Interstitial lung fibrosis is more frequently idiopathic and fatal. TGF-? is one of the main players in idiopathic pulmonary fibrosis (IPF). P311 is a highly conserved RNA binding protein that stimulates TGF-?s 1-3 translation through its interaction with eukaryotic translation initiation factor 3b. P311 is expressed in alveolar epithelial cells (AEC) and mesenchymal cells (MCs) in IPF and in bleomycin (BLM)-induced pulmonary fibrosis in mice, but not in normal lung. Our studies indicated that P311 knock out mice (P311 KOs) are highly protected against BLM-induced pulmonary fibrosis and have lower TGF-?s 1-3 than WT controls, while P311 transgenic mice (P311 TGs) develop spontaneous lung fibrosis and have high TGF-?s 1-3. Furthermore, BLM induces P311 expression in AEC and MS cultures. In silico analysis disclosed conserved binding sites for eleven transcription factors (TFs) in the promoter region of P311 and a pilot study suggested involvement of at least one of them in BLM-induced P311 expression in AECs but not in MCs. Initial studies showed that a synthetic peptide composed of five amino acids (aas) of the P311 RNA recognition motif (RRM) and the cell penetrating peptide TAT47-57 aa sequence (P311RRM-TAT) penetrated lung AECs and downregulated TGF-?s translation. Furthermore, a P311 siRNA conjugated to TAT47-57 (P311siRNA-TAT) protected from BLM-induced lung fibrosis. Based on the above, we hypothesize that P311 induces pulmonary fibrosis by differentially acting on AECs and MCs; that it does so in part by stimulating TGF-?s translation; that factors upstream and downstream of P311, other than TGF-?s, are involved in P311-mediated fibrosis; and that intratracheal delivery of P311RRM-TAT or P311siRNA-TAT will protect mice from experimentally-induced fibrosis .
The specific aims to test our hypotheses are: 1. To fully characterize the involvement of P311 in experimentally induced pulmonary fibrosis. We will perform the basic studies needed to solidify the main tenet of our working hypothesis. Acute and chronic models of lung fibrosis in wild type (WT), P311 KOs and P311 TGs and standard fibrosis read outs will be used to accomplish this aim. 2. To determine the effect of P311 on AECs and MCs responses associated with fibrosis. The read outs will include cell proliferation, death, migration, collagens production, extracellular matrix (ECM) proteinases expression, ECM proteolysis, and myofibroblast differentiation. 3. To identify upstream and downstream P311 effectors with a role in pulmonary fibrosis. The involvement of TGF-?s 1-3 as downstream P311 effectors will be determined in animal models of lung fibrosis by gain and loss of function experiments. To identify other P311 downstream effectors we will perform CLIPseq assays. To identify P311 upstream effectors we will perform loss and gain of function studies targeting the TFs suggested by the in silico analysis. The involvement of selected P311 effectors on mouse lung fibrosis will be determined. 4. To determine whether intratracheally delivered P311RRM-TAT and P311siRNA-TAT are effective in the treatment of experimentally-induced pulmonary fibrosis.
This project will establish the importance of P311, a small intracellular protein, in lung scarring; unveil its mechanistic pathway, and assess whether two novel compounds targeting P311 can treat lung scarring in mice.
| Cheng, Tao; Yue, Michael; Aslam, Muhammad Nadeem et al. (2017) Neuronal Protein 3.1 Deficiency Leads to Reduced Cutaneous Scar Collagen Deposition and Tensile Strength due to Impaired Transforming Growth Factor-?1 to -?3 Translation. Am J Pathol 187:292-303 |
