Traumatic joint injury is a significant cause of osteoarthritis. At the cellular level, injurious mechanical compression to articular cartilage leads to decreased proteoglycan synthesis and increased chondrocyte proliferation. The molecular pathways leading to these biochemical and cellular changes remain poorly understood. We explored the role of the ERK 1/2 pathway following injurious mechanical compression to bovine articular cartilage explants. Specifically, we investigated the involvement of the ERK 1/2 pathway in chondrocyte proliferation and proteoglycan synthesis.
Methods Following explantation and processing, bovine articular cartilage explants were cultured with and without the ERK 1/2 pathway inhibitor PD98059. Explants were then statically loaded to 40% strain at a strain rate of 1-sec to produce peak stresses of ˜35 MPa. Loading was maintained for 5 seconds to simulate an acute injurious event. Control explants were cultured under similar conditions but were not loaded. Thus, four experimental groups were created; 1) no load without inhibitor, 2) loaded without inhibitor, 3) no load with the inhibitor PD98059, and 4) loaded with the inhibitor PD98059. Explants were cultured for 0-120 hours after loading. Explants were then analyzed both biochemically and immunohistochemicaly. Western blot was used to determine the presence of phospho-ERK 1/2 at selected time points. 35SO4 incorporation was utilized as a measure of proteoglycan biosynthesis. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) was used as a marker of cellular proliferation
Results Injurious mechanical compression induced activation of the ERK 1/2 pathway via phosphorylation, with activation beginning at 30 minutes and returning to baseline at 48 hours. This activation was attenuated with the ERK 1/2 pathway inhibitor PD98059. Loading of explants resulted in increased chondrocyte proliferation and decreased proteoglycan biosynthesis. Addition of PD98059 to the culture medium of loaded explants led to significantly less chondrocyte proliferation following loading. This was observed at all time points. Furthermore, with application of inhibitor, the decrease in proteoglycan synthesis seen following loading was reversed.
Conclusions The ERK 1/2 pathway is an integral component of the chondrocyte proliferative response and proteoglycan biosynthetic response following acute mechanical injury to articular cartilage.