Department of Experimental Rheumatology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands.
Department of Experimental Rheumatology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands.
Osteoarthritis Cartilage. 2019 Aug;27(8):1197-1207. doi: 10.1016/j.joca.2019.04.014. Epub 2019 May 2.
Transforming growth factor-β (TGF-β) is an important homeostatic regulator of cartilage. In contrast, interleukin-6 (IL-6) is a pro-inflammatory cytokine implicated in cartilage degeneration. Cross-talk between TGF-β and IL-6 is reported in tissues other than articular cartilage. Here, we investigated regulation of IL-6 signaling by TGF-β in articular chondrocytes.
Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with TGF-β1 or interleukin-1β (IL-1β). Expression of IL-6 and IL-6 receptor (IL-6R) was determined on mRNA and protein level. TGF-β regulation of IL-6 signaling via phosho-STAT3 (p-STAT3) was determined using Western blot, in presence of inhibitors for IL-6R, and Janus kinase(JAK)- and activin receptor-like kinase ALK)5 kinase activity. Furthermore, induction of STAT3-responsive genes was used as a read-out for IL-6 induced gene expression.
TGF-β1 increased IL-6 mRNA and protein expression in both G6 and primary chondrocytes. Moreover, TGF-β1 stimulation clearly induced p-STAT3), which was abolished by inhibition of either IL-6R, JAK- or ALK5 kinase activity. However, TGF-β1 did not increase expression of the STAT3-responsive gene SOCS3 and pre-treatment with TGF-β1 even inhibited induction of p-STAT3 and SOCS3 by rhIL-6. Interestingly, TGF-β1 potently decreased IL-6R expression. In contrast, IL-1β did increase IL-6 levels, but did not affect IL-6R expression. Finally, addition of recombinant IL-6R abolished the inhibitory effect of TGF-β1 on IL-6-induced p-STAT3 and downstream SOCS3, BCL3, SAA1 and MMP1 expression.
In this study we show that TGF-β decreases IL-6R expression, thereby dampening IL-6 signaling in chondrocytes. This reveals a novel effect of TGF-β, possibly important to restrict pro-inflammatory IL-6 effects to preserve cartilage homeostasis.
转化生长因子-β（TGF-β）是软骨内稳态调节的重要因子。相反，白细胞介素-6（IL-6）是一种与软骨退化有关的促炎细胞因子。TGF-β 和 IL-6 之间的串扰在关节软骨以外的组织中已有报道。在这里，我们研究了 TGF-β 在关节软骨细胞中对 IL-6 信号的调节。
用 TGF-β1 或白细胞介素-1β（IL-1β）刺激人原代软骨细胞和人 G6 软骨细胞系。在存在 IL-6R 抑制剂、Janus 激酶（JAK）和激活素受体样激酶 ALK5 激酶活性抑制剂的情况下，通过 Western blot 确定 TGF-β 对 IL-6 信号的调节作用。此外，将 STAT3 反应基因的诱导用作 IL-6 诱导基因表达的读出。
TGF-β1 增加了 G6 和原代软骨细胞中 IL-6 mRNA 和蛋白的表达。此外，TGF-β1 刺激明显诱导了 p-STAT3），这被 IL-6R、JAK 或 ALK5 激酶活性抑制剂所阻断。然而，TGF-β1 并没有增加 STAT3 反应基因 SOCS3 的表达，而且 TGF-β1 预处理甚至抑制了 rhIL-6 诱导的 p-STAT3 和 SOCS3 的诱导。有趣的是，TGF-β1 强烈降低了 IL-6R 的表达。相反，IL-1β 确实增加了 IL-6 水平，但不影响 IL-6R 的表达。最后，添加重组 IL-6R 消除了 TGF-β1 对 IL-6 诱导的 p-STAT3 和下游 SOCS3、BCL3、SAA1 和 MMP1 表达的抑制作用。
在这项研究中，我们表明 TGF-β 降低了 IL-6R 的表达，从而抑制了软骨细胞中的 IL-6 信号。这揭示了 TGF-β 的一种新作用，可能对限制促炎的 IL-6 效应以维持软骨内稳态很重要。
