Characterization of the metalloprotease ADAMTS16 and its role in fibronectin assembly

Authors: Schnellmann, Rahel;
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Abstract

The A Disintegrin And Metalloproteinase with Thrombospondin Motif (ADAMTS) family is a family of extracellular metalloproteinases involved in the degradation and reorganization of the extracellular matrix (ECM). These proteinases play important roles during embryogenesis, wound healing and cancer progression. During cancer progression the degradation of the ECM by proteinases is a key step allowing cancer cells to leave the primary tumor site and invade the surrounding tissue. ADAMTS16 is a poorly characterized family member but although nothing is known about its substrates or its mechanism of action, several studies showed that ADAMTS16 plays a role during renal and gonadal development. In addition to its role during embryogenesis, ADAMTS16 has been implicated in blood pressure regulation. Moreover, several studies linked ADAMTS16 expression to cancer progression and metastasis. Thus, ADAMTS16 is well-connected to morphogenesis and human diseases, but without knowledge of its characteristics, substrates, and the molecular pathways in which it participates. We developed an assay using a decellularized ECM and liquid chromatography-mass spectrometry (LC-MS/MS) to identify substrates of ADAMTS16 and to gain further insights into its mechanism of action. Using this approach we identified fibronectin (FN) as a substrate of ADAMTS16. FN is one of the most abundant proteins within the ECM. FN fibers allow binding and fibrillogenesis of other ECM proteins such as fibrillins, collagens, tenascin-C and TGFβ-binding proteins and are therefore important components for the formation of an intact ECM. We showed that cleavage of FN near its N-terminus by ADAMTS16, leads to inhibition of FN fibril maturation, thereby having a strong impact on ECM assembly. Furthermore, we observed that ectopic ADAMTS16 expression in the epithelial cell line MDCKI strongly altered morphology when cultured in 3D collagen gels. MDCKI spheroids lacked FN in their matrix, resulting in smaller size and the formation of multiple lumina. FN is, however, not only important for ECM formation, but has a crucial role in cell adhesion, migration and cell signaling via binding to cell surface integrins. We could show that ADAMTS16 expression and the resulting cleavage of FN upregulates MMP3 expression in MDCKI cells, therefore creating an intriguing dual protease feed-forward loop that may serve to limit and fine-tune FN assembly and control tubular morphogenesis.