The Endoplasmic Reticulum Chaperone Protein GRP94 Is Required for Maintaining Hematopoietic Stem Cell Interactions with the Adult Bone Marrow Niche

Abstract

Hematopoietic stem cell (HSC) homeostasis in the adult bone marrow (BM) is regulated by both intrinsic gene expression products and interactions with extrinsic factors in the HSC niche. GRP94, an endoplasmic reticulum chaperone, has been reported to be essential for the expression of specific integrins and to selectively regulate early T and B lymphopoiesis. In GRP94 deficient BM chimeras, multipotent hematopoietic progenitors persisted and even increased, however, the mechanism is not well understood. Here we employed a conditional knockout (KO) strategy to acutely eliminate GRP94 in the hematopoietic system. We observed an increase in HSCs and granulocyte-monocyte progenitors in the Grp94 KO BM, correlating with an increased number of colony forming units. Cell cycle analysis revealed that a loss of quiescence and an increase in proliferation led to an increase in Grp94 KO HSCs. This expansion of the HSC pool can be attributed to the impaired interaction of HSCs with the niche, evidenced by enhanced HSC mobilization and severely compromised homing and lodging ability of primitive hematopoietic cells. Transplanting wild-type (WT) hematopoietic cells into a GRP94 null microenvironment yielded a normal hematology profile and comparable numbers of HSCs as compared to WT control, suggesting that GRP94 in HSCs, but not niche cells, is required for maintaining HSC homeostasis. Investigating this, we further determined that there was a near complete loss of integrin α4 expression on the cell surface of Grp94 KO HSCs, which showed impaired binding with fibronectin, an extracellular matrix molecule known to play a role in mediating HSC-niche interactions. Furthermore, the Grp94 KO mice displayed altered myeloid and lymphoid differentiation. Collectively, our studies establish GRP94 as a novel cell intrinsic factor required to maintain the interaction of HSCs with their niche, and thus regulate their physiology.

MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress

“…Previously, we and others found that MZB1 directly associates with the PDI ERp57 and the chaperones BiP and GRP94, suggesting that MZB1 does not act on its own but rather functions in collaboration with folding factors (Shimizu et al 2009;van Anken et al 2009;Flach et al 2010). GRP94 is a substrate-specific chaperone that serves a rather small subset of client proteins, including mHCs, TLRs, and integrins (Melnick et al 1994;Staron et al 2010;Luo et al 2011). Notably, we found that the associations of MZB1 with both GRP94 and mHC are markedly enhanced by induction of a UPR in tunicamycin-treated cells.…”

Deficiency of GRP94 in the Hematopoietic System Alters Proliferation Regulators in Hematopoietic Stem Cells

“…f/f ; Mx-1-Cre mice in a mixed C57BL/6; 129/Sv background were generated, and genotyping was performed as previously described [30]. Littermates that were negative for Cre transgene were used as controls.…”

“…Littermates that were negative for Cre transgene were used as controls. Mice that were 5.5 to 6.5 weeks old were subjected to pI.pC-induced deletion of Grp94 primarily in the hematopoietic system performed as previously described [30].…”

“…As a chaperone that assists in the folding, assembly, and secretion of a selective collection of client proteins, GRP94 performs unique functions in the ER, and controls specific pathways critical for cell growth, differentiation, organ homeostasis, and immune functions [27][28][29]. Our previous study using an Mx-1-Cre-mediated inducible knockout mouse model identified GRP94 as a regulator for HSC physiology, as loss of GRP94 in the hematopoietic system leads to an expansion of the hematopoietic stem and progenitor cell pool through increased proliferation [30]. This increased proliferation could, in part, be attributed to detachment of HSCs from the BM niche, as GRP94-null HSCs show increased mobilization, as well as impaired homing and engraftment [30].…”

“…Our previous study using an Mx-1-Cre-mediated inducible knockout mouse model identified GRP94 as a regulator for HSC physiology, as loss of GRP94 in the hematopoietic system leads to an expansion of the hematopoietic stem and progenitor cell pool through increased proliferation [30]. This increased proliferation could, in part, be attributed to detachment of HSCs from the BM niche, as GRP94-null HSCs show increased mobilization, as well as impaired homing and engraftment [30]. Furthermore, this impaired homing and engraftment was cell autonomous, as transplanting wild-type hematopoietic cells into a GRP94-null microenvironment yielded a normal hematopoietic profile with comparable numbers of HSCs as compared with controls.…”