TRPM7 Regulates Cell Adhesion by Controlling the Calcium-dependent Protease Calpain
2006; Elsevier BV; Volume: 281; Issue: 16 Linguagem: Inglês
10.1074/jbc.m512885200
ISSN1083-351X
AutoresLi-Ting Su, Maria Agapito, Mingjiang Li, William Simonson, Anna Huttenlocher, Raymond Habas, Lixia Yue, Loren W. Runnels,
Tópico(s)Trace Elements in Health
Resumom-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes. m-Calpain is a protease implicated in the control of cell adhesion through focal adhesion disassembly. The mechanism by which the enzyme is spatially and temporally controlled is not well understood, particularly because the dependence of calpain on calcium exceeds the submicromolar concentrations normally observed in cells. Here we show that the channel kinase TRPM7 localizes to peripheral adhesion complexes with m-calpain, where it regulates cell adhesion by controlling the activity of the protease. Our research revealed that overexpression of TRPM7 in cells caused cell rounding with a concomitant loss of cell adhesion that is dependent upon the channel of the protein but not its kinase activities. Knockdown of m-calpain blocked TRPM7-induced cell rounding and cell detachment. Silencing of TRPM7 by RNA interference, however, strengthened cell adhesion and increased the number of peripheral adhesion complexes in the cells. Together, our results suggest that the ion channel TRPM7 regulates cell adhesion through m-calpain by mediating the local influx of calcium into peripheral adhesion complexes. TRPM7 is one of only two ion channels to possess its own kinase domain (1Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (621) Google Scholar). It is a member of the transient receptor potential ion channel family with the closest similarity to its bifunctional homologue TRPM6 as well as to melastatin (TRPM1), whose reduced expression has been used as a prognosis marker for metastasis in patients with localized melanoma (2Fleig A. Penner R. Novartis Found. Symp. 2004; 258: 248-266Crossref PubMed Google Scholar, 3Nilius B. Voets T. Novartis Found Symp. 2004; 258 (263-266): 140-159Crossref PubMed Google Scholar, 4Clapham D.E. 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Biol. 1999; 9: R43-R45Abstract Full Text Full Text PDF PubMed Google Scholar, 10Ryazanov A.G. FEBS Lett. 2002; 514: 26-29Crossref PubMed Scopus (126) Google Scholar). Annexin I has been identified as a substrate for the kinase, but the functional significance of annexin I phosphorylation by TRPM7 is not yet understood (11Dorovkov M.V. Ryazanov A.G. J. Biol. Chem. 2004; 279: 50643-50646Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Autophosphorylation of the channel does not alter channel activity (12Matsushita M. Kozak J.A. Shimizu Y. McLachlin D.T. Yamaguchi H. Wei F.Y. Tomizawa K. Matsui H. Chait B.T. Cahalan M.D. Nairn A.C. J. Biol. Chem. 2005; 280: 20793-20803Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, phospholipase C inactivates TRPM7 channel activity through hydrolysis of phosphatidylinositol 4,5-bisphosphate, which is presumably gating the channel (13Kozak J.A. Matsushita M. Nairn A.C. Cahalan M.D. J. Gen. Physiol. 2005; 126: 499-514Crossref PubMed Scopus (113) Google Scholar, 14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (455) Google Scholar). Magnesium ions block channel activity (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar, 15Kerschbaum H.H. Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 2293-2305Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Kozak J.A. Cahalan M.D. Biophys. J. 2003; 84: 922-927Abstract Full Text Full Text PDF PubMed Google Scholar, 17Prakriya M. Lewis R.S. J. Gen. Physiol. 2002; 119: 487-507Crossref PubMed Scopus (265) Google Scholar), and, more recently, TRPM7 current has been shown to be potentiated by protons (18Jiang J. Li M. Yue L. J. Gen. Physiol. 2005; 126: 137-150Crossref PubMed Scopus (151) Google Scholar). Despite these recent advances in understanding TRPM7 channel regulation, the physiological role of this unique bifunctional protein still remains unclear. The passage of Mg2+ by TRPM7 has linked it to the regulation of magnesium homeostasis in mammalian cells (19Schmitz C. Perraud A.L. Johnson C.O. Inabe K. Smith M.K. Penner R. Kurosaki T. Fleig A. Scharenberg A.M. Cell. 2003; 114: 191-200Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar). Its capacity to carry calcium, in contrast, has been associated with calcium overload during anoxic cell death (20Aarts M. Iihara K. Wei W.L. Xiong Z.G. Arundine M. Cerwinski W. MacDonald J.F. Tymianski M. Cell. 2003; 115: 863-877Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar), calcium-dependent regulation of the cell cycle (21Hanano T. Hara Y. Shi J. Morita H. Umebayashi C. Mori E. Sumimoto H. Ito Y. Mori Y. Inoue R. J. Pharmacol. Sci. 2004; 95: 403-419Crossref PubMed Scopus (169) Google Scholar), and most recently, skeletogenesis and kidney stone formation in zebrafish (22Elizondo M.R. Arduini B.L. Paulsen J. Macdonald E.L. Sabel J.L. Henion P.D. Cornell R.A. Parichy D.M. Curr. Biol. 2005; 15: 667-671Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). An early study by Nadler et al. (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar) showed that overexpression of TRPM7 caused HEK-293 cells to detach and die, suggesting that the channel may have a role in controlling cell adhesion. Here we present evidence that TRPM7 is a potent regulator of m-calpain. Fourteen distinct members of the mammalian calpain family have been identified, but only two are well characterized: μ-calpain, which is activated by μm calcium concentrations (in vitro), and m-calpain, which is activated by millimolar concentrations of Ca2+ (in vitro) (23Glading A. Lauffenburger D.A. Wells A. Trends Cell Biol. 2002; 12: 46-54Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Both isoforms are thought to play significant roles in the regulation of cell adhesion (23Glading A. Lauffenburger D.A. Wells A. Trends Cell Biol. 2002; 12: 46-54Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 24Carragher N.O. Westhoff M.A. Riley D. Potter D.A. Dutt P. Elce J.S. Greer P.A. Frame M.C. Mol. Cell. Biol. 2002; 22: 257-269Crossref PubMed Scopus (97) Google Scholar, 25Franco S.J. Huttenlocher A. J. Cell Sci. 2005; 118: 3829-3838Crossref PubMed Scopus (397) Google Scholar, 26Wells A. Huttenlocher A. Lauffenburger D.A. Int. Rev. Cytol. 2005; 245: 1-16Crossref PubMed Scopus (79) Google Scholar). μ-Calpain is involved in the activation of Rac during focal complex formation during cell spreading (27Kulkarni S. Saido T.C. Suzuki K. Fox J.E. J. Biol. Chem. 1999; 274: 21265-21275Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 28Bialkowska K. Kulkarni S. Du X. Goll D.E. Saido T.C. Fox J.E. J. Cell Biol. 2000; 151: 685-696Crossref PubMed Scopus (91) Google Scholar), whereas m-calpain has been implicated in adhesion complex disassembly and deadhesion (29Glading A. Chang P. Lauffenburger D.A. Wells A. J. Biol. Chem. 2000; 275: 2390-2398Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 30Glading A. Uberall F. Keyse S.M. Lauffenburger D.A. Wells A. J. Biol. Chem. 2001; 276: 23341-23348Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 31Glading A. Bodnar R.J. Reynolds I.J. Shiraha H. Satish L. Potter D.A. Blair H.C. Wells A. Mol. Cell. Biol. 2004; 24: 2499-2512Crossref PubMed Scopus (230) Google Scholar). We found that expression of TRPM7 in HEK-293 cells produced cell rounding and a loss of cell adhesion that was dependent upon m-calpain. TRPM7-dependent cell rounding occurred without significantly elevating cytosolic calcium concentrations, suggesting that calcium influx through the channel was creating local calcium gradients to activate the protease. Indeed, TRPM7 colocalized with m-calpain at peripheral vinculin-containing adhesion complexes, where presumably, as one of its physiological roles, TRPM7 controls the activity of m-calpain to regulate cell adhesion. Recently, the bifunctional channel has been shown to play a key role in anoxic cell death. The control of m-calpain by TRPM7 may also contribute to some of the cellular events that occur during ischemia, as well as to other pathologies associated with the calcium-dependent protease (32Starling A. de Paula F. Silva H. Vainzof M. Zatz M. J. Mol. Neurosci. 2003; 21: 233-236Crossref PubMed Scopus (20) Google Scholar, 33Zatz M. Starling A. N. Engl. J. Med. 2005; 352: 2413-2423Crossref PubMed Scopus (193) Google Scholar). Materials—All of the chemicals, unless otherwise stated, were from Sigma. The calpain inhibitor ALLM, 2The abbreviations used are: ALLM, N-acetyl-leucyl-leucyl-methioninal; Z, benzyloxycarbonyl; FMK, fluoromethyl ketone; HA, hemagglutinin; GST, glutathione S-transferase; FRT, Flp recombination target. the caspase 3 inhibitor Z-DEVD-FMK, and the Rho kinase inhibitor Y27632 were from Calbiochem. TRPM7-expressing Cell Lines—TRPM7, kinase mutants, and small hairpin RNA-expressing cell lines were made using the Flp-In system (Invitrogen) and the commercially available Flp-In T-Rex 293 cells following the manufacturer's instructions. The Flp-In T-Rex 293 cell line expresses the tetracycline repressor protein (TetR), which in the absence of tetracycline blocks transcription from the cytomegalovirus promoter containing control elements from the bacterial tetracycline resistance operon. The Flp-In T-Rex 293 cell line contains a single, stably integrated FRT site at a transcriptionally active genomic locus, so that target integration of a Flp-In expression vector ensures reproducible isogenic high level gene expression. To make the cell lines, an expression vector (pcDNA5/FRT/TO) containing amino-terminal hemagglutinin (HA)-tagged murine TRPM7 (GenBank™ accession number AF376052), TRPM7-K1645A, TRPM7-G1618D, TRPM7ΔKIN, GFP-KIN, or GFP-CTKIN was cotransfected into the parental cell line with the pOG44 plasmid that expresses Flp recombinase. The respective coding sequences were then integrated into the genome via Flp recombinase-mediated DNA recombination at the specific genomic location. To test the function of the kinase domain, we created cell lines expressing a version of TRPM7 in which the catalytic kinase domain has been removed (293-TRPM7ΔKIN) or one in which the kinase domain has been rendered catalytically inactive (293-TRPM7-K1645A, 293-TRPM7-G1618D). The truncation of the kinase domain was made by changing the TCG codon encoding serine 1501 into a stop codon. The K1645A substitution renders the kinase inactive by mutating a critical invariant lysine to alanine. The G1618D mutation disrupts kinase activity by blocking ATP binding to the P-loop in the catalytic domain. Control experiments have shown that a GST fusion of the kinase domain harboring either the K1645A or G1618D substitutions was soluble but lacked catalytic activity (data not shown). TRPM7-K1645A, TRPM7-G1618D, and TRPMΔKIN were generated using QuikChange (Stratagene) with the following primers: TRPM7-K1645A, 5′-CCT GAA GTC AGG GCA TCT CTA TAT CAT TGC GTC ATT TCT TCC TGA GGT G-3′ and 5′-CAC CTC AGG AAG AAA TGA CGC AAT GAT ATA GAG ATG CCC TGA CTT CAG G-3′; TRPM7-G1618D, 5′-GTA AAG AGG AAA TGG GAG ATG GTT TAC GAA GAG CAG-3′ and 5′-CTG CTC TTC GTA AAC CAT CTC CCA TTT CCT CTT TAC-3′; and TRPM7ΔKIN, 5′-CTG TAG TAG AAG AGC GTA GAC GGA AGA CTCT CCA G-3′ and 5′-CTG GAG AGT CTT CCG TCT ACG CTC TTC TAC TAC AG-3′. shRNA Cell Lines—To fully test the hypothesis that TRPM7 is involved in regulating cell adhesion, we created 293 cells in which native TRPM7 protein levels were lowered by expression of shRNAs that target TRPM7 (34Matsukura S. Jones P.A. Takai D. Nucleic Acids Res. 2003; 31: e77Crossref PubMed Scopus (163) Google Scholar). We designed six variant shRNAs to target either human, mouse, or rat TRPM7 from the following sequences: M7shRNA1, 5′-GCA AAT GGA GTT ACC CAA AC-3′; M7shRNA2, 5′-GCA TAA ATT CCT TAC CAT TC-3′; M7shRNA3, 5′-GGT TGG ATC CTT GGA ACA AGC-3′; M7shRNA4, 5′-GGA ACA AGC TAT GCT TGA TGC-3′; M7shRNA5, 5′-GGA AAT CTT CCT CCA GGA TAT-3′; and M7shRNA6, 5′-GCA CTC CTC AGT TGC GAA AGA-3′. Cell lines were constructed by first cloning double-stranded oligonucleotides that encoded the shRNAs into the pENTR/H1/TO vector (Invitrogen). Expression from the pENTR/H1/TO vector is driven by RNA polymerase III off a H1 promoter modified to contain two tetracycline operator 2 (TetO2) sites. We screened the pENTR/H1/TO TRPM7 shRNA clones by transfecting them into 293-TRPM7ΔKIN-expressing cells and then evaluated their ability to attenuate TRPM7 expression (supplemental Fig. S1). Four of the best constructs were then used to make TRPM7-shRNA-expressing cell lines. First, pcDNA5/FRT/TO/GATEWAY was created using the Gateway Vector Conversion System (Invitrogen) by blunt end ligation of the Gateway cassette into the NruI and EcoRV sites of pcDNA5/FRT/TO (which removed the cytomegalovirus promotor). A Gateway LR recombination reaction (Invitrogen) was then performed to introduce the shRNA expression cassette into pcDNA5/FRT/TO/GATEWAY. The final GATEWAY vectors expressing shRNAs against TRPM7 (pcDNA5/FRT/TO/GATEWAY-M7shRNA-2, M7shRNA-3, M7shRNA-5, and M7shRNA-6) were used to generate stable cell lines employing the Flp-In system (Invitrogen). A nonsilencing sequence (5′-AAT TCT CCG AAC GTG TCA CGT-3′) was used to make the control cell line 293-shRNA-C (20Aarts M. Iihara K. Wei W.L. Xiong Z.G. Arundine M. Cerwinski W. MacDonald J.F. Tymianski M. Cell. 2003; 115: 863-877Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Anti-TRPM7 Antibodies—The TRPM7-specific PLIKC47 antibody (α-C47), which recognizes residues 1816-1863 from rat TRPM7, has been previously described (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (455) Google Scholar). A second rabbit polyclonal TRPM7 (α-CTERM) was generated using a GST fusion protein with residues 1384-1506 of murine TRPM7 as the antigen. The α-CTERM antibody was purified following protocols that have been described (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (455) Google Scholar). Both α-C47 and α-CTERM are specific and suitable for immunocytochemistry, Western blotting, and immunoprecipitation experiments (supplemental Fig. S2). Western Blotting and Immunoprecipitation Experiments—293-TRPM7 cells expressing recombinant HA-tagged TRPM7 or channel and kinase variants were lysed after a 24-h treatment with tetracycline using 2 ml of ice-cold radioimmune precipitation assay buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Igepal CA-630, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, and 10 mm iodoacetamide). The proteins were immunoprecipitated overnight from cell lysates from a 60-mm dish with an anti-HA affinity matrix. In the case of GFP-KIN and GFP-CTKIN, expressed proteins were immunoprecipitated using a monoclonal anti-GFP antibody (Roche Applied Science) bound to protein G-agarose. The samples were washed three times in TBST (50 mm Tris-Cl, pH 7.6, 150 mm NaCl, 0.05% Tween 20), eluted by boiling in 2× SDS-PAGE sample buffer, and then resolved by SDS-PAGE and Western blotting following standard protocols. The monoclonal 12CA5 anti-HA antibody or the monoclonal 7.1 and 13.1 anti-GFP antibodies were used as the primary antibodies (Roche Applied Science). The SuperSignal West Dura maximum sensitive substrate (Pierce) was used for immunochemiluminescence detection. To detect native TRPM7 in the HEK-293 line, cells were lysed from a 10-cm dish using 2 ml of ice-cold radioimmune precipitation assay buffer. TRPM7 was immunoprecipitated overnight from cell lysates using 10 μg of α-C47 antibody absorbed to protein A-agarose (Santa Cruz Biotechnology). The samples were washed three times in TBST buffer, eluted in 2× SDS-PAGE sample buffer, and then resolved by SDS-PAGE and Western blotting using the second anti-TRPM7 antibody (α-CTERM). The SuperSignal West Dura maximum sensitive substrate (Pierce) was used for immunochemiluminescence detection. Detection of Talin Cleavage—Talin was detected using a primary monoclonal antibody from Upstate Biotechnology, Inc. (clone TA205), which recognizes an epitope within the head domain of human talin (amino acids 139-433). Immunokinase Assay—The immunokinase assay was performed as follows. Briefly, 10-cm dishes of cells were grown to confluence and allowed to express the individual proteins by the addition of tetracycline (1 μg/ml) to the medium. After 24 h, the cells were lysed in radioimmune precipitation assay buffer, and the specific proteins were immunoprecipitated as described above. The immunocaptured proteins were washed four times with ice-cold phosphate-buffered saline containing 0.1% polyoxyethylenesorbitan monolaurate (Tween 20). The wash buffer was replaced by ice-cold reaction buffer (KIN-DET) containing 50 mm HEPES (pH 7.0), 50 mm NaCl, 5 mm MgCl2, 3.5 mm MnCl2, 0.1% Tween 20, 0.5 mm ATP, and 2 μCi of [γ-32P]ATP (specific activity of 3000 Ci/mmol). The samples were then incubated at 37 °C for 30 min in a 50-μl reaction, before being terminated by the addition of 10 μl of 6× SDS sample buffer. The samples were then resolved on a 6% SDS-PAGE gel. The gels were dried, and γ-32P incorporation was visualized by autoradiography. Immunofluorescence and Confocal Microscopy—293-TRPM7 cells were plated onto polylysine coated coverslips and treated with tetracycline for 20 h to analyze the cellular distribution of HA-tagged TRPM7. The cells were fixed at room temperature for 10 min in phosphate-buffered saline (pH 7.4) with 4% paraformaldehyde (Electron Microscopy Sciences) and permeabilized in phosphate-buffered saline with 0.1% Saponin (Sigma). Primary antibodies against TRPM7 (described above) or the monoclonal 12CA5 anti-HA (Roche Applied Science) were used to visualize TRPM7 by immunofluorescence. A monoclonal antibody against vinculin (HVIN-1 clone; Sigma) was used to image peripheral adhesion complexes. A rabbit polyclonal antibody against m-calpain (Triple Point Biologics, Inc.) was used to visualize the cellular distribution of the protease. A 1:2000 dilution of Alexa Fluor 488 or Alexa Fluor 568 goat antibody to rabbit or mouse (Molecular Probes) was used as the secondary antibody. The images were obtained from a Zeiss LSM 410 confocal microscope using a 488-nm excitation wavelength and a 512-nm band pass emission filter. The pinhole size used was 30 Airy Units, and the contrast/brightness settings were kept the same for each image. Electrophysiological Recordings and Calcium Imaging—The voltage clamp technique was used to evaluate the whole cell currents of TRPM7 expressed in HEK-293 cells as described (14Runnels L.W. Yue L. Clapham D.E. Nat. Cell Biol. 2002; 4: 329-336Crossref PubMed Scopus (455) Google Scholar). Briefly, whole cell current recordings of TRPM7-expressing cells were elicited by voltage stimuli lasting 250 ms delivered every 1 s using voltage ramps from -120 to +100 mV. The data were digitized at 2 or 5 kHz and digitally filtered off-line at 1 kHz. The internal pipette solution for macroscopic current recordings contained 145 mm cesium methanesulfonate, 8 mm NaCl, 10 mm EGTA, and 10 mm HEPES, pH adjusted to 7.2 with CsOH. The extracellular solution for whole cell recordings contained 140 mm NaCl, 5mm KCl, 2 mm CaCl2, 20 mm HEPES, and 10 mm glucose, pH adjusted to 7.4 (NaOH). Calcium imaging was conducted using an IonOptix ratio calcium imaging system. In brief, the cells were plated on 25-mm glass coverslips and incubated with Fura 2/acetoxymethyl (5 μm) for 50 min. After the extracellular Fura 2/acetoxymethyl was washed away, the cells were incubated for an additional 30 min. Fluorescence intensity at 510 nm with 340- and 380-nm excitation was collected at a rate of 1 Hz using a intensified CCD camera (Ionoptix), and the data were analyzed using Ionwizard (Ionoptix). 1 μm ionomycin was applied to each cell and was used as a reference to normalize the changes in the F340/F380 ratio. For Ca2+ imaging the extracellular Tyrode's solution contained 140 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 20 mm HEPES, and 10 mm glucose, pH adjusted to 7.4 (NaOH). Cell Rounding Assay—Changes in 293 cell morphology were scored manually employing the following criteria. The cells that had a fully round cell body with no membrane extension processes were given 1 point. Partially rounded cells with one or two membrane extensions were assigned half a point. Nonrounded cells having three or four membrane extension processes, with a cell morphology similar to wild type HEK-293 cells, were given 0 points. Adhesion Assay—Cell adhesion was measured using a trypsinization assay (36Ortiz-Urda S. Garcia J. Green C.L. Chen L. Lin Q. Veitch D.P. Sakai L.Y. Lee H. Marinkovich M.P. Khavari P.A. Science. 2005; 307: 1773-1776Crossref PubMed Scopus (113) Google Scholar). Briefly, ∼2.5×106 cells were plated onto a 60-mm Falcon tissue culture dish for 24 h and then treated with 0.5 ml of 0.05% trypsin-EDTA for 4 min to stimulate cell detachment. Detached cells in the trypsin-EDTA solution were collected, and the trypsinization was terminated by the addition of 3 ml of culture medium. The number of detached cells was then manually counted using a hemocytometer and expressed as a percentage of the total number of cells on the plate. Rho Activity Assay—Detection of activated Rho was accomplished using a modified GST pull-down purification assay. To detect activated Rho, the pull-down was performed using GST fused to the Rhotekin Rho-binding domain (37Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1359) Google Scholar). The assay was performed by incubating cell lysates with partially purified GST fused to the Rhotekin Rho-binding domain bound to glutathione beads for 1 h (Amersham Biosciences), washing the beads, and then resolving bound Rho by SDS-PAGE and Western blotting as described (38Habas R. Kato Y. He X. Cell. 2001; 107: 843-854Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). The cells transfected with ephexin, a Rho-GEF, were used as a positive control. The ephexin expression vector was a gift of Michael Greenberg (Division of Neuroscience, Children's Hospital, Boston, MA). Calpain 2 shRNA Blockade in LTRPC7 Cells—RNA interference of m-calpain (calpain 2) expression was achieved by expression of a shRNA in the pSUPERretro vector specific to human m-calpain. To make the pSUPERretro-CAPN2 vector, the following oligonucleotides were cloned into the BglII-HindIII sites of pSUPERretro (sense, 5′-GAT CCC CGG CAT ACG CCA AGA TCA ACT TCA AGA GAG TTG ATC TTG GCG TAT GCC TTT TTG GAA A-3′; antisense, 5′-AGC TTT TCC AAA AAG GCA TAC GCC AAG ATC AAC TCT CTT GAA GTT GAT CTT GGC GTA TGC CGG G-3′), in which the m-calpain target sequence is 5′-AAG GCA UAC GCC AAG AUC AAC-3′. pSUPERretro-CAPN2 was transiently transfected using Lipofectamine 2000 (Invitrogen) into LTRPC7 cells (generously provided by Dr. Andrew Scharenberg, University of Washington) (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar). Cells expressing the shRNA targeting m-calpain were selected using 5 μg/ml puromycin for 3 days prior to expression of TRPM7 by the addition of tetracycline (1 μg/ml) to the media. Control experiments showed that nontransfected cells treated with puromycin died within 3 days. Silencing of m-calpain was assessed by SDS-PAGE and Western blotting of cell lysates, using a rabbit polyclonal antibody against m-calpain (Triple Point Biologics, Inc.). A monoclonal antibody against μ-calpain (Alexis Biochemicals) was used to show that pSUPERretro-CAPN2 did not affect μ-calpain protein levels. TRPM7 Regulates Cell Adhesion—A previous study had showed that overexpression of TRPM7 induces cell detachment and subsequent cell death (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar). We therefore sought to determine whether TRPM7 is involved in the control of cell adhesion. To study the cellular function of TRPM7, we employed the Flp-In system and Flp-In T-Rex cells (Invitrogen) to generate a HEK-293 cell line that could inducibly overexpress TRPM7 with the addition of tetracycline to the growth medium (293-TRPM7). 293-TRPM7 cells expressed high levels of channel activity and produced whole cell currents with activation kinetics and current-voltage relationships similar to those described earlier (see Fig. 2A) (1Runnels L.W. Yue L. Clapham D.E. Science. 2001; 291: 1043-1047Crossref PubMed Scopus (621) Google Scholar, 8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar). As previously reported, HEK-293 cells that express TRPM7 rounded up within 18-24 h and became loosely attached to tissue culture plates (Fig. 1A) (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar, 20Aarts M. Iihara K. Wei W.L. Xiong Z.G. Arundine M. Cerwinski W. MacDonald J.F. Tymianski M. Cell. 2003; 115: 863-877Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). We tested whether expression of TRPM7 in 293-TRPM7 cells was toxic, because an earlier study by Nadler et al. (8Nadler M.J.S. Hermosura M.C. Inabe K. Perraud A.-L. Zhu Q. Stokes A.J. Kurosaki T. Kine J.-P. Penner R. Scharenberg A.M. Fleig A. Nature. 2001; 411: 590-595Crossref PubMed Scopus (797) Google Scholar) found that overexpression of TRPM7 in their LTRPC7 cell line led to cell rounding, detachment, and subsequent cell death. Nonexpressing 293-TRPM7 cells had comparable amounts of cell death (less than 10%) to cells expressing TRPM7 for at least 72 h, as assessed by trypan blue exclusion analysis (data not shown). However, expression of TRPM7 in the original LTRPC7 cell line, which expresses two or three times more channel activity than 293-TRPM7 cells, did cause significant cell death (∼25%) (data not shown). In addition, the effect of TRPM7 on cell morphology and adhesion is specific. Expression of TRPM1, TRPC5, lymphocyte α-kinase, and TRPM6 (a second TRPM family member with a kinase domain) in HEK-293 cells failed to produce the morphological changes that were visible when TRPM7 was expressed (data not shown). This finding is consistent with a recent report showing that TRPM6 and TRPM7 are functionally nonredundant (39Schmitz C. Dorovkov M.V. Zhao X. Davenport B.J. Ryazanov A.G. Perraud A.L. J. Biol. Chem. 2005; 280: 37763-37771Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).FIGURE 1TRPM7 regulates cell adhesion in HEK-293 cells. A, application of tetracycline (TET) induced expression of HA-TRPM7 and produced cell rounding of 293-TRPM7 cells. Knockdown of native TRPM7 in 293-M7shRNA2 cells produced cells that were more spread and had 50% longer extensions than a control cell line expressing a nonsilencing shRNA (293-shRNA-C). The same magnification was used in all four images. B, 293-M7shRNA2 cells adhered more strongly to the substratum than 293-shRNA-C or 293-TRPM7-expressing cells. Adhesion was measured using a trypsinization assay in which ∼2.5 × 106 cells were plated onto 60-mm Falcon tissue culture dishes for 24 h and then treated with 0.05% trypsin-EDTA for 4 min to stimulate cell detachment. The number of detached cells was counted using a hemocytometer and expressed as a percentage
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