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Fibroblast Growth Factor-2 Stimulates Proliferation of Human Adipose-Derived Stem Cells via Src Activation

Background: Human adipose-derived stem cells (hASCs) is a subset of mesenchymal stem cells (MSCs), it has been regarded as one of the most promising stem cells. Fibroblast growth factor-2 (FGF-2) is one member of FGF families, which has multifunctions such as mitogenesis, migration, morphogenesis, angiogenesis, organ development, organ regeneration, and wound healing. The FGF-2 pathway is one of the most significant regulators in proliferation of human embryonic stem cell (hESC) and oncogenesis of tumor. Our previous study found that FGF-2 induced the proliferation and differentiation of hASC. However the signaling pathways of hASCs activated by FGF-2 remain unclear. Our present study aims to found part of signaling pathways involve in FGF-2 mediated proliferation.

Methods: hASCs were cultured with different concentration of FGF-2 (0, 1, 5, 10 and 20 ng/ml), and assessed by WST-8 assay. We assessed FGF Receptor (FGFR) inhibitor (NVP-BGJ398) effect on the proliferation of hASCs via cell cycle test and WST-8. Various protein kinases such as ERK1/2 inhibitor (PD98059), PI3K/Akt inhibitor (LY294002), JNK inhibitor (SP600125), and p38 MAPK inhibitor (SB203580) and Src inhibitor (PP1) effect on the proliferation of hASCs was assessed by WST-8. At the same time, we assessed the effect of FGFR inhibitor on different signaling pathway such as ERK1/2, JNK, p38, Src, MEK1/2, and Akt, and the effect of Src inhibitor on signaling pathway such as Src, MEK1/2 in protein level.

Result: The proliferation of hASCs was increased by treatment with 1 ng/ml FGF-2 (0.01 < P < 0.05 vs control) and 5 ng/ml FGF-2 stimulated cell proliferation to a greater extent (P < 0.01 vs control), NVP-BGJ398 (0.05/0.1/1 uM) and various signaling pathway inhibitors (PD98059, 5 uM, SP600125, 10 uM, SB203580, 20 uM, LY294002, 10 uM, PP1 1/2/5 uM) suppressed this phenomenon (P < 0.01 vs control). The result of flow cytometry showed the percent of cells treated with FGF-2 in the S and G2/M phase (24.56 ± 0.65% and 4.20 ± 0.32%, respectively) was significantly higher than control (16.26 ± 0.47% and 2.02 ± 0.23%, respectively) (P < 0.01 vs control) while the percent of cells treated with FGF-2 plus NVP-BGJ398 (1 uM) in the S and G2/M phases (11.4 ± 1.43% and 0.96 ± 0.34%, respectively) was also significantly different from control (16.26 ± 0.47% and 2.02 ± 0.23%, respectively) (P < 0.01 vs control). The phosphorylation of Erk, JNK, p38 MAPK, and Akt in FGF-2 treated cells increased, however NVP-BGJ398 inhibited the phosphorylation of these enzymes even in the presence of FGF-2. The level of p-Src and p-MEK was increased by FGF-2 mediated proliferation, and NVP-BGJ398 and PP1 (5 uM) inhibited the phosphorylation induced by FGF-2.

Conclusion: FGF-2 at concentrations lower than 10 ng/ml enhanced hASCs proliferation. FGF-2 effects the proliferation of hASCs was mediated by different signaling pathways. Src activation can be essential for the activation of down-stream multiple pathways in FGF-2-mediated proliferation on ASCs.