Hypertrophic scars (HSs) occur at a high incidence after burns and lack effective treatment. TGF-?, a powerful profibrogenic growth factor, plays an important role in HS development. We recently demonstrated that P311, an 8 kDa intracellular protein present in human HSs, controls TGF-?1-3 level/activity by stimulating their translation, and preliminary work suggested that P311 directly binds to eukaryotic translation initiation factor 3b (eIF3b). We developed a murine model of HS by topical application of bleomycin (BLM) on skin excisional wounds. P311 is expressed in these HSs, but not in normal scars. Pilot studies suggested that P311 KO mice are protected against BLM-induced HSs, while P311 transgenic mice (P311 TGs) develop HSs in the absence of BLM. P311 has a conserved, PEST-like motif, known to mediate protein-protein interactions. A peptide representing the P311 PEST-like motif or a P311 siRNA, each of them conjugated to the cellular penetrating peptide TAT47-57 (TAT47-57P311PEST and TAT47-57P311siRNA), targeted P311 and reduced TGF-?s levels in vitro, moreover, the TAT47-57P311siRNA ameliorated BLM-induced HSs. Based on the above, we hypothesize that P311 promotes HSs by binding to eIF3b and thereby stimulating TGF-?s translation;and that topical application of TAT47-57P311PEST or TAT47-57P311siRNA will protect mice from developing HSs.
The specific aims to test this hypothesis are: 1. To determine the involvement of P311 in mouse HSs and whether it is mediated by the P311 PEST-like domain. We will characterize the BLM-treated scars of P311 KO mice and WTs, the scars of P311 TGs, and the effect of P311 and P311 PEST mutants intradermally delivered at the wound site. The read-outs will include histological quantification of fibrosis using the Aperio system pls immunoblot/densitometry;quantification of myofibroblasts;and determination of P311 and TGF-?1-3 mRNAs and protein levels. Mouse and human keratinocyte (KT) and dermal fibroblast (DF) response to P311 will be studied in primary tissue cultures 2. To determine the effect of P311 and its PEST-like domain in TGF-?s translation by mouse and human keratinocytes (KTs) and dermal fibroblasts (DFs), and whether it is mediated by P311 interaction with eIF3b. We will employ KT and DF primary cultures, P311 and P311 PEST mutants delivered by lentivirus transfer, and purified P311 and eIF3b. Among the read outs will be TGF-? 1-3 translation reporter assays, co-immunoprecipitations, protein pull-downs, use of surface plasmon resonance, RNA immunoprecipitation assays and TGF-?1-3 translation reporter assays after eIF3b knockdown by RNA interference. 3. To determine whether topically-delivered TAT47-57P311PEST and TAT47-57P311siRNA are effective in preventing BLM-induced HSs. Protocols for the topical delivery of each of these two compounds in P311 TG mice wounds/scars and BLM-treated wounds/scars will be optimized and their effect in preventing the development of HSs will be determined/compared. Read-outs will be the same as for aim #1.
This project will establish the importance of P311, a small intracellular protein, in the production of excessive skin scarring;unveil the mechanism used by P311 to control a growth factor called TGF-beta, which is critical to produce scarring, and assess whether two novel compounds targeting P311 can treat excessive skin scarring in mice.