Fibrogenesis, the formation of fibrous connective tissue, is essential in development and wound healing. When unrestrained, however, pathologic fibrogenesis contributes to progressive fibrosis of the lungs and other organs leading to organ failure. Diffuse, progressive fibrosis of the lungs is a hallmark of idiopathic pulmonary fibrosis (IPF), a condition that is relentlessly progressive and ultimately fatal. While recent trials have shown that pirfenidone and nintedanib can slow the rate of decline in lung function, novel mechanism-based therapies that not only slow the progression of fibrosis but resolve established fibrosis are urgently needed. We have discovered that protein tyrosine phosphatase-? (PTP?) promotes TGF?-dependent fibrogenic responses in lung fibroblasts, representing a key checkpoint in the fibrogenic pathway. This project will address the hypothesis that PTP? promotes fibrosis in the lung by indirectly controlling the phosphorylation state of tyrosine residues in the cytoplasmic tail of TGF? receptor (T?R)II thus enhancing Smad-dependent fibrogenic signals in fibroblasts. Our experimental construct is that PTP? amplifies and prolongs fibrogenic signals from T?Rs and the ECM in the context of integrin-based focal adhesions thus enhancing production of collagen and fibronectin leading to tissue fibrosis. Using a combination of pharmacological, biochemical (mass spec, immunoprecipitation, in vitro analysis of recombinant proteins, and phospho-proteomic analysis), and molecular (siRNA gene silencing, RNA Seq) approaches in cultured human and murine fibroblasts, we will determine how PTP? is recruited to the TGF? receptor complex and regulates tyrosine phosphorylation of T?Rs and associated molecules in the receptor complex indirectly through Src tyrosine kinases. We will then ascertain the effects of PTP? on downstream Smad-dependent expression of profibrotic genes including collagen, fibronectin, ?-SMA, and miR-29. We will assess the importance of PTP? in myofibroblast differentiation, proliferation, and apoptosis. We will then determine how PTP? is recruited to focal adhesions and integrates signals from a mechanically stiff `fibrotic' ECM with signals transduced through Src, T?Rs, and FAK culminating in fibrogenic responses. These studies will employ molecular and imaging-based approaches with expression of fluorescent fusion proteins in fibroblasts grown on ECM-coated polyacrylamide hydrogels of varying stiffness. We will then test our hypothesis in preclinical animal models of pulmonary fibrosis. We will determine the effect of fibroblast-specific genetic deletion of PTP? in our Ptpraf/f mice using Cre driven by fibroblast-specific promoters (DERMO1, Col1a1, and Col1a2) in three models of pulmonary fibrosis: (i) adenoviral expression of recombinant TGF-?; (ii) single dose and (iii) multiple dose intratracheal bleomycin. The role of Src kinases will be assessed in these models using gene-targeted mice and pharmacological inhibitors. Ultimately, this knowledge will be used to develop small molecule or biological approaches selectively targeting these profibrotic pathways to treat pulmonary fibrosis in humans.
Progressive fibrosis of the lungs typifies idiopathic pulmonary fibrosis (IPF), a condition that is relentlessly progressive and ultimately fatal and, while recent trials have shown that medications can slow the rate of decline in lung function, new treatments that prevent or reverse established fibrosis are urgently needed. We have discovered that protein tyrosine phosphatase-? (PTP?) functions as a key checkpoint in the fibrogenic pathway in fibroblasts and that genetic deletion of PTP? prevents pulmonary fibrosis in animal models. This proposal seeks to elucidate the mechanisms by which PTP? controls lung fibrosis and how it can be targeted therapeutically to treat patients with progressive pulmonary fibrosis.
| Aschner, Yael; Downey, Gregory P (2018) The Importance of Tyrosine Phosphorylation Control of Cellular Signaling Pathways in Respiratory Disease: pY and pY Not. Am J Respir Cell Mol Biol 59:535-547 |
| Wang, Qin; Delcorde, Julie; Tang, Tracy et al. (2018) Regulation of IL-1 signaling through control of focal adhesion assembly. FASEB J 32:3119-3132 |
| Downey, Gregory P; Aschner, Yael (2017) Taking It Off: New Insights into the Role of Tyrosine Phosphorylation-dependent Pathways in the Pathogenesis of Pulmonary Fibrosis. Am J Respir Crit Care Med 195:418-420 |
| Aschner, Yael; Downey, Gregory P (2016) Transforming Growth Factor-?: Master Regulator of the Respiratory System in Health and Disease. Am J Respir Cell Mol Biol 54:647-55 |
