Abstract
RUNX2 is a member of the runt family of DNA-binding transcription factors. RUNX2 mediates endothelial cell migration and invasion during tumor angiogenesis and is expressed in metastatic breast and prostate tumors. Our published studies showed that RUNX2 DNA-binding activity is low during growth arrest, but elevated in proliferating endothelial cells. To investigate its role in cell proliferation and cell cycle regulation, RUNX2 was depleted in human bone marrow endothelial cells using RNA interference. Specific RUNX2 depletion inhibited DNA-binding activity as measured by electrophoretic mobility shift assay resulting in inhibition of cell proliferation. Cells were synchronized at the G(1)/S boundary with excess thymidine or in mitosis (M phase) with nocodazole. Endogenous or ectopic RUNX2 activity was maximal at late G(2) and during M phase. Inhibition of RUNX2 expression by RNA interference delayed entry into and exit out of the G(2)/M phases of the cell cycle. RUNX2 was coimmunoprecipitated with cyclin B1 in mitotic cells, which further supported a role for RUNX2 in cell cycle progression. Moreover, in vitro kinase assays using recombinant cdc2 kinase showed that RUNX2 was phosphorylated at Ser(451). The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNA-binding activity during mitosis and the RUNX2 mutant S451A exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G(2) and M phases, thus promoting endothelial cell proliferation required for tumor angiogenesis.
Highlights
The RUNX gene family of transcriptional regulators is the mammalian homologue of the Drosophila transcription factor runt
The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNAbinding activity during mitosis and the RUNX2 mutant Ser451 to alanine (S451A) exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G2 and M phases, promoting endothelial cell proliferation required for tumor angiogenesis
human bone marrow endothelial (HBME) cells were transfected with plasmids expressing RNAi or mutant RNAi corresponding to specific regions of the RUNX2 gene and cultured for 1, 2, or 3 days
Summary
The RUNX gene family of transcriptional regulators is the mammalian homologue of the Drosophila transcription factor runt. RUNX1 may promote proliferation and cell cycle progression through G1 phase and entry into S phase by controlling the gene expression of cyclin D2, D3, and the CDK inhibitor p21Cip in hematopoietic cells. Serum deprivation of HBME cells resulted in growth arrest at the G1/S boundary of the cell cycle and reduced RUNX2 protein levels and DNA-binding activity [43]. The RUNX2 Ser451 mutant was less potent than wild type RUNX2 at stimulating growth in soft agar These data demonstrate that RUNX2 may promote cell proliferation in endothelial cells by regulating cell cycle progression. We have identified cdc as one of the kinases responsible for post-translational modification of RUNX2 These findings support a mechanism for cell proliferation involving RUNX2 phosphorylation by a cellular cdk, which may promote angiogenesis and/or tumorigenesis
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