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The cellular mechanisms by which articular cartilage responds to load are poorly understood, but such responses may involve regulation at the level of protein translation rather than synthesis of mRNA. We investigated the role of translational control in cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz) and statically loaded porcine articular cartilage explants. Messenger RNA was extracted for real time polymerase chain reaction (RT-PCR) and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase), eIF2a (eukaryotic initiation factor 2a), eEFs (eukaryotic elongation factors), and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF) receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. These results show that translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load.

Original publication

DOI

10.22203/ecm.v022a14

Type

Journal article

Journal

Eur cell mater

Publication Date

20/09/2011

Volume

22

Pages

178 - 189

Keywords

Animals, Cartilage, Articular, Cells, Cultured, Chondrocytes, Elongation Factor 2 Kinase, Extracellular Matrix Proteins, Intracellular Signaling Peptides and Proteins, Phosphoinositide-3 Kinase Inhibitors, Phosphorylation, Protein Biosynthesis, RNA, Messenger, Real-Time Polymerase Chain Reaction, Receptors, Fibroblast Growth Factor, Signal Transduction, Stress, Mechanical, Swine, eIF-2 Kinase