Roles of Growth Factors on Corneal Morphogenesis

生长因子对角膜形态发生的作用

• 批准号: 7743750
• 负责人: WINSTON W KAO
• 金额: $ 46.47万
• 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/7743750
• 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; Figs - dietary; Fluorescein; Fluoresceins; 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; Transforming Growth Factors; Transgenes; Tumor Suppressor Proteins; Variant; Virus; Vision; Western Blotting; Wild Type Mouse; Wound Healing; activating transcription factor; activating transcription factor 1; base; cell motility; corneal epithelium; design; effective therapy; feeding; healing; in vivo; inhibitor/antagonist; injured; member; migration; mouse model; mutant; neutralizing antibody; overexpression; public health relevance; receptor; repaired; research study; restoration; small hairpin RNA; therapy design; time interval; transcription factor

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.

性状（申请人提供）：转化生长因子？(TGF-?)在角膜伤口愈合中具有关键作用。以往的研究表明，p38 MAPK和Smad信号通路的激活在介导TGF-β 1和TGF-β 2的表达中具有不同的作用。角膜上皮清创术和角膜切除术的信号传导。这种差异可以解释的事实，清创愈合上皮迁移的基底膜，而角膜切除术迁移的剥脱基质的胶原基质，导致不同的整合素表达模式在迁移上皮损伤1小时内。因此，我们假设，不同的整合素与TGF-？受体占的差异，TGF-？信号通路，即，上皮清创术中p38 MAPK的激活和角膜切除术中Smads级联反应（假设1）。它也被发现，抑制细胞增殖和激活转录因子2（ATF 2）是独立的TGF-？上皮清创后的信号传导。因此，通过替代途径，即，JNK及其随后形成的活化蛋白-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）。具体目标1将确定和整合素在TGF-？在使用三转基因Cre-LoxP小鼠模型的角膜上皮清创术和角膜切除术的愈合期间的信号传导途径，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基因在多西环素诱导后在角膜上皮中特异性消融，从而可以确定在Tbr 2和Smad 4（Aim 1A）存在和不存在的情况下信号通路的潜在变化，以检查整合素在介导TGF-？受体信号传导（Aim 1B）和检测p38 MAPK 1和Smad 7在创伤愈合期间对细胞迁移和增殖的调节的功效（Aim 1C）。具体目标2将通过使用免疫沉淀和蛋白质印迹分析鉴定角膜上皮清创术和角膜切除术的愈合上皮中的ATF 2和/或AP-1复合物来阐明ATF 2和AP-1在角膜伤口愈合期间抑制细胞增殖中的作用（目标2A），通过显性阴性表达的过度表达，确定在上皮清创愈合过程中ATF 2参与抑制细胞增殖ATF 2和？N-ATF 2突变蛋白（Aim 2B），并确定JNK和p38 MAPK抑制剂对角膜伤口愈合期间ATF 2活化的影响（Aim 2C）。这些实验将为通过干预受损角膜的TBR 2和ATF 2信号通路来恢复正常视力提供有用的信息。公共卫生相关性拟议的研究将探讨TGF-？在调节角膜功能的实验动物，有条件地过度表达显性负突变和/或野生型信号转导分子的TGF-？受体介导的途径，例如，ATF 2、p38 MAPK、Smad 7的表达，以及基因的条件性切除，即，三转基因小鼠角膜上皮中的Tbr 2、Smad 4和cJun，多西环素诱导后的Krt 12 rtTA/rtTA/tet-O-Cre/Tbr 2f/f、Krt 12 rtTA/rtTA/tet-O-Cre/Smad 4f/f和Krt 12 rtTA/rtTA/tet-O-Cre/cJunf/f小鼠。拟议的研究将填补空白，我们的理解TGF-？在伤口愈合期间以及在成人中的内环境稳定中对角膜形态发生的信号传导。所获得的数据将产生有用的信息，更好地了解角膜疾病的分子和细胞水平在体内，并作为基础设计治疗方案的角膜伤口愈合。