Roles of Growth Factors on Corneal Morphogenesis

描述（由申请人提供）：转化生长因子？（TGF-?）在角膜创面愈合中起关键作用。既往研究表明，p38MAPK和Smad信号通路的激活在介导TGF-？分别为角膜上皮清创和角膜切除术的信号。这种差异可以解释为清创的愈合上皮在基底膜上迁移，而角膜切除术的愈合上皮在脱落基质的胶原基质上迁移，导致损伤后1小时内迁移上皮中整合素的表达模式不同。因此，我们假设不同整合素与TGF-？受体与TGF-？信号通路，即p38MAPK在角膜切除术中上皮清创和Smads级联中的激活（假设1）。还发现细胞增殖的抑制和活化转录因子2 （ATF2）的激活不依赖于TGF-？上皮清创后的信号。因此，通过另一途径，即JNK激活ATF2，并随后形成活化蛋白-1转录因子（AP-1）复合物，在角膜损伤早期愈合阶段抑制上皮细胞增殖中起关键作用（假设2）。特异性目标1将识别并描述整合素在TGF-？使用三转基因Cre-LoxP小鼠模型，即Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f和Krt12rtTA/rtTA/tet-O-Cre/Smad4f/f，在多西环素诱导下特异性消融角膜上皮中固定的Tbr2和Smad4基因，从而确定信号通路在Tbr2和Smad4缺失和存在时的潜在变化（Aim 1A），以检验整合素在介导TGF-？受体信号（Aim 1B），并研究p38MAPK1和Smad7在伤口愈合过程中调节细胞迁移和增殖的功效（Aim 1C）。特异性目的2将阐明ATF2和AP-1在角膜创面愈合过程中抑制细胞增殖的作用，通过免疫沉淀和免疫印迹分析（Aim 2A）在角膜上皮清创和角膜切除术的愈合上皮中鉴定ATF2和/或AP-1复合物（Aim 2A），通过过表达显性阴性ATF2和？N-ATF2突变蛋白（Aim 2B），并确定JNK和p38MAPK抑制剂在角膜创面愈合过程中对ATF2激活的影响（Aim2C）。这些实验将为通过干预损伤角膜的TBR2和ATF2信号通路来恢复正常视力提供有用的信息。拟议的研究将检验TGF-？TGF-?的显性负突变型和/或野生型信号转导分子有条件过表达的实验动物调节角膜功能受体介导的途径，如ATF2， p38MAPK， Smad7通过腺病毒载体传递的转基因，以及有条件地消融三转基因小鼠角膜上皮中的基因，即Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f， Krt12rtTA/rtTA/tet-O-Cre/Smad4f/f和Krt12rtTA/rtTA/tet-O-Cre/cJunf/f，在多西环素诱导下。提出的研究将填补我们对TGF-？成人创面愈合过程中角膜形态发生的信号传导及体内平衡。获得的数据将为更好地了解角膜疾病在体内的分子和细胞水平提供有用的信息，并作为设计角膜伤口愈合治疗方案的基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE 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.