Function of the receptor tyrosine kin ÄSE, HTK/EPHB4 and its ligand, HTKL/EPHRIN-B2 in erythropoiesis

HTK/EphB4 is a receptor tyrosine kinase of the Eph family. We have previously shown that HTK is exclusively expressed on committed erythroid progenitors and its ligand, HTKL/ephrin-B2 is expressed on stromal cells in human bone marrow (Blood 89-.2151-2765, 1997) HTKL is a transmembrane protein and is inducibly phosphorylated upon binding HTK. To analyze the functions of HTK and HTKL in erythropoiesis, we co-cultured human bone marrow cells with HTKLtransfected stromal cells. Full-length HTKL (MS5/HTKL) or truncated HTKL which lacks its cytoplasmic domain (MS5/A267-333) was transfected into mouse stromal cell line, MS5. Both the full-length and the truncated HTKLs were able to induce the phosphorylation of HTK. Sorted c-Kit-positive cells from human bone marrow were cocultured with these stromal cell lines supplemented with stem cell factor, IL-3 and erythropoietin. Hematopoietic cells were grown in 12 days of co-culture with MS5/HTKL. Especially, the number of erythroblasts was twice more than the control (MS5/Neo). However. MS5/A267-333 did not enhance the erythropoiesis significantly. These results suggest that the phosphorylation of HTKL may modify the stromal cell function to enhance erythropoiesis. We are now investigating the molecular changes of the stromal cell upon the HTKHTKL binding. This study is a key to solving the mechanism of "erythroblastic island" formation or hematopoietic microenvironments.