Vascular Endothelial Growth Factor (VEGF) Increases KCC4 Expression In Erythroid Cells Via Signaling Pathways Involving HIF-1α

Abstract 2039

The K-Cl co-transporters are key components of volume regulation in human reticulocytes. K-Cl transport activity is increased in sickle red blood cells (SS RBC) and is thought to contribute to SS RBC dehydration, a process that potentiates sickling. Four mammalian KCC isoforms have been cloned and characterized, of which three are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Studies in sickle and normal reticulocytes indicate substantial variation in the relative expression of these genes among individuals, suggesting a potential source of phenotypic variability in sickle cell disease (SCD). Relatively little is known about the regulation of KCC expression. Previous studies have shown an increase in mRNA levels of KCC3a in HUVEC cells treated with the angiogenic factor vascular endothelial growth factor (VEGF) (Hiki, et.al. (1999) J. Biol. Chem. 274, 10661–10667). Because VEGF and the related placenta growth factor (PlGF) levels are elevated in SCD, we evaluated the potential effects of the vascular growth factors on KCC expression in erythroid cells. RT-PCR revealed that erythroid K562 cells expressed the VEGF receptor-1 (VEGF-R1, or Flt-1) but not VEGF receptor-2, (VEGF-R2 or Flk-1). Treatment of K562 cells with VEGF (50 ng/ml) showed an increase in KCC4 mRNA levels at 8 hours, with no significant change in KCC1, KCC3a and KCC3b expression. An inhibitor specific for the VEGF receptor-1 (VEGF-R1), SU5416, ablated the effect of VEGF, suggesting a role for this receptor in the signaling pathway. Studies with pharmacological inhibitors for specific kinases indicated that VEGF-stimulated KCC4 expression involves PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), which have been implicated in other VEGF effects. Analysis of the KCC4 promoter revealed binding sites for transcription factor SP-1 at positions -35 to -44 and -56 to -64, relative to the transcriptional start site. In addition, seven putative binding sites for transcription factor HIF-1α were found in the KCC4 promoter, suggesting a role for HIF-1α in VEGF-stimulated KCC4 expression, which occurs under non-hypoxic conditions. We examined KCC4 promoter activity using KCC4 luciferase promoter constructs expressed in K562 cells. Luciferase activity was stimulated by VEGF treatment, with maximum activity from the promoter constructs spanning 1200 bp and 875 bp from the start site. Minimal promoter activity was seen in the -65bp construct. Additionally, a mutation in the HIF-1α binding site at -73 to -76bp significantly inhibited promoter activity. Site-directed mutagenesis of the SP-1 sites at position -35 to -44 and position -56 to -64 also attenuated promoter activities upon VEGF treatment. These results suggest that activation of VEGF-R1 by VEGF, and presumably its other ligand, PlGF, leads to non-hypoxic activation of HIF-1α and SP-1-mediated up-regulation of KCC4 expression in erythroid K562 cells via its canonical signaling pathways. Variation in KCC gene expression and its modulation by cytokines and growth factors may be a source of inter-individual variation in SS RBC volume regulation and thus of phenotypic variability of SCD. Identifying the factors that modulate transcriptional control of KCC4 expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC.