Abstract 16344: LMO2 Modulates SPHK1 and Promotes Endothelial Cell Migration
Introduction: Lim-domain only (LMO) 2 is a transcription factor that regulates hematopoiesis and angiogenesis. Only a few LMO2 modulated genes have been described. In ChIP-Seq analysis we identified potential LMO2 binding sites on the Sphingosine Kinase (SPHK)1, the enzyme that phosphorylates sphingosine to sphingosine-1-phosphate.
Hypothesis: In the present study we describe the novel relationship between LMO2 and SPHK1 in endothelial cells and how this affects endothelial cell migration.
Methods: LMO2 and SPHK1 loss-of-function (LOF) in-vivo was performed by morpholino injection in tg(fli1:gfp)y1 zebrafish at 1-2 cell stage. LMO2 modified mRNA was used for rescue. The embryonic phenotype was described at 24 and 48 hpf. Endothelial cell proliferation in-vivo was analysed by BrdU immunostaining in whole-mounted and enzymatically digested tg(fli;gfp)y1 embryos and FACS analysis. In-vitro, LMO2 LOF was performed in stable LMO2 KD human endothelial cells generated by infection with virus containing LMO2 shRNA, followed by migration assay. ChIP-PCR analysis was performed for probing LMO2-SPHK1 interactions. Gene and protein expression were assessed by real time PCR and western blotting, respectively.
Results: LMO2 LOF study in zebrafish showed a significant reduction in the number and the length of intersegmental vessels (ISV), phenotype that was rescued by injection of LMO2 modified mRNA. SPHK1 LOF showed a similar, although less extensive, phenotype. FACS analysis on disgregated embryos did not show difference in the percentage of GFP+ cells in LMO2 LOF group compared to control. BrdU immunostaining showed an accumulation of BrdU-positive nuclei at the ISV sprouting origin and a very low presence of these along the impaired ISV compared to control, suggesting low migration. LMO2 LOF human endothelial cells also showed a significant reduction in the level of migration, a reduced level of SPHK1 expression and SPHK1 DNA in ChIP-PCR assay performed with LMO2 antibody.
Conclusions: Our data showed that LMO2 is necessary for SPHK1 gene expression in endothelial cells. LMO2 LOF reduced LMO2 protein-SPHK1 gene interaction, impaired ISV formation and reduced cell migration. We identified for the first time SPHK1 as downstream effector of LMO2.
Author Disclosures: G. Matrone: None. S. Meng: None. J.P. Cooke: None.
- © 2016 by American Heart Association, Inc.