Burn injuries claim the lives of 4000 United States civilians each year and send another 500,000 to seek medical attention. This accounts for over 900,000 hospital days per year and more than $1 billion per year in associated costs, including loss of productivity. Thus it is an unmet need to develop a therapy to limit burn injury progression and promote robust wound healing which is not addressed by any FDA approved therapy. P12 is a small fibronectin peptide with shown promise limiting injury progression. The overall goal of this project is to investigate the mechanism of P12 promoting cell survival by in vitro models created to mimic the compromised condition in peri-burn tissue. This is a logical extension of previous biochemical experiments demonstrating P12's synergistic effects with platelet derived growth factor-BB (PDGF-BB) to promote cell survival. Our first hypothesis is that P12 functions through augmenting PDGF-BB survival signals as a PDGF- BB co-factor.
Thus Aim1 is to determine the effect of P12 on PDGF-BB-stimulated signal transduction pathways, particularly the PI3K/Akt pathway, in AHDF under nutrient deprivation. For this aim, nutrient deprivation system will be employed to mimic the ischemic condition in the peri-burn tissue. PDGFR effectors upstream and downstream PI3K-Akt pathway will be probed by western blot to determine the effectors of P12's pro-survival activity under nutrient deprivation. The structure of P12 is very similar to commonly used protein transduction domains (PTDs) that promote internalization of bound cargoes into mammalian cells. So it is possible that P12 can bring PDGF /PDGFR complex into the cell. By entering the cell through different internalization pathways, PDGF can produce different signaling leading to migration, proliferation or survival. Also, growth factor receptors trigger different signaling complex and signaling pathways when they are present in endosomal and cell membrane compartments. Based on the binding between PDGF-BB and P12, the second hypothesis is that P12 either enhances internalization or changes trafficking of PDGFR, thereby its pro-survival signaling.
Thus Aim 2 is to determine whether P12 modulates PDGF-BB/PDGFR-? Internalization/ trafficking. For this aim, PDGF/P12 induced PDGFR internalization will be measured by reversible biotinylation. Then, Inhibitors for different internalization pathways will be employed to determine the pathway the P12 modulates. Finally, a inhibitory peptide will be used to interrupt the binding between P12 and PDGF-BB to determine whether their binding is important for P12 pro-survival activity, downstream signaling events, and PDGFR internalization. 1
Burn injuries claim the lives of 4000 people in the United States each year and send another 500,000 to seek medical attention. Burn injuries also account for over 900,000 hospital days per year and more than $1 billion per year in associated costs, including loss of productivity. The purpose of this grant is to investigate the mechanism of a novel bioactive peptide as a therapy to limit burn injury progression and promote robust wound healing which is not addressed by any FDA approved therapy.
|Zhu, Jia; Lin, Fubao; Brown, Deborah A et al. (2014) A fibronectin peptide redirects PDGF-BB/PDGFR complexes to macropinocytosis-like internalization and augments PDGF-BB survival signals. J Invest Dermatol 134:921-929|
|Zhu, Jia; Clark, Richard A F (2014) Fibronectin at select sites binds multiple growth factors and enhances their activity: expansion of the collaborative ECM-GF paradigm. J Invest Dermatol 134:895-901|