Immune Suppressor Factor Confers Enhanced Supporting Activity for Hematopoietic Stem Cells in Bone Marrow Stroma.

Immune suppressor factor (ISF) is a subunit of vacuolar ATPase proton pump that plays a critical role for proton transfer to extracellular space or subcellular organs. Physiological importance of vacuolar ATPase was evident in a variety of cellular functions such as re-absorption of bones by osteoclasts, acidification of lysosomes in macrophages, and acidification of urine in kidney. However, a role for vacuolar ATPase in hematopoiesis is still unknown. We have previously identified a short form of ISF (ShIF) as a stromal cell derived factor that supports factor-independent growth of a mutant subline of Ba/F3, an IL-3 dependent murine hematipoietic cell line. In this study, we addressed whether ISF supports self-renewal and expansion of primary hematopoietic stem cells (HSC). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) enhanced their colony-forming activity as revealed by the numbers and the size of the colonies. In addition, the numbers of long-term culture initiating cell (LTC-IC) were markedly increased by co-culture on MS10/ISF or MS10/ShIF. Moreover, competitive repopulating activity of c-Kit+Sca-1+Lin- HSC was significantly maintained without any added cytokines when they were co-cultured with MS10/ISF or MS10/ShIF compared with a mock control. In order to check whether these activities were dependent on the proton pump function of ISF, we generated a mutant ISF or ShIF whose proton pump activity was abolished by point mutation. Interestingly, all stem cell supporting activities described above were abolished in the mutant ISF or ShIF, indicating that proton transfer across cellular or endosomal membrane was critical. Analysis of gene expression profile of mock and ISF-transfected cell lines revealed that any cytokines or growth factors previously known to affect hematopoiesis are not modulated at mRNA level. However, downregulation of secreted frizzled related protein (sFRP)-1, an antagonist for Wnt, and up-regulation of matrix metalloproteinase-3 (MMP-3) were clearly noted in ISF/ ShIF-overexpressing cell line, suggesting that relative increase of Wnt activity and the modulation of extracellular matrix are the key molecular events underlying the enhanced supporting activity for HSC. These results propose a novel role for ISF in self-renewal and expansion of HSC in vivo.