SMAD4 regulation of colon epithelial cell inflammatory responses

项目摘要 虽然许多研究已经确定了炎症是如何发生和维持的，但对炎症的发生和维持知之甚少。 如何解决。我们已经发现结肠上皮是加重或抑制炎症的关键。 众所周知，微生物群和免疫细胞向上皮细胞发出信号以增加微生物群的产生。 趋化因子和促炎细胞因子，可以加速炎症，但我们已经发现， 上皮内通过SMAD 4的信号传导抑制促炎转录网络。后 在成年小鼠结肠上皮中的条件性Smad 4基因缺失，我们发现了显著的证据， 上皮区室内的炎性信号传导伴随炎性浸润的增加。 在机制上，TGF β 1和/或BMP 2抑制TNF、IL-10和/或BMP-2对多种炎性基因的转录诱导。 1b，或LPS处理小鼠和人培养的结肠上皮细胞。此外，>75%的成年小鼠- Smad 4基因在肠上皮中的起始缺失发展为浸润性粘液腺癌， 葡聚糖硫酸钠（DSS）诱导结肠炎后3个月内远端结肠，而未发现肿瘤 无结肠炎或SMAD 4+对照。在人类中，我们发现结肠炎中SMAD 4丢失的发生率要高得多- 相关癌（CAC）比散发性结直肠癌。根据我们的观察，我们假设 通过SMAD 4的TGF β信号传导部分地通过抑制促炎性细胞因子而起到肿瘤抑制剂的作用。 肠上皮细胞的细胞因子反应。本研究的主要目的是确定 TGF β超家族信号通过什么途径调节结肠炎症反应以及这种调节是如何联系在一起的 肿瘤抑制。我们将通过以下具体目标来检验我们的假设：目标1。确定 SMAD 4介导的信号传导调节结肠上皮细胞中细胞因子信号传导途径的机制。 工作假设：SMAD 4介导的信号转导抑制了炎症刺激的转录反应， 结肠上皮细胞通过特异性抑制复合物对尼古丁诱导的基因。我们将决定 基因直接受SMAD 4调节，以及这种调节如何改变对促炎性信号的反应， 通过RNA-seq、ATAC-seq和ChIP-seq实验的组合在培养的细胞和体内进行。目标二。 确定失调的细胞因子信号传导如何调节Smad 4缺失结肠上皮的肿瘤发生。 工作假设：需要SMAD 4介导的上皮炎症反应基因抑制， 预防结肠炎期间的肿瘤发生。我们建议在体内研究SMAD 4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识别这些调控途径，以了解细胞特异性的 SMAD 4在结肠上皮中的作用，并将评估SMAD 4在抑制上皮细胞增殖中的作用。 在人类CRC中，特别是在CAC中，存在炎症基因。我们的工作将有助于更好地了解炎症 调节结肠上皮细胞，并有可能确定新的调控目标的干预策略。

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.