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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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Effect of cKO microenvironment on HSC maintenance.A) Scheme of creating chimeric mice with WT hematopoietic cells transplanted into WT or cKO microenvironment. B) Complete blood count with tail peripheral blood from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. C) Quantitation of spleen weights from WT-WT (n = 4) and WT-cKO (n = 5) chimeric mice. D) Representative flow cytometric analysis with BM from WT-WT and WT-cKO chimeric mice using Lin, c-Kit and Sca-1. E) Summary of percentage of LSK cells in the BM from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. All data are presented as mean ± s.e..
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pone-0020364-g009: Effect of cKO microenvironment on HSC maintenance.A) Scheme of creating chimeric mice with WT hematopoietic cells transplanted into WT or cKO microenvironment. B) Complete blood count with tail peripheral blood from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. C) Quantitation of spleen weights from WT-WT (n = 4) and WT-cKO (n = 5) chimeric mice. D) Representative flow cytometric analysis with BM from WT-WT and WT-cKO chimeric mice using Lin, c-Kit and Sca-1. E) Summary of percentage of LSK cells in the BM from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. All data are presented as mean ± s.e..

Mentions: In the competitive repopulation and competitive lodgment assay, where cKO cells are transplanted into a WT microenvironment, we observed impairment in the function of the cells indicating that Grp94 deletion in HSCs at least partially contributes to the disruption of the HSC-niche interaction. However, in our model system Grp94 was knocked out in the whole BM, therefore the disruption of HSC-niche interactions could also be attributed to the niche cells. To further explore a possible extrinsic role of GRP94 niche cells in HSC homeostasis disturbance in cKO mice, we created a chimeric mouse model with WT hematopoietic cells and a GRP94 microenvironment. WT BM cells were transplanted into lethally irradiated WT or cKO recipient mice. Following 8 weeks to allow for full reconstitution of WT hematopoietic system, Grp94 deletion was induced by pI.pC administration (Figure 9A). According to previous reports, BM stromal cells do not engraft after BM transplantation [48], therefore, Grp94 KO only occurred in the hematopoietic cells. Chimeras with WT hematopoietic cells and cKO microenvironment showed a normal hematology profile and spleen size as WT control chimeras (Figure 9B and 9C). Flow cytometric analysis revealed a similar LSK percentage in BM from cKO chimeras and WT controls (Figure 9D and 9E). These findings provide direct evidence that GRP94 in HSCs is required for their interaction with the niche, and depleting GRP94 in BM microenvironment itself is not sufficient to affect the regulation of HSCs.


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)

Effect of cKO microenvironment on HSC maintenance.A) Scheme of creating chimeric mice with WT hematopoietic cells transplanted into WT or cKO microenvironment. B) Complete blood count with tail peripheral blood from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. C) Quantitation of spleen weights from WT-WT (n = 4) and WT-cKO (n = 5) chimeric mice. D) Representative flow cytometric analysis with BM from WT-WT and WT-cKO chimeric mice using Lin, c-Kit and Sca-1. E) Summary of percentage of LSK cells in the BM from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. All data are presented as mean ± s.e..
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020364-g009: Effect of cKO microenvironment on HSC maintenance.A) Scheme of creating chimeric mice with WT hematopoietic cells transplanted into WT or cKO microenvironment. B) Complete blood count with tail peripheral blood from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. C) Quantitation of spleen weights from WT-WT (n = 4) and WT-cKO (n = 5) chimeric mice. D) Representative flow cytometric analysis with BM from WT-WT and WT-cKO chimeric mice using Lin, c-Kit and Sca-1. E) Summary of percentage of LSK cells in the BM from WT-WT (n = 5) and WT-cKO (n = 8) chimeric mice. All data are presented as mean ± s.e..
Mentions: In the competitive repopulation and competitive lodgment assay, where cKO cells are transplanted into a WT microenvironment, we observed impairment in the function of the cells indicating that Grp94 deletion in HSCs at least partially contributes to the disruption of the HSC-niche interaction. However, in our model system Grp94 was knocked out in the whole BM, therefore the disruption of HSC-niche interactions could also be attributed to the niche cells. To further explore a possible extrinsic role of GRP94 niche cells in HSC homeostasis disturbance in cKO mice, we created a chimeric mouse model with WT hematopoietic cells and a GRP94 microenvironment. WT BM cells were transplanted into lethally irradiated WT or cKO recipient mice. Following 8 weeks to allow for full reconstitution of WT hematopoietic system, Grp94 deletion was induced by pI.pC administration (Figure 9A). According to previous reports, BM stromal cells do not engraft after BM transplantation [48], therefore, Grp94 KO only occurred in the hematopoietic cells. Chimeras with WT hematopoietic cells and cKO microenvironment showed a normal hematology profile and spleen size as WT control chimeras (Figure 9B and 9C). Flow cytometric analysis revealed a similar LSK percentage in BM from cKO chimeras and WT controls (Figure 9D and 9E). These findings provide direct evidence that GRP94 in HSCs is required for their interaction with the niche, and depleting GRP94 in BM microenvironment itself is not sufficient to affect the regulation of HSCs.

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