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Endocrinology Vol. 141, No. 11 4185-4193
Copyright © 2000 by The Endocrine Society

ARTICLES

Obligate Mitogen-Activated Protein Kinase Activation in Parathyroid Hormone Stimulation of Calcium Transport But Not Calcium Signaling1

W. Bruce Sneddon, Fengming Liu, Frank A. Gesek and Peter A. Friedman

Departments of Pharmacology (W.B.S., P.A.F.) and of Medicine (P.A.F.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Department of Pharmacology and Toxicology, Dartmouth Medical School (F.L., F.A.G.), Hanover, New Hampshire 03755

Address all correspondence and requests for reprints to: Peter A. Friedman, Ph.D., University of Pittsburgh School of Medicine, Department of Pharmacology, E1347 Biomedical Science Tower, Pittsburgh, Pennsylvania 15261. E-mail: paf10{at}pitt.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH regulates calcium homeostasis through direct actions on its cognate type I receptor in the kidney and bone. PTH inhibits phosphate transport in renal proximal (PCT) tubules and stimulates calcium absorption by distal convoluted tubules (DCT). We examined PTH activation of the mitogen-activated protein kinase (MAPK) cascade raf-MEK-ERK in PCT and DCT cells and its effects on calcium transport and signaling. In DCT cells, PTH stimulates phosphorylation of ERK2 and activation of ERK2 kinase and is blocked by the MEK inhibitor PD98059. In DCT cells, stimulation of calcium entry with ionomycin did not activate ERK2 or augment PTH-stimulated ERK2 activity, indicating that MAPK activation lies upstream of calcium entry. ERK2 activation by PTH was blocked by the protein kinase C inhibitor calphostin-C but was unaffected by the protein kinase A inhibitor Rp-cAMPs. PD98059 abolished the increase of intracellular calcium induced by PTH demonstrating that ERK2 activation is directly involved in the increase of intracellular calcium activated by PTH in the DCT. Thus, PTH- stimulated ERK2 activation is PKC dependent and calcium independent. PTH also induced ERK2 phosphorylation in PCT cells. However, this effect is not involved in the transient rise of intracellular calcium because PD98059 did not inhibit the PTH-stimulated rise of intracellular calcium but abolished ERK2 activation. In conclusion, PTH activates MAPK in both distal and proximal renal tubule cells. However, the rise of [Ca2+]i depends upon MAPK activation only in distal cells. Thus, a common PTH1R exhibits differential signaling along the nephron that contributes to the ability to regulate distinct physiological actions of PTH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH REGULATES calcium homeostasis through actions on kidney and bone. In the kidney, PTH reduces calcium excretion by stimulating calcium absorption by distal convoluted tubules (DCT) (1) in most species and, in the case of rabbit, by connecting tubules (1, 2, 3). These effects are mediated by the type I PTH/PTH-related protein (PTH/PTHrP) receptor (PTH1R), which belongs to a subfamily of class 2 G protein-coupled receptors (GPCR) that includes, in part, receptors for calcitonin, secretin, GH-releasing factor, vasoactive intestinal peptide, glucagon-like peptide, and glucagon (4). Molecular cloning of the PTH receptor established three important points. First, PTH and PTHrP bind to the PTH1R with comparable affinity; second, the PTH1R receptor couples, or at least is able to couple, to two different signaling pathways involving adenylyl cyclase and phosphatidylinositol-specific phospholipase C (PI-PLC); and third, both signaling events occur through a common receptor (5, 6). However, in some instances, occupancy of the PTH1R receptor activates only one signaling pathway. In keratinocytes (7, 8, 9), cardiac myocytes (10, 11), lymphocytes (12, 13, 14), for example, the PTH1R receptor activates PI-PLC but not adenylyl cyclase. Conversely, in vascular smooth muscle cells the PTH1R receptor activates adenylyl cyclase but not PI-PLC (15, 16). Other signaling pathways involving PLA2 (17, 18, 19) or PLD (20, 21) have also been described though the details are less well characterized. In mouse DCT cells, stimulation of calcium transport by PTH requires activation of protein kinase A (PKA) and protein kinase C (PKC) (22). In contrast, only PKC activation is required for stimulation of calcium absorption by PTH in mixed cultures of rabbit connecting and cortical collecting tubule cells (23).

There are also important qualitative differences in PTH actions and signaling pathways between proximal and distal tubules. In proximal tubules (PCT), PTH actions such as inhibition of phosphate transport involve calcium signaling but do not stimulate calcium transport. PTH activates PI-PLC with attendant inositol trisphosphate formation, which causes a transient rise of intracellular calcium ([Ca2+]i). In contrast, in DCT cells, PTH stimulates calcium transport without calcium signaling (22). PTH activates adenylyl cyclase and induces a sustained elevation of [Ca2+]i that is dependent on PKC activation (22) that arises through stimulation of phospholipase D (PLD) with the subsequent formation of diacylglycerol (DAG) (21). The basis for the cell-specific signaling patterns exhibited by the PTH1R in the nephron is the subject of the present study.

Mitogen-activated protein kinases (MAPK) are a group of protein serine and threonine kinases that play important roles in the regulation of cell growth and differentiation (24, 25, 26). Extracellular regulated kinases 1 and 2 (ERK1/2), jun-N-terminal kinase (JNK), and p38 kinase are MAPKs that lie at the end of parallel protein kinase cascades. The best-characterized pathway of ERK1/2 signaling occurs in response to activation by the small G protein ras in its GTP bound state. Activated ras interacts with raf-1 and translocates it to the plasma membrane, where it is activated by phosphorylation (27). Activated raf-1 phosphorylates and activates MAPK kinase (MEK), leading to the phosphorylation and activation of ERK. MAPKs are activated in response to the stimulation of many different classes of cell surface receptors including GPCRs (28, 29).

Emerging evidence indicates that PTH can regulate MAPK activity in a cell-specific and G protein type-dependent manner. For example, in Chinese hamster ovary R15 cells, PTH activation of MAPK is mediated by cAMP and is independent of ras (30). In contrast, in osteoblastic UMR 106 cells, PTH inhibits MAPK activation by EGF (31), FGF, and PDGF (32) in a PKA-dependent manner. Other GPCRs employ different means to activate MAPK. Gi-coupled receptors, through the release of Gß{gamma}, activate MAPK in a ras-dependent fashion. For receptors coupled to Gq, ß{gamma}-dependent and -independent activation of MAPK has been reported; ß{gamma}-mediated activation occurs via ras, whereas the Gq{alpha}-induced activation can occur through PKC activation, independent of ras (33, 34).

Although both PKA and PKC activation are required for PTH-stimulated calcium entry in mouse DCT cells, the steps lying downstream of each of these kinases and how their respective signals are integrated has yet to be defined. As outlined above, PTH can activate the raf-MEK-ERK cascade through both PKA and PKC, depending on the cellular context. In the present study, the role of ERK1/2 activation on PTH-dependent calcium transport or calcium signaling was examined in DCT and PCT cells. The results demonstrate that PTH stimulates ERK2 activation in DCT cells in a PKC-dependent and PKA-independent manner and that such activation is required for stimulating calcium entry and a sustained elevation of [Ca2+]i. By contrast, in PCT cells, PTH increases ERK1/2 activity but this plays no role in the transient rise of [Ca2+]i.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
bPTH (1–84) was obtained from the National Hormone and Pituitary Program, NIDDK, NICHHD, USDA. The following were purchased from commercial sources: 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA, Sigma, St. Louis, MO), calphostin-C (Cal-C, Calbiochem, La Jolla, CA), fura-2 AM (Molecular Probes, Inc., Eugene, OR), hPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Bachem Bioscience, Inc., King of Prussia, PA), ionomycin (Calbiochem), salmon calcitonin (CT, Sigma), PD98059 (Calbiochem), Rp-cAMPs (Sigma), phorbol 12-myristate 13-acetate (PMA, Sigma), nifedipine (Sigma). Cell culture materials were obtained as follows: 50:50 mix of DMEM and Ham’s F12 (DMEM/F12, Life Technologies, Inc.), FBS (Life Technologies, Inc.), antibiotic mixture (50 µg penicillin, 50 µg streptomycin, 100 µg neomycin/100 ml media (PSN), (Life Technologies, Inc.). Supplies for Western analysis were purchased as follows: Hybond-ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ), monoclonal phosphospecific p44/42 MAP kinase antibody (New England Biolabs, Inc. (NEB), Beverly, MA), horseradish peroxidase (HRP)-conjugated rabbit secondary antibody (NEB), goat polyclonal ß-actin primary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HRP-conjugated rabbit antigoat secondary antibody (Santa Cruz), LumiGLO chemiluminescent detective solution (NEB). A commercial kit was purchased to assay ERK1/2 activity (NEB).

Cell culture
The preparation, subcloning, characterization, and culture conditions of DCT cells have been described (35). DCT cells exhibit a phenotype that includes: 1) stoichiometric sodium and chloride uptake that is inhibited by chlorothiazide but not bumetanide (35); 2) stimulation of calcium transport by PTH and chlorothiazide (35, 36); and, 3) activation of adenylyl cyclase by PTH (36). PCT cells exhibit Na-Pi cotransport and PTH-stimulated adenylyl cyclase (37, 38). Cells were grown on 100-mm dishes (Corning, Inc. Glass Works, Corning, NY) in a 50:50 mix of DMEM/F12, supplemented with 5% heat-inactivated (56 C for 20 min) FBS, and PSN in a humidified atmosphere of 95% air-5% CO2 at 37 C. Unless stated otherwise, cells were switched to serum-free DMEM/F12 media 16 h before use.

Western analysis of MAP kinase phosphorylation
Cells were grown on 100-mm dishes in DMEM/F12 containing 5% heat-inactivated FBS until the cells were 50% confluent. For 24 h and then again 2 h before treatment, cells were switched to DMEM/F12 containing 0.5% FBS. The medium was then aspirated and the cells were treated by adding fresh 0.5% FBS medium containing PTH(1–84) (10-8 M) with or without the specified inhibitor at 37 C, for the times indicated. After incubation, the medium was aspirated and the cells were washed three times with ice-cold PBS . SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue) (100 µl) was added to each dish. The cells were immediately scraped and the extract was transferred to a 1.5-ml microfuge tube on ice. The sample was sonicated for five pulses at a duty cycle setting of 40% using a Branson Sonifier 450 (Branson Ultrasonics, Danbury, CT) and transferred to a 0.5-ml microfuge tube. The sample was heated at 100 C for 5 min, put on ice briefly and then microcentrifuged at 14,000 rpm for 30 sec. Thirty microliters of the supernatant were loaded onto a 7.5% SDS-PAGE gel. After electrophoresis, the protein was electrotransferred to Hybond-ECL nitrocellulose. The membrane was blocked in Tris-buffered saline (20 mM Tris, 140 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST) and 5% milk at room temperature for 2 h. The membrane was then incubated with the primary antibody, a monoclonal phosphospecific p44/42 MAP kinase at a 1:1000 dilution in TBST with 5% milk overnight at 4 C. The blot was washed three times for 10 min in TBST at room temperature and then incubated with a horseradish peroxidase (HRP)-conjugated rabbit secondary antibody in TBST containing 5% milk at a 1:2000 dilution at room temperature for 1 h. After washing three times with TBST, the membrane was incubated with LumiGLO chemiluminescent detective solution for 1 min and exposed to Kodak X-Omat Blue XB-1 film. A duplicate blot was probed with a goat polyclonal ß-actin primary and an HRP-conjugated rabbit antigoat secondary antibody.

MAP kinase activity
ERK1/2 activity was determined with a commercial kit (NEB). DCT cells were grown to confluence in 6-well plates in DMEM/F12 containing 5% FBS. Cells were switched to serum-free DMEM/F12 24 h before protein isolation. Hormones and inhibitors were added, as indicated, in serum-free DMEM/F12. Cell lysates were prepared as outlined by the manufacturer. Active ERK1/2 was immunoprecipitated overnight at 4 C using an immobilized agarose-conjugated monoclonal antibody to phospho-p44/42 MAP kinase and used in an in vitro kinase assay to phosphorylate a GST-Elk1 (codons 307–428) fusion protein at 30 C for 30 min in a volume of 50 µl. To terminate the kinase reaction, 15 µl of 4' SDS-PAGE loading buffer (see above) was added. The sample was incubated at 95 C for 5 min and then microcentrifuged for 2 min. 20 µl of the supernatant were electrophoresed on a 12.5% SDS-PAGE gel. The protein was electrotransferred to Hybond-ECL nitrocellulose and probed as outlined above using a rabbit polyclonal IgG to phospho-Elk-1 (Ser 383) included in the kit. The secondary antibody was an HRP-conjugated goat antirabbit antibody.

Free intracellular calcium ([Ca2+]i)
Cells were grown to near confluence on 25-mm glass coverslips and rinsed three times with a saline buffer consisting of: 140 mM NaCl, 4.6 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 18 mM Tris base that was adjusted to pH 7.40 ± 0.01 and 295 ± 2 mosmol/kg H2O. Cells on coverslips were incubated for 60 min at 37 C with the Ca2+-sensitive indicator fura-2 AM (10-5 M). The coverslips were then rinsed several times and mounted in a microincubation system (Narishige, Greenvale, NY) maintained at 37 C on the stage of a Nikon Diaphot microscope. Fluorescence emission intensity was measured with a Nikon Photoscan-2 (Nikon, Inc., Natick, MA) as described previously (35). Clusters of 1–3 cells were selected with a shutter assembly mounted in front of the photomultiplier tube. Periods of control fluorescence were recorded before the addition of hormone or drugs. Solutions containing hormones or drugs were maintained at 37 C within a water-jacketed chamber attached to a heating circulator and directed to the coverslip with a peristaltic perfusion pump. Fluorescence intensity was sampled at a rate of 60 Hz. Absolute values of [Ca2+]i were determined by in situ calibrations, which were performed at the end of each experiment as described (35, 39).

45Ca2+ uptake
Cells were suspended in saline buffer as outlined previously (36), and nonspecific binding of isotope to filters and cells was determined in the presence of LiCl-HEPES buffer added to the cells before addition of 45Ca2+. Studies were performed by pretreatment with PTH or sCT and blockers for 15 min with 45Ca2+ added for the final minute. Results were normalized for protein content, which was measured by the Lowry method on 50 µl aliquots of cells (40). Tracer uptakes are expressed as nanomoles per minute mg protein (nmol min-1 mg protein-1).

Quantitation and statistical analysis
Autoradiographs of Western blots were optically scanned and bands of interest were quantified using ImageQuant software (Amersham Pharmacia Biotech, Piscataway, NJ). Data are presented as means ± SE, where n indicates the number of independent observations. Comparisons between control and experimental treatment groups were evaluated by ANOVA and posthoc analysis of multiple comparisons using the Dunnet method (Instat; GraphPad Software, Inc., San Diego, CA). Values of P = 0.05 were assumed to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTH stimulated phosphorylation of ERK2 in mouse DCT cells in a time-dependent fashion. Maximal stimulation of 3.1-fold was achieved after 15 min (Fig. 1Go), returning to baseline by 30 min. In contrast, PTH did not affect ERK1 levels over the same period. To determine if the elevated phospho-ERK2 was accompanied by enhanced ERK2-kinase activity, in vitro kinase assays were performed on DCT cells that had been treated with hormone for 15 min. As shown in Fig. 2Go, PTH augmented ERK2-kinase by 3.1-fold over control. FBS, a positive control, increased ERK2-kinase activity by 5.3-fold (Fig. 2Go). CT, which also stimulates calcium absorption and increases [Ca2+]i in DCT cells, stimulated ERK2 activity 2.7-fold (Fig. 3Go).



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  		Figure 1. PTH induces ERK2 phosphorylation in DCT cells. DCT cells were incubated for the indicated times with 10-8 M bPTH(1–84). Proteins from treated cells were isolated and analyzed by Western blot as outlined in Materials and Methods. A, A representative experiment. B, A composite of all data were quantified as outlined in Materials and Methods and normalized with respect to ß-actin expression. Data are presented as fold changes from control (MAPK activation in the absence of PTH) and represent the mean ± SEM of three to five experiments. *, P < 0.05.

 


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  		Figure 2. PTH induces ERK2 kinase activity in DCT cells. DCT cells were incubated with 10-8 M hPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) or 10% FCS for 15 min. Active ERK2 was immunoprecipitated and in vitro kinase reactions were assayed by Western blots as detailed in Materials and Methods. A, A representative experiment shows the effect of PTH. B, Average results of five independent experiments are presented as fold changes from control. *, P < 0.05.

 


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  		Figure 3. The MEK inhibitor PD98059 suppresses both baseline and CT-stimulated ERK2 activity in DCT cells. DCT cells were incubated for 15 min with 10-8 M sCT, 10-5 M PD98059, or both as indicated. Active ERK2 was immunoprecipitated and in vitro kinase reactions were assayed by Western blot analysis. A, A representative experiment. B, Average results from three to five experiments were quantified as outlined in Materials and Methods. Data are presented as fold changes from control. *, P < 0.05.

 
Calcium entry in DCT cells stimulated by PTH or CT is accompanied by a sustained rise of [Ca2+]i that is due to influx of calcium from extracellular sources (36, 41). To determine if calcium entry is sufficient for ERK2 activation in DCT cells, the following experiment was performed. Addition of the calcium ionophore, ionomycin (10 µM), increased [Ca2+]i immediately (data not shown) but had no effect on ERK2-kinase activity (Fig. 4Go) and did not augment PTH-stimulated ERK2-kinase activation (Fig. 4Go). Because PTH does not release calcium from intracellular stores in DCT cells (22), ERK2-kinase activation by PTH occurs independently of, or before, PTH-induced increases of [Ca2+]i. Furthermore, the calcium channel blocker nifedipine at 10 µM had no effect on PTH-stimulated ERK2 activity (Table 1Go), consistent with the observation that calcium entry is not required. Chelating [Ca2+]i with BAPTA also had no effect on PTH-stimulated ERK2 (Table 1Go). Therefore, elevation of [Ca2+]i is not required for ERK2 activation.



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  		Figure 4. PTH-induced ERK2 activity does not depend on calcium entry in DCT cells. DCT cells were incubated for 15 min with 10-8 M hPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), 10-5 M ionomycin, or both as indicated. Active ERK2 was immunoprecipitated and in vitro kinase reactions were assayed by Western blots as outlined in Materials and Methods. A, A representative experiment. B, Summary data of five independent experiments were quantified as outlined in Materials and Methods. Data are presented as fold changes from control. *, P < 0.05.

 

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  		Table 1. Effects of Nifedipine and BAPTA on ERK2 activity in DCT cells

 
To test the hypothesis that PTH-stimulated ERK2 activity proceeds through the raf-MEK-ERK pathway, the effect of the specific MEK inhibitor PD98059 (42) was examined. PD98059 (10 µM) suppressed both PTH-stimulated ERK2-kinase activity (Fig. 5Go) and CT-stimulated ERK2 kinase activity (Fig. 3Go). Baseline activity was also reduced (Figs. 3Go and 5Go). Because the raf-MEK-ERK pathway can be activated in a PKC-dependent manner (43), we tested whether ERK2 activation by PTH in DCT cells occurs downstream of PTH-stimulated PKC activation. Calphostin-C (100 nM), an inhibitor of PKC, blocked PTH-stimulated ERK2-kinase activity (Fig. 6Go) but had no effect on baseline ERK2-kinase activity. Down-regulation of PKC activity with 1 nM PMA for 16 h similarly blocked PTH-stimulated ERK2 kinase activity but had no effect on baseline activity. PTH-stimulated ERK2 activity in the absence and presence of PMA was (3.6 ± 0.9) and (0.8 ± 0.2)-fold, respectively (n = 4). Basal levels of ERK2 activity in DCT cells, therefore, are likely set by a PKC-independent mechanism. In contrast, the PKA inhibitor, Rp-cAMPs (300 µM), had no effect on ERK2 activity (Fig. 6Go). Furthermore, activation of adenylyl cyclase by forskolin (1 µM) had no effect on ERK2 kinase activity in DCT cells (data not shown). Thus, PTH stimulates elevated levels of ERK2 activity through the raf-MEK-ERK pathway in a PKC-dependent manner in DCT cells.



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  		Figure 5. The MEK inhibitor PD98059 suppresses both baseline and PTH-stimulated ERK2 activity in DCT cells. DCT cells were incubated for 15 min with 10-8 M hPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), 10-5 M PD98059, or both as indicated. Active ERK2 was immunoprecipitated, and in vitro kinase reactions were assayed by Western blot analysis. A, A representative experiment. B, Summary data were quantified as outlined in Materials and Methods. Data are presented as fold changes from control and represent the mean ± SEM of five experiments. *, P < 0.05.

 


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  		Figure 6. PTH activates ERK2 in a PKC-dependent and PKA-independent manner. DCT cells were incubated for 15 min with 10-8 M hPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), 10-7 M calphostin-C (Cal-C), 3 ' 10-4 M Rp-cAMPs alone or in combination as indicated. Active ERK2 was immunoprecipitated and analyzed by in vitro kinase reactions as described in Materials and Methods. A, A representative experiment. B, The results of five independent experiments are presented as fold changes from control. *, P < 0.05.

 
ERK2 can also be activated in DCT cells in a PKC-independent manner. The a1-adrenergic agonist, phenylephrine, caused a 5-fold increase in ERK2 phosphorylation (n = 3) in DCT cells and calphostin-C (100 nM) had no effect on this response. It is, therefore, unlikely that calphostin-C inhibits PTH stimulated ERK2 activity by means of a direct action on ERK2.

PTH stimulates calcium entry and increases [Ca2+]i in DCT cells by a mechanism that requires activation of both PKA and PKC (22). PTH-stimulated ERK2 activation, as shown here, proceeds through a PKC-dependent mechanism. Therefore, we tested the theory that PTH-stimulated increases of [Ca2+]i and PTH-induced calcium uptake depend upon ERK2 activation. The MEK inhibitor PD98059 blocked the PTH-induced rise of [Ca2+]i in DCT cells (Fig. 7Go) and PTH- and CT-stimulated calcium uptake by DCT cells (Fig. 8Go), thereby confirming the view that ERK2 activation is required for both PTH- and CT-induced stimulation of calcium transport in DCT cells.



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  		Figure 7. The MEK inhibitor, PD98059, blocks the PTH-stimulated increase of [Ca2+]i in DCT cells. Cells were grown on coverslips and loaded with the calcium-sensitive dye, fura-2 AM. [Ca2+]i was measured as detailed in Materials and Methods and elsewhere (35 ). Each trace depicts the results from a single cell or small cluster of cells. Where PTH was examined alone, 10-8 M bPTH(1–84) was added at 2 min and [Ca2+]i was monitored for the time indicated. A single representative control experiment is shown for comparison. In the other three experiments, 10 µM PD98059 was added at 2 min. 10-8 M bPTH(1–84) was added at 15 min.

 


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  		Figure 8. The MEK inhibitor, PD98059, blocks the PTH- and CT-stimulated calcium uptake in DCT cells. DCT cells were treated for 15 min with 10-8 M bPTH(1–84) or 10-8 M sCT, alone or in combination with PD98059 (10-5 M), as indicated. At the end of this treatment, 45Ca2+ uptake was measured for 1 min, as detailed in Materials and Methods. Data are presented as fold changes from control and represent mean ± SEM of three experiments. *, P < 0.05.

 
In renal proximal tubules, PTH also increases [Ca2+]i, but this effect is transient and is due to release of calcium from intracellular stores (22, 44). We examined PTH-induced phosphorylation of both ERK1 and ERK2 in PCT cells to determine if PTH-induced changes of [Ca2+]i require activation of MAPK. As in DCT cells, PTH activated ERK2 (Fig. 9Go). Notably, and in contrast to DCT cells, blockade of MEK with PD98059 inhibited phosphorylation of MAPK (Fig. 9Go), but had no effect on PTH-stimulated increases of [Ca2+]i (Fig. 10Go). MAPK activation by PTH, therefore, does not play a role in calcium release by PCT cells.



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  		Figure 9. PTH induces ERK2 phosphorylation in PCT cells. PCT cells were incubated for 15 min with 10-8 M bPTH(1–84) in the presence or absence of 10 µM PD98059. Proteins from treated cells were isolated and analyzed by Western blot using a monoclonal phosphospecific p44/p42 MAPK antibody as outlined in Materials and Methods. A duplicate blot was analyzed by Western blotting using a polyclonal goat ß-actin antibody.

 


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  		Figure 10. The MEK inhibitor, PD98059, has no effect on the PTH-stimulated increase of [Ca2+]i in PCT cells. Cells were grown on coverslips and [Ca2+]i was measured as described in Materials and Methods and elsewhere (35 ). Each recording represents a single cell or small cluster of cells. A typical experiment is shown in which 10 µM PD98059 was added at 2 min and 10-8 M bPTH(1–84) was added at 12 min and again at 20 min. A control experiment, where PTH was added alone, is shown for comparison.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
PTH signaling, physiological actions, and associated changes of [Ca2+]i vary both qualitatively and quantitatively among nephron segments and between other PTH target tissues such as bone, heart, or adipocytes (45, 46, 47, 48). For example, in proximal renal tubules, PTH activates PI-PLC and induces a transient rise of [Ca2+]i that results from the release of calcium from intracellular stores (49). These transient changes in [Ca2+]i are thought to be involved in the reduction of phosphate transport by PTH. In contrast, in DCT cells (36), PTH activates both PKA and PKC (22), leading to calcium entry with a concomitant and sustained rise of [Ca2+]i. This action is consistent with the physiological role of PTH in stimulating renal calcium absorption. Inhibition of either PKA with Rp-cAMPS, or PKC with calphostin C abolishes PTH-stimulated calcium entry (22). Likewise, calcium transport is initiated only when both PKA and PKC are stimulated exogenously. Moreover, in DCT cells, PTH activates PLD, not PI-PLC, with subsequent diacylglycerol formation and PKC activation that is independent of inositol trisphosphate formation and a transient rise of [Ca2+]i (21). The role of ras and the G protein components of this signaling cascade leading to increased [Ca2+]i by PTH in DCT are not yet characterized. The participation of PLD in this process may also be significant because it can both activate and be activated by PKC (50). The present studies were initiated to elucidate further the signal transduction pathways employed by the PTH1R receptor.

The MAPK pathway can serve as a point of integration for different signal transduction cascades, including those involving GPCRs, tyrosine kinases, PKC, and PKA (28, 29). The hypothesis examined in this study was that the raf-MEK-ERK MAPK pathway is activated in two PTH renal target tissues but is uniquely involved in the mechanism of PTH action on calcium transport by distal tubule cells. The data show that, in DCT cells, PTH activated ERK2 (Figs. 1Go and 2Go) and inhibition of ERK2 (Figs. 5Go and 7Go) blocked PTH-stimulated increases of [Ca2+]i. This contrasts with the role of ERK activation by PTH in proximal tubule cells, where increases of [Ca2+]i and ERK activity could be dissociated (Figs. 9Go and 10Go). The present findings that activation of MAPK by PTH is required for calcium transport by DCT cells but is unrelated to the release of [Ca2+]i by PCT cells support and extend the observations of Cole that PTH activates MAPK in proximal tubule-like opossum kidney (OK) cells (51). A recent study by Lederer and colleagues supplements these observations, implicating MAPK activation in PTH-dependent inhibition of phosphate transport by OK cells (52).

PTH-stimulated ERK2 activation in DCT cells employs a PKC-dependent mechanism (Fig. 6Go) that is independent of PKA (Fig. 6Go) and of calcium entry (Fig. 4Go, Table 1Go). PTH can activate MAPK activity in several cell-specific ways. Stimulation of MAPK by PTH in OK cells, for instance, can occur through PKA or PKC and is calcium dependent (51). In Chinese Hamster ovary (CHO) cells overexpressing the rat PTH1R, PTH-stimulates MAPK activation by PKA through a ras-independent mechanism (30). Occupancy of the PTH1R in DCT cells activates PKA and PKC in parallel. However, ERK2 activation arises through the PKC limb of the bifurcating signaling pathways employed by the PTH1R. Inhibition of adenylyl cyclase did not interfere with PTH activation of MAPK (Fig. 6Go) and, conversely, blockade of MEK with PD98059 did not prevent adenylyl cyclase activation by PTH (data not shown). Therefore, in DCT cells, PTH activates PKA and MAPK through independent pathways, although both are required for the stimulatory effect on calcium transport.

MAPK activation occurs downstream of PKC stimulation in a number of distinct cell types (28). For example, in cardiac myocytes, hypertrophic stimuli increase [Ca2+]i by a mechanism that employs PKC activation of ras with subsequent raf-MEK-ERK activation (43). PTH-induced ERK2 activation is similarly downstream of PKC in DCT cells (Fig. 6Go). The calcitonin receptor (CTR), a related GPCR that can activate both adenylyl cyclase and PI-PLC (53, 54, 55), activates ERK via PKC in a calcium-dependent manner in human embryonic kidney 293 cells overexpressing the rabbit CTR C1a isoform (56). The activation of MAPK by PTH in DCT is calcium independent. Therefore, cell-specific differences contribute to the role of calcium in PKC-dependent MAPK activation. These differences may reflect true cell-specific modifications or result from differences in receptor number or other consequences of heterologous receptor expression.

PTH and CT stimulate calcium transport by DCT cells in a manner that requires prior activation of MAPK (Fig. 8Go). It is uncertain whether these additional signaling steps, along with presumptive downstream nuclear events, may contribute to the latency of the action of PTH compared with CT (41). It is as yet unclear how MAPK activation relates to the observation that inhibition of protein synthesis with cycloheximide blocks PTH-stimulated calcium transport but not CT-stimulated transport by DCT cells (41). These findings are consistent with the hypothesis that protein translation is necessary for activation of calcium transport by PTH but not by CT.

In conclusion, the present results show PTH activates MAPK in PCT and DCT cells. Blockade of the transient PTH-stimulated ERK2 activation prevented the increase of [Ca2+]i in DCT cells but not in PCT cells. Thus, the rise of [Ca2+]i depends upon MAPK activation only in DCT but not in PTH-sensitive PCT cells. The signal transduction cascade leading to MAPK activation and subsequent calcium entry by PTH in DCT is mediated through PKC, and inhibition of PKC or its down-regulation likewise prevent activation by PTH. Two important points emerge from this work. First, specialization along the nephron allows a single canonical PTH1R, in part through different signaling pathways, to inhibit phosphate absorption by proximal tubules, while stimulating calcium transport by distal tubules. Second, these findings more generally establish the ability of the PTH1R, and perhaps other GPCRs, to elicit adaptive signaling mechanisms in a cell-specific fashion.


   Footnotes
 
1 This work was supported by a grant from the National Institutes of Health (DK-54171). Back

Received December 21, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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