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

项目概要/摘要 骨形态发生蛋白1-样蛋白酶（BMP 1-LPs）在许多物种的形态发生中发挥作用， 假设依赖于BMP 1-LP生物合成加工蛋白质前体成成熟蛋白质的能力， 细胞外基质（ECM）的成分，并激活某些生长因子。Bmp 1基因编码 BMP 1-LP BMP 1和mRNA的选择性剪接RNA，而基因Tll 1编码BMP 1-LP TLL 1。这些 BMP 1-LPs是在皮肤中表达的，它们的一些预测底物表明对皮肤的重要性 形成、维持和病理学。然而，Bmp 1-/-和Tll 1-/-小鼠的早期致死性先前阻碍了 体内研究。为了克服早期致死性障碍，我们制造了具有floxed Bmp 1和Tll 1等位基因的小鼠， 开始通过在单一小鼠品系中同时/普遍诱导切除两种基因（“BTKO”）来进行研究 小鼠），以避免功能重叠的潜在问题和组织特异性敲低的可能补偿 通过循环BMP 1-LP。发现BTKO小鼠具有明显异常的皮肤， 缺乏形态学可识别的真皮白色脂肪组织（dWAT）、异常胶原蛋白和 伤口愈合和少量候选ECM底物的处理中-证明 后者是实际的体内BMP 1-LP底物。然而，全面的皮肤补充观 需要依赖于BMP 1-LP进行生物合成加工的蛋白质，以确定体内的全部范围。 BMP 1-LP在皮肤中的作用。还有待确定的是哪种BMP 1-LP负责皮肤中的哪种底物， 对于先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 两个单独的小鼠品系，其中Bmp 1或Tll 1可以被单独条件性切除。我们将利用这些 新菌株用于确定哪个基因导致BTKO皮肤中观察到的哪个缺陷。我们最近还 发现条件性敲除BMP 1-LP底物胶原V（colV）的小鼠表型模仿BTKO， 皮肤/脂肪表型。后一个发现，沿着colV在维持干细胞库中的作用， 提出高风险高影响研究的动力，以检验我们的假设，即BMP 1-LPs可以影响WAT 生物学通过关键的生物合成colV处理，允许colV细胞自主作用， 干细胞（ASC）池作为干细胞龛的一部分。还提出了FACS分选的前脂肪细胞的研究 和来自上述新小鼠品系的CD 24 + ASC，以确定严重dWAT 减少是由于这些脂肪细胞谱系细胞的增殖和/或分化被破坏 人口。研究还将包括一个细胞/ECM亚分级管道结合尖端的高 分辨率/定量质谱法，以获得1）正常和正常的BMP 1-LP底物的全局视图， 受伤的皮肤和前脂肪组织，和2）不是BMP 1-LP底物的蛋白质，但其水平 基因型之间的差异显著，提供了对BMP 1-LPs次要影响途径的见解。 还将提供一个高风险高影响的尝试，以获得罕见的CD 24 + ASC的蛋白质组。

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.