LMO2 Regulates Angiogenesis through TGFB1 and HGF

Abstract

Background LIM domain only 2 (LMO2) is a key transcription factor of angiogenesis during embryonic development. However, role of LMO2 in adult endothelial angiogenesis and the possible mechanism involved is largely unknown. Methods and Results: Loss-of-function studies were performed in human umbilical vein endothelial cells (HUVEC) with knock down (KD) of LMO2 with Lentiviral shRNA targeting LMO2 gene. LMO2 KD in HUVECs resulted in 70% reduction in both LMO2 mRNA and protein level. KD of LMO2 significantly impaired tube formation compared with ECs transfected with control shRNA. After screening of 84 potential angiogenesis related molecular targets using Angiogenesis PCR array, transforming growth factor beta 1 (TGF-β1) and Hepatocyte growth factor (HGF) were found downregulated by LMO2 KD. ELISA assay further demonstrated that secreted TGF-β1 and HGF level was decreased in LMO2 KD cells. Supplement of exogenous TGF-β1 or HGF reversed the impaired angiogenesis. Bioinformatics prediction suggests two potential LMO2 binding sites (-518 and -4295) on TGF-β1 promoter and one distal LMO2 binding site on HGF promoter. ChIP assay demonstrates that LMO2 binds to both -518 and -4295 position of TGF-β1 promoter. However, LMO2 binding only decreased at -518 position in LMO2 KD ECs, suggesting LMO2 binding at this position is dynamic and potentially regulated TGF-β1 expression. LMO2 binding to HGF promoter is not significantly changed in LMO2 KD cells. Conclusion LMO2 regulates angiogenesis through TGF-β1 and HGF. TGF-β1 level was regulated by LMO2 through its binding to -518 position of TGF-β1 promoter. LMO2 regulates HGF through TF binding independent manner.