VEGF Mediated Regulation of KCl Cotransporters Gene Expression in Erythroid Cells,

Abstract 3163

The KCl co-transporter (KCC) family of proteins catalyzes the electroneutral, coupled movement of K⁺ and Cl⁻ ions across the plasma membrane, thereby mediating transepithelial ion transport and regulating cell volume. These proteins play an important role in disease states such as cancer, numerous neurological conditions as well as sickle cell disease (SCD). KCC activity is increased in sickle red blood cells and contributes to their dehydration, which potentiates sickling. The mechanisms of increased KCC activity and its abnormal regulation are not understood. Of the four mammalian KCC isoforms, KCC 1, 3 and 4 are expressed in erythroid cells (Crable et al. Exp Hematol. 2005; 33:624). Hiki et. al. showed that the angiogenic factor vascular endothelial growth factor (VEGF) increased KCC 3a expression in HUVEC cells (J.B.C. 274, 10661–10667, 1999). As levels of VEGF and related family member, placenta growth factor (PlGF) are elevated in sickle cell patients, we hypothesized that VEGF and PlGF may influence 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). Additionally, flow cytometric analysis of WT C57Bl6 mouse bone marrow showed the presence of the Flt-1 receptor, but not Flk-1 or Flk-3 in erythroid progenitors and expression decreased with maturation. VEGF treatment (50 ng/ml) of K562 cells increased KCC 1, 3a, 3b and 4 mRNA levels; PlGF treatment increased KCC 1, 3a and 4 mRNA levels but not KCC 3b. The VEGF receptor inhibitor, SU5416, ablated the effect of VEGF. VEGF-stimulated KCC 4 expression was blocked by pharmacological inhibitors that implicated PI3 kinase, p38 MAP kinase, mTOR, JNK kinase and the transcription factor hypoxia inducible factor-1α (HIF-1α), as with other VEGF effects. Analysis of the KCC 4 promoter showed that the −875 and −90 bp promoter luciferase constructs exhibited similar levels of activity as the −1200 bp promoter construct, when compared to the promoterless reporter plasmid. Deleted constructs corresponding to −65 bp from transcription start site showed ∼90% reduced promoter activity. In silico analysis of the −90 bp region of the KCC 4 promoter showed potential binding sites for transcription factor SP-1 and HIF-1α. Binding sites for transcription factor SP-1 at positions −35 to −44 bp and −56 to −64 bp were shown to be active by site directed mutagenesis. Mutation of the HIF-1α binding site at −73 to −76 bp significantly inhibited promoter activity, whereas mutation of the HIF-1α binding site at position −21 bp to −18 bp did not have any effect on activity. Similar analysis of the KCC 3a promoter indicate potential binding sites for SP-1 at positions −8 to −4 bp and a HIF-1α binding site at position −23 to −20 bp, and the KCC 3b promoter has binding sites for HIF-1α at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at position −13 to −10 bp. Luciferase assays with KCC 3b promoter constructs indicated that the −190 bp promoter region containing HIF-1α sites at –9 to −6 bp and −49 to −46 bp and an AP-1 binding site at −13 to −10 bp contained minimal promoter required for transcription activity. Mutations within both HIF-1α binding sites attenuated promoter activity indicating a role for HIF-1α in regulating KCC 3b activity, as well. EMSA and ChIP assays with the KCC 4 promoter demonstrated that VEGF treatment of K562 cells increased HIF-1α binding to the HIF-1α sites, which was abrogated by mutating these sites. Similar results were obtained for the KCC 3a and 3b promoters.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 KCC3a, 3b and 4 expressions 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 KCC expression is important to understanding volume regulation in reticulocytes and its dysregulation in SS RBC.