Proteomic analysis of early-response to mechanical stress in neonatal rat mandibular condylar chondrocytes.
The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four-point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 microstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2-DE and MALDI-TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling.
Li
Huang
H
Stomatological Hospital and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China. lihuang76@nju.edu.cn
Yang
Han-Shuo
HS
Wu
Tuo-Jiang
TJ
Zhang
Xiang-Yu
XY
Jiang
Wen-Hui
WH
Ma
Qiao-Lin
QL
Chen
Yang-Xi
YX
Xu
Yun
Y
Li
Song
S
Hua
Zi-Chun
ZC
D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002453:Cell Cycle; D048430:Cell Shape; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D003599:Cytoskeleton / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D008335:Mandibular Condyle / Q000166:cytology*; D013058:Mass Spectrometry; D008954:Models, Biological; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013314:Stress, Mechanical; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D014746:Vimentin; D048049:Extracellular Signal-Regulated MAP Kinases