Roles of Growth Factors on Corneal Morphogenesis

生长因子对角膜形态发生的作用

• 批准号: 8204626
• 负责人: WINSTON W KAO
• 金额: $ 45.94万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204626
• 关键词: Abbreviations; Ablation; Accounting; Activating Transcription Factor 2; Adenovirus Vector; Adult; Alleles; Animal Experimentation; Animals; Antibodies; Basement membrane; Binding; Bromodeoxyuridine; Caliber; Cell Death; Cell Proliferation; Cell physiology; Collaborations; Complex; Cornea; Corneal Diseases; Corneal Injury; DNA Double Strand Break; Data; Debridement; Dimerization; Dominant-Negative Mutation; Doxycycline; Epithelial Cell Proliferation; Epithelium; Eye; Family; Fluorescein; Gene Deletion; Genes; Genetic; Growth Factor; Healed; Homeostasis; Hour; Immunohistochemistry; Immunoprecipitation; In Vitro; Injury; Integrins; Intracellular translocation; Luciferases; MAP Kinase Gene; MAPK8 gene; Measures; Mediating; Molecular; Molecular Biology; Morphogenesis; Mus; Pathway interactions; Pattern; Phase; Phosphorylation; Play; Protein Biosynthesis; Proteins; Receptor Signaling; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; SP600125; Signal Pathway; Signal Transduction; Staining method; Stains; Stress; Tetanus Helper Peptide; Transcription Factor AP-1; Transforming Growth Factors; Transgenes; Tumor Suppressor Proteins; Variant; Virus; Vision; Western Blotting; Wild Type Mouse; Wound Healing; base; cell motility; corneal epithelium; design; effective therapy; feeding; healing; in vivo; inhibitor/antagonist; injured; member; migration; mouse model; mutant; neutralizing antibody; overexpression; receptor; repaired; research study; restoration; small hairpin RNA; therapy design; time interval; transcription factor

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.

高文斯顿Y. 转化生长因子（TGF-β）在角膜伤口愈合中具有关键作用。先前的研究显示， p38 MAPK和Smad信号通路的激活在介导TGF-β信号通路中具有不同的作用， 角膜上皮清创术和角膜切除术。这种差异可以解释为 清创术的愈合上皮在基底膜上迁移，而角膜切除术的愈合上皮在基底膜上迁移， 在裸露的间质胶原基质上迁移，导致不同的整合素表达模式， 在损伤后1小时内迁移上皮。因此，我们假设不同整合素与 TGF-β受体解释了TGF-β信号通路的差异，即，p38 MAPK的激活 角膜切除术中的上皮清创和Smads级联（假设1）。还发现 细胞增殖的抑制和活化转录因子2（ATF 2）的活化不依赖于 上皮清创后的TGF-β信号传导。因此，通过旁路途径激活ATF 2， 也就是说，JNK及其随后形成的活化蛋白-1转录因子（AP-1）复合物在调节细胞凋亡中起着关键作用。 在角膜损伤早期愈合阶段抑制上皮细胞增殖中的作用（假设 2）。具体目标1将确定和表征整合素在TGF-β信号通路中的作用， 使用三转基因Cre-LoxP小鼠模型的角膜上皮清创和角膜切除术的愈合，即， Krt 12 rtTA/rtTA/tet-O-Cre/Tbr 2f/f和Krt 12 rtTA/rtTA/tet-O-Cre/Smad 4f/f，其中floxed Tbr 2和Smad 4基因是 在多西环素诱导后在角膜上皮中特异性消融， 在Tbr 2和Smad 4（Aim 1A）存在和不存在的情况下，信号通路的变化，以检查 整合素在介导TGF-β受体信号传导（Aim 1B）中的作用，并检测p38 MAPK β和 Smad 7对伤口愈合期间细胞迁移和增殖的调节（Aim 1C）。具体目标2将 通过以下方法阐明ATF 2和AP-1在角膜伤口愈合过程中抑制细胞增殖的作用： 在角膜上皮清创术的愈合上皮中鉴定ATF 2和/或AP-1复合物， 使用免疫沉淀和蛋白质印迹分析（Aim 2A）进行角膜切除术，确定ATF 2的参与 在上皮清创愈合过程中，通过过表达显性 阴性ATF 2和<$N-ATF 2突变蛋白（Aim 2B），并确定JNK和p38 MAPK的作用 在角膜伤口愈合过程中抑制ATF 2的活化（Aim 2C）。 这些实验将产生有用的信息，用于通过干预TBR 2和 损伤角膜的ATF 2信号通路。