Transforming growth factor ? (TGF-?) has a pivotal role in corneal wound healing. Previous studies revealed that activation of p38MAPK and Smad signaling pathway have distinct roles in mediating TGF-? signaling of corneal epithelium debridement and keratectomy, respectively. Such differences can be explained by the fact that healing epithelium of debridement migrates on basement membrane, whereas that of keratectomy migrates on collagenous matrix of denuded stroma, resulting in distinct integrin expression patterns in migrating epithelium within 1 hour of injuries. Thus, we hypothesize that interaction of different integrins with TGF-? receptors accounts for the difference in TGF-? signaling pathways, i.e., activation of p38MAPK in epithelium debridement and Smads cascades in keratectomy (Hypothesis 1). It has also been found that suppression of cell proliferation and activation of Activating Transcription Factor 2 (ATF2) are independent of TGF-? signaling following epithelium debridement. Thus, the activation of ATF2 by an alternative pathway, i.e., JNK, and its subsequent formation of Activating Protein-1 transcription factor (AP-1) complex plays a key role in the suppression of epithelial cell proliferation in the early healing phase of corneal injury (Hypotheisis 2).
Specific Aim 1 will identify and characterize roles of integrins in TGF-? signaling pathways during the healing of corneal epithelium debridement and keratectomy using tritransgenic Cre-LoxP mouse models, i.e., Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f and Krt12rtTA/rtTA/tet-O-Cre/Smad4f/f in which floxed Tbr2 and Smad4 genes are ablated specifically in corneal epithelium upon doxycycline induction so that one can determine potential variations in signaling pathways in the absence and presence of Tbr2 and Smad4 (Aim 1A), to examine roles of integrins in mediating TGF-? receptor signaling (Aim 1B) and to examine efficacy of p38MAPK1 and Smad7 on modulation of cell migration and proliferation during wound healing (Aim 1C).
Specific Aim 2 will elucidate roles of ATF2 and AP-1 in suppression of cell proliferation during corneal wound healing by identification of ATF2 and/or AP-1 complexes in healing epithelium of corneal epithelium debridement and keratectomy using immunoprecipitation and western blot analysis (Aim 2A), determine involvement of ATF2 in suppression of cell proliferation during healing of epithelium debridement by overexpression of dominant negative ATF2 and ?N-ATF2 mutant proteins (Aim 2B), and to determine effects of JNK and p38MAPK inhibitors on activation of ATF2 during corneal wound healing (Aim2C). These experiments will yield useful information for restoration of normal vision by intervening TBR2 and ATF2 signaling pathways of injured corneas.
The proposed studies will examine the roles of TGF-? in modulating corneal functions using experimental animals that conditionally over express dominant negative mutant and/or wild type signal transduction molecules of TGF-? receptor mediated pathways, e.g., ATF2, p38MAPK, Smad7 by transgenes delivered with Adenoviral vectors, and conditional ablation of genes, i.e., Tbr2, Smad4 and cJun in corneal epithelium of tritransgenic mice, i.e., Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f, Krt12rtTA/rtTA/tet-O-Cre/Smad4f/f and Krt12rtTA/rtTA/tet-O-Cre/cJunf/f mice upon doxycycline induction. The proposed studies will fill gaps of our understanding of TGF-? signaling on corneal morphogenesis during wound healing as well as homeostasis in adults. Data obtained will yield useful information for a better understanding of corneal diseases at molecular and cellular levels in vivo and serve as basis for designing treatment regiments for corneal wound healing.
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