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. Am. J. Hematol. 89:273–281, 2014. © 2013 Wiley Periodicals, Inc.

Erythropoietin-mediated expression of placenta growth factor is regulated via activation of hypoxia-inducible factor-1α and post-transcriptionally bymiR-214in sickle cell disease

“…It is pertinent to note that shRNAs for PI-3 kinase and MAP kinase (ERK-2) were effective in attenuating expression of these proteins by 50 μ as shown in the Supplementary Figures S1(A) and S1(B). Taken together, these data showed EPO-mediated signalling for PlGF expression involved PI3 kinase as well as previously shown p38 MAP kinase, MAP kinase and JNK pathways [30]. …”

“…K562, a human erythroleukaemia cell line, was cultured in Iscove’s modified DMEM (Dulbecco’s Modified Eagle’s medium; IMDM), supplemented with 10 %heat-inactivated FBS, 100 μ g/ml streptomycin and 100 units/ml penicillin, [30]. Cells were kept in serum-free medium overnight prior to treatment with EPO (3 units/ml).…”

The Zn 2+ -sensing receptor, ZnR/GPR39, upregulates colonocytic Cl − absorption, via basolateral KCC1, and reduces fluid loss

“…This result is consistent with previous studies showing that the neuronal KCC2 is up-regulated by PKC-dependent phosphorylation of serine 940 [68]. In addition, MAPK-dependent upregulation of KCC1 activity was shown in red blood cells [69,70]. In addition, synaptically-released Zn 2+ activates ZnR/GPR39-dependent MAPK signaling that upregulates the neuronal KCC2 [44].…”

K+-Cl− cotransporter 1 (KCC1): a housekeeping membrane protein that plays key supplemental roles in hematopoietic and cancer cells

“…There is evidence to suggest that hypoxia might still explain why K + -Cl − coefflux is increased in HbS cells as it could do so through systemic rather than local effects. Indeed, placental growth factor (PIGF) has been found at high circulating levels in sickle cell anemia, probably as a result of HIF1α upregulation in ischemic tissues, and to increase KCC1 expression in an erythroid RTC type cell line [66]. Given, however, that PIGF is also upregulated in normal RTC by low pO 2i [67], its synthesis by non-erythroid cells would have to be sufficiently important to bypass any inhibitory effects that low pO 2i might exert on KCC in HbS cells (Fig.…”