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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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Conditional knockout of Grp94 in the bone marrow.A) Schematic drawings of the Grp94 wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) Grp94 mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of internal control 18S RNA. The data are presented as mean ± s.e., ***p<0.001.
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pone-0020364-g001: Conditional knockout of Grp94 in the bone marrow.A) Schematic drawings of the Grp94 wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) Grp94 mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of internal control 18S RNA. The data are presented as mean ± s.e., ***p<0.001.

Mentions: To study the in vivo function of GRP94, we created Grp94 mutant mice with the floxed or knockout allele (Figure 1A). Deletion of exon 2 flanked by the loxP-FRT sites led to an early frameshift mutation resulting in the inactivation of the Grp94 allele. To determine the role of GRP94 in the hematopoietic system, we bred Grp94F/F mice with transgenic mice bearing the Mx-1-Cre transgene that allows GRP94 to be acutely eliminated in the hematopoietic system in an inducible manner by administration of pI.pC. In these studies, Grp94 was deleted in 5.5 to 6.5 week old Grp94F/F; Mx-1-Cre (referred to below as cKO) mice. Littermates lacking the Cre transgene (Grp94F/F), which are phenotypically equivalent to animals with wild-type (WT) Grp94 alleles, were also injected with pI.pC and served as controls for any side effects of pI.pC injection. These control mouse cohorts are referred to below as WT mice. The status of the Grp94 allele deletion was validated by PCR in isolated BM cells (Figure 1B). Real-time quantitative PCR analysis with mouse BM cells 11 days after 7 shots of pI.pC injection showed that Grp94 transcripts were mostly depleted from the BM cells of the cKO mice (Figure 1C).


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)

Conditional knockout of Grp94 in the bone marrow.A) Schematic drawings of the Grp94 wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) Grp94 mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of internal control 18S RNA. The data are presented as mean ± s.e., ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020364-g001: Conditional knockout of Grp94 in the bone marrow.A) Schematic drawings of the Grp94 wild-type (WT) allele, the floxed allele and the knockout (KO) allele. The exons are boxed and numbered. The loxP sites (closed triangle) and the FRT site (open triangle) and expected PCR products for genotyping is indicated. B) Representative BM PCR genotyping results of mice with indicated genotypes after pI.pC injection. C) Grp94 mRNA expression measured by quantitative real-time PCR from WT (n = 16) and cKO (n = 18) mouse BM after pI.pC injection. The level of Grp94 mRNA was normalized against the level of internal control 18S RNA. The data are presented as mean ± s.e., ***p<0.001.
Mentions: To study the in vivo function of GRP94, we created Grp94 mutant mice with the floxed or knockout allele (Figure 1A). Deletion of exon 2 flanked by the loxP-FRT sites led to an early frameshift mutation resulting in the inactivation of the Grp94 allele. To determine the role of GRP94 in the hematopoietic system, we bred Grp94F/F mice with transgenic mice bearing the Mx-1-Cre transgene that allows GRP94 to be acutely eliminated in the hematopoietic system in an inducible manner by administration of pI.pC. In these studies, Grp94 was deleted in 5.5 to 6.5 week old Grp94F/F; Mx-1-Cre (referred to below as cKO) mice. Littermates lacking the Cre transgene (Grp94F/F), which are phenotypically equivalent to animals with wild-type (WT) Grp94 alleles, were also injected with pI.pC and served as controls for any side effects of pI.pC injection. These control mouse cohorts are referred to below as WT mice. The status of the Grp94 allele deletion was validated by PCR in isolated BM cells (Figure 1B). Real-time quantitative PCR analysis with mouse BM cells 11 days after 7 shots of pI.pC injection showed that Grp94 transcripts were mostly depleted from the BM cells of the cKO mice (Figure 1C).

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