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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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GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.
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pone-0020364-g004: GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.

Mentions: To examine the underlying cause for the increase in primitive hematopoietic cells in the BM of the cKO mice, we analyzed the cell cycle distribution in LSK cells by Hoechst/Pyronin Y staining (Figure 4A). While approximately 75% of the LSK cells in both genotypes were in G1 phase, the proportion of cells in G0 decreased from 15% in the control to 7.7% in the cKO mice (p<0.001), whereas the amount of S+G2+M cells increased from 10% in the control to 17% for cKO mice (p<0.01) (Figure 4B). Furthermore, the expansion of the Grp94 KO primitive hematopoietic cell pool was not a consequence of altered cell survival as analysis of the level of apoptotic cells using Annexin V did not reveal significant differences between WT and cKO LSK cells (p = 0.324) (Figure 4C). These findings suggest a loss of quiescence and increase in proliferation account at least in part for the primitive hematopoietic cell expansion observed in cKO mice, similar to what has been shown using other models of HSC function [45].


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)

GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.
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Related In: Results  -  Collection

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

pone-0020364-g004: GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.A) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. To examine early effects of GRP94 depletion on HSC proliferation, BM was extracted from WT and cKO mice 3 days after 4 shots of pI.pC injection every other day. B) Summary of cell cycle distribution of LSK cells from WT and cKO mice (n = 7). C) Summary of flow cytometric analysis of apoptotic LSK cells using Annexin V and 7AAD (n = 5 for WT, n = 8 for cKO) (p = 0.324). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.
Mentions: To examine the underlying cause for the increase in primitive hematopoietic cells in the BM of the cKO mice, we analyzed the cell cycle distribution in LSK cells by Hoechst/Pyronin Y staining (Figure 4A). While approximately 75% of the LSK cells in both genotypes were in G1 phase, the proportion of cells in G0 decreased from 15% in the control to 7.7% in the cKO mice (p<0.001), whereas the amount of S+G2+M cells increased from 10% in the control to 17% for cKO mice (p<0.01) (Figure 4B). Furthermore, the expansion of the Grp94 KO primitive hematopoietic cell pool was not a consequence of altered cell survival as analysis of the level of apoptotic cells using Annexin V did not reveal significant differences between WT and cKO LSK cells (p = 0.324) (Figure 4C). These findings suggest a loss of quiescence and increase in proliferation account at least in part for the primitive hematopoietic cell expansion observed in cKO mice, similar to what has been shown using other models of HSC function [45].

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