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Disabled-2 (Dab2) Mediates Transforming Growth Factor β(TGFβ)-stimulated Fibronectin Synthesis through TGFβ-activatedKinase 1 and Activation of the JNKPathway

2005; Elsevier BV; Volume: 280; Issue: 27 Linguagem: Inglês

10.1074/jbc.m501150200

1083-351X

Barbara A. Hocevar, Céline Prunier, Philip H. Howe,

Cancer-related gene regulation

The multifunctional cytokine transforming growth factor β (TGFβ)exerts many of its effects through its regulation of extracellular matrixcomponents, including fibronectin (FN). Although expression of both TGFβand FN are essential for embryonic development and wound healing in the adult,overexpression leads to excessive deposition of extracellular matrix observedin many fibroproliferative disorders. We previously have demonstrated thatTGFβ-stimulated FN induction requires activation of the c-Jun N-terminalkinase (JNK) pathway; however, the signaling molecules that link the TGFβreceptors to the JNK pathway remain unknown. We show here that the cytosolicadaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereasstable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mousefibroblasts and A10 rat aortic smooth muscle cells demonstrates that itsexpression is required for TGFβ-mediated FN induction. We demonstratethat TGFβ treatment stimulates the association of Dab2 with themitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellularTAK1 levels by transient double-stranded RNA oligonucleotide transfection aswell as overexpression of kinase-deficient TAK1 leads to abrogation ofTGFβ-stimulated FN induction. Furthermore, cell migration, anotherJNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We,therefore, delineate a signaling pathway proceeding from the TGFβreceptors to Dab2 and TAK1, leading to TGFβ-stimulated JNK activation, FNexpression, and cell migration. The multifunctional cytokine transforming growth factor β (TGFβ)exerts many of its effects through its regulation of extracellular matrixcomponents, including fibronectin (FN). Although expression of both TGFβand FN are essential for embryonic development and wound healing in the adult,overexpression leads to excessive deposition of extracellular matrix observedin many fibroproliferative disorders. We previously have demonstrated thatTGFβ-stimulated FN induction requires activation of the c-Jun N-terminalkinase (JNK) pathway; however, the signaling molecules that link the TGFβreceptors to the JNK pathway remain unknown. We show here that the cytosolicadaptor protein disabled-2 (Dab2) directly stimulates JNK activity, whereasstable small interfering RNA-mediated ablation of Dab2 in NIH3T3 mousefibroblasts and A10 rat aortic smooth muscle cells demonstrates that itsexpression is required for TGFβ-mediated FN induction. We demonstratethat TGFβ treatment stimulates the association of Dab2 with themitogen-activated protein kinase kinase kinase, TAK1. Attenuation of cellularTAK1 levels by transient double-stranded RNA oligonucleotide transfection aswell as overexpression of kinase-deficient TAK1 leads to abrogation ofTGFβ-stimulated FN induction. Furthermore, cell migration, anotherJNK-dependent response, is attenuated in NIH3T3-siDab2-expressing clones. We,therefore, delineate a signaling pathway proceeding from the TGFβreceptors to Dab2 and TAK1, leading to TGFβ-stimulated JNK activation, FNexpression, and cell migration. Fibronectin (FN), 1The abbreviations used are: FN, fibronectin; ECM, extracellular matrix;TGF, transforming growth factor; TAK1, TGFβ-activated kinase 1; TAB1,TAK1-binding protein; PTB, phosphotyrosine binding domain; si-, smallinterfering; TAKKW, kinase-deficient form of TAK1; Dab2, Disabled-2; MAPK,mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; TβRI andTβRII, TGFβ type I and type II, respectively; HA, hemagglutinin;GST, glutathione S-transferase.1The abbreviations used are: FN, fibronectin; ECM, extracellular matrix;TGF, transforming growth factor; TAK1, TGFβ-activated kinase 1; TAB1,TAK1-binding protein; PTB, phosphotyrosine binding domain; si-, smallinterfering; TAKKW, kinase-deficient form of TAK1; Dab2, Disabled-2; MAPK,mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; TβRI andTβRII, TGFβ type I and type II, respectively; HA, hemagglutinin;GST, glutathione S-transferase.a widely expressed component of the extracellular matrix (ECM), is a dimericglycoprotein whose ability to promote cell adhesion and migration plays a keyrole in signaling events that regulate cell growth and differentiation(1Geiger B. 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Consistent with thisobservation, mouse fibroblasts derived from Smad2-, Smad3-, andSmad4-deficient embryos retain the ability to induce FN synthesis afterTGFβ stimulation (23Piek E. Ju W.J. Heyer J. Escalante-Alcalde D. Stewart C.L. Weinstein M. Deng C. Kucherlapati R. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2001; 276: 19945-19953Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar,24Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itié A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Furthermore, efficientactivation of the Smad pathway may require activation of JNK, in thatfibroblasts that express antisense JNK oligonucleotides show decreasedTGFβ-stimulated Smad2 phosphorylation(25Utsugi M. Dobashi K. Ishizuka T. Masubuchi K. Shimizu Y. Nakazawa T. Mori M. Am. J. Respir. CellMol. Biol. 2003; 28: 754-761Crossref PubMed Scopus (77) Google Scholar), whereas JNK has beenshown to directly phosphorylate Smad3, leading to its activation and nuclearaccumulation (26Engel M. McDonnell M. Law B.K. Moses H.L. J. Biol. Chem. 1999; 274: 37413-37420Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar).Activation of JNK is triggered by dual phosphorylation of a TPY motif bythe upstream MAPK kinases MKK7 and MKK4, whereas activation of these MAPKkinases can be mediated through multiple MAPK kinase kinases including membersof the MAPK/extracellular signal-regulated kinase kinase (MEK) kinase (MEKK),mixed-lineage kinase, and apoptosis signal-regulating kinase families, Tpl2,and TGFβ-activated kinase 1 (TAK1)(27Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3593) Google Scholar). TAK1 was initiallyidentified as a kinase whose activity was stimulated by TGFβ and BMP4, aTGFβ superfamily member, resulting in phosphorylation of MKK4(28Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1169) Google Scholar). TAK1 has subsequentlybeen shown to be involved in interleukin 1- and Wnt-mediated signaltransduction(29Ishitani T. Kishida S. Hyodo-Miura J. Ueno N. Yasuda J. Waterman M. Shibuya H. Moon R.T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. Biol. 2003; 23: 131-139Crossref PubMed Scopus (469) Google Scholar, 30Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1010) Google Scholar, 31Smit L. Baas A. Kuipers J. Korswagen H. van de Wetering M. Clevers H. J. Biol. Chem. 2004; 279: 17232-17240Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar)and can activate the p38 pathway by phosphorylation of MKK3 and MKK6(32Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Activation of TAK1requires interaction with TAB1 (TAK1-binding protein)(33Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (516) Google Scholar), which also binds to theX-linked inhibitor of apoptosis(34Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar). TAB1 has been shown toassociate with the type I bone-morphogenetic protein receptor when theX-linked inhibitor of apoptosis is co-expressed, suggesting a possible link ofthe receptor complex to TAK1. However, this association may involve anotherinteracting protein since yeast two-hybrid analysis failed to demonstrate adirect interaction between X-linked inhibitor of apoptosis and type I BMPreceptor (34Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar).Dab2, a member of the Disabled gene family, is a widely expressed adaptormolecule shown to be involved in several receptor-mediated signaling pathways(11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar,35Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar,36Oleinikov A.V. Zhao J. Makker S.P. Biochem. J. 2000; 347: 613-621Crossref PubMed Scopus (131) Google Scholar). Disabled family memberspossess a highly conserved N-terminalphosphotyrosine-interacting/phosphotyrosine binding domain (PID/PTB), recentlyrenamed the DAB homology domain(37Yun M. Keshvara L. Park C-G. Zhang Y.-M. Dickerson J.B. Zheng J. Rock C.O. Curran T. Park H.-W. J. Biol. Chem. 2003; 278: 36572-36581Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), linked to divergentC-terminal sequences. Dab2 directly binds to several members of thelipoprotein receptor family through its PTB domain(35Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar,36Oleinikov A.V. Zhao J. Makker S.P. Biochem. J. 2000; 347: 613-621Crossref PubMed Scopus (131) Google Scholar,38Gotthardt M. Tromsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar), whereas its associationwith clathrin, the clathrin adaptor protein AP2, and myosin VI, mediated bysequences in the linker and C-terminal regions of Dab2, facilitatesclathrin-coated pit assembly and receptor-mediated endocytosis(35Morris S.M. Cooper J.A. Traffic. 2001; 2: 111-123Crossref PubMed Scopus (217) Google Scholar,39Mishra S.K. Keyel P.A. Hawryluk M.J. Agostinelli N.R. Watkins S.C. Traub L.M. EMBOJ. 2002; 21: 4915-4926Crossref PubMed Scopus (249) Google Scholar,40Morris S.M. Arden S.D. Roberts R.C. Kendrick-Jones J. Cooper J.A. Luzio J.P. Buss F. Traffic. 2002; 3: 331-341Crossref PubMed Scopus (198) Google Scholar). We have recentlydemonstrated that Dab2 serves as an adaptor molecule to link the TGFβreceptors to activation of the Smad pathway(11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar). Expression of Dab2restored Smad-dependent responses to a TGFβ-signaling-deficient mutantcell line. In addition, Dab2 was found to augment TGFβ-stimulated FNinduction, a JNK-dependent and Smad-independent response(11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar). We, therefore, wished toexplore the possibility that Dab2 could directly mediate TGFβ-stimulatedJNK activity, leading to induction of FN.We report here that stable small interfering RNA (siRNA)-mediated silencingof Dab2 in NIH3T3 mouse fibroblasts leads to decreased TGFβ-stimulated FNinduction, concomitant with decreased TGFβ-mediated JNK activation. Dab2is shown to directly stimulate JNK activity, which is blocked by expression ofa kinase-deficient form of TAK1, TAKKW. We also report that abrogation of TAK1expression or TAKKW overexpression leads to decreased TGFβ-stimulated FNprotein and mRNA induction, respectively. At a biological level we provideevidence suggesting that Dab2 is required for TGFβ-stimulated cellularmigration, a JNK-dependent response. Dab2, thus, provides a link between theactivated TGFβ receptors and TAK1, leading to activation of theJNK-signaling pathway.MATERIALS AND METHODSReagents—Recombinant TGFβ2 was generously provided byGenzyme (Cambridge, MA). SP600125 (JNK Inhibitor II) and SB203580 werepurchased from Calbiochem, and PD98059 was purchased from Alexis. Thefollowing primary antibodies were purchased from Santa Cruz Biotechnology:mouse α-HA (sc-7392), mouse α-myc (sc-40), rabbit α-TAK1(sc-7162), mouse α-TAK1 (sc-7967), rabbit α-HSP 90 (sc-7947), andrabbit α-JNK1 (sc-474). Mouse monoclonal antibody to Disabled-2/p96 waspurchased from BD Transduction Laboratories. The mouse α-FLAG-M2antibody and gelatin-agarose was obtained from Sigma. Rabbit polyclonalphospho-c-Jun (Ser63) antibody II was purchased from Cell SignalingTechnology, and rabbit α-rat FN antibody was from Invitrogen.Plasmid Construction—Construction of the FLAG-tagged humanDab2 in the RK5 plasmid has been described previously(11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar). The various domainconstructs of human Dab2 were generated by standard PCR methods using thefull-length construct as template and inserted into the RK5 vector containinga FLAG epitope. Sequences used for PCR amplification are available uponrequest. For construction of the stable siDab2 construct, forward andcomplementary reverse primers corresponding to mouse p96(5′-CAAATGGAGTCACCTCCTG-3′) flanked by AA at the 5′-end andTT at the 3′-end were synthesized, annealed, and ligated into the pSUPERexpression vector as described(41Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Crossref PubMed Scopus (3942) Google Scholar). The FLAG-tagged JNKconstruct was provided by Roger Davis(42Derijard B. Hibi M. Wu I.-H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2949) Google Scholar), and the HA-taggedwild-type TAK1, kinase-deficient TAK1 (TAKK63W), constitutively active TAK1(TAK1dN) (29Ishitani T. Kishida S. Hyodo-Miura J. Ueno N. Yasuda J. Waterman M. Shibuya H. Moon R.T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. Biol. 2003; 23: 131-139Crossref PubMed Scopus (469) Google Scholar), and Myc-taggedTAB1 (34Yamaguchi K. Nagai S. Ninomiya-Tsuji J. Nishita M. Tamai K. Irie K. Ueno N. Nishida E. Shibuya H. Matsumoto K. EMBO J. 1999; 18: 179-187Crossref PubMed Scopus (324) Google Scholar) were provided by E.Nishida. The pPur vector was purchased from Clontech.Cell Culture and Generation of Cell Lines—NIH3T3 and COS7cells were cultured in Dulbecco's modified Eagle's medium supplemented with10% newborn calf serum. A10 cells were cultured in Dulbecco's modified Eagle'smedium supplemented with 10% fetal calf serum. To generate cells that stablyexpress the siDab2 construct, NIH3T3 and A10 cells in 100-mm-diameter plateswere transfected with the siDab2 construct (10 μg) and pPur (2 μg)utilizing FuGENE 6 (Roche Diagnostics) or Lipofectamine PLUS (Invitrogen),respectively, as per the manufacturer's instructions. Stable transfectantswere selected in serum-containing Dulbecco's modified Eagle's mediumsupplemented with 1 μg/ml puromycin and maintained in the same media eitheras a pool (A10) or individual clones (NIH3T3). Expression levels of Dab2 wereassessed by Western analysis utilizing the monoclonal α-p96 antibody. Togenerate the 3T3-TAKKW cell line, NIH3T3 cells were co-transfected with TAKKW(10 μg) and pPur (2 μg) constructs using FuGENE 6 and selected andmaintained as a pool in Dulbecco's modified Eagle's medium containing 10%newborn calf serum and 1 μg/ml puromycin. Expression of the TAKKW constructwas verified by Western analysis utilizing a monoclonal α-HAantibody.Preparation of Cell Lysates, Immunoprecipitation, and Protein KinaseAssays—For immunoprecipitation and Western blot analysis, cellswere lysed in buffer D (20 mm Tris, pH 7.5, 1% Triton X-100, 10%glycerol, 137 mm NaCl, 2 mm EDTA, 25 mmβ-glycerophosphate, 1 mm Na3V04, andComplete EDTA-free protease inhibitor mixture; Roche Diagnostics), andimmunoprecipitation was carried out as previously described(21Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar). For JNK assays in COS7cells, transient transfection of the indicated constructs was performedutilizing FuGENE 6 (Roche Diagnostics) according to the manufacturer'sprotocols. After 48 h cells were lysed in buffer D. Equal amounts of cellularprotein (250 μg) were immunoprecipitated with α-FLAG antibody andcaptured on protein G-Sepharose beads. The beads were subsequently washedtwice with buffer D followed by 1 wash in kinase assay buffer (25mm HEPES, pH 7.5, 25 mm β-glycerophosphate, 25mm MgCl2, 2 mm dithiothreitol, 0.1mm Na3VO4, and Complete EDTA-free proteaseinhibitor mixture; Roche Diagnostics). Kinase reactions were performed in 30μl of kinase buffer containing 1 μg GST-c-Jun (Stratagene), 50μm ATP, and 2 μCi of [γ-32P]ATP. Reactionswere incubated at 30 °C for 20 min and terminated by the addition of anequal volume of 2× Laemmli sample buffer. Samples were resolved on 10%SDS/PAGE gels, dried, and visualized by autoradiography. The JNK assay inNIH3T3 and 3T3-siDab2 clones was performed as described previously(32Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) after stimulation with 5ng/ml TGFβ for various times. Reaction products were resolved on 10%SDS/PAGE gels, transferred to Immobilon membranes, and subjected to Westernblot analysis with α-phospho-c-Jun (Ser-63) antibody II.Fibronectin Protein Assay—TGFβ-stimulated FN inductionwas determined as previously described(21Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar), with the exception thatin some assays gelatin-agarose beads (Sigma) were used to precipitate35S-labeled FN from the media of labeled cells. Samples wereresolved on 6% SDS/PAGE gels, subjected to fluorography using Enhance(Amersham Biosciences), and visualized by autoradiography. The inhibitorsSP600125 (10 μm and 50 μm), SB203580 (10μm), or PD98059 (10 μm) were added simultaneouslywith TGFβ (2 ng/ml) at the initiation and maintained throughout the FNassay.Transient siRNA-mediated Silencing of TAK1—The negativecontrol, TAK1 pool, and individual double-stranded RNA oligos to TAK1 werepurchased from Dharmacon RNA Technologies (SMARTpool Plus, Q-004500-00-09) andresuspended in reagents provided by the manufacturer. NIH3T3 cells weretransfected in 6-well plates (2 × 105 cells/well) usingOligofectamine reagent (Invitrogen) with 200 nm concentrations eachof double-stranded RNA oligo per manufacturer's instructions. After 48 h ofsiRNA treatment, the FN assay was initiated and performed as described above.FN protein synthesis was analyzed from the media, whereas cell lysates fromthe same wells were analyzed for TAK1 expression by Western analysis.Northern Blot Analysis—Total RNA was isolated from NIH3T3,3T3-siDab2 clone 8, and 3T3-TAKKW cells after treatment with 5 ng/ml TGFβfor the various times indicated using TRIzol reagent (Invitrogen) per themanufacturer's instructions. Total RNA (40 μg) of each sample was resolvedin a 1.2% formaldehyde/agarose gel, transferred to nitrocellulose, andsubjected to Northern analysis utilizing probes to FN(45Tong L. Pav S. White D.M. Rogers S. Crane K.M. Cywin C.L. Brown M.L. Pargellis C.A. Nat.Struct. Biol. 1997; 4: 311-316Crossref PubMed Scopus (393) Google Scholar) and cyclophilin (1B15)(43Danielson P.E. Forss-Petter S. Brow M.A. Calavetta L. Douglass J. Milner R.J. Sutcliffe J.G. DNA (N. Y.). 1988; 7: 261-267Crossref PubMed Scopus (1032) Google Scholar) as previously described(21Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar).Wound Closure Assay—For the wound closure assay, confluentmonolayer cells in 60-mm plates were wounded by manual scraping with a pipettetip. Plates were washed several times to remove non-adherent cells and placedin complete media with or without TGFβ (5 ng/ml). Assessment of cellmigration into the wounded area was performed by microscopy after 24 h. Cellmigration of NIH3T3 and 3T3-siDab2 cells was quantitated utilizing theQCM™ 24-well colorimetric cell migration assay (Chemicon International)as suggested by the manufacturer. Briefly, 2 × 105 cellsresuspended in serum-free media were added to the upper chamber and allowed tomigrate through a membrane insert with an 8-μm pore size for 4 h toward 10%fetal bovine serum-containing media in the absence or presence of 5 ng/mlTGFβ. After the incubation period, the cells on the upper surface of themembrane were removed, cells that had migrated through the membrane werestained, and 5 random fields were counted by microscopy at 250×magnification.RESULTSDab2 Expression Is Required for TGFβ-stimulated FNInduction—We have previously shown that TGFβ-stimulated FNinduction is greatly abrogated in a TGFβ-signaling-deficient cell line,derived from the human HT1080 fibrosarcoma cell line, which expresses a mutantform of Dab2 (11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar,21Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar). We, therefore, wished totest whether Dab2 expression is required for TGFβ-mediated FN inductionin two different cell types that respond to TGFβ stimulation, namelyfibroblast and smooth muscle cells. To ablate the expression of Dab2, we choseto use stable expression of a siRNA specifically targeted to decreaseexpression of the p96 splice-form of Dab2, which we previously have shown torestore TGFβ signaling to a mutant cell line(11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar). We have previouslycharacterized this siRNA-Dab2 vector in several cell lines with regard to itsspecificity for the p96 form of Dab2(71Prunier C. Howe P.H. J.Biol. Chem. 2005; 280: 17540-17548Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Stable introduction ofthe siDab2 construct into mouse fibroblast NIH3T3 and rat A10 aortic smoothmuscle cells was performed. As shown by Western analysis inFig. 1A, the siDab2construct was able to decrease the p96, but not p67, form of Dab2 expressionin individual clones of NIH3T3 cells (left panel) and in a pool ofA10 (right panel) cells to varying extents. Analysis of these clonesfor induction of FN synthesis after TGFβ stimulation reveals a dosedependence of Dab2 expression for both basal and TGFβ-mediated FNinduction, in that clones that express the lowest levels of Dab2(i.e. clone 8 and 9) also demonstrate the lowest basal andTGFβ-stimulated FN protein induction(Fig. 1B). FN proteinexpression was also assessed in wild-type and the siDab2-expressing A10 cellsafter stimulation with TGFβ for 4 and 24 h. Similarly to NIH3T3 cells,TGFβ-mediated FN induction was markedly attenuated in A10 cells withdecreased Dab2 expression (Fig.1C). These results, thus, demonstrate that Dab2expression is required for TGFβ-mediated FN induction in two differentcell types that can be physiologically stimulated to express FN afterTGFβ treatment, which are fibroblast and smooth muscle cells.Dab2 Directly Stimulates JNK Activity—We have previouslyshown that TGFβ stimulation of human HT1080 fibrosarcoma cells leads toactivation of the JNK pathway and that TGFβ-stimulated induction of FNcould be blocked by overexpression of dominant-negative JNK and MKK4(21Hocevar B.A. Brown T.L. Howe P.H. EMBO J. 1999; 18: 1345-1356Crossref PubMed Google Scholar). We, therefore, wished toassess whether Dab2 could directly activate the JNK pathway, since we observedthat TGFβ-stimulated FN induction was dependent on expression of Dab2. Inaddition, we wished to assess the contribution of the individual domains ofDab2 to JNK activation, because we have previously demonstrated thatrestoration of TGFβ signaling to a TGFβ-signaling-deficient cellline requires both the N-terminal PTB and C-terminal proline-rich domains ofDab2 (11Hocevar B.A. Smine A Xu X.-X. Howe P.H. EMBO J. 2001; 20: 2789-2801Crossref PubMed Scopus (200) Google Scholar). To assess this,COS7 cells were transiently transfected with JNK along with full-length Dab2or various constructs containing the different domains of Dab2(Fig. 2, A andC). As shown in Fig.2B, expression of full-length Dab2 is capable ofefficiently stimulating JNK activity that was assessed by the ability of JNKto phosphorylate GST-c-Jun in an in vitro kinase assay. Furthermore,the construct bearing only the C-terminal portion of Dab2, designated #323consisting of amino acids 323–770, stimulates JNK activity to the sameextent as full-length (Fig.2B). Expression of a construct bearing the proline-richdomain of Dab2, #534, consisting of amino acids 534–770, however, is notsufficient to stimulate JNK activity even though it is expressed at the samelevel as full-length Dab2 (Fig.2C). These results demonstrate that Dab2 can directlymediate JNK activation and that this activity can be localized to theC-terminal domain of Dab2.Fig. 2Dab2 directly stimulates JNK activity. A, diagrammaticrepresentation of the various constructs of Dab2. Depicted are the full-lengthand deletion constructs of Dab2 containing the N-ter