Integrin-linked Kinase Controls Neurite Outgrowth in N1E-115 Neuroblastoma Cells
2001; Elsevier BV; Volume: 276; Issue: 46 Linguagem: Inglês
10.1074/jbc.m105198200
ISSN1083-351X
AutoresToshiaki Ishii, Eiki Satoh, Masakazu Nishimura,
Tópico(s)Signaling Pathways in Disease
ResumoMouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against β1 integrin inhibits neurite outgrowth. Thus, β1 integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of β1 integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells. Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against β1 integrin inhibits neurite outgrowth. Thus, β1 integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of β1 integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells. extracellular matrix integrin-linked kinase nerve growth factor mitogen-activated protein extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase green fluorescent protein phosphatidylinositol p21-activated kinase dominant negative Src homology polyacrylamide gel electrophoresis polymerase chain reaction fetal bovine serum Dulbecco's modified Eagle's medium Cell interactions with extracellular matrix (ECM)1 proteins are mediated primarily by integrins that function as cell-surface receptors composed of heterodimers of the α and β subunits. Such interactions are important in the regulation of cell proliferation and differentiation (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). Clustering of integrins on the cell surface in contact with the ECM induces focal adhesion that recruits numerous mitogenic signaling proteins, such as growth factor receptors (2Plopper G.E. McNamee H.P. Dike L.E Bojanowski K. Ingber D.E. Mol. Biol. Cell. 1995; 6: 1349-1365Crossref PubMed Scopus (473) Google Scholar), mitogen-activated protein (MAP) kinase (3Miyamoto S. Teramoto H. Gutkind J.S. Yamada K.M. J. Cell Biol. 1996; 135: 1633-1642Crossref PubMed Scopus (680) Google Scholar), and small GTP-binding proteins (4Hotchin N.A. Hall A. J. Cell Biol. 1995; 131: 1857-1865Crossref PubMed Scopus (370) Google Scholar) to integrin receptors. Thus, integrin-associated focal adhesions serve as signaling centers where adhesive and mitogenic pathways can integrate. Numerous physical interactions between integrins or focal adhesion components and mitogenic signaling proteins have been demonstrated (5Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar). These studies indicate a biochemical coupling between integrin and growth factor signaling pathways; however, the functional significance of these interactions in the context of the regulation of proliferation and differentiation is not well understood. The intracellular signaling cascades that are activated when integrins bind to their ECM ligands are varied in many different cell types (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar). The specific intracellular pathways differ depending on the specific integrin/ECM ligand interaction and/or on the specific integrin/intracellular signal proteins coupling in different cell types. In neuronal cells, neurite formation in response to differentiation signals is strongly promoted by ECM ligands such as laminin, fibronectin, or collagen (6Turner D.C. Flier L.A. Dev. Neurosci. 1989; 11: 300-312Crossref PubMed Scopus (13) Google Scholar, 7Turner D.C. Flier L.A. Carbonetto S. J. Neurosci. 1989; 9: 3287-3296Crossref PubMed Google Scholar). Mouse N1E-115 neuronal cells exhibit neurite outgrowth in response to serum deprivation (8Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (577) Google Scholar, 9Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar). Neurite outgrowth in serum-starved N1E-115 cells is enhanced when the cells are grown on a laminin matrix (10Sarner S. Kozma R. Ahmed S. Lim L. Mol. Cell. Biol. 2000; 20: 158-172Crossref PubMed Scopus (108) Google Scholar). On the other hand, neurite outgrowth of PC12 cells, which are induced to differentiate into sympathetic neuron-like cells by growth factors such as nerve growth factor (NGF), is also affected by cell adhesion to ECM (11Rossino P. Gavazzi I. Timpl R. Aumailley M. Abbadini M. Giancotti F. Silengo L. Marchisio P.C. Tarone G. Exp. Cell Res. 1990; 189: 100-108Crossref PubMed Scopus (69) Google Scholar, 12Zhang Z. Tarone G. Turner D.C. J. Biol. Chem. 1993; 268: 5557-5565Abstract Full Text PDF PubMed Google Scholar). The mechanisms by which the ECM regulates neuronal differentiation, however, are currently poorly understood. Integrin-linked kinase (ILK) is a cytoplasmic protein serine/threonine kinase that interacts with β1 integrin (13Hannigan G.H. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar). Recent biochemical and functional studies indicate that ILK serves as a mediator in integrin- and growth factor-mediated signal transduction (14Dedhar S. Williams B. Hannigan G. Trends Cell Biol. 1999; 9: 319-323Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 15Huang Y. Wu C. Int. J. Mol. Med. 1999; 3: 563-572PubMed Google Scholar). Overexpression of ILK in epithelial cells results in an altered cellular morphology, a reduction in cell adhesion to ECM, and also stimulation of anchorage-independent (13Hannigan G.H. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar), but not serum-independent, growth (16Radeva G. Petrocelli T. Behrend E. Leung-Hagesteijn C. Filmus J. Slingerland J. Dedhar S. J. Biol. Chem. 1997; 272: 13937-13944Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The present study investigated the roles and potential mechanisms of ILK in the control of neuronal differentiation of N1E-115 neuroblastoma cells. We demonstrated that ILK activation is an early and important event in the integrin-mediated signal pathway and is necessary for neurite outgrowth in serum-starved N1E-115 cells on laminin. We also report that activation of p38 MAP kinase, but neither MEK nor ERK, is critically involved in the ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth. LY294002 was obtained from Sigma. The rabbit polyclonal anti-ILK IgG (UB 06-550 and UB 06-592) and myelin basic protein were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-phospho-p38 MAP kinase, anti-p38 MAP kinase, anti-phospho-MEK, and anti-MEK antibodies were obtained from New England Biolabs (Beverly, MA). Anti-phospho-p44/42 MAP kinase (anti-phospho-ERK1/ERK2) and anti-p44/42 MAP kinase (anti-ERK1/ERK2) antibodies were obtained from Promega (Madison, WI). All other chemicals were of analytical grade and were obtained from Sigma or Wako Pure Chemical Co. (Osaka, Japan) unless otherwise specified. Kinase assays were performed as described by Delcommenne et al. (17Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar). Cells were lysed in 50 mm Hepes buffer (pH 7.5) containing 150 mmNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 μg/ml leupeptin, 2.5 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 5 mm sodium fluoride, and 1 mm sodium orthovanadate. The lysates were incubated with anti-ILK antibody (UB 06-592) at 4 °C for 12 h. After incubation, the lysates were precleared and immune complexes were collected with Protein A-Sepharose. The immunoprecipitated ILK was incubated for 20 min at 30 °C in the presence or absence of 10 μg of the exogenous substrate myelin basic protein in a total volume of 50 μl of kinase reaction buffer (50 mm HEPES, pH 7.0, 10 mmMnCl2, 10 mm MgCl2, 2 mm NaF, 1 mm Na3VO4) containing 10 μCi of [γ-32P]ATP (6000 Ci/mmol, NEG-502Z, NEN Life Sciences). The reaction was stopped by the addition of an equal volume of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The kinase reaction products were analyzed using SDS-PAGE (5–20% polyacrylamide) and autoradiography. For detection of the immunoprecipitated ILK and DN-ILK proteins, the precipitated proteins were released from the immunobeads by boiling in 80 μl of SDS-PAGE sample buffer for 5 min. Equal volumes of the samples were loaded onto SDS-PAGE. Total ILK and DN-ILK proteins were detected by immunoblotting with an anti-ILK antibody (UB 06-550) that recognizes both ILK and DN-ILK proteins. A cDNA library was constructed in Uni-ZAP XR using cDNA synthesis kits (nos. 200400–200402, Stratagene, La Jolla, CA). Poly(A)+ RNA prepared from the whole brain of an adult guinea pig was converted to cDNA by oligo(dT)-primed reverse transcription. The obtained cDNAs were ligated between EcoRI and XhoI sites of Uni-ZAP XR as described by the manufacturer. This cDNA library was screened with 32P-labeled antisense oligonucleotides, 3′-ATACGTGGACGGACCCAT-5′ and 3′-GGACTTCTGTGTTTGTCT-5′, both of which were designed to bind to the encoding sequences for the amino acids sequences, 351YAPAWV356 and365PEDTNR370, respectively, within the catalytic domain of human ILK (13Hannigan G.H. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar). One positive clone containing a 1.8-kilobase pair insert, which binds both oligonucleotide probes, was isolated from 2 × 105 plaques. After excision of the pBluescript phagemid vector from the Uni-ZAP XR, the cDNA insert was cloned into the pBluescript SK plasmid as described by the manufacturer and sequenced. The nucleotide sequence of guinea pig ILK has been submitted to the GenBank™ data base with accession numberAF256520. The encoded protein has a 98.9% amino acid homology with the human ILK. The kinase-deficient ILK (DN-ILK) was generated by site-directed mutagenesis (Glu to Lys) at amino acid residue 359 within the kinase domain using the polymerase chain reaction (PCR) as follows. A 1153-kilobase pair cDNA containing the 5′-untranslated sequence and the coding region for Met1–Glu366 of guinea pig ILK, in which Glu359 was mutated to Lys, was synthesized using PCR with two oligonucleotide primers, 5′-TGGCGGCCGCTCTAGAACTAGTGGAT-3′ (pBluescript vector primer, the NotI site is underlined) and 5′-TTCAGGTTTCTTCTGCAAAGCTT(T)AGGGGCTA-3′ (a uniqueHindIII site of ILK cDNA is underlined, and a single mutation site (C to T) is shown in parentheses), and ILK cDNA in pBluescript vector as a template. The PCR fragment was digested withNotI/HindIII restriction enzymes, and was then placed back into a full-length ILK cDNA in a pBluescript vector. The nucleotide sequence of the PCR fragment was determined using a standard DNA sequencing technique (18Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). Wild type ILK and DN ILK cDNAs were ligated into the polylinkers in a mammalian expression vector, pTracerTM-CMV2 (V885-01, Invitrogen Corp., Carlsbad, CA), and were introduced into N1E-115 cells. DN-ILK cDNA was transfected into N1E-115 cells (5 × 105cells/100-mm culture dish) using the calcium/phosphate precipitation method as described by Graham and van der Eb (19Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6499) Google Scholar), and 48 individual zeocin-resistant cell lines were isolated over the next 4–5 weeks. Among them, three different cell lines were selected on the basis of detection of the GFP fluorescence and confirmation of gene transcription using reverse transcriptase-PCR. The cloned cell lines were maintained in Dulbecco's modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS; HyClone, Logan, UT) and zeocin (0.5 mg/ml). N1E-115 cells were seeded at a density of 5 × 104 cells/dish onto 35-mm noncoated culture dishes and grown in DMEM containing 10% FBS. At 20 h following plating, cells were transfected with the plasmid containing the wild type ILK cDNA using LipofectAMINE transfection reagent (Life Technologies, Inc.). Control cells were transfected with the empty plasmid. Briefly, cells were washed in serum-free medium and incubated in serum-free medium for 1 h. During this time, the plasmid DNAs were mixed with the LipofectAMINE reagent and incubated for 30 min at room temperature and then added to the cells. Ten percent FBS was added to the cells 6 h after addition of the transfection mix. At 18 and 36 h following the transfection, the morphological changes produced by the transfection of the plasmid of interest were examined using fluorescence microscopy. Cells that had positively expressed green fluorescent protein (GFP) were assumed to be expressing the protein of interest cDNA. Cells that have a process longer than 2-fold the length of a cell body were categorized as being neurite-bearing cells, and neurite-bearing cell was assessed as the percentage of the total number of GFP-expressing cells. Cells were solubilized in five volumes of buffer containing 1% Triton X-100, 150 mm NaCl, 50 mm Tris-HCl (pH 7.4), 5 mm EGTA, and 2 mm phenylmethylsulfonyl fluoride at 4 °C. The solubilized materials were subjected to SDS-PAGE (5–20% gradient, 6.5 or 10% polyacrylamide) and transferred onto nitrocellulose membranes at 4 °C in 25 mm Tris-HCl (pH 8.4), 192 mmglycine, 20% methanol, and 0.025% SDS. After blocking, the blots were probed with anti-ILK polyclonal antibody (UB 06-550) for the detection of endogenous ILK in phosphate-buffered saline containing 0.05% Tween 20, followed by goat anti-rabbit IgG conjugated to horseradish peroxidase. The final protein-IgG complexes were visualized following the reaction to 3,3′-diaminobenzidine tetrahydrochloride. For detection of the phosphorylation status of p38 MAP kinase and MEK, polyclonal antibodies reactive with the phosphorylated form of p38 MAP kinase and MEK were purchased from New England Biolabs. For detection of the phosphorylation status of p44/42 MAP kinase (ERK1/ERK2), polyclonal antibodies reactive with the phosphorylated form of p44/42 MAP kinase (ERK1/ERK2) were purchased from Promega. Preparation of cell lysate and protein blotting for detection of these phosphorylated MAP kinases were conducted according to the manufacturer's instructions. Mouse N1E-115 neuroblastoma cells exhibit neurite outgrowth in response to serum deprivation (8Jalink K. van Corven E.J. Hengeveld T. Morii N. Narumiya S. Moolenaar W.H. J. Cell Biol. 1994; 126: 801-810Crossref PubMed Scopus (577) Google Scholar, 9Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar), which is strongly affected by the substrates to which the cells adhere in the extracellular matrix (10Sarner S. Kozma R. Ahmed S. Lim L. Mol. Cell. Biol. 2000; 20: 158-172Crossref PubMed Scopus (108) Google Scholar, 20van Leeuwen F.N. Kain H.E.T. van der Kammen R.A. Michiels F. Kranenburg O.W. Collard J.G. J. Cell Biol. 1997; 139: 797-807Crossref PubMed Scopus (319) Google Scholar). We observed neurite outgrowth of cells grown on laminin-coated plates under serum-free conditions. In this condition, cells became flattened and then gradually extended neurites within 8 h, and ∼88% of the cells possessed neurites after 16 h (Fig. 1 B; see also Fig. 3). The number of neurite-bearing cells grown on noncoated plates, however, was quite low (8.6 ± 3.0% at 8 h and 18.3 ± 5.2% at 16 h). Sarner et al. (10Sarner S. Kozma R. Ahmed S. Lim L. Mol. Cell. Biol. 2000; 20: 158-172Crossref PubMed Scopus (108) Google Scholar) found that pre-treatment of N1E-115 cells with β1 integrin antibody blocks adhesion and neurite outgrowth of the cells plated on laminin-coated glass slide in serum-free condition, suggesting that β1 integrin is involved in both adhesion and neuritogenesis of N1E-115 cells grown on a laminin matrix. We also examined the effect of β1 integrin antibody on neurite outgrowth in serum-starved N1E-115 cells grown on the laminin-coated plastic plate. Cells were briefly pretreated for 7 min with varying concentrations of an antibody directed against β1integrin and then seeded on laminin-coated plates under serum-free conditions. As shown in Fig. 1, higher concentrations of antibody significantly blocked adhesion. On the other hand, lower concentrations of antibody (0.3 and 1.0 μg/ml) did not affect cell adhesion but significantly inhibited neurite outgrowth 8 and 16 h after antibody-treatment. Our observations are similar to those of Sarneret al. (10Sarner S. Kozma R. Ahmed S. Lim L. Mol. Cell. Biol. 2000; 20: 158-172Crossref PubMed Scopus (108) Google Scholar). Thus, β1 integrin is involved in both adhesion and neuritogenesis of N1E-115 cells grown on a laminin matrix under serum-free conditions.Figure 3Time course of neurite outgrowth in parental and DN-ILK-transfected N1E-115 cells after seeding on laminin-coated plates in the presence and absence of serum. Photomicrographs were taken 8 h (b and e) and 16 h (a, c, d, and f) after plating. Parental (a–c) and DN-ILK-transfected (d-f) cells were seeded on laminin-coated plates and cultured in the presence of 20% FBS (a and d) and in the absence of FBS (b, c, e, and f). Parental cells cultured in serum-free conditions exhibited extensive neurite outgrowth. In contrast, DN-ILK-transfected cells cultured in serum-free medium exhibited less neurite outgrowth.View Large Image Figure ViewerDownload (PPT) ILK is a serine/threonine protein kinase that interacts directly with the cytoplasmic domain of the β1 integrin subunit (13Hannigan G.H. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar) and whose kinase activity is modulated by cell-extracellular matrix interactions and insulin in a PI 3-kinase-dependent manner (17Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar). ILK has important roles in integrin- and growth factor-mediated signal transduction in several different cells, leading to regulation of cell adhesion, growth, migration, survival, proliferation, and differentiation (13Hannigan G.H. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (970) Google Scholar, 16Radeva G. Petrocelli T. Behrend E. Leung-Hagesteijn C. Filmus J. Slingerland J. Dedhar S. J. Biol. Chem. 1997; 272: 13937-13944Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 21Huang Y. Li J. Zhang Y. Wu C. J. Cell Biol. 2000; 150: 861-871Crossref PubMed Scopus (68) Google Scholar, 22Persad S. Attwell S. Gray V. Delcommenne M. Troussard A. Sanghera J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3207-3212Crossref PubMed Scopus (381) Google Scholar, 23Wu C. Keightley S.Y. Leung-Hagesteijn C. Radeva G. Coppolino M. Goicoechea S. McDonald J.A. Dedhar S. J. Biol. Chem. 1998; 273: 528-536Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Little is known, however, regarding the role of ILK in neuronal cells. Because β1 integrin is involved in both adhesion and neuritogenesis of N1E-115 cells grown on the laminin matrix (Fig. 1), we further examined whether ILK is expressed in the cells. We found that ILK is highly expressed in N1E-115 cells and also that the expression level of ILK did not change during neuronal differentiation (Fig.2). We first examined whether endogenous ILK is involved in integrin-dependent neurite outgrowth. To inactivate the endogenous ILK, cells were stably transfected with a kinase-deficient mutant of ILK (DN-ILK) that behaves as a dominant negative (17Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (950) Google Scholar). Based on the results obtained from immunoblotting (Fig. 4, inset), the expression level of DN-ILK protein in DN-ILK-transfected cells could be estimated to be at least twice more than that of endogenous ILK protein. As shown in Figs. 3 and4, neurite outgrowth of the DN-ILK-transfected cells was significantly inhibited compared with that of untransfected parental cells 8 and 16 h after seeding on the laminin matrix under serum-free conditions. This result suggests that the kinase activity of endogenous ILK is critical for neurite outgrowth in serum-starved N1E-115 cells grown on a laminin matrix. We also examined the effect of transient expression of the wild type ILK on neurite outgrowth of the cells. Overexpression of the wild type ILK stimulated neurite outgrowth in the cells even grown on noncoated plates (Fig. 5). We next examined whether ILK is activated after seeding on a laminin matrix under serum-free conditions. As shown in Fig. 6, the ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells did not change after cell attachment to the laminin. Maximal stimulation of ILK activity in the parental cells occurred 60 min after plating, and then rapidly disappeared. Moreover, ILK activation was prevented by treatment of the cells with LY294002, a specific inhibitor of PI 3-kinase (24Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Thus, ILK activation following cell attachment to the laminin under serum-free conditions is PI 3-kinase-dependent. These results suggest that transient activation of ILK activity after cell attachment to a laminin matrix is required for neurite outgrowth in serum-starved N1E-115 cells.Figure 5Effect of transient expression of wild type ILK on neurite outgrowth in N1E-115 cells grown on the noncoated plate. N1E-115 cells were seeded at a density of 5 × 104/dish onto 35-mm noncoated culture dishes and grown in DMEM containing 10% FBS. At 20 h following plating, cells were transfected with the plasmid containing the wild type ILK cDNA (Wild-type ILK) using LipofectAMINE transfection reagent, as described under "Experimental Procedures." Control cells were transfected with the empty plasmid (Vector). At 18 and 36 h following the transfection, the morphological changes produced by the transfection of the plasmid were examined using fluorescence microscopy. At least 100 randomly selected GFP-positive cells were assessed, and the cells that have a process longer than 2-fold the length of a cell body were categorized as being neurite bearing cells. The neurite bearing cell was assessed as the percentage of the total number of GFP-expressing cells. Each experiment was repeated at least three times. Values are the means ± S.D.View Large Image Figure ViewerDownload (PPT)Figure 6Stimulation of ILK activity after cell adhesion on laminin in serum-free condition. Cells were seeded on laminin-coated plates in serum-free medium and cultured for the indicated time periods. The cells were lysed, and ILK was immunoprecipitated from cell extracts. ILK activity was determined using myelin basic protein as an exogenous substrate, as described under "Experimental Procedures" (top). To examine the involvement of PI 3-kinase in the stimulation of ILK activity, cells were treated with 40 μm LY294002, a specific inhibitor of PI 3-kinase, for 1 h by direct addition to the culture medium. Total ILK and DN-ILK proteins in the immunoprecipitates were detected by immunoblotting with an anti-ILK antibody that recognizes both ILK and DN-ILK proteins, as described under "Experimental Procedures" (bottom).View Large Image Figure ViewerDownload (PPT) Activation of the MEK/ERK pathway is required for NGF-induced neuronal differentiation in PC12 cells (25Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1854) Google Scholar,26Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Unlike the NGF-induced neuronal differentiation in PC12 cells, however, activation of MEK and/or ERK was not detected in the integrin-dependent neuronal differentiation of N1E-115 cells (Fig. 7, A andB). We next examined the possible involvement of another member of the MAP kinase family, p38 MAP kinase, in the signaling pathway of integrin-dependent neuronal differentiation of N1E-115 cells. As shown in Fig. 7 (A and C), the p38 MAP kinase in the parental control cells was transiently activated after seeding on the laminin matrix under serum-free conditions. The activation of p38 MAP kinase in the parental cells reached a maximum level within 60 min and then declined rapidly. Weak activation was still detected 90 min after plating. Thus, the time course of the activation of p38 MAP kinase was very similar to that of the ILK. In contrast, activation of p38 MAP kinase in the DN-ILK-transfected cells was not detected (Fig. 7, A and C). Thus, endogenous ILK is somehow involved in the activation of p38 MAPK. These results suggest that the ILK activation after cell attachment to laminin is necessary for the activation of p38 MAP kinase in serum-starved N1E-115 cells. To evaluate the specific role of the p38 MAP kinase signaling pathway in integrin-dependent neurite outgrowth of N1E-115 cells, the cells were treated for 3 h with varying concentrations of SB203580, a specific inhibitor of p38MAP kinase (27Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1981) Google Scholar). SB203580 was applied after seeding the cells on laminin-coated plates under serum-free conditions. Treatment was terminated by changing the medium. As shown in Fig. 8, treatment of the cells with SB203580 markedly, but not completely, inhibited integrin-dependent neurite outgrowth in a dose-dependent manner with a maximal inhibition obtained at 10 μm. PD98059, a specific inhibitor of MEK (26Pang L. Sawada T. Decker S.J. Saltiel A.R. J. Biol. Chem. 1995; 270: 13585-13588Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar), did not affect neurite outgrowth (Fig. 8). On the other hand, the same treatment of DN-ILK-transfected cells with SB203580 did not affect neurite outgrowth (Fig. 9), suggesting that p38 MAP kinase is not involved in ILK-independent neurite outgrowth. Moreover, SB203580 (10 μm) maximally blo
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