Proteomic analysis of early‐response to mechanical stress in neonatal rat mandibular condylar chondrocytes

Abstract

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.

Endoplasmic Reticulum Stress Regulates Rat Mandibular Cartilage Thinning under Compressive Mechanical Stress

“…Furthermore, many studies have indicated that cartilage thinning is important in the pathogenesis of TMJ diseases (14, 28 -31). Teramoto et al (14) demonstrated a significant decrease in the proliferation of condylar cartilage and the amount of extracellular matrix under compressive force, and we also previously demonstrated reduced proliferation of mandibular chondrocytes in response to mechanical stress in vitro (11). The results of the present study are consistent with these studies.…”

“…This approach has been successfully utilized to examine chondrocyte differentiation and cartilage pathophysiology (9,10). Our previous study of the response of mandibular chondrocytes to mechanical stress in vitro demonstrated a transient but inhibitory effect on cell cycle progression, accompanied by cytoskeleton remodeling and MAPK pathway activation (11). However, the molecular mechanisms regulating mandibular cartilage responses to mechanical stress in vivo have not been thoroughly investigated, and the determinants mediating cartilage loss induced through mechanical stress remain largely unknown.…”

YAP and ERK mediated mechanical strain‐induced cell cycle progression through RhoA and cytoskeletal dynamics in rat growth plate chondrocytes

“…Cyclic tensile strain (CTS) was applied to growth plate chondrocytes plated on culture plates by using the four-point bending system which has been characterized and described in detail elsewhere. 26 In brief, when cells had reached 80% confluence, the medium was changed to serum-free media and cultured for overnight, and the cells were exposed to CTS at a frequency of 0.5 Hz. The unstrained control group was cultured on similar plates and kept in the same incubator for the same time period.…”

The PI3K/Akt signalling pathway may play an internal role related to abnormal condylar growth: a preliminary study

“…21 Under aseptic conditions, the cartilage was removed from the head of the condyle. In brief, the condylar cartilage was minced and digested with 0.25% trypsin and 0.2% collagenase, and then the chondrocytes were rinsed three times and prepared as a single cell suspension in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1% L-glutamine, and 100 mg/ml kanamycin.…”