PTH secretion by Cao2+ remains poorly understood (3, 4, 5, 6). In addition to the control of hormone secretion, a possible role for the CaR in the regulation of cell growth has been suggested by the association between abnormal CaR sensitivity and parathyroid cell proliferation present in human parathyroid diseases and animal and in vitro models (2, 7, 8).
The MAPKs are serine/threonine-specific protein kinases and include ERK1 and ERK2, the c-Jun NH2-terminal kinases and the p38-MAPKs (9, 10). In particular, phosphorylated ERK1/2 participates in the regulation of cell growth and differentiation through phosphorylation and activation of nuclear transcription factors, such as Elk-1. Whereas in the past MAPK pathway was thought to represent the intracellular cascade specifically activated by growth factors, it is now well established that several G protein-coupled receptors are able to stimulate ERK1/2 by different mechanisms (11, 12, 13). Recently, experimental data in different cell systems expressing CaR, including Rat-1 and NIH/3T3 fibroblasts, human embryonic kidney cells CaR, and bovine parathyroid cells, demonstrated the ability of CaR to activate MAPK cascade (14, 15, 16). However, to date, no information on the ability of CaR to modulate MAPK activity in human parathyroid cells is available.
The present study shows that in normal parathyroid cells, CaR activation caused a significant stimulation of ERK1/2 activity that in turn participated in the inhibition of PTH secretion by Cao2+. On the contrary, Cao2+-induced stimulation of ERK1/2 activity was lost in parathyroid adenomas.
Materials and Methods
Cultures of human parathyroid cells
Normal parathyroid cells were obtained from 11 biopsies of normal parathyroid glands incidentally removed from normocalcemic patients during surgery for various thyroid diseases because of their intrathyroidal location. Eight parathyroid adenomas and one normal parathyroid gland from patients with primary hyperparathyroidism and five hyperplastic glands from patients with secondary renal hyperparathyroidism were also included in the study. Tissues removed at surgery were in part placed in sterile medium for cell culture and in part snap frozen in liquid nitrogen and stored at -70 C until analysis. Dispersed parathyroid cells were prepared by 2-h collagenase (2 mg/ml) digestion at 37 C, as previously described (17). The study was approved by the local ethical committee, and informed consent was obtained from each patient.
Determination of ERK1/2 phosphorylation and activity
For ERK1/2 activity determination dispersed cells were used after 4-h incubation in HAM-F10 (containing 0.5 mM Cao2+ and 0.5 mM Mg2+), supplemented with 0.5% heat-inactivated FCS and penicillin/streptomycin (1%) in a humidified atmosphere of 5% CO2 at 37 C. Subsequent incubation with various reagents and with increasing Cao2+ were performed in HAM-F10 + 0.5% FCS at 37 C for 10 min. In the media, Cao2+ concentrations were controlled by direct electric potential method (EML-100, Radiometer, Copenhagen, Denmark). Incubation with different stimuli was stopped by placing the cells on ice. The medium was removed and cells were treated with 500 µl ice-cold lysis buffer in the presence of protease and phosphatase inhibitors, as previously described (18). Briefly, active ERK was selectively immunoprecipitated from cell lysates by using an immobilized anti-phospho-p44/42 ERK monoclonal antibody (Thr202 and Tyr204), and the resulting immunoprecipitates were then incubated with a Elk-1 fusion protein (2 µg) in the presence of ATP (200 µM) and kinase buffer for 30 min at 37 C, which allows immunoprecipitated active MAPK to phosphorylate Elk-1. Phosphorylation of Elk-1 at Ser383 was measured by Western blotting using an anti-phospho-Elk-1 (Ser383) antibody. For the determination of total and phosphorylated ERK 1/2 levels, Western blot analysis of cell lysates was performed using anti-p44/42 ERK and anti-phospho-p44/42 ERK polyclonal antibodies diluted 1:1000 (New England Biolabs, Inc., Beverly, MA) and detected by chemiluminescent method (Phototope-HRP Western detection kit, New England Biolabs, Inc.). In the majority of experiments, samples from normal, hyperplastic, and adenomatous parathyroid tissues were run simultaneously. The densitometric readings of the resulting bands were evaluated using a GS-670 imaging densitometer (Bio-Rad Laboratories, Inc.). Experiments were repeated at least twice.
CaR protein expression
CaR protein was detected using an antiserum raised in rabbits (Primm srl, Milan, Italy) against a peptide corresponding to amino acids 345–359 within the predicted extracellular domain of the bovine parathyroid CaR, as previously described (17, 19). Proteins from normal, hyperplastic, and adenomatous parathyroid tissues were run simultaneously. Briefly, 20 µg total proteins, measured using the Micro BCA protein reagent kit (Pierce Chemical Co., Rockford, IL) were separated by SDS-PAGE at 7.5% and transferred to nitrocellulose membranes. The filters were subsequently incubated overnight at 4 C with CaR antiserum at 1:750 dilution, visualized by Phototope-HRP Western detection kit (New England Biolabs, Inc.) and then evaluated, using an imaging densitometer. Experiments were repeated at least twice.
PTH secretion experiments
Dispersed cells were plated in 24-well plastic cluster dishes at a density of 2 x 105 cells/ml and cultured in HAM-F10 supplemented with 10% FCS for 18–22 h, as previously described (17). Cells were then starved in HAM-F10 + 0.5% FCS for 4 h and tested with and without 50 µM PD98059 and various Cao2+ for 1 h in triplicate. Actual Cao2+ concentrations were controlled by photometrical technique. At the end of incubation, medium was removed and stored at -20 C for determination of intact PTH 1–84, using a chemiluminescent assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The intra- and interassay coefficients of variation were less than 5.7% and less than 6.7%, respectively, and the sensitivity was 2 pg/ml.
Materials
Phospho-p42/44 ERK (Thr 202 and Tyr 204) monoclonal antibody, p42/44 ERK monoclonal antibody, and phospho-Elk-1 (Ser 383) antibody were obtained from New England Biolabs, Inc. The phototope-HRP-enhanced chemiluminescence kit (LumiGLO) was purchased from New England Biolabs, Inc. PD98059 (2'-amino-3'-methoxyflavone), Calphostin C, H89, wortmannin, HAM-F10, collagenase, 3-isobytyl-1-methylxanthine, forskolin, neomycin, and gadolinium were obtained from Sigma (St. Louis, MO).
Statistical analysis
Data are presented as the mean ± SD. Statistical analysis were carried out using the unpaired t test. P less than 0.05 was considered as statistically significant.
Results
ERK1/2 activity in human normal parathyroid cells
The ERK1/2 activity was determined in each cell preparation (n = 6) by evaluating the phosphorylation of Elk-1 at Ser383 that represents the major phosphorylation site for ERK1/2 required for Elk-1-dependent transcriptional activity. In normal parathyroid cell cultures, increasing Cao2+ from 0.5 (basal condition) to 1 and 2 mM caused ERK1/2 activation, the mean percent stimulation being 545 ± 140 and 800 ± 205, respectively (Fig. 1
). Similar results were obtained by measuring the levels of phosphorylated p44-p42 ERK1/2 at increasing Cao2+ concentrations in two experiments (data not shown). In normal parathyroid cells from one patient affected with primary hyperparathyroidism, high Cao2+ induced a 6-fold increase in ERK1/2 activity, suggesting that hypercalcemia did not alter ERK1/2 response to Cao2+. Incubation of parathyroid cells with the CaR agonist gadolinium (Gd3+, at 30 and 300 µM) and neomycin (100 µM) caused an ERK1/2 activation similar to that induced by Cao2+ (Fig. 1), thus confirming CaR involvement.
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Hyperplastic parathyroid cells obtained from five patients affected with hyperparathyroidism secondary to chronic renal failure showed basal ERK1/2 levels that were 2- to 4-fold higher than that observed in normal parathyroid cells in the same experimental conditions (Fig. 2). With the exception of one hyperplastic cell preparation that was unresponsive to Cao2+, the pattern of ERK1/2 regulation by Cao2+ in hyperplastic cells was similar to that operating in normal cells, the percent stimulation being 290 ± 71 and 350 ± 73 at 1 and 2 mM Cao2+, respectively (Fig. 2). Similar effects were induced by Gd3+ (Fig. 2). Accordingly, Cao2+ caused a similar increase of phosphorylated p44-p42 ERK1/2 levels in two experiments (data not shown). Because of the high number of cells obtained from secondary hyperplasia in comparison with that from normal biopsies, the mechanism of ERK1/2 activation by Cao2+ was investigated in details in responsive hyperplastic cells. Preincubation for 1 h with PD98059 (50 µM), a compound that has been shown to inhibit specifically MEK1 and MEK2, which in turn phosphorylate ERK1/2, abolished both basal and Cao2+-stimulated ERK1/2 activity (Fig. 2). The role of PKC on CaR-mediated ERK1/2 activation was investigated by preincubating the cells with calphostin C, a specific PKC inhibitor, for 30 min. This agent, at concentrations higher than 10 nM, markedly reduced both basal and Cao2+-stimulated ERK1/2 activity, the maximal effect being observed at 1 µM (Fig. 3). In addition to PKC, a minor role for the PI3K pathway in the Cao2+-induced ERK1/2 activation was suggested by the observation that 1-h preincubation with wortmannin (100 nM), a PI3K inhibitor known to prevent the stimulatory effect of G protein ß subunit on MAPK cascade, caused a 30% reduction of ERK1/2 activation by 2 mM Cao2+ (Fig. 3). By contrast, adenylyl cyclase activation by forskolin (1 µM) as well as PKA inhibition by the selective PKA blocker H89 (10 µM for 1 h) did not modify basal and Cao2+-stimulated ERK1/2 activity (Fig. 3; data not shown).
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In adenomatous parathyroid cells from eight patients affected with primary hyperparathyroidism, basal ERK1/2 activity appeared significantly higher than that observed in normal cells and similar to the hyperplastic ones. Conversely, Western blot analysis showed similar amounts of total ERK1/2 protein in normal and adenomatous tissues (data not shown). In adenomatous cells increasing Cao2+ from 0.5 to 1 and 2 mM did not result in any change of ERK1/2 activity (Fig. 4). Similar data were obtained with Gd3+ at the maximally effective concentrations (300 µM) (Fig. 4). ERK1/2 activity largely depended on PKC pathway because preincubation with the specific PKC inhibitor calphostin C (0.1 and 1 µM) markedly reduced the activity observed in resting conditions (Fig. 4). In these cells, forskolin, H89, and wortmannin were not able to affect MAPK activity (data not shown).
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The expression of CaR protein was evaluated by immunoblotting analysis. The antiserum recognized a protein of about 120 kDa, detectable as a single band in our experimental conditions as previously described (14) (Fig. 5). CaR protein was detected in all samples. When compared with the CaR expression observed in normal parathyroid tissues, neoplastic tissues showed a variable reduction in CaR protein levels that was not statistically significant, probably because of the low number of tissues considered (OD arbitrary units: 1.74 ± 0.97 in normal vs. 0.91 ± 0.54 in tumoral samples, P = 0.11, Fig. 5).
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In normal parathyroid cell preparations (n = 5), basal PTH release was reduced by 25.7% ± 11.1% in the presence of physiological concentrations of Cao2+ (1.5 mM) (Fig. 6). Treatment of cell cultures with the specific inhibitor of MEK PD98059 (50 µM) did not modify PTH release observed in basal conditions (0.5 mM Cao2+) but totally abrogated the inhibitory effect of 1.5 mM Cao2+ (Fig. 6).
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This study first demonstrates that human parathyroid cells respond to physiological Cao2+ concentrations with an increase in the activity of the mitogen-activated protein kinases ERK1/2. This activation occurred through the well-characterized ERK cascade because it was totally abrogated by preincubating the cells with PD98059, a compound that has been shown to inhibit specifically the upstream MAPK kinases MEK1 and MEK2, which in turn phosphorylate ERK1/2 (20, 21). Moreover, Cao2+ increased ERK1/2 activity by specifically interacting with the CaR because a similar effect was induced by the CaR agonist Gd3+. As reported in other cell lines (14, 15, 16), in parathyroid cells the activation of PKC resulted in being the main pathway determining ERK1/2 activity in resting conditions as well as in response to increasing Cao2+, likely as a consequence of the Gq/11-dependent increase in calcium mobilization and diacylglicerol formation caused by Cao2+ (3, 4). In addition to PKC, a minor role for the PI3K pathway was suggested by the observation that preincubation with wortmannin, a PI3K inhibitor known to prevent the stimulatory effect of Gi protein ß subunit on MAPK cascade (22), slightly reduced ERK1/2 activation induced by Cao2+. Therefore, in human parathyroid cells, the two G proteins that have been demonstrated to be coupled to CaR, that is Gq/11 and Gi (3, 4, 23, 24), cooperate in determining ERK1/2 response to Cao2+ by activating PKC and PI3K, respectively. In agreement with a poor, if any, role of cAMP in the modulation of PTH release by Cao2+ (4), changes in PKA activity did not result in any ERK1/2 modification.
Evidence indicates that the biological response to CaR activation largely depends on the cell type expressing the receptor. In fibroblast Rat-1 cells expressing an endogenous CaR, high Cao2+ was demonstrated to induce cell proliferation by activating proliferation-associated signals, including ERK1/2 phosphorylation (14). Similarly, evidence for the existence of a pathway linking CaR to cell proliferation were provided in NIH3T3 fibroblasts expressing an activating mutant of the CaR (Thr151Met) that was isolated from a family affected with autosomic dominant hypocalcemia, in which particularly aggressive neoplasia were present (15). By contrast, in parathyroid cells CaR seems to directly or indirectly mediate the inhibition of cell proliferation (1, 2, 5, 8, 25). This is strongly suggested by the occurrence of marked parathyroid hyperplasia in both neonatal severe hyperparathyroidism because of loss-of-function mutations of the CaR gene and in homozygous CaR knockout mice (2, 7). Accordingly, the potent and selective CaR activators NPS R-467 and NPS R-568 have been demonstrated to inhibit both PTH secretion (25) and parathyroid proliferation (26). To date, no information on the possible role of MAPK pathway on PTH secretion is available. In this study ERK1/2 activation was found to participate in the inhibition of PTH release induced by increasing Cao2+ because this effect was totally abrogated by inhibiting the upstream MAPK kinases MEK1 and MEK2. These findings strongly suggest that ERK1/2 activation represents one of the steps downstream PKC required for the inhibition of PTH release in response to high Cao2+.
The ERK1/2 activation induced by increasing Cao2+ was maintained in cells obtained from almost all hyperplastic glands from patients with secondary renal hyperparathyroidism, whereas this response was lost in parathyroid adenomas. The absent ERK1/2 activation in parathyroid adenomas was not secondary to CaR desensitization because of hypercalcemia because normal parathyroid cells from one patient with primary hyperparathyroidism showed the expected ERK1/2 activation by Cao2+. Moreover, the lack of Cao2+ effect on ERK1/2 was not simply because of absent expression of CaR because adenomas of the present series had receptor protein levels that were only slightly lower than those detected in normal parathyroid cells. Similarly, no difference in the levels of ERK protein among the different tissues was detected, suggesting that the amounts of ERK protein available for phosphorylation by MEK are similar in the different tissues. The cellular events preventing the activation of ERK1/2 by Cao2+ in adenomatous cells remain at present unknown, because it has been reported that CaR expressed in parathyroid adenomas was normally coupled to Gq/11-dependent generation of intracellular effectors, although increased levels of Cao2+ were generally required to generate the response (15, 23, 27). Therefore, the absent ERK1/2 activation by Cao2+ observed in adenomatous cells might represent a component of the altered set-point for Cao2+ that characterizes parathyroid neoplasia and probably involves low receptor expression as well as postreceptor defects. Because this pathway was shown to mediate at least in part the inhibition of PTH release by high Cao2+ in normal parathyroid cells, it is tempting to speculate that the lack of ERK1/2 activation in parathyroid tumors may represent an additional alteration leading to defective Cao2+ sensing. Alternatively, the lack of ERK1/2 activation by Cao2+ in parathyroid tumors might be owing to the constitutive activation of this pathway by other agents coupled to PKC, including growth factors that have been demonstrated to be specifically expressed together with the receptor in parathyroid adenomas (28, 29, 30). This view is consistent with the elevated levels of ERK1/2 activity observed in tumoral cells in resting conditions.
In conclusion, these data demonstrate that CaR activation, through the PKC pathway and to a lesser extent PI3K, increased ERK1/2 activity in normal and hyperplastic parathyroid cells, and this pathway seems to be involved in the inhibition of PTH secretion by Cao2+. The pathogenetic implications of the different pattern of MAPK activity and regulation in parathyroid tumors remain to be investigated.
Footnotes
This work was partially supported by MURST (Rome) and Ricerca Corrente Funds of Ospedale Maggiore IRCCS.
Abbreviations: Cao2+, Extracellular calcium; CaR, calcium-sensing receptor; Gd3+, gadolinium.
Received August 27, 2001.
Accepted February 6, 2002.
References
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, P < 0.05 vs. ERK1/2 at 2 mM Cao2+.
