The capability of hematopoietic stem cells (HSC) to enter bone marrow and to proliferate and differentiate is critically dependent on expression of adhesion molecules that mediate specific cell-cell and cell-matrix adhesive interactions. CD44, an adhesion molecule known to bind to extracellular matrix elements, is an important mediator of leukocyte migration and of adhesive interactions in the marrow. The selectins (E-, P- and L-selectin) are a class of carbohydrate binding proteins that also regulate cellular trafficking and adhesive interactions in the marrow. In the prior funding period of this project, we determined that Hematopoietic Cell L-selectin Ligand (HCLL), a novel L-selectin ligand that had been previously defined by functional criteria, is a specialized glycoform of CD44. Moreover, we found that this CD44 glycoform is also an E-selectin ligand (HCEL), thereby now defining this molecule as Hematopoietic Cell E-/L- selectin Ligand (HCELL). We hypothesize that HCELL plays a critical role in the growth and differentiation of hematopoietic progenitor cells and in the physiologic migration of these cells into the bone marrow. In this proposal, we intend to define the discrete structural determinant(s) on HCELL that confer E- and L-selectin ligand activities and elucidate the role of HCELL/L-selectin and HCELL/E-selectin interactions in hematopoiesis. We will analyze the HCELL protein regions displaying relevant carbohydrate binding determinants for both L- and E-selectin adherence and elucidate the component carbohydrate structures. Antibody reagents specific for the ligand determinant(s) and small molecule antagonists of the E- and L-selectin binding activity of HCELL will be developed. The precursor-product processing of HCLL and HCEL CD44 phenotypes will be investigated, and the effects of cytokines on this process will be explored. We will determine if CD44 on murine HC expresses HCELL phenotype, and, if so, we will analyze E- and L-selectin ligand expression in HC of mice deficient in CD44 and in various glycosyltransferases. Moreover, the expression of HCELL among normal and leukemic hematopoietic cells will be analyzed and its distribution within the marrow of normal and pathologic specimens will be characterized. To analyze the role(s) of HCELL interactions with E- and L-selectin in hematopoiesis, we will perform in vitro clonogenic assays and studies in murine models to delineate the role(s) of HCELL in progenitor cell migration to bone marrow and in hematopoietic events in vivo. It is anticipated that the results of these studies will provide fundamental insights into the role(s) of HCELL in mediating HSC entry into bone marrow and in the proliferation and differentiation of these cells. This information will contribute greatly to our understanding of the adhesive systems that regulate steady-state and pathologic hematopoiesis, as well as hematopoietic recovery following stem cell transplantation.
Showing the most recent 10 out of 35 publications
