Functional Interactions of NANOG with Fetal Liver Kinase‐1 (FLK1) and CYCLIN‐D1 Regulate Wnt‐Mediated Angiogenesis

RATIONALE Angiogenesis occurs during development and plays a critical role in many diseases including cardiovascular disease and cancer. As the activation of the Wnt ( wingless ) signaling pathway contributes not only to stem cell self‐renewal and proliferation, but also angiogenesis, we investigated the functional interactions of NANOG with FLK1 and CYCLIN‐D1 expressions in endothelial cells (ECs). RESULTS We demonstrate that expression of NANOG, FLK1 and CYCLIN‐D1 proteins increased in HUVECs stimulated with WNT3A. We show that in basal conditions and in response to WNT3A stimulation, NANOG binding to ATTA sites (minimal NANOG binding sequence) in the FLK1 promoter induced expression of FLK1 in ECs. Moreover, luciferase‐reporter assays show functional interaction of NANOG to the FLK1‐ promoter following WNT stimulation. NANOG‐depletion resulted in decreased FLK1 and CYCLIN‐D1 proteins essential for proliferation in vitro and angiogenesis in vivo . Nevertheless, re‐expression of FLK1 into NANOG knockdown ECs partially restored the effect of NANOG‐depletion in these cells. CONCLUSION These findings functionally connect canonical WNT signaling to NANOG expression in regulating FLK1 and CYCLIN‐D1 expression that is crucial to the angiogenic phenotypes of ECs. Supported by NIH (R01HL079356), American Heart Association, T32GM070388 and T32HL072742 NIH training grants.