Blood Res.  2015 Dec;50(4):218-226. 10.5045/br.2015.50.4.218.

Expression and functional roles of the chemokine receptor CXCR7 in acute myeloid leukemia cells

• Kim HY
• Lee SY
• Kim DY
• Moon JY
• Choi YS
• Song IC
• Lee HJ
• Yun HJ
• Kim S
• Jo DY

• KMID: 2133756
• DOI: http://doi.org/10.5045/br.2015.50.4.218

Abstract

BACKGROUND
The C-X-C chemokine receptor 7 (CXCR7) has been shown to be a decoy receptor for CXCR4 in certain cell types. We investigated the expression status and functional roles of CXCR7 in acute myeloid leukemia (AML) cells in vitro.
METHODS
CXCR7 mRNA was knocked down in AML cells by using small interfering RNA (siRNA) technology, and subsequent biological alterations in the cells were evaluated in vitro.
RESULTS
All AML cell lines examined in this study (U937, K562, KG1a, HL-60, and MO7e) and primary CD34+ cells obtained from patients with AML expressed CXCR7 mRNA at various levels. Western blotting showed that all AML cells produced CXCR7. Furthermore, all AML cells expressed CXCR7 in both the cytoplasm and on the cell surface at various levels. Stromal cell-derived factor-1 (SDF-1; C-X-C motif ligand 12 (CXCL12)) induced internalization of cell surface CXCR7. However, neither hypoxia nor the examined hematopoietic growth factors (interleukin-1beta (IL-1beta), IL-3, IL-6, granulocyte-colony-stimulating factor, granulocyte, macrophage-colony-stimulating factor, and stem cell factor) and proinflammatory cytokines (interferon-gamma, transforming growth factor-beta, and tumor necrosis factor-alpha) were found to alter cell surface CXCR7 expression. The transfection of AML cells with CXCR4 siRNA, but not CXCR7 siRNA, significantly impaired the CXCL12-induced transmigration of the cells. The transfection of AML cells with CXCR7 siRNA did not affect the survival or proliferation of these cells. Knockdown of CXCR7, but not CXCR4, induced the upregulation of CXCL12 mRNA expression and CXCL12 production in AML cells.
CONCLUSION
CXCR7 is involved in the regulation of autocrine CXCL12 in AML cells.

Keyword

Acute myeloid leukemia; Apoptosis; Cell proliferation; Stromal cell-derived factor-1; CXCL12; CXCR7

MeSH Terms

Anoxia
Apoptosis
Blotting, Western
Cell Line
Cell Proliferation
Cytokines
Cytoplasm
Granulocytes
Humans
Intercellular Signaling Peptides and Proteins
Interleukin-3
Interleukin-6
Leukemia, Myeloid, Acute*
Necrosis
RNA, Messenger
RNA, Small Interfering
Stem Cells
Transfection
Up-Regulation
Cytokines
Intercellular Signaling Peptides and Proteins
Interleukin-3
Interleukin-6
RNA, Messenger
RNA, Small Interfering

Figure

• 

  Fig. 1 Acute myeloid leukemia (AML) cells express and produce C-X-C chemokine receptor 7 (CXCR7). (A) Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in AML cell lines. (B) Western blot analysis in AML cell lines. (C) Flow cytometric analysis for CXCR7 in AML cell lines. (D) Flow cytometric analysis after dual staining of CXCR4 and CXCR7 in AML cell lines. (E) Flow cytometric analysis for CXCR7 in CD34+ cells obtained from the bone marrow of 5 patients with AML. (F) Immunofluorescence staining after permeabilization for CXCR4 and CXCR7 in U937 cells.Abbreviations: CXCL12, C-X-C motif ligand 12; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 2 C-X-C motif ligand 12 (CXCL12) induces internalization of cell surface C-X-C chemokine receptor 7 (CXCR7) in acute myeloid leukemia (AML) cells. Flow cytometric analysis for cell surface CXCR7 in U937 cells before and after a 2-hr incubation with CXCL12 (200 ng/mL).

• 

  Fig. 3 Cytokines and hypoxia do not alter C-X-C chemokine receptor 7 (CXCR7) expression in acute myeloid leukemia (AML) cells. (A) AML cell lines were treated with cytokines at various concentrations for up to 72 hr and then subjected to flow cytometric analysis. Representative results (upper panel) and fold-increase of mean fluorescence intensity (MFI) of the experimental cells from that in the controls (no cytokine treatment) (lower panel) from 3 independent experiments, in which U937 cells were treated with 10 µg of each cytokine for 24 hr, are shown. Cells were incubated with 5% CO2 and 1% O2, balanced with N2 gas, at 37℃ for up to 12 hr, and then subjected to flow cytometric analysis (B) and western blot analysis (C).Abbreviations: IL-6, interleukin 6; GM-SCF, granulocyte-macrophage colony-stimulating factor; TGF-β, tumor growth factor-β; INF-γ, interferon-γ; TNF-α, tumor necrosis factor-α; G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor; TPO, thrombopoietin; Con, control.

• 

  Fig. 4 C-X-C chemokine receptor 7 (CXCR7) does not affect migration, serum-deprivation-induced apoptosis, or spontaneous proliferation of acute myeloid leukemia (AML) cells. Using small interfering RNA (siRNA) technology, CXCR4 and CXCR7 were knocked down in MO7e and U937 cells and subsequent biological alterations occurring in the cells were evaluated. (A, B) Transmembrane migration of the cells. Data are expressed as mean±SD of the percentages of migrated cells. (C, D) Data are expressed as mean±SD of the Annexin V-positive apoptotic cells analyzed using flow cytometry. (E, F) Data are expressed as mean±SD of the relative proliferation index at indicated time. All data are from 3 independent experiments.

• 

  Fig. 5 Knock-down of C-X-C chemokine receptor 7 (CXCR7) upregulates C-X-C motif ligand 12 (CXCL12) expression in acute myeloid leukemia (AML) cells. Using small interfering RNA (siRNA) technology, CXCR4 and CXCR7 were knocked down in U937 cells and then subjected to quantitative reverse transcription polymerase chain reaction (RT-PCR) (A, B) and western blot analysis (C, D). Data are expressed as mean±SD of 3 independent experiments.

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