The K+Cl- cotransporter (KCC) plays a significant role in the maintenance of red cell volume. Activity of the cotransporter is higher in sickle (SS) compared to normal (AA) reticulocytes, and contributes to SS dehydration. Thus, KCC is considered a potential modifier gene for sickle cell disease (SCD). We have demonstrated the presence of transcripts for KCC1, KCC3, and KCC4 in human reticulocytes (
) and shown that one splice variant of KCC1 (KCC1ex1b), which codes for a protein with a small (7aa) alternative N-terminal exon, is detected in AA, but not SS reticulocytes. Studies with murine KCC1 have demonstrated that proteins produced by N-terminal truncation are inactive for K+Cl- cotransport, and function as dominant negative regulators of full-length KCC1 and KCC3 proteins. Since high level expression of this variant in AA cells compared to SS cells might explain the relatively low KCC activity in AA reticulocytes, we have identified the promoter for the KCC1ex1b transcript and investigated the regulatory elements that control its expression. Here we report the involvement of TNFα and NF-ΚB in the transcriptional regulation of the KCC1ex1b variant. Although KCC1ex1b is not expressed in reticulocytes isolated from sickle cell patients, we found that SS erythroid precursor cells cultured in vitro express this variant. SS and AA peripheral blood mononuclear cells were cultured in semi-liquid media with stem cell factor and erythropoietin, and collected after 5, 10, and 14 days in culture. Cells harvested at 14 days and isolated by binding to micromagnetic beads coated with transferrin receptor antibody were 95–98% positive for glycophorin A. RNA was extracted and analyzed by semi-quantitative RT-PCR, using primers for KCC1ex1 and KCC1ex1b. In both AA and SS cells, the transcript level for KCC1ex1b rose over the time in culture, while the KCC1ex1 transcript was constant. This difference between the in vitro and in vivo expression patterns for the KCC1ex1b variant could be explained by regulation via an external factor, such as a cytokine present in the blood of sickle cell patients, but absent in the in vitro culture system. The levels of numerous cytokines, including TNF, VEGF, and various interleukins, are elevated in SCD. We therefore assayed the effect of TNFα on endogenous KCC1ex1b expression in K562 cells by RT-PCR analysis at 24 and 48 hours after the addition of TNFα to the tissue culture medium. The steady-state mRNA levels of the KCC1ex1b variant decreased approximately 40% in response to TNF treatment. The transcription factor NF-ΚB is activated by TNF signaling, and an NF-ΚB consensus site is present in the KCC1ex1b promoter region. We assayed the effect of co-expressing NF-ΚB and our KCC1ex1b promoter constructs in K562 cells. NF-ΚB expression produced an 8-fold decrease in luciferase activity from these promoter constructs indicating NF-ΚB transcriptionally represses this promoter, either directly or indirectly. Our current model proposes that induction or modulation of the expression of the KCC1ex1bvariant could be an important factor in the control of red cell hydration.
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