Surface Expression of ASIC2 Inhibits the Amiloride-sensitive Current and Migration of Glioma Cells

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes. Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes. Acid-sensing ion channels (ASICs) 2The abbreviations used are: ASIC, acid-sensing ion channel; ENaC, ephithelial Na+ channel; DEG, degenerin; GBM, glioblastoma multiforme; CFTR, cystic fibrosis transmembrane conductance regulator; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; ER, endoplasmic reticulum; RT, reverse transcription; Endo H, endoglycosidase H. are proton gated cation channels of the epithelial Na+ channel/degenerin (ENaC/DEG) superfamily. ASICs have been discovered in sensory neurons originating from dorsal root ganglia in many parts of the body including the heart (1Sutherland S.P. Benson C.J. Adelman J.P. McCleskey E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 711-716Crossref PubMed Scopus (331) Google Scholar). Not only have ASICs been implicated in pH detection, but they also play a role in normal touch sensation (2Price M.P. McIlwrath S.L. Xie J. Cheng C. Qiao J. Tarr D.E. Sluka K.A. Brennan T.J. Lewin G.R. Welsh M.J. Neuron. 2001; 32: 1071-1083Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar). These same channels have also been found in the brain, although their function there is less clear. ASICs are present in hippocampal neurons and, because of the pH fluctuations that are known to occur during synaptic activity (3Chesler M. Kaila K. Trends Neurosci. 1992; 15: 396-402Abstract Full Text PDF PubMed Scopus (477) Google Scholar), this channel has been hypothesized to be involved in synaptic plasticity (4Baron A. Waldmann R. Lazdunski M. J. Physiol. (Lond.). 2002; 539: 485-494Crossref Scopus (185) Google Scholar, 5Wemmie J.A. Chen J. Askwith C.C. Hruska-Hageman A.M. Price M.P. Nolan B.C. Yoder P.G. Lamani E. Hoshi T. Freeman Jr., J.H. Welsh M.J. Neuron. 2002; 34: 463-477Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 6Leonard A.S. Yermolaieva O. Hruska-Hageman A. Askwith C.C. Price M.P. Wemmie J.A. Welsh M.J. Proc. 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In situ hybridization studies in mouse brain revealed ASIC expression in the Purkinje, the granular cell layers of the cerebellum, dentate regions of hippocampus, and the olfactory bulb (12Garcia-Anoveros J. Derfler B. Neville-Golden J. Hyman B.T. Corey D.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1459-1464Crossref PubMed Scopus (296) Google Scholar). Thus, these channels are primarily found in neurons. One exception to this generalization is the presence of ASICs in cells obtained from high grade brain gliomas (13Bubien J.K. Keeton D.A. Fuller C.M. Gillespie G.Y. Reddy A.T. Mapstone T.B. Benos D.J. Am. J. Physiol. 1999; 276: C1405-C1410Crossref PubMed Google Scholar). These tumors are of astrocyte cell origin. In grade IV gliomas, also called glioblastoma multiforme (GBM), there exists a constitutively activated, amiloride-sensitive inward Na+ current. This glioma cation current is mediated by mixed ASIC and ENaC components, including ASIC1 and ASIC2 (14Berdiev B.K. Xia J. McLean L.A. Markert J.M. Gillespie G.Y. Mapstone T.B. Naren A.P. Jovov B. Bubien J.K. Ji H.L. Fuller C.M. Kirk K.L. Benos D.J. J. Biol. Chem. 2003; 278: 15023-15034Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Interestingly, this current is absent in normal astrocytes and low grade gliomas. We hypothesize that GBM cells express this basal current because of the lack of ASIC2 in the plasma membrane. If this current could be blocked either pharmacologically or by manipulation of its regulatory pathway, the high rate of migration of the GBM cells, proliferation, and invasiveness could be corrected. The chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, are known to stabilize protein conformation, increase the rate of protein refolding, and accelerate oligomeric protein assembly (15Skach W.R. Kidney Int. 2000; 57: 825-831Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Zeitlin P.L. Diener-West M. Rubenstein R.C. Boyle M.P. Lee C.K. Brass-Ernst L. Mol. Ther. 2002; 6: 119-126Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Brown C.R. Hong-Brown L.Q. Welch W.J. J. Clin. Invest. 1997; 99: 1432-1444Crossref PubMed Scopus (161) Google Scholar, 18Brown C.R. Hong-Brown L.Q. Welch W.J. J. Bioenerg. Biomembr. 1997; 29: 491-502Crossref PubMed Scopus (54) Google Scholar, 19Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 20Tamarappoo B.K. Yang B. Verkman A.S. J. Biol. Chem. 1999; 274: 34825-34831Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). They have been used successfully to promote the membrane insertion of a misfolded mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), ΔF508-CFTR (19Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 21Rubenstein R.C. Egan M.E. Zeitlin P.L. J. Clin. Invest. 1997; 100: 2457-2465Crossref PubMed Scopus (330) Google Scholar, 22Cheng S.H. Fang S.L. Zabner J. Marshall J. Piraino S. Schiavi S.C. Jefferson D.M. Welsh M.J. Smith A.E. Am. J. Physiol. 1995; 268: L615-L624PubMed Google Scholar). Sodium 4-phenylbutyrate has also been shown to have inhibitory effects on glioma cell proliferation, migration, and invasiveness (23Engelhard H.H. Homer R.J. Duncan H.A. Rozental J. J. Neurooncol. 1998; 37: 97-108Crossref PubMed Scopus (40) Google Scholar, 24Baker M.J. Brem S. Daniels S. Sherman B. Phuphanich S. J. Neurooncol. 2002; 59: 239-242Crossref PubMed Scopus (17) Google Scholar) and to promote differentiation of tumor cells in vivo and in vitro (25Carducci M.A. Nelson J.B. Chan-Tack K.M. Ayyagari S.R. Sweatt W.H. Campbell P.A. Nelson W.G. Simons J.W. Clin. Cancer Res. 1996; 2: 379-387PubMed Google Scholar). However, the mechanism underlying the action of sodium 4-phenylbutyrate in glioma cells is unknown. Using a combination of electrophysiological, cell biological, and biochemical techniques, we have tested the hypothesis that glycerol and sodium 4-phenylbutyrate promote the delivery of ASIC2 to the surface of tumor cells. Electrophysiological studies demonstrated that the movement of ASIC2 to the surface inhibits the basally active, amiloride-sensitive, inward cation current of glioma cells. Cell proliferation and migration assays demonstrated that the translocation of ASIC2 to the plasma membrane inhibits the proliferation and mobility of GBM cells. The results obtained in this study support the hypothesis that a basally active, multimeric ASIC/ENaC constitutes the conduction pathway responsible for inward Na+ current seen in high grade gliomas. Cell Culture—Primary cultures of normal human astrocytes and primary cultured human glioblastoma cells (GBM) were obtained from the University of Alabama at Birmingham, Neurosurgery Brain Tissue Bank (Institutional Review Board approval X030403011). U87-MG cells were purchased from ATCC. SK-MG-1 and D54-MG cells were gifts from Dr. Gregory Caincross, University of Calgary and Dr. Darell Bigner, Duke University, respectively. The cells were cultured in Dulbecco's modified Eagle's medium/F-12 (1:1) supplemented with 10% fetal bovine serum. All cells were maintained at 37 °C in a 95% O2 and 5% CO2 humidified incubator. RT-PCR—Total RNA was isolated from tissue culture cells in log phase using TRIzol (Invitrogen) following the manufacturer's instructions. The ASIC1 and ASIC2 forward and reverse primer sequences were as follows: ASIC1 5′-GGACTCGGATTTGGATT-3′ (forward primer) and 5′-TGTTGGCAGCGTATGT-3′ (reverse primer); ASIC2, 5′-CCGTCACTGTGTGCAACAA-3′ (forward primer) and 5′ATCCTCGCCTGAGTTAAACATG-3′ (reverse primer). These to from as to genomic was using the with of RNA and a primer of were by negative RNA was to as a of All assays were at The of was by of the and and the sequences and or the and from were used for and normal GBM, and glioblastoma cell were with phosphate-buffered and pH The cells were with of of pH sodium and supplemented with were and into they were with a from the for at was with of either or at °C on a by the of protein were with and were with pH glycerol, 5% and at °C for and were were on with were polyvinylidene were blocked with 10% at and with the were with either a or and using either or the of the for was demonstrated by of for the primary Cell and normal astrocytes were on for were with and with in pH with were once with in and for in with were with PBS, in PBS, and at at They were in and on at at were to a and the of of a of were for at were by and of the were to the were with pH 10% were by in on and cells were either with or were with Endo to manufacturer's and at 37 cell the of cells by the of into The obtained is to the activity of functional and the of cells. were with a in and at and for human primary GBM, and D54-MG cells, and for human normal in cells were with of either glycerol or sodium The was The cell culture was with of mixed with of a in The cells were for of in The was at in a The of the with cells was as This was cell and GBM cells were in culture at a of one was and with and cells were using a The were with glycerol and sodium 4-phenylbutyrate were at and and as This was cell cells were on culture at a of they they were with a and with cell culture They were with either glycerol, sodium 4-phenylbutyrate, or and in a on the of a to a to were maintained cell culture Dulbecco's modified Eagle's medium/F-12 (1:1) supplemented with 10% at 37 °C using a and of 95% O2 and 5% CO2 using were with phase at were for We the at the of the and it from the at the of the and to the to the of cell This was cells were in culture and with either glycerol or sodium 4-phenylbutyrate for They were and by at for at The plasma membrane protein was used to the manufacturer's to the plasma membrane from the The plasma membrane was in in PBS, and plasma membrane protein was using the protein were with either glycerol or sodium 4-phenylbutyrate for They were in a with culture and and on the of the cells to the of the that were to and with and at a pH of were the plasma membrane of cells. the and the cells were obtained by to the the were to the cells, a was to the membrane the the This a low to the This is the In this the plasma membrane be and conductance be the been the of was and during by the membrane to and negative The cells were at a membrane potential of and for to membrane of to at of to the potential of for The in the effects of as and as a for the of the The were and in using the a was on at cells. U87-MG cell the migration was were obtained from The of the was with in a migration was in with with bovine in The U87-MG cells were from their culture and in Dulbecco's modified Eagle's medium/F-12 with bovine cells were to the The cells were at 37 °C for with in PBS, with PBS, and with on the of the were with a that the to the of the were in of This was of ASIC1 and of was to the hypothesis that ASIC1 and ASIC2 are present in astrocytes and glioma cells. this we used and to for the presence of ASIC1 and ASIC2 in normal human human primary GBM cells, and cell and Our results that ASIC1 and ASIC2 and protein are present in normal human primary GBM, and D54-MG cells, but only ASIC1 was found in U87-MG cells. ASIC2 in in that ASIC1 ASIC2 were in the plasma membrane, surface by of ASIC1 and ASIC2, were on normal human and human primary GBM tumor cells. of the ASIC1 and ASIC2 be However, in the cell surface of SK-MG-1 D54-MG and human primary GBM only ASIC1 is In human normal ASIC1 and ASIC2 are present at the plasma membrane ASIC2 in the in GBM the of cells were and ASIC The was with the endoglycosidase Endo H, and by We the of ASIC1 and ASIC2 in SK-MG-1 cells. The of to ASIC1 and ASIC2 the pathway. shown in Endo H, the characteristic of the but or in the a in the of ASIC2 with ASIC2 in the However, ASIC1 was to Endo that it is the of and Sodium on Cell cell was used to the of glycerol and sodium 4-phenylbutyrate on cell for The function is to cells in the We found that glycerol was sodium 4-phenylbutyrate we used glycerol and sodium of sodium 4-phenylbutyrate on cell for sodium 4-phenylbutyrate in D54-MG cells with the for glioma cell at cell Sodium 4-phenylbutyrate was in the of They from of and Sodium on Cell on the results obtained from the we tested glycerol and sodium 4-phenylbutyrate cell or cell proliferation of glioma cells. We found that compounds proliferation at and it in cell glycerol and sodium 4-phenylbutyrate proliferation, cell at these human astrocytes and proliferation with the with glycerol normal astrocytes to glioma cells sodium 4-phenylbutyrate and sodium 4-phenylbutyrate not normal human astrocytes and sodium for normal astrocytes cells. were with at and for glycerol and sodium 4-phenylbutyrate with the They from in and Sodium the membrane of the cells was using the Electrophysiological were in human primary GBM, and normal human astrocytes with glycerol or sodium In cells, inward that are were However, glycerol or sodium 4-phenylbutyrate the amiloride-sensitive current in the human primary GBM, and SK-MG-1 cells was present In glycerol and sodium 4-phenylbutyrate less on amiloride-sensitive current in the U87-MG cells, not express ASIC2 We have shown that the constitutively active, amiloride-sensitive current in glioma cells is to Na+ and J.K. Ji H.L. Gillespie G.Y. Fuller C.M. Markert J.M. Mapstone T.B. Benos D.J. Am. J. Physiol. 2004; PubMed Scopus Google Scholar). the amiloride-sensitive current present in these to normal human not have this amiloride-sensitive not present in current in the presence of glycerol or sodium 4-phenylbutyrate and human primary GBM D54-MG SK-MG-1 U87-MG and normal human astrocytes were with either glycerol or sodium 4-phenylbutyrate for They were and in from potential of for basal following with current the of cells and human primary GBM D54-MG SK-MG-1 U87-MG and normal human astrocytes were with either glycerol or sodium 4-phenylbutyrate for They were and in from potential of for basal following with current the of cells and human primary GBM D54-MG SK-MG-1 U87-MG and normal human astrocytes were with either glycerol or sodium 4-phenylbutyrate for They were and in from potential of for basal following with current the of cells and human primary GBM D54-MG SK-MG-1 U87-MG and normal human astrocytes were with either glycerol or sodium 4-phenylbutyrate for They were and in from potential of for basal following with current the of cells of and Sodium on Cell the migration and proliferation ability of SK-MG-1 cells with glycerol, sodium 4-phenylbutyrate, or by using a migration cells migrate into and it However, SK-MG-1 cells to glycerol, sodium 4-phenylbutyrate, or only of the the hypothesis that the presence of ASIC2 a role in the migration and proliferation of glioma cells, we U87-MG cells, lack ASIC2 and migration assays were used to U87-MG cell migration in the presence of glycerol, sodium 4-phenylbutyrate, or This migration that glycerol and sodium 4-phenylbutyrate not have on U87-MG cells migration However, is of migration in cell migration of U87-MG cells. U87-MG cells were with glycerol, sodium 4-phenylbutyrate, or for that the were and on a that from the migration of the cells, and it was glycerol and sodium 4-phenylbutyrate not have on U87-MG cells. and Sodium the of ASIC2 to the that surface delivery of ASIC2 is to glycerol and sodium 4-phenylbutyrate we on a plasma membrane of D54-MG cells. shown in ASIC2 was absent from the plasma membrane of cells. However, a to glycerol or sodium 4-phenylbutyrate, ASIC2 in the plasma membrane of D54-MG cells ASIC1 was present at the plasma membrane of and cells These results support the hypothesis that compounds stabilize ASIC2 and to the delivery of ASIC2 to the plasma membrane. The polyvinylidene were with to that of protein were to of that we were with plasma membrane were for a of the plasma membrane. The was in membrane The plasma membrane obtained from cells with sodium 4-phenylbutyrate increase in the of with previous demonstrated that sodium 4-phenylbutyrate expression J. Am. J. Physiol. 1999; Google Scholar). that the obtained plasma membrane was not with were using a mouse is a of and a for the of membrane were negative for a membrane with These results that only plasma membrane The of to their be by PubMed Scopus Google Scholar). ion channels as ENaC and as a of their plasma membrane the is not only the their and but is also the for insertion into the plasma membrane. are many in the to that these are or If they are they are R.R. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of this of with the in that results in the of the protein to of the to the plasma membrane (15Skach W.R. Kidney Int. 2000; 57: 825-831Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). of as low glycerol, and sodium 4-phenylbutyrate have been used to stabilize the ΔF508-CFTR of it to the plasma membrane it channel function P.L. Diener-West M. Rubenstein R.C. Boyle M.P. Lee C.K. Brass-Ernst L. Mol. Ther. 2002; 6: 119-126Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 18Brown C.R. Hong-Brown L.Q. Welch W.J. J. Bioenerg. Biomembr. 1997; 29: 491-502Crossref PubMed Scopus (54) Google Scholar, 19Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). The results obtained in this study support the hypothesis that ASIC2 is not present in the plasma membrane of GBM cells, in and protein In ASIC1 and ASIC2 are present in the plasma membrane of normal human cells in basal amiloride-sensitive current be We also tested the hypothesis that lack of plasma membrane expression of ASIC2 is a of of this that ASIC2 is not the in glioma cells. In this we biochemical and that of human primary GBM, and D54-MG glioma cells that were from high grade ASIC2 to glycerol and sodium 4-phenylbutyrate results in the delivery of functional ASIC2 to the plasma membrane. is not known ASIC2 is intracellularly into functional channel and to the surface membrane or ASIC2 the plasma membrane and into as has been for of the ENaC family J.A. C.M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the the is to active channel characteristic of high grade gliomas into characteristic of normal human astrocytes. Thus, these results are with the hypothesis that abolishing the amiloride-sensitive inward Na+ current promotes a reversion of a high grade glioma cell to a normal The results obtained from the glioma cell that was in the presence of glycerol and sodium 4-phenylbutyrate, suggest that the cells phenotype resembling a normal astrocyte the results also show that the presence of ASIC2 a role in the of the amiloride-sensitive current because the U87-MG cells, lack ASIC2, were not by glycerol or sodium The results suggest that in the of ASIC2, glycerol and sodium 4-phenylbutyrate to promote a reversion of glioma cell to a normal phenotype as by migration and The in this study are with the hypothesis that the movement of Na+ channels the growth and migration of glioma cells. these results previous that a of high grade gliomas, for ASIC2, constitutively activated inward (14Berdiev B.K. Xia J. McLean L.A. Markert J.M. Gillespie G.Y. Mapstone T.B. Naren A.P. Jovov B. Bubien J.K. Ji H.L. Fuller C.M. Kirk K.L. Benos D.J. J. Biol. Chem. 2003; 278: 15023-15034Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Our results are also with the hypothesis that by of these amiloride-sensitive ion channels growth and migration gliomas are tumors that the normal brain, we hypothesize that the expression of these ion channels that tumor cell movement the in the brain, because of their in tumor cell either the ion channel or its regulatory a or for or of these cells. We of and University of Alabama at and of University of Alabama at for cell culture

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