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Originally published In Press as doi:10.1074/jbc.M405079200 on July 19, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40419-40430, September 24, 2004
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Calcium Restriction Allows cAMP Activation of the B-Raf/ERK Pathway, Switching Cells to a cAMP-dependent Growth-stimulated Phenotype*

Tamio Yamaguchi{ddagger}, Darren P. Wallace{ddagger}, Brenda S. Magenheimer§, Scott J. Hempson{ddagger}, Jared J. Grantham{ddagger}§, and James P. Calvet§

From the §Departments of Biochemistry and Molecular Biology and {ddagger}Internal Medicine, the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas 66160

Received for publication, May 7, 2004 , and in revised form, July 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
cAMP can be either mitogenic or anti-mitogenic, depending on the cell type. We demonstrated previously that cAMP inhibited the proliferation of normal renal epithelial cells and stimulated the proliferation of cells derived from the cysts of polycystic kidney disease (PKD) patients. The protein products of the genes causing PKD, polycystin-1 and polycystin-2, are thought to regulate intracellular calcium levels, suggesting that abnormal polycystin function may affect calcium signaling and thus cause a switch to the cAMP growth-stimulated phenotype. To test this hypothesis, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 collecting duct cells and primary cultures of human kidney epithelial cells with calcium channel blockers and by lowering extracellular calcium with EGTA. Calcium restriction for 3–5 h converted both cell types from a normal cAMP growth-inhibited phenotype to an abnormal cAMP growth-stimulated phenotype, characteristic of PKD. In M-1 cells, we showed that calcium restriction was associated with an elevation in B-Raf protein levels and cAMP-stimulated, Ras-dependent activation of B-Raf and ERK. Moreover, the activity of Akt, a negative regulator of B-Raf, was decreased by calcium restriction. Inhibition of Akt or phosphatidylinositol 3-kinase also allowed cAMP-dependent activation of B-Raf and ERK in normal calcium. These results suggest that calcium restriction causes an inhibition of the phosphatidylinositol 3-kinase/Akt pathway, which relieves the inhibition of B-Raf to allow the cAMP growth-stimulated phenotypic switch. Finally, M-1 cells stably overexpressing an inducible polycystin-1 C-terminal cytosolic tail construct were shown to exhibit a cAMP growth-stimulated phenotype involving B-Raf and ERK activation, which was reversed by the calcium ionophore A23187 [GenBank] . We conclude that disruption of calcium mobilization in cells that are normally growth-inhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium channel blockers are used widely, either alone or in combination with other drugs, for the treatment of hypertension and other forms of cardiovascular disease including stable angina and acute coronary artery disease (1, 2). Their mechanism of action is to restrict the entry of calcium into cells by blocking L-type calcium channels, a class of voltage-gated calcium channels found on the plasma membrane of both excitable and nonexcitable cells (3). Calcium influx into cells modulates the activities of calcium-binding proteins (4–6) that can regulate signal transduction pathways leading to changes in gene expression and the control of cell growth and differentiation (7–11). Short term responses to changes in calcium, as seen in muscle contraction, synaptic transmission, or neuroendocrine secretion, are usually accompanied by calcium-induced posttranslational events including protein phosphorylation/dephosphorylation mediated by protein kinases, protein phosphatases, or calcium-sensitive adenylate cyclases (12–18). By contrast, long term calcium responses can involve changes in gene expression that result from the modulation of transcription factor activity leading to changes in cell proliferation or to changes in the phenotypic state of a cell (14, 15, 18–23).

Polycystic kidney disease (PKD)1 is characterized by the abnormal growth of benign cystic tumors from renal tubular epithelial cells (24–26). PKD is caused by loss of function mutations in either of two genes, PKD1 or PKD2, thus resulting in insufficient levels of their protein products, polycystin-1 or polycystin-2. It is thought that each cyst is the result of the abnormal growth of a single cell, which proliferates out of control, expanding the tubule wall until the cyst pinches off and continues to grow to a large size by continued cell proliferation and by the secretion of fluid into the cyst lumen. The progressive enlargement of numerous cysts in affected kidneys leads to renal failure in about half of PKD patients.

cAMP may have a central role in cyst growth by stimulating both fluid secretion and cell proliferation (24, 27–29). It has been demonstrated that normal renal epithelial cells are growth-inhibited by cAMP, whereas cyst epithelial cells are growth-stimulated. These studies have shown that cultured normal renal tubular epithelial cells have decreased rates of cell proliferation in response to treatment with cAMP, which inhibits the Ras/Raf-1/MEK/ERK pathway at the level of Raf-1 (30). In contrast, cultured cyst epithelial cells from PKD kidneys show increased rates of cell proliferation in response to cAMP, which activates B-Raf instead of inhibiting Raf-1. B-Raf then activates the MEK/ERK pathway and cell proliferation. This so-called "PKD phenotype" can be mimicked in mouse M-1 cortical collecting duct cells by stable transfection and inducible overexpression of a short, polycystin-1 cytosolic C-terminal tail construct (31), suggesting that this cAMP-responsive PKD phenotype involves disruption of polycystin function in these cells.

Polycystin-1 and polycystin-2 are thought to be components of a multiprotein signaling complex that is involved in regulating intracellular calcium levels in response to yet-to-be-determined extracellular signals (32). Polycystin-2 is a calciumpermeable, nonselective cation channel, which has been shown to function both in calcium entry and in calcium release (33–36); it is possible that polycystin-1 is a regulator of polycystin-2 activity (36–38). Indeed, polycystin-1 has been shown to interact directly with polycystin-2 (39) and to couple with heterotrimeric G proteins (40–42), making possible several mechanisms for polycystin-1 to regulate either polycystin-2 calcium channel activity or other calcium-mediated signaling pathways. We considered the possibility that disruption of polycystin function, either by loss of a functional PKD gene or by overexpression of a polycystin-1 C-tail construct, might affect calcium mobilization and thus induce the cAMP growth-stimulated phenotype characteristic of PKD cells.

To test this idea, we disrupted intracellular calcium mobilization by treating immortalized mouse M-1 cells and primary cultures of normal human kidney epithelial cells with calcium channel blockers or EGTA to lower intracellular calcium. These conditions were found to convert the cells from a normal cAMP growth-inhibited phenotype to a cAMP growth-stimulated phenotype. Cells treated with calcium-lowering reagents showed cAMP-dependent activation of B-Raf and ERK, suggesting that the cAMP growth stimulation is dependent on B-Raf activation. Because B-Raf can be inhibited by Akt in a PI3K- and calcium-dependent manner, we tested whether pharmacological inhibition of PI3K and Akt had an effect on the cAMP-dependent cell-growth phenotype. We found that these inhibitors allowed cAMP-dependent activation of B-Raf and ERK in the presence of normal levels of calcium, thus supporting a role for PI3K and Akt in suppressing cAMP-dependent B-Raf signaling and preventing the phenotypic switch. Finally, M-1 cells stably expressing the polycystin-1 C-tail fragment were shown to exhibit cAMP-dependent activation of B-Raf and ERK and a cAMP growth-stimulated phenotype that was reversed by tonic calcium repletion. We conclude from these experiments that disruption of calcium mobilization in cells that are normally growth-inhibited by cAMP can derepress the B-Raf/ERK pathway, thus converting these cells to a phenotype that is growth-stimulated by cAMP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—M-1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 (50:50) with 5% fetal bovine serum (FBS) and penicillin/streptomycin as described previously (31). M-1 cells (clone 20) stably transfected with the C-tail of polycystin-1 were maintained in G418-containing medium as described previously (31). Human kidney cortex (HKC) cells were cultured from a freshly obtained nephrectomy specimen (K129) as described previously (29, 30). The kidney retrieval protocol was approved by the Institutional Review Board of the University of Kansas Medical Center.

Cell Proliferation Assays—2 x 103 M-1 cells or 4 x 103 HKC cells were seeded onto individual chambers of a 96-well culture plate. The cells were incubated in DMEM/F-12 with 1% heat-inactivated FBS and penicillin/streptomycin. After 24 h, the FBS was reduced to 0.002%. 24 h later, cells were treated with dexamethasone (DEX), 8-Br-cAMP, calcium channel blockers, EGTA, or the calcium ionophore A23187 [GenBank] (see figure legends) for another 48 (M-1 cells) or 72 h (HKC cells). Cell proliferation was determined by counting cell numbers in a hemocytometer or by the Promega Cell Titer 96 MTT assay method, which measures the optical density of a proliferation-dependent reaction product (29, 31).

Western Blot Assays—Cells were seeded onto 100-mm diameter plastic dishes containing DMEM/F-12 with 1% FBS and penicillin/streptomycin. At 75–80% confluence, FBS was reduced to 0.002%, and the cells were grown for 24 h. The cells were then treated with various combinations of DEX, calcium channel blockers, EGTA, A23187 [GenBank] , Akt inhibitor, Bay-K8644, H89, LY294002, PD98059, and/or PP1. 8-Br-cAMP was added for the final 15 min. Cells were lysed in 500 µl of ice-cold lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM {beta}-glycerol phosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 2 mM Na2HPO4, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM benzamidine, and 0.5 mM dithiothreitol). Insoluble cell lysate was removed by centrifugation. Aliquots of soluble cellular protein were measured by the BCA protein assay kit. Cell lysate (20 µg of protein) was then heated (95–100 °C) in SDS sample buffer, separated by 10% SDS-PAGE, and transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences). After transfer, the membranes were blocked with 5% powdered milk in TBS-T, pH 8.0 (20 mM Tris-HCl, 137 mM NaCl, and 0.05% Tween 20), for 1 h at room temperature. Blocked membranes were incubated with primary antibodies (see figure legends) in 5% powdered milk in TBS-T for 2 h at room temperature or overnight at 4 °C. Membranes were then washed three times with TBS-T and incubated with secondary antibodies with 5% milk in TBS-T for 1 h at room temperature. The membranes were washed three times with TBS-T, and proteins were visualized by using an enhanced chemiluminescence system (ECL; Amersham Biosciences). Intensity was detected and quantitatively analyzed by the Fluor-S MAX multi-imager system (Bio-Rad).

Transient Transfection Assays—M-1 cells were plated in DMEM/F-12 plus 5% FBS at a density of 2 x 105 cells per well in a 6-well plate. After 24 h, the cells were transfected with LipofectAMINE 2000 (Invitrogen) in serum-free DMEM/F-12 containing 0.5 µg per well HA-p44ERK-1 DNA (from J. Kyriakis) plus 0.5 µg per well of one of the following DNAs: Rap1A G12V (constitutively active) 2xMYC; Rap1B G12V 2xMYC; H-Ras G12V 2xMYC; Rap1A S17N (dominant negative) 2xMYC; Rap1B S17N 2xMYC; H-Ras S17N 2xMYC, all from the Guthrie cDNA Resource Center (Sayre, PA). After 5 h, the transfection solution was replaced with DMEM/F-12 plus 0.002% FBS, and the cells were cultured overnight and were either left untreated or were treated with verapamil for an additional 5 h. 8-Br-cAMP was added for the final 15 min as indicated. Cells were lysed as described above, and the lysates were incubated with anti-HA antibody conjugated to agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). The beads were boiled in loading buffer (without dithiothreitol or mercaptoethanol); the supernatants were collected and re-boiled in loading buffer with dithiothreitol and mercaptoethanol, and the samples were electrophoresed and blotted as described above. Blocked membranes were incubated with primary and secondary antibodies, washed, and visualized as described above.

B-Raf and Raf-1 Kinase Assays—M-1 cells (wild-type or clone 20) were cultured as described above with or without DEX, inhibitors, calcium channel blockers, EGTA, and/or 8-Br-cAMP (see figure legends). The in vitro B-Raf kinase assay was modified from that of Erhardt et al. (43). 500 µg of clarified cellular extract was immunoprecipitated for 2 h with gentle rotation at 4 °C with anti-B-Raf antibody covalently coupled to protein A/G Plus-agarose beads (Santa Cruz Biotechnology). Immunoprecipitates were washed and resuspended in 20 µl of 0.5 mM {beta}-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM dithiothreitol, 0.03% Brij 35. For the radioactive assay, reaction mixtures containing 20 µl of 16 µl of 50 mM MgCl2, 1 µl of 1 mM ATP, 10 µCi of [{gamma}-32P]ATP (6,000 Ci/mM; PerkinElmer Life Sciences), and 0.5 µg of human full-length MEK-1 fusion protein (Santa Cruz Biotechnology) were mixed with 20 µl of the resuspended beads, incubated at 30 °C for 30 min, and stopped with SDS sample buffer. Samples were heated (95–100 °C); beads were sedimented; supernatants were separated by 10% SDS-PAGE, and the gels were dried and placed under x-ray film. For the nonradioactive assay, reaction mixtures containing 20 µl of 16 µl of 50 mM MgCl2, 2 µl of 1 mM ATP, and 2 µg of human MEK-1 fusion protein were mixed with 20 µl of the resuspended beads, incubated at 30 °C for 30 min, and stopped with SDS sample buffer. After electrophoresis and transfer, the membranes were blocked with 5% powdered milk in TBS-T, pH 8.0, for 1 h at room temperature. Blocked membranes were incubated with phospho-MEK antibody in 5% powdered milk in TBS-T overnight at 4 °C. Membranes were then washed three times with TBS-T and incubated with secondary antibody with 5% milk in TBS-T for 1 h at room temperature. The membranes were washed three times with TBS-T, and proteins were visualized using chemiluminescence. Phospho-MEK was detected and quantitated with the Fluor-S MAX multi-imager system. Raf-1 kinase activity was determined using the nonradioactive assay and anti-Raf-1 antibody covalently coupled to protein A/G Plus-agarose beads (Santa Cruz Biotechnology).

Measurement of Intracellular Calcium—Changes of intracellular calcium and the magnitude of calcium release from thapsigargin-sensitive calcium stores were determined using Fura-2. M-1 cells were grown on glass coverslips (25 mm diameter) as subconfluent monolayers in DMEM/F-12 supplemented with 1% FBS. Monolayers receiving the same growth conditions were incubated in either control medium or medium containing 1 µM verapamil for 24 h prior to the experiment. Cells were loaded with 10 µM Fura-2/AM for 60 min at room temperature, and then rinsed in control medium for 45 min at 37 °C. Coverslips were mounted in a thermal-controlled chamber on the stage of a Nikon inverted microscope equipped with a monochromator for selecting excitation wavelengths of 340 and 380 nm. The chamber was continuously perfused with DMEM/F-12 equilibrated with 5% CO2, 95% air at 37 °C. Emitted light was measured at 510 nm with a photomultiplier detection system (Photo Technology International, South Brunswick, NJ). Felix 32 analysis software (Photo Technology International) controlled the monochromator and data acquisition to generate the 340:380 excitation ratio. For each monolayer, a steady-state level of intracellular calcium was established, and 1 µM thapsigargin was then added to deplete calcium stores.

Antibodies—Anti-ERK1 (C-16), -ERK2 (C-14), -phospho-ERK (E-4), -B-Raf (C-19), -Raf-1 (C-12), and -Akt (C-20) antibodies were purchased from Santa Cruz Biotechnology. Anti-phospho-MEK-1 and -2 (Ser-222) and anti-phospho-Akt (Ser-473) were from BIOSOURCE (Camarillo, CA). Anti-rabbit, -mouse, -rat, or -goat IgG-conjugated horseradish peroxidase secondary antibodies were from Santa Cruz Biotechnology.

Reagents—8-Br-cAMP, gadolinium, nifedipine, verapamil, A23187 [GenBank] , EGTA, and Bay-K8644 were obtained from Sigma. PP1, LY294002, H89, and Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (44–46) were obtained from Calbiochem. PD98059 was obtained from New England Biolabs (Beverly, MA).

Statistics—Mean and S.E. were calculated, and levels of significant difference were determined by unpaired t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We demonstrated previously that HKC cells are growth-inhibited by cAMP agonists, whereas cyst wall epithelial cells from PKD kidneys are growth-stimulated by cAMP (29). This cAMP-proliferative phenotype can also be demonstrated in mouse M-1 cortical collecting duct cells by stable transfection and overexpression of a dexamethasone (DEX)-inducible polycystin-1 C-tail construct, which appears to act in a dominant negative fashion to inhibit endogenous polycystin function (31). Because the polycystins are thought to regulate calcium mobilization, we reasoned that this phenotypic switch may be caused by a disruption of calcium signaling in these stably transfected cells. If so, the PKD-like cAMP growth-stimulated phenotype may be mimicked in parental (wild-type) M-1 cells by treatment with calcium channel blockers. As seen in Fig. 1A, the rate of cell proliferation of control parental M-1 cells was decreased following treatment with cAMP. In contrast, the rate of proliferation of these cells was increased with cAMP in the presence of the nonspecific calcium channel blocker gadolinium and the L-type calcium channel blockers nifedipine and verapamil, and the effect of the calcium channel blockers was diminished by the calcium ionophore A23187 [GenBank] . Normal HKC cells were also growth-stimulated by cAMP in the presence of the calcium channel blockers (Fig. 1B). This effect was also seen in M-1 cells following incubation in media containing calculated free calcium concentrations ranging from 0.46 to 0.26 mM with the addition of EGTA (Fig. 1C). To show that this effect was because of the decreased free calcium rather than the EGTA per se, the EGTA concentration was increased to >1 mM (Fig. 1C, rightmost bar) in the presence of an excess of calcium. As shown, a high level of extracellular calcium, even in the presence of EGTA, was sufficient to confer a normal, growth-inhibited response to cAMP. To demonstrate that calcium channel blockers are able to decrease intracellular calcium levels, M-1 cells were incubated for 24 h in 1 µM verapamil. The cells were loaded with Fura-2/AM, and steady-state levels of intracellular calcium were measured. As shown in Fig. 1D, steady-state intracellular calcium levels and the capacity of thapsigargin-sensitive calcium stores were decreased by calcium restriction. Thus, it appears that a normal, cAMP growth-inhibited phenotype requires normal levels of calcium and that a cAMP growth-stimulated PKD phenotype can be caused by decreased intracellular calcium.



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  		FIG. 1.
Proliferative responses of cells treated with calcium channel blockers and EGTA. Cells were cultured in medium with 1% FBS for 24 h and then with 0.002% FBS for 24 h before treatment. A, wild-type M-1 cells were treated with the calcium channel blockers gadolinium (Gad) (25 µM), nifedipine (Nif) (0.1 µM), or verapamil (Ver) (1 µM), and 8-Br-cAMP (cAMP) (100 µM) and/or A23187 [GenBank] (1 nM) as indicated, and the cells were cultured for another 48 h in this medium (containing 0.002% FBS). Cell proliferation rates were determined by the Promega Cell Titer 96 MTT assay and compared with control cells (Con) that were not treated with cAMP. B, HKC cells were treated with the calcium channel blockers nifedipine (0.1 µM) or verapamil (1 µM) and 8-Br-cAMP (100 µM) as indicated, and the cells were cultured for another 72 h in this medium (containing 0.002% FBS). Cell numbers were determined by counting cells in a hemocytometer and were compared with control cells that were not treated with cAMP. C, wild-type M-1 cells were placed in medium containing 0.26–3.18 mM calcium (by varying EGTA and total calcium concentrations) and 8-Br-cAMP (100 µM) as indicated, and the cells were cultured for another 48 h in this medium (containing 0.002% FBS). Cell numbers were determined by cell counting in a hemocytometer and were compared with control cells that were not treated with EGTA and cAMP. D, intracellular calcium levels and calcium release from thapsigargin-sensitive calcium stores. M-1 cells were grown on glass coverslips as subconfluent monolayers in DMEM/F-12 supplemented with 1% FBS. Cells were incubated in either control medium or medium containing 1 µM verapamil for 24 h. Cells were loaded with 10 µM Fura-2/AM for 60 min at room temperature and rinsed, and the coverslips were mounted in a thermal controlled chamber on the stage of a Nikon inverted microscope equipped with a monochromator for selecting excitation wavelengths of 340 and 380 nm. The chamber was continuously perfused with DMEM/F-12 equilibrated with 5% CO2, 95% air at 37 °C. Emitted light was measured at 510 nm and data acquisition generated a 340: 380 excitation ratio. For each monolayer, a steady-state level of intracellular calcium was established, and 1 µM thapsigargin was then added to deplete calcium stores. The figure shows a representative experiment. For the cell growth experiments, one asterisk indicates a significant difference at p < 0.05; two asterisks indicate p < 0.01.

 
In normal renal epithelial cells, as in many cell types, cAMP treatment decreases ERK activation (47). By contrast, in PKD cells, cAMP treatment activates ERK (29). As shown in Fig. 2A, cAMP treatment of M-1 cells decreased the level of phosphorylated (activated) ERK when the cells were cultured in extracellular free calcium concentrations ranging from 1.06 to 0.56 mM. When the cells were incubated in lower extracellular free calcium concentrations (ranging from 0.46 mM down to 0.26 mM; Fig. 2A, boxed), cAMP increased the level of phospho-ERK. These were the same free calcium concentrations in which cAMP stimulated an increase in cell proliferation (Fig. 1C). ERK was also activated by cAMP in the presence of calcium channel blockers, in both M-1 cells (Fig. 2B, boxed) and HKC cells (Fig. 2C, boxed), and this activation was prevented by pretreatment with the L-type calcium channel activator Bay-K8644 or by the calcium ionophore A23187 [GenBank] (Fig. 2B). Thus, both the cAMP-dependent activation of ERK and the resulting growth-stimulated phenotype can be caused by conditions that restrict intracellular calcium.



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  		FIG. 2.
Activation of ERK in response to calcium channel blockers and EGTA. Cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. A, wild-type M-1 cells were placed in calcium/EGTA-containing medium (0.0–1.06 mM EGTA) producing calculated free concentrations of 0.26–3.18 mM calcium (3.18 mM calcium was achieved by supplementing with additional CaCl2). The cells were cultured in this medium (containing 0.002% FBS) for 7 h; the medium was changed to wash out autocrine factors, and the cells were cultured for an additional 1 h prior to a 15-min treatment with 8-Br-cAMP (cAMP) (100 µM) as indicated. B, wild-type M-1 cells were placed in medium containing the L-type calcium channel activator Bay-K8644 (1 µM) or the calcium ionophore A23187 [GenBank] (1 nM) for 30 min prior to addition of the calcium channel blockers nifedipine (0.01 µM), verapamil (1 µM), or EGTA (0.7 mM, calculated free calcium 0.36 mM; control medium contains 1.06 mM). The cells were cultured in this medium (containing 0.002% FBS) for 7 h; the medium was changed to wash out autocrine factors, and the cells were cultured for an additional 1 h prior to a 15-min treatment with 8-Br-cAMP (cAMP) (100 µM) as indicated. C, HKC (K129) cells were placed in medium containing the calcium channel blockers nifedipine (0.01 µM), verapamil (1 µM), or gadolinium (25 µM). The cells were cultured in this medium (containing 0.002% FBS) for 24 h; the medium was changed to wash out autocrine factors, and the cells were cultured for an additional 1 h prior to a 15-min treatment with 8-Br-cAMP (100 µM) as indicated. Cell lysates were prepared for Western blotting with anti-phospho-ERK (P-ERK) and anti-ERK (Total ERK) antibodies. The upper and lower bands in each blot are p44 and p42 ERK, respectively. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to controls (1.00) that received no treatment.

 
To determine whether the phenotypic switch is caused by short term or long term decreases in calcium levels, a time course study was carried out. Fig. 3A shows the level of activated ERK in control M-1 cells without treatment with cAMP, and in cAMP-treated cells in low, medium, or high concentrations of verapamil for 1, 3, 5, or 8 h. Phosphorylated ERK was elevated by cAMP in cells treated for 5 h with medium and high verapamil concentrations. The highest level of ERK phosphorylation occurred at 8 h. No further increase occurred when the cells were incubated for 24 h in verapamil (data not shown). Thus, ERK activation was dose- and time-dependent, requiring up to 8 h of treatment for a maximal effect. Fig. 3B shows that ERK activation by EGTA treatment required a minimum of 3 h. Fig. 3C shows that the continuous presence of the calcium channel blocker is required for ERK activation, as a 1-h verapamil treatment followed by 7 h in its absence (1 h verapamil, 8 h total treatment; see Fig. 3C, boxed) was insufficient for ERK activation. This cAMP growth-stimulated phenotype can persist for up to 8–12 h following withdrawal of verapamil (Fig. 3D). These results suggest that a switch to the cAMP growth-stimulated phenotype requires calcium decreases of several hours, perhaps involving changes in gene transcription and new protein synthesis, and that these changes persist for several hours after restoring calcium before eventually diminishing.



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  		FIG. 3.
Time course of ERK activation in response to calcium channel blockers and EGTA. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. A, M-1 cells were placed in medium containing the calcium channel blocker verapamil at low (L), medium (M), and high (H) concentrations (0.1, 1, and 10 µM, respectively) for 1–8 h, as indicated, prior to a 15-min treatment with 8-Br-cAMP (cAMP) (100 µM). Control (Con) cells were cultured for 8 h without treatment. B, M-1 cells were placed in EGTA-containing medium (0.7 mM, calculated free calcium 0.36 mM; control medium contains 1.06 mM). The cells were cultured for 0.5–8 h, as indicated, prior to a 15-min treatment with 8-Br-cAMP (100 µM). Control cells were cultured for 8 h either with no treatment or with treatment with cAMP alone. C, M-1 cells were placed in medium containing the calcium channel blocker verapamil (1 µM) either for 1 h followed by 7-h washout (total 8 h treatment), for 8 h, or for only 1 h prior to a 15-min treatment with 8-Br-cAMP (100 µM). Control cells were cultured for 8 h without treatment. Two separate experiments are shown. D, M-1 cells were placed in medium containing the calcium channel blocker verapamil (1 µM) for 8 h and then transferred to verapamil-free medium (Washout) for 0–12 h, as indicated, prior to a 15-min treatment with 8-Br-cAMP (100 µM). Control cells were cultured for 8 h without treatment. Two separate experiments are shown. Cell lysates were prepared for Western blotting with anti-phospho-ERK (P-ERK) and anti-ERK (Total ERK) antibodies. The upper and lower bands are p44 and p42 ERK, respectively. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to controls that received no treatment.

 
Activation of ERK by cAMP can be PKA-dependent (47) or PKA-independent (48). To determine which is the case in M-1 cells undergoing the cAMP-dependent phenotypic switch, cells were treated with calcium channel blockers in the presence of the PKA inhibitor, H89 (Fig. 4A, boxed). As shown, ERK activation was dependent on PKA. PKA has been shown to phosphorylate Src on Ser-17, leading to activation of Rap1 and B-Raf and ultimately ERK (47). As shown in Fig. 4B (boxed), treatment with the Src inhibitor PP1 led to an almost complete inactivation of ERK, suggesting that Src is required for cAMP-dependent ERK activation in M-1 cells.



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  		FIG. 4.
PKA and Src dependence of ERK activation in response to calcium channel blockers. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. A, M-1 cells were placed in medium containing the calcium channel blocker verapamil (1 µM) for 8 h. H89 (10 µM) was added for the last 45 min, and 8-Br-cAMP (cAMP) (100 µM) was added for the last 15 min. Control cells were cultured for 8 h without treatment. B, M-1 cells were placed in medium containing the calcium channel blockers nifedipine (0.01 µM) or verapamil (1 µM) for 8 h. PP1 (10 µM) or PD98059 (50 µM) were added for the last 45 min, and 8-Br-cAMP (100 µM) was added for the last 15 min. Control cells were cultured for 8 h without treatment. Cell lysates were prepared for Western blotting with antiphospho-ERK (P-ERK) and anti-ERK (Total ERK) antibodies. The upper and lower bands are p44 and p42 ERK, respectively. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to controls.

 
Treatment of cells with cAMP, in addition to activating Rap1 (47), can also activate Ras (48). To determine whether either of these small G proteins is required for ERK activation in M-1 cells undergoing the cell-growth phenotypic switch, cells were transfected with constitutively active (G12V) Ras or Rap1 or with dominant negative (S17N) Ras or Rap1, treated with verapamil, and assayed for the level of phospho-ERK. In the three experiments shown in Fig. 5A (left), constitutively active Ras allowed verapamil-dependent ERK activation either in the absence of cAMP (2.12, 1.19, 2.02; average = 1.78) or in the presence of cAMP (2.23, 1.30, 2.14; average = 1.89). Activation by verapamil alone suggested that cAMP is dispensable in the presence of an activated Ras, which is consistent with cAMP acting upstream of Ras (the slightly higher levels of phospho-ERK in the cAMP treated cells can be attributed to the activation of endogenous Ras). In contrast to these results, dominant negative Ras prevented this verapamil (and cAMP) activation of ERK (Fig. 5A, right). Transfection of the constitutively active and dominant negative Rap1 constructs did not activate or block the cAMP and verapamil activation of ERK (Fig. 5B, left and right). In fact, the results in Fig. 5B look very similar to those for control cells transfected with the HA-p44ERK-1 construct alone (Fig. 5C). These results suggest that cAMP activation of ERK is mediated by Ras rather than Rap1. The verapamil dependence observed for the ERK activation suggests that calcium restriction acts at a step downstream of Ras.



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  		FIG. 5.
Ras dependence of ERK activation in response to verapamil. M-1 cells were transfected with LipofectAMINE in serum-free DMEM/F-12 containing 0.5 µg per well HA-p44ERK-1 DNA plus 0.5 µg per well of one of the following DNAs. A, constitutively active (Con. Act.) H-Ras G12V (left 4 lanes); dominant negative (Dom. Neg.) H-Ras S17N (right 4 lanes) (three independent experiments are shown); B, constitutively active Rap1A G12V (upper blot, left 4 lanes) or Rap1B G12V (lower blot, left 4 lanes); dominant negative Rap1A S17N (upper blot, right 4 lanes) or Rap1B S17N (lower blot, right 4 lanes). C, HA-p44ERK-1 alone (four independent experiments). After 5 h, the transfection solution was replaced with DMEM/F-12 plus 0.002% FBS, and the cells were cultured overnight and were either left untreated or were treated with verapamil (1 µM) for an additional 5 h. 8-Br-cAMP (cAMP) (100 µM) was added for the final 15 min where indicated. Cell lysates were incubated with anti-HA antibody conjugated to agarose beads, and the samples were collected, electrophoresed, and blotted as described under "Experimental Procedures" with the anti-phospho-ERK (HA-P-ERK) antibody. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to untreated (no cAMP or verapamil) controls (1.00) (left lane in each set).

 
Previously, B-Raf was found to be activated in PKD cells by treatment with cAMP, which then led to activation of MEK and ERK (30). To determine whether B-Raf is activated in M-1 cells undergoing the cell-growth phenotypic switch, an in vitro kinase assay was employed to determine the activity of endogenous immunoprecipitated B-Raf, using MEK as a substrate. As shown in Fig. 6A, treatment with cAMP alone had little or no effect on B-Raf activity, whereas treatment with verapamil, nifedipine, or EGTA gave rise to a pronounced increase in B-Raf activity that was further increased by cAMP. By contrast, the activity of immunoprecipitated Raf-1 was unaffected by calcium restriction either in the absence or presence of cAMP (data not shown). These results suggest that cAMP in the presence of normal intracellular calcium levels cannot activate B-Raf but that calcium restriction alone is able to do so.



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  		FIG. 6.
B-Raf activation in response to inhibition of Akt. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. A, cells were placed in medium containing verapamil (1 µM), nifedipine (0.01 µM), or EGTA (0.7 mM, calculated free calcium 0.36 mM; control medium contains 1.06 mM) for 8 h. 8-Br-cAMP (cAMP) (100 µM) was added for the last 15 min. Control cells were cultured for 8 h without treatment. B, cells were placed in medium containing the PI3K inhibitor LY294002 (20 µM) or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (44–46) (5 µM) and cultured for 5 h. 8-Br-cAMP (100 µM) was added for the last 15 min. Control cells were incubated for 5 h but received no treatment. C, cells were placed in medium containing the PI3K inhibitor LY294002 (20 µM) or the Akt inhibitor (5 µM) and cultured for either 45 min (45 m) or 5 h (5 h). 8-Br-cAMP (cAMP) (100 µM) was added for the last 15 min. Control cells received no treatment. Cell lysates were prepared for B-Raf in vitro kinase assay using MEK as a substrate. B-Raf activity was assessed by Western blotting with an anti-phospho-MEK (P-MEK) antibody. Shown in A and B are representative Western blots. Band intensities were quantitated with the Fluor-S MAX multi-imager system. The bars represent the averages of three separate experiments ± S.E. compared with controls.

 
Akt, which is known to inhibit B-Raf (49–51), can be activated in some cells by calcium-dependent signaling (52–57). If this inability of cAMP to activate B-Raf is due to phosphorylation by Akt on an inhibitory site on B-Raf, pharmacological inhibition of Akt or its upstream activator PI3K might mimic calcium restriction. Fig. 6B shows that inhibition of PI3K and Akt can indeed activate B-Raf and that cAMP can augment this effect. Although a maximal induction of B-Raf activity required a long term treatment (5 h) with the PI3K and Akt inhibitors (Fig. 6, B and C), it was also possible to see a partial increase with only a short term treatment (45 min) (Fig. 6C). To determine whether calcium restriction can inhibit Akt activity, phospho-Akt levels were assayed in cells following treatment with verapamil and nifedipine. As shown in Fig. 7, the calcium channel blockers were able to reduce Akt phosphorylation to an extent comparable with that seen with a direct Akt inhibitor. Fig. 7 also shows that calcium acts upstream of PI3K, since the calcium ionophore A23187 [GenBank] was able to restore Akt phosphorylation in the presence of the calcium channel blockers but not in the presence of the PI3K inhibitor.



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  		FIG. 7.
Akt inhibition by calcium channel blockers. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. The cells were placed in medium containing the calcium ionophore A23187 [GenBank] (10 nM) and cultured for 8 h, 30 min. At 8 h before harvesting (following 30 min of preincubation with A23187 [GenBank] ) the Akt inhibitor (5 µM), the PI3K inhibitor LY294002 (20 µM), or the calcium channel blockers verapamil (0.1 µM) or nifedipine (0.01 µM) were added. Control cells were incubated for 8 h, 30 min but received no treatment. Cell lysates were prepared for Western blotting with anti-phospho-Akt (Ser-473) antibody (P-Akt) or anti-Akt antibody (Total Akt). Shown is a representative Western blot. Band intensities were quantitated with the Fluor-S MAX multi-imager system and compared with the control (1.00) that received no treatment.

 
The requirement for long term exposure to calcium channel blockers or Akt inhibitors for maximal activation of B-Raf suggests that B-Raf protein levels may be affected by these treatments. To assess B-Raf protein levels in M-1 cells, Western blot analysis was carried out following short and long term treatment with nifedipine and verapamil. As shown in Fig. 8A, treatment with the calcium channel blockers resulted in increases in B-Raf levels of over 2-fold after 24 h, suggesting that they may affect the synthesis and/or turnover rate of B-Raf protein. Fig. 8B shows that pharmacological inhibition of PI3K and Akt can also increase B-Raf levels. An increase in B-Raf protein may partially explain the increased cAMP-stimulated ERK activation seen with calcium restriction and with Akt inhibition.



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  		FIG. 8.
B-Raf protein levels in response to calcium channel blockers. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. A, cells were placed in medium containing nifedipine (Nif) (0.01 µM) or verapamil (Ver) (0.1 µM) for 30 min (30 m) or 24 h (24 h). Control (Con) cells were not treated. B, cells were placed in medium containing the PI3K inhibitor LY294002 (20 µM), the Akt inhibitor (5 µM), nifedipine (0.01 µM), or verapamil (0.1 µM) for 24 h. Control cells were treated with Me2SO (DMSO) only. Cell lysates were prepared for Western blotting with anti-B-Raf p95 (B-Raf) and anti-ERK (Total ERK) antibodies. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to controls.

 
To determine whether inhibition of the PI3K/Akt pathway contributes to the mitogenic effect of cAMP, ERK activation was assessed following treatment with PI3K and Akt inhibitors in the absence of calcium channel blockers. As shown in Fig. 9, ERK was indeed activated by cAMP following treatment with the PI3K and Akt inhibitors (Fig. 9, boxed). Thus, the mitogenic action of cAMP appears to require an inhibition of Akt either directly or by calcium restriction, causing derepression of B-Raf and increased B-Raf protein levels, together with a cAMP-driven PKA, Src, and Ras-dependent activation of this B-Raf.



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  		FIG. 9.
PI3K and Akt inhibition of cAMP-mediated ERK activation. Wild-type M-1 cells were cultured in medium with 1% FBS for 48 h and then with 0.002% FBS for 24 h before treatment. The cells were placed in medium containing the PI3K inhibitor LY294002 (20 µM) or the Akt inhibitor (5 µM) and cultured for 8 h. 8-Br-cAMP (cAMP) (100 µM) was added for the last 15 min. Control cells were incubated for 8 h but received no treatment. Cell lysates were prepared for Western blotting with anti-phospho-ERK (P-ERK) and anti-ERK (Total ERK) antibodies. The upper and lower bands are p44 and p42 ERK, respectively. Shown is a representative Western blot. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to the control (1.00) that received no treatment.

 
We demonstrated previously that M-1 cells stably transfected with a dexamethasone (DEX)-inducible polycystin-1 C-tail construct are growth-stimulated by cAMP (31). To determine whether this cell-growth phenotype involves activation of B-Raf and ERK, the M-1 cells were incubated in the absence and presence of cAMP and DEX, and the levels of activated B-Raf and phospho-ERK were assayed. As shown in Fig. 10A, fusion protein induction resulted in a pronounced cAMP-dependent activation of B-Raf and ERK phosphorylation. To determine whether the phenotypic switch seen in these stably transfected M-1 cells could be prevented by an influx of calcium, the cells were treated with a calcium ionophore. As shown in Fig. 10B, treatment of the DEX-induced cells with cAMP resulted in growth stimulation. By contrast, cAMP in the presence of a low concentration of the calcium ionophore A23187 [GenBank] prevented the growth stimulation phenotype, suggesting that the stably transfected cells have impaired intracellular calcium mobilization and can be "rescued" by calcium influx.



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  		FIG. 10.
Response of M-1 cells expressing a polycystin-1 C-tail construct to cAMP and A23187 [GenBank] . M-1 cells (clone 20) stably transfected with a DEX-inducible polycystin-1 C-tail construct (31) were cultured in medium with 1% FBS for 24 h and then with 0.002% FBS for 24 h. A, cells were then cultured for 24 h in medium containing 0.002% FBS in the absence or presence of 1 µM DEX. 8-Br-cAMP (cAMP) (100 µM) was added as indicated 15 min prior to harvesting. Cell lysates were prepared for B-Raf in vitro kinase assay and for Western blotting. B-Raf activity (32P-MEK) was determined by in vitro kinase assay using immunoprecipitated B-Raf and MEK as a substrate for [{gamma}-32P]ATP. Western blotting was carried out with anti-phospho-ERK (P-ERK) and anti-ERK (Total ERK) antibodies. The upper and lower bands are p44 and p42 ERK, respectively. Band intensities were quantitated with the Fluor-S MAX multi-imager system and are reported as density units relative to controls that received no treatment. B, 8-Br-cAMP (100 µM) and/or A23187 [GenBank] (1 nM) were added to the cultures, and the cells were cultured with or without DEX (1 µM) for another 48 h in medium containing 0.002% FBS. Cell proliferation rates were determined by the Promega Cell Titer 96 MTT assay and compared with control cells (Con) that were not treated with cAMP. Without DEX induction (–DEX), cAMP-mediated decreased cell proliferation was not altered by A23187 [GenBank] . With DEX induction (+DEX), cAMP-mediated increased cell proliferation was prevented by A23187 [GenBank] . Asterisk indicates a significant difference at p < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that both primary cultures and immortalized renal epithelial cells can respond to calcium channel blockers by switching their phenotype. In our experiments, the untreated cells responded to cAMP with a decrease in cell proliferation, whereas the same cells following treatment with calcium channel blockers responded to cAMP with an increase in cell proliferation. L-type calcium channels, which are targeted by two of the calcium channel blockers used in this study (nifedipine and verapamil), are expressed in many nonexcitable cell types including renal proximal tubule cells (3) and principal cells of the collecting duct (58). The effects of these calcium channel blockers were both time- and dose-dependent, with relatively low levels of the drugs being effective. EGTA and gadolinium were also effective, suggesting that the switch to a cAMP growth-stimulated phenotype is caused by a generalized loss of calcium homeostasis. The effects of the calcium channel blockers were reversed by the calcium ionophore A23187 [GenBank] and the L-type calcium channel activator Bay-K8644 (Figs. 1A, 2B, and 10B), suggesting that tonic repletion of intracellular calcium can maintain M-1 cells in their normal, cAMP growth-suppressed state. Our experiments contrast with reports using other cell types that showed an inhibition of cell proliferation by calcium channel blockers (59–62). As such, the effects of calcium channel blockers may be cell type-specific or may be a more general property of a wide variety of cells, but requiring co-stimulation with cAMP to activate cell proliferation.

The possibility that the phenotypic switch is calcium-based was tested in M-1 and normal HKC cells by demonstrating that calcium restriction using EGTA and the calcium channel blockers led to a cAMP-dependent stimulation of cell proliferation (Fig. 11). This increased cell proliferation was associated with PKA- and Src-dependent activation of the mitogen-activated protein kinase (ERK) pathway (Fig. 4). The small G protein Rap1 has been implicated in cAMP-stimulated, PKA-, and Src-dependent ERK activation but does not appear to be required in this M-1 cell pathway (Fig. 5B). Instead, we showed that cAMP-dependent ERK activation was completely blocked by dominant negative Ras in either the absence or presence of verapamil (Fig. 5A, right). This suggests that the cAMP-driven pathway goes through Ras, rather than Rap1. PKA is likely to be upstream of Ras because ERK could be activated by calcium channel inhibition in the absence of cAMP in cells transfected with constitutively active Ras (Fig. 5A, left, 3rd lane). This is in contrast to what was observed in untransfected cells containing only wild-type Ras where there was a clear dependence on cAMP (Figs. 2C, 3C, and 3D). Others have shown that activation of ERK by cAMP can be either PKA-dependent/Rap1-dependent (47) or PKA-independent/Ras-dependent (48). Our results suggest a novel pathway in which activation of ERK by cAMP is PKA-dependent/Ras-dependent but Rap1-independent (Fig. 11). We have not determined where Src lies in the pathway; however, others have shown that PKA-activated Src can be required for Rap1-independent activation of ERK (63).



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  		FIG. 11.
Pathways from cAMP and calcium to ERK and cell proliferation. Left, basal cell proliferation. Growth factors in the culture medium stimulate the Ras/Raf-1/MEK/ERK pathway causing a low rate of cell proliferation. The presence of calcium in the medium contributes to the activation of PI3K and Akt, keeping B-Raf inhibited. Although not shown in the figure, it is also possible that calcium acts through other pathways to inhibit B-Raf, bypassing PI3K and Akt. Middle, anti-mitogenic action of cAMP in the presence of normal levels of calcium. cAMP activates PKA, which inhibits Raf-1 causing decreased ERK activation (downward ERK arrow) and a decreased rate of cell proliferation compared with the basal rate. Right, mitogenic action of cAMP in the presence of calcium channel blockers. cAMP activates PKA, which activates Ras. Ras can now activate the B-Raf/MEK/ERK (upward ERK arrow) pathway because Akt no longer blocks B-Raf activation, causing an increased rate of cell proliferation compared with the basal rate. Dark symbols and arrows indicate active pathways; gray symbols and arrows indicate inactive or inhibited pathways.

 
In vitro kinase assays identified B-Raf (Fig. 6), rather than Raf-1, as the downstream target of cAMP-activated Ras (Fig. 11). We found that although Raf-1 was active under basal growth conditions and could be further activated by epidermal growth factor or serum stimulation, it was inhibited by cAMP treatment (data not shown), consistent with observations in other cell types (64–68). The verapamil dependence of the ERK activation in cells transfected with constitutively active Ras (Fig. 5A, left) suggests that the target for calcium restriction is downstream of Ras, perhaps at B-Raf. In fact, B-Raf is known to be inhibited by Akt in a PI3K- and calcium-dependent fashion (49–51). Thus, it is possible that with normal calcium levels, Akt is maintained in a sufficiently active state to keep B-Raf inactive, but in decreased calcium B-Raf is derepressed. We observed that Akt activity was high (Fig. 7) and B-Raf activity was low (Fig. 6) under basal growth conditions. When the cells were treated with calcium channel blockers, the level of active Akt decreased to ~35% of normal (Fig. 7), whereas the activity of B-Raf increased 6–12-fold (Fig. 6A). As such, it appears that B-Raf lies at the nexus of two pathways, one regulated by calcium and the other by cAMP, and thus is in a position to integrate the cAMP- and calcium-dependent proliferative and anti-proliferative signals regulated by these pathways.

Calcium channel inhibition alone, in the absence of increased cAMP, was ineffective in activating ERK (Figs. 2C, 3C, and 3D) or in increasing the rate of cell proliferation (Fig. 1, A and B), whereas it did activate B-Raf to a degree (Fig. 6A). Apparently, the modest (3–4-fold) increase in B-Raf activity with the calcium channel blockers alone was insufficient to trigger an increase in ERK activity. It is also important that increased cAMP alone was ineffective in activating ERK. In fact, cAMP caused an inhibition of ERK phosphorylation (Figs. 2, 3, 4 and 9) and cell proliferation (Fig. 1), probably by inhibiting Raf-1. cAMP alone was unable to activate B-Raf (Fig. 6), due perhaps to the effective suppression of B-Raf by the PI3K/Akt pathway. Together, these results demonstrate that the combination of increased cAMP and decreased calcium acts synergistically to up-regulate the Ras/B-Raf/MEK/ERK pathway to increase cell proliferation, since neither alone is capable of doing so.

Treatment of M-1 cells with calcium channel blockers or with EGTA lowers intracellular calcium within minutes, as we have confirmed by Fura-2 fluorescence measurements (data not shown), and only a 15-min treatment with cAMP is required to see activation of ERK (Figs. 2, 3, 4, 5). Yet a 3–5 h treatment with the calcium channel blockers or with EGTA was required before cAMP was able to activate ERK (Fig. 3). This suggests that activation of B-Raf may only be partially responsible for the phenotypic switch. The requirement for a prolonged decrease in calcium (Fig. 3) or prolonged treatment with PI3K and Akt inhibitors for maximal activation of B-Raf (Fig. 6C) and the observation that the effect of the calcium channel blockers persists for hours following washout (Fig. 3D) suggest that changes in gene expression are associated with the phenotypic switch. Evidence for such a change in gene expression is an increase in B-Raf protein levels following a 24-h treatment with calcium channel blockers or with inhibitors of the PI3K/Akt pathway (Fig. 8). Thus, calcium restriction appears to have at least two effects that augment cAMP-induced ERK activation; it decreases Akt inhibition of B-Raf activity and it increases the level of B-Raf protein.

Our previous studies (29) showed that normal renal tubular epithelial cells (HKC cells) have decreased rates of cell proliferation in response to cAMP. In contrast, cyst wall epithelial cells from patients with PKD have increased rates. In PKD cells, cAMP treatment leads to B-Raf activation (30). B-Raf then activates MEK, which in turn activates ERK and cell proliferation. This so-called PKD phenotype is mimicked in M-1 cells by stable transfection and overexpression of a polycystin-1 C-tail construct (31). Thus, one possibility is that a switch in the cell-growth phenotype of these M-1 cells results from a disruption of the endogenous polycystin signaling pathway, caused by overexpression of the truncated polycystin-1 construct. Because the polycystins are thought to regulate intracellular calcium, the effect of the polycystin-1 construct may be that it disrupts normal calcium homeostasis. This hypothesis was supported by demonstrating that the polycystin-transfected M-1 cells could be rescued by treatment with a calcium ionophore (Fig. 10B).

Polycystin-1 has been implicated in the activation of PI3K and Akt (76). This activation of PI3K may involve a calcium-dependent mechanism controlled by polycystin-mediated calcium entry or release. As such, a loss of the polycystins would be expected to result in decreased Akt activation, which in turn could lead to increased B-Raf and ERK activation and cell proliferation in the presence of cAMP stimulation. Loss of polycystin-1 could also lead to a decrease in Gi-mediated signaling (40–42) with a concomitant increase in adenylyl cyclase activity. Loss of polycystin-modulated intracellular calcium could further increase cAMP by decreasing calcium inhibition of the type 5/6 isoforms of adenylyl cyclase (69, 70) and by decreasing calcium stimulation of the PDE1 isoform of cAMP phosphodiesterases (71), both of which are found in renal tubular epithelial cells. Indeed, treatment of PKD animal models with a vasopressin V2 receptor antagonist, which lowered renal cAMP, slowed disease progression or caused regression (72, 73). Thus, a number of therapeutic targets for treating PKD are suggested by these studies, which include inhibiting Src, Ras, or B-Raf, decreasing cAMP, or increasing intracellular calcium.

BRAF mutations (affecting the B-Raf protein) have been identified in human lung adenocarcinoma (K438T), lung small cell carcinoma (T439P), and malignant melanoma (K438Q), all affecting a consensus threonine phosphorylation sequence at Thr-439. Mutation of this sequence prevents B-Raf from being phosphorylated by Akt, leading to loss of Akt-mediated B-Raf inhibition (74). These so-called "activating" mutations in BRAF result in constitutively active ERK (75). Thus, unscheduled activation of B-Raf resulting either by direct BRAF mutation, by loss of polycystin function, or by a combination of calcium restriction and elevated cAMP in otherwise normal cells can lead to activation of ERK and ultimately to abnormal cell proliferation. It is also conceivable that BRAF is a modifier of PKD, with some alleles increasing the susceptibility of renal epithelial cells to threshold levels of calcium or cAMP and thus promoting the initiation of cyst formation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK53763, DK57301 (to J. J. G. and J. P. C.), and DK064756 (to D. P. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, the Kidney Institute, University of Kansas Medical Center, MS3030, Kansas City, KS 66160. Tel.: 913-588-7424; Fax: 913-588-7440; E-mail: jcalvet{at}kumc.edu.

1 The abbreviations used are: PKD, polycystic kidney disease; HKC, human kidney cortex; FBS, fetal bovine serum; PI3K, phosphatidylinositol 3-kinase; 8-Br-cAMP, 8-bromo-cAMP; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/ERK kinase; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; PKA, cAMP-dependent protein kinase; DEX, dexamethasone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Robin Maser for helpful comments on the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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D. Zhang, G. Zhang, M.S. Hayden, M.B. Greenblatt, C. Bussey, R.A. Flavell, S. Gosh, M. Leist, P. Ghezzi, G. Grasso, et al.
A Novel Molecule Designed to Keep Bacteria out of the Urinary Tract: A Toll-Like Receptor that Prevents Infection by Uropathogenic Bacteria. Science 303: 1522-1526, 2004
J. Am. Soc. Nephrol., January 1, 2005; 16(1): 4 - 7.
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