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. 2013 Dec 1;22(23):3062-73.
doi: 10.1089/scd.2013.0181. Epub 2013 Aug 20.

Deficiency of GRP94 in the hematopoietic system alters proliferation regulators in hematopoietic stem cells

Biquan Luo  1 , Chun-Chih Tseng, Gregor B Adams, Amy S Lee
Affiliations

Deficiency of GRP94 in the hematopoietic system alters proliferation regulators in hematopoietic stem cells

Biquan Luo et al. Stem Cells Dev. .

Abstract

We have previously reported that acute inducible knockout of the endoplasmic reticulum chaperone GRP94 led to an expansion of the hematopoietic stem and progenitor cell pool. Here, we investigated the effectors and mechanisms for this phenomenon. We observed an increase in AKT activation in freshly isolated GRP94-null HSC-enriched Lin(-) Sca-1(+) c-Kit(+) (LSK) cells, corresponding with higher production of PI(3,4,5)P3, indicative of PI3K activation. Treatment of GRP94-null LSK cells with the AKT inhibitor MK2206 compromised cell expansion, suggesting a causal relationship between elevated AKT activation and increased proliferation in GRP94-null HSCs. Microarray analysis demonstrated a 97% reduction in the expression of the hematopoietic cell cycle regulator Ms4a3 in the GRP94-null LSK cells, and real-time quantitative PCR confirmed this down-regulation in the LSK cells but not in the total bone marrow (BM). A further examination comparing freshly isolated BM LSK cells with spleen LSK cells, as well as BM LSK cells cultured in vitro, revealed specific down-regulation of Ms4a3 in freshly isolated BM GRP94-null LSK cells. On examining cell surface proteins that are known to regulate stem cell proliferation, we observed a reduced expression of cell surface connexin 32 (Cx32) plaques in GRP94-null LSK cells. However, suppression of Cx32 hemichannel activity in wild-type LSK cells through mimetic peptides did not lead to increased LSK cell proliferation in vitro. Two other important cell surface proteins that mediate HSC-niche interactions, specifically Tie2 and CXCR4, were not impaired by Grp94 deletion. Collectively, our study uncovers novel and unique roles of GRP94 in regulating HSC proliferation.

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Figures

FIG. 1.
FIG. 1.
Expanded primitive hematopoietic cell pools on GRP94 deficiency in the BM. (A) Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1, CD41, CD48, and CD150. (B) Quantification of flow cytometric analysis of LSKCD41−CD48−CD150+ LT-HSCs and LSKCD41−CD48−CD150− ST-HSCs (n= 12 for Grp94f/f, n= 8 for cGrp94f/f). (C) Quantification of flow cytometric analysis of LSK cells in BM (n= 12 for Grp94f/f, n= 8 for cGrp94f/f). All data are presented as mean±S.E., ***P<0.001. BM, bone marrow; HSCs, hematopoietic stem cells; LSK, Lin− Sca-1+ c-Kit+; LT-HSCs, long-term HSCs; ST-HSCs, short-term HSCs. Color images available online at www.liebertpub.com/scd
FIG. 2.
FIG. 2.
In vitro culture of GRP94-null LSK cells. (A) Scheme of culturing Grp94f/f and cGrp94f/f LSK cells in vitro. (Upper panel) with growth factors and cytokines only; (lower panel) with stromal cells, growth factors, and cytokines. CD45 staining was used to determine hematopoietic cells derived from the LSK cells. (B) Cell numbers from cultures without stromal cells determined by a hemocytometer. (C) Flow cytometric analysis using CD45 as a marker for hematopoietic cells derived from LSK cells co-cultured with stromal cells. The green line represents Grp94f/f cells, and the red line indicates cGrp94f/f cells. (D) Quantification of hematopoietic cells under co-culture conditions with stromal cells, based on the number of stromal cells seeded and the proportion of CD45+ cells determined by flow cytometric analysis. All data are presented as mean±S.E., **P<0.01. Color images available online at www.liebertpub.com/scd
FIG. 3.
FIG. 3.
Increased AKT activation is required for cGrp94f/f LSK cell proliferation. (A) Fluorescence staining of phosphorylated-AKT (pAKT, Ser473; in green) on freshly isolated BM LSK cells from the indicated genotypes. The corresponding nuclei were stained by DAPI in blue. Scale bar represents 10 μm. (B) Quantification of pAKT levels in Grp94f/f and cGrp94f/f LSK cells with ImageJ (n= 41 for Grp94f/f, n= 54 for cGrp94f/f). ***P<0.001. (C) Fluorescence staining of total AKT (in red) on freshly isolated BM LSK cells. (D) Quantification of total AKT levels in Grp94f/f and cGrp94f/f LSK cells with ImageJ (n= 45 for Grp94f/f, n= 64 for cGrp94f/f). *P<0.05. (E) Representative PI(3,4,5)P3 staining on freshly isolated BM LSK cells from Grp94f/f and cGrp94f/f mice. The corresponding nuclei were stained by DAPI in blue. Scale bar represents 5 μm. (F) Fold change in cell numbers from cultured LSK cells that were treated with either DMSO or MK2206 for 2 days at the indicated concentrations, with the number of cells seeded set as one. All data are presented as mean±S.E. Color images available online at www.liebertpub.com/scd
FIG. 4.
FIG. 4.
Context-dependent expression of Ms4a3 in LSK cells. (A) Ms4a3 mRNA expression measured by quantitative real-time PCR on BM LSK cells, whole BM cells, Gr-1+, F4/80+ and B220+ BM cells, spleen LSK cells, and BM LSK cells cultured in vitro for 2 days. The levels of Ms4a3 mRNA were normalized against 18S RNA. (B) Western blot analysis of GRP94 and β-actin expression in the human promyelocytic leukemia cells (HL60) infected with lentiviral shCtrl or shGrp94. (C) Ms4a3 mRNA expression from HL60 cells infected with shCtrl and shGrp94 normalized against 18S RNA. The experiments were repeated two to four times. All data are presented as mean±S.E., *P<0.05, **P<0.01.
FIG. 5.
FIG. 5.
Reduction of Cx32 cell surface plaques on Grp94 deletion. (A) Immunofluorescent Cx32 staining on freshly isolated Grp94f/f and cGrp94f/f BM LSK cells. White arrows indicate Cx32 plaques on the cell surface. Scale bar represents 2 μm. (B) The number of Cx32 plaques was counted from Z-stack images for whole cells, and the percentages of Grp94f/f (left) and cGrp94f/f (right) BM LSK cells with different numbers of Cx32 plaques on the cell surface are presented. Color images available online at www.liebertpub.com/scd
FIG. 6.
FIG. 6.
Effect of Cx32 mimetic peptides on LSK cell proliferation in vitro. (A) Validation of the ability of 32Gap27 in blocking gap-junctional intercellular communication (GJIC) by scrape-loading and dye transfer assay. HeLa cells transfected with empty vector or Cx32 expression vector were scraped and loaded with the mixture of Lucifer yellow and Rhodamine dextran dyes. The level of intracellular communication, as indicated by the ratio between the cells receiving Lucifer yellow from neighboring cells and the cells initially labeled under various experimental conditions, is shown. (B) Scheme of experimental design for treatment and assay with mimetic (32Gap27) or scrambled peptides. (C) Cell numbers from the cultures subjected to the different treatment conditions as indicated next. The concentrations of the peptides in μg/mL are shown. NT, not treated. All data are presented as mean±S.E.
FIG. 7.
FIG. 7.
Lack of effect of GRP94 depletion on Tie2 and CXCR4 surface expression. (A) Western blot analysis of GRP94 expression in the Grp94f/f and cGrp94f/f BM, with β-actin serving as loading control. (B) Cell surface Tie2 expression on Grp94f/f and cGrp94f/f BM LSK cells determined by flow cytometric analysis. The green line represents Grp94f/f cells, and the red line indicates cGrp94f/f cells. (C) GRP94 expression in the U266 multiple myeloma cells infected with lentiviral shCtrl or shGrp94 determined by western blot. (D) Representative flow cytometric analysis on cell surface CXCR4 expression on the U266 cells. The gray area represents isotype control staining; the blue line indicates NT cells; the green line represents U266 cells infected with shCtrl; and the red line indicates U266 cells infected with shGrp94. Color images available online at www.liebertpub.com/scd
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