The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m503707200
ISSN1083-351X
AutoresTatsuya Ozawa, Norie Araki, Shunji Yunoue, Hiroshi Tokuo, Liping Feng, Siriporn Patrakitkomjorn, Toshihiro Hara, Yasuko Ichikawa, Kunio Matsumoto, Kiyotaka Fujii, Hideyuki Saya,
Tópico(s)Neurofibromatosis and Schwannoma Cases
ResumoNeurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients. Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients. Neurofibromatosis type 1 (NF1), 2The abbreviations used are:NF1neurofibromatosis type 1DNdominant negativeGAPGTPase-activating proteinGRDGAP-related domainLIMKLIM-kinaseMAPKmitogen-activated protein kinasePI3Kphosphatidylinositol 3-kinaseRTreverse transcriptaseADFactin-depolymerizing factorROCKRho-associated, coiled-coilforming protein kinasesiRNAsmall interfering RNALPAlysophosphatidic acid 2The abbreviations used are:NF1neurofibromatosis type 1DNdominant negativeGAPGTPase-activating proteinGRDGAP-related domainLIMKLIM-kinaseMAPKmitogen-activated protein kinasePI3Kphosphatidylinositol 3-kinaseRTreverse transcriptaseADFactin-depolymerizing factorROCKRho-associated, coiled-coilforming protein kinasesiRNAsmall interfering RNALPAlysophosphatidic acid also called von Recklinghausen disease, is autosomal dominant and one of the most common inherited disorders, affecting 1 in 3500 individuals (1Stephens K. Riccardi V.M. Rising M. Ng S. Green P. Collins F.S. Rediker K.S. Powers J.A. Parker C. Donis-Keller H. Genomics. 1987; 1: 353-357Crossref PubMed Scopus (21) Google Scholar). The phenotype of NF1 is highly variable, with several organ systems being affected, including bones, skin, irises, and central and peripheral nervous systems. The disease commonly manifests with "café au lait" macules in the skin, iris Lisch nodules, and learning disability (2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 3Bernards A. Biochim. Biophys. Acta. 1995; 1242: 43-59PubMed Google Scholar). The hallmark of NF1 is benign tumors that develop in the peripheral nervous system accompanied by an increased risk of malignancies.The NF1 gene lies on chromosome 17q11.2 and encodes a large 2818- amino acid protein, termed neurofibromin. Sequence analysis of neurofibromin revealed that a region centered around the 360 amino acids encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP), which interacts with Ras and promotes hydrolysis of Rasbound GTP (active form) to GDP (inactive form), resulting in inactivation of the Ras protein. Accordingly, loss and/or mutations of neurofibromin elevate Ras activity and are followed by activation of various Ras effectors. Ras activation has been considered to be the causative event for tumor formation and other clinical manifestations in NF1 patients (2Cichowski K. Jacks T. Cell. 2001; 104: 593-604Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar, 3Bernards A. Biochim. Biophys. Acta. 1995; 1242: 43-59PubMed Google Scholar).Recent studies have suggested that neurofibromin has additional functions besides regulating cell proliferation via Ras pathways. For instance, mast cells in Nf+/- mice exhibited increased motility through hyperactivation of the Ras-PI3K-Rac2 pathway in response to KitL, which is secreted by homozygous Nf1 mutant (Nf1-/-) Schwann cells, leading to formation of cell aggregations, which become neurofibromas (4Yang F.C. Ingram D.A. Chen S. Hingtgen C.M. Ratner N. Monk K.R. Clegg T. White H. Mead L. Wenning M.J. Williams D.A. Kapur R. Atkinson S.J. Clapp D.W. J. Clin. Invest. 2003; 112: 1851-1861Crossref PubMed Google Scholar, 5Ingram D.A. Hiatt K. King A.J. Fisher L. Shivakumar R. Derstine C. Wenning M.J. Diaz B. Travers J.B. Hood A. Marshall M. Williams D.A. Clapp D.W. J. Exp. Med. 2001; 194: 57-69Crossref PubMed Scopus (112) Google Scholar). In addition, as we have shown previously, neurofibromin regulates neuronal differentiation via its GAP function. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after nerve growth factor stimulation and were correlated with the down-regulation of Ras activity during neurite extension (6Yunoue S. Tokuo H. Fukunaga K. Feng L. Ozawa T. Nishi T. Kikuchi A. Hattori S. Kuratsu J. Saya H. Araki N. J. Biol. Chem. 2003; 278: 26958-26969Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Although cell migration and neurite extension are known to be regulated by cytoskeletal reorganization in cells, the molecular mechanisms by which neurofibromin is involved in these cytoskeletal dynamics are understood poorly.Coordinated regulation of actin cytoskeleton dynamics is required for several fundamental cellular events such as cell movement, cell adhesion, and cytokinesis. Actin reorganization is often initiated by extracellular stimuli and is regulated by diverse actin-binding proteins. Among these, the actin-depolymerizing factor (ADF)/cofilin family proteins play an essential role in promoting actin depolymerization at pointed ends and severing long actin filaments, which leads to rapid turnover of actin filaments (7Pollard T.D. Borisy G.G. Cell. 2003; 112: 453-465Abstract Full Text Full Text PDF PubMed Scopus (3234) Google Scholar). In mammals, the activity of ADF/cofilin is repressed by phosphorylation at Ser3, leading to actin cytoskeletal reorganization. Four kinases responsible for this phosphorylation have been identified: LIM-kinase 1 and 2 (LIMK1/2) and TES-kinase 1 and 2. LIMK1/2 are serine/threonine/tyrosine kinases characterized structurally by two NH2-terminal LIM domains and a PDZ domain. Recent studies have revealed that activities of LIMK are regulated by the downstream effectors of Rho family GTPases. LIMK1 is phosphorylated specifically and activated by p21-activated kinase 1 (PAK1) that is downstream of the Rac1 and Cdc42 GTPases. On the other hand, LIMK2 is specifically phosphorylated and activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) downstream of RhoA. In addition, both LIMK are also activated by myotonic dystrophy kinase-related Cdc42-binding kinase α, which is downstream of Cdc42. Taken together, the Rho family-associated signal transduction pathways, such as Cdc42/Rac-PAK1-LIMK1, Rho-ROCK-LIMK2, and Cdc42-myotonic dystrophy kinaserelated Cdc42-binding kinase α-LIMK, play distinct roles in the regulation of cofilin-mediated actin dynamics (8Sumi T. Matsumoto K. Takai Y. Nakamura T. J. Cell Biol. 1999; 147: 1519-1532Crossref PubMed Scopus (310) Google Scholar, 9Sumi T. Matsumoto K. Shibuya A. Nakamura T. J. Biol. Chem. 2001; 276: 23092-23096Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 10Edwards D.C. Sanders L.C. Bokoch G.M. Gill G.N. Nat. Cell Biol. 1999; 1: 253-259Crossref PubMed Scopus (835) Google Scholar, 11Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar, 12Yang N. Higuchi O. Ohashi K. Nagata K. Wada A. Kangawa K. Nishida E. Mizuno K. Nature. 1998; 393: 809-812Crossref PubMed Scopus (1049) Google Scholar, 13Arber S. Barbayannis F.A. Hanser H. Schneider C. Stanyon C.A. Bernard O. Caroni P. Nature. 1998; 393: 805-809Crossref PubMed Scopus (1148) Google Scholar, 14Toshima J. Toshima J.Y. Takeuchi K. Mori R. Mizuno K. J. Biol. Chem. 2001; 276: 31449-31458Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 15Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Mol. Biol. Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (220) Google Scholar).In this study, to further elucidate the functions of neurofibromin, we attempted to reduce the expression of neurofibromin by applying RNA interference in cultured human cells. Our results demonstrated that the acute knockdown of neurofibromin stimulates phosphorylation of cofilin by LIMK2 and induces characteristic morphological changes with abnormal actin stress fiber reorganization and formation of gross cell aggregates in matrix. We propose that neurofibromin plays a significant role in cell motility by regulating the dynamics and reorganization of actin filaments via the Rho-ROCK-LIMK2-cofilin pathway.EXPERIMENTAL PROCEDURESCell Culture and Transfections—HeLa and HT1080 cells were cultured under 5% CO2 at 37 °C in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (Sigma) with 10% fetal bovine serum. For transient transfection of plasmids, cells in 6-well plates were transfected using a FuGene6 transfection reagent following the manufacturer's instructions (Roche Applied Science).Plasmids and Constructs—Mammalian expression plasmids for NF1-GTPase-activating protein-related domain (GRD) types 1 and 2 (pcDNA3-FLAG-GRD1 and pcDNA3-FLAG-GRD2) were prepared as described previously (6Yunoue S. Tokuo H. Fukunaga K. Feng L. Ozawa T. Nishi T. Kikuchi A. Hattori S. Kuratsu J. Saya H. Araki N. J. Biol. Chem. 2003; 278: 26958-26969Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). An expression plasmid for a nonphosphorylatable form of cofilin (pcDNA3-FLAG-CofilinS3A) was previously constructed (8Sumi T. Matsumoto K. Takai Y. Nakamura T. J. Cell Biol. 1999; 147: 1519-1532Crossref PubMed Scopus (310) Google Scholar). The pEF-BOS-hemagglutinin RhoN19 plasmid and the pBj-1/DN-Ras(S17N) plasmid were kindly provided by Dr. K. Kaibuchi (Nagoya University, Japan) and Dr. A. Kikuchi (Hiroshima University, Japan), respectively.Antibodies and Chemicals—Polyclonal antibodies against the C-terminal (D) portion of neurofibromin, LIM kinase 2 (LIMK2), RhoA, phospho-cofilin (Ser3), and a monoclonal antibody against H-Ras were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An anti-hemagglutinin monoclonal antibody was obtained from Roche Applied Science. Polyclonal antibodies against Akt, phospho-Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, LIM kinase 1 (LIMK1), cofilin, and phospho-cofilin (Ser3) were purchased from Cell Signaling Technology. Monoclonal antibody against paxillin was purchased from Transduction Laboratories. Monoclonal antibody against Ras and RalA were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal antibodies against the β-actin and polyclonal antibodies against LIMK1, cofilin, and FLAG came from Sigma. Secondary antibodies linked to horseradish peroxidase and Cy5 were purchased from Amersham Biosciences. Texas Red, rhodamin phalloidin, and Alexa Fluor 488 phalloidin were from Molecular Probes, Inc. (Eugene, OR). Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitors U0126 and PD98059 were purchased from Promega and Cell Signaling Technology, respectively. The PI3K inhibitor LY294002 and wortmannin were from Sigma. ROCK inhibitors Y27632 and HA1077 were supplied by Calbiochem and Upstate Biotechnology, respectively. Dispase was purchased from BD Biosciences.siRNA—We designed the following three target sequences for human NF1 siRNA: 609AAC TTC GGA ATT CTG CCT CTG629 and 827AAG GCG GTT CAG TTA GCA GTT847 and 3924AAC TAG CTC GAG TTC TGG TTA3944. Annealing of the component strands of each siRNA and transfection were performed as described (16Elbashir S.M. Lendeckel W. Tuschl T. Genes Dev. 2001; 15: 188-200Crossref PubMed Scopus (2678) Google Scholar, 17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8081) Google Scholar). Transfection was performed using Oligofectamine reagent (Invitrogen) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The above three siRNA oligonucleotides gave similar results. In addition, we designed target sequences for LIMK1 siRNA (1276AAG AGC ATG GAC AGC CAG TAC1296) for LIMK2 siRNA (460AAC TAC GCC ACC ACT GTG CAA480) and for RalA siRNA (157AAG AAG GTA GTG CTA GAT GGG177). Double-stranded RNA targeting the luciferase gene (GL-2) (17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8081) Google Scholar) and the rodent NF1 gene (rNF1 siRNA; 249AAC AAG GAG TGT CTG ATC AAC269) were used as controls.RT-PCR Analysis of siRNA—To evaluate NF1 mRNA expression after RNA interference, we performed RT-PCR using a set of primers: 5′-CGTGAAGGAAACCAGCATGCAGCT-3′ (NFS1-S) and 5′-TGTCAGCTGCCTACTTCCTCCATG-3′ (NFS1-AS). β-Actin primers, 5′-TCCCTGGAGAAGAGCTACGAGC-3′ (ACT-S) and 5′-GTAGTTTCGTGGATGCCACAGG-3′ (ACT-AS), were used as internal controls. First strand cDNA, which served as the PCR template, was synthesized from 2 μg of total RNA purified using an RNeasy minikit (Qiagen). The reverse transcription (RT) reaction was performed using an oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen). PCR was performed with 1 μl of the RT reaction, 1.25 units of rTaq DNA polymerase (Takara), 2 mm MgCl2, and 0.8 mm dNTP mixture in a final volume of 50 μl. PCR conditions were 94 °C, involving a 2-min initial denaturation step followed by 25 cycles at 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min. PCR products were resolved by electrophoresis in a 1.5% agarose gel with ethidium bromide.Small G-protein Pull-down Assays—Ras activity was measured by detecting Ras protein bound to the Ras-binding domain of Raf-1 immobilized to agarose with a Ras activation assay kit (Upstate Biotechnology) used according to the manufacturer's instructions and as described previously (18de Rooij J. Bos J.L. Oncogene. 1997; 14: 623-625Crossref PubMed Scopus (420) Google Scholar). The activity of Ral was also measured by a method similar to the Ras activity assay. In place of the Raf-1 Ras-binding domain, the Ral binding domain of Ral-BP1 (Ral-BP1 Ral-binding domain) was used to measure Ral activity (Upstate Biotechnology).Immunofluorescence—HeLa or HT1080 cells grown on a 35-mm culture dish were fixed with 4% paraformaldehyde/phosphate-buffered saline for 15 min at room temperature and then permeabilized with 0.2% Triton X-100/phosphate-buffered saline for 5 min. After being washed with phosphate-buffered saline, the cells were incubated with primary antibodies diluted in phosphate-buffered saline containing 0.2% bovine serum albumin, followed by a secondary antibody conjugated with a fluorescent dye for 60 min at room temperature, respectively. Analysis was performed by confocal microscopy (Fluoview, FV300; Olympus).Matrigel Assays—HeLa or HT1080 cells were transfected with siRNA and/or plasmids. At 24 h after transfection, ∼5000 cells were placed on a layer of Matrigel (3.5 mg/ml in culture medium; BD Biosciences) in a 35-mm culture dish. The thickness of the Matrigel layer was usually ∼1000 μm. Cells on the Matrigel were observed by phase-contrast microscopy.For time lapse video analysis, cells were placed on Matrigel in ΔT 0.15-mm dishes (Biopteches). Before observation, the culture medium was replaced with dye-free L-15 medium, pH 7.2 (Sigma), supplemented with 10% fetal calf serum and overlaid with mineral oil. Dishes were maintained at 37 °C using the ΔT Culture Dish System (Biopteches) and imaged on an Olympus IX 70 microscope equipped with a sensitive SenSys-1401E CCD camera (Roper Scientific). Images were obtained using a ×20 UPlan Apo objective (Olympus). The camera, shutters, and filter wheel were controlled by MetaMorph imaging software (Universal Imaging), and the images were collected every 5 min with exposure times of 100 ms.RESULTSSuppression of NF1 Expression Leads to Changes in Cell Shape and Cytoskeletal Organization—To examine the roles of neurofibromin in cell growth and morphology, we attempted to reduce the expression of neurofibromin in HeLa cells by an RNA interference strategy. The levels of both NF1 mRNA and protein were suppressed beginning at 9 h after siRNA transfection and were reduced up to 95% at 72 h. Three siRNA oligonucleotides against NF1 showed similar effects in reducing expression of neurofibromin (Fig. 1, A and B). In addition, immunocytochemical analysis also confirmed the reduction of neurofibromin expression in cytoplasm by NF1 siRNA transfection, whereas siRNA controls showed no effects (Fig. 1C).Immunocytochemical analysis showed that NF1 siRNA induced dynamic morphological changes according to the level of neurofibromin suppression with time. Neurofibromin-depleted HeLa cells tended to display the flattened shapes having excessive actin stress fiber formation from 12 h after transfection, and these changes were enhanced from 24 to 48 h (Fig. 2, J-O). Control siRNA induced no discernible alterations in cell morphology and cytoskeletal structure during the observation period (Fig. 2, D-I). Similar morphological changes and actin stress fiber formation were observed when other siRNA oligonucleotides against NF1 were used (supplemental Fig. S1).FIGURE 2Morphological changes and excessive actin stress fiber formation in neurofibromin-depleted cells. HeLa and HT1080 cells were transfected with control or NF1 (827) siRNA and stained with Alexa 488 phalloidin and anti-paxillin antibody at 24 h (D-F, d-f, J-L, j-l) or 48 h (G-I, g-i, M-O, m-o) after treatment. Cells without siRNA treatment are also shown in A-C and a-c, respectively. The white arrows indicate excessive actin stress fiber formation in neurofibromin-depleted cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The formation of actin stress fibers is usually accompanied by assembly of focal adhesions at cell margins. Immunostaining of paxillin, a major component of focal adhesions, revealed that NF1 siRNA promotes formation of focal adhesions at 24 and 48 h after transfection, when excess actin stress fibers were formed (Fig. 2, K and N). In contrast, control siRNA had no effect on formation of stress fibers and focal adhesions (Fig. 2, E and H).We examined the effects of neurofibromin depletion on another NF1-relevant tumor cell, HT1080 human fibrosarcoma. HT1080 fibrosarcoma cells exhibit similar characteristics to malignant peripheral nerve sheath tumors (MPNSTs) frequently found in NF1 patients. As observed in HeLa cells, siRNA treatment effectively reduced the expression of neurofibromin (supplemental Fig. S2) and induced dynamic morphological changes with the robust stress fiber formation and focal adhesion assembly in HT1080 cells (Fig. 2, j-o). These observations suggest that neurofibromin plays a role in regulation of cytoskeletal organization.Effect of NF1 siRNA on Cell Migration in Extracellular Matrix—Migration of cells in the extracellular matrix requires morphological changes with dynamic remodeling of the actin cytoskeleton. Therefore, we performed a Matrigel assay to assess the growth and motility of neurofibromin-depleted cells in the extracellular matrix. The growth of neurofibromin-depleted HeLa cells was about 1.5-2.0 times faster than that of control cells after contact with Matrigel for 3 days (data not shown). However, the number of colonies formed in the Matrigel was obviously fewer in neurofibromin-depleted cells compared with control cells (Fig. 3A), because neurofibromin-depleted cells tended to generate cell aggregation. After culturing for 24 h, the control cells became rounded shape and migrated into the Matrigel (Fig. 3B, a and e) and then formed a small spherical mass in Matrigel at 96 h in culture (Fig. 3B, i and m). In contrast, ∼30% of the neurofibromin-depleted cells showed an elongated morphology (Fig. 3B, b-d and f-h). The neurofibromin-depleted cells migrated into the Matrigel and formed gross irregular and insular spherical masses around 96 h in culture (Fig. 3B, j-i and n-p). Time lapse, real time recording analysis revealed that neurofibromin-depleted cells actively move and rapidly invade in the Matrigel and aggregate to form gross spherical masses (Fig. 3C, lower panels, and supplemental Movie 2), whereas control cells move slowly and experience less aggregation (Fig. 3C, upper panels, and supplemental Movie 1). HT1080 cells treated with NF1 siRNA also tended to form cell aggregation in the Matrigel matrix (Supplemental Fig. S3). These results indicate that the reduction of neurofibromin by siRNA promotes peculiar cell migration and cell-cell adhesion, forming cell aggregates in the Matrigel matrix.FIGURE 3Depletion of neurofibromin promotes the formation of gross spherical cell aggregates in Matrigel matrix.A, the number of colonies that formed on Matrigel matrix. HeLa cells were transfected with either control or three NF1 siRNA on normal culture dish. After 24 h, 5000 cells were seeded onto a Matrigel-precoated dish. The number of colonies formed at 96 h after the replating was counted under the phase-contrast microscope. Each datum represents the average of three independent experiments. The error bars represent S.D. B, neurofibromin-depleted cells formed gross irregular spherical masses. Transfected cells replated on the Matrigel matrix were examined by the phase-contrast microscope at × 100 and × 200 magnifications at 24 h (a-h) and 96 h (i-p) after replating. C, the process of aggregation of neurofibromin-depleted cells on the Matrigel matrix was observed by time lapse DIC video microscopy (supplemental Movies 1 and 2). Cells were transfected with control siRNA (upper panels; Movie 1) and NF1 (609) siRNA (lower panels; Movie 2) on culture dishes and then were seeded onto Matrigel-precoated dishes. Time lapse movie recording was started 12 h after the replating, and images were collected every 5 min for a period of 2 days.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Morphological Changes Induced by Reduction of NF1 Expression Are Due to Cofilin Phosphorylation—The morphological changes and active motility of neurofibromin-depleted cells suggested that neurofibromin is involved in regulation of actin cytoskeletal reorganization. Thus, we investigated various signal molecules that regulate actin cytoskeletal reorganization in the neurofibromin-depleted cells and found that phosphorylation of ADF/cofilin is significantly elevated with time in the cells, whereas their levels in control siRNA transfectants had no changes (Fig. 4A and supplemental Fig. S4). The activity of ADF/cofilin is negatively regulated by phosphorylation at Ser3 through a RhoGTPase-ROCK-LIMK pathway that is activated in response to extracellular stimuli, such as lysophosphatidic acid (LPA), and followed by stabilization and accumulation of actin filaments (11Maekawa M. Ishizaki T. Boku S. Watanabe N. Fujita A. Iwamatsu A. Obinata T. Ohashi K. Mizuno K. Narumiya S. Science. 1999; 285: 895-898Crossref PubMed Scopus (1269) Google Scholar). Therefore, we hypothesized that the excessive actin stress fiber formation in neurofibromin-depleted cells was due to the enhancement of ADF/cofilin phosphorylation through Rho activation. It was shown that components of serum, such as LPA, activate Rho-GTPase, leading to the formation of actin stress fibers and focal adhesion, and subsequently Rho become inactivated in adherent cells (19Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar, 20Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1355) Google Scholar). Therefore, we investigated whether LPA and serum stimulation affect the level of phosphocofilin (Fig. 4B) and the formation of actin stress fibers in neurofibromin-depleted cells (Fig. 4C). Control cells after serum stimulation exhibited prompt cofilin dephosphorylation after transient phosphorylation with the enhancement of cortical actin (Fig. 4, B (upper panels) and C (b and c)), followed by attenuation to be consistent with the dephosphorylation of cofilin. In contrast, after serum stimulation, neurofibromin-depleted cells exhibited sustained levels of phosphorylated cofilin with the enhancement of actin stress fibers in the cell body (Fig. 4, B (upper panels) and C (g and h)). The induced actin stress fibers persisted, consistent with the sustained phosphorylation of cofilin. In addition, when cells were stimulated by LPA, control cells exhibited biphasic cofilin phosphorylation (dephosphorylation at 10 min and rephosphorylation at 60 min after the LPA treatment) with enhancement of cortical actin and fine stress fibers formed in the cell body (Fig. 4, B (lower panels) and C (d and e)), followed by the attenuation consistent with the dephosphorylation of cofilin. In contrast, neurofibromin-depleted cells exhibited a high level of phosphorylated cofilin with enhancement of actin stress fiber polymerization in the center of the cell body (Fig. 4, B (lower panels) and C (i and j)). These results supported our hypothesis that the sustained cofilin phosphorylation by serum and LPA stimulation induces the formation of stable actin stress fibers in cells where neurofibromin was down-regulated.FIGURE 4Promotion of cofilin phosphorylation in neurofibromin-depleted cells.A, effect of neurofibromin depletion on the levels of phosphocofilin. Either control or NF1 siRNA were transfected into HeLa cells. After 24 and 48 h, cells were harvested and examined for the levels of Ser(P)3-cofilin by immunoblotting analysis. B, neurofibromin-depleted cells exhibited a high level of phosphorylated cofilin. After 24 h of siRNA transfection in a serum-free condition, HeLa cells were stimulated with 10% serum or 10 μm LPA for the indicated periods and then examined for levels of Ser(P)3-cofilin by immunoblot analysis. C, enhancement of actin stress fiber formation in neurofibromin-depleted cells. After 24 h of siRNA transfection under a serum-free condition, HeLa cells were stimulated with serum or LPA at the indicated times and then stained with rhodamin phalloidin to visualize the actin cytoskeleton. D, the nonphosphorylatable cofilin S3A mutant suppresses excessive stress fiber formation in neurofibromin-depleted cells. HeLa cells co-transfected with FLAG-cofilin S3A and either control or NF1 siRNA were stained with Alexa 488 phalloidin and anti-FLAG antibody at 24 h after transfection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nonphosphorylatable Cofilin-S3A Mutant Rescues the Phenotypes of Neurofibromin-depleted Cells—To confirm whether NF1 siRNA-induced morphological effects are attributable to inactivation of cofilin, we co-transfected NF1 siRNA with a cofilin-S3A mutant that functions as a constitutively active form of cofilin. Immunocytochemical analysis after 24 h of co-transfection revealed that the cofilin-S3A mutant significantly suppresses excess fo
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