Radboud University Medical Center, Department of Rheumatology, Experimental Rheumatology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Radboud University Medical Center, Department of Rheumatology, Experimental Rheumatology, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Cell Signal. 2017 Dec;40:190-199. doi: 10.1016/j.cellsig.2017.09.010. Epub 2017 Sep 21.
Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications.
Co-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA-luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA-luciferase activity.
TGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation.
间充质干细胞（MSC）的软骨分化需要转化生长因子β（TGFβ）信号。TGFβ与 I 型受体激活素样激酶（ALK）5 结合，导致 C 端 SMAD2/3 磷酸化（pSMAD2/3C）。反过来，pSMAD2/3C 易位到细胞核并调节靶基因表达。已知炎症介质对 MSC 分化有抑制作用。在这项研究中，我们研究了白细胞介素 1β（IL1β）对 SMAD2/3 信号转导动力学和翻译后修饰的影响。
MSC 与 TGFβ 和 IL1β 共同刺激不会影响刺激后 1 小时的峰值 pSMAD2C 水平。令人惊讶的是，与 TGFβ 单独刺激相比，CAGA-荧光素酶报告基因构建体确定的 SMAD3 转录活性通过 TGFβ 和 IL1β 的共同刺激得到增强。此外，IL1β 刺激以 SMAD 依赖的方式诱导 CAGA-荧光素酶活性。由于 SMAD 功能可以通过 SMAD 连接域独立于经典 TGFβ 信号进行调节，因此我们研究了 SMAD2 连接域磷酸化在特定苏氨酸和丝氨酸残基上的情况。在 TGFβ、IL1β 或 TGFβ 和 IL1β 刺激后 1 小时内观察到 SMAD2 连接域 threonine 和 serine 修饰。在共刺激时，修饰的 SMAD2 在前细胞质中积累，并且在 2-4 小时时 SMAD2/3 靶基因转录（ID1、JUNB）受到抑制。对 IL1β 诱导的 SMAD2 连接域修饰的详细时间过程分析显示，与 TGFβ 相比具有独特的时空调控模式。与两种因子的共刺激导致与 TGFβ 单独刺激相似的动力学特征。然而，IL1β 在刺激后 8 至 24 小时之间略微改变了 TGFβ 诱导的 pSMAD2C 水平，这反映在 TGFβ 靶基因表达（PAI1、JUNB）中。通过过表达 SMAD3 或 SMAD3 连接域磷酸突变体，获得了 SMAD3 连接域修饰对 IL1β 对 TGFβ 信号影响的重要性的直接证据。最后，进行了抑制剂筛选以鉴定参与 SMAD2/3 连接域修饰的激酶。我们确定 TAK1 激酶活性对于 IL1β 诱导的 SMAD2 连接域修饰和 CAGA-荧光素酶活性至关重要。
TGFβ 和 IL1β 信号在人原代 MSC 中在 SMAD2/3 水平上相互作用。IL1β 独立于 C 端 SMAD2 磷酸化抑制下游 TGFβ 靶基因。我们证明 SMAD2/3 连接域修饰是这种相互作用所必需的，并确定 TAK1 是 IL1β 诱导的 TGFβ 信号调节的关键介质。
