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

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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.