Detection of beta1 integrin activation in chondrocytes

Abstract

Purpose: Integrins are heterodimeric transmembrane receptors linking the actin cytoskeleton to the ECM. Their activity is regulated via an allosteric switch between low- and high-affinity states, induced by effector binding to the cytoplasmic tail of the α- and β-subunits or by ligand binding to the ectodomain. β1 integrin activity in chondrocytes has been shown to increase during osteoarthritis leading to increased catabolism, however β1 integrin signalling is required during chondrogenic differentiation. Integrin signalling is involved in mechanisms of cell survival, growth, differentiation and matrix remodelling, however the exact mechanisms by which β1 integrin signalling may interact with other homeostatic signalling pathways in chondrocytes remains unknown. Here, we aim to examine the expression pattern of β1 integrins within their activated state in chondrocytes. Methods: Costal chondrocytes obtained from 8 to 12-week-old male wild type (WT) mice were cultured under standard conditions. β1 integrin within an active conformation was detected by immunofluorescence staining using a Purified Rat Anti-Mouse CD29 Clone 9EG7 antibody. Activation levels were compared in chondrocytes plated for 24 hours on either fibronectin, collagen type II or uncoated glass coverslips. Chondrocytes in non-coated wells were stimulated in a time course with 1 mM Mn2+ to act as a positive control. To further investigate β1 integrin activation as part of attachment behaviour, chondrocytes were added to fibronectin coated and uncoated coverslips for 1 hour, 6 hours and 24 hours before β1 integrin activation was assessed by immunocytochemistry. Integrin-specific downstream signalling was analysed by immunoblotting of p-FAK/FAK and PKC in lysates of treated chondrocytes. Results: β1 integrins within an active conformation could be detected in wild type murine chondrocytes in vitro. The 9EG7 epitope was detected firstly at low levels in chondrocytes grown in standard conditions, however activation levels were found to increase significantly when chondrocytes were cultured on either fibronectin or type II collagen coatings. Mn2+ added to chondrocytes cultured in uncoated wells as a positive control led to increased levels of activated β1 integrin. Activated integrin β1 in chondrocytes grown on fibronectin and type II collagen exhibited a specific activation pattern, which was not found in chondrocytes grown on uncoated surfaces. Immunoblotting demonstrated that activation of β1 integrins via manganese or through interaction with RGD sequence containing ECM substrates led to increased phosphorylation of the focal adhesion kinase and protein kinase C. Conclusions: β1 integrins within an active conformation were able to be detected in in vitro cultured chondrocytes using immunocytochemistry for the 9EG7 epitope of β1 integrin. The activated state was increased both by Mn2+ and when chondrocytes were grown on either fibronectin or type II collagen substrates. This detection of β1 integrin activation state allows for the further investigation of integrin signalling within the context of interactions with homeostatic growth factor and chemokine signalling pathways.