ADAMTS12 is a stromal modulator in chronic liver disease.

Adamalysins, a family of metalloproteinases containing a disintegrin and metalloproteinases (ADAMs) and ADAM with thrombospondin motifs (ADAMTSs), belong to the matrisome and play important roles in various biological and pathological processes, such as development, immunity and cancer. Using a liver cancer dataset from the International Cancer Genome Consortium, we developed an extensive in silico screening that identified a cluster of adamalysins co-expressed in livers from patients with hepatocellular carcinoma (HCC). Within this cluster, ADAMTS12 expression was highly associated with recurrence risk and poorly differentiated HCC signatures. We showed that ADAMTS12 was expressed in the stromal cells of the tumor and adjacent fibrotic tissues of HCC patients, and more specifically in activated stellate cells. Using a mouse model of carbon tetrachloride-induced liver injury, we showed that Adamts12 was strongly and transiently expressed after a 24 h acute treatment, and that fibrosis was exacerbated in Adamts12-null mice submitted to carbon tetrachloride-induced chronic liver injury. Using the HSC-derived LX-2 cell line, we showed that silencing of ADAMTS12 resulted in profound changes of the gene expression program. In particular, genes previously reported to be induced upon HSC activation, such as PAI-1, were mostly down-regulated following ADAMTS12 knock-down. The phenotype of these cells was changed to a less differentiated state, showing an altered actin network and decreased nuclear spreading. These phenotypic changes, together with the down-regulation of PAI-1, were offset by TGF-ß treatment. The present study thus identifies ADAMTS12 as a modulator of HSC differentiation, and a new player in chronic liver disease.

The transcription factor c-Jun inhibits RBM39 to reprogram pre-mRNA splicing during genotoxic stress.

Genotoxic agents, that are used in cancer therapy, elicit the reprogramming of the transcriptome of cancer cells. These changes reflect the cellular response to stress and underlie some of the mechanisms leading to drug resistance. Here, we profiled genome-wide changes in pre-mRNA splicing induced by cisplatin in breast cancer cells. Among the set of cisplatin-induced alternative splicing events we focused on COASY, a gene encoding a mitochondrial enzyme involved in coenzyme A biosynthesis. Treatment with cisplatin induces the production of a short isoform of COASY lacking exons 4 and 5, whose depletion impedes mitochondrial function and decreases sensitivity to cisplatin. We identified RBM39 as a major effector of the cisplatin-induced effect on COASY splicing. RBM39 also controls a genome-wide set of alternative splicing events partially overlapping with the cisplatin-mediated ones. Unexpectedly, inactivation of RBM39 in response to cisplatin involves its interaction with the AP-1 family transcription factor c-Jun that prevents RBM39 binding to pre-mRNA. Our findings therefore uncover a novel cisplatin-induced interaction between a splicing regulator and a transcription factor that has a global impact on alternative splicing and contributes to drug resistance.

The Dual Function of RhoGDI2 in Immunity and Cancer.

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases. It is highly expressed in hematopoietic cells but is also present in a large array of other cell types. RhoGDI2 has been implicated in multiple human cancers and immunity regulation, where it can display a dual role. Despite its involvement in various biological processes, we still do not have a clear understanding of its mechanistic functions. This review sheds a light on the dual opposite role of RhoGDI2 in cancer, highlights its underappreciated role in immunity and proposes ways to explain its intricate regulatory functions.

Cleavage of LOXL1 by BMP1 and ADAMTS14 Proteases Suggests a Role for Proteolytic Processing in the Regulation of LOXL1 Function.

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.

Lipin-1, a Versatile Regulator of Lipid Homeostasis, Is a Potential Target for Fighting Cancer.

The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated.

Differential cleavage of lysyl oxidase by the metalloproteinases BMP1 and ADAMTS2/14 regulates collagen binding through a tyrosine sulfate domain.

Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.

Proteomic discovery of substrates of the cardiovascular protease ADAMTS7.

The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729-Val-730 and Glu-732-Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-ß-binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1-binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.

Integration of miRNA-regulatory networks in hepatic stellate cells identifies TIMP3 as a key factor in chronic liver disease.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSC) is a critical process involved in liver fibrosis. Several miRNAs are implicated in gene regulation during this process but their exact and respective contribution is still incompletely understood. Here we propose an integrative approach of miRNA-regulatory networks to predict new targets. METHODS: miRNA regulatory networks in activated HSCs were built using lists of validated miRNAs and the CyTargetLinker tool. The resulting graphs were filtered according to public transcriptomic data and the reduced graphs were analysed through GO annotation. A miRNA network regulating the expression of TIMP3 was further studied in human liver samples, isolated hepatic cells and mouse model of liver fibrosis. RESULTS: Within the up-regulated miRNAs, we identified a subnetwork of five miRNAs (miR-21-5p, miR-222-3p, miR-221-3p miR-181b-5p and miR-17-5p) that target TIMP3. We demonstrated that TIMP3 expression is inversely associated with inflammatory activity and IL1-ß expression in vivo. We further showed that IL1-ß inhibits TIMP3 expression in HSC-derived LX-2 cells. Using data from The Cancer Genome Atlas (TCGA), we showed that, in hepatocellular carcinoma (HCC), TIMP3 expression is associated with survival (P < .001), while miR-221 (P < .05), miR-222 (P < .01) and miR-181b (P < .01) are markers for a poor prognosis. CONCLUSIONS: Several miRNAs targeting TIMP3 are up-regulated in activated HSCs and down-regulation of TIMP3 expression is associated with inflammatory activity in liver fibrosis and poor prognosis in HCC. The regulatory network including specific miRNAs and TIMP3 is therefore central for the evolution of chronic liver disease.

Design of Degradable Polyphosphoester Networks with Tailor-Made Stiffness and Hydrophilicity as Scaffolds for Tissue Engineering.

In the recent decades, biodegradable and biocompatible polyphosphoesters (PPEs) have gained wide attention in the biomedical field as relevant substitutes for conventional aliphatic polyesters. These amorphous materials of low glass transition temperature offer promise for the design of soft scaffolds for tissue engineering. Advantageously, the easy variation of the nature of the lateral pendant groups of PPEs allows the insertion of pendent unsaturations valuable for their further cross-linking. In addition, varying the length of the pendent alkyl chains allows tuning their hydrophilicity. The present work aims at synthesizing PPE networks of well-defined hydrophilicity and mechanical properties. More precisely, we aimed at preparing degradable materials exhibiting identical hydrophilicity but different mechanical properties and vice versa. For that purpose, PPE copolymers were synthesized by ring-opening copolymerization of cyclic phosphate monomers bearing different pendent groups (e.g., methyl, butenyl, and butyl). After UV irradiation, a stable and well-defined cross-linked material is obtained with the mechanical property of the corresponding polymer films controlled by the composition of the starting PPE copolymer. The results demonstrate that cross-linking density could be correlated with the mechanical properties, swelling behavior, and degradation rate of the polymers network. The polymers were compatible to human skin fibroblast cells and did not exhibit significant cytotoxicity up to 0.5 mg mL-1. In addition, degradation products appeared nontoxic to skin fibroblast cells and showed their potential as promising scaffolds for tissue engineering.

Platelet-rich plasma (PRP) and tendon healing: comparison between fresh and frozen-thawed PRP.

Platelet-rich plasma (PRP) is increasingly used in the treatment of musculoskeletal diseases. Its preservation by freezing it for the realization of multiple injections in clinical use has never been discussed. Calcaneal tendons of rats were surgically sectioned. Platelet concentration of the PRP was 2.5 x 106/µl with autologous plasma of rats. Frozen-thawed PRP was prepared by performing two cycles of freezing and thawing on PRP aliquots. Both platelet preparations were injected in the lesion. Biomechanical and histological evaluations were carried out after 7, 20 or 40 days post surgery. After 7 and 40 days, no significant difference was observed between the PRP and the frozen-thawed PRP group. There is however a difference 20 days after surgery: the ultimate tensile strength (UTS) was greater in the fresh PRP group. No obvious difference with histological aspect was observed between the two groups. In conclusion, fresh PRP and frozen-thawed PRP injections can lead to similar results in the healing process of section calcaneal tendons of rats. Improvements with fresh PRP are slight. PRP could thus be frozen to be preserved if multiple injections are needed (e.g. osteoarthritis).