K-Cl Cotransporter Gene Expression during Human and Murine Erythroid Differentiation
Molecular and Cell Therapy Program, Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
07/2011; 286(35):30492-503. DOI: 10.1074/jbc.M110.206516
The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.
Available from: Yoshinori Marunaka
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ABSTRACT: Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K(+) and Cl(-), and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl(-)content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.
Cellular Physiology and Biochemistry 07/2012; 30(3):538-51. DOI:10.1159/000341436 · 2.88 Impact Factor
Available from: Kenneth Bradley Gagnon
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ABSTRACT: The homologous genes encoding the electroneutral solute carrier family 12A (SLC12A) were identified more than 20 years ago, however, over the last few years, it has become clear that each of the genes within this family potentially encode for more than one cation-chloride cotransporter (CCC). Even more surprising, despite more than 30 years of functional studies and a wealth of knowledge on the activators, inhibitors, ion affinities, and kinetics of these cotransporters, we still cannot sufficiently explain why some cells express only one CCC isoform, while others express two, three, or more CCC isoforms. In 2009, Drs. Alvarez-Leefmans and Di Fulvio published an extensive in silico molecular analysis of the potential splice variants of the Na(+)-dependent cation-chloride cotransporters. In this review, we will look at the exceptionally large variety of potential splice variants within the Na(+)-independent cation-chloride cotransporter (SLC12A4-SLC12A7) genes, their initial tissue identification, and their physiological relevance. © 2014 S. Karger AG, Basel.
Cellular Physiology and Biochemistry 12/2013; 32(7):14-31. DOI:10.1159/000356621 · 2.88 Impact Factor
Available from: Clinton H Joiner
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ABSTRACT: The potassium chloride co-transporters (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. The KCC activity exhibit is high in reticulocytes, and contributes to 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 over-expression 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 SCD.
American Journal of Hematology 03/2014; 89(3). DOI:10.1002/ajh.23631 · 3.80 Impact Factor
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