Acute inducible ablation of GRP78 reveals its role in hematopoietic stem cell survival, lymphogenesis and regulation of stress signaling

Abstract

GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes Grp78 in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with Grp78 deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, xbp-1 splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.

Figure 1. GRP78 deficiency leads to lymphopenia.

A: GRP78 expression in WT BM subpopulations. (Left) Grp78 mRNA expression in WT BM subpopulations measured by quantitative real-time PCR. The experiments were performed in duplicates; each replicate contains pooled BM from two WT mice. (Right) GRP78 expression in LSKCD34⁻ and LSKCD34⁺ subpopulations in WT mice (n = 4) measured by flow cytometry. The bar graph represents the medium intensities of GRP78 staining with LSK cells set as 1. B: (Upper) Representative PCR genotyping results from 78^f/f and c78^f/f BM 6 days post completion of pI.pC treatment. (Lower) Western blot results for detection of GRP78 protein level in the BM performed in duplicates. C: Organ size and morphology from mice of the indicated genotypes. Arrows on top of the heart indicate thymus. D: Quantitation of the thymus cellularity (n = 4 for 78^f/f, n = 4 for c78^f/f) and spleen weight (n = 14 for 78^f/f, n = 20 for c78^f/f). E: H&E staining of paraffin sections of thymus and spleen of 78^f/f and c78^f/f mice. Arrows indicate megakaryocytes in the spleen. The scale bar represents 200 µm in thymus and 20 µm in spleen. F: Peripheral lymphocyte count using complete blood count analysis with tail peripheral blood from (Left) 78^f/f (n = 12), c78^f/f (n = 16) mice and (Right) 78^f/f(t) (n = 3), c78^f/f(t) (n = 3) chimeric mice. All data are presented as mean ± s.e (**P<0.01, ***P<0.001, Student’s t test).

Figure 2. Deletion of GRP78 in the hematopoietic system leads to altered hematopoiesis.

A: Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) from 78^f/f (n = 6) and c78^f/f (n = 6) mice. B: Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) from 78^f/f(t) (n = 3) and c78^f/f(t) (n = 3) mice. C: Quantitation of lymphoid and myeloid cells from flow cytometric analysis with BM cells in 78^f/f and c78^f/f mice using lineage markers B220, CD3, Gr-1 and Mac-1 (n = 4 for each genotype). D: Quantitation of lymphoid and myeloid cells from flow cytometric analysis with BM cells in 78^f/f(t) and c78^f/f(t) mice using lineage markers B220, CD3, Gr-1 and Mac-1 (n = 3 for each genotype). All data are presented as mean ± s.e (*P<0.05, **P<0.01, ***P<0.001, Student’s t test).

Figure 3. GRP78 deficiency in the BM reduced HSC-enriched population through increased cell death.

A: Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1 and CD34. B: ?Left) Quantitation of flow cytometric analysis of Lin^-c-Kit⁺Sca-1⁺CD34⁻ (LT-HSC) and Lin^-c-Kit⁺Sca-1⁺CD34⁺ (ST-HSC) populations in the BM (n = 6 for 78^f/f, n = 6 for c78^f/f). (Right) Quantitation of flow cytometric analysis of HSC-enriched LSK population in the BM (n = 12 for 78^f/f, n = 16 for c78^f/f, n = 4 for c78^f/+). C: Total BM cell number from 78^f/f (n = 11) and c78^f/f (n = 16) mice. D: Quantitation of LSK percentage and total BM cell number from 78^f/f(t) and c78^f/f(t) mice (n = 3 for each analysis). E: (Left) Representative flow cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD. (Right) Summary of apoptotic LSK cells (Annexin V⁺7-AAD^–) (n = 5 for 78^f/f, n = 6 for c78^f/f). F: (Left) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. (Right) Summary of cell cycle distribution of LSK cells from 78^f/f (n = 4) and c78^f/f (n = 4) mice. All data are presented as mean ± s.e (*P<0.05, **P<0.01, Student’s t test).

Figure 4. Knockout of GRP78 in BM cells activates UPR signaling pathways.

A: Western blot results using BM cell lysates (n = 3 for each genotype) for detection of GRP78, phospho-eIF2α, total eIF2α, CHOP, ATF6 (p50), ATF6 (p90), calreticulin, pro-caspase-7 intermediate, cleaved caspase-7 and β-actin. B: (Upper panel) RT-PCR results for detection of xbp-1 spliced [xbp-1(s)], xbp-1 unspliced [xbp-1(u)] and β-actin mRNA levels from BM cells of 78^f/f and c78^f/f mice (n = 3 for each genotype). The PCR image was inverted for better clarity. (Lower panel) Quantitation of the ratio of xbp-1(s) to xbp-1(u). The average ratio of xbp-1(s)/xbp-1(u) in 78^f/f was set as 1. The data is presented as mean ± s.e. (*P<0.05, Student’s t test).

Figure 5. Differential cytokine and chemokine expression in serum of GRP78 deficient mice.

(Upper, middle, lower) bar graphs demonstrating the differential expression of cytokines and chemokines in the serum of 78^f/f (n = 3) and c78^f/f (n = 3) mice. The ones that exhibit major difference between the c78^f/f and 78^f/f mice are circled and bold highlighted. The data is presented as mean ± s.e. The P values are indicated (***P<0.001, Student’s t test).

Figure 6. Summary diagram of alteration of hematopoiesis, UPR signaling and apoptosis in Grp78 conditional knockout in the hematopoietic system.

GRP78 depletion in the hematopoietic system leads to altered hematopoiesis, activated UPR signaling and enhanced apoptosis. Open arrows represent an increased level and closed arrows represent a decreased level.