FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024254525> ?p ?o ?g. }

• W2024254525 endingPage "40430" @default.
• W2024254525 startingPage "40419" @default.
• W2024254525 abstract "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. 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. 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. 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. 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 (1Abernethy D.R. Schwartz J.B. N. Engl. J. Med. 1999; 341: 1447-1457Crossref PubMed Scopus (363) Google Scholar, 2Kizer J.R. Kimmel S.E. Arch. Intern. Med. 2001; 161: 1145-1158Crossref PubMed Scopus (46) Google Scholar). 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 (3Zhang M.I. O'Neil R.G. J. Membr. Biol. 1996; 154: 259-266Crossref PubMed Scopus (32) Google Scholar). Calcium influx into cells modulates the activities of calcium-binding proteins (4Sallese M. Iacovelli L. Cumashi A. Capobianco L. Cuomo L. De Blasi A. Biochim. Biophys. Acta. 2000; 1498: 112-121Crossref PubMed Scopus (74) Google Scholar, 5Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (670) Google Scholar, 6Haeseleer F. Imanishi Y. Sokal I. Filipek S. Palczewski K. Biochem. Biophys. Res. Commun. 2002; 290: 615-623Crossref PubMed Scopus (139) Google Scholar) that can regulate signal transduction pathways leading to changes in gene expression and the control of cell growth and differentiation (7Berridge M.J. BioEssays. 1995; 17: 491-500Crossref PubMed Scopus (458) Google Scholar, 8Hardingham G.E. Bading H. Biometals. 1998; 11: 345-358Crossref PubMed Scopus (63) Google Scholar, 9Spitzer N.C. Lautermilch N.J. Smith R.D. Gomez T.M. BioEssays. 2000; 22: 811-817Crossref PubMed Scopus (126) Google Scholar, 10Munaron L. Int. J. Mol. Med. 2002; 10: 671-676PubMed Google Scholar, 11McKinsey T.A. Zhang C.L. Olson E.N. Trends Biochem. Sci. 2002; 27: 40-47Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). 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 (12Cooper D.M. Mons N. Karpen J.W. Nature. 1995; 374: 421-424Crossref PubMed Scopus (556) Google Scholar, 13Mosior M. Epand R.M. Mol. Membr. Biol. 1997; 14: 65-70Crossref PubMed Scopus (17) Google Scholar, 14Anderson K.A. Kane C.D. Biometals. 1998; 11: 331-343Crossref PubMed Scopus (44) Google Scholar, 15Cruzalegui F.H. Bading H. Cell. Mol. Life Sci. 2000; 57: 402-410Crossref PubMed Scopus (63) Google Scholar, 16Maier L.S. Bers D.M. J. Mol. Cell. Cardiol. 2002; 34: 919-939Abstract Full Text PDF PubMed Scopus (241) Google Scholar, 17Agell N. Bachs O. Rocamora N. Villalonga P. Cell. Signal. 2002; 14: 649-654Crossref PubMed Scopus (351) Google Scholar, 18Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar). 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 (14Anderson K.A. Kane C.D. Biometals. 1998; 11: 331-343Crossref PubMed Scopus (44) Google Scholar, 15Cruzalegui F.H. Bading H. Cell. Mol. Life Sci. 2000; 57: 402-410Crossref PubMed Scopus (63) Google Scholar, 18Shibasaki F. Price E.R. Milan D. McKeon F. Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar, 19Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Crossref PubMed Scopus (641) Google Scholar, 20Cruzalegui F.H. Hardingham G.E. Bading H. EMBO J. 1999; 18: 1335-1344Crossref PubMed Scopus (63) Google Scholar, 21Wu H. Naya F.J. McKinsey T.A. Mercer B. Shelton J.M. Chin E.R. Simard A.R. Michel R.N. Bassel-Duby R. Olson E.N. Williams R.S. EMBO J. 2000; 19: 1963-1973Crossref PubMed Scopus (377) Google Scholar, 22Johnson C.M. Hill C.S. Chawla S. Treisman R. Bading H. J. Neurosci. 1997; 17: 6189-6202Crossref PubMed Google Scholar, 23Schaefer A. Magocsi M. Fandrich A. Marquardt H. Biochem. J. 1998; 335: 505-511Crossref PubMed Scopus (31) Google Scholar). Polycystic kidney disease (PKD) 1The 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. is characterized by the abnormal growth of benign cystic tumors from renal tubular epithelial cells (24Calvet J.P. Grantham J.J. Semin. Nephrol. 2001; 21: 107-123Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 25Somlo S. Ehrlich B. Curr. Biol. 2001; 11: R356-R360Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 26Igarashi P. Somlo S. J. Am. Soc. Nephrol. 2002; 13: 2384-2398Crossref PubMed Scopus (446) Google Scholar). 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 (24Calvet J.P. Grantham J.J. Semin. Nephrol. 2001; 21: 107-123Abstract Full Text PDF PubMed Scopus (144) Google Scholar, 27Sullivan L.P. Wallace D.P. Grantham J.J. Physiol. Rev. 1998; 78: 1165-1191Crossref PubMed Scopus (176) Google Scholar, 28Hanaoka K. Guggino W.B. J. Am. Soc. Nephrol. 2000; 11: 1179-1187PubMed Google Scholar, 29Yamaguchi T. Pelling J.C. Ramaswamy N.T. Eppler J.W. Wallace D.P. Nagao S. Rome L.A. Sullivan L.P. Grantham J.J. Kidney Int. 2000; 57: 1460-1471Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). 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 (30Yamaguchi T. Nagao S. Wallace D.P. Belibi F.A. Cowley B.D. Pelling J.C. Grantham J.J. Kidney Int. 2003; 63: 1983-1994Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). 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 (31Sutters M. Yamaguchi T. Maser R.L. Magenheimer B.S. St. John P.L. Abrahamson D.R. Grantham J.J. Calvet J.P. Kidney Int. 2001; 60: 484-494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), 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 (32Delmas P. Nauli S.M. Li X. Coste B. Osorio N. Crest M. Brown D.A. Zhou J. FASEB J. 2004; 18: 740-742Crossref PubMed Scopus (132) Google Scholar). Polycystin-2 is a calciumpermeable, nonselective cation channel, which has been shown to function both in calcium entry and in calcium release (33Vassilev P.M. Guo L. Chen X.Z. Segal Y. Peng J.B. Basora N. Babakhanlou H. Cruger G. Kanazirska M. Ye C. Brown E.M. Hediger M.A. Zhou J. Biochem. Biophys. Res. Commun. 2001; 282: 341-350Crossref PubMed Scopus (200) Google Scholar, 34Gonzalez-Perret S. Kim K. Ibarra C. Damiano A.E. Zotta E. Batelli M. Harris P.C. Reisin I.L. Arnaout M.A. Cantiello H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1182-1187Crossref PubMed Scopus (395) Google Scholar, 35Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (560) Google Scholar, 36Ikeda M. Guggino W.B. Curr. Opin. Nephrol. Hypertens. 2002; 11: 539-545Crossref PubMed Scopus (21) Google Scholar); it is possible that polycystin-1 is a regulator of polycystin-2 activity (36Ikeda M. Guggino W.B. Curr. Opin. Nephrol. Hypertens. 2002; 11: 539-545Crossref PubMed Scopus (21) Google Scholar, 37Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (669) Google Scholar, 38Xu G.M. Gonzalez-Perrett S. Essafi M. Timpanaro G.A. Montalbetti N. Arnaout M.A. Cantiello H.F. J. Biol. Chem. 2003; 278: 1457-1462Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Indeed, polycystin-1 has been shown to interact directly with polycystin-2 (39Qian F. Germino F.J. Cai Y. Zhang X. Somlo S. Germino G.G. Nat. Genet. 1997; 16: 179-183Crossref PubMed Scopus (558) Google Scholar) and to couple with heterotrimeric G proteins (40Parnell S.C. Magenheimer B.S. Maser R.L. Rankin C.A. Smine A. Okamoto T. Calvet J.P. Biochem. Biophys. Res. Commun. 1998; 251: 625-631Crossref PubMed Scopus (193) Google Scholar, 41Delmas P. Nomura H. Li X. Lakkis M. Luo Y. Segal Y. Fernandez-Fernandez J.M. Harris P. Frischauf A.M. Brown D.A. Zhou J. J. Biol. Chem. 2002; 277: 11276-11283Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 42Parnell S.C. Magenheimer B.S. Maser R.L. Zien C.A. Frischauf A.M. Calvet J.P. J. Biol. Chem. 2002; 277: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), 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. 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 (31Sutters M. Yamaguchi T. Maser R.L. Magenheimer B.S. St. John P.L. Abrahamson D.R. Grantham J.J. Calvet J.P. Kidney Int. 2001; 60: 484-494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). M-1 cells (clone 20) stably transfected with the C-tail of polycystin-1 were maintained in G418-containing medium as described previously (31Sutters M. Yamaguchi T. Maser R.L. Magenheimer B.S. St. John P.L. Abrahamson D.R. Grantham J.J. Calvet J.P. Kidney Int. 2001; 60: 484-494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Human kidney cortex (HKC) cells were cultured from a freshly obtained nephrectomy specimen (K129) as described previously (29Yamaguchi T. Pelling J.C. Ramaswamy N.T. Eppler J.W. Wallace D.P. Nagao S. Rome L.A. Sullivan L.P. Grantham J.J. Kidney Int. 2000; 57: 1460-1471Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 30Yamaguchi T. Nagao S. Wallace D.P. Belibi F.A. Cowley B.D. Pelling J.C. Grantham J.J. Kidney Int. 2003; 63: 1983-1994Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The kidney retrieval protocol was approved by the Institutional Review Board of the University of Kansas Medical Center. Cell Proliferation Assays—2 × 103 M-1 cells or 4 × 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 (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 (29Yamaguchi T. Pelling J.C. Ramaswamy N.T. Eppler J.W. Wallace D.P. Nagao S. Rome L.A. Sullivan L.P. Grantham J.J. Kidney Int. 2000; 57: 1460-1471Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 31Sutters M. Yamaguchi T. Maser R.L. Magenheimer B.S. St. John P.L. Abrahamson D.R. Grantham J.J. Calvet J.P. Kidney Int. 2001; 60: 484-494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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, 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 β-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 × 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. (43Erhardt P. Troppmair J. Rapp U.R. Cooper G.M. Mol. Cell. Biol. 1995; 15: 5524-5530Crossref PubMed Scopus (121) Google Scholar). 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 β-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 [γ-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, 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 (44Hu Y. Qiao L. Wang S. Rong S.B. Meuillet E.J. Berggren M. Gallegos A. Powis G. Kozikowski A.P. J. Med. Chem. 2000; 43: 3045-3051Crossref PubMed Scopus (199) Google Scholar, 45Chodniewicz D. Zhelev D.V. Blood. 2003; 101: 1181-1184Crossref PubMed Scopus (65) Google Scholar, 46Zhang R. Xu Y. Ekman N. Wu Z. Wu J. Alitalo K. Min W. J. Biol. Chem. 2003; 278: 51267-51276Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) 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. 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 (29Yamaguchi T. Pelling J.C. Ramaswamy N.T. Eppler J.W. Wallace D.P. Nagao S. Rome L.A. Sullivan L.P. Grantham J.J. Kidney Int. 2000; 57: 1460-1471Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). 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 (31Sutters M. Yamaguchi T. Maser R.L. Magenheimer B.S. St. John P.L. Abrahamson D.R. Grantham J.J. Calvet J.P. Kidney Int. 2001; 60: 484-494Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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. 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. In normal renal epithelial cells, as in many cell types, cAMP treatment decreases ERK activation (47Stork P.J. Schmi" @default.
• W2024254525 created "2016-06-24" @default.
• W2024254525 creator A5001840552 @default.
• W2024254525 creator A5006804331 @default.
• W2024254525 creator A5007219151 @default.
• W2024254525 creator A5032242069 @default.
• W2024254525 creator A5050373233 @default.
• W2024254525 creator A5069611407 @default.
• W2024254525 date "2004-09-01" @default.
• W2024254525 modified "2023-09-30" @default.
• W2024254525 title "Calcium Restriction Allows cAMP Activation of the B-Raf/ERK Pathway, Switching Cells to a cAMP-dependent Growth-stimulated Phenotype" @default.
• W2024254525 cites W1492104840 @default.
• W2024254525 cites W1507667128 @default.
• W2024254525 cites W1545554770 @default.
• W2024254525 cites W1821092064 @default.
• W2024254525 cites W1849480711 @default.
• W2024254525 cites W1963957029 @default.
• W2024254525 cites W1964152667 @default.
• W2024254525 cites W1978996010 @default.
• W2024254525 cites W1980614440 @default.
• W2024254525 cites W1980647755 @default.
• W2024254525 cites W1984362855 @default.
• W2024254525 cites W1990499518 @default.
• W2024254525 cites W1994109113 @default.
• W2024254525 cites W1994188885 @default.
• W2024254525 cites W1994860376 @default.
• W2024254525 cites W1999954329 @default.
• W2024254525 cites W2000253969 @default.
• W2024254525 cites W2000732162 @default.
• W2024254525 cites W2006666150 @default.
• W2024254525 cites W2009034762 @default.
• W2024254525 cites W2009376981 @default.
• W2024254525 cites W2011182719 @default.
• W2024254525 cites W2013056080 @default.
• W2024254525 cites W2013620388 @default.
• W2024254525 cites W2014264816 @default.
• W2024254525 cites W2014604277 @default.
• W2024254525 cites W2022926357 @default.
• W2024254525 cites W2036507877 @default.
• W2024254525 cites W2040190828 @default.
• W2024254525 cites W2043524463 @default.
• W2024254525 cites W2045876058 @default.
• W2024254525 cites W2047364183 @default.
• W2024254525 cites W2051950569 @default.
• W2024254525 cites W2060420349 @default.
• W2024254525 cites W2063640156 @default.
• W2024254525 cites W2064576105 @default.
• W2024254525 cites W2065603011 @default.
• W2024254525 cites W2069941772 @default.
• W2024254525 cites W2069985598 @default.
• W2024254525 cites W2070023258 @default.
• W2024254525 cites W2071001965 @default.
• W2024254525 cites W2073637975 @default.
• W2024254525 cites W2079238835 @default.
• W2024254525 cites W2079437966 @default.
• W2024254525 cites W2081437948 @default.
• W2024254525 cites W2087197643 @default.
• W2024254525 cites W2089902775 @default.
• W2024254525 cites W2091166353 @default.
• W2024254525 cites W2091553865 @default.
• W2024254525 cites W2110396329 @default.
• W2024254525 cites W2110487056 @default.
• W2024254525 cites W2111432014 @default.
• W2024254525 cites W2113479676 @default.
• W2024254525 cites W2114366598 @default.
• W2024254525 cites W2114742085 @default.
• W2024254525 cites W2121692329 @default.
• W2024254525 cites W2122228538 @default.
• W2024254525 cites W2133041598 @default.
• W2024254525 cites W2134914006 @default.
• W2024254525 cites W2136477311 @default.
• W2024254525 cites W2145949182 @default.
• W2024254525 cites W2146041633 @default.
• W2024254525 cites W2149183853 @default.
• W2024254525 cites W2153066126 @default.
• W2024254525 cites W2157522734 @default.
• W2024254525 cites W2164020057 @default.
• W2024254525 cites W2260330619 @default.
• W2024254525 cites W2324185390 @default.
• W2024254525 cites W2335012920 @default.
• W2024254525 cites W4254047100 @default.
• W2024254525 cites W4254607679 @default.
• W2024254525 doi "https://doi.org/10.1074/jbc.m405079200" @default.
• W2024254525 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15263001" @default.
• W2024254525 hasPublicationYear "2004" @default.
• W2024254525 type Work @default.
• W2024254525 sameAs 2024254525 @default.
• W2024254525 citedByCount "308" @default.
• W2024254525 countsByYear W20242545252012 @default.
• W2024254525 countsByYear W20242545252013 @default.
• W2024254525 countsByYear W20242545252014 @default.
• W2024254525 countsByYear W20242545252015 @default.
• W2024254525 countsByYear W20242545252016 @default.
• W2024254525 countsByYear W20242545252017 @default.
• W2024254525 countsByYear W20242545252018 @default.
• W2024254525 countsByYear W20242545252019 @default.
• W2024254525 countsByYear W20242545252020 @default.
• W2024254525 countsByYear W20242545252021 @default.

• next