Defining Key Roles for BMP1-like proteases and ECM in the formation, maintenance, and pathologies of skin and white adipose tissue

定义 BMP1 样蛋白酶和 ECM 在皮肤和白色脂肪组织的形成、维护和病理学中的关键作用

• 批准号: 9893628
• 负责人: DANIEL S GREENSPAN
• 金额: $ 20.46万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893628
• 关键词: Ablation; Adipocytes; Adipose tissue; Adult; Affect; Alleles; Alternative Splicing; Biological Assay; Biology; Birth; Cell Lineage; Cell physiology; Cells; Collaborations; Collagen; Communities; Complement; Cutaneous; Data; Defect; Dermal; Dermis; Desiccation; Development; Diabetes Mellitus; Disease; Epidermis; Epithelial; Epithelium; Excision; Extracellular Matrix; Family; Fatty acid glycerol esters; Fibroblasts; Financial compensation; Fractionation; Genes; Genotype; Growth; Growth Factor; Hair; Health; Heat Losses; Homeostasis; Immunosuppression; In Vitro; Infection; Knock-out; Knockout Mice; LoxP-flanked allele; Maintenance; Mass Spectrum Analysis; Modeling; Molecular; Morphology; Mouse Strains; Mus; Obesity; Pathologic; Pathology; Pathway interactions; Patients; Peptide Hydrolases; Phenocopy; Phenotype; Play; Population; Process; Protein Precursors; Proteins; Proteome; Research; Resolution; Resources; Role; Sampling; Signal Pathway; Skin; Skin injury; Skin repair; Skin wound healing; Technology; Testing; Therapeutic Intervention; Thinness; Tissues; Transgenes; Ubiquitin C; Vascular blood supply; adipocyte biology; adipocyte differentiation; antimicrobial peptide; base; conditional knockout; extracellular; high risk; in vivo; injury and repair; innovation; insight; interest; knock-down; novel; precursor cell; prevent; procollagen C-endopeptidase; promoter; skin disorder; skin wound; stem cell niche; stem cells; wound healing

Project Summary/Abstract Bone morphogenetic protein 1-like proteases (BMP1-LPs) play morphogenetic roles in many species, roles hypothesized to rely on the ability of BMP1-LPs to biosynthetically process protein precursors into mature components of the extracellular matrix (ECM), and to activate certain growth factors. The gene Bmp1 encodes alternatively spliced RNAs for BMP1-LPs BMP1 and mTLD, while gene Tll1 encodes BMP1-LP TLL1. These are the BMP1-LPs expressed in skin, and some of their predicted substrates suggest importance to skin formation, maintenance and pathologies. However, early lethality of Bmp1-/- and Tll1-/- mice previously impeded in vivo studies. To overcome early lethality barriers, we created mice with floxed Bmp1 and Tll1 alleles, and began studies by simultaneous/ubiquitous induced excision of both genes in a single mouse strain (“BTKO” mice), to avoid potential issues of functional overlap and possible compensation of tissue-specific knockdowns by circulating BMP1-LPs. BTKO mice were found to have markedly aberrant skin with severe thinning, seeming absence of morphologically identifiable dermal white adipose tissue (dWAT), abnormal collagen, and deficits in wound healing and in processing of the small number of candidate ECM substrates examined - demonstrating the latter to be actual in vivo BMP1-LP substrates. However, a global view of the full complement of skin proteins dependent on BMP1-LPs for biosynthetic processing is needed, to determine the full range of in vivo BMP1-LP roles in skin. Also to be determined is which BMP1-LP is responsible for which substrate in skin and for which previously observed deficits of uninjured or wounded BTKO skin. Recently, we successfully produced two separate mouse strains in which Bmp1 or Tll1 can be singly conditionally excised. We will employ these novel strains to determine which gene is responsible for which deficit observed in BTKO skin. We also recently found that mice with conditional knockout of the BMP1-LP substrate collagen V (colV) phenocopy the BTKO skin/adipose phenotype. The latter finding, along with findings of a colV role in maintaining stem cell pools, are the impetus for proposed high risk high impact studies testing our hypothesis that BMP1-LPs can affect WAT biology via crucial biosynthetic colV processing, allowing a colV cell-autonomous role in sustaining adipocyte stem cell (ASC) pools as part of the stem cell niche. Also proposed are studies of FACS-sorted preadipocytes and CD24+ ASCs from the above novel mouse strains, to determine the extent to which severe dWAT diminishment is due to disruption of proliferation and/or differentiation of these adipocyte-lineage cell populations. Studies will also include a cell/ECM sub-fractionation pipeline combined with cutting-edge high resolution/quantitative mass spectrometry to obtain global views of 1) BMP1-LP substrates of normal and wounded skin, and of preadipoctyes, and 2) proteins that are not BMP1-LP substrates, but have levels that significantly differ between genotypes, providing insights into pathways secondarily affected by BMP1-LPs. Also provided will be a high risk high impact attempt to obtain the proteome of rare CD24+ ASCs.

项目摘要/摘要 骨形态发生蛋白1类样蛋白（BMP1-LPS）在许多物种中扮演形态学作用，角色 假设依靠BMP1-LP的生物合成能力将蛋白质前体处理成成熟的能力 细胞外基质（ECM）的成分，并激活某些生长因子。基因BMP1编码 或者，用于BMP1-LPS BMP1和MTLD的剪接RNA，而基因TLL1编码BMP1-LP TLL1。这些 是在皮肤中表达的BMP1-LPS吗？它们的某些预测底物表明对皮肤很重要 形成，维护和病理。但是，BMP1 - / - 和TLL1 - / - 小鼠的早期致死性先前阻碍 体内研究。为了克服早期的致死性障碍，我们用Floxed BMP1和TLL1等位基因创建了小鼠，以及 通过简单/无处不在的单个小鼠菌株中这两个基因的惊喜开始研究（“ btko” 小鼠），以避免功能重叠的潜在问题和组织特异性敲低的补偿 通过循环BMP1-LP。发现BTKO小鼠的皮肤明显异常，稀疏，似乎 缺乏形态学上可识别的皮肤白色脂肪组织（DWAT），异常胶原蛋白，并定义 伤口愈合和处理少量候选ECM底物的处理 - 证明 后者是实际体内BMP1-LP底物。但是，全球景观完全完成皮肤 需要取决于BMP1-LP的蛋白质进行生物合成处理，以确定体内的全范围 BMP1-LP在皮肤中的作用。还要确定哪个BMP1-LP负责皮肤和 对于以前观察到的未受伤或受伤的BTKO皮肤的缺陷。最近，我们成功地生产了 BMP1或TLL1可以单独出色的两个单独的小鼠菌株。我们将采用这些 新型菌株以确定哪种基因负责在BTKO皮肤中观察到的缺陷。我们最近也 发现有条件敲除BMP1-LP底物胶原蛋白V（COLV）表观的小鼠BTKO 皮肤/脂肪表型。后来的发现，以及Colv在维持干细胞库中的作用的发现，是 提出的高风险高影响研究的推动力测试了我们的假设，即BMP1-LP会影响WAT 通过关键的生物合成COLV加工生物学，允许COLV细胞自主在维持脂肪细胞中的作用 干细胞（ASC）池作为干细胞生态位的一部分。还提出的是对FACS分级前脂肪细胞的研究 和上述新型小鼠菌株的CD24+ ASC，以确定严重的DWAT的程度 由于这些脂肪细胞细胞的增殖和/或分化的破坏是由于破坏的原因 人群。研究还将包括一个细胞/ECM子分割管道，并结合最前沿 分辨率/定量质谱法以获得1）正常和正常的BMP1-LP底物 受伤的皮肤和前脂肪的皮肤和2）不是BMP1-LP底物的蛋白质，但具有该水平 基因型之间的显着差异，提供了对受BMP1-LPS影响的途径的见解。 还提供了高风险的高风险尝试，以获取稀有CD24+ ASC的蛋白质组。