GRP94 Regulates Stem Cell-Niche Interactions Figure 8. Grp94 KO HSCs failed to reconstitute the hematopoietic system in the presence of WT competitors. A) Scheme of the in vivo competitive repopulation assay. B) Representative flow cytometric analysis with tail blood from recipient mice 4 weeks after BM transplantation. CD45.1+ cells represent blood cells from WT competitor, while CD45.2+ cells represent progenies from WT or cKO LSK cells. Tail blood from recipient mice 8, 12, and 24 weeks after BM transplantation yielded the same results (data not shown). C) Summary of the percentage contribution of WT or cKO LSK cells to the peripheral blood MNCs (n = 5). The data are presented as mean 6 s.e., ***p,0.001. doi:10.1371/journal.pone.0020364.g008 them from differentiation [3]. Here we determined that Grp94 KO primitive cells are less efficient in contacting with BM niche, with the consequence that the mutant LSK cells can exit quiescence and enter cell cycle. While the precise mechanisms by which GRP94 affects this awaits future investigation, one plausible mechanism may be attributed to the requirement of GRP94 to facilitate processing of factors such as integrins specifically required for HSC-niche adhesion [19]. Secretory factors and cell-surface proteins are synthesized in the rough ER, and translocated into the ER where they are folded into their functional conformation by ER chaperone proteins [30]. GRP94 has been reported to be essential for the proper folding of several integrins, including a4 [40]. This has been reported to mediate the interaction of HSCs with niche cells and the extracellular matrix, as loss of integrin a4 results in impaired homing and reduced efficiency of HSCs to lodge to the endosteal surface [19,49]. While integrin a4 and a5 are both important for HSC retention in the niche [17,51], GRP94 is specifically required for processing integrin a4 but not a5 in HSCs. The failure of the Grp94 KO HSCs to express integrin a4 on the cell surface and its inability to bind to fibronectin are supportive of this notion. It is also possible that GRP94 is required for the proper folding of soluble ligands mediating the cross talk from HSCs to the niche. Other mechanisms may include cell cycle regulation by GRP94 and the ability of GRP94 to regulate ER Ca2+ efflux and signaling. As GRP94 is also recently localized to the cell surface and also identified as a transmembrane ER protein [30], it may also control signaling pathways initiated from the cell surface and/or the ER and cytosol interphase affecting stem cell proliferation. Our studies raise the general issue concerning the roles of protein folding chaperones in hematopoiesis and its relationship to the unfolded protein response (UPR). Hematopoiesis is tightly regulated by growth factors, cytokines and their receptors, which are secretory and cell membrane proteins [52]. Secretory factors PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20364