Lymphoid enhancer-binding factor 1, a representative of vertebrate-specific Lef1/Tcf1 sub-family, is a Wnt-beta-catenin pathway target gene in human endothelial cells which regulates matrix metalloproteinase-2 expression and promotes endothelial cell invasion

Journal of Angiogenesis Research

Randall F Holcombe¹  Kestutis Planutis¹  Marina Planutiene¹ 

[1] The Tisch Cancer Institute at The Mount Sinai Medical Center, One Gustave L Levy Place, New York, NY 10029-6500, USA

关键词: Wnt signaling pathway;    siRNA;    MMP2;    Lef1;    invasion assay;    endothelial cells;    cancer;    β-catenin;    angiogenesis;   

Others  :  802155
DOI  :  10.1186/2045-824X-3-28

 received in 2011-09-09, accepted in 2011-12-14,  发布年份 2011

【 摘 要 】

Background

Wnt signaling is activated in many types of cancer and normal physiological processes. Various Wnt-related secreted factors may influence angiogenesis both in the tumor microenvironment and in normal tissues by direct action on endothelial cells. The mechanism of this Wnt action in angiogenesis is not well defined. We hypothesize that endothelial cells are responsive to Wnt signals and that Lef1, a member of the vertebrate-specific Wnt/beta-catenin throughput-inducing transcription factors' sub-family Lef1/Tcf1, mediates this responsiveness and promotes endothelial cell invasion.

Methods

A human endothelial cell line, EAhy926 was exposed to Wnt3a or directly transfected with Lef1. Readouts included assessment of nuclear beta-catenin, Wnt throughput with a SuperTOPflash reporter assay, induction of Lef1 transcription, induction of matrix metalloproteinase (MMP)-2 transcription, cell proliferation and cell invasion through a matrix in vitro. The effects on MMP2 were also evaluated in the presence of Lef1 silencing siRNA.

Results

Wnt3a increased nuclear beta-catenin and up-regulated Wnt/beta-catenin throughput. Wnt3a increased Lef1 transcription and activity of the Lef1 promoter. Both Wnt3a treatment and Lef1 overexpression induced MMP2 transcription but this effect was completely abrogated in the presence of Lef1 siRNA. Inhibition of Lef1 also reduced basal MMP2 levels suggesting that Lef1 regulates MMP2 expression even in the absence of exogenous Wnt pathway activation. Lef1 slightly increased proliferation of EAhy926 cells and increased invasion by more than two-fold.

Conclusions

EAhy926 cells activate canonical Wnt signaling in response to Wnt3a ligand. The Wnt target Lef1 specifically regulates MMP2 expression in these cells and promotes endothelial cell invasion. The EAhy926 cell line provides a convenient alternative to primary human umbilical vein endothelial cells (HUVEC) in the study of angiogenesis and the role of Wnt signaling on endothelial cell function.

【 授权许可】

   
2011 Planutiene et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Giles RH, van Es JH, Clevers H: Caught up in a Wnt storm: Wnt signaling in cancer. Biochimica Et Biophysica Acta-Reviews on Cancer 2003, 1653(1):1-24.
• [2]Hall C, Bafico A, Dai J, Aaronson S, Keller E: Prostate cancer cells promote osteoblastic bone metastases through Wnts. Cancer Res 2005, 65(17):7554-7560.
• [3]Holcombe RF, Marsh JL, Waterman ML, Lin F, Milovanovic T, Truong T: Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma. Journal of Clinical Pathology-Molecular Pathology 2002, 55(4):220-226.
• [4]Zi X, Guo Y, Simoneau A, Hope C, Xie J, Holcombe R, Hoang B: Expression of Frzb/secreted frizzled-related protein 3, a secreted Wnt antagonist, in human androgen-independent prostate cancer PC-3 cell suppresses tumor growth and cellular invasiveness. CANCER RESEARCH 2005, 65(21):9762-9770.
• [5]Finak G, Bertos N, Pepin F, Sadekova S, Souleimanova M, Zhao H, Chen H, Omeroglu G, Meterissian S, Omeroglu A, et al.: Stromal gene expression predicts clinical outcome in breast cancer. Nature Medicine 2008, 14(5):518-527.
• [6]Huang CL, Liu D, Nakano J, Ishikawa S, Kontani K, Yokomise H, Ueno M: Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor - An expression in non-small-cell lung cancer. Journal of Clinical Oncology 2005, 23(34):8765-8773.
• [7]Dejana E: The role of wnt signaling in physiological and pathological angiogenesis. Circ Res 2010, 107(8):943-952.
• [8]Planutis K, Planutiene M, Moyer MP, Nguyen AV, Perez CA, Holcombe RF: Regulation of norrin receptor frizzled-4 by Wnt2 in colon-derived cells. BMC Cell Biology 2007., 8Article No.: 12
• [9]Goodwin AM, Sullivan KM, D'Amore PA: Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling. Developmental Dynamics 2006, 235(11):3110-3120.
• [10]Wright M, Aikawa M, Szeto W, Papkoff J: Identification of a Wnt-responsive signal transduction pathway in primary endothelial cells. Biochemical and Biophysical Research Communications 1999, 263(2):384-388.
• [11]Zerlin M, Julius MA, Kitajewski J: Wnt/Frizzled signaling in angiogenesis. Angiogenesis 2008, 11(1):63-69.
• [12]Goodwin AM, Kitajewski J, D'Amore PA: Wnt1 and Wnt5a affect endothelial proliferation and capillary length; Wnt2 does not. Growth Factors 2007, 25(1):25-32.
• [13]Levy L, Neuveut C, Renard CA, Charneau P, Branchereau S, Gauthier F, Van Nhieu JT, Cherqui D, Petit-Bertron AF, Mathieu D, et al.: Transcriptional activation of interleukin-8 by beta-catenin-Tcf4. Journal of Biological Chemistry 2002, 277(44):42386-42393.
• [14]Masckauchan TN, Shawber CJ, Funahashi Y, Li CM, Kitajewski J: Wnt/beta-catenin signaling induces proliferation, survival and interleukin-8 in human endothelial cells. Angiogenesis 2005, 8(1):43-51.
• [15]Gherghe CM, Duan J, Gong J, Rojas M, Klauber-Demore N, Majesky M, Deb A: Wnt1 is a proangiogenic molecule, enhances human endothelial progenitor function, and increases blood flow to ischemic limbs in a HGF-dependent manner. FASEB J 2011, 25(6):1836-1843.
• [16]Chenga CW, Smith SK, Charnock-Jones DS: Wnt-1 signaling inhibits human umbilical vein endothelial cell proliferation and alters cell morphology. Experimental Cell Research 2003, 291(2):415-425.
• [17]Samarzija I, Sini P, Schlange T, Macdonald G, Hynes NE: Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways. Biochem Biophys Res Commun 2009, 386(3):449-454.
• [18]Hanai J, Dhanabal M, Karumanchi SA, Albanese C, Waterman M, Chan B, Ramchandran R, Pestell R, Sukhatme VP: Endostatin causes G(1) arrest of endothelial cells through inhibition of cyclin D1. Journal of Biological Chemistry 2002, 277(19):16464-16469.
• [19]Ezan J, Leroux L, Barandon L, Dufourcq P, Jaspard W, Moreau C, Allieres C, Daret D, Couffinhal T, Duplaa C: FrzA/sFRP-1, a secreted antagonist of the Wnt-Frizzled pathway, controls vascular cell proliferation in vitro and in vivo. Cardiovascular Research 2004, 63(4):731-738.
• [20]Klingel S, Morath I, et al.: Subfunctionalization and neofunctionalization of vertebrate Lef/Tcf transcription factors. Wnt Meeting Proceedings 2011, 33.
• [21]Gradl D, König A, Wedlich D: Functional diversity of Xenopus lymphoid enhancer factor/T-cell factor transcription factors relies on combinations of activating and repressing elements. J Biol Chem 2002, 277(16):14159-14171.
• [22]Roose J, Huls G, van Beest M, Moerer P, van der Horn K, Goldschmeding R, Logtenberg T, Clevers H: Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1. Science 1999, 285(5435):1923-1926.
• [23]Jamieson C, Sharma M, Henderson BR: Regulation of β-Catenin Nuclear Dynamics by GSK-3β Involves a LEF-1 Positive Feedback Loop. Traffic 2011, 12(8):983-999.
• [24]Nguyen A, Rosner A, Milovanovic T, Hope C, Planutis K, Saha B, Chaiwun B, Lin F, Imam SA, Marsh JL, et al.: Wnt pathway component Lef1 mediates tumor cell invasion and is expressed in human and murine breast cancers lacking ErbB2 (her-2/neu) overexpression. International Journal of Oncology 2005, 27(4):949-956.
• [25]Medici D, Hay ED, Olsen BR: Snail and Slug Promote Epithelial-Mesenchymal Transition through beta-Catenin-T-Cell Factor-4-dependent Expression of Transforming Growth Factor-beta 3. Molecular Biology of the Cell 2008, 19(11):4875-4887.
• [26]Edgell CJ, McDonald CC, Graham JB: PERMANENT CELL-LINE EXPRESSING HUMAN FACTOR-VIII-RELATED ANTIGEN ESTABLISHED BY HYBRIDIZATION. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 1983, 80(12):3734-3737.
• [27]Planutis K, Planutiene M, Holcombe RF: Coculture methodologies for the study of Wnt signals. Methods Mol Biol 2008, 468:255-261.
• [28]Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003, 423(6938):448-452.
• [29]Wu B, Crampton S, Hughes C: Wnt signaling induces matrix metalloproteinase expression and regulates T cell transmigration. Immunity 2007, 26(2):227-239.
• [30]Albini A, Benelli R: The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation. Nature Protocols 2007, 2(3):504-511.
• [31]Vacca A, Ribatti D, Presta M, Minischetti M, Iurlaro M, Ria R, Albini A, Bussolino F, Dammacco F: Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma. Blood 1999, 93(9):3064-3073.
• [32]Goodwin A, D'Amore P: Wnt signaling in the vasculature. Angiogenesis 2002, 5(1-2):1-9.
• [33]Atcha F, Munguia J, Li T, Hovanes K, Waterman M: A new beta-catenin-dependent activation domain in T cell factor. J Biol Chem 2003, 278(18):16169-16175.
• [34]Waterman ML: Lymphoid enhancer factor/T cell factor expression in colorectal cancer. Cancer Metastasis Rev 2004, 23(1-2):41-52.
• [35]Davis G, Senger D: Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res 2005, 97(11):1093-1107.
• [36]Masckauchán TN, Agalliu D, Vorontchikhina M, Ahn A, Parmalee NL, Li CM, Khoo A, Tycko B, Brown AM, Kitajewski J: Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2. Mol Biol Cell 2006, 17(12):5163-5172.
• [37]Ju R, Cirone P, Lin S, Griesbach H, Slusarski DC, Crews CM: Activation of the planar cell polarity formin DAAM1 leads to inhibition of endothelial cell proliferation, migration, and angiogenesis. Proc Natl Acad Sci USA 2010, 107(15):6906-6911.
• [38]Hovanes K, Li TW, Munguia JE, Truong T, Milovanovic T, Lawrence Marsh J, Holcombe RF, Waterman ML: Beta-catenin-sensitive isoforms of lymphoid enhancer factor-1 are selectively expressed in colon cancer. Nat Genet 2001, 28(1):53-57.