Identification of PCPE-2 as the endogenous specific inhibitor of human BMP-1/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-ß superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.

Comparison of extracellular matrix enrichment protocols for the improved characterization of the skin matrisome by mass spectrometry.

A striking feature of skin organization is that the extracellular matrix (ECM) occupies a larger volume than the cells. Skin ECM also directly contributes to aging and most cutaneous diseases. In recent years, specific ECM enrichment protocols combined with in silico approaches allowed the proteomic description of the matrisome of various organs and tumor samples. Nevertheless, the skin matrisome remains under-studied and protocols allowing the efficient recovery of the diverse ECM found in skin are still to be described. Here, we compared four protocols allowing the enrichment of ECM proteins from adult mouse back skin and found that all protocols led to a significant enrichment (up to 65%) of matrisome proteins when compared to total skin lysates. The protocols based on decellularization and solubility profiling gave the best results in terms of numbers of proteins identified and confirmed that skin matrisome proteins exhibit very diverse solubility and abundance profiles. We also report the first description of the skin matrisome of healthy adult mice that includes 236 proteins comprising 95 core matrisome proteins and 141 associated matrisome proteins. These results provide a reliable basis for future characterizations of skin ECM proteins and their dysregulations in disease-specific contexts. SIGNIFICANCE: Extracellular matrix proteins are key players in skin physiopathology and have been involved in several diseases such as genetic disorders, wound healing defects, scleroderma and skin carcinoma. However, skin ECM proteins are numerous, diverse and challenging to analyze by mass spectrometry due to the multiplicity of their post-translational modifications and to the heterogeneity of their solubility profiles. Here, we performed the thorough evaluation of four ECM enrichment protocols compatible with the proteomic analysis of mouse back skin and provide the first description of the adult mouse skin matrisome in homeostasis conditions. Our work will greatly facilitate the future characterization of skin ECM alterations in preclinical mouse models and will inspire new optimizations to analyze the skin matrisome of other species and of human clinical samples.

BMP-1 disrupts cell adhesion and enhances TGF-ß activation through cleavage of the matricellular protein thrombospondin-1.

Bone morphogenetic protein 1 (BMP-1) is an important metalloproteinase that synchronizes growth factor activation with extracellular matrix assembly during morphogenesis and tissue repair. The mechanisms by which BMP-1 exerts these effects are highly context dependent. Because BMP-1 overexpression induces marked phenotypic changes in two human cell lines (HT1080 and 293-EBNA cells), we investigated how BMP-1 simultaneously affects cell-matrix interactions and growth factor activity in these cells. Increasing BMP-1 led to a loss of cell adhesion that depended on the matricellular glycoprotein thrombospondin-1 (TSP-1). BMP-1 cleaved TSP-1 between the VWFC/procollagen-like domain and the type 1 repeats that mediate several key TSP-1 functions. This cleavage induced the release of TSP-1 C-terminal domains from the extracellular matrix and abolished its previously described multisite cooperative interactions with heparan sulfate proteoglycans and CD36 on HT1080 cells. In addition, BMP-1-dependent proteolysis potentiated the TSP-1-mediated activation of latent transforming growth factor-ß (TGF-ß), leading to increased signaling through the canonical SMAD pathway. In primary human corneal stromal cells (keratocytes), endogenous BMP-1 cleaved TSP-1, and the addition of exogenous BMP-1 enhanced cleavage, but this had no substantial effect on cell adhesion. Instead, processed TSP-1 promoted the differentiation of keratocytes into myofibroblasts and stimulated production of the myofibroblast marker α-SMA, consistent with the presence of processed TSP-1 in human corneal scars. Our results indicate that BMP-1 can both trigger the disruption of cell adhesion and stimulate TGF-ß signaling in TSP-1-rich microenvironments, which has important potential consequences for wound healing and tumor progression.