Background/Purpose: Previous studies have shown that activation of the chondrocyte PI-3K-Akt signaling pathway by IGF-1 promotes chondrocyte survival and matrix synthesis. However, other studies have shown activation of this pathway by cytokines such IL-1 plus oncostatin M (OSM) can promote chondrocyte MMP production. Based on the latter studies, some investigators have proposed targeting this pathway for treatment of OA. The purpose of the present study was to clarify the function of this important signaling pathway in chondrocytes by directly comparing the effects of anabolic and catabolic stimuli.    

Methods: Human articular chondrocytes were isolated from normal adult ankle (tali) and knee articular cartilage and from knee cartilage removed at the time of joint replacement for OA (n=3 donors each for normal and OA). Primary confluent cultures were made serum-free and stimulated with IGF-1 (100ng/ml), IL-1β (0.02ng/ml), OSM (10ng/ml) or IL-1β+OSM (0.02ng/ml+10ng/ml). The doses of IL-1 and OSM were based on previous studies demonstrating increased MMP production stimulated by the combination of the two. Stimulus independent activation of Akt was achieved by lentiviral infection of constitutively active (CA) Akt. Activation of signaling proteins was measured at 30 minutes by immunoblotting of cell lysates and MMP-13 production was measured in conditioned media after overnight stimulation. Immunoblots were quantified by densitometry and normalized to total Akt and MMP-2 respectively. Proteoglycan (PG) synthesis was measured by sulfate incorporation and collagen production by ELISA and results normalized to cell numbers.

Results: In normal chondrocytes (ankle or knee), IGF-1 stimulated strong Akt phosphorylation while IL-1β did not stimulate pAkt and the others were weak (OSM 23% and IL-1β+OSM 24% of IGF-1 stimulation). The pattern was similar in OA cells except IGF stimulated pAkt was less than in normal. Production of MMP-13 was stimulated in normal cells by OSM and IL-1β+OSM and in OA cells by IL-1β+OSM but not by either cytokine alone. Despite strong pAkt, IGF-1 did not stimulate MMP-13 production. Inhibition of PI-3 kinase by LY294002 (5μM) inhibited by 88% the OA cell production of MMP-13 by IL-1β +OSM and completely inhibited normal chondrocyte production of MMP-13. Overexpression of CA-Akt markedly stimulated PG and collagen synthesis but did not increase MMP-13 production. Inhibition of Akt1 and Akt2 using Akt inhibitor XIII (1μM) did not affect ILβ +OSM stimulated MMP-13 production.  

Conclusion: The function of the PI-3 kinase-Akt pathway in articular chondrocytes is stimulus dependent. IGF-1 stimulation of this pathway is pro-anabolic and activity of Akt alone is sufficient for this effect. IL-1β+OSM weakly activate Akt phosphorylation and Akt activation alone is not sufficient to induce MMP production. The inhibition of IL-1β+OSM induced MMP-13 by the PI-3K inhibitor suggests that PI3K acts in concert with additional pathways, such as the MAP kinases, to stimulate MMP-13 production. Inhibition of these pathways, rather than PI-3K-Akt would be a better therapeutic target given the pro-anabolic and survival functions of Akt.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/function-of-the-chondrocyte-pi-3-kinase-akt-signaling-pathway-is-stimulus-dependent/