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,§
*
Department of Medicine University of Louisville School of Medicine,
Louisville, Kentucky.
§
Veterans Affairs Medical Center, Louisville, Kentucky.
Department of Biochemistry, University of Louisville School of Medicine,
Louisville, Kentucky.
Department of Molecular Biology, University of Louisville School of
Medicine, Louisville, Kentucky.
Correspondence to Dr. Eleanor D. Lederer, Kidney Disease Program, University of Louisville, 615 South Preston Street, Louisville, KY 40202. Phone: 502-852-5757; Fax: 502-852-7643; E-mail: elederer{at}kdppl.kdp-baptist.louisville.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially identified as a signaling pathway stimulated by receptor tyrosine
kinases, ERK are now well documented to be activated by receptors coupled to
heterotrimeric G proteins. Several different signal transduction pathways are
reported to activate ERK, of which more than one may exist in a single cell
type. These pathways converge at the level of MAPK/ERK kinases (MEK), dual
specificity kinases that activate ERK by threonine/tyrosine phosphorylation
(8). MEK is activated by
serine/threonine phosphorylation by one of a family of raf kinases through
ras-dependent or ras-independent mechanisms or by MEK kinases (MEKK-1)
(9,
10,
11,
12,
13,
14). For Gi-coupled receptors
(
2 adrenergic receptors, M2 muscarinic receptors, and lysophosphatidic
acid receptors), activation of ERK is dependent on ß
subunits
(15,
16,
17,
18,
19). Gß stimulates
tyrosine phosphorylation of an adaptor protein Shc, which leads to the
association of Shc with Grb2 and the ras guanine nucleotide exchange factor
Sos. Sos stimulates GTP binding to ras, a process required for ras-mediated
activation of raf kinases. M1 muscarinic receptors, 1 adrenergic
receptors, and platelet-activating factor receptors are coupled to Gq or Go
and stimulate ERK activity through a subunit-mediated activation of
phospholipase C (20,
21,
22). Phospholipase C generates
diacylglycerol from phosphatidylinositol leading to activation of protein
kinase C, which directly activates raf kinases. Lev et al.
(23,
24,
25) described a separate
pathway whereby the phospholipase C-mediated increase in intracellular calcium
activates a tyrosine kinase, PYK2, leading to ras-dependent ERK activity.
Winston et al. (12)
described a pathway for ERK activation by tumor necrosis factor- in
mouse macrophages, where MEK is activated directly by MEKK-1 in a raf
kinase-independent manner.
Recently, several laboratories have reported hormone-stimulated ERK activity in renal cells. Terada et al. (26) reported angiotensin II-stimulated ERK activity in OK cells, a model for renal proximal tubule, that was partially blocked by inhibition of protein kinase C, suggesting a protein kinase C-dependent pathway for activation of ERK in OK cells. Quamme et al. (7) reported PTH-stimulated ERK activity in three OK cell clones. The first clone responded to PTH with an increase in cAMP, an increase in intracellular calcium, and a decrease in sodium-phosphate cotransport. In the second clone, PTH stimulated an increase in cAMP, but not intracellular calcium, and a decrease in sodium-phosphate cotransport. The third clone showed the increase in cAMP and intracellular calcium, but failed to decrease sodium-phosphate cotransport in response to PTH. PTH stimulated ERK activity in the first two clones, but only a minimal increase in ERK activity occurred in the third clone. These findings suggest that PTH stimulates ERK activity independent of protein kinase A and protein kinase C activation, that PTH-stimulated ERK activity is not dependent on an increase in intracellular calcium, and that ERK regulates phosphate transport. The purpose of the present study was to determine the pathway(s) of PTH-stimulated ERK activity in OK cells and to assess the role of ERK activity in regulation of sodium-phosphate cotransport.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-[32P]ATP were obtained
from ICN Biomedicals (Irvine, CA).
Cell Culture Technique
OK cells were grown to confluence as monolayers in 175-cm2
flasks in culture medium consisting of Eagle's medium with Earle's salts (Life
Technologies, Grand Island, NY) supplemented with 10% heat-inactivated fetal
calf serum, 4 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml
streptomycin, pH 7.4. Cells were maintained in a humidified atmosphere with 5%
CO2 at 37°C. The medium was changed three times per week, and
the cells were split 1:3 or 1:4 once per week by brief trypsinization and
dispersal. The cells from passages 82 to 88 were used for experiments at 100%
confluence.
Phosphate Uptake
Sodium-dependent phosphate uptake was measured by uptake of radiolabeled
phosphate into OK cell monolayers. The cells were seeded onto 96-well tissue
culture plates. Twenty-four hours before the uptake experiments, the culture
medium was replaced by serum-free medium. Assays were initiated by aspiration
of serum-free medium and addition of serum-free medium containing agonist. The
cells were incubated with PTH or diluent at 37°C for 2 h then washed three
times with transport medium (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2,
1.2 mM MgSO4, and 0.1 mM KH2PO4). Measurement
of phosphate uptake was initiated by addition of transport medium containing
32P-radiolabeled phosphoric acid (2 µCi/ml, pH 7.4) at room
temperature. Uptake was terminated after 10 min by aspiration of the transport
medium and washing the cells with ice-cold, radionuclide-free transport medium
in which NaCl was replaced with N-methylglucamine. This time point is
within the linear range of phosphate uptake. The cell monolayers were
solubilized by addition of 125 µl of 0.5% Triton X-100 per well. After a
90-min incubation at room temperature, 100 µl from each well was pipetted
into scintillant, and the radioactivity was counted by liquid scintillation
spectroscopy. Each assay was performed in sextuplicate, and the mean values of
counts per minute per assay were calculated. Absolute values of phosphate
uptake were calculated from standards run in triplicate with each assay.
Nonspecific phosphate binding was determined and subtracted from all other
counts. Because sodium-independent phosphate uptake accounts for <10% of
the total uptake (data not shown), the calculations were not corrected for
sodium-independent uptake. Results are expressed as percent change in
phosphate uptake and absolute phosphate uptake in picomoles per milligram
protein per minute. Protein determination was performed on 12 wells chosen
randomly from each 96-well plate.
ERK Assay
ERK activity was measured by in vitro phosphorylation of myelin
basic protein in partially purified cell lysates. Cells were seeded onto 10-mm
Petri dishes and grown to confluence. Twenty-four hours before the assay, the
culture medium was replaced with serum-free, phenol-free medium. Assays were
initiated by aspiration of the incubation medium and replacement with
serum-free, phenol-free medium containing agonist or carrier. The assay was
continued for the indicated time at 37°C and terminated by aspiration of
the medium and replacement with ice-cold lysis buffer containing 0.5% Triton
X-100, 5 µg/ml leupeptin, 0.2 U/ml aprotinin, 50 mM
ß-glycerophosphate, pH 7.2, 0.1 mM sodium orthovanadate, 2 mM
MgCl2, 1 mM dithiothreitol, and 1 mM
ethyleneglycol-bis(ß-aminoethyl ether)-N,N'-tetra-acetic
acid (EGTA). The cells were placed on ice, scraped into microcentrifuge tubes,
and centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was
adsorbed onto 0.5 ml of diethylaminoethyl/Sephacel bead columns that had been
prewashed with buffer containing 50 mM ß-glycerophosphate, pH 7.2, 0.1 mM
sodium orthovanadate, 1 mM dithiothreitol, and 1 mM EGTA. The columns were
washed with 3 ml of buffer. ERK activity was eluted with 0.5 ml of buffer
containing 500 mM NaCl. Twenty-microliter aliquots from each elution were
incubated at 30°C for 15 min with 20 µl of a reaction mixture that
contained 50 mM ß-glycerophosphate, pH 7.2, 0.1 mM sodium orthovanadate,
0.2 mM ATP, 20 mM MgCl2, and 1 mM EGTA, as well as 0.5 µl of
[-32P]ATP, 1 mg/ml myelin basic protein, and 50 µg/ml
IP-20 (protein kinase A inhibitor). The reaction was terminated by spotting 30
µl from each assay onto P81 Whatman filter paper (Clifton, NJ). The filters
were washed in 150 mM phosphoric acid three times and once in acetone, dried,
and placed in scintillation vials. A total of 4 ml of scintillation fluid was
added, and the radioactivity was counted by scintillation spectroscopy.
Nonspecific binding was determined by assaying the reaction mixture with
elution buffer and that value was subtracted from each experimental result.
Each assay was performed in triplicate and the results were averaged. The
results are expressed as fold increase in counts per minute compared with
control nonstimulated cells.
Peptide Antibody Production
A peptide identical to the carboxy-terminal 12 amino acid sequence of
NaPi-4 (27) (CGVLSQHNATRL) was
generated and conjugated to keyhole limpet hemocyanin (Genosys
Biotechnologies, Woodlands, TX). Peptide (100 µg) was mixed in Freund's
complete adjuvant and injected subcutaneously into New Zealand White rabbits.
The rabbits were given booster injections in incomplete Freund's adjuvant on a
monthly basis. After immunization, 50 ml of blood was drawn and the serum was
separated and frozen three times monthly.
Immunoblot Analysis
Cells grown to confluence in 6-well trays were washed with calcium- and
magnesium-free phosphate-buffered saline and with Hepes-KOH (5 mM Hepes, pH
7.4). A total of 1.5 ml of Hepes-KOH containing 4 mM EDTA and 1 mM
phenylmethylsulfonyl fluoride was added to each well, and the cells were
scraped into microcentrifuge tubes and homogenized five times with a 22-gauge
needle. The cell lysates were centrifuged at 2000 rpm for 10 min, the pellet
was discarded, and the supernatant was centrifuged at 16,000 rpm for 60 min at
4°C. The pellet (crude membrane fraction) was resuspended in 60 µl of
50 mM mannitol and 10 mM Hepes-Tris, pH 7.2. Fifty micrograms of the suspended
membranes was solubilized in Laemmli sodium dodecyl sulfate (SDS) sample
buffer, boiled, subjected to 10% SDS-polyacrylamide gel electrophoresis, and
transferred electrophoretically to PVDF. Nonspecific binding was blocked by
incubating the PVDF for 1 h in 5% milk in Tris-buffered saline with 0.05%
Tween 20 (TTBS). The PVDF was then incubated overnight at 4°C in a 1:2000
dilution of polyclonal antisera against NaPi-4 in 5% milk in TTBS. The
membrane was then washed and incubated for 1 h at room temperature in a
1:10,000 dilution of goat anti-rabbit IgG in 5% milk in TTBS. Bound antibody
was detected by chemiluminescence (Renaissance, DuPont-New England Nuclear,
Boston, MA). The films were scanned using a Personal Densitometer SI
(Molecular Dynamics, Sunnyvale, CA), and the areas of interest were identified
and quantified by integrated software (ImageQUANT; Molecular Dynamics). The
results of NaPi-4 expression were expressed as arbitrary densitometric
units.
Confocal Imaging
OK cell monolayers were trypsinized, seeded lightly onto 8-well chamber
slides, and incubated overnight in serum-free medium containing agonist. On
the morning of the assay, the cells were washed in calcium- and magnesium-free
Hanks' balanced salt solution (HBSS) and fixed in 4% paraformaldehyde in
Ca/Mg-free HBSS for 1 h at room temperature. The cells were rinsed, incubated
in rabbit anti-NaPi-4 antisera 1:100 in Ca/Mg-free HBSS for 1 to 2 h at room
temperature, rinsed again, and incubated in FITC-coupled goat anti-rabbit IgG
1:500 in HBSS for 1 h at room temperature in the dark. Fluorescence was
visualized by confocal microscopy (Meridian Laboratories, Okemos, MI). With
online computer software (InSight Plus; Meridian Laboratories), individual
cells were optically sectioned horizontally from the base to the apex.
Statistical Analyses
The model system demonstrated day-to-day variability in phosphate transport
and ERK activation by agonists; however, on a single given day, both
parameters varied little between separate well or trays of cells. Therefore,
the experiments were designed specifically to pair the data by the day on
which the experiments were performed. Data were analyzed by paired t
test or ANOVA as indicated, using SigmaStat software. The 95% confidence
limits were a priori determined to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish that the measured kinase activity was due to ERK, the effect of the specific MEK inhibitor PD098059 on PTH-stimulated kinase activity was examined (28). An optimally inhibitory concentration of PD098059 was determined in OK cells by pretreatment with PD098059 at 10-7 to 10-4 M for 30 min followed by stimulation with phorbol myristate acetate (PMA) for 30 min. PMA-stimulated ERK activity was decreased from an 8.1-fold increase to a 2.7-fold increase by 10-4 M PD098059 (data not shown). Lower concentrations of PD098059 had little effect on PMA-stimulated ERK. Pretreatment with 10-4 M PD098059 for 30 min before addition of PTH completely blocked PTH-stimulated ERK activity at both 10 and 20 min (Figure 2). Therefore, we conclude that the phosphorylation of myelin basic protein detected in this assay is mediated by ERK activity.
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Signal Transduction Pathways Leading to the Early and Late ERK
Stimulation by PTH
The effect of pretreatment with pertussis toxin on PTH-stimulated ERK
activity was determined by preincubating OK cells in medium containing 50
ng/ml pertussis toxin overnight, a concentration that we have previously
demonstrated to inhibit pertussis toxin-sensitive responses
(29). At 10 min incubation,
10-7 M PTH stimulated a 2.4 ± 0.3-fold increase in ERK
activity in untreated cells and a 2.0 ± 0.5-fold increase in OK cells
pretreated with pertussis toxin (n = 3, P = 0.7, NS). At 20
min incubation, 10-7 M PTH stimulated a 3.1 ± 0.5-fold
increase in ERK activity in untreated cells and a 3.0 ± 0.8-fold
increase in pretreated cells (n = 4, P = 0.9, NS). Thus, a
pertussis toxin-sensitive G protein does not participate in PTH-mediated ERK
activation.
PTH stimulates both protein kinase A and protein kinase C. In various cell types, protein kinase A activity exerts either a stimulatory or inhibitory effect on ERK (30, 31, 32, 33, 34). Protein kinase A activation decreases angiotensin II-stimulated ERK activity in OK cells (26), and PTH inhibits epidermal growth factor-stimulated ERK activity in bone cells (35). Therefore, the role of protein kinase A in PTH-stimulated ERK activity in OK cells was examined. As shown in Figure 3A, direct stimulation of protein kinase A with either 10-4 M forskolin or 10-4 M 8-bromo-cAMP had no effect on PTH-stimulated ERK activity at either 10 or 20 min. Figure 3B shows the effect of inhibition of protein kinase A on both early and late stimulation of ERK activity by PTH. Pretreatment for 30 min with 10-4 M dideoxyadenosine, a P site antagonist, or for 60 min with 10-6 M H89, a direct protein kinase A antagonist, had no effect on PTH-stimulated ERK activity at either time point. In experiments not shown, we demonstrated that pretreatment with dideoxyadenosine and H89 under the described conditions inhibited 8-bromo-cAMP-stimulated protein kinase A activation. These data suggest that PTH-stimulated ERK activity is independent of protein kinase A activation.
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Lefkowitz and colleagues have suggested that Gq protein-coupled receptor proteins stimulate ERK in a pertussis toxin-independent, protein kinase C-dependent manner (21). Direct activation of protein kinase C in OK cells by 10-6 M phorbol myristate acetate stimulated ERK activity with a peak 6.1 ± 0.6-fold increase in activity after 30 min (n = 8). Figure 4 shows the effect of inhibition of protein kinase C on PTH-stimulated ERK activity. PTH stimulated a 1.6 ± 0.5-fold increase in ERK activity at 10 min and a 2.7 ± 0.2-fold increase in ERK activity at 20 min. Pretreatment for 60 min with 10-6 M calphostin C, an inhibitor of protein kinase C, had no effect on PTH-stimulated ERK activity at 10 min. However, calphostin C significantly decreased the increase in PTH-stimulated ERK at 20 min. These data suggest that the later peak of ERK activity stimulated by PTH is mediated by protein kinase C, while the early peak is PKC-independent.
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Both nonreceptor tyrosine kinases (23, 24) and phosphoinositide 3-kinase (PI-3K) (36, 37, 38, 39) can participate in ERK activation. To determine whether the stimulation of ERK activity by PTH was mediated through nonreceptor tyrosine kinases, OK cells were pretreated for 30 min with 10-4 M genistein or 10-3 M herbimycin A. As shown in Figure 5, pretreatment with genistein or herbimycin A significantly reduced PTH stimulation of ERK activity at 10 min from 4.2 ± 0.2-fold to 1.5 ± 0.2-fold and 2.1 ± 0.5-fold, respectively. PTH-stimulated ERK activity at 20 min was also significantly reduced. To determine whether the effects of genistein on PTH-stimulated ERK were due to inhibition of tyrosine kinase activity or to an unrelated effect, PTH-stimulated ERK was measured in cells treated with daidzein, an analogue of genistein that does not exhibit tyrosine kinase inhibitory activity (40). Daidzein also significantly inhibited the early and late peaks of PTH-stimulated ERK activation. At all concentrations ranging from 10-7 to 10-4 M, daidzein completely abolished PTH-stimulated ERK activation (data not shown). Despite the effect of daidzein, the congruent effects of two different tyrosine kinase inhibitors support the conclusion that a nonreceptor tyrosine kinase participates in the early and late peaks of PTH-stimulated ERK activity.
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To determine whether PTH stimulation of ERK activity was dependent on PI-3K activation, OK cells were pretreated for 30 min with 100 nM wortmannin or 5 x 10-5 M LY294002. Figure 6 shows that PTH stimulated a 3.3 ± 0.4-fold increase in ERK activity at 10 min that was decreased significantly to 1.1 ± 0.1-fold after pretreatment with wortmannin and to 1.5 ± 0.2-fold increase after pretreatment with LY294002. Pretreatment with wortmannin and LY294002 also significantly inhibited PTH-stimulated ERK activity at 20 min. These data suggest that PI-3K also participates in the early and late phases of PTH stimulation of ERK activity.
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Effect of ERK on Phosphate Transport and NaPi-4 Expression
One of the major actions of PTH in proximal tubule cells is to inhibit
sodium-dependent phosphate uptake. To determine whether PTH-stimulated ERK
activity participates in the regulation of phosphate transport, the effect of
pretreatment with 10-4 M PD098059 on PTH inhibition of phosphate
uptake in OK cells was measured. Treatment of OK cells with 10-7 M
PTH for 2 h inhibited sodium-dependent phosphate uptake by 35.6 ± 1.4%.
After pretreatment with PD098059, PTH inhibition of phosphate uptake was
reduced to 24.5 ± 2.6% (Figure
7). To exclude the possibility that this attenuating effect of
PD098059 on phosphate uptake was due to an increase in sodium-independent
phosphate uptake, phosphate uptake in sodium-containing or sodium-free medium
was measured in cells treated with vehicle or PD098059. In sodium-containing
medium, phosphate uptake was 3.45 and 3.24 nmol/mg per min in vehicle-treated
and PD098059-treated cells, respectively. In sodium-free medium, phosphate
uptake was 0.08 nmol/mg per min in both groups of cells, representing
approximately 1% of the total phosphate uptake (n = 2). These
findings confirm that PD098059 directly affects PTH inhibition of phosphate
uptake.
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One mechanism by which PTH regulates sodium-phosphate uptake in OK cells is by decreasing the expression of the cotransporter NaPi-4 (2). To determine whether ERK activation regulates the expression of NaPi-4, immunoblot analysis of membrane proteins using polyclonal antisera directed against the C-terminal peptide of NaPi-4 was performed (41,42). Figure 8 shows an immunoblot of OK cell membranes from control cells, cells treated for 2 h with 10-7 M PTH, cells treated for 2 h with PTH preceded by treatment with 10-4 M PD098059 for 30 min, and cells treated for 2.5 h with PD098059 alone. Densitometric analysis of the immunoblot data demonstrates that PTH decreases NaPi-4 by 47.7 ± 3.5%. Pretreatment with PD098059 did not affect PTH inhibition of the expression of NaPi-4. Membranes from PD098059-treated cells showed slightly reduced expression of NaPi-4 (20% decrease in NaPi-4 density). These data suggest that PTH-stimulated ERK activity contributes to inhibition of phosphate uptake by decreasing the function but not the membrane expression of the sodium-phosphate cotransporter in OK cells. To confirm that inhibition of ERK activation did not affect PTH regulation of NaPi-4 expression, the effect of PD098059 on surface expression of OK cells was examined by confocal microscopy. Pretreatment with PD098059 had no effect on immunofluorescent identification of NaPi-4 in control or PTH-treated cells when compared to cells that were not pretreated with PD098059. Figure 9 shows apical membrane and mid-cell sections of confluent OK cells stained for NaPi-4. As can be seen, 2 h treatment with PTH decreases apical expression of NaPi-4, with more fluorescence appearing in the mid-cell section (bottom left two panels) compared to control cells (top left two panels). Pretreatment with PD098059 had no effect on basal apical NaPi-4 expression (top right two panels) or on downregulation of apical NaPi-4 expression by PTH (bottom right two panels).
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To determine which phase of PTH-stimulated ERK activity contributed to regulation of phosphate transport, PTH inhibition of phosphate uptake was measured in cells pretreated with either 10-4 M genistein, 10-7 M wortmannin, or 10-6 M calphostin C. All three inhibitors alone significantly inhibited phosphate uptake, preventing interpretation of their effects on PTH inhibition of phosphate uptake.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mechanisms of protein kinase C-dependent activation of ERK activity have
been defined by transient transfection of receptors into COS and Chinese
hamster ovary cells. Receptors coupled to Go or Gq proteins activate
phospholipase C through their subunits. Phospholipase C generates the
second messenger diacylglycerol, which activates protein kinase C
(20,
21,
22). Protein kinase C,
bypassing ras, directly activates raf, leading to MEK then ERK activation. In
OK cells, angiotensin II activates raf kinase and ERK through a protein kinase
C-dependent mechanism, establishing the presence of this pathway for ERK
activation by a G protein-coupled receptor in these cells
(26). PTH receptor activation
of the late peak of ERK activity is also protein kinase C-dependent,
suggesting that PTH might use this pathway. However, unlike
angiotensin-stimulated ERK activation, PTH-stimulated ERK is not pertussis
toxin-sensitive and is tyrosine kinase- and PI-3K-dependent. The failure of
pertussis toxin to inhibit PTH-stimulated ERK is not surprising, since PTH
receptors couple to Gs (3) and
Gq (4), but not Gi proteins.
These findings suggest that PTH may activate ERK through a different protein
kinase C-dependent pathway than that activated by angiotensin II.
Depending on the cell type, protein kinase A activity has been shown to either inhibit or stimulate ERK activation (30, 31, 32, 33, 34). In OK cells, protein kinase A activation inhibits ERK activation by angiotensin II (26) and in osteosarcoma cells, PTH-stimulated protein kinase A inhibits epidermal growth factor-stimulated ERK activation (35), demonstrating the presence of a protein kinase A-dependent negative regulatory pathway for ERK activation. Our data show that protein kinase A activity neither stimulates nor inhibits PTH-stimulated ERK activation, indicating that PTH-stimulated ERK activity in OK cells occurs through a pathway independent of protein kinase A activation.
Receptors coupled to Gi proteins stimulate ERK activity through a
ß subunit-dependent activation of nonreceptor tyrosine kinases and
PI-3K, a pathway that also includes Shc, Grb2, Ras, and Raf kinases as
components (18). Although the
precise sequence of activation steps has not been definitively determined,
recent studies suggest that ß subunits directly activate PI-3K,
initiating the pathway (37).
ERK activation by PTH was Gi protein-independent, but the ability of
inhibitors of nonreceptor tyrosine kinases and PI-3K to block PTH-stimulated
ERK suggests that this pathway may be activated by PTH receptors. The ability
of daidzein, an analogue of genistein that does not inhibit tyrosine kinases,
to inhibit PTH-stimulated ERK suggests that PTH-stimulated ERK is not tyrosine
kinase-dependent. The tyrosine kinase dependence of PTH-stimulated ERK,
however, is supported by the ability of another unrelated tyrosine kinase
inhibitor to block PTH-stimulated ERK. Daidzein has been reported to have
independent effects on cell function in other models
(40).
Quamme et al. (7) demonstrated PTH-stimulated ERK activity in OK cells and presented indirect evidence suggesting that PTH stimulation of ERK may be linked to PTH inhibition of phosphate uptake. The present study provides further evidence that PTH-stimulated ERK activity participates in PTH inhibition of sodium-dependent phosphate uptake. Pretreatment of OK cells with the MEK inhibitor PD098059 completely blocked PTH-stimulated ERK activity and partially attenuated the ability of PTH to inhibit phosphate uptake. Because the kinase inhibitors genistein, wortmannin, and calphostin C all significantly inhibited basal phosphate uptake, we could not determine whether the early phase, the late phase, or both phases of PTH-stimulated ERK activity were important for regulation of phosphate uptake. The mechanism by which ERK participates in PTH regulation of phosphate remains to be defined. Immunoblot analysis of the expression of NaPi-4 in OK cell membranes suggests that ERK activation does not play a role in the PTH-mediated downregulation of NaPi-4 in these cells. One possible mechanism for ERK regulation of NaPi-4 activity is that ERK phosphorylate NaPi-4 directly or phosphorylate regulatory proteins, which inhibit transport function. Alternatively, PTH-stimulated ERK activation could decrease the activity of Na-K-ATPase, decreasing phosphate transport by reducing the driving force for sodium entry into the cells. This latter hypothesis is particularly attractive because our data show that PTH-stimulated ERK is protein kinase C-dependent and PTH-stimulated protein kinase C has been shown to inhibit proximal tubule cell Na-K-ATPase (2). Additionally, we have recently demonstrated that activation of protein kinase C in OK cells inhibits sodium-phosphate cotransport but does not cause downregulation of NaPi-4 expression (46). Thus, ERK may be the intracellular signal activated by protein kinase C to inhibit Na-K-ATPase resulting in inhibition of sodium-phosphate co-transport.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
rapidly activates the mitogen-activated protein
kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent
fashion in mouse macrophages. Proc Natl Acad Sci USA 92
: 1614-1618, 1995 subunits. Nature
369: 418
-420, 1994[Medline]
activation of p21ras. Proc Natl Acad Sci
USA 91: 12706-12710, 1994 subunit
actiates Ras, Raf, and MAP kinase in HEK 293 cells. FEBS Lett 368
: 183-187, 1995[Medline]
subunit-mediated activation of mitogen-activated protein
kinases. J Biol Chem 271: 19443
-19450, 1996-subunits activate mitogen-activated protein kinase via
a novel protein kinase C-dependent mechanism. J Biol Chem 271
: 1266-1269, 1996
subunits of heterotrimeric G proteins stimulate the mitogen-activated protein
kinase pathway in COS-7 cells. J Biol Chem 269
: 7851-7854, 1994-mediated mitogen-activated protein kinase signaling pathway. J Biol Chem
271: 12133
-12136, 1996This article has been cited by other articles:
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H. S. Tenenhouse and H. Murer Disorders of Renal Tubular Phosphate Transport J. Am. Soc. Nephrol., January 1, 2003; 14(1): 240 - 247. [Full Text] [PDF]
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 T. A. Doggett, J. T. Swarthout, S. C. Jefcoat Jr., D. Wilhelm, A. Dieckmann, P. Angel, and N. C. Partridge Parathyroid Hormone Inhibits c-Jun N-Terminal Kinase Activity in Rat Osteoblastic Cells by a Protein Kinase A-Dependent Pathway Endocrinology, May 1, 2002; 143(5): 1880 - 1888. [Abstract] [Full Text] [PDF]
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S. J. Khundmiri and E. Lederer PTH and DA regulate Na-K ATPase through divergent pathways Am J Physiol Renal Physiol, March 1, 2002; 282(3): F512 - F522. [Abstract] [Full Text] [PDF]
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 P. L. Dudas, A. R. Villalobos, G. Gocek-Sutterlin, G. Laverty, and J. L. Renfro Regulation of transepithelial phosphate transport by PTH in chicken proximal tubule epithelium Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2002; 282(1): R139 - R146. [Abstract] [Full Text] [PDF]
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M. M. Friedlaender, H. Wald, M. Dranitzki-Elhalel, H. K. Zajicek, M. Levi, and M. M. Popovtzer Vitamin D reduces renal NaPi-2 in PTH-infused rats: complexity of vitamin D action on renal Pi handling Am J Physiol Renal Physiol, September 1, 2001; 281(3): F428 - F433. [Abstract] [Full Text] [PDF]
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W. Tian, Z. Zhang, and D. M. Cohen MAPK signaling and the kidney Am J Physiol Renal Physiol, October 1, 2000; 279(4): F593 - F604. [Abstract] [Full Text] [PDF]
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