CD61/ Integrin β3 Ligation Contributes to the Thrombopoietin-Mediated Niche Function of Mouse Hematopoietic Stem Cells.

Abstract 383

Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P<0.0001). HSCs per se appeared to be responsible for these results because CD61-/- CD34-KSL cells but not CD61-/- CD34+KSL cells showed accelerated cell-cycle progression compared to those of WT, suggesting that CD61 might contribute to maintenance of quiescent state of HSCs within the BM. It is known that thrombopoietin (TPO) acts on not only potent hematopoietic differentiation from HSCs but also for maintaining quiescent state of HSCs. Accordingly, we examined the relationship between TPO effect and integrin signaling through CD61 in HSCs. The in vitro single cell culture system showed the decreased cell proliferation and survival in CD61-/- CD34-KSL cells that were cultured in the presence of TPO (50 ng/mL), compared to the WT CD34-KSL cells (P<0.01). In contrast, CD61-deficency did not influence the phenotype of cultured CD34-KSL cells in the presence of stem cell factor (SCF, 50 ng/ml). Interestingly, these results were mirrored by CD61Y747A KI CD34-KSL cells and their littermate control CD34-KSL cells. Furthermore, when 40 CD34-KSL cells after 5 days culture in the presence of TPO and they were then applied into long-term competitive repopulation assay, CD61Y747A KI CD34-KSL cells showed less LTRA than that of WT (P<0.01). These results indicated that tyrosine at 747 of the β3 integrin subunit was required for the TPO-dependent expansion/maintenance of HSC in vitro. Whereas, to initiate integrin outside-in signaling in the presence of TPO, CD34-KSL cells were cultured on a well of 96-well plates pre-coated with various CD61 ligands (either vitronectin, 5 μg/ml or osteopontin, 5 μg/mL), resulting in the enhancement of their LTRA, compared to without these ligands. Since these ligands could interact with integrin receptors besides CD61, we utilized the antibody to CD61, 2C9.G2 that is capable of phosphorylating CD61 to mimic the actions of outside-in signaling. Using CD34-KSL cells were cultured with 2C9.G2, we further confirmed that CD61 signaling allowed CD34-KSL cells to maintain undivided state in vitro and increase LTRA in vivo, which were dependent upon the presence of TPO but neither SCF nor other growth factors including insulin growth factor-2 and fibroblast growth factor-1. More interestingly, both stimulation with CD61 ligation and TPO influenced ex vivo expansion of HSCs when started at 40 CD34-KSL cells in a 96-well culture well. Collectively, our results indicate that CD61/β3 integrin signaling plays important roles in TPO-mediated expansion/maintenance of HSC within the BM niche, which are possibly mediated through the tyrosine phosphorylation at 747 tyrosine residue of the β3 integrin cytoplasmic domain.