Identification of signalling pathways triggered by changes in the mechanical environment in rat chondrocytes

Abstract

Aim: The aim of this work was to determine the pathways implicated in the mechanosensing of chondrocytes.

Methods: Rat chondrocytes were cultured in collagen hydrogels of different stiffness (2-20 Pa) in normoxia and hypoxia, in monolayer and embedded inside hydrogels. First, chondrocyte were cultured on hydrogels in the presence of antibodies to block integrins. Second, custom RT-PCR array plates and western blot were used to detect changes in expression of genes implicated in downstream signalling pathways.

Results: The results allowed us to demonstrate the mechanosensing of chondrocytes for changes in stiffness in the range of Pascals. We also identified Non-Muscle Myosin II (NMMII) and integrins α1, β1 and β3 as participants in the mechanosensing, since their blockade inhibits the sensing of the stiffness, and they are up-regulated in the process. RT-PCR arrays and western blot detected up-regulation of Paxillin, RhoA, Fos, Jun and Sox9. We detected no expression of Src in the monolayer cultures, but we found a role for this protein in 3D. The expression of HIF-1α was not modified under normoxia but was found to participate under hypoxia. Focal Adhesion Kinase (FAK), showed a direct relationship with the expression of Aggrecan in hypoxia and an inverse one in normoxia. Finally, immunofluorescence analysis located the expression of factors AP-1, Sox-9 and HIF-1α inside the cell nuclei and RhoA, Src, Paxillin and FAK close to the cytoplasmic membrane.

Conclusions: We determined here some of the genes that are up-regulated during the process of chondrocyte mechanosensing.