Zhiyi Hu1, Ning Zhang, Guoyong Yin. 1. Department of Orthopaedics, the First Affiliated Hospital of Nanjing Medical University, Nanjing Jiangsu, 210029, PR China.
Abstract
OBJECTIVE: To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2) localization in osteoblasts. METHODS: Primary osteoblasts were isolated and cultured from cranial bone of 10 mice at the age of 3 days, weighting 6-9 g without limitation in male and female. The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 micromol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation. The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton induced by PDGF. RESULTS: Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibited. CONCLUSION: Src activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.
OBJECTIVE: To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signal-regulated kinase 1/2 (pERK1/2) localization in osteoblasts. METHODS: Primary osteoblasts were isolated and cultured from cranial bone of 10 mice at the age of 3 days, weighting 6-9 g without limitation in male and female. The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroups according to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 micromol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation. The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton induced by PDGF. RESULTS: Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibited. CONCLUSION:Src activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.