Focal Adhesion Modulation of IL-1 Signaling: Importance in Pulmonary Fibrosis

描述(由申请人提供)：特发性肺纤维化(IPF)是一种进行性疾病，通常是致命的，目前还没有有效的治疗策略。目前的建议集中在白介素1(IL-1)，一种强大的促炎细胞因子，在成纤维细胞中诱导多种信号级联反应。这些信号用于宿主防御，但矛盾的是，可能会导致炎症组织损伤和肺和其他器官的纤维化。IL-1通过丝裂原活化蛋白(MAP)激酶途径刺激细胞外基质降解，促进细胞内钙离子释放和多种细胞因子和炎症因子的表达。目前，IL-1诱导的信号调节或终止的机制还不完全清楚。到目前为止，我们的研究已经确定，在成纤维细胞中，IL-1诱导的信号需要局灶性粘连(FA)，并且我们开发的FA分散肽可以消散IL-1信号。我们最近的研究表明，两种蛋白酪氨酸磷酸酶(PTP)SHP-2和PTP()在调节IL-1诱导的FA成熟和IL-1诱导的信号转导中具有重要作用。我们发现SHP-2介导局灶性粘连和内质网之间的功能相互作用，这对内质网钙的释放是至关重要的；这些相互作用与局灶性粘连一起是IL-1信号转导的中心决定因素。我们的假设是，PTP(在FA中)介导了FA对IL-1的反应的成熟和重塑。在这些多分子信号平台中，PTP(与SHP-2相互作用并使其去磷酸化，导致额外的信号和支架分子的招募和激活，这些分子是从内质网释放钙离子、信号转导ERK以及分泌基质金属蛋白酶-1和3所必需的。在特定的目标1中，我们将确定PTP如何调节依赖FA的IL-1信号导致ERK激活和基质金属蛋白酶-1和3的分泌。我们将使用培养的正常人和IPF肺成纤维细胞和来自SHP-2或PTP(-空胚胎)的小鼠成纤维细胞作为体外模型来研究FA限制的信号转导。ERK的激活和MMP1和3的释放将作为IL-1信号转导的结果来评估SHP-2和PTP(。在特定的目标2中，我们将确定PTP(通过局灶性粘连和内质网调节IL-1信号)的作用。我们将研究PTP(和SHP-2)相互作用的分子决定因素，以及它们如何调节IP3受体和其他导致MMP表达的FA依赖信号。在具体目标3中，我们将评估PTP(和MMP3在体内)在博莱霉素诱导的小鼠肺纤维化模型中的作用。我们还将检测肺纤维化患者银行肺组织样本中PTP(和SHP-2)表达和/或激活的潜在变化。这些样本将被用来确定这些患者肺中SHP2和PTP(mRNA和蛋白)的表达是否存在与肺纤维化严重程度相关的变化。由于IL-1是肺纤维化等慢性炎症性疾病的重要介质，阐明IL-1信号通路是确定有效抗炎药的特定靶点的基础。这一点以特定PTP的小分子抑制剂和旨在选择性干扰促纤维化途径的多肽为例，作为潜在的治疗药物。项目简介：肺纤维化(肺部疤痕形成)是一种进行性疾病，通常是致命的，目前还没有有效的治疗方法。目前认为，疤痕形成过程是由肺(上皮)衬里细胞受损开始的，可能是由于病毒感染或环境污染物造成的。我们已经发现，白介素1，一种由肺内细胞分泌的小分子，强烈促进导致肺内疤痕形成的途径。我们建议对这些途径进行表征，目的是确定特定的治疗靶点，以遏制这一毁灭性的过程。

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis (IPF) is a progressive and frequently fatal disorder for which there are currently no effective treatment strategies. The current proposal focuses on Interleukin-1 (IL-1), a potent, pro-inflammatory cytokine that induces multiple signaling cascades in fibroblasts. These signals serve in host defense but, paradoxically, may contribute to inflammatory tissue injury and to fibrosis of the lung and other organs. IL-1 stimulates Ca2+ release and expression of multiple cytokines and inflammatory factors such as matrix metalloproteinases (MMPs) that drive extracellular matrix degradation via mitogen activated protein (MAP) kinase pathways. Currently, the mechanisms by which IL-1-induced signals are moderated or terminated are incompletely understood. Our studies to date have determined that in fibroblasts, IL-1-induced signaling requires focal adhesions (FA) and that IL-1 signals are dissipated by FA dispersing peptides that we have developed. Our recent studies indicate the importance of two protein tyrosine phosphatases (PTP), SHP- 2 and PTP(, in the regulation of IL-1-induced FA maturation and IL-1 induced signals. We discovered that SHP-2 mediates functional interactions between focal adhesions and the endoplasmic reticulum that are crucial for ER Ca2+ release; these interactions, together with focal adhesions, are central determinants of IL-1 signaling. Our hypothesis is that the PTP( in FA mediates maturation and remodeling of FA in response to IL- 1. In these multi-molecular signaling platforms, PTP( interacts with and dephosphorylates SHP-2, leading to recruitment and activation of additional signaling and scaffold molecules that are essential for Ca2+ release from the endoplasmic reticulum, signaling to ERK, and MMP-1 and 3 secretion. In Specific Aim 1 we will determine how PTP( regulates FA-dependent IL-1 signaling leading to ERK activation and MMP-1 and 3 secretion. We will use cultured human lung fibroblasts from normals and IPF lungs and murine fibroblasts from SHP-2 or PTP(-null embryos, reconstituted with wild type or mutant proteins, as in vitro models to study FA-restricted signaling. ERK activation and MMP1 and 3 release will be used as outcomes of IL-1 signaling to assess the impact of SHP-2 and PTP(. In Specific Aim 2, we will determine the role of PTP( in regulating IL-1 signaling through focal adhesions and the ER. We will examine the molecular determinants of PTP( and SHP-2 interactions and how they regulate the IP3 receptor and other FA-dependent signals leading to MMP expression. In Specific Aim 3, we will assess the roles of PTP( and MMP3 in vivo in a murine model of bleomycin-induced pulmonary fibrosis. We will also examine potential alterations in PTP( and SHP-2 expression and/or activation in samples of banked lung tissue from patients with pulmonary fibrosis. These samples will be used to determine if there are alterations in the expression of SHP2 and PTP( mRNA and protein in the lungs of these patients that are associated with severity of pulmonary fibrosis. As IL-1 is a critical mediator of chronic inflammatory conditions such as pulmonary fibrosis, elucidation of IL-1 signaling pathways is fundamental in the identification of specific targets for effective anti-inflammatory agents. This is exemplified by small molecule inhibitors of specific PTP and peptides designed to selectively interfere with pro-fibrotic pathways as potential therapeutics. PROJECT NARRATIVE: Pulmonary fibrosis (scarring of the lung) is a progressive and usually fatal disorder for which there is currently no effective therapy. It is currently believed that the scarring process is started by damage to the lining cells of the lung (epithelium), perhaps as the result of a viral infection or from environmental pollutants. We have discovered that Interleukin-1, a small molecule secreted by the cells lining the lung, strongly promotes pathways leading to scarring in the lung. We propose to characterize these pathways with the aim of identifying specific targets for therapies to curtail this devastating process.