The Endoplasmic Reticulum Chaperone Protein GRP94 Is Required for Maintaining Hematopoietic Stem Cell Interactions with the Adult Bone Marrow Niche Biquan Luo 1 , Ben S. Lam2, Sung Hyung Lee3, Shiuan Wey1, Hui Zhou 1 , Miao Wang1, Si-Yi Chen 3 , Gregor B. Adams 2 , Amy S. Lee1* 1 Department of Biochemistry and Molecular Biology and the USC Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California, United States of America, 2 Department of Cell and Neurobiology, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, University of Southern California Keck School of Medicine, Los Angeles, California, United States of America, 3 Department of Molecular Microbiology and Immunology, University of Southern California Keck School of Medicine, Los Angeles, California, United States of America 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 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 a4 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. Citation: Luo B, Lam BS, Lee SH, Wey S, Zhou H, et al. (2011) The Endoplasmic Reticulum Chaperone Protein GRP94 Is Required for Maintaining Hematopoietic Stem Cell Interactions with the Adult Bone Marrow Niche. PLoS ONE 6(5): e20364. doi:10.1371/journal.pone.0020364 Editor: Christopher B. Doering, Emory University School of Medicine, United States of America Received January 31, 2011; Accepted April 26, 2011; Published May 24, 2011 Copyright: 2011 Luo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported from the National Institutes of Health CA027607 (ASL) and the American Society of Hematology (GBA).The USC core facilities described were supported in part by P30CA014089 from the National Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: amylee@ccnt.usc.edu Introduction In the adult hematopoietic system, hematopoietic stem cell (HSC) regulation of self-renewal and differentiation is at both the intrinsic and extrinsic level, and is tightly regulated under physiological conditions [1,2]. HSCs reside in a specific anatomic location in the bone marrow (BM) known as the stem cell niche [3,4]. Signaling cues from neighboring cells in the niche are key in dictating the function of the cell to maintain the hematopoietic system of the individual [5–7]. At the endosteal surface [8], which is the interface between bone and the BM, specialized osteoblasts represent the main components of HSC niche [9,10]. It has been proposed that this heterogeneous group of cells regulates HSC survival, self- renewal, migration, differentiation, and quiescence [11,12] through several soluble factors and their receptors such as angiopoietin/Tie2 [13], osteopontin [14,15] and Ca2+/CaR [16], as well as direct contact through extracellular matrix and cell surface proteins [17,18], such as integrins [19,20]. HSC engraftment is a multistep process involving homing, transmarrow migration, and lodging within the BM niche [15], all of which is controlled by adhesion molecules, soluble ligands and their receptors [3]. It is also clear that the decision of the HSC to self-renew or differentiate is dependent upon the extrinsic signaling mecha- nisms controlling the expression of intrinsic determinants of HSC function. Previous studies have determined that a number of cell cycle regulators [1,21] such as the early G1-phase checkpoint regulator p18INK4C [22], and the later G1-phase checkpoint regulator P21cip1/waf1 [23], are critical to maintain HSC quiescence. Retinoblastoma family protein [24] and PTEN [25,26] also play crucial roles in maintaining HSC homeostasis. Transcription factors such as Zinc-finger repressor Gfi1 [27,28] and chromatin-associated factors like Bmi1 [29] have been implicated as key regulators in maintaining HSC self-renewal. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20364