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

<p>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 <i>78^f/f</i>, n = 6 for <i>c78^f/f</i>). (Right) Quantitation of flow cytometric analysis of HSC-enriched LSK population in the BM (n = 12 for <i>78^f/f</i>, n = 16 for <i>c78^f/f</i>, n = 4 for <i>c78^f/+</i>). C: Total BM cell number from <i>78^f/f</i> (n = 11) and <i>c78^f/f</i> (n = 16) mice. D: Quantitation of LSK percentage and total BM cell number from <i>78^f/f</i>(t) and <i>c78^f/f</i>(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 <i>78^f/f</i>, n = 6 for <i>c78^f/f</i>). 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 <i>78^f/f</i> (n = 4) and <i>c78^f/f</i> (n = 4) mice. All data are presented as mean ± s.e (*P<0.05, **P<0.01, Student’s <i>t</i> test).</p

Summary diagram of alteration of hematopoiesis, UPR signaling and apoptosis in <i>Grp78</i> conditional knockout in the hematopoietic system.

<p>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.</p

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

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

Acute Inducible Ablation of GRP78 Reveals Its Role in Hematopoietic Stem Cell Survival, Lymphogenesis and Regulation of Stress Signaling

<div><p>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 <em>Grp78</em> 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 <em>Grp78</em> 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, <em>xbp-1</em> 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.</p> </div

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

<p>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 <i>78^f/f</i> (n = 6) and <i>c78^f/f</i> (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 <i>78^f/f</i>(t) (n = 3) and <i>c78^f/f</i>(t) (n = 3) mice. C: Quantitation of lymphoid and myeloid cells from flow cytometric analysis with BM cells in <i>78^f/f</i> and <i>c78^f/f</i> 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 <i>78^f/f</i>(t) and <i>c78^f/f</i>(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 <i>t</i> test).</p

Knockout of GRP78 in BM cells activates UPR signaling pathways.

<p>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 <i>xbp-1</i> spliced [<i>xbp-1(s)</i>], <i>xbp-1</i> unspliced [<i>xbp-1(u)</i>] and <i>β-actin</i> mRNA levels from BM cells of <i>78^f/f</i> and <i>c78^f/f</i> mice (n = 3 for each genotype). The PCR image was inverted for better clarity. (Lower panel) Quantitation of the ratio of <i>xbp-1(s)</i> to <i>xbp-1(u)</i>. The average ratio of <i>xbp-1(s)</i>/<i>xbp-1(u)</i> in <i>78^f/f</i> was set as 1. The data is presented as mean ± s.e. (*P<0.05, Student’s <i>t</i> test).</p

GRP78 deficiency leads to lymphopenia.

<p>A: GRP78 expression in WT BM subpopulations. (Left) <i>Grp78</i> 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 <i>78^f/f</i> and <i>c78^f/f</i> 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 <i>78^f/f</i>, n = 4 for <i>c78^f/f</i>) and spleen weight (n = 14 for <i>78^f/f</i>, n = 20 for <i>c78^f/f</i>). E: H&E staining of paraffin sections of thymus and spleen of <i>78^f/f</i> and <i>c78^f/f</i> 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) <i>78^f/f</i> (n = 12), <i>c78^f/f</i> (n = 16) mice and (Right) <i>78^f/f</i>(t) (n = 3), <i>c78^f/f</i>(t) (n = 3) chimeric mice. All data are presented as mean ± s.e (**P<0.01, ***P<0.001, Student’s <i>t</i> test).</p

GRP94-deficient LSK cells displayed increased proliferation and loss of quiescence.

<p>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.</p

<i>Grp94</i> KO mice displayed altered myeloid and lymphoid differentiation.

<p>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.</p

Conditional knockout of <i>Grp94</i> in the bone marrow.

<p>A) Schematic drawings of the <i>Grp94</i> 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) <i>Grp94</i> 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 <i>Grp94</i> mRNA was normalized against the level of internal control <i>18S RNA</i>. The data are presented as mean ± s.e., ***p<0.001.</p