Roles of Growth Factors on Corneal Morphogenesis

作者：Winston W.-Y. 转化生长因子在角膜创面愈合中起着关键作用。先前的研究表明 P38MAPK和Smad信号通路的激活在介导转化生长因子-β信号转导中具有不同的作用 分别行角膜上皮清创术和角膜切削术。这种差异可以用以下事实来解释： 清创术的愈合上皮在基底膜上迁移，而角膜切削术的愈合上皮在基底膜上迁移 在裸露基质的胶原基质上迁移，导致不同的整合素在 损伤后1小时内上皮移行。因此，我们假设不同整合素与 转化生长因子-β受体是转化生长因子-β信号通路不同的原因，即p38MAPK的激活 角膜切削术中的上皮清创和SMADS级联(假设1)。还发现， 抑制细胞增殖和激活转录因子2(ATF2)不依赖于 上皮清创术后的转化生长因子-β信号传导。因此，通过另一种途径激活ATF2， 即JNK，其随后形成的激活蛋白-1转录因子(AP-1)复合体起着关键作用 在角膜损伤愈合早期抑制上皮细胞增殖中的作用 2)。具体目标1将确定和表征整合素在转化生长因子-β信号通路中的作用 用三基因Cre-loxP小鼠模型修复角膜上皮清创和角膜切除，即 Krt12rtTA/rtTA/tet-O-cre/Tbr2f/f和Krt12rtTA/rtTA/tet-O-cre/Smad4f/f，其中Tbr2和Smad4基因是 在多西环素诱导下特定地在角膜上皮细胞中消融，以便确定潜力 在没有和存在Tbr2和Smad4的情况下信号通路的变化(目标1A) 整合素在介导转化生长因子受体信号转导中的作用(Aim 1B)及检测p38MAPK和p38MAPK的作用 Smad7关于伤口愈合过程中细胞迁移和增殖的调节(目标1C)。特定目标2将 ATF2和AP-1在角膜创伤愈合过程中抑制细胞增殖中的作用 角膜上皮清创愈合上皮中ATF2和/或AP-1复合体的鉴定 应用免疫沉淀和免疫印迹分析(Aim 2A)进行角膜切除术，确定ATF2的参与 优势基因过表达抑制上皮清创愈合过程中的细胞增殖 阴性的ATF2和N-ATF2突变蛋白(目标2B)，并确定JNK和p38MAPK的作用 角膜创伤愈合过程中ATF2激活的抑制剂(Aim2C)。 这些实验将为干预TBR2和TbR2恢复正常视力提供有用的信息。 角膜损伤后的ATF2信号转导通路。

Kao, Winston W.-Y. 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 p38MAPK¿ 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.