GRP94 Regulates Stem Cell-Niche Interactions Figure 6. Increased mobilization of Grp94 KO HSCs. A) Representative flow cytometric analysis of splenocytes from WT (n = 9) and cKO (n = 9) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). In these studies, splenocytes were extracted from WT and cKO mice 5 days after 7 injections of pI.pC (to examine HSC mobilization before the spleen was enlarged). B) Representative flow cytometric analysis of blood MNCs from WT (n = 11) and cKO (n = 10) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). All data are presented as mean 6 s.e., ***p,0.001. doi:10.1371/journal.pone.0020364.g006 loss of GRP94 abrogates integrin a4 but not a5 expression on BM cells [40]. To test specific interactions of the primitive hematopoietic cells with adhesion molecules known to be present at the endosteal surface and play a role in mediating HSC-niche interactions, we examined the ability of the cells to bind to fibronectin in vitro, the binding partner of integrin a4, with binding to BSA as a negative control. While WT LSK cells were able to bind to fibronectin, we did not detect binding of the cKO cells to fibronectin above the negative control level (Figure 10B). Collectively, these findings demonstrate that acute depletion of GRP94 in HSCs disrupts the interaction of HSCs with niche, possibly through the disruption of adhesion to key extracellular matrix molecules mediated by integrin a4. Deletion of Grp94 in the hematopoietic system leads to altered myeloid and lymphoid differentiation To determine the consequence of acute depletion of GRP94 following Mx-1-Cre induction, we examined the hematopoietic phenotypes of cKO mice compared to control siblings. Complete blood count analysis demonstrated a general leukocytosis and severe thrombocytopenia, while red blood cell and hemoglobin counts were normal. Monocytes and granulocytes in peripheral blood were increased 8-fold and 10-fold, respectively, in cKO mice compared to control mice (Figure 11A), which is consistent with the increased GMP in the cKO BM (Figure 3A). Analysis of blood smears further confirmed the leukocytosis in peripheral blood (Figure 11B). Compared with WT littermates, cKO mice showed significantly lower thymus and lymph node cellularities (Figure 11C), consistent with smaller thymus and peripheral lymph nodes (data not shown). The increase in myeloid cells and decrease in lymphoid cells corresponded with a higher proportion of granulocytes and macrophages and a lower proportion of T- cells and B-cells in the spleen of the cKO mice (Figure 11D and E). The expansion of myeloid cells was also evident in the BM, where Gr-1+ cells were increased and B220+ cells were decreased in cKO mice. The proportion of CD4+ and CD8+ cells in the BM of cKO mice were comparable to the control mice (Figure 11F and G). Discussion GRP94 is the most abundant glycoprotein in the ER and is emerging as an important player in protein processing. Its unique interaction with specific client proteins may allow it to specifically regulate cellular processes distinct from other major chaperones in the ER. In particular, recent model studies suggested that GRP94 is critically required for the functional expression of secretory and/ or membrane proteins that enable the integration of cells into tissues [32]. Here we examined the role of GRP94 in the HSC interactions with the adult BM niche. The model system that we PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20364