SUMMARY Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal, in a process that requires PARP activity. Whether regressed forks can efficiently restart and the factors required to restart fork progression after fork reversal are still unknown. Here we combined biochemical and electron microscopy approaches with single-molecule DNA fiber analysis, to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition, not shared by other human RecQ proteins. We show that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.

Vascular remodelling is a hallmark of pulmonary hypertension (PH) and is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates a crucial role of transcription factors in the vascular remodelling processes. Here, we characterize the involvement of meprin β, a novel activator protein-1 (AP-1) effector molecule, in PH. Fra-2 transgenic (TG) mice exhibited increased right ventricular systolic pressure (RVSP), accompanied by vascular remodelling and activation of the pro-proliferative and pro-fibrotic AKT pathway. Microarray studies revealed the collagen-processing metalloprotease meprin β as the most up-regulated gene in Fra-2 TG mice. Its expression, increased at all investigated time points, preceded the decreased expression of MMPs and increased TGFβ, followed by collagen deposition. Correspondingly, remodelled pulmonary arteries from explanted idiopathic pulmonary arterial hypertension (IPAH) patients' lungs exhibited pronounced expression of meprin β. Fra-2 and meprin β expression in human PASMCs was regulated by PDGF-BB and TGFβ in a complementary fashion. Importantly, PDGF-BB-dependent proliferation was attenuated by silencing AP-1 expression or by meprin β inhibition. This study delineates a novel molecular mechanism underlying PASMCs proliferation and extracellular matrix (ECM) deposition by identifying meprin β as an important mediator in regulating vascular remodelling processes. Thus, meprin β may represent a new molecule that can be targeted in pulmonary hypertension.

Lung fibrosis is a severe disease characterized by epithelial cell injury, inflammation and collagen deposition. The metalloproteases meprinα and meprinβ have been shown to enhance collagen maturation and inflammatory cell infiltration via cleavage of cell-cell contact molecules; therefore we hypothesized that meprins could play a role in lung fibrosis. An exhaustive characterization of bleomycin-treated meprinα, meprinβ and the double meprinsαβ knock-out (KO) with respective wt-littermates was performed by using several different methods. We observed no difference in lung function parameters and no change in inflammatory cells infiltrating the lung between wt and all meprins KO mice after 14 days bleomycin. No difference in epithelial integrity as assessed by e-cadherin protein level was detected in bleomycin-treated lungs. However, morphological analysis in the bleomycin-treated mice revealed decrease collagen deposition and tissue density in meprinβ KO, but not in meprinα and meprinαβ KO mice. This finding was accompanied by localization of meprinβ to epithelial cells in regions with immature collagen in mice. Similarly, in human IPF lungs meprinβ was mostly localized in epithelium. These findings suggest that local environment triggers meprinβ expression to support collagen maturation. In conclusion, our data demonstrate the in vivo relevance of meprinβ in collagen deposition in lung fibrosis.

ABSTRACT:The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin b and subsequent intramembrane proteolysis by g-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin b with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin b would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin b induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin b in Mep1b 2/2 mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin b could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin b inhibitortreated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin b and promotes transendothelial cell migration. FASEB J. 31, 1226-1237 (2017). www.fasebj.orgRegulated intramembrane proteolysis (RIP) of cell adhesion molecules, such as junctional adhesion molecule (JAM)-A, intercellular adhesion molecule (ICAM)-1, and L-selectin, was shown to be essential for transendothelial migration (TEM) of inflammatory or cancer cells (1). Meprin b, a multidomain type I transmembrane metalloprotease, is an initiator of RIP, and structural studies revealed dimeric formation of the protease with the active site in proximity to the cell surface (2-4). In addition, meprin b can be shed from the cell surface by ADAM10/ 17, resulting in a soluble active protease, which for instance is important for mucus detachment in the small intestine (5). Meprin b is characterized by a unique cleavage specificity, with a preference for negatively charged amino acids (6). These structural features provide all requirements that meprin b must have to act as an ectodomain sheddase at the cell surface. Indeed, membrane-bound amyloid precursor protein (APP), for instance, is cleaved by meprin b, resulting in the release of sAPP-b fragments and neurotoxic Ab peptides (4,7,8). Many of the known substrates of meprin b have been identified by mass spectrometry (MS)-based proteomic approaches (9).