The K-Cl cotransporter family (KCC) plays a crucial role in cell volume regulation, and KCC1 and KCC3 have been reported to participate in cell growth events (

Shen MR, PNAS 98, 2001
;
Shen MR JBC 278, 2003
). Expression of KCC1, KCC3 and KCC4 has been reported in erythroid cells. In β-thalassemic red blood cells (RBCs), K-Cl cotransport activity is abnormally activated and contributes to red cell loss of water and K. This study evaluated the gene expression of two KCC gene products and the effects of the KCC inhibitor [(dihydroindoenyl)oxy]alkanoic acid (DIOA) on in vitro liquid-culture expansion of human normal and β thalassemic (β thal) erythroid precursors from peripheral blood CD34+ cells. Cells from normal subjects and from β thalassemia major patients (cod39cod39) were cultured for 7 days (to the pro-normobast stage) and 14 days (to the eythroblast stage) in the presence or absence of 10 mM DIOA, At each time point the following parameters were evaluated;

  • cells counts;

  • cytospins stained with Wright-Giemsa to assess differential cell counts and morphology,

  • cell cycle stage by fluorescence-activated cell sorting after propidium iodide staining;

  • KCC protein expression by Western-blot analysis with antibody to the shared KCC carboxy-terminus;

  • mRNA by real time-PCR analysis.

KCC protein expression increased during erythropoiesis in both normal and β thal cells, and was higher in β thal cells than in normal controls. KCC1 mRNA level was increased only in β thal cells at day 14, whereas KCC3 mRNA level was increased at day 14 in both normal and β thal cells. DIOA significantly reduced the number of both normal and β thal cells, parallelled by increases in the percentage of polychromatophilic normoblasts among normal progenitors and of basophilic normoblasts among b thal progenitors. We further investigated the inhibitory effects of DIOA on cell growth by FACS evaluation of cell cycle distributions and by determination ofCycD, p21, Casp3 and Casp8 gene expression. At day 14 DIOA exposure was associated with:

  • significant reduction in the percentage of β thal cells in S-phase compared to either untreated cells or DIOA-treated normal controls;

  • up-regulation of CycD gene expression in both normal and β thal cells;

  • down-regulation of p21 in β thal cells;

  • up-regulation of casp3 and casp8 in both normal and β thal cells. These data suggest that KCC is involved in the late phase of erythropoiesis mainly in β thal cells, and support a novel role of KCC in erythroid cell growth.

Topics:
antibodies, caspase-3, caspase-8, cell separation, cooley's anemia, erythroid progenitor cells, giemsa stain, iodides, pharmacology, polymerase chain reaction

Author notes

Corresponding author

2005, The American Society of Hematology
2005
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