Glucose‐activated RUNX2 phosphorylation promotes endothelial cell proliferation and an angiogenic phenotype

Abstract

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 (in vitro tube formation), monolayer wound healing, and proliferation. These results define a novel relationship between glucose-activated RUNX2 phosphorylation, cell cycle progression, and EC differentiation. These data suggest that inhibition of RUNX2 expression or DNA binding may be a useful strategy to inhibit EC proliferation in tumor angiogenesis.

Matrigel: From discovery and ECM mimicry to assays and models for cancer research

“…In some cases, the importance of a gene in angiogenesis has been evaluated using the assay in genetically modified mice. For example, RUNX2 is a DNA-binding transcription factor that has been implicated in skeletal morphogenesis, tumor metastasis, and angiogenesis [144] Using a Runx2 overexpressing mouse, VEGF was found to promote more angiogenesis in the BME/Matrigel plug assay than that observed in the heterozygous animals (Fig. 7).…”

NEU1 Sialidase Regulates the Sialylation State of CD31 and Disrupts CD31-driven Capillary-like Tube Formation in Human Lung Microvascular Endothelia

“…In Vitro EC Capillary-like Tube Formation Assay-Each well of a 96-well plate was coated with 50 l of Matrigel (10 mg/ml; BD Biosciences) as described with modifications (52,53). Briefly, Matrigel (50 l on ice) was allowed to polymerize for 1 h at room temperature in each of the wells, followed by 1 h at 37°C in a humidified atmosphere of 5% CO 2 .…”

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion

“…In addition, the pro-angiogenic effects of RUNX2 suggest a major role for this transcription factor in tumor promotion. These effects include endothelial cell proliferation, migration and invasion [15; 16], induction of VEGF expression and physical and functional interactions between RUNX2 and another major pro-angiogenic factor, hypoxia-inducible factor 1-a (HIF1-a) [17; 18]. …”