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• W2049649964 abstract "Neovascularization promotes wound healing, tumor growth, and arthritis. Endothelial cell migration and survival during neovascularization are regulated by adhesion proteins, including integrin α5β1. Integrin α5β1 is poorly expressed on normal quiescent blood vessels, but its expression is induced on tumor blood vessels and in response to angiogenic factors such as basic fibroblast growth factor, interleukin-8, tumor necrosis factor-α, and the angiomatrix protein Del-1. We show here that α5β1 expression, and hence function, during angiogenesis is regulated by the transcription factor Hox D3, a homeobox gene that also controls the expression of endothelial cell integrin αvβ3 and urokinase-type plasminogen activator. Hox D3 expression in endothelial cells enhances integrin α5 protein and message expression, whereas Hox D3 antisense inhibits its expression. Hox D3 promotes α5expression during angiogenesis in vivo, whereas inhibition of α5 expression by Hox D3 antisense suppresses angiogenesis. Hox D3 binds directly to the promoters of the integrin α5 and β3 subunits, inducing subunit expression. As Hox D3, integrin αvβ3, and integrin α5β1 are expressed on tumor blood vessels but not on normal quiescent vessels, these studies suggest that Hox D3 coordinately regulates the expression of integrin α5β1 and integrin αvβ3 during angiogenesis in vivo. These studies also suggest that Hox D3 inhibition could be a useful approach to inhibit tumor angiogenesis. Neovascularization promotes wound healing, tumor growth, and arthritis. Endothelial cell migration and survival during neovascularization are regulated by adhesion proteins, including integrin α5β1. Integrin α5β1 is poorly expressed on normal quiescent blood vessels, but its expression is induced on tumor blood vessels and in response to angiogenic factors such as basic fibroblast growth factor, interleukin-8, tumor necrosis factor-α, and the angiomatrix protein Del-1. We show here that α5β1 expression, and hence function, during angiogenesis is regulated by the transcription factor Hox D3, a homeobox gene that also controls the expression of endothelial cell integrin αvβ3 and urokinase-type plasminogen activator. Hox D3 expression in endothelial cells enhances integrin α5 protein and message expression, whereas Hox D3 antisense inhibits its expression. Hox D3 promotes α5expression during angiogenesis in vivo, whereas inhibition of α5 expression by Hox D3 antisense suppresses angiogenesis. Hox D3 binds directly to the promoters of the integrin α5 and β3 subunits, inducing subunit expression. As Hox D3, integrin αvβ3, and integrin α5β1 are expressed on tumor blood vessels but not on normal quiescent vessels, these studies suggest that Hox D3 coordinately regulates the expression of integrin α5β1 and integrin αvβ3 during angiogenesis in vivo. These studies also suggest that Hox D3 inhibition could be a useful approach to inhibit tumor angiogenesis. Angiogenesis, the formation and differentiation of blood vessels from pre-existing vessels or endothelial progenitor cells, is important in both health and disease (1Varner J. Goldberg I. Rosen E.M. Regulation of Angiogenesis. Birkhauser Verlag Basel, Switzerland1997: 361-390Google Scholar, 2Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7531) Google Scholar, 3Boudreau N. Myers C. Breast Cancer Res. 2003; 5: 140-146Crossref PubMed Scopus (115) Google Scholar, 4Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar, 5Eliceiri B.P. Cheresh D.A. J. Clin. Investig. 1999; 103: 1227-1230Crossref PubMed Scopus (619) Google Scholar, 6Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3298) Google Scholar). Neovascularization is an important process during embryonic development, wound healing, and reproduction. It also plays an important role in the development of tumors and other diseases such as diabetic retinopathy, age-related macular degeneration, and psoriasis (1Varner J. Goldberg I. Rosen E.M. Regulation of Angiogenesis. Birkhauser Verlag Basel, Switzerland1997: 361-390Google Scholar, 2Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7531) Google Scholar, 3Boudreau N. Myers C. Breast Cancer Res. 2003; 5: 140-146Crossref PubMed Scopus (115) Google Scholar, 4Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7235) Google Scholar, 5Eliceiri B.P. Cheresh D.A. J. Clin. Investig. 1999; 103: 1227-1230Crossref PubMed Scopus (619) Google Scholar, 6Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3298) Google Scholar). Because neovascularization promotes cancer and other diseases, it is important to gain an understanding of the mechanisms by which angiogenesis is regulated. The integrin family of cell adhesion proteins mediates cell attachment to the extracellular matrix and promotes the survival, proliferation, and motility of ECs 1The abbreviations used are: ECendothelial cellsbFGFbasic fibroblast growth factorILinterleukinTNF-αtumor necrosis factor-αCAMschorioallantoic membraneVEGFsvascular endothelial growth factorsuPAurokinase-type plasminogen activatorRTreverse transcriptaseGADPHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent proteinCMVcytomegalovirusHAhemagglutininHMECimmortalized human microvascular endothelial cells. during angiogenesis (7Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9026) Google Scholar, 8Cheresh D. Adv. Mol. Cell Biol. 1993; 6: 225-252Crossref Scopus (63) Google Scholar, 9Meredith Jr., J.E. Fazeli B. Schwartz M.A. Mol. Biol. Cell. 1993; 4: 953-961Crossref PubMed Scopus (1405) Google Scholar, 10Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2185) Google Scholar, 11Boudreau N. Werb Z. Bissell M.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3509-3513Crossref PubMed Scopus (279) Google Scholar, 12Kim S. Bakre M. Yin H. Varner J. J. Clin. Investig. 2002; 110: 933-941Crossref PubMed Scopus (160) Google Scholar, 13Stupack D.G. Puente X.S. Butsaboualoy S. Storgard C.M. Cheresh D.A. J. Cell Biol. 2001; 155: 459-470Crossref PubMed Scopus (443) Google Scholar, 14Cheresh D.A. Stupack D.G. Nat. Med. 2002; 8: 1-2Crossref PubMed Scopus (102) Google Scholar, 15Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2750) Google Scholar, 16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar). In fact, at least three integrins receptors for provisional matrix proteins (αvβ3, αvβ5, and α5β1) play important roles in angiogenesis (10Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2185) Google Scholar, 12Kim S. Bakre M. Yin H. Varner J. J. Clin. Investig. 2002; 110: 933-941Crossref PubMed Scopus (160) Google Scholar, 13Stupack D.G. Puente X.S. Butsaboualoy S. Storgard C.M. Cheresh D.A. J. Cell Biol. 2001; 155: 459-470Crossref PubMed Scopus (443) Google Scholar, 16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 18Friedlander M. Brooks P.C. Shaffer R.W. Kincaid C.M. Varner J.A. Cheresh D.A. Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1225) Google Scholar, 19Kloss C. Werner A. Klein M. Shen J. Menuz K. Probst J.C. Kreutzberg G.W. Raivitch G. J. Comp. Neurol. 1999; 411: 162-178Crossref PubMed Scopus (113) Google Scholar, 20Clark R.A.F. Tonneson M.G. Gailit J. Cheresh D.A. Am. J. Pathol. 1996; 148: 1407-1421PubMed Google Scholar, 21Drake C.J. Cheresh D.A. Little C.D. J. Cell Sci. 1995; 108: 2655-2661Crossref PubMed Google Scholar, 22Friedlander M. Theesfield C.L. Sugita M. Fruttiger M. Thomas M.A. Chang S. Cheresh D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9764-9769Crossref PubMed Scopus (440) Google Scholar). Integrin α5β1 plays a key role in the regulation of angiogenesis, as antagonists of this integrin inhibit angiogenesis (12Kim S. Bakre M. Yin H. Varner J. J. Clin. Investig. 2002; 110: 933-941Crossref PubMed Scopus (160) Google Scholar, 16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Both α5β1 and its ligand fibronectin are poorly expressed in quiescent endothelium but strongly expressed in proliferating endothelium (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Expression of integrin α5β1 is up-regulated on human tumor vasculature in breast and colon tumors (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). It is also up-regulated on blood vessels in the brain during wound healing (19Kloss C. Werner A. Klein M. Shen J. Menuz K. Probst J.C. Kreutzberg G.W. Raivitch G. J. Comp. Neurol. 1999; 411: 162-178Crossref PubMed Scopus (113) Google Scholar). Once expressed, α5β1 regulates the survival and migration of endothelial cells in vitro and in vivo (12Kim S. Bakre M. Yin H. Varner J. J. Clin. Investig. 2002; 110: 933-941Crossref PubMed Scopus (160) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Loss of the gene encoding the integrin α5 subunit is embryonic lethal and is associated with vascular and cardiac defects, as well as with a complete absence of the posterior somites (23Yang J.T. Rayburn H. Hynes R.O. Development. 1993; 119: 1093-1105Crossref PubMed Google Scholar, 24Goh K.L. Yang J.T. Hynes R.O. Development. 1997; 124: 4309-4319PubMed Google Scholar). As expression of integrin α5β1 is modulated during angiogenesis, thereby affecting its function during angiogenesis, it is important to delineate the mechanisms by which its expression in the endothelium is controlled. endothelial cells basic fibroblast growth factor interleukin tumor necrosis factor-α chorioallantoic membrane vascular endothelial growth factors urokinase-type plasminogen activator reverse transcriptase glyceraldehyde-3-phosphate dehydrogenase green fluorescent protein cytomegalovirus hemagglutinin immortalized human microvascular endothelial cells. Like the integrin α5β1, integrin αvβ3 is poorly expressed by quiescent endothelium, but its expression is significantly up-regulated in response to angiogenic growth factors (15Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2750) Google Scholar). The expression of αvβ3 during angiogenesis is regulated by the transcription factor Hox D3 (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar, 26Taniguchi Y. Komatsu N. Moriuchi T. Blood. 1995; 85: 2786-2794Crossref PubMed Google Scholar, 27Uyeno L.A. Newman-Keagle J.A. Cheung I. Hunt T.K. Young D.M. Boudreau N. J. Surg. Res. 2001; 100: 46-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar), a homeobox-containing transcription factor that converts endothelial cells from the quiescent to the proliferative state (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar, 26Taniguchi Y. Komatsu N. Moriuchi T. Blood. 1995; 85: 2786-2794Crossref PubMed Google Scholar, 27Uyeno L.A. Newman-Keagle J.A. Cheung I. Hunt T.K. Young D.M. Boudreau N. J. Surg. Res. 2001; 100: 46-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar, 28Zhong J. Eliceiri B. Stupack D. Penta K. Sakamoto G. Hynes R. Coleman M. Quertermous T. Boudreau N. Varner J. J. Clin. Investig. 2003; 112: 30-41Crossref PubMed Scopus (104) Google Scholar). Homeobox genes are master transcription factors discovered for their roles in regulating the development of the body plan during embryogenesis (29Holland P.W.H. Fernandez J. Dev. Biol. 1996; 173: 382-395Crossref PubMed Scopus (361) Google Scholar). The hoxd-3 gene, and the paralogous members of its chromosomal linkage group, hoxa-1 and hoxb-3, control the development of mesenchyme-derived structures during development; loss of these structures causes various defects in the establishment of the body axis and inappropriate vessel development (29Holland P.W.H. Fernandez J. Dev. Biol. 1996; 173: 382-395Crossref PubMed Scopus (361) Google Scholar, 30Condle B.G. Capecchi M.R. Development. 1993; 119: 579-595PubMed Google Scholar, 31Manley N. Capecchi M.R. Dev. Biol. 1997; 192: 274-288Crossref PubMed Scopus (139) Google Scholar). In the adult, Hox D3 is expressed in vascular endothelium in response to angiogenic growth factors, such as bFGF and Del-1, in healing wounds, and in tumors (27Uyeno L.A. Newman-Keagle J.A. Cheung I. Hunt T.K. Young D.M. Boudreau N. J. Surg. Res. 2001; 100: 46-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar). This transcription factor subsequently promotes expression of several genes associated with the angiogenic phenotype including cyclin D1, integrin αvβ3, and uPA (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar, 26Taniguchi Y. Komatsu N. Moriuchi T. Blood. 1995; 85: 2786-2794Crossref PubMed Google Scholar, 28Zhong J. Eliceiri B. Stupack D. Penta K. Sakamoto G. Hynes R. Coleman M. Quertermous T. Boudreau N. Varner J. J. Clin. Investig. 2003; 112: 30-41Crossref PubMed Scopus (104) Google Scholar, 32Hidai C. Zupancic T. Penta K. Mikhail A. Kawana M. Quertermous E.E. Aoka Y. Fukagawa M. Matsui Y. Platika D. Auerbach R. Hogan B.L.M. Snodgrass R. Quertermous T. Genes Dev. 1998; 12: 21-33Crossref PubMed Scopus (200) Google Scholar, 33Penta K. Varner J.A. Liaw L. Hidai H. Schatzman R. Quertermous T. J. Biol. Chem. 1999; 274: 11101-11109Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 34Aoka Y. Schatzman R. Hirata K.-I. Hidai C. Varner J. Quertermous T. Microvasc. Res. 2002; 64: 148-161Crossref PubMed Scopus (75) Google Scholar). When overexpressed in vivo, Hox D3 promotes a hemangioma-like proliferation of blood vessels (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar, 28Zhong J. Eliceiri B. Stupack D. Penta K. Sakamoto G. Hynes R. Coleman M. Quertermous T. Boudreau N. Varner J. J. Clin. Investig. 2003; 112: 30-41Crossref PubMed Scopus (104) Google Scholar). In contrast, Hox D3 antisense inhibits angiogenesis and suppresses expression of integrin αvβ3 and uPA (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar, 26Taniguchi Y. Komatsu N. Moriuchi T. Blood. 1995; 85: 2786-2794Crossref PubMed Google Scholar). In the current studies, we found that Hox D3 regulates integrin α5β1 expression. As integrin α5β1 and Hox D3 are both expressed by tumor endothelium but not by normal endothelium, these results suggest that Hox D3 regulates integrin α5β1 expression in tumor endothelium. Thus, Hox D3 may provide a switch to activate a program of angiogenesis that includes expression of both during αvβ3 and α5β1 angiogenesis. Once integrins α5β1 and αvβ3 are expressed, angiogenesis depends on each integrin as antagonists of each can block angiogenesis in vivo (10Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2185) Google Scholar, 15Brooks P.C. Clark R.A. Cheresh D.A. Science. 1994; 264: 569-571Crossref PubMed Scopus (2750) Google Scholar, 16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 35Stoeltzing O. Liu W. Reinmuth N. Fan F. Parry G.C. Parikh A.A. McCarty M.F. Bucana C.D. Mazar A.P. Ellis L.M. Int. J. Cancer. 2003; 104: 496-503Crossref PubMed Scopus (196) Google Scholar). CAM Assays—Chicken embryos (McIntyre Poultry, Ramona, CA) were stimulated with 3 μg of recombinant murine Del-1 and 30 ng of recombinant human bFGF, IL-8, TNFα, or human VEGF (Genzyme, Cambridge, MA) or saline in 30 μl as described (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Unfixed CAMs were flash-frozen in OCT, sectioned, and stained with anti-integrin α5β1 or αvβ5 and von Willebrand factor antibodies or homogenized in ice-cold RIPA buffer prior to analysis of protein expression for integrins α5β1 or αvβ5 by Western blotting. In some studies, 2 μg of purified plasmid DNA of pCHG (Hox D3 sense) (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar) or pCMV-D3AS (Hox D3 antisense) (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar) and/or 2 μg of green fluorescent protein plasmid (N1-GFP) were applied to Del-1 or bFGF-stimulated CAMs. 500 μl of 3.7% paraformaldehyde were applied to CAMs prior to excision for counting vessel branch points (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Ten embryos were used per treatment group. Statistical analyses were performed using Student's t test. In Vitro Cell Culture—Endothelial cells (human microvascular endothelial cells) were cultured in EGM (complete growth medium containing bFGF, VEGF and serum from Clonetics, San Diego, CA). Cell lysates were prepared by as described (17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). HMEC-1 immortalized human microvascular endothelial cells (36Ades W. Candal F.J. Swerlick R.A. George V.G. Summers S. Bosse D.C. Lawley T.J. J. Investig. Dermatol. 1992; 99: 683-690Abstract Full Text PDF PubMed Google Scholar) were a gift from T. Lawley, Emory University. Cell adhesion and ligand binding assays were performed as described (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). HMEC-1 cells were cultured as described previously (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar). HMEC-1 were transfected with HA/Hox D3 or CMVβgal using Effectene (Qiagen, Valencia, CA), and stable pools of transfected cells were selected using 35 μg/ml G418. The HA/Hox D3 expression plasmid was constructed by cloning the Hox D3 coding sequence into a CMV-driven expression plasmid (pcDNA3). The insert plus promoter were excised using Kpn/NotI and inserted in-frame into the pHM6 epitope expression vector under control of the CMV promoter (Roche Applied Science). The sequence of the resulting HA/Hox D3 expression vector was confirmed by ABI sequencing at the Biomolecular Resource Center at the University of California, San Francisco. RT-PCR—RNA was extracted from CAMs using Qiagen (Valencia, CA) RNA easy kits. Semi-quantitative RT-PCR was performed using Quantum™ RNA 18 S internal competitive standards (Ambion, Woodward, TX). Specific cDNA primers were chicken GAPDH forward (5′-CTACACACGGACACTTCAAGGGCA-3′), chicken GAPDH reverse (5′-TCCAGACGGCAGGTCAGGTCAACA-3′), and chicken Hox D3 forward (5′-AAAGAGATACACGGGGACAGCA-3′), chicken Hox D3 reverse (5′ AGAGATGAGTTAGACCAAAGAT-3′). Products were chicken GAPDH (598 bp) and chicken GAPDH (170 bp). Immunoprecipitation of DNA Bound to Hox Proteins—Hox-bound DNA was recovered by a modification of methods described previously (37Fragoso G. Hager G.L. Methods. 1997; 11: 246-252Crossref PubMed Scopus (33) Google Scholar, 38Orlando V. Paro R. Cell. 1993; 75: 1187-1198Abstract Full Text PDF PubMed Scopus (301) Google Scholar). 8 × 106 HMEC-1 were treated with 1% formaldehyde for 1, 5, 30, and 60 min; 5 min of fixation yielded the best results and was used for further studies (37Fragoso G. Hager G.L. Methods. 1997; 11: 246-252Crossref PubMed Scopus (33) Google Scholar). Cells were then solubilized in cold phosphate-buffered saline, and cytoplasmic and nuclear fractions were prepared as described (37Fragoso G. Hager G.L. Methods. 1997; 11: 246-252Crossref PubMed Scopus (33) Google Scholar). Nuclei were confirmed to be intact using a hemocytometer and 10-20 μg of intact nuclei were resuspended in Workman and Langman's buffer (37Fragoso G. Hager G.L. Methods. 1997; 11: 246-252Crossref PubMed Scopus (33) Google Scholar). The intact nuclei were restriction-digested with 100 units of HaeIII or PvuII. Following digestion, nuclei were lysed by shearing through a 21-gauge needle in RIPA, and 10 μl of anti-HA (Roche Applied Science), anti-HoxB3 (Covance, Princeton, NJ), or control IgG was added overnight at 4 °C. Immune complexes were precipitated by addition of 50 μl of 10% w/v protein A-Sepharose. Pellets were then washed in 5 times in RIPA, and formaldehyde cross-links were reversed by heating for 1 h at 60 °C. Proteinase K was added for 1 h to digest associated proteins, and the remaining DNA was purified by extraction with phenol/chloroform. A fraction of the recovered DNA was end labeled with [32P]dCTP to confirm both length and presence of DNA remaining at this step. Analysis of Isolated Genomic Sequences—To screen for the presence of promoter sequences in Hox D3-bound genomic DNA, 1 μg of pellet or supernatant DNA was amplified with the following primers: β3 integrin-F, 5′-atgtggtcttgccctcaaca-3′ corresponding to bp 9-26, and R 5′-ctcgcatctcgtccgcct 3′-corresponding to bp 574-591 of the published sequence (GenBank™ accession number L28832). Amplification was for 35 cycles at an annealing temperature of 50 °C. The α5 integrin promoter was amplified using the following primers: F 5′-ttaggagctgaaggtttgggt-3′ corresponding to bp 11-32 and R 5′-cagggaagagcgctatg-3′ corresponding to bp 933-953 of published sequence (GenBank™ accession number U48214). Amplification was for 35 cycles at an annealing temperature of 55 °C. The sequences of the resulting PCR products were subsequently confirmed by Big Dye Terminator at the Biomolecular Resource Center, University of California, San Francisco. Slot blot analysis was performed using 1 ng of genomic DNA obtained from pellets or 10 ng from supernatants following Hox immunoprecipitation as described (38Orlando V. Paro R. Cell. 1993; 75: 1187-1198Abstract Full Text PDF PubMed Scopus (301) Google Scholar). DNA was diluted in 10× SSC and spotted onto Hybond N+ nylon membranes using a slot blot apparatus. DNA was then denatured in 1.5 m NaCl and 0.5 m NaOH and subsequently neutralized with 1.5 m NaCl and 0.5 m Tris, pH 7.2. Integrin β3 promoter (600 bp), integrin α5 promoter (900 bp), or MMP14 promoter (1.4 kb) probes were labeled with [32P]dCTP by random priming. Blots were hybridized with 1 × 106 cpm of probe/ml of hybridization buffer (Hybridsol I, Oncor, Gaithersburg, MD) overnight at 45 °C and washed with 1% SSC, 0.1% SDS, and 0.2% SSC and 0.5% SDS at 45 and 68 °C, respectively, and exposed to x-ray film overnight. Promoter Assays—The 600-bp PCR product corresponding to the β3 integrin promoter was cloned into the PGL3 luciferase reporter vector (Promega, Madison, WI). Site-directed mutagenesis of two adjacent ATTA Hox consensus sites in the β3 promoter were introduced using a QuikChange Mutagenesis kit (Stratagene, La Jolla, CA) with the following primer, 5′-ggcaagaaaaaacttagtgaagcttaaaggactgaaccgg-3′. A 1.4-kb region of the MMP-14 promoter cloned into the PGL3 luciferase reporter vector was a gift from J. Madri (Yale University, New Haven CT). Luciferase assays were performed 72 h following transient transfection of HMEC-1 with promoter/reporter constructs. Luciferase activity was detected using the Luciferase Assay kit (Promega, Madison, WI). Transfection efficiency was quantified by co-transfection with CMV-LacZ, and β-galactosidase activity was determined using the Galactolight kit (Tropix, Bedford MA). In Situ Hybridization and Fluorescence in Situ Hybridization—7 μm paraffin-embedded human breast tissue sections were deparaffinized by heating at 80 °C for 30 min followed by two washes in xylene for 5 min as described (27Uyeno L.A. Newman-Keagle J.A. Cheung I. Hunt T.K. Young D.M. Boudreau N. J. Surg. Res. 2001; 100: 46-56Abstract Full Text PDF PubMed Scopus (38) Google Scholar). Sections were rehydrated through an ethanol series, post-fixed for 5 min with 4% paraformaldehyde, digested with 1 μg/ml proteinase K (Sigma) for 10 min, and hybridized using 800 ng/ml digoxigenin-labeled Hox D3 riboprobes generated by using a Roche Applied Science RNA Digoxigenin labeling kit with either T7 or Sp6 RNA polymerase as described (25Boudreau N. Andrews C. Srebow A. Ravanpay A. Cheresh D.A. J. Cell Biol. 1997; 139: 257-264Crossref PubMed Scopus (195) Google Scholar). Expression of Integrin α5β1during Tumor Angiogenesis—The integrin α5β1 plays an important in role in the regulation of angiogenesis (12Kim S. Bakre M. Yin H. Varner J. J. Clin. Investig. 2002; 110: 933-941Crossref PubMed Scopus (160) Google Scholar, 16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 17Kim S. Harris M. Varner J. J. Biol. Chem. 2000; 275: 33920-33928Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 35Stoeltzing O. Liu W. Reinmuth N. Fan F. Parry G.C. Parikh A.A. McCarty M.F. Bucana C.D. Mazar A.P. Ellis L.M. Int. J. Cancer. 2003; 104: 496-503Crossref PubMed Scopus (196) Google Scholar). Our previous studies (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar) demonstrated that integrin α5β1 is expressed on blood vessels in breast and colon tumors but not on blood vessels in normal colon or breast. As α5β1 plays a key role in regulating tumor angiogenesis (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, 35Stoeltzing O. Liu W. Reinmuth N. Fan F. Parry G.C. Parikh A.A. McCarty M.F. Bucana C.D. Mazar A.P. Ellis L.M. Int. J. Cancer. 2003; 104: 496-503Crossref PubMed Scopus (196) Google Scholar), we examined the expression of this integrin on a variety of tumor types. Immunohistochemical staining was performed on frozen biopsies of various tumors to detect integrin α5β1 (red) and von Willebrand factor (green), a marker of vascular endothelium. We found that integrin α5β1 is expressed on many of the smaller vessels and some of the larger vessels in each tumor type so far examined (seen as yellow in merged images of α5β1 and von Willebrand factor staining), including squamous cell, colon, ovarian, non-small cell lung, bladder, and breast carcinomas, as well as glioblastoma and melanoma (Fig. 1). Some integrin α5β1 expression is also observed on tumor cells in melanoma and bladder carcinoma. In contrast, α5β1 is not expressed on blood vessels in normal tissues, such as normal ovary (Fig. 1). Thus, integrin α5β1 expression is up-regulated on vascular endothelium in a wide variety of tumors. Growth Factor Regulation of α5β1Expression—To evaluate the mechanisms regulating α5β1 expression in tumor angiogenesis, we examined the expression of this integrin in response to various growth factors known to promote tumor angiogenesis. We previously observed that bFGF, but not VEGF, up-regulates integrin α5β1 expression in vivo (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). To evaluate further the roles of various angiogenic growth factors in the regulation of α5β1 expression, chick chorioallantoic membranes were stimulated with saline, IL-8, TNFα, or the angiogenic extracellular matrix protein Del-1, which is also expressed in many tumors (32Hidai C. Zupancic T. Penta K. Mikhail A. Kawana M. Quertermous E.E. Aoka Y. Fukagawa M. Matsui Y. Platika D. Auerbach R. Hogan B.L.M. Snodgrass R. Quertermous T. Genes Dev. 1998; 12: 21-33Crossref PubMed Scopus (200) Google Scholar). Frozen sections of these CAMs were immunostained to detect von Willebrand factor, a marker of blood vessels (green), and integrin α5β1 expression (red). Integrin α5β1 expression on blood vessels is observed as yellow in the merged images. We found that integrin α5β1 expression is up-regulated in vivo in response to the angiogenic growth factors bFGF (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar), IL-8, and TNFα, whereas CAMs exposed to saline or VEGF (16Kim S. Bell K. Mousa S. Varner J.A. Am. J. Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text" @default.
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• W2049649964 title "The Homeobox Transcription Factor Hox D3 Promotes Integrin α5β1 Expression and Function during Angiogenesis" @default.
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