MEOX2 regulates nuclear factor-kappaB activity in vascular endothelial cells through interactions with p65 and IkappaBbeta.
OBJECTIVE
Tumours secrete proangiogenic factors to induce the ingrowth of blood vessels, the end targets of which are vascular endothelial cells (ECs). The MEOX2 homeoprotein inhibits nuclear factor-kappaB (NF-kappaB) signalling and EC activation in response to serum and proangiogenic factors. We hypothesize that MEOX2 interacts with components of this pathway in vascular ECs to modulate NF-kappaB activity and EC activation and that these interactions depend upon specific domains within the MEOX2 protein.


RESULTS
To test our hypothesis, we transduced ECs with MEOX2 expression constructs. MEOX2 protein localized to the nuclear fraction, as did IkappaBbeta and p65. By co-immunoprecipitation, MEOX2 bound to both p65 and IkappaBbeta. Immunofluorescence demonstrated that MEOX2 colocalizes in the nucleus with both p65 and IkappaBbeta and that this colocalization requires the MEOX2 homeodomain and N-terminal domain. Finally, promoter assays revealed that MEOX2 expression has a biphasic effect on NF-kappaB-dependent promoters. At low levels, MEOX2 stimulates NF-kappaB activity, whereas at high levels, it represses, effects that also depend upon the homeodomain and the N-terminal domain.


CONCLUSIONS
Our results represent the first report of an interaction between a homeobox protein and IkappaBbeta and suggest that MEOX2 modulates the activity of the RelA complex through direct interaction with its components. These observations implicate MEOX2 as a potentially important regulatory gene inhibiting not only the angiogenic response of ECs to proangiogenic factors, but also their response to chronic inflammatory stimulation that normally activates NF-kappaB, suggesting MEOX2 as a possible molecular target for the therapy of angiogenesis-dependent diseases such as cancer.
Chen
Yun
Y
Division of Surgical Oncology, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ, USA.
Rabson
Arnold B
AB
Gorski
David H
DH
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D047468:Immunoprecipitation; D051798:Inhibitor of Differentiation Protein 1 / Q000235:genetics / Q000378:metabolism; D051796:Inhibitor of Differentiation Proteins / Q000235:genetics / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D051996:Transcription Factor RelA / Q000378:metabolism*; D014162:Transfection
D018398:Homeodomain Proteins; C497453:ID1 protein, human; D051798:Inhibitor of Differentiation Protein 1; D051796:Inhibitor of Differentiation Proteins; D015850:Interleukin-6; C484193:MEOX2 protein, human; C081336:Meox2 protein, rat; D009124:Muscle Proteins; D009363:Neoplasm Proteins; C498143:RELA protein, human; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1; C075640:ID3 protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human