Home LiteratureArticle Details
PMID: 16775010 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

Molecular biology of the cell ·Vol. 17 ·No. 9 ·2006-09-00 ·Pages 3819-31

Zhao BM, Hoffmann FM

Abstract

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

MeSH Terms
Amino Acid Sequence Animals Aptamers, Peptide/chemistry,metabolism Cell Nucleus/metabolism Cells, Cultured Cytoplasm/metabolism Epithelial Cells/cytology,drug effects Extracellular Signal-Regulated MAP Kinases/metabolism Humans Intracellular Signaling Peptides and Proteins/chemistry,metabolism Mesoderm/cytology,drug effects Mice Molecular Sequence Data Multiprotein Complexes/metabolism Phosphorylation/drug effects Protein Binding Protein Transport/drug effects Proto-Oncogene Proteins c-akt/metabolism Serine Endopeptidases/chemistry,metabolism Signal Transduction/drug effects Smad Proteins/metabolism Thioredoxins/chemistry,metabolism Transforming Growth Factor beta/pharmacology Transforming Growth Factor beta1 p38 Mitogen-Activated Protein Kinases/metabolism
Chemicals
Aptamers, Peptide Intracellular Signaling Peptides and Proteins Multiprotein Complexes Smad Proteins TGFB1 protein, human Tgfb1 protein, mouse Transforming Growth Factor beta Transforming Growth Factor beta1 Thioredoxins Proto-Oncogene Proteins c-akt Extracellular Signal-Regulated MAP Kinases p38 Mitogen-Activated Protein Kinases ZFYVE16 protein, human Serine Endopeptidases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zhao Bryan M
McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI 53706, USA.
Hoffmann F Michael
References (95)
95 references, click to expand
  1. EBNA-1, a bifunctional transcriptional activator.
    Mol Cell Biol. 2003 Oct;23(19):6901-8 PMID: 12972608
  2. Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells.
    J Biol Chem. 2003 Sep 26;278(39):37610-21 PMID: 12842895
  3. Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
    Nature. 2003 Oct 9;425(6958):577-84 PMID: 14534577
  4. Distinct domain utilization by Smad3 and Smad4 for nucleoporin interaction and nuclear import.
    J Biol Chem. 2003 Oct 24;278(43):42569-77 PMID: 12917407
  5. Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice.
    Development. 2003 Dec;130(24):6143-53 PMID: 14597578
  6. Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction.
    J Clin Invest. 2003 Nov;112(10):1486-94 PMID: 14617750
  7. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development.
    Mech Dev. 2003 Nov;120(11):1351-83 PMID: 14623443
  8. Epithelial-mesenchymal transitions in development and pathologies.
    Curr Opin Cell Biol. 2003 Dec;15(6):740-6 PMID: 14644200
  9. Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer.
    Nat Rev Cancer. 2003 Nov;3(11):807-21 PMID: 14557817
  10. Molecular aspects of epithelial cell plasticity: implications for local tumor invasion and metastasis.
    Mutat Res. 2004 Jan;566(1):9-20 PMID: 14706509
  11. GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor.
    J Cell Biol. 2004 Jan 19;164(2):291-300 PMID: 14718519
  12. Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury.
    Am J Pathol. 2004 Feb;164(2):651-63 PMID: 14742269
  13. SB-505124 is a selective inhibitor of transforming growth factor-beta type I receptors ALK4, ALK5, and ALK7.
    Mol Pharmacol. 2004 Mar;65(3):744-52 PMID: 14978253
  14. The role of epithelial-to-mesenchymal transition in renal fibrosis.
    J Mol Med (Berl). 2004 Mar;82(3):175-81 PMID: 14752606
  15. Inhibition of mammalian cell proliferation by genetically selected peptide aptamers that functionally antagonize E2F activity.
    Oncogene. 1999 Jul 29;18(30):4357-63 PMID: 10439043
  16. Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis.
    J Clin Invest. 2004 Nov;114(9):1308-16 PMID: 15520863
  17. Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro.
    Neoplasia. 2004 Sep-Oct;6(5):603-10 PMID: 15548370
  18. Development of TGF-beta signalling inhibitors for cancer therapy.
    Nat Rev Drug Discov. 2004 Dec;3(12):1011-22 PMID: 15573100
  19. Selective inhibition of activin receptor-like kinase 5 signaling blocks profibrotic transforming growth factor beta responses in skin fibroblasts.
    Arthritis Rheum. 2004 Dec;50(12):4008-21 PMID: 15593186
  20. The nuclear import function of Smad2 is masked by SARA and unmasked by TGFbeta-dependent phosphorylation.
    Nat Cell Biol. 2000 Aug;2(8):559-62 PMID: 10934479
  21. Phosphatidylinositol 3-kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition and cell migration.
    J Biol Chem. 2000 Nov 24;275(47):36803-10 PMID: 10969078
  22. Transforming growth factor-beta1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism.
    Mol Biol Cell. 2001 Jan;12(1):27-36 PMID: 11160820
  23. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation.
    Mol Cell. 2000 Dec;6(6):1365-75 PMID: 11163210
  24. Smurf1 interacts with transforming growth factor-beta type I receptor through Smad7 and induces receptor degradation.
    J Biol Chem. 2001 Apr 20;276(16):12477-80 PMID: 11278251
  25. Smad7 inhibits the survival nuclear factor kappaB and potentiates apoptosis in epithelial cells.
    Oncogene. 2001 Feb 15;20(7):879-84 PMID: 11314022
  26. Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung.
    Am J Physiol Lung Cell Mol Physiol. 2001 Jun;280(6):L1327-34 PMID: 11350814
  27. Filamin associates with Smads and regulates transforming growth factor-beta signaling.
    J Biol Chem. 2001 May 25;276(21):17871-7 PMID: 11278410
  28. TGF-beta signaling in tumor suppression and cancer progression.
    Nat Genet. 2001 Oct;29(2):117-29 PMID: 11586292
  29. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells.
    Nat Med. 2001 Oct;7(10):1118-22 PMID: 11590434
  30. Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition.
    Bioessays. 2001 Oct;23(10):912-23 PMID: 11598958
  31. Effects of anti-TGF-beta type II receptor antibody on experimental glomerulonephritis.
    Kidney Int. 2001 Nov;60(5):1745-55 PMID: 11703592
  32. Smad3 as a mediator of the fibrotic response.
    Int J Exp Pathol. 2004 Apr;85(2):47-64 PMID: 15154911
  33. SB-431542, a small molecule transforming growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility.
    Mol Cancer Ther. 2004 Jun;3(6):737-45 PMID: 15210860
  34. Roles for the MH2 domain of Smad7 in the specific inhibition of transforming growth factor-beta superfamily signaling.
    J Biol Chem. 2004 Jul 23;279(30):31568-74 PMID: 15148321
  35. Smad and p38-MAPK signaling mediates apoptotic effects of transforming growth factor-beta1 in human airway epithelial cells.
    Am J Physiol Lung Cell Mol Physiol. 2004 Sep;287(3):L515-24 PMID: 15132952
  36. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression.
    J Clin Invest. 2004 Aug;114(4):569-81 PMID: 15314694
  37. Transforming the TGFbeta pathway: convergence of distinct lead generation strategies on a novel kinase pharmacophore for TbetaRI (ALK5).
    Curr Opin Drug Discov Devel. 2004 Jul;7(4):437-45 PMID: 15338953
  38. Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.
    Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2909-14 PMID: 8610141
  39. Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2.
    Nature. 1996 Apr 11;380(6574):548-50 PMID: 8606778
  40. TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.
    Genes Dev. 1996 Oct 1;10(19):2462-77 PMID: 8843198
  41. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes.
    Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11400-6 PMID: 8876147
  42. MADR2 is a substrate of the TGFbeta receptor and its phosphorylation is required for nuclear accumulation and signaling.
    Cell. 1996 Dec 27;87(7):1215-24 PMID: 8980228
  43. Interaction between Smad7 and beta-catenin: importance for transforming growth factor beta-induced apoptosis.
    Mol Cell Biol. 2005 Feb;25(4):1475-88 PMID: 15684397
  44. TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition.
    Mol Biol Cell. 2005 Apr;16(4):1987-2002 PMID: 15689496
  45. Inhibition of gene markers of fibrosis with a novel inhibitor of transforming growth factor-beta type I receptor kinase in puromycin-induced nephritis.
    J Pharmacol Exp Ther. 2005 Jun;313(3):943-51 PMID: 15769863
  46. Selective inhibition of TGF-beta responsive genes by Smad-interacting peptide aptamers from FoxH1, Lef1 and CBP.
    Oncogene. 2005 Jun 2;24(24):3864-74 PMID: 15750622
  47. TGF-(beta) type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells.
    J Cell Sci. 1999 Dec;112 ( Pt 24):4557-68 PMID: 10574705
  48. Structural basis of Smad2 recognition by the Smad anchor for receptor activation.
    Science. 2000 Jan 7;287(5450):92-7 PMID: 10615055
  49. Microtubule binding to Smads may regulate TGF beta activity.
    Mol Cell. 2000 Jan;5(1):27-34 PMID: 10678166
  50. NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events.
    J Biol Chem. 2000 Mar 31;275(13):9501-9 PMID: 10734098
  51. Smad7 mediates apoptosis induced by transforming growth factor beta in prostatic carcinoma cells.
    Curr Biol. 2000 May 4;10(9):535-8 PMID: 10801443
  52. Long-term prevention of renal insufficiency, excess matrix gene expression, and glomerular mesangial matrix expansion by treatment with monoclonal antitransforming growth factor-beta antibody in db/db diabetic mice.
    Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8015-20 PMID: 10859350
  53. Engineered protein scaffolds for molecular recognition.
    J Mol Recognit. 2000 Jul-Aug;13(4):167-87 PMID: 10931555
  54. Integrin beta 1 signaling is necessary for transforming growth factor-beta activation of p38MAPK and epithelial plasticity.
    J Biol Chem. 2001 Dec 14;276(50):46707-13 PMID: 11590169
  55. A nuclear antagonistic mechanism of inhibitory Smads in transforming growth factor-beta signaling.
    J Biol Chem. 2002 Feb 8;277(6):4176-82 PMID: 11711531
  56. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling.
    Genes Cells. 2002 Mar;7(3):321-31 PMID: 11918675
  57. Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.
    EMBO J. 2004 Mar 10;23(5):1155-65 PMID: 14976548
  58. The interaction of specific peptide aptamers with the DNA binding domain and the dimerization domain of the transcription factor Stat3 inhibits transactivation and induces apoptosis in tumor cells.
    Mol Cancer Res. 2004 Mar;2(3):170-82 PMID: 15037656
  59. Smad7 is required for TGF-beta-induced activation of the small GTPase Cdc42.
    J Cell Sci. 2004 Apr 1;117(Pt 9):1835-47 PMID: 15075243
  60. TGF-beta signaling and the fibrotic response.
    FASEB J. 2004 May;18(7):816-27 PMID: 15117886
  61. New insights into TGF-beta-Smad signalling.
    Trends Biochem Sci. 2004 May;29(5):265-73 PMID: 15130563
  62. Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.
    Nature. 1997 Oct 9;389(6651):631-5 PMID: 9335507
  63. Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo.
    Curr Biol. 1997 Nov 1;7(11):860-9 PMID: 9382809
  64. Chimeric extracellular domain type II transforming growth factor (TGF)-beta receptor fused to the Fc region of human immunoglobulin as a TGF-beta antagonist.
    Eur J Biochem. 1998 Jun 15;254(3):505-13 PMID: 9688260
  65. Physical and functional interaction of murine and Xenopus Smad7 with bone morphogenetic protein receptors and transforming growth factor-beta receptors.
    J Biol Chem. 1998 Sep 25;273(39):25364-70 PMID: 9738003
  66. TGFbeta signaling is necessary for carcinoma cell invasiveness and metastasis.
    Curr Biol. 1998 Nov 19;8(23):1243-52 PMID: 9822576
  67. Topical application of a peptide inhibitor of transforming growth factor-beta1 ameliorates bleomycin-induced skin fibrosis.
    J Invest Dermatol. 2005 Sep;125(3):450-5 PMID: 16117784
  68. Specificity and versatility in tgf-beta signaling through Smads.
    Annu Rev Cell Dev Biol. 2005;21:659-93 PMID: 16212511
  69. Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads.
    Mol Cell Biol. 2005 Nov;25(22):9845-58 PMID: 16260601
  70. Smad transcription factors.
    Genes Dev. 2005 Dec 1;19(23):2783-810 PMID: 16322555
  71. An artificial cell-cycle inhibitor isolated from a combinatorial library.
    Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14272-7 PMID: 9826690
  72. SARA, a FYVE domain protein that recruits Smad2 to the TGFbeta receptor.
    Cell. 1998 Dec 11;95(6):779-91 PMID: 9865696
  73. Dominant-negative Smad2 mutants inhibit activin/Vg1 signaling and disrupt axis formation in Xenopus.
    Dev Biol. 1999 Mar 15;207(2):364-79 PMID: 10068469
  74. Regulation of cell proliferation by Smad proteins.
    J Cell Physiol. 2002 Apr;191(1):1-16 PMID: 11920677
  75. Inhibition of transforming growth factor (TGF)-beta1-induced extracellular matrix with a novel inhibitor of the TGF-beta type I receptor kinase activity: SB-431542.
    Mol Pharmacol. 2002 Jul;62(1):58-64 PMID: 12065755
  76. 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 Jul;62(1):65-74 PMID: 12065756
  77. Metastasis is driven by sequential elevation of H-ras and Smad2 levels.
    Nat Cell Biol. 2002 Jul;4(7):487-94 PMID: 12105419
  78. TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses.
    EMBO J. 2002 Jul 15;21(14):3749-59 PMID: 12110587
  79. p38 mitogen-activated protein kinase is required for TGFbeta-mediated fibroblastic transdifferentiation and cell migration.
    J Cell Sci. 2002 Aug 1;115(Pt 15):3193-206 PMID: 12118074
  80. Smad3 allostery links TGF-beta receptor kinase activation to transcriptional control.
    Genes Dev. 2002 Aug 1;16(15):1950-63 PMID: 12154125
  81. Antitumor activity of a recombinant soluble betaglycan in human breast cancer xenograft.
    Cancer Res. 2002 Aug 15;62(16):4690-5 PMID: 12183427
  82. Epithelial-mesenchymal transitions in tumour progression.
    Nat Rev Cancer. 2002 Jun;2(6):442-54 PMID: 12189386
  83. Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFbeta signaling complexes in the cytoplasm and nucleus.
    Mol Cell. 2002 Aug;10(2):271-82 PMID: 12191473
  84. Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-beta receptor activity.
    Mol Cell. 2002 Aug;10(2):283-94 PMID: 12191474
  85. Nuclear convergence of the TGFbeta and cAMP signal transduction pathways in murine embryonic palate mesenchymal cells.
    Cell Signal. 2003 Feb;15(2):235-42 PMID: 12464395
  86. Elucidation of Smad requirement in transforming growth factor-beta type I receptor-induced responses.
    J Biol Chem. 2003 Feb 7;278(6):3751-61 PMID: 12446693
  87. Transforming growth factor-beta1 (TGF-beta)-induced apoptosis of prostate cancer cells involves Smad7-dependent activation of p38 by TGF-beta-activated kinase 1 and mitogen-activated protein kinase kinase 3.
    Mol Biol Cell. 2003 Feb;14(2):529-44 PMID: 12589052
  88. Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover.
    Nat Cell Biol. 2003 May;5(5):410-21 PMID: 12717440
  89. Attenuation of the TGF-beta-Smad signaling pathway in pancreatic tumor cells confers resistance to TGF-beta-induced growth arrest.
    Oncogene. 2003 Jun 12;22(24):3698-711 PMID: 12802277
  90. The gatekeeper effect of epithelial-mesenchymal transition regulates the frequency of breast cancer metastasis.
    Cancer Res. 2003 Jun 15;63(12):3386-94 PMID: 12810675
  91. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.
    Cell. 2003 Jun 13;113(6):685-700 PMID: 12809600
  92. Targeting the TGF beta signaling network in human neoplasia.
    Cancer Cell. 2003 Jun;3(6):531-6 PMID: 12842082
  93. Cooperative inhibition of bone morphogenetic protein signaling by Smurf1 and inhibitory Smads.
    Mol Biol Cell. 2003 Jul;14(7):2809-17 PMID: 12857866
  94. Evaluation of anti-TGF-beta2 antibody as a new postoperative anti-scarring agent in glaucoma surgery.
    Invest Ophthalmol Vis Sci. 2003 Aug;44(8):3394-401 PMID: 12882787
  95. Role for integrin-linked kinase in mediating tubular epithelial to mesenchymal transition and renal interstitial fibrogenesis.
    J Clin Invest. 2003 Aug;112(4):503-16 PMID: 12925691
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e05-10-0990
Published
2006-09-00
Epub
2006-00-14
Pages
3819-31
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC1556379
Subset
IM
Grants
NCI NIH HHS · P30 CA014520 · United States
NCI NIH HHS · R01 CA090875 · United States
NCI NIH HHS · T32 CA009135 · United States
NCI NIH HHS · T32-CA09135 · United States
External Links
PubMed source DOI: 10.1091/mbc.e05-10-0990 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2