SMAD4 regulation of colon epithelial cell inflammatory responses

项目摘要 虽然许多研究已经定义了炎症是如何产生和维持的，但对此知之甚少 它是如何解决的。我们发现，结肠上皮是加重或抑制炎症的关键。 众所周知，微生物区系和免疫细胞向上皮细胞发出信号，以增加 趋化因子和促炎细胞因子可以加速炎症，但我们发现TGFb 通过Smad4在上皮细胞内的信号转导抑制了促炎转录网络。之后 在成年小鼠结肠上皮细胞中条件性Smad4基因缺失，我们发现了显著的证据 上皮室内的炎性信号伴随着炎性浸润物的增加。 从机制上讲，TGFb1和/或BMP2可抑制肿瘤坏死因子、白介素2等多种炎症基因的转录诱导。 1B，或脂多糖处理培养的小鼠和人结肠上皮细胞。此外，75%的成年小鼠- 肠上皮中Smad4基因的起始缺失发展为浸润性粘液腺癌 葡聚糖硫酸钠(DSS)诱导的结肠炎后3个月内远端结肠，未发现肿瘤 没有结肠炎或Smad4+对照组。在人类中，我们发现结肠炎中Smad4缺失的发生率要高得多- 相关癌(CAC)高于散发性结直肠癌。根据我们的观察，我们假设 通过Smad4的TGFb信号作为肿瘤抑制因子发挥作用，部分是通过抑制促炎作用。 肠上皮细胞的细胞因子反应。本研究的主要目的是确定其作用机制。 TGFb超家族信号通过什么调控结肠炎症反应以及这一调控是如何联系的 对肿瘤的抑制。我们将通过以下具体目标来检验我们的假设：目标1.确定 Smad4介导的信号调节结肠上皮细胞细胞因子信号通路的机制。 工作假说：Smad4介导的信号转导抑制对炎症刺激的转录反应 结肠上皮细胞通过细胞因子诱导基因的特异性抑制复合体。我们将确定哪一个 基因是由Smad4直接调节的，以及这种调节如何改变对促炎信号的反应 培养细胞和体内通过RNA-SEQ、ATAC-SEQ和CHIP-SEQ相结合的实验。目标2. 确定失调的细胞因子信号如何调节Smad4缺失的结肠上皮的肿瘤发生。 工作假说：Smad4介导的上皮炎性反应基因抑制是必需的 预防结肠炎期间的肿瘤形成。我们建议在体内研究Smad4如何调节特定的炎症- 相关的信号通路和基因表达，以及这些通路如何有助于抑制CAC。 我们将使用单细胞分析方法来确定这些调控途径，以了解细胞特异性 Smad4在结肠上皮细胞中的作用，并将评估Smad4在抑制上皮细胞中的作用 人类结直肠癌中的炎性基因，特别是在CAC中。我们的工作将更好地理解炎症性疾病 在结肠上皮细胞的调控，并可能确定新的干预策略的调控靶点。

Project Summary While a number of studies have defined how inflammation arises and is maintained, much less is known about how it is resolved. We have found that colon epithelium is key to either exacerbating or suppressing inflammation. It is well-established that microbiota and immune cells signal to epithelial cells to increase production of chemokines and pro-inflammatory cytokines that can accelerate inflammation, but we have found that TGFb signaling via SMAD4 within the epithelium suppresses a pro-inflammatory transcriptional network. After conditional Smad4 gene deletion in the adult murine colonic epithelium, we found striking evidence for increased inflammatory signaling within the epithelial compartment concomitant with an increase in inflammatory infiltrate. Mechanistically, TGFb1 and/or BMP2 inhibit transcriptional induction of multiple inflammatory genes by TNF, IL- 1b, or LPS treatment in mouse and human cultured colon epithelial cells. Furthermore, >75% of mice with adult- onset deletion of the Smad4 gene in intestinal epithelium developed invasive mucinous adenocarcinomas of the distal colon within 3 months after dextran sodium sulfate (DSS)-induced colitis, while no tumors were found without colitis or in the SMAD4+ controls. In humans, we found a much higher incidence of SMAD4 loss in colitis- associated carcinoma (CAC) than in sporadic colorectal cancer. Based on our observations, we hypothesize that TGFb signaling via SMAD4 functions as a tumor suppressor, in part, through the inhibition of pro-inflammatory cytokine responses in intestinal epithelial cells. The major goal of this research is to determine the mechanisms by which TGFb superfamily signaling regulates colonic inflammatory responses and how this regulation is linked to tumor suppression. We will examine our hypothesis through the following specific aims: Aim 1. Determine the mechanism by which SMAD4-mediated signaling modulates cytokine signaling pathways in colon epithelial cells. Working hypothesis: SMAD4-mediated signaling inhibits transcriptional responses to inflammatory stimuli in colonic epithelial cells via specific inhibitory complexes on cytokine-induced genes. We will determine which genes are directly regulated by SMAD4 and how this regulation alters responses to pro-inflammatory cues in cultured cells and in vivo through a combination of RNA-seq, ATAC-seq and ChIP-seq experiments. Aim 2. Determine how dysregulated cytokine signaling regulates tumorigenesis in Smad4 null colonic epithelium. Working hypothesis: SMAD4-mediated suppression of epithelial inflammatory response genes is required to prevent tumorigenesis during colitis. We propose to examine in vivo how SMAD4 regulates specific inflammation- associated signaling pathways and gene expression and how those pathways contribute to suppression of CAC. We will identify these regulatory pathways using single cell analytical approaches to understand the cell-specific roles of SMAD4 within colon epithelium and will evaluate the role of SMAD4 in suppressing epithelial inflammatory genes in human CRC, particularly in CAC. Our work will yield better understanding of inflammatory regulation in colonic epithelium and is likely to identify novel regulatory targets for interventional strategies.