Roles of Growth Factors on Corneal Morphogenesis

高，温斯顿 W.-Y。 转化生长因子 ¿ (TGF-¿) 在角膜伤口愈合中具有关键作用。之前的研究表明 p38MAPK 和 Smad 信号通路的激活在介导 TGF-¿ 信号传导中具有不同的作用 分别进行角膜上皮清创术和角膜切除术。这种差异可以用以下事实来解释： 清创术的愈合上皮迁移到基底膜上，而角膜切除术的愈合上皮迁移到基底膜上 在裸露基质的胶原基质上迁移，导致不同的整合素表达模式 受伤后 1 小时内迁移上皮。因此，我们假设不同的整合素与 TGF-¿ 受体解释了 TGF-¿ 信号通路的差异，即 p38MAPK 的激活 角膜切除术中上皮清创和 Smads 级联反应（假设 1）。还发现 细胞增殖的抑制和激活转录因子 2 (ATF2) 的激活独立于 上皮清创后的 TGF-¿ 信号传导。因此，通过替代途径激活 ATF2， 即 JNK 及其随后形成的激活蛋白 1 转录因子 (AP-1) 复合物起着关键作用 在角膜损伤早期愈合阶段抑制上皮细胞增殖的作用（假设 2）。具体目标 1 将识别并表征整合素在 TGF-¿ 信号通路中的作用 使用三转基因 Cre-LoxP 小鼠模型进行角膜上皮清创和角膜切除术的愈合，即 Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f 和 Krt12rtTA/rtTA/tet-O-Cre/Smad4f/f，其中 floxed Tbr2 和 Smad4 基因 在多西环素诱导后，在角膜上皮中特异性消融，以便可以确定潜在的 在 Tbr2 和 Smad4 不存在和存在的情况下信号通路的变化（目标 1A），以检查 整合素在介导 TGF-¿ 受体信号传导（目标 1B）中的作用并检查 p38MAPK¿ 的功效和 Smad7 对伤口愈合过程中细胞迁移和增殖的调节（目标 1C）。具体目标 2 将 阐明 ATF2 和 AP-1 在角膜伤口愈合过程中抑制细胞增殖的作用 鉴定 ATF2 和/或 AP-1 复合物在角膜上皮清创和愈合上皮中的作用 使用免疫沉淀和蛋白质印迹分析（目标 2A）进行角膜切除术，确定 ATF2 的参与 在上皮清创愈合过程中通过显性基因的过度表达来抑制细胞增殖 阴性 ATF2 和 ¿N-ATF2 突变蛋白（目标 2B），并确定 JNK 和 p38MAPK 的影响 角膜伤口愈合过程中 ATF2 激活的抑制剂 (Aim2C)。 这些实验将为通过干预 TBR2 和 受伤角膜的 ATF2 信号通路。

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.