Production of VEGF induced by GMCSF via ERK-NF-KB singling 'pathway in human fibroblasts during wound healing / 中华创伤杂志

ABSTRACT

ObjectiveTo observe production of vascular endothelial growth factor (VEGF) induced by granulocyte/macrophage colony-stimulating factor (GMCSF)via ERK nerve growth factor (NF)-κB singling pathway in human fibroblasts during wound healing and explore relating mechanism.MethodsHuman fibroblasts from the injured skin were used for this study and treated with GMCSF.RT-PCR was used for analyzing the protein and mRNA levels of VEGF and Western blotting was employed to determine the phosphorylation of ERK. The fibroblasts were pre-treated with ERK specific inhibitor PD98059 and further treated with GMCSF, then the fibroblasts and the supernatant were collected for detection of protein level of VEGF by means of Western blot. ERK signal pathway was inhibited to detect the activation of NF-κB by means of immunofluorescence staining. Furthermore, the nuclear and cytoplasmic extraction kit was used to separate the cytoplasm and nucleus and Western blot employed for observation of the NF-κB activation. ResultsThe mRNA level and protein level of VEGF were increased significantly with treatment with higher concentration of GMCSF in a dose-dependent manner. VEGF mRNA level was increased two hours after administration with GMCSF and reached peak at 4-6 hours. GMCSF could remarkably activate the ERK phosphorylation. Compared with GMCSF, the ERK specific inhibitor PD98059inhibited significantly the effect of GMCSF in inducing VEGF expression (P ＜ 0.05). Western blot and immunofluorescence staining analyses showed that the activation of NF-ΚB was inhibited with reduced production of VEGF after GMCSF treatment.Conclusion GMCSF up-regulates production of VEGF through activating NF-κB via ERK signal pathway in the human fibroblasts.