Angiogenic growth factors augment K-Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α

Abstract

The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease.

Figure 1

VEGF increases expression of the KCC genes in K562 cells: (A) VEGF-induced mRNA expression of the KCC isoforms in K562 cells after 8 hr of treatment. (B) VEGF induced protein expression of the KCC isoforms in K562 cells after 24 hr of treatment, using the NanoPro 1000 System as described in “Materials and Methods” and antibodies specific for KCC1, KCC3a, KCC3b KCC4, and anti-ERK as a loading control. Bar graph represents the ratio of the normalized signals in VEGF treated samples to control samples. (C) PlGF-induced expression of the KCC isoforms after 4 hr of treatment. (D) PlGF-induced expression of the KCC proteins in K562 cells (24 hr treatment). Bar graph represents the ratio of normalized signals in PlGF-treated samples to that of controls. (E,F) VEGF-induced and (G,H) PlGF-induced mRNA expression of KCC4, in K562 cells treated with the indicated pharmacological inhibitors (30 min). mRNA expression was normalized to GAPDH mRNA levels. Data are expressed as mean ± SD of three independent experiments. ***P < 0.001, **P < 0.01,*P < 0.05, ns, P > 0.05.

Figure 2

VEGF-induced KCC4 and KCC3b mRNA expression in K562 cells: K562 cells were transfected with siRNA for p38 MAP kinase, p47^phox, JNK kinase, and scrambled scRNA control, prior to stimulation with VEGF for 8 hr. (A) Effect of VEGF on KCC4 mRNA levels. (B) Effect of VEGF on KCC3b mRNA levels. (C,D) Effect of knock-down and overexpression of HIF-1α on KCC4 and KCC3b mRNA levels. Data is represented as mean ± SD of three, independent experiments. Data are expressed as mean ± SD of three independent experiments. ***P < 0.001, **P < 0.01,*P < 0.05, ns, P > 0.05. (E) VEGF treatment (8 hr) increases HIF-1α protein expression in K562 cells, which is attenuated by pharmacological inhibitors for PI3-kinase (LY294002), MAP kinase (PD98059), VEGF-R1 (SU5416), and HIF-1α (R59949). Western blots for HIF-1α were performed as described in “Materials and Methods”. Blots were stripped and probed with β-actin to confirm equal loading. Data are representative of three independent experiments.

Figure 3

KCC4 and KCC3b promoter activity requires the activity of HIF-1α. (A) Schematic of the KCC4 (−875/+12 bp) promoter region, containing two HRE sites and two SP-1 binding sites. (B) Deletion analysis of KCC4 promoter utilizing reporter luciferase assay. (C) Analysis of HRE and SP-1 binding sites in the KCC4 promoter. (D) Schematic of the KCC3b (−1,100/+142 bp) promoter region containing two HRE sites. (E) Promoter analysis of the KCC3b (−1,100/+142 bp) promoter by reporter luciferase assay. Data are mean ± SD of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, ns P > 0.05.

Figure 4

VEGF and PlGF induce binding of HIF-1α to the KCC4 and KCC3b promoters in K562 cells. (A,B) ChIP analysis of HIF-1α binding to the KCC4 and KCC3b promoter in K562 cells treated without and with VEGF for 4 hr. (C,D) ChIP analysis of the KCC4 and KCC3b promoter in K562 cells treated with PlGF. HIF-1α antibody (top panel) or control rabbit IgG (middle panel) was used for immunoprecipitation of soluble chromatin. The bottom panel represents the amplification of input DNA before immunoprecipitation. Primers used to amplify the PCR products are indicated in Supporting Information Table 1. Data are representative of three independent experiments. (E) Ter199⁺ bone marrow progenitors were sorted by flow cytometry based on CD44 expression and forward scatter into I: Proerythroblast and basophilic normoblasts, II: Basophilic and polychromatophilic normoblasts, III: Polychromatophilic and orthochromatic normoblasts, and IV: Orthochromatic normoblasts, reticulocytes, and RBCs (Supporting Information Fig. 1A). KCC mRNA expression, as determined by qRT-PCR, was normalized to corresponding GAPDH mRNA levels. qRT-PCR data from cells from PlGF overexpressing mice were further normalized to values obtained from control mice overexpressing GFP, to yield fold-changes in KCC mRNA expression. Data are expressed as mean ± SD of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, ns, P > 0.05