Endostatin Causes G1 Arrest of Endothelial Cells through Inhibition of Cyclin D1

Endostatin, a type XVIII collagen fragment, is a potent antiangiogenic molecule that inhibits endothelial cell migration, promotes apoptosis, and induces cell cycle arrest in vitro. We have investigated the mechanism by which endostatin causes G₁ arrest in endothelial cells. Endostatin decreased the hyperphosphorylated retinoblastoma gene product and down-regulated cyclin D1 mRNA and protein. Importantly, endostatin was unable to arrest cyclin D1 overexpressing endothelial cells, suggesting that cyclin D1 is a critical target for endostatin action. Next, we analyzed cyclin D1 promoter activity in endothelial cells and found that endostatin down-regulated the cyclin D1 promoter. Using a series of deletion and mutant promoter constructs, we identified the LEF1 site in the cyclin D1promoter as essential for the inhibitory effect of endostatin. Finally, we showed that endostatin can repress cyclin D1 promoter activity in cells over-expressing β-catenin but not in cells over-expressing a transcriptional activator that functions through the LEF1 site and is insensitive to β-catenin. Collectively, our data pointed to a role for cyclin D1, and in particular, transcription through the LEF1 site as critical for endostatin actionin vitro and suggest that β-catenin is a target for endostatin.