• Regulation of glucose-activated RUNX2 phosphorylation: effects on endothelial cell proliferation and an angiogenic phenotype

      Pierce, Adam Daniel; Passaniti, Antonino (2011)
      The runt-related protein-2 (RUNX2) is a DNA-binding transcription factor that regulates bone formation, tumor cell metastasis, endothelial cell (EC) proliferation, and angiogenesis. RUNX2 DNA binding is glucose and cell cycle regulated. We propose that glucose may activate RUNX2 through changes in post-translational phosphorylation that are cell cycle-specific and will regulate EC function. Glucose increased cell cycle progression in EC through both G2/M and G1 phases with entry into S-phase occurring only in subconfluent cells. In the absence of nutrients and growth factors (starvation), subconfluent EC were delayed in G1 when RUNX2 expression was reduced. RUNX2 phosphorylation, activation of DNA binding, and pRb phosphorylation were stimulated by glucose and were necessary to promote cell cycle progression. Glucose increased RUNX2 localization at focal subnuclear sites, which co-incided with RUNX2 occupancy of the cyclin-dependent kinase (cdk) inhibitor p21Cip1 promoter, a gene normally repressed by RUNX2. Mutation of the RUNX2 cdk phosphorylation site in the C-terminal domain (S451A.RUNX2) reduced RUNX2 phosphorylation and DNA binding. Expression of this cdk site mutant in EC inhibited glucose-stimulated differentiation, monolayer wound healing, and proliferation. We also found that the methionine and cysteine residues of RUNX2 were sensitive to reactive oxygen species (ROS) that directly regulated RUNX2 DNA-binding activity in ECs. Functional screening of RUNX2-specific inhibitors identified the vitamin D3 prohormone to have a novel role in EC proliferation mediated through the regulation of RUNX2 activity. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data also suggest that regulation of RUNX2 DNA-binding activity by vitamin D3 or ROS may be useful to inhibit EC proliferation in tumor angiogenesis.