ABSTRACT. A major pathway for K+ efflux in human reticulocytes and young RBCs is K:Cl cotransport (K:Cl-CT). The activity of K:Cl-CT is increased in pathologic RBCs containing hemoglobins S and C and may contribute to the abnormal dehydration state of these cells. Human K:Cl-CT (gene product KCC1) has been recently sequenced from human (hKCC1), rabbit and rat tissue by Gillen et al. (J Biol Chem 271:16237, 1996). We report here the sequence of KCC1 from human and mouse erythroleukemic cells (K562 and MEL cells, respectively). The cDNA for human erythroid-KCC1 is 100% identical to hKCC1 and the cDNA for mouse erythroid-KCC1 shares 89% identity with hKCC1, which translates to 96% identity at the amino acid level. Mammalian KCC1 is strongly conserved with >95% identity between human, rabbit, rat, and mouse KCC1 proteins. We did not detect any full-length mRNA transcripts of human erythroid-KCC1 in circulating reticulocytes. We detected two mRNA isoforms of human erythroid-KCC1 that resulted in C-terminal truncated proteins (73 amino acid and 17 amino acids, respectively). Human and mouse erythroid-KCC1 differed at several consensus sites including a predicted PKC phosphorylation site at (108)threonine and a predicted CK2 phosphorylation site at (51)serine,within the predicted cytoplasmic N-terminal, that are present in human but not mouse erythroid-KCC1. Expression of MEL-KCC1 mRNA increases substantially upon DMSO-induced differentiation opening the possibility that erythroid-KCC1 plays a role in early erythroid maturation events. The molecular identification of erythroid-KCC1 is an important step towards understanding the physiologic role mediated by this protein in young and pathologic RBCs and during erythropoiesis, as well as providing a new tool for the elucidation of pathways and signals involved in RBC volume regulation.

Keywords: Erythrocyte, cation transport, cell volume, erythropoiesis, K562, MEL.

Reprint requests to: Robert S. Schwartz, Ph.D., Associate Professor of Medicine, Division of Hematology, Albert Einstein College of Medicine-Montefiore Medical Center, 111 East 210th Street, Bronx, NY 10467-22490, phone: (718) 920-5135, fax: (718) 881-7108, e-mail: rschwart@worldnet.att.net.

ABSTRACT: We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells. Following transfer to appropriate medium all of the pathway enzymes are induced within three days. Unlike differentiating mouse erythroleukemia cells (Lake-Bullock, H. and Dailey, H.A. Mol Cell Biol 13:7122-7132, 1993), all of the enzymes appear to be induced simultaneously and not sequentially in differentiating ES cells. The role of erythroid 5-aminolevulinate synthase (ALAS-2) in this differentiation process was examined by disruption of the ALAS-2 gene. The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable neomycin resistance gene. The resulting genetically modified (ALAS-2 knockout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodies (EBs) of the two cell lines were observed. ES cells exhibited morphology of typical EBs with a dark field (blood island) in the center, while ALAS-2 knockout ES cells developed very poorly both in size and shape. At 8 days of differentiation, only 3% of all EBs contained visible erythropoietic cells (i.e., stained positively for hemoglobin) in the ALAS-2 knockout cell line, compared with 50% in ES cells. Most of the genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS-2 knockout cell line failed to significantly increase the level of expression of these genes. Fetal beta-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation. These results suggest that ALAS-2 is necessary for ES cell erythroid differentiation and that there is an interrelationship between heme and globin synthesis in differentiating ES cells.

Keywords: Heme biosynthesis, ALA synthase, erythropoiesis, regulation.

Reprint requests to: Harry A. Dailey, Ph.D., Department of Biochemistry and Molecular Biology, University of Georgia, Athens GA 30602-7229, phone: (706) 542-2690, fax: (706) 542-7567, e-mail: dailey@bscr.uga.edu.