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Originally published In Press as doi:10.1074/jbc.M305190200 on November 10, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4862-4868, February 6, 2004
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The Homeobox Transcription Factor Hox D3 Promotes Integrin {alpha}5{beta}1 Expression and Function during Angiogenesis*

Nancy J. Boudreau{ddagger} and Judith A. Varner§

From the {ddagger}Department of Surgery, University of California, San Francisco, California 94143 and the §Department of Medicine and Comprehensive Cancer Center, University of California, San Diego, California 92093

Received for publication, May 17, 2003 , and in revised form, November 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neovascularization promotes wound healing, tumor growth, and arthritis. Endothelial cell migration and survival during neovascularization are regulated by adhesion proteins, including integrin {alpha}5{beta}1. Integrin {alpha}5{beta}1 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-{alpha}, and the angiomatrix protein Del-1. We show here that {alpha}5{beta}1 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 {alpha}v{beta}3 and urokinase-type plasminogen activator. Hox D3 expression in endothelial cells enhances integrin {alpha}5 protein and message expression, whereas Hox D3 antisense inhibits its expression. Hox D3 promotes {alpha}5expression during angiogenesis in vivo, whereas inhibition of {alpha}5 expression by Hox D3 antisense suppresses angiogenesis. Hox D3 binds directly to the promoters of the integrin {alpha}5 and {beta}3 subunits, inducing subunit expression. As Hox D3, integrin {alpha}v{beta}3, and integrin {alpha}5{beta}1 are expressed on tumor blood vessels but not on normal quiescent vessels, these studies suggest that Hox D3 coordinately regulates the expression of integrin {alpha}5{beta}1 and integrin {alpha}v{beta}3 during angiogenesis in vivo. These studies also suggest that Hox D3 inhibition could be a useful approach to inhibit tumor angiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis, the formation and differentiation of blood vessels from pre-existing vessels or endothelial progenitor cells, is important in both health and disease (1-6). 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 (1-6). 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 ECs1 during angiogenesis (7-18). In fact, at least three integrins receptors for provisional matrix proteins ({alpha}v{beta}3, {alpha}v{beta}5, and {alpha}5{beta}1) play important roles in angiogenesis (10, 12, 13, 16-22). Integrin {alpha}5{beta}1 plays a key role in the regulation of angiogenesis, as antagonists of this integrin inhibit angiogenesis (12, 16, 17). Both {alpha}5{beta}1 and its ligand fibronectin are poorly expressed in quiescent endothelium but strongly expressed in proliferating endothelium (16). Expression of integrin {alpha}5{beta}1 is up-regulated on human tumor vasculature in breast and colon tumors (16). It is also up-regulated on blood vessels in the brain during wound healing (19). Once expressed, {alpha}5{beta}1 regulates the survival and migration of endothelial cells in vitro and in vivo (12, 17). Loss of the gene encoding the integrin {alpha}5 subunit is embryonic lethal and is associated with vascular and cardiac defects, as well as with a complete absence of the posterior somites (23, 24). As expression of integrin {alpha}5{beta}1 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.

Like the integrin {alpha}5{beta}1, integrin {alpha}v{beta}3 is poorly expressed by quiescent endothelium, but its expression is significantly up-regulated in response to angiogenic growth factors (15). The expression of {alpha}v{beta}3 during angiogenesis is regulated by the transcription factor Hox D3 (25-27), a homeobox-containing transcription factor that converts endothelial cells from the quiescent to the proliferative state (25-28). Homeobox genes are master transcription factors discovered for their roles in regulating the development of the body plan during embryogenesis (29). 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 (29-31). 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 (27). This transcription factor subsequently promotes expression of several genes associated with the angiogenic phenotype including cyclin D1, integrin {alpha}v{beta}3, and uPA (25, 26, 28, 32-34). When overexpressed in vivo, Hox D3 promotes a hemangioma-like proliferation of blood vessels (25, 28). In contrast, Hox D3 antisense inhibits angiogenesis and suppresses expression of integrin {alpha}v{beta}3 and uPA (25, 26).

In the current studies, we found that Hox D3 regulates integrin {alpha}5{beta}1 expression. As integrin {alpha}5{beta}1 and Hox D3 are both expressed by tumor endothelium but not by normal endothelium, these results suggest that Hox D3 regulates integrin {alpha}5{beta}1 expression in tumor endothelium. Thus, Hox D3 may provide a switch to activate a program of angiogenesis that includes expression of both during {alpha}v{beta}3 and {alpha}5{beta}1 angiogenesis. Once integrins {alpha}5{beta}1 and {alpha}v{beta}3 are expressed, angiogenesis depends on each integrin as antagonists of each can block angiogenesis in vivo (10, 15-17, 35).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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, TNF{alpha}, or human VEGF (Genzyme, Cambridge, MA) or saline in 30 µl as described (16). Unfixed CAMs were flash-frozen in OCT, sectioned, and stained with anti-integrin {alpha}5{beta}1 or {alpha}v{beta}5 and von Willebrand factor antibodies or homogenized in ice-cold RIPA buffer prior to analysis of protein expression for integrins {alpha}5{beta}1 or {alpha}v{beta}5 by Western blotting. In some studies, 2 µg of purified plasmid DNA of pCHG (Hox D3 sense) (25) or pCMV-D3AS (Hox D3 antisense) (25) 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 (16). 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 (17). HMEC-1 immortalized human microvascular endothelial cells (36) were a gift from T. Lawley, Emory University. Cell adhesion and ligand binding assays were performed as described (16). HMEC-1 cells were cultured as described previously (25). HMEC-1 were transfected with HA/Hox D3 or CMV{beta}gal 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 QuantumTM 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 (37, 38). 8 x 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 (37). Cells were then solubilized in cold phosphate-buffered saline, and cytoplasmic and nuclear fractions were prepared as described (37). Nuclei were confirmed to be intact using a hemocytometer and 10-20 µg of intact nuclei were resuspended in Workman and Langman's buffer (37). 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: {beta}3 integrin-F, 5'-atgtggtcttgccctcaaca-3' corresponding to bp 9-26, and R 5'-ctcgcatctcgtccgcct 3'-corresponding to bp 574-591 of the published sequence (GenBankTM accession number L28832 [GenBank] ). Amplification was for 35 cycles at an annealing temperature of 50 °C. The {alpha}5 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 (GenBankTM accession number U48214 [GenBank] ). 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 (38). DNA was diluted in 10x 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 {beta}3 promoter (600 bp), integrin {alpha}5 promoter (900 bp), or MMP14 promoter (1.4 kb) probes were labeled with [32P]dCTP by random priming. Blots were hybridized with 1 x 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 {beta}3 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 {beta}3 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 {beta}-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 (27). 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 (25).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Integrin {alpha}5{beta}1 during Tumor Angiogenesis—The integrin {alpha}5{beta}1 plays an important in role in the regulation of angiogenesis (12, 16, 17, 35). Our previous studies (16) demonstrated that integrin {alpha}5{beta}1 is expressed on blood vessels in breast and colon tumors but not on blood vessels in normal colon or breast. As {alpha}5{beta}1 plays a key role in regulating tumor angiogenesis (16, 35), 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 {alpha}5{beta}1 (red) and von Willebrand factor (green), a marker of vascular endothelium. We found that integrin {alpha}5{beta}1 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 {alpha}5{beta}1 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 {alpha}5{beta}1 expression is also observed on tumor cells in melanoma and bladder carcinoma. In contrast, {alpha}5{beta}1 is not expressed on blood vessels in normal tissues, such as normal ovary (Fig. 1). Thus, integrin {alpha}5{beta}1 expression is up-regulated on vascular endothelium in a wide variety of tumors.



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  		FIG. 1.
Integrin {alpha}5{beta}1 is expressed on human tumor endothelium but not normal endothelium. Five-micron sections of human tumor biopsy specimens were immunostained to detect integrin {alpha}5{beta}1 and von Willebrand factor, a marker of vascular endothelium. x200 magnification images of squamous cell carcinoma, colon carcinoma, ovarian carcinoma, non-small cell lung carcinoma, bladder carcinoma, ductal breast carcinoma in situ, glioblastoma, melanoma and normal ovary are shown. von Willebrand factor staining is observed in green, integrin {alpha}5{beta}1 in red, and co-localization of the two markers in yellow.

 
Growth Factor Regulation of {alpha}5{beta}1 Expression—To evaluate the mechanisms regulating {alpha}5{beta}1 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 {alpha}5{beta}1 expression in vivo (16). To evaluate further the roles of various angiogenic growth factors in the regulation of {alpha}5{beta}1 expression, chick chorioallantoic membranes were stimulated with saline, IL-8, TNF{alpha}, or the angiogenic extracellular matrix protein Del-1, which is also expressed in many tumors (32). Frozen sections of these CAMs were immunostained to detect von Willebrand factor, a marker of blood vessels (green), and integrin {alpha}5{beta}1 expression (red). Integrin {alpha}5{beta}1 expression on blood vessels is observed as yellow in the merged images. We found that integrin {alpha}5{beta}1 expression is up-regulated in vivo in response to the angiogenic growth factors bFGF (16), IL-8, and TNF{alpha}, whereas CAMs exposed to saline or VEGF (16) did not express this integrin (Fig. 2A). Integrin {alpha}5{beta}1 is also up-regulated in response to Del-1 (Fig. 2A).



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  		FIG. 2.
Integrin {alpha}5{beta}1 is up-regulated in response to angiogenic growth factors, including Del-1. A, 5-µm cryosections of chick chorioallantoic membranes stimulated with saline, IL-8, TNF{alpha}, or Del-1 were immunostained to detect integrin {alpha}5{beta}1 and von Willebrand factor, a marker of endothelium. x200 magnification images depict von Willebrand factor staining of vascular endothelium in green, integrin {alpha}5{beta}1 staining in red, and co-localization of the two proteins in yellow. B, 5-µm cryosections of CAMs stimulated with Del-1 for 0-36 h were immunostained to detect integrin {alpha}5{beta}1 or integrin {alpha}v{beta}5 and von Willebrand factor, a marker of endothelium. x200 magnification images depict von Willebrand factor staining in green, integrin staining in red, and co-localization of the two in yellow.

 
Integrin {alpha}5{beta}1 expression can be detected within 12 h after stimulation by Del-1 and expression peaks 24 h after stimulation by Del-1 (Fig. 2B) or by bFGF (16). In fact, the expression of this integrin parallels that of integrin {alpha}v{beta}3, which is first detected 12 h after exposure to Del-1 (28). In contrast, expression of another vascular integrin, integrin {alpha}v{beta}5, is not changed by exposure to growth factors (Fig. 2B). These studies suggest that transcription plays a role in the expression of integrin {alpha}5{beta}1 in tumor vessels and that many of the angiogenic factors expressed by tumors can up-regulate integrin {alpha}5{beta}1 expression.

Integrin {alpha}5{beta}1 Promotes Del-1 and Tumor-mediated Angiogenesis—As previous studies (16) indicated a functional role for integrin {alpha}5{beta}1 in angiogenesis, {alpha}5{beta}1 expression on blood vessels may correlate closely with a functional role in angiogenesis. For example, antagonists of {alpha}5{beta}1 inhibit angiogenesis induced in chick chorioallantoic membranes and human skin by bFGF, IL-8, and TNF{alpha} but not angiogenesis induced by VEGF (16). Antagonists of {alpha}5{beta}1 also inhibit Del-1-mediated angiogenesis (Fig. 3A). Importantly, the present studies indicate that bFGF, TNF{alpha}, IL-8, and Del-1 induce {alpha}5{beta}1 expression and function during angiogenesis. Together with our previous observations that {alpha}5{beta}1 promotes endothelial cell survival during angiogenesis, these results suggest that once expressed, integrin {alpha}5{beta}1 may function as a survival factor for vascular endothelium during angiogenesis (12). Thus, {alpha}5{beta}1 expression on blood vessels correlates with a functional role in angiogenesis.



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  		FIG. 3.
Integrin {alpha}5{beta}1 regulates Del-1 and tumor-mediated angiogenesis. A, the CAMs of 10-day-old chicken embryos were stimulated for 24 h with saline or Del-1 and then treated with saline, 25 µg of function blocking anti-{alpha}5{beta}1 or non-function blocking anti-{alpha}5{beta}1 antibodies (control IgG), 50 µM cyclic peptide inhibitor of {alpha}5{beta}1 (CRRETAWAC), or 50 µM of a scrambled cyclic peptide control (CATAERWRC), n = 10. After 48 h, blood vessel branch points were counted. Fifty-mg fragments of DU145 prostate (B), MCF7 breast (C), and Hep3 epidermoid carcinoma (D) tumors cultured on CAMs were treated systemically by intravenous injection with 10 µM active (CRRETAWAC) and inactive control (CATAERWRC) peptide inhibitors of {alpha}5{beta}1 or with antibody antagonists of {alpha}5{beta}1 and control IgG (n = 10). Five days later, tumors were excised, and mean tumor weights per treatment group (mean ± S.E.) were determined. E, 2,000,000 {alpha}5{beta}1 negative HT29 colon carcinoma cells were subcutaneously inoculated in severe combined immunodeficient mice. When tumors measured 200 mm3 (3 weeks later), mice were treated by 3 times weekly intravenous injections of 10 µM active and inactive peptides described above. After 3 weeks of treatment, tumors were excised and weighed. Ten animals were in each treatment group. Statistical significance was determined by Student's t test, p < 0.03.

 
As integrin {alpha}5{beta}1 expression and function are induced during growth factor-mediated angiogenesis, its expression on vascular endothelium in tumors suggests that it promotes tumor angiogenesis and growth. In fact, antagonists of {alpha}5{beta}1 block tumor growth of a wide variety of tumor types. For example, {alpha}5{beta}1 function-blocking antibodies or peptide antagonists block the growth of colon carcinoma (16), prostate carcinoma (Fig. 3A), breast carcinoma (Fig. 3B), and squamous cell carcinoma (Fig. 3C) on chick chorioallantoic membranes (Fig. 3). Integrin {alpha}5{beta}1 antagonists also block tumor growth in mouse models of tumor growth (Fig. 3D) (35). As integrin {alpha}5{beta}1 expression and function promote growth factor and tumor-induced angiogenesis, an understanding of how its expression is regulated could identify new means to control tumor angiogenesis.

Hox D3 Promotes {alpha}5 Expression in Vitro—The expression of integrins {alpha}v{beta}3 and {alpha}5{beta}1 are stimulated by the same growth factors during angiogenesis; therefore, their expression patterns may be regulated by the same transcription factors. The homeobox containing transcription factor Hox D3 promotes integrin {alpha}v{beta}1 expression during angiogenesis (25, 27). Therefore, we examined the role of Hox D3 in the regulation of integrin {alpha}5{beta}1 expression. HMEC-1 endothelial cells were stably transfected with pCMV-D3, a Hox D3 expression vector, leading to enhanced Hox D3 expression (Fig. 4A). Integrin {alpha}5 mRNA (Fig. 4B) and protein levels (Fig. 4C) were significantly up-regulated by expression of Hox D3 (pCMV-D3) in microvascular endothelial cells in vitro. Conversely, expression of HOX D3 antisense inhibits HOX D3, but not HOX B3 or HOX A3, gene expression (Fig. 4D) as well as {alpha}5 mRNA expression (Fig. 4E). Hox D3 had no effect on expression of the {beta}1 integrin subunit (not shown). These studies indicate that the transcription factor Hox D3 promotes integrin {alpha}5 message and protein expression.



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  		FIG. 4.
Hox D3 regulates integrin {alpha}5 subunit expression. A, ethidium bromide staining of Hox D3 cDNA amplified by RT-PCR from total RNA of HMEC-1 stably transfected with control or Hox D3 expression plasmid. 18 S rRNA band is shown as loading control. B, Northern blot analysis for {alpha}5 integrin mRNA in control or Hox D3-transfected HMEC. Ethidium bromide-stained total ribosomal RNA is shown as loading control. C, Western blot analysis of {alpha}5 integrin, ephrin A1, and actin levels in control or Hox D3-expressing HMEC-1. D, ethidium bromide staining of cDNA of Hox D3, Hox A3, Hox B3, or 18 S RNA amplified by semi-quantitative RT-PCR from total RNA of HMEC-1 stably transfected with control or Hox D3 antisense (HoxAS) plasmids. Amplification was semi-quantitatively performed using 18 S RNA competimers according to the manufacturer's directions (Ambion, Inc.). E, Northern blot analysis of {alpha}5 integrin mRNA isolated from control or Hox D3 antisense (HoxAS) transfected HMEC-1. Ethidium bromide-stained total ribosomal RNA is shown as loading control.

 
To determine whether Hox D3 directly binds to and activates the integrin {alpha}5 promoter, we performed chromatin cross-linking and immunoprecipitation studies in HMEC stably expressing an HA/Hox D3 expression plasmid. Anti-HA antibodies immunoprecipitated HA-Hox D3 from HA-Hox D3-transfected but not control-transfected cells (Fig. 5A). As Hox D3 regulates integrin {beta}3 expression (28), we first determined whether Hox D3 binds to the {beta}3 promoter. Following protein/DNA cross-linking and immunoprecipitation with either anti-HA, control IgG, or Hox B3 antibodies, we performed PCR analysis for the {beta}3 integrin promoter in genomic DNA recovered in both pellets and supernatants. The promoter for {beta}3 integrin was enriched in DNA pelleted with anti-HA antibodies but was depleted from the corresponding supernatants (Fig. 5B). In contrast, the {beta}3 promoter remained in the supernatants following cross-linking and immunoprecipitation with control IgG. Importantly, the {beta}3 integrin promoter was not recovered by immunoprecipitation with antibodies against the paralogous gene HOX B3, which does not alter {alpha}v{beta}3 expression (39). To demonstrate further that only Hox D3-regulated genes are recovered by anti-HA/Hox D3 immunoprecipitation, we probed DNA recovered from the pellets and supernatants with probes for the {beta}3 integrin promoter or the promoter corresponding to MMP-14, a gene whose expression is not affected by Hox D3 (Fig. 5C). We observed that the {beta}3 integrin promoter was present in pellets following immunoprecipitation with anti-HA antibodies, whereas the promoter for MMP-14 remained in supernatants (Fig. 5D).



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  		FIG. 5.
Hox D3 binds to and activates integrin promoters. A, HA-tagged Hox D3 was immunoprecipitated from pHM6-HA-Hox D3 and control (pHM6) transfected HMEC-1 and immunoblotted with anti-HA antibodies. B, PCR amplification of a 600-bp region of the integrin {beta}3 promoter from pellets (p) and supernatants (s) of genomic DNA recovered from anti-HA-Hox D3, control IgG, and anti-Hox B3 immunoprecipitates from HA-Hox D3-transfected HMEC. C, Northern blot of integrin {beta}3 and MMP-14 message levels in control or HA-Hox D3-transfected cells. Ethidium bromide-stained total ribosomal RNA is shown as loading control. D, slot blots of DNA recovered from supernatants or pellets of anti-HA immunoprecipitates from HA/Hox D3-transfected HMEC-1 were probed with [32P]dCTP-labeled promoters of integrin {beta}3 or MMP-14. E, PCR amplification of integrin {alpha}5 promoter in genomic DNA recovered from pellets (p) or supernatants (s) following cross-linking and immunoprecipitation with anti-HA/Hox D3. F, slot blot analysis of genomic DNA recovered in the pellet or supernatants following immunoprecipitation with anti-HA/Hox D3 or control IgG. Blotted DNA was hybridized with a [32P]dCTP-labeled probe corresponding to {alpha}5 integrin. G, sequence of the wild-type {beta}3 integrin promoter with two putative Hox consensus bindings sites underlined; the corresponding mutated {beta}3 integrin promoter sequence ({beta}3 mutant 1) is shown below with mutated base pairs shown in boldface. H, relative luciferase activity of both the wild-type and mutant {beta}3 promoter/reporter constructs after transient transfection into control (open bars) or Hox D3-expressing HMEC-1 (filled bars). I, relative luciferase activity of the MMP-14 promoter after transient transfection into control (open bars) or Hox D3 (filled bars)-expressing HMEC-1.

 
To determine whether the promoter for the {alpha}5 integrin subunit is also a direct target of Hox D3, we performed chromatin cross-linking and immunoprecipitation with anti-HA antibodies. PCR analysis of recovered DNA shows that a 900-bp fragment of the {alpha}5 promoter was present in the pellet but not the supernatant (Fig. 5E). Subsequent slot blot analysis confirmed that the {alpha}5 promoter was enriched in the DNA pelleted with Hox D3 and not in DNA recovered following immunoprecipitation with control IgG (Fig. 5F). Together these studies indicate that the transcription factor Hox D3 induces expression of integrin {alpha}5 mRNA and protein and that both {beta}3 and {alpha}5 integrins promoters are direct targets of Hox D3 in endothelial cells.

As Hox D3 directly binds to the integrin subunit promoters and also induces their up-regulation in endothelial cells, Hox D3 is likely to directly regulate the expression of these integrins by binding to their promoters and initiating transcription. To test this possibility, a luciferase expression construct under control of the integrin {beta}3 promoter was transfected into HMEC stably expressing Hox D3 (Fig. 5G). Transcriptional activation of the {beta}3 promoter was significantly higher in Hox D3-expressing HMEC as compared with control HMEC (Fig. 5H). Mutation of two putative Hox-binding sites within the {beta}3 promoter resulted in the loss of Hox D3-dependent transcriptional activity (Fig. 5H). In contrast, activity of the MMP-14 promoter was similar in both control and Hox D3-expressing HMEC (Fig. 5I). Together these results demonstrate that the Hox D3 directly binds and activates an integrin promoter.

Hox D3 Promotes {alpha}5 Expression in Vivo—Angiogenic growth factors such as bFGF induce integrin {alpha}v{beta}3 and {alpha}5{beta}1 expression (15, 16) as well as Hox D3 expression in vivo (28). Recent studies (28) demonstrated that Hox D3 regulates integrin {beta}3 expression in vivo, as Hox D3 expression stimulates and Hox D3 antisense inhibits integrin {beta}3 expression during Del-1- and bFGF-induced angiogenesis in vivo. To determine whether Hox D3 regulates integrin {alpha}5{beta}1 expression in vivo, chick chorioallantoic membranes stimulated with saline, Del-1, or bFGF were transfected with Hox D3 sense or antisense constructs. Application of either bFGF (Fig. 6, A and B) or Del-1 (Fig. 6, C and D) to CAMs stimulates angiogenesis (Fig. 6, A and C) and induces {alpha}5{beta}1 expression (Fig. 6, B and D). Integrin {alpha}v{beta}5 expression remains unchanged upon treatment with bFGF or Del-1, however. Hox D3 antisense suppresses {alpha}5 expression in vivo (Fig. 6, B and D). Expression of Hox D3 antisense suppresses chicken Hox D3 but not chicken Hox B3, indicating that the human Hox D3 antisense construct suppresses chicken Hox D3 expression and thereby integrin expression (Fig. 6E). These results indicate that Hox D3 regulates integrin {alpha}5 expression in vitro and in vivo.



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  		FIG. 6.
Hox D3 promotes {alpha}5{beta}1 expression during angiogenesis in vivo. A, CAMs of 10-day-old chicken embryos stimulated by saline or bFGF were transfected with either N1-GFP reporter vector (-) or Hox D3 sense (S) or Hox D3 antisense (AS) DNA expression constructs and N1-GFP. CAMs were fixed prior to excision, and blood vessel branch points were quantified. PBS, phosphate-buffered saline. B, expression levels of integrin {alpha}5 and {beta}5 in CAMs from B were determined by densitometry, and the ratio of {alpha}5/{alpha}v{beta}5 expression was determined. C, CAMs of 10-day-old chicken embryos stimulated by saline or Del-1 were transfected with either N1-GFP reporter vector (-) or Hox D3 sense (S) or Hox D3 antisense (AS) DNA expression constructs and N1-GFP. CAMs were fixed prior to excision, and blood vessel branch points were quantified. D, expression levels of integrin {alpha}5 and {beta}5 in CAMs from C were determined by Western blotting. Average expression levels for each integrin were determined by densitometry, and the ratio of {alpha}5/{alpha}v{beta}5 expression was determined. E, expression levels of the chicken (ck) Hox D3, chicken Hox B3, and chicken GAPDH genes in treated CAM tissues were determined by RT-PCR. Amplification was semi-quantitatively performed using 18 S RNA competimers according to the manufacturer's directions (Ambion, Inc.). * indicates statistically significant differences, with p < 0.01.

 
To determine whether Hox D3, like integrin {alpha}5{beta}1 and {alpha}v{beta}3, is expressed in tumor vessels, we performed in situ hybridization for Hox D3 in normal and tumorigenic human breast tissue. We observed little or no Hox D3 transcript in microvessels in normal breast tissue. However, expression of Hox D3 is markedly increased in angiogenic vessels associated with high grade ductal carcinoma in situ and invasive ductal carcinoma (Fig. 7). As integrin {alpha}5{beta}1 expression is up-regulated on tumor vessels, these studies indicate that Hox D3 may play a key role in regulating tumor angiogenesis. Thus, therapies based on inhibiting integrin {alpha}5{beta}1 or {alpha}v{beta}3 expression during angiogenesis by inhibiting Hox D3 expression may be valuable approaches to inhibiting tumor angiogenesis and thereby tumor growth.



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  		FIG. 7.
Expression of Hox D3 tumor endothelium. In situ hybridization for Hox D3 in normal human breast tissue (left), high grade ductal carcinoma in situ (middle), and invasive ductal carcinoma (right). Left panel shows little or no expression of Hox D3 mRNA in capillary endothelial cells (arrowheads). Middle panel shows strong positive hybridization (black) to Hox D3 mRNA in microvessels adjacent to high grade ductal carcinoma in situ. Right panel shows positive hybridization of Hox D3 in stromal capillaries from invasive ductal carcinoma.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Integrin {alpha}5{beta}1 plays a key role in regulating tumor angiogenesis (12, 16, 17). In the studies presented here, we show that the role of this integrin in angiogenesis is regulated by its expression in vivo. Furthermore, we show that {alpha}5 expression during angiogenesis is regulated in vivo and in vitro by Hox D3, a transcription factor that converts the vascular endothelium from the quiescent state to a proliferative state (25). As Hox D3 expression as well as angiogenesis can be suppressed by Hox D3 antisense, these results suggest a potential new target for clinical angiogenesis inhibition.

These and previous studies show that integrins {alpha}5{beta}1 and {alpha}v{beta}3 play key roles in regulating tumor angiogenesis (10, 15, 16, 22, 40). Furthermore, expression of both integrins is low in quiescent endothelium and markedly up-regulated in tumor-induced angiogenesis. In the studies presented here, we show that HOX D3 co-ordinately regulates expression of these key vascular cell integrins. Accordingly, we found that expression of Hox D3 is also low in quiescent endothelium but increased in tumor-associated vessels.

The expression of integrins {alpha}5{beta}1 and {alpha}v{beta}3 is up-regulated in response to similar angiogenic growth factors including TNF{alpha}, bFGF, and Del-1 (10, 15, 16, 22, 28). Antagonists of both of these integrins suppress angiogenesis induced by a variety of growth factors (10, 15, 16). Hox D3 expression can be induced by many of the same pro-angiogenic factors including bFGF, TNF{alpha}, and Del-1 (25, 28). Furthermore, when Hox D3 expression is blocked by using antisense strategies, these growth factors fail to induce expression of {alpha}v{beta}3 (25, 28) or {alpha}5{beta}1, underscoring a central role for Hox D3 in mediating growth factor-induced integrin expression and angiogenesis. Interestingly, expression of Hox D3 increases expression of {alpha}v{beta}3 integrin, promotes angiogenesis, and facilitates wound repair in diabetic tissues exhibiting compromised angiogenesis after wounding (27). These studies thus indicate a key role for Hox D3 in promoting angiogenesis in vivo through its modulation of integrins and other factors.

Interestingly neither integrin {alpha}v{beta}3 nor {alpha}5{beta}1 is up-regulated by VEGF, and antagonists of neither integrin are able to inhibit VEGF-induced angiogenesis (15, 16, 22). Importantly, we have observed that VEGF also does not induce expression of Hox D3 in EC (data not shown). Although a wide variety of tumors produce abundant VEGF, and early intervention with VEGF antagonists can block tumor angiogenesis and growth (41, 42), many tumors do not respond well to anti-VEGF treatment as other angiogenic pathways predominate (43). Our findings that expression of Hox D3 and its direct target genes, {alpha}v{beta}3 and {alpha}5{beta}1, are independent of VEGF but are directly downstream of bFGF, TNF{alpha}, and Del-1 suggest that targeting Hox D3 may provide a means to combat angiogenesis in tumors that are refractory to VEGF therapy.

Our studies show that the Hox D3 transcription factor can bind directly to the promoters of the {beta}3 and {alpha}5 integrins in intact endothelial cells by using in vivo immunoprecipitation assays. Like many other transcription factors, the minimal Hox binding consensus sequence ATTA (TAAT) is expressed frequently in the mammalian genome. Therefore, the regulation of Hox protein-target interaction is thought to be mediated both by the availability of Hox binding co-factors as well as accessibility to the target sequence in the context of chromatin (44). For example, although the promoter for MMP-14 also contains several putative Hox-binding sites, Hox D3 does not bind or activate the MMP-14 promoter in endothelial cells. In these studies, we expressed an epitope-tagged Hox D3 fusion protein to identify target genes because there are no antibodies to the Hox D3 protein. However, as antisense against endogenous Hox D3 blocks in vitro and in vivo expression of both {alpha}5 and {beta}3 integrins, these studies show that genes are physiologically relevant targets of Hox D3.

Although our studies indicate that integrin {alpha}5{beta}1 and {alpha}v{beta}3 can be similarly regulated during angiogenesis, they do not exclude the possibility that the two integrins are differentially regulated during angiogenesis in specific tissues or in other cell types. Currently, antagonists of both integrins are under investigation in separate clinical trials for cancer therapy. Future experimental results and clinical results will determine whether these integrins have identical or overlapping functions during angiogenesis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants CA83133 (to J. A. V.) and CA85249 (to N. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: 9500 Gilman Dr., La Jolla, CA 92093-0912. Tel.: 858-822-0086; Fax: 858-822-1325; E-mail: jvarner{at}ucsd.edu.

1 The abbreviations used are: EC, endothelial cells; bFGF, basic fibroblast growth factor; IL, interleukin; TNF-{alpha}, tumor necrosis factor-{alpha}; CAMs, chorioallantoic membrane; VEGFs, vascular endothelial growth factors; uPA, urokinase-type plasminogen activator; RT, reverse transcriptase; GADPH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; CMV, cytomegalovirus; HA, hemagglutinin; HMEC, immortalized human microvascular endothelial cells. Back


   ACKNOWLEDGMENTS
 
We thank Connie Myers for helpful advice with the chromatin immunoprecipitation and Kitty Cheung for help with transcriptional analysis of the {beta}3 promoter.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Varner, J. (1997) in Regulation of Angiogenesis (Goldberg, I., and Rosen, E. M., eds) pp. 361-390, Birkhauser Verlag Basel, Switzerland
  2. Carmeliet, P., and Jain, R. K. (2000) Nature 407, 249-257[CrossRef][Medline] [Order article via Infotrieve]
  3. Boudreau, N., and Myers, C. (2003) Breast Cancer Res. 5, 140-146[CrossRef][Medline] [Order article via Infotrieve]
  4. Folkman, J. (1995) Nat. Med. 1, 27-31[CrossRef][Medline] [Order article via Infotrieve]
  5. Eliceiri, B. P., and Cheresh, D. A. (1999) J. Clin. Investig. 103, 1227-1230[Medline] [Order article via Infotrieve]
  6. Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J., and Holash, J. (2000) Nature 407, 242-248[CrossRef][Medline] [Order article via Infotrieve]
  7. Hynes, R. O. (1992) Cell 69, 11-25[CrossRef][Medline] [Order article via Infotrieve]
  8. Cheresh, D. (1993) Adv. Mol. Cell Biol. 6, 225-252
  9. Meredith, J. E., Jr., Fazeli, B., and Schwartz, M. A. (1993) Mol. Biol. Cell 4, 953-961[Abstract]
  10. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994) Cell 79, 1157-1164[CrossRef][Medline] [Order article via Infotrieve]
  11. Boudreau, N., Werb, Z., and Bissell, M. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3509-3513[Abstract/Free Full Text]
  12. Kim, S., Bakre, M., Yin, H., and Varner, J. (2002) J. Clin. Investig. 110, 933-941[CrossRef][Medline] [Order article via Infotrieve]
  13. Stupack, D. G., Puente, X. S., Butsaboualoy, S., Storgard, C. M., and Cheresh, D. A. (2001) J. Cell Biol. 155, 459-470[Abstract/Free Full Text]
  14. Cheresh, D. A., and Stupack, D. G. (2002) Nat. Med. 8, 1-2[CrossRef][Medline] [Order article via Infotrieve]
  15. Brooks, P. C., Clark, R. A., and Cheresh, D. A. (1994) Science 264, 569-571[Abstract/Free Full Text]
  16. Kim, S., Bell, K., Mousa, S., and Varner, J. A. (2000) Am. J. Pathol. 156, 1345-1362[Abstract/Free Full Text]
  17. Kim, S., Harris, M., and Varner, J. (2000) J. Biol. Chem. 275, 33920-33928[Abstract/Free Full Text]
  18. Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M., Varner, J. A., and Cheresh, D. A. (1995) Science 270, 1500-1502[Abstract/Free Full Text]
  19. Kloss, C., Werner, A., Klein, M., Shen, J., Menuz, K., Probst, J. C., Kreutzberg, G. W., and Raivitch, G. (1999) J. Comp. Neurol. 411, 162-178[CrossRef][Medline] [Order article via Infotrieve]
  20. Clark, R. A. F., Tonneson, M. G., Gailit, J., and Cheresh, D. A. (1996) Am. J. Pathol. 148, 1407-1421[Abstract]
  21. Drake, C. J., Cheresh, D. A., and Little, C. D. (1995) J. Cell Sci. 108, 2655-2661[Abstract]
  22. Friedlander, M., Theesfield, C. L., Sugita, M., Fruttiger, M., Thomas, M. A., Chang, S., and Cheresh D. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9764-9769[Abstract/Free Full Text]
  23. Yang, J. T., Rayburn, H., and Hynes, R. O. (1993) Development 119, 1093-1105[Abstract]
  24. Goh, K. L., Yang, J. T., and Hynes, R. O. (1997) Development 124, 4309-4319[Abstract]
  25. Boudreau, N., Andrews, C., Srebow, A., Ravanpay, A., and Cheresh, D. A. (1997) J. Cell Biol. 139, 257-264[Abstract/Free Full Text]
  26. Taniguchi, Y., Komatsu, N., and Moriuchi, T. (1995) Blood 85, 2786-2794[Abstract/Free Full Text]
  27. Uyeno, L. A., Newman-Keagle, J. A., Cheung, I., Hunt, T. K., Young, D. M., and Boudreau, N. (2001) J. Surg. Res. 100, 46-56[CrossRef][Medline] [Order article via Infotrieve]
  28. Zhong, J., Eliceiri, B., Stupack, D., Penta, K., Sakamoto, G., Hynes, R., Coleman, M., Quertermous, T., Boudreau, N., and Varner, J. (2003) J. Clin. Investig. 112, 30-41[CrossRef][Medline] [Order article via Infotrieve]
  29. Holland, P. W. H., and Fernandez, J. (1996) Dev. Biol. 173, 382-395[CrossRef][Medline] [Order article via Infotrieve]
  30. Condle, B. G., and Capecchi, M. R. (1993) Development 119, 579-595[Abstract]
  31. Manley, N., and Capecchi, M. R. (1997) Dev. Biol. 192, 274-288[CrossRef][Medline] [Order article via Infotrieve]
  32. Hidai, 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., and Quertermous, T. (1998) Genes Dev. 12, 21-33[Abstract/Free Full Text]
  33. Penta, K., Varner, J. A., Liaw, L., Hidai, H., Schatzman, R., and Quertermous, T. (1999) J. Biol. Chem. 274, 11101-11109[Abstract/Free Full Text]
  34. Aoka, Y., Schatzman, R., Hirata, K.-I., Hidai, C., Varner, J., and Quertermous, T. (2002) Microvasc. Res. 64, 148-161[CrossRef][Medline] [Order article via Infotrieve]
  35. Stoeltzing, O., Liu, W., Reinmuth, N., Fan, F., Parry, G. C., Parikh, A. A., McCarty, M. F., Bucana, C. D., Mazar, A. P., and Ellis, L. M. (2003) Int. J. Cancer 104, 496-503[CrossRef][Medline] [Order article via Infotrieve]
  36. Ades, W., Candal, F. J., Swerlick, R. A., George, V. G., Summers, S., Bosse, D. C., and Lawley, T. J. (1992) J. Investig. Dermatol. 99, 683-690[CrossRef][Medline] [Order article via Infotrieve]
  37. Fragoso, G., and Hager, G. L. (1997) Methods 11, 246-252[CrossRef][Medline] [Order article via Infotrieve]
  38. Orlando, V., and Paro, R. (1993) Cell 75, 1187-1198[CrossRef][Medline] [Order article via Infotrieve]
  39. Myers, C., Charboneau, A., and Boudreau, N. (2000) J. Cell Biol. 148, 343-352[Abstract/Free Full Text]
  40. Brooks, P. C., Stromblad, S., Klemke, R., Visscher, D., Sarkur, F. H., and Cheresh, D. A. (1995) J. Clin. Investig. 96, 1815-1822[Medline] [Order article via Infotrieve]
  41. Ferrara, N. (2002) Semin. Oncol. 29, 10-14[Medline] [Order article via Infotrieve]
  42. Dvorak, H. F. (2002) J. Clin. Oncol. 20, 4368-4380[Abstract/Free Full Text]
  43. Yoshiji, H., Harris, S. R., and Thorgeirsson, U. P. (1997) Cancer Res. 57, 3924-3928[Abstract/Free Full Text]
  44. Zappavigna, V., Falciola, L., Helmer-Citterich, M., Mavilio, F., and Bianchi, M. E. (1996) EMBO J. 15, 4981-4991[Medline] [Order article via Infotrieve]

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