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）是一种进行性且经常致命的疾病，目前尚无有效的治疗策略。目前的建议集中在白细胞介素-1 (IL-1)，一种有效的促炎细胞因子，可诱导成纤维细胞中的多种信号级联反应。这些信号在宿主防御中起作用，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刺激Ca2+的释放和多种细胞因子和炎症因子的表达，如基质金属蛋白酶（MMPs），通过丝裂原活化蛋白（MAP）激酶途径驱动细胞外基质降解。目前，il -1诱导的信号被调节或终止的机制尚不完全清楚。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粘连（FA），而IL-1信号被我们开发的FA分散肽所消散。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诱导的信号中的重要性。我们发现SHP-2介导局灶粘连和内质网之间的功能相互作用，这对ER Ca2+释放至关重要；这些相互作用以及局灶黏附是IL-1信号传导的主要决定因素。我们的假设是，在IL- 1的作用下，FA中的PTP介导了FA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募集和激活，这些分子对于Ca2+从内质网释放、向ERK发出信号以及MMP-1和3分泌至关重要。在Specific Aim 1中，我们将确定PTP如何调节fa依赖性IL-1信号传导导致ERK激活和MMP-1和3分泌。我们将使用来自正常和IPF肺的培养人肺成纤维细胞和来自SHP-2或PTP（-null）胚胎的小鼠成纤维细胞，用野生型或突变蛋白重组，作为体外模型来研究fa限制性信号传导。ERK的激活和MMP1和3的释放将作为IL-1信号的结果来评估SHP-2和PTP（）的影响。在Specific Aim 2中，我们将确定PTP在通过局灶粘连和内质网调节IL-1信号传导中的作用。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。在特异性目标3中，我们将评估PTP（和MMP3）在博莱霉素诱导肺纤维化小鼠模型中的作用。我们还将检测肺纤维化患者肺组织样本中PTP（和SHP-2）表达和/或激活的潜在改变。这些样本将用于确定这些患者肺中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改变。由于IL-1是肺纤维化等慢性炎症的重要介质，阐明IL-1信号通路对于确定有效抗炎药的特定靶点至关重要。特定PTP的小分子抑制剂和肽被设计为选择性地干扰促纤维化途径作为潜在的治疗方法。项目描述：肺纤维化（肺瘢痕）是一种进行性且通常致命的疾病，目前尚无有效的治疗方法。目前认为，疤痕形成的过程是由肺上皮细胞的损伤开始的，可能是病毒感染或环境污染物的结果。我们已经发现，白细胞介素-1，一种由肺内壁细胞分泌的小分子，强烈地促进了导致肺部瘢痕形成的途径。我们建议表征这些途径，目的是确定治疗的具体目标，以减少这一毁灭性的过程。

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.