Roles of Growth Factors on Corneal Morphogenesis

生长因子对角膜形态发生的作用

• 批准号: 7583309
• 负责人: WINSTON W KAO
• 金额: $ 46.18万
• 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/7583309
• 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.

描述(申请人提供)：转化生长因子？(转化生长因子？)在角膜伤口愈合中起着关键作用。以往的研究表明，p38MAPK和Smad信号通路的激活在介导转化生长因子？角膜上皮清创术和角膜切削术的信号。造成这种差异的原因是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剥脱基质的胶原基质上迁移，导致在损伤后1小时内迁移上皮中整合素的表达模式不同。因此，我们假设不同的整合素与转化生长因子β？受体是导致转化生长因子-β的差异的原因。信号通路，即P38MAPK在上皮清除中的激活和Smads在角膜切除术中的级联(假设1)。也有研究发现，抑制细胞增殖和激活转录因子2(ATF2)不依赖于转化生长因子-β。上皮清创术后发出信号。因此，ATF2通过另一种途径即JNK激活，并随后形成激活蛋白-1转录因子(AP-1)复合体，在角膜损伤愈合早期抑制上皮细胞增殖中起关键作用(Hypotheisis 2)。具体目标1将确定和表征整合素在转化生长因子？Krt12rtTA/rtTA/tet-O-cre/Tbr2f/f和Krt12rtTA/rtTA/tet-O-cre/Smad4f/f三基因转基因小鼠角膜上皮清创和角膜切除过程中的信号转导通路研究：在多西环素诱导下，Tbr2和Smad4基因在角膜上皮细胞中被特异性地去除，以便人们能够确定在没有和存在Tbr2和Smad4(Aim 1A)的情况下信号转导通路的潜在变化，以研究整合素在介导转化生长因子？受体信号转导(Aim 1B)，并检测p38MAPK1和Smad7在伤口愈合过程中调节细胞迁移和增殖的有效性(Aim 1C)。通过免疫沉淀和免疫印迹分析鉴定ATF2和/或AP-1复合体，阐明ATF2和AP-1在角膜创伤愈合过程中细胞增殖抑制中的作用(Aim 2A)，确定ATF2通过过表达显性阴性的ATF2和？N-ATF2突变蛋白抑制角膜创伤愈合过程中细胞增殖的作用(Aim 2B)，以及JNK和p38MAPK抑制剂对角膜创伤愈合过程中ATF2激活的影响(Aim2C)。这些实验将通过干预损伤角膜的TBR2和ATF2信号通路，为恢复正常视力提供有用的信息。与公共卫生的相关性拟议的研究将检查转化生长因子-？利用有条件地过表达显性负性突变体和/或野生型信号转导分子的实验动物来调节角膜功能。腺病毒载体介导的受体介导的ATF2、p38MAPK、Smad7通路；多西环素诱导Krt12rtTA/rtTA/tet-O-Cre/Tbr2f/f、Krt12rtTA/RTTA/tet-O-Cre/Smad4f/f和Krt12rtTA/RTTA/tet-O-Cre/cJunf/f等转基因小鼠角膜上皮细胞中Tbr2、Smad4和cjun基因的条件性切除。建议的研究将填补我们对转化生长因子-？创伤愈合过程中角膜形态发生的信号转导以及成人的动态平衡。所获得的数据将为从分子和细胞水平更好地了解活体角膜疾病提供有用的信息，并为设计角膜伤口愈合的治疗方案提供依据。