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The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche.

Luo B, Lam BS, Lee SH, Wey S, Zhou H, Wang M, Chen SY, Adams GB, Lee AS - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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.

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Inability of Grp94 KO HSCs to express surface integrin α4 and bind to fibronectin.A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2− and LSKFlk2+ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean ± s.e..
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pone-0020364-g010: Inability of Grp94 KO HSCs to express surface integrin α4 and bind to fibronectin.A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2− and LSKFlk2+ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean ± s.e..

Mentions: To investigate the potential mechanism by which GRP94 maintains the interaction between HSCs and the niche, we examined the cell surface expression of integrin α4 and α5, which are known to be important for the homing and adhesion of HSCs to the endosteal niche [49], [50]. Flow cytometric analysis with mouse BM LSKFlk2− and LSKFlk2+ populations demonstrated significantly lower expression of cell surface CD49d (integrin α4/β1) on cKO than WT cells, whereas CD49e (integrin α5/β1) expression was comparable between the two groups (Figure 10A). These results establish that GRP94 is required for the expression of integrin α4 on the cell surface of hematopoietic stem and progenitor cells, which is consistent with the recent finding that loss of GRP94 abrogates integrin α4 but not α5 expression on BM cells [40].


The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche.

Luo B, Lam BS, Lee SH, Wey S, Zhou H, Wang M, Chen SY, Adams GB, Lee AS - PLoS ONE (2011)

Inability of Grp94 KO HSCs to express surface integrin α4 and bind to fibronectin.A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2− and LSKFlk2+ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean ± s.e..
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101259&req=5

pone-0020364-g010: Inability of Grp94 KO HSCs to express surface integrin α4 and bind to fibronectin.A) Representative flow cytometric analysis of CD49d and CD49e with BM LSKFlk2− and LSKFlk2+ cells from WT and cKO mice. Grey-filled histogram represents isotype control staining; dashed green line represents WT cells; solid red line indicates cKO cells. B) The percentage of WT and cKO LSK cells bound to fibronectin in vitro. The number of cells binding to BSA was subtracted from that binding to fibronectin, the results then were normalized against the number of WT cells bound to BSA. The experiments were performed twice in duplicate; each replicate contained pooled BM from 2 to 4 WT or cKO mice. The data are presented as mean ± s.e..
Mentions: To investigate the potential mechanism by which GRP94 maintains the interaction between HSCs and the niche, we examined the cell surface expression of integrin α4 and α5, which are known to be important for the homing and adhesion of HSCs to the endosteal niche [49], [50]. Flow cytometric analysis with mouse BM LSKFlk2− and LSKFlk2+ populations demonstrated significantly lower expression of cell surface CD49d (integrin α4/β1) on cKO than WT cells, whereas CD49e (integrin α5/β1) expression was comparable between the two groups (Figure 10A). These results establish that GRP94 is required for the expression of integrin α4 on the cell surface of hematopoietic stem and progenitor cells, which is consistent with the recent finding that loss of GRP94 abrogates integrin α4 but not α5 expression on BM cells [40].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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 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.

Show MeSH
Related in: MedlinePlus