SMAD4: A predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV

International Journal of Cancer

Volumn 126, Issue 12, 2010, Pages 2914-2927

  Dempe, Sebastian ^a   Stroh Dege, Alexandra Y ^a   Schwarz, Elisabeth ^a   Rommelaere, Jean ^a,b   Dinsart, Christiane ^a,b  

^a GERMAN CANCER RESEARCH CENTER DKFZ   (Germany)

^b Institut National de la Sante et de la Recherche Medicale U701   (Germany)

Author keywords

Autonomous parvovirus; Cancer therapy; Pancreatic carcinoma; Transcription factor SMAD4

Indexed keywords

ONCOLYTIC VIRUS; PROTEIN C JUN; SMAD4 PROTEIN; LUCIFERASE; MESSENGER RNA; NS1 PROTEIN, PARVOVIRUS; TUMOR MARKER; VIRUS PROTEIN;

ARTICLE; BINDING SITE; CONTROLLED STUDY; GENE DELETION; GENE EXPRESSION; GENE MUTATION; GENE REPLICATION; GENE SEQUENCE; H-1 PARVOVIRUS; HUMAN; HUMAN CELL; IMMUNOBLOTTING; PANCREAS ADENOCARCINOMA; PARVOVIRUS INFECTION; PRIORITY JOURNAL; ADENOCARCINOMA; DRUG ANTAGONISM; GENE EXPRESSION REGULATION; GENE VECTOR; GENETIC TRANSFECTION; GENETICS; METABOLISM; METASTASIS; MUTATION; PANCREAS CARCINOMA; PANCREAS TUMOR; PATHOLOGY; PHYSIOLOGY; PROGNOSIS; PROMOTER REGION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; TUMOR CELL CULTURE; VIROLOGY; VIRUS REPLICATION;

ADENOCARCINOMA; CARCINOMA, PANCREATIC DUCTAL; GENE EXPRESSION REGULATION, NEOPLASTIC; GENETIC VECTORS; H-1 PARVOVIRUS; HUMANS; LUCIFERASES; MUTATION; PANCREATIC NEOPLASMS; PARVOVIRIDAE INFECTIONS; PROGNOSIS; PROMOTER REGIONS, GENETIC; REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION; RNA, MESSENGER; SMAD4 PROTEIN; TRANSFECTION; TUMOR CELLS, CULTURED; TUMOR MARKERS, BIOLOGICAL; VIRAL NONSTRUCTURAL PROTEINS; VIRUS REPLICATION;

EID: 77951933403     PISSN: 00207136     EISSN: 10970215     Source Type: Journal    
DOI: 10.1002/ijc.24992     Document Type: Article

References (83)

1. Bardeesy N, DePinho RA. Pancreatic cancer biology and genetics. Nat Rev Cancer 2002;2:897-909.
2. Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor A, Feuer EJ, Thun MJ. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10-30.
3. Beauchamp RD, Lyons RM, Yang EY, Coffey RJ, Jr., Moses HL. Expression of and response to growth regulatory peptides by two human pancreatic carcinoma cell lines. Pancreas 1990;5:369-380 (Pubitemid 20214971)
4. Cardenes HR, Chiorean EG, Dewitt J, Schmidt M, Loehrer P. Locally advanced pancreatic cancer: current therapeutic approach. Oncologist 2006;11:612-623 (Pubitemid 43967616)
5. Li D, Xie K, Wolff R, Abbruzzese JL. Pancreatic cancer. Lancet 2004;363:1049-1057
6. Safran H, Iannitti D, Ramanathan R, Schwartz JD, Steinhoff M, Nauman C, Hesketh P, Rathore R, Wolff R, Tantravahi U, Hughes M, Maia C, et al. Herceptin and gemcitabine for metastatic pancreatic cancers that overexpress HER-2/neu. Cancer Invest 2004;22:706-712
7. Xiong HQ, Rosenberg A, LoBuglio A, Schmidt W, Wolff RA, Deutsch J, Needle M, Abbruzzese JL. Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic cancer: a multicenter phase II trial. J Clin Oncol 2004;22:2610-2616 (Pubitemid 41103748)
8. Kindler HL, Friberg G, Singh DA, Locker G, Nattam S, Kozloff M, Taber DA, Karrison T, Dachman A, Stadler WM, Vokes EE. Phase II trial of bevacizumab plus gemcitabine in patients with advanced pancreatic cancer. J Clin Oncol 2005;23:8033-8040 (Pubitemid 46657405)
9. Van Cutsem E, van de Velde H, Karasek P, Oettle H, Vervenne WL, Szawlowski A, Schoffski P, Post S, Verslype C, Neumann H, Safran H, Humblet Y, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004;22:1430-1438
10. Kasuya H, Takeda S, Nomoto S, Nakao A. The potential of oncolytic virus therapy for pancreatic cancer. Cancer Gene Ther 2005;12:725-736 (Pubitemid 41208612)
11. Kuhlmann KF, Gouma DJ, Wesseling JG. Adenoviral gene therapy for pancreatic cancer: where do we stand? Dig Surg 2008;25:278-292
12. Clary BM, Coveney EC, Philip R, Blazer DG, III, Morse M, Gilboa E, Lyerly HK. Inhibition of established pancreatic cancers following specific active immunotherapy with interleukin-2 gene-transduced tumor cells. Cancer Gene Ther 1997;4:97-104. (Pubitemid 127667104)
13. Fioretti F, Fradelizi D, Stoppacciaro A, Ramponi S, Ruco L, Minty A, Sozzani S, Garlanda C, Vecchi A, Mantovani A. Reduced tumorigenicity and augmented leukocyte infiltration after monocyte chemotactic protein-3 (MCP-3) gene transfer: perivascular accumulation of dendritic cells in peritumoral tissue and neutrophil recruitment within the tumor. J Immunol 1998;161:342-346
14. Kimura M, Yoshida Y, Narita M, Takenaga K, Takenouchi T, Yamaguchi T, Saisho H, Sakiyama S, Tagawa M. Acquired immunity in nude mice induced by expression of the IL-2 or IL-4 gene in human pancreatic carcinoma cells and anti-tumor effect generated by in vivo gene transfer using retrovirus. Int J Cancer 1999;82:549-555 (Pubitemid 29343455)
15. Wetzel K, Menten P, Opdenakker G, Van Damme J, Grone HJ, Giese N, Vecchi A, Sozzani S, Cornelis JJ, Rommelaere J, Dinsart C. Transduction of human MCP-3 by a parvoviral vector induces leukocyte infiltration and reduces growth of human cervical carcinoma cell xenografts. J Gene Med 2001;3:326-337
16. Giese NA, Raykov Z, DeMartino L, Vecchi A, Sozzani S, Dinsart C, Cornelis JJ, Rommelaere J. Suppression of metastatic hemangiosarcoma by a parvovirus MVMp vector transducing the IP-10 chemokine into immunocompetent mice. Cancer Gene Ther 2002;9:432-442 (Pubitemid 34438585)
17. Moehler M, Blechacz B, Weiskopf N, Zeidler M, Stremmel W, Rommelaere J, Galle PR, Cornelis JJ. Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors. Cancer Gene Ther 2001;8:158-167 (Pubitemid 32322255)
18. Cornelis JJ, Lang SI, Stroh-Dege AY, Balboni G, Dinsart C, Rommelaere J. Cancer gene therapy through autonomous parvovirus-mediated gene transfer. Curr Gene Ther 2004;4:249-261 (Pubitemid 39162387)
19. Herrero YCM, Cornelis JJ, Herold-Mende C, Rommelaere J, Schlehofer JR, Geletneky K. Parvovirus H-1 infection of human glioma cells leads to complete viral replication and efficient cell killing. Int J Cancer 2004;109:76-84. (Pubitemid 38160665)
20. Raykov Z, Grekova S, Galabov AS, Balboni G, Koch U, Aprahamian M, Rommelaere J. Combined oncolytic and vaccination activities of parvovirus H-1 in a metastatic tumor model. Oncol Rep 2007;17:1493-1499
21. Wetzel K, Struyf S, Van Damme J, Kayser T, Vecchi A, Sozzani S, Rommelaere J, Cornelis JJ, Dinsart C. MCP-3 (CCL7) delivered by parvovirus MVMp reduces tumorigenicity of mouse melanoma cells through activation of T lymphocytes and NK cells. Int J Cancer 2007;120:1364-1371 (Pubitemid 46256792)
22. Enderlin M, Kleinmann EV, Struyf S, Buracchi C, Vecchi A, Kinscherf R, Kiessling F, Paschek S, Sozzani S, Rommelaere J, Cornelis JJ, Van Damme J, et al. TNF-alpha and the IFN-gamma-inducible protein 10 (IP-10/CXCL-10) delivered by parvoviral vectors act in synergy to induce antitumor effects in mouse glioblastoma. Cancer Gene Ther 2009;16:149-160
23. Berns KI, Giraud C. Biology of adeno-associated virus. Curr Top Microbiol Immunol 1996;218:1-23.
24. Spalholz BA, Tattersall P. Interaction of minute virus of mice with differentiated cells: strain-dependent target cell specificity is mediated by intracellular factors. J Virol 1983;46:937-943 (Pubitemid 13092500)
25. Dupressoir T, Vanacker JM, Cornelis JJ, Duponchel N, Rommelaere J. Inhibition by parvovirus H-1 of the formation of tumors in nude mice and colonies in vitro by transformed human mammary epithelial cells. Cancer Res 1989;49:3203-3208 (Pubitemid 19162125)
26. Rommelaere J, Cornelis JJ. Antineoplastic activity of parvoviruses. J Virol Methods 1991;33:233-251
27. Faisst S, Guittard D, Benner A, Cesbron JY, Schlehofer JR, Rommelaere J, Dupressoir T. Dose-dependent regression of HeLa cell-derived tumours in SCID mice after parvovirus H-1 infection. Int J Cancer 1998;75:584-589 (Pubitemid 28094105)
28. Cornelis JJ, Salome N, Dinsart C, Rommelaere J. Vectors based on autonomous parvoviruses: novel tools to treat cancer? J Gene Med 2004;6(suppl 1): S193-S202.
29. Rhode SL, III, Paradiso PR. Parvovirus genome: nucleotide sequence of H-1 and mapping of its genes by hybrid-arrested translation. J Virol 1983;45:173-184
30. Li X, Rhode SL, III. Mutation of lysine 405 to serine in the parvovirus H-1 NS1 abolishes its functions for viral DNA replication, late promoter trans activation, and cytotoxicity. J Virol 1990;64:4654-4660 (Pubitemid 20329780)
31. Nüesch JPF. Regulation of non-structural protein functions by differential synthesis, modification and trafficking. In: Bloom ME, Cotmore SF, Linden RM, Parrish CR, Kerr JR, editors. Parvoviruses. London: Hodder Arnold, 2006. 275-286
32. Cotmore SF, Tattersall P. High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin. J Virol 1998;72:8477-8484
33. Nuesch JP, Cotmore SF, Tattersall P. Sequence motifs in the replicator protein of parvovirus MVM essential for nicking and covalent attachment to the viral origin: identification of the linking tyrosine. Virology 1995;209:122-135
34. Wilson GM, Jindal HK, Yeung DE, Chen W, Astell CR. Expression of minute virus of mice major nonstructural protein in insect cells: purification and identification of ATPase and helicase activities. Virology 1991;185:90-98
35. Rhode SL, III. trans-Activation of parvovirus P38 promoter by the 76K noncapsid protein. J Virol 1985;55:886-889
36. Ahn JK, Gavin BJ, Kumar G, Ward DC. Transcriptional analysis of minute virus of mice P4 promoter mutants. J Virol 1989;63:5425-5439
37. Gu Z, Plaza S, Perros M, Cziepluch C, Rommelaere J, Cornelis JJ. NF-Y controls transcription of the minute virus of mice P4 promoter through interaction with an unusual binding site. J Virol 1995;69:239-246
38. Perros M, Deleu L, Vanacker JM, Kherrouche Z, Spruyt N, Faisst S, Rommelaere J. Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells. J Virol 1995;69:5506-5515
39. Deleu L, Pujol A, Faisst S, Rommelaere J. Activation of promoter P4 of the autonomous parvovirus minute virus of mice at early S phase is required for productive infection. J Virol 1999;73:3877-3885
40. Fuks F, Deleu L, Dinsart C, Rommelaere J, Faisst S. Ras oncogene-dependent activation of the P4 promoter of minute virus of mice through a proximal P4 element interacting with the Ets family of transcription factors. J Virol 1996;70:1331-1339
41. Klimstra DS, Longnecker DS. K-ras mutations in pancreatic ductal proliferative lesions. Am J Pathol 1994;145:1547-1550
42. Schneider G, Schmid RM. Genetic alterations in pancreatic carcinoma. Mol Cancer 2003;2:15.
43. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271:350-353
44. Massague J, Gomis RR. The logic of TGFbeta signaling. FEBS Lett 2006;580:2811-2820
45. Massague J, Blain SW, Lo RS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell 2000;103:295-309.
46. Angelova AL, Aprahamian M, Grekova SP, Hajri A, Leuchs B, Giese NA, Dinsart C, Herrmann A, Balboni G, Rommelaere J, Raykov Z. Improvement of gemcitabinebased therapy of pancreatic carcinoma by means of oncolytic parvovirus H-1PV. Clin Cancer Res 2009;15:511-519
47. Cornelis JJ, Spruyt N, Spegelaere P, Guetta E, Darawshi T, Cotmore SF, Tal J, Rommelaere J. Sensitization of transformed rat fibroblasts to killing by parvovirus minute virus of mice correlates with an increase in viral gene expression. J Virol 1988;62:3438-3444
48. Wrzesinski C, Tesfay L, Salome N, Jauniaux JC, Rommelaere J, Cornelis J, Dinsart C. Chimeric and pseudotyped parvoviruses minimize the contamination of recombinant stocks with replication-competent viruses and identify a DNA sequence that restricts parvovirus H-1 in mouse cells. J Virol 2003;77:3851-3858 (Pubitemid 36297305)
49. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999;6:973-985 (Pubitemid 29295202)
50. Haag A, Menten P, Damme JV, Dinsart C, Rommelaere J, Cornelis JJ. Highly efficient transduction and expression of cytokine genes in human tumor cells by means of autonomous parvovirus vectors: generation of antitumor responses in recipient mice. Hum Gene Ther 2000;11:597-609. (Pubitemid 30134303)
51. Kestler J, Neeb B, Struyf S, Van Damme J, Cotmore SF, D'Abramo A, Tattersall P, Rommelaere J, Dinsart C, Cornelis JJ. Cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses. Hum Gene Ther 1999;10:1619-1632 (Pubitemid 29328206)
52. Zhang Y, Musci T, Derynck R. The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function. Curr Biol 1997;7:270-276 (Pubitemid 27176853)
53. Lagna G, Hata A, Hemmati-Brivanlou A, Massague J. Partnership between DPC4 and SMAD proteins in TGF-beta signalling pathways. Nature 1996;383:832-836 (Pubitemid 26372668)
54. Brockhaus K, Plaza S, Pintel DJ, Rommelaere J, Salome N. Nonstructural proteins NS2 of minute virus of mice associate in vivo with 14-3-3 protein family members. J Virol 1996;70:7527-7534
55. Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang XF. Tumor suppressor Smad4 is a transforming growth factor beta-inducible DNA binding protein. Mol Cell Biol 1997;17:7019-7028
56. Wilentz RE, Su GH, Dai JL, Sparks AB, Argani P, Sohn TA, Yeo CJ, Kern SE, Hruban RH. Immunohistochemical labeling for dpc4 mirrors genetic status in pancreatic adenocarcinomas: a new marker of DPC4 inactivation. Am J Pathol 2000;156:37-43.
57. Schutte M, Hruban RH, Hedrick L, Cho KR, Nadasdy GM, Weinstein CL, Bova GS, Isaacs WB, Cairns P, Nawroz H, Sidransky D, Casero RA, Jr, et al. DPC4 gene in various tumor types. Cancer Res 1996;56:2527-2530
58. Wan M, Huang J, Jhala NC, Tytler EM, Yang L, Vickers SM, Tang Y, Lu C, Wang N, Cao X. SCF(beta-TrCP1) controls Smad4 protein stability in pancreatic cancer cells. Am J Pathol 2005;166:1379-1392 (Pubitemid 40586509)
59. Moore PS, Sipos B, Orlandini S, Sorio C, Real FX, Lemoine NR, Gress T, Bassi C, Kloppel G, Kalthoff H, Ungefroren H, Lohr M, et al. Genetic profile of 22 pancreatic carcinoma cell lines. Analysis of. K-ras, p53, p16 and. DPC4/Smad4. Virchows Arch 2001;439:798-802.
60. Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1998;1:611-617 (Pubitemid 128374697)
61. Johnson K, Kirkpatrick H, Comer A, Hoffmann FM, Laughon A. Interaction of Smad complexes with tripartite DNA-binding sites. J Biol Chem 1999;274:20709-20716 (Pubitemid 129519559)
62. Ghosh-Choudhury N, Singha PK, Woodruff K, St Clair P, Bsoul S, Werner SL, Choudhury GG. Concerted action of Smad and CREB-binding protein regulates bone morphogenetic protein-2-stimulated osteoblastic colony-stimulating factor-1 expression. J Biol Chem 2006;281:20160-20170
63. Massague J, Seoane J, Wotton D. SMAD transcription factors. Genes Dev 2005;19:2783-2810
64. Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 1998;394:909-913
65. Baldus SE, Schwarz E, Lohrey C, Zapatka M, Landsberg S, Hahn SA, Schmidt D, Dienes HP, Schmiegel WH, Schwarte-Waldhoff I. Smad4 defficiency in cervical carcinoma cells. Oncogene 2005;24:810-819 (Pubitemid 40216089)
66. Inman GJ, Nicolas FJ, Callahan JF, Harling JD, Gaster LM, Reith AD, Laping NJ, Hill CS. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4. ALK5, and. ALK7. Mol Pharmacol 2002;62:65-74.
67. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitortype 1 gene. EMBO J 1998;17:3091-3100
68. Shi Y, Wang YF, Jayaraman L, Yang H, Massague J, Pavletich NP. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 1998;94:585-594
69. Song CZ, Siok TE, Gelehrter TD. Smad4/DPC4 and Smad3 mediate transforming growth factor-beta (TGF-beta) signaling through direct binding to a novel TGF-beta-responsive element in the human plasminogen activator inhibitor-1 promoter. J Biol Chem 1998;273:29287-29290
70. Zhang W, Ou J, Inagaki Y, Greenwel P, Ramirez F. Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription. J Biol Chem 2000;275:39237-39245
71. Lai CF, Feng X, Nishimura R, Teitelbaum SL, Avioli LV, Ross FP, Cheng SL. Transforming growth factor-beta upregulates the beta 5 integrin subunit expression via Sp1 and Smad signaling. J Biol Chem 2000;275:36400-36406
72. Kucich U, Rosenbloom JC, Abrams WR, Rosenbloom J. Transforming growth factor-beta stabilizes elastin mRNA by a pathway requiring active Smads, protein kinase C-delta, and p38. Am J Respir Cell Mol Biol 2002;26:183-188
73. Shiou SR, Datta PK, Dhawan P, Law BK, Yingling JM, Dixon DA, Beauchamp RD. Smad4-dependent regulation of urokinase plasminogen activator secretion and RNA stability associated with invasiveness by autocrine and paracrine transforming growth factor-beta. J Biol Chem 2006;281:33971-33981
74. Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 2002;7:1191-1204 (Pubitemid 36024597)
75. Stettner MR, Nance JA, Wright CA, Kinoshita Y, Kim W-K, Morgello S, Rappaport J, Khalili K, Gordon J, Johnson EM. SMAD proteins of oligodendroglial cells regulate transcription of JC virus early and late genes coordinately with the Tat protein of human immunodefficiency virus type 1. J Gen Virol 2009;90:2005-2014
76. Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999;96:319-328
77. Lohr M, Trautmann B, Gottler M, Peters S, Zauner I, Maier A, Kloppel G, Liebe S, Kreuser ED. Expression and function of receptors for extracellular matrix proteins in human ductal adenocarcinomas of the pancreas. Pancreas 1996;12:248-259
78. Linder S, Castanos-Velez E, von Rosen A, Biberfeld P. Immunohistochemical expression of extracellular matrix proteins and adhesion molecules in pancreatic carcinoma. Hepatogastroenterology 2001;48:1321-1327 (Pubitemid 33010416)
79. Subramanian G, Schwarz RE, Higgins L, McEnroe G, Chakravarty S, Dugar S, Reiss M. Targeting endogenous transforming growth factor beta receptor signaling in SMAD4-deficient human pancreatic carcinoma cells inhibits their invasive phenotype1. Cancer Res 2004;64:5200-5211 (Pubitemid 39006539)
80. Simeone DM, Pham T, Logsdon CD. Disruption of TGFbeta signaling pathways in human pancreatic cancer cells. Ann Surg 2000;232:73-80. (Pubitemid 30456553)
81. Kleeff J, Ishiwata T, Friess H, Buchler MW, Korc M. Concomitant over-expression of activin/inhibin beta subunits and their receptors in human pancreatic cancer. Int J Cancer 1998;77:860-868
82. Ishida W, Hamamoto T, Kusanagi K, Yagi K, Kawabata M, Takehara K, Sampath TK, Kato M, Miyazono K. Smad6 Is a Smad1/5-induced Smad inhibitor. Characterization of bone morphogenetic protein-responsive element in the mouse. Smad6 promoter. J Biol Chem 2000;275:6075-6079
83. Kusanagi K, Kawabata M, Mishima HK, Miyazono K. alpha-Helix 2 in the amino-terminal Mad homology 1 domain is responsible for specific DNA binding of Smad3. J Biol Chem 2001;276:28155-28163