Effect of Interleukin-1β on Osteogenic Protein 1–Induced Signaling in Adult Human Articular Chondrocytes

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Objective.Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1β (IL-1β)–induced cartilage degeneration to investigate the effect of IL-1β on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes.Methods.Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1β on BMP receptors was studied by reverse transcription–polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1β (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1β and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1β activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38).Results.IL-1β reduced the number of activin receptor–like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1–mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1β offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38.Conclusion.These findings describe new mechanisms of the crosstalk between OP-1 and IL-1β in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes.

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