Role of the GRP78-c-Src signaling pathway on osteoblast differentiation of periodontal ligament fibroblasts induced by cyclic mechanical stretch

Abstract

Objectives: This study aims to investigate the influence of glucose regulated protein (GRP) 78 on osteoblast differentiation in periodontal ligament fibroblasts (PDLFs) under cyclic mechanical stretch and determine the underlying mechanism.

Methods: FlexCell 5000 cell mechanical device was applied to simulate the stress environment of orthodontic teeth. GRP78High and GRP78Low subpopulation were obtained by flow sorting. Gene transfection was performed to knockdown GRP78 and c-Src expression and overexpress c-Src. Western blot analysis was used to detect the protein expression of Runt-related gene 2 (RUNX2), Osterix, osteocalcin (OCN), and osteopontin (OPN). Immunoprecipitation assay was used to determine the interaction of GRP78 with c-Src. The formation of cellular mineralized nodules was determined by alizarin red staining.

Results: GRP78 was heterogeneously expressed in PDLFs, and GRP78High and GRP78Low subpopulations were obtained by flow sorting. The osteogenic differentiation ability and phosphorylation level of c-Src kinase in the GRP78High subpopulation were significantly increased compared with those in GRP78Low subpopulation after cyclic mechanical stretch (P<0.05). GRP78 interacted with c-Src in PDLFs. The overexpression c-Src group showed significantly increased osteogenic differentiation ability than the vector group (P<0.05), and the sic-Src group showed significantly decreased osteogenic differentiation ability (P<0.05) after cyclic mechanical stretch.

Conclusions: GRP78 upregulates c-Src expression by interacting with c-Src kinase and promotes osteogenic differentiation under cyclic mechanical stretch in PDLFs.

Keywords: cyclic mechanical stretch; glucose regulated protein 78; osteogenic differentiation; periodontal ligament fibroblasts.

图 1. PDLFs的鉴定 免疫荧光染色 × 400

Fig 1 Identification of PDLFs immunofluorescence staining × 400 A：角蛋白染色阴性；B：波形蛋白染色阳性；C：成纤维细胞特异性蛋白1染色阳性。

图 2. 周期性牵张力对PDLFs成骨分化的影响

Fig 2 Effect of cyclic mechanical stretch on osteogenic differentiation of PDLFs A：ALP活性检测；B：蛋白质印迹实验RUNX2、Osterix、OPN、OCN蛋白表达；C：ALP染色 倒置荧光显微镜 × 200；D：茜素红染色 倒置荧光显微镜 × 200。*P<0.05，**P<0.01。

图 3. 获取PDLFs GRP78High细胞和GRP78Low细胞

Fig 3 Obtained GRP78High subpopulation and GRP78Low subpopulation in PDLFs A：PDLFs转染红色绿色双荧光启动子质粒 倒置荧光显微镜 × 200；B：流式细胞术细胞分选；C：分选后鉴定GRP78蛋白表达。

图 4. GRP78High细胞成骨分化能力高于GRP78Low细胞

Fig 4 GRP78High subpopulation demonstrated superior osteogenic potential compared with GRP78Low subpopulation A：ALP染色 倒置荧光显微镜 × 200；B：茜素红染色 倒置荧光显微镜 × 200；C：蛋白质印迹实验RUNX2、Osterix、OPN、OCN蛋白表达。*P<0.05。

图 5. GRP78对c-Src激酶磷酸化水平的影响

Fig 5 The effect of GRP78 on the phosphorylation Levels of the c-Src kinase A： c-Src蛋白表达；B：抑制GRP78表达对c-Src蛋白表达的影响。*P<0.05，**P<0.01。

图 6. 免疫共沉淀实验检测GRP78与c-Src相互作用

Fig 6 Co-immunoprecipitation assay was applied for detecting the interaction between GRP78 and c-Src input为阳性对照；IP:IgG为同型对照（阴性对照），免疫沉淀IgG抗体；IP:GRP78为免疫沉淀GRP78抗体。

图 7. 过表达c-Src对GRP78Low细胞成骨分化的影响

Fig 7 Effect of overexpression of c-Src on osteogenic differentiation of GRP78Low subpopulation A：GRP78Low细胞过表达c-Src；B：蛋白质印迹实验检测RUNX2、Osterix、OPN、OCN蛋白表达；C：ALP染色 倒置荧光显微镜 × 200；D：茜素红染色 倒置荧光显微镜 × 200。*P<0.05，**P<0.01。

图 8. 沉默c-Src对GRP78High细胞成骨分化的影响

Fig 8 Effect of knockdown c-Src on osteogenic differentiation of GRP78High subpopulation A：GRP78High细胞敲减c-Src；B：蛋白质印迹实验检测RUNX2、Osterix、OPN、OCN蛋白表达；C：ALP染色 倒置荧光显微镜 × 200；D：茜素红染色 倒置荧光显微镜 × 200。*P<0.05，**P<0.01。