Mechanical signals upregulate articular chondrocyte (AC) proliferation via SOX-9, VEGF, and c-Myc mRNA expression and ERK1/2 activation. ACs were exposed to no treatment or to treatment with interleukin-1-beta (IL-1β), dynamic strain (DS) alone, or DS and IL-1β. Subsequently, ACs were subjected to DS for 90 minutes per day for 3 days. (a) On day 4, the rate of cell proliferation was assessed by MTT assay. ACs were treated either with medium alone or with PD98059 (2 μM) for 30 minutes. Cells were exposed to the treatment regimens above for 3 hours, and the mRNA expression for c-Myc (b), SOX-9 (c), and VEGF (d) was analyzed by real-time polymerase chain reaction. (e) Western blot analysis showing ERK1/2 phosphorylation using phospho-Thr202/Tyr204 ERK1/2 (P-ERK1/2) and total ERK1/2 (T-ERK1/2) antibodies. (f) Immunofluorescence analysis showing minimal phospho-ERK1/2 in control cells [a], cells stained with secondary antibody alone [b], optimal phosphorylation of ERK1/2 and its nuclear translocation in response to IL-1β at 10 and 30 minutes [c,d], and nuclear translocation and cytoplasmic redistribution of p-ERK1/2 in response to DS in the absence [e,f] and presence [g,h] of IL-1β. Cells were counterstained with fluorescein isothiocyanate-phalloidin to show β-actin. Experiments in (a,c-e) were performed in triplicate and were repeated three times in (b) and two times in (f). The error bars represent standard error of the mean (standard error of the mean in a-d). Gels in (e) represent one of three experiments with similar results. Φ P < 0.05 as compared with untreated controls; *P < 0.05 as compared with cells treated with DS or with DS and IL-1. C, control; Cont, control; ERK1/2, extracellular receptor kinase 1/2; MTT, 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; SOX-9, SRY-related protein-9; VEGF, vascular endothelial cell growth factor.
Perera et al. Arthritis Research & Therapy 2010 12:R106 doi:10.1186/ar3039