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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL060528-09
Application #
6942361
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Thomas, John
Project Start
1997-09-22
Project End
2007-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
9
Fiscal Year
2005
Total Cost
$432,500
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
030811269
City
Boston
State
MA
Country
United States
Zip Code
02115
Videira, Paula A; Silva, Mariana; Martin, Kyle C et al. (2018) Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-Derived Dendritic Cells Programs Transendothelial Migration. J Immunol 201:1030-1043
Sackstein, Robert (2016) Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology. Glycobiology 26:560-70
Merzaban, Jasmeen S; Imitola, Jaime; Starossom, Sarah C et al. (2015) Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis. Glycobiology 25:1392-409
Abdi, Reza; Moore, Robert; Sakai, Shinobu et al. (2015) HCELL Expression on Murine MSC Licenses Pancreatotropism and Confers Durable Reversal of Autoimmune Diabetes in NOD Mice. Stem Cells 33:1523-31
Sackstein, Robert; Fuhlbrigge, Robert (2015) Western blot analysis of adhesive interactions under fluid shear conditions: the blot rolling assay. Methods Mol Biol 1312:399-410
Sackstein, Robert (2012) Engineering cellular trafficking via glycosyltransferase-programmed stereosubstitution. Ann N Y Acad Sci 1253:193-200
Sackstein, Robert (2012) Glycoengineering of HCELL, the human bone marrow homing receptor: sweetly programming cell migration. Ann Biomed Eng 40:766-76
Sackstein, Robert (2011) The biology of CD44 and HCELL in hematopoiesis: the 'step 2-bypass pathway' and other emerging perspectives. Curr Opin Hematol 18:239-48
Merzaban, Jasmeen S; Burdick, Monica M; Gadhoum, S Zeineb et al. (2011) Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells. Blood 118:1774-83
Jacobs, Pieter P; Sackstein, Robert (2011) CD44 and HCELL: preventing hematogenous metastasis at step 1. FEBS Lett 585:3148-58

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