Transforming growth factor-β dampens interleukin-6 signaling in chondrocytes by decreasing the interleukin-6 receptor

Abstract

Purpose: Interleukin-6 (IL-6) is a pro-inflammatory cytokine present in increased levels in serum and synovial fluid from patients with osteoarthritis (OA) and is even considered to be predictive for disease progression. Previous studies implicate IL-6 in articular cartilage destruction, which is the main hallmark of OA. In contrast, transforming growth factor-β (TGF-β) is an important homeostatic regulator of cartilage that can decrease the catabolic effects of pro-inflammatory cytokines in chondrocytes. Multiple mechanisms of cross-talk between TGF-β and IL-6 are reported in other tissues. In this study, we investigated whether TGF-β is able to control IL-6 signaling in chondrocytes. Methods: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with rhTGF-β1 (1.0 ng/ml). We determined mRNA expression levels of IL-6 and IL-6 receptor (IL-6R) using qPCR, measured IL-6 protein release using Luminex multianalyte technology and IL-6R protein levels by Western Blot. rhIL-1β was used as a positive control for IL-6 induction. Induction of signal transducer and activator of transcription (STAT)3 phosphorylation by TGF-β was determined using Western Blot, in presence of the IL-6R inhibitor Tocilizumab to study IL-6R dependency. Induction of suppressor of cytokine signaling (SOCS)3 gene expression was used as a read-out for IL-6/STAT3 mediated transcriptional activity. Results: TGF-β increased both IL-6 mRNA expression and protein release in G6 and primary chondrocytes. Moreover, we observed activation of the main IL-6R downstream signaling protein STAT3 after TGF-β exposure, which could be completely prevented by blockade of the IL-6R, suggesting IL-6 dependency. We further explored whether TGF-β mediated IL-6/p-STAT3 signaling resulted in activation of target genes, and measured expression of SOCS3, which is a well-known IL-6 target gene as well as an important regulator of IL-6 signaling. Surprisingly however, TGF-β did not increase SOCS3 expression, despite the increased IL-6 and p-STAT3 levels we observed in these cells. This unexpected finding suggests regulation of STAT3 mediated transcriptional activity by TGF-β. The effect of exogenously added rhIL-6 was also inhibited by TGF-β, as 6h pre-treatment with TGF-β prevented rhIL-6-induced SOCS3 expression. At the level of STAT3, TGF-β decreased the response to rhIL-6 as well, although p-STAT3 levels were not reduced to baseline level. This points towards a crucial role for TGF-β in the regulation of IL-6 effects upstream of STAT3. Interestingly, we observed decreased expression of the IL-6R in both primary and G6 chondrocytes after exposure to TGF-β, which might explain the decreased intracellular IL-6 signaling. This effect was not dependent on IL-6 itself, as IL-1β did not affect IL-6R expression, despite being a strong inducer of IL-6. Finally, we showed that rescuing IL-6R levels using soluble IL-6R dose-dependently restored induction of SOCS3 and p-STAT3 by rhIL-6 in the presence of TGF-β. This confirms that TGF-β inhibition of IL-6 effects is indeed dependent on reduced IL-6R levels. Conclusions: Our study reveals a novel, protective effect of TGF-β that could help preserve cartilage homeostasis under inflammatory conditions by dampening IL-6 signaling via IL-6R down regulation.