Chronic allograft rejection shares a common pathophysiological feature in each organ studied: accumulations of mesenchymal cells in critical lumena leading to organ dysfunction. In the course of experiments examining the ability of HMG-CoA reductase inhibitors to selectively induce apoptosis in fibroblasts with deregulated growth control, we discovered a key role for the cap-dependent translation initiation apparatus in the regulation of fibroblast viability. In mammals, a bimolecular complex designated eIF4F initiates cap-dependent translation. It consists of three proteins: eIF4E, which binds the 7-methyl guanosine cap at the 5' mRNA terminus and is generally considered to be present in rate limiting amounts; eIF4A, an mRNA helicase; and eIF4G, which serves as a docking protein binding eIF4E and eIF4A as well as the adapter protein eIF3 which targets the intact eIF4F complex to the 40S subunit of the ribosome. Translation initiation factor 4E is regulated by a family of repressor proteins, the 4E-BPs, which share a motif with eIF4G allowing them to sequester eIF4E in a competitive manner. Phosphorylation of the 4E-BPs decreases their affinity for eIF4E, freeing it to bind eIF4G and initiate translation. Most importantly from a therapeutic point of view, we have found that fibroblasts with deregulated growth have an increased requirement for cap-dependent translation to suppress apoptosis in vitro and in vivo, when compared to normal fibroblasts. Thus, here we propose to take the first step toward developing therapies for the lumenal airway fibrosis characteristic of lung allograft rejection. We will test the hypothesis that transfer of genes encoding translational repressors operating at the apical step of cap-dependent translation initiation will selectively chemosensitize fibroblasts in airway allograft fibrosis to safe doses of HMG-CoA reductase inhibitors, without harming desirable parenchymal bystanders. We propose 2 Specific Aims: 1) Use the known crystal structure of eIF4E to develop, validate and transfer gene constructs in vitro that lead to loss of translation initiation factor 4E (eIF4E) function; and test their ability to chemosensitize fibroblasts to HMG-CoA reductase inhibitor-induced apoptosis; 2) Starting with wild type 4E-BP1, systematically test whether transfer of genes encoding those translational repressors found to be proapoptotic in Aim 1 will collaborate with HMG-CoA reductase inhibitors to diminish airway lumenal narrowing in a well established murine allograft model of airway fibroproliferation. The proposed experiments are designed to test """"""""proof of concept"""""""", an essential first step toward identification of novel molecular targets for drug discovery.
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