Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (19): 2959-2964.doi: 10.3969/j.issn.2095-4344.1240

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Lysophosphatidic acid affects the expression of connective tissue growth factor  in osteoblasts

Hu Guang, Zhang Kaiwei, Xu Yuankun   

  1.  (Department of Orthopedics, the First Affiliated Hospital of Guiyang University of Chinese Medicine, Guiyang 550001, Guizhou Province, China)
  • Received:2019-01-31 Online:2019-07-08 Published:2019-07-08
  • Contact: Zhang Kaiwei, MD, Chief physician, Department of Orthopedics, the First Affiliated Hospital of Guiyang University of Chinese Medicine, Guiyang 550001, Guizhou Province, China
  • About author:Hu Guang, Master, Associate chief physician, Department of Orthopedics, the First Affiliated Hospital of Guiyang University of Chinese Medicine, Guiyang 550001, Guizhou Province, China

Abstract:

BACKGROUND: Lysophosphatidic acid stimulates the expression of connective tissue growth factor 2 in 
epithelial and myoblast cells, but the molecular mechanisms involved are still unclear. Osteoblasts are important cells in bone development and fracture healing, the expression of the lysophosphatidic acid receptor, whereas fracture local high expression of lysophosphatidic acid and connective tissue growth factor 2 in primary osteoblasts and fracture healing.
OBJECTIVE: To investigate the molecular mechanism in which lysophosphatidic acid stimulates the expression of connective tissue growth factor 2 in osteoblasts.
METHODS: (1) MC3T3-E1 cells were cultivated in vitro, cells were divided into control (mineralized solution) and experimental (mineralized solution plus 20 μmol/L lysophosphatidic acid) groups for 0.5, 1, 2, 4, 6 and 8 and 12 hours. Then, total RNAs were extracted and the expression of connective tissue growth factor 2 was tested. (2) MC3T3-E1 cells were pretreated by protein kinase A inhibitor H-89 and protein kinase C inhibitor staurosporine for 0.5 hours, followed by adding lysophosphatidic acid. The expression levels of connective tissue growth factor 2 mRNA and protein were detected by quantitative PCR and western blot assay. The translocation of protein kinase C was detected by western blot and immunofluorescence.
RESULTS AND CONCLUSION: (1) Lysophosphatidic acid could trigger connective tissue growth factor 2 mRNA and protein expression transiently in MC3T3-E1 cells, which peaked at 2 hours after stimulation, followed by a decrease, and restored to the baseline level at 8 and 12 hours. (2) Staurosporine could reduce the ability of lysophosphatidic acid stimulating connective tissue growth factor 2 in osteoblasts, while H-89 increased the ability of lysophosphatidic acid stimulating connective tissue growth factor 2 in osteoblasts. (3) The amount protein kinase C expressed in the cell membrane in MC3T3-E1 cells treated with lysophosphatidic acid was increased significantly. At the same time, the amount of protein kinase C expressed in cytoplasm was reduced. (4) To conclude, lysophosphatidic acid induces the expression of connective tissue growth factor may be via protein kinase A and C pathways.

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松组织工程

Key words: lysophosphatidic acid, fracture healing, osteoblasts, osteogenesis, connective tissue growth factor, protein kinase A, protein kinase C, bone defect

CLC Number: