Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy.
Department of Surgery, University of Rome Tor Vergata, Rome, Italy.
Front Immunol. 2018 Aug 10;9:1854. doi: 10.3389/fimmu.2018.01854. eCollection 2018.
In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-β1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut.
在炎症性肠病（IBD）黏膜中，由于转录后机制增强了 Smad7 的乙酰化状态，从而阻止了该蛋白的泛素化介导的蛋白酶体降解，导致抑制性细胞因子转化生长因子-β1 的细胞内抑制剂 Smad7 过度表达。IBD 相关炎症还伴有 Sirt1 的表达缺陷，Sirt1 是一种 III 类 NAD+-依赖性去乙酰化酶，可促进各种细胞内蛋白的泛素化介导的蛋白酶体降解，并引发抗炎信号。我们的研究目的是确定在 IBD 中 Smad7 和 Sirt1 是否存在相互调节。通过 Western blot 和免疫组化检测 IBD 患者和正常对照者的黏膜样本中的 Smad7 和 Sirt1，通过荧光测定法评估 Sirt1 活性。为了确定 Smad7 是否受 Sirt1 调节，分别用 Sirt1 抑制剂（Ex527）或激活剂（Cay10591）培养正常或 IBD 固有层单核细胞（LPMC）。为了确定 Smad7 是否控制 Sirt1 的表达，用 Smad7 反义或正义寡核苷酸处理 IBD 黏膜外植体的器官培养物。此外，在给予葡聚糖硫酸钠（DSS）的 Smad7 转基因小鼠的 LPMC 中评估 Sirt1 的表达。在 IBD 患者的上皮和固有层中均可见 Smad7 的上调，这与 Sirt1 的表达和活性降低有关。用 Cay10591 激活 IBD LPMC 中的 Sirt1 可降低 Smad7 的乙酰化并增强泛素化驱动的蛋白酶体介导的降解，而用 Ex527 抑制正常 LPMC 中的 Sirt1 激活可增加 Smad7 的表达。在 IBD 黏膜外植体中敲低 Smad7 可增强 Sirt1 的表达，从而表明 Smad7 对 Sirt1 诱导有负性影响。一致地，Smad7 转基因小鼠的黏膜 T 细胞中 Sirt1 水平降低，该缺陷在诱导 DSS 结肠炎时被放大。数据表明 Smad7 和 Sirt1 之间存在相互调节机制，这可能有助于放大肠道中的炎症信号。
