The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche - PubMed (original) (raw)
The endoplasmic reticulum chaperone protein GRP94 is required for maintaining hematopoietic stem cell interactions with the adult bone marrow niche
Biquan Luo et al. PLoS One. 2011.
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 null 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.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. 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.
Figure 2. GRP94 deficiency in the bone marrow expanded the primitive cell pool.
A) Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1, Flk2 and CD34. B) Quantitation of flow cytometric analysis of primitive cell proportions. Left panel shows the percentage of LSKFlk2−CD34− in BM and right panel shows LSKFlk2−CD34+, LSKFlk2+ (n = 5 for WT, n = 8 for cKO) and total LSK (n = 16 for WT, n = 22 for cKO) cells in BM using Lin, c-Kit, Sca-1. C) Total BM cell number from WT (n = 19) and cKO (n = 20) mice (p = 0.373). All data are presented as mean ± s.e., **p<0.01, ***p<0.001.
Figure 3. GRP94 deficiency led to increased granulocyte–monocyte progenitors in the bone marrow.
A) Quantitation of flow cytometric analysis of myeloid and lymphoid progenitors including common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP), megakaryocyte-erythroid progenitor (MEP) and common lymphoid progenitor (CLP) from WT (n≥7) and cKO (n≥10) mice. B) Quantitation of colonies formed from unfractioned BM from WT (n = 4) and cKO (n = 7) mice in methylcellulose medium. All data are presented as mean ± s.e., *p<0.05, **p<0.01.
Figure 4. 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.
Figure 5. Increase extramedullary hematopoiesis in the spleen of Grp94 KO mice.
A) Representative spleen size and morphology of WT and cKO mice. B) Average spleen weights of WT (n = 19) and cKO (n = 25) mice. The data are presented as mean ± s.e., ***p<0.001. C) Representative H&E staining of paraffin sections of spleen from WT and cKO mice. Hematopoietic cells in the red pulp are indicated by the area between the two arrows. Scale bar represents 2 mm.
Figure 6. Increased mobilization of Grp94 KO HSCs.
A) Representative flow cytometric analysis of splenocytes from WT (n = 9) and cKO (n = 9) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). In these studies, splenocytes were extracted from WT and cKO mice 5 days after 7 injections of pI.pC (to examine HSC mobilization before the spleen was enlarged). B) Representative flow cytometric analysis of blood MNCs from WT (n = 11) and cKO (n = 10) mice using Lin, c-Kit and Sca-1 (left) and quantitation (right). All data are presented as mean ± s.e., ***p<0.001.
Figure 7. Grp94 KO HSCs displayed impaired interaction with the niche.
A) Scheme of the in vivo competitive lodgment assay. B) Representative flow cytometric analysis of CFSE-labeled WT LK cells and SNARF-labeled cKO LK cells with host BM and spleen cells. C) Summary of LK cells homed to the BM and spleen (n = 4 for BM, n = 2 for spleen), with the level of WT cells in BM and spleen set as 1. D) Bone section of a recipient femur. WT LK cells were labeled with CFSE (green); cKO LK cells were labeled with SNARF (red); and nuclei were stained with DAPI (blue). Solid arrows indicate cells lodged in the endosteal region (within two cell diameters from the endosteal surface), while open arrows indicate cells located in the central marrow. Scale bar represents 1 mm. E) Summary of the percentage of labeled LK cells found at the endosteal region among those homed to BM from 3 independent experiments. All data are presented as mean ± s.e., *p<0.05.
Figure 8. Grp94 KO HSCs failed to reconstitute the hematopoietic system in the presence of WT competitors.
A) Scheme of the in vivo competitive repopulation assay. B) Representative flow cytometric analysis with tail blood from recipient mice 4 weeks after BM transplantation. CD45.1+ cells represent blood cells from WT competitor, while CD45.2+ cells represent progenies from WT or cKO LSK cells. Tail blood from recipient mice 8, 12, and 24 weeks after BM transplantation yielded the same results (data not shown). C) Summary of the percentage contribution of WT or cKO LSK cells to the peripheral blood MNCs (n = 5). The data are presented as mean ± s.e., ***p<0.001.
Figure 9. 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..
Figure 10. 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..
Figure 11. Grp94 KO mice displayed altered myeloid and lymphoid differentiation.
A). Complete blood count of peripheral blood from WT (n = 31) and cKO (n = 37) mice. B) Representative Wright-Giemsa staining of blood smear with tail peripheral blood from WT and cKO mice. Scale bar represents 500 µm. C) Total thymus cell number (left) and total left and right axillary lymph nodes cell number (right) from WT and cKO mice (n = 7 for each group). D) Representative flow cytometric analysis of splenocytes from WT and cKO mice using lineage markers Gr-1 and CD3 (left), F4/80 and B220 (right). E) Quantitation of (D) from WT (n = 4) and cKO (n = 7) mice. F) Representative flow cytometric analysis with BM cells using lineage markers Gr-1 and B220 (left) and CD4 and CD8a (right). G) Quantitation of (F). Gr-1 and B220 (n = 7 for WT and n = 9 for cKO mice); CD4 and CD8a (n = 7 for each genotype). All data are presented as mean ± s.e., ***p<0.001.
References
- Zon LI. Intrinsic and extrinsic control of haematopoietic stem-cell self-renewal. Nature. 2008;453:306–313. - PubMed
- Wagers AJ, Christensen JL, Weissman IL. Cell fate determination from stem cells. Gene Ther. 2002;9:606–612. - PubMed
- Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol. 2006;6:93–106. - PubMed
- Jones DL, Wagers AJ. No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol. 2008;9:11–21. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Medical
Molecular Biology Databases
Research Materials
Miscellaneous