Abstract
BackgroundIn osteoarthritis, chondrocytes adopt an abnormal hypertrophic morphology and upregulate the expression of the extracellular matrix-degrading enzymes, MMP-13 and ADAMTS-5. The activation of the hedgehog signalling pathway has been established in osteoarthritis and is thought to influence both of these processes. However, the role of this pathway in the initiation and progression of osteoarthritis is unclear as previous studies have been unable to isolate the effects of hedgehog pathway activation from other pathological processes. In this study we test the hypothesis that hedgehog pathway activation causes cartilage degradation in healthy cartilage and in an in vitro model of inflammatory arthritis.MethodsIsolated articular chondrocytes from the bovine metacarpal-phalangeal joint were stimulated for up to 24 hours with the agonist, recombinant Indian hedgehog (r-Ihh). ADAMTS-5 and MMP-13 gene expression was quantified by real-time PCR. In addition, healthy bovine cartilage explants were treated with r-Ihh or the hedgehog antagonist, cyclopamine, and sGAG release into the media was measured over 72 hours. Studies were repeated using chondrocytes and cartilage explants from human knee joint. Finally, studies were conducted to determine the effect of hedgehog pathway activation on matrix catabolism in the presence of the pro-inflammatory cytokine, IL-1β.ResultsAddition of r-Ihh activated hedgehog signalling, confirmed by upregulation of Gli1 and Ptch1 expression, but did not increase ADAMTS-5 or MMP-13 expression in bovine or human chondrocytes. Furthermore, r-Ihh did not induce sGAG release in healthy bovine or human cartilage explants. IL-1β treatment induced sGAG release, but this response was not altered by the stimulation or inhibition of hedgehog signalling. Hedgehog pathway activation was downregulated by IL-1β. Conversely, r-Ihh weakly suppressed IL-1β-induced ADAMTS-5 expression.ConclusionOur results show for the first time that Indian hedgehog does not cause extracellular matrix degradation in healthy ex vivo cartilage or in the presence of IL-1β and that IL-1β downregulates Indian hedgehog induced signalling. Thus, we suggest reported hedgehog induced matrix catabolism in osteoarthritis must be due to its interaction with pathological factors other than IL-1β. Hence, hedgehog signalling and its downstream effects are highly context-dependent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0891-z) contains supplementary material, which is available to authorized users.
Highlights
In osteoarthritis, chondrocytes adopt an abnormal hypertrophic morphology and upregulate the expression of the extracellular matrix-degrading enzymes, matrix metalloproteinase (MMP)-13 and ADAMTS-5
Hedgehog pathway modulation does not alter cartilage degradation in bovine cartilage explants The induction of mRNA does not always correlate with catabolic effects, the effect of hedgehog pathway activation on cartilage degradation was directly investigated in bovine cartilage explants
Explants were treated with r-Indian hedgehog (Ihh) for 24, 48 and 72 hrs and cartilage degradation monitored by measuring the release of sulphated glycosaminoglycan (sGAG) into the culture media (Fig. 2a)
Summary
Chondrocytes adopt an abnormal hypertrophic morphology and upregulate the expression of the extracellular matrix-degrading enzymes, MMP-13 and ADAMTS-5. In OA, chondrocytes exhibit a phenotypic shift to a more terminal growth-plate-like state, adopting a similar hypertrophic morphology and upregulating the expression of hypertrophic markers, including type X collagen, matrix metalloproteinase (MMP)-13 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 [14]. Hedgehog blockade reduces the expression of OA-related genes, including ADAMTS5 and MMP13, in mice with surgically induced OA and in human OA cartilage samples [5, 6]. Further evidence for the role of hedgehog pathway activation in OA stems from the increased expression of Indian hedgehog (Ihh) in cartilage and synovial fluid in human OA, with a 5.2-fold and a 1.71-fold increase, respectively, compared to their normal controls [4, 15]
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