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. ? ? ?
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