HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells

Abstract

The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-␣)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp ؊210 to ؊221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-␣-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-␣-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.

HOXA9 Methylation by PRMT5 Is Essential for Endothelial Cell Expression of Leukocyte Adhesion Molecules

“…Mammalian expression vectors containing appropriate cDNA, including HOXA9, were subcloned as described previously (4). A Myc tag epitope sequence was introduced upstream of the initiating ATG codon in the PRMT5 cDNA purchased from Origene Technologies (Rockville, MD) by PCR and subcloned into pcDNA3.…”

“…cDNA constructs were verified for protein expression using a TNT-coupled in vitro translation system from Promega Corporation (Madison, WI). Reporter luciferase constructs containing a 510-bp fragment of the E-selectin promoter (E-selectin-510) were generated as described earlier (4). The plasmid pCMV␤ (Clontech) containing the ␤-galactosidase gene driven by the cytomegalovirus (CMV) promoter was used as a control in the transfection experiments.…”

“…Human umbilical vein EC (HUVEC) were routinely cultured in MCDB 107 medium supplemented with 15% fetal bovine serum (FBS), 150 g/ml endothelial cell growth supplement (ECGS), and 90 g/ml heparin (54). Transfections of effector, reporter, and control plasmids were carried out with either Lipofectin (Life Technologies, Inc.) or Targefect F-2/peptide enhancer (Targeting Systems, CA) in cultured cells as described earlier (4). Cells were replenished with fresh culture medium and allowed to recover for 16 to 20 h before harvest or TNF-␣ treatment.…”

“…Exposure of EC to an inflammatory signal causes histone modifications and chromatin remodeling to allow both sequence-specific DNA binding and associations among transcription factors/cofactors for the induction of these molecules, including Eselectin (19). The homeobox gene HOXA9 plays a key transcriptional role in this activation (4,44), which also requires other necessary transcription factors/cofactors, including NF-B, ATF-2/Jun, HMG-I(Y), and p300 (14,22,50).…”

“…Importantly, they play a regulatory role in EC function, including differentiation from embryonic stem cells (3), EC maturation and vascular development from mesoderm-derived precursor cells (23,66), proliferation, migration, and angiogenesis (9,10,12,13,39,51). Recently, the pursuit of HOXA9 in EC gene regulation has led to the recognition of important new concepts (4,8,44,53,63). Apart from its role in E-selectin (4) or VCAM-1 induction, HOXA9 is necessary for endothelial tube formation during angiogenesis (8) and is a key regulator of adult progenitor cell commitment to the endothelial lineage (53).…”