Role of P311 in the development of skin hypertrophic scars

P311 在皮肤增生性疤痕形成中的作用

• 批准号: 8917276
• 负责人: Lucia Schuger
• 金额: $ 30.11万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8917276
• 关键词: 5' Untranslated Regions; Amino Acid Sequence; Antibodies; Binding; Biological Assay; Bleomycin; Burn injury; Caliber; Cicatrix; Co-Immunoprecipitations; Collagen; Complex; D-Aspartic Acid; Data; Densitometry; Deposition; Dermal; Dermis; Development; Epidermis; Eukaryotic Initiation Factors; Fibroblasts; Fibrosis; Glutamic Acid; Growth Factor; Healed; Health; Histologic; Human; Hyperplasia; Hypertrophic Cicatrix; Immunoblotting; Immunohistochemistry; Immunoprecipitation; In Vitro; Incidence; Injury; Lead; Mediating; Messenger RNA; Modeling; Mus; Myofibroblast; Peptides; Pilot Projects; Play; Primary Cell Cultures; Production; Proline; Proteins; Protocols documentation; RNA; RNA Interference; Reading; Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine; Site; Skin; Small Interfering RNA; Subcutaneous Tissue; Subfamily lentivirinae; Surface Plasmon Resonance; System; Testing; Thick; Threonine; Topical application; Transforming Growth Factor beta; Transgenic Mice; Translations; Trichrome stain; Untranslated Regions; Western Blotting; Work; Wound Healing; base; biglycan; decorin; effective therapy; healing; in vivo; keratinocyte; member; mouse development; mutant; novel; overexpression; peptide A; prevent; protein protein interaction; response; tissue culture; tissue repair; vector; wound

DESCRIPTION (provided by applicant): Hypertrophic scars (HSs) occur at a high incidence after burns and lack effective treatment. TGF-�, a powerful profibrogenic growth factor, plays an important role in HS development. We recently demonstrated that P311, an 8 kDa intracellular protein present in human HSs, controls TGF-�1-3 level/activity by stimulating their translation, and preliminary work suggested that P311 directly binds to eukaryotic translation initiation factor 3b (eIF3b). We developed a murine model of HS by topical application of bleomycin (BLM) on skin excisional wounds. P311 is expressed in these HSs, but not in normal scars. Pilot studies suggested that P311 KO mice are protected against BLM-induced HSs, while P311 transgenic mice (P311 TGs) develop HSs in the absence of BLM. P311 has a conserved, PEST-like motif, known to mediate protein-protein interactions. A peptide representing the P311 PEST-like motif or a P311 siRNA, each of them conjugated to the cellular penetrating peptide TAT47-57 (TAT47-57P311PEST and TAT47-57P311siRNA), targeted P311 and reduced TGF-�s levels in vitro, moreover, the TAT47-57P311siRNA ameliorated BLM-induced HSs. Based on the above, we hypothesize that P311 promotes HSs by binding to eIF3b and thereby stimulating TGF-�s translation; and that topical application of TAT47-57P311PEST or TAT47-57P311siRNA will protect mice from developing HSs. The specific aims to test this hypothesis are: 1. To determine the involvement of P311 in mouse HSs and whether it is mediated by the P311 PEST-like domain. We will characterize the BLM-treated scars of P311 KO mice and WTs, the scars of P311 TGs, and the effect of P311 and P311 PEST mutants intradermally delivered at the wound site. The read-outs will include histological quantification of fibrosis using the Aperio system pls immunoblot/densitometry; quantification of myofibroblasts; and determination of P311 and TGF-�1-3 mRNAs and protein levels. Mouse and human keratinocyte (KT) and dermal fibroblast (DF) response to P311 will be studied in primary tissue cultures 2. To determine the effect of P311 and its PEST-like domain in TGF-�s translation by mouse and human keratinocytes (KTs) and dermal fibroblasts (DFs), and whether it is mediated by P311 interaction with eIF3b. We will employ KT and DF primary cultures, P311 and P311 PEST mutants delivered by lentivirus transfer, and purified P311 and eIF3b. Among the read outs will be TGF-� 1-3 translation reporter assays, co-immunoprecipitations, protein pull-downs, use of surface plasmon resonance, RNA immunoprecipitation assays and TGF-�1-3 translation reporter assays after eIF3b knockdown by RNA interference. 3. To determine whether topically-delivered TAT47-57P311PEST and TAT47-57P311siRNA are effective in preventing BLM-induced HSs. Protocols for the topical delivery of each of these two compounds in P311 TG mice wounds/scars and BLM-treated wounds/scars will be optimized and their effect in preventing the development of HSs will be determined/compared. Read-outs will be the same as for aim #1.

描述（适用提供）：肥厚疤痕（HSS）发生在烧伤后的高事件中，缺乏有效的治疗。 TGF-是一种强大的能力生长因子，在HS发育中起着重要作用。我们最近证明，p311是人类HSS中存在的8 kDa细胞内蛋白，通过刺激其翻译来控制TGF-�1-3水平/活性，并且初步工作表明p311直接与真核翻译起始因子结合。 3B（EIF3B）。我们通过局部应用博来霉素（BLM）在皮肤上的伤口上开发了HS的鼠模型。 P311在这些HSS中表达，但在正常疤痕中不表示。试点研究表明，P311 KO小鼠受到BLM诱导的HSS的保护，而p311转基因小鼠（P311 TGS）在没有BLM的情况下会发展HSS。 p311具有保守的害虫样基序，已知可以介导蛋白质 - 蛋白质相互作用。代表p311害虫状的ob或p311 siRNA的胡椒粉，每个siRNA都与细胞穿透性肽Tat47-57（Tat47-57p311pest和Tat47-57p311sirna）相结合，靶向p311，并降低了TGF-s水平的tgf-5 HSS。基于上述内容，我们假设p311通过与EIF3B结合并刺激TGF-的翻译来促进HSS。 tat47-57p311pest或tat47-57p311sirna的局部应用将保护小鼠免受发展HSS的影响。检验该假设的具体目的是：1。确定p311在小鼠HSS中的参与以及它是否由p311害虫样域介导。我们将表征P311 KO小鼠和WTS的BLM处理的疤痕，p311 TGS的疤痕以及在伤口部位递送的p311和p311害虫突变体的效果。读出将包括使用Aperio System PLS免疫印迹/光密度测定法进行纤维化的组织学定量；定量肌纤维细胞；以及p311和TGF-�1-3mRNA和蛋白质水平的测定。 Mouse and human keratinocyte (KT) and dermal fibroblast (DF) response to P311 will be studiod in primary tissue cultures 2. To determine the effect of P311 and its PEST-like domain in TGF-�s translation by mouse and human keratinocytes (KTs) and dermal fibroblasts (DFs), and whether it is mediated by P311 interaction with EIF3B。我们将采用KT和DF原发性培养物，P311和P311害虫突变体，该突变体通过慢病毒转移传递，以及纯化的P311和EIF3B。在读取中，将包括TGF-�1-3翻译报告基因测定，共免疫沉淀，蛋白质下拉，使用表面等离子体共振，RNA免疫沉淀测定和TGF-�1-3Transertation Reporter分析eIF3B在EIF3B敲低后，由RNA造成RNA干扰。 3。确定局部传递的Tat47-57p311pest和Tat47-57p311sirna是否有效防止BLM诱导的HSS。 P311 TG小鼠伤口/疤痕和BLM治疗的伤口/疤痕中这两种化合物中每种化合物的局部递送方案将得到优化，并将确定/比较它们在防止HSS发展的效果。读出将与AIM＃1相同。