Korean J Hematol.  2007 Mar;42(1):24-32. 10.5045/kjh.2007.42.1.24.

Role of Pertussis Toxin-sensitive G Protein-coupled Receptor Signaling in the Proliferation of Bone Marrow Mesenchymal Stem Cells

• Kim SW
• Hwang JH
• Jin SA
• Yun GW
• Yang YJ
• Park NW
• Lee HJ
• Yun HJ
• Jo DY
• Kim S

• KMID: 2252265
• DOI: http://doi.org/10.5045/kjh.2007.42.1.24

Abstract

BACKGROUND: Bone marrow (BM) mesenchymal stem cells (MSCs) can be expanded over 20~30 cell doublings in vitro even in the absence of any growth factors. However, the mechanisms that govern MSC proliferation are not well understood.
METHODS
We investigated the role of signaling of the pertussis toxin (PTX)-sensitive G protein-coupled receptor in the proliferation of BM MSCs.
RESULTS
PTX inhibited the proliferation of human BM MSCs and murine BM stromal MS-5 cells in a dose-dependent manner. Among the chemokines produced by the BM stromal cells, stromal cell-derived factor-1 (SDF-1) enhanced the proliferation of BM MSCs, while MIP-1alpha, MCP-3 or RANTES did not. PTX also inhibited the proliferation of some fibroblasts, such as MRC-5 and NIH-3T3, but did not affect the proliferation of HeLa and HSF cells. HSF cells did not express CXCR4 mRNA, but did produce SDF-1. In contrast, HeLa cells expressed CXCR4 strongly on the cell surface, but did not produce SDF-1. BM MSCs, MS-5, MRC-5, and NIH-3T3 cells all expressed CXCR4 minimally on the cell surface. These cells, however, had abundant CXCR4 protein in their cytoplasm, which was demonstrated by flow cytometric analysis performed after permeabilization of the cells. In addition, an ELISA performed on the culture supernatants of the cells revealed that these cells constitutively produce and secrete SDF-1.
CONCLUSION
These results indicate that the signaling through the PTX-sensitive G protein-coupled receptor, which is induced by autocrine factors, plays an important role in the proliferation of BM MSCs and in some fibroblasts, and that SDF-1 is the most probable candidate for the autocrine growth factor.

Keyword

Bone marrow; Mesenchymal stem cells; G protein-coupled receptor; Pertussis toxin; Stromal cell-derived factor-1; Cell proliferation

MeSH Terms

Bone Marrow*
Cell Proliferation
Chemokine CCL3
Chemokine CCL5
Chemokines
Cytoplasm
Enzyme-Linked Immunosorbent Assay
Fibroblasts
HeLa Cells
Humans
Intercellular Signaling Peptides and Proteins
Mesenchymal Stromal Cells*
NIH 3T3 Cells
Pertussis Toxin
RNA, Messenger
Stromal Cells
Whooping Cough*
Chemokine CCL3
Chemokine CCL5
Chemokines
Intercellular Signaling Peptides and Proteins
Pertussis Toxin
RNA, Messenger

Figure

• 

  Fig. 1 Pertussis toxin (PTX) inhibits the proliferation of human bone marrow mesenchymal stem cells (MSCs) in a dose-dependent manner. MSCs were incubated in IMDM (A) or IMDM supplemented with 10% FBS (B) in the absence (control) or presence of PTX (0.2～200ng/mL) or SDF-1 (100ng/mL). After 3 days, the cultures were observed under the phase contrast microscope. A representative result of two independent experiments is shown.

• 

  Fig. 2 Effects of pertussis toxin (PTX) on the apoptosis of human bone marrow mesenchymal stem cells (MSCs). MSCs were incubated in serum-free X-VIVO medium in the absence (A) or presence (B) of PTX (200ng/mL). After 24 hours of incubation, the cells were stained with propium iodide (PI) and annexin V, and then subjected to a flow cytometric analysis. A representative result of three independent experiments is shown.

• 

  Fig. 3 SDF-1, but not other chemokines, enhances the proliferation of human bone marrow mesenchymal stem cells (MSCs). MSCs were incubated in serum-free medium X-VIVO in the absence (control) or presence of various chemokines (100ng/mL) for 3 days. Data are the mean and S.D. of number of the cells expressed in percentage of control from three independent experiments. ∗P＜0.05.

• 

  Fig. 4 Pertussis toxin (PTX) inhibits the proliferation not only of human bone marrow mesenchymal stem cells (BM MSC) (C) but also of other several fibroblasts (A, B, D) in a dose-dependent manner. Cells were incubated in serum-free medium X-VIVO in the absence (control) or presence of PTX (0.2～200ng/mL) or SDF-1 (100ng/mL) for up to 4 days. A representative result of two independent experiments is shown.

• 

  Fig. 5 Pertussis toxin (PTX) does not inhibit the proliferation of HSF and HeLa cells. Cells were incubated in serum-free medium X-VIVO in the absence (control) or presence of PTX (200ng/mL) for 4 days. Data are the mean and S.D. of number of the cells expressed in percentage of control from three independent experiments. ∗P＜0.05.

• 

  Fig. 6 Expression of CXCR4 and production SDF-1 in bone marrow mesenchymal stem cells (MSCs) and some fibroblasts. (A) Expression of mRNA for SDF-1 and CXCR4 analyzed by RT-PCR in various cell types. (B) Concentrations of SDF-1 in the supernatants of three-day culture of the cells analyzed by ELISA. (C) Cell surface expression CXCR4 in human MSCs analyzed by flow cytometry. (D) Flow cytometric analysis on CXCR4 expression in various cell types, before and after permeabilization.

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