Lmo2 (LIM-Domain-Only 2) Modulates Sphk1 (Sphingosine Kinase) and Promotes Endothelial Cell Migration

Abstract

Objective Lim-domain only (Lmo)2 transcription factor is involved in hematopoiesis and vascular remodeling. Sphingosine kinase (Sphk)1 phosphorylates sphingosine to sphingosine-1-phosphate (S1P). We hypothesized that Lmo2 regulates Sphk1 to promote endothelial cell (EC) migration and vascular development. Approach and Results Lmo2 and Sphk1 knockdown (KD) were performed in Tg(fli1:EGFP)y1 zebrafish and in HUVEC. Rescue of phenotypes or overexpression of these factors were achieved using mRNA encoding Lmo2 or Sphk1. Endothelial cell proliferation in vivo was assessed by BrdU immunostaining and FACS analysis of dissociated Tg(fli1:EGFP)y1 embryos. Cell migration was assessed by scratch assay in HUVEC and mouse aortic rings. Lmo2 interactions with Sphk1 promoter were assessed by ChIP-PCR. Lmo2 or Sphk1 KD reduced number and length of intersegmental vessels (ISVs). There was no reduction in the numbers of GFP+ endothelial cells following Lmo2 KD. However, reduced numbers of BrdU+GFP+ nuclei were observed along the dysmorphic ISVs, accumulating instead at the sprouting origin of the ISVs. This anomaly was likely due to impaired EC migration, which was confirmed in migration assays using Lmo2 KD HUVECs and mouse aortic rings. Both in vivo and in vitro, Lmo2 KD reduced Sphk1 gene expression, associated with less Lmo2 binding to the Sphk1 promoter as assessed by ChIP-PCR. Sphk1 mRNA rescued the Lmo2 KD phenotype. Conclusions Our data showed that Lmo2 is necessary for Sphk1 gene expression in endothelial cells. Lmo2 KD reduced Lmo2-Sphk1 gene interaction, impaired ISVs formation and reduced cell migration. We identified for the first time Sphk1 as downstream effector of Lmo2.

Molecular mechanisms of regulation of sphingosine kinase 1

“…Additionally, LMO2 has been shown to promote angiogenic traits in glioma stem cells [23]. It has been shown in zebrafish, that knockdown of Lmo2 , via morpholino, resulted in defective intersegmental vessels [24]. Lmo2 knockdown impaired migration of human umbilical vein endothelial cells (HUVECs) [24].…”

“…It has been shown in zebrafish, that knockdown of Lmo2 , via morpholino, resulted in defective intersegmental vessels [24]. Lmo2 knockdown impaired migration of human umbilical vein endothelial cells (HUVECs) [24]. Furthermore, knockdown or overexpression of LMO2 decreased or increased, respectively, SK1 protein levels [24].…”

“…Lmo2 knockdown impaired migration of human umbilical vein endothelial cells (HUVECs) [24]. Furthermore, knockdown or overexpression of LMO2 decreased or increased, respectively, SK1 protein levels [24]. Finally, LMO2 was found to bind to the Sphk1 gene using ChIP assays [24].…”

LMO2 (LIM Domain Only 2) and Endothelial Cell Migration in Developmental and Postnatal Angiogenesis

“…This applies to the paper by Matrone et al 1 in this issue of ATVB , where the authors' delineated a novel signaling pathway within the domain of endothelial migration. LIM domain only 2 (LMO2) was first discovered as a recurrent translocation partner of T-cell receptor (TCR) loci in a subset of patients with T cell acute lymphoblastic leukemia in 1981 2, 3 .…”

“…Studies on LMO2 have now shown that LMO2 not only functions as a proto-oncogenic gene in T-cells but also in hematopoiesis, vascular remodeling and stem cell biology 4-9 . Matrone et al 1 showed that LMO2 levels in zebra fish embryos regulate intersegmental vessel (ISV) formation. The evidence for a migration defect is the absence of endothelial cells in ISVs in the elegant photomicrographs.…”