Meryem Blati

Cartilage-specific deletion of mTOR upregulates autophagy and protects mice from osteoarthritis

Objectives Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo. Methods Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51–like kinase 1 (ULK1) siRNA to determine mTOR signalling. Results mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage. Conclusions This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.

PPARγ deficiency results in severe, accelerated osteoarthritis associated with aberrant mTOR signalling in the articular cartilage

Objectives We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. Methods Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. Results Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. Significance PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.

Corrigendum: ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis

This corrects the article DOI: 10.1038/nm.4419

ADAM10-mediated ephrin-B2 shedding promotes myofibroblast activation and organ fibrosis.

Maladaptive wound healing responses to chronic tissue injury result in organ fibrosis. Fibrosis, which entails excessive extracellular matrix (ECM) deposition and tissue remodeling by activated myofibroblasts, leads to loss of proper tissue architecture and organ function; however, the molecular mediators of myofibroblast activation have yet to be fully identified. Here we identify soluble ephrin-B2 (sEphrin-B2) as a new profibrotic mediator in lung and skin fibrosis. We provide molecular, functional and translational evidence that the ectodomain of membrane-bound ephrin-B2 is shed from fibroblasts into the alveolar airspace after lung injury. Shedding of sEphrin-B2 promotes fibroblast chemotaxis and activation via EphB3 and/or EphB4 receptor signaling. We found that mice lacking ephrin-B2 in fibroblasts are protected from skin and lung fibrosis and that a disintegrin and metalloproteinase 10 (ADAM10) is the major ephrin-B2 sheddase in fibroblasts. ADAM10 expression is increased by transforming growth factor (TGF)-β1, and ADAM10-mediated sEphrin-B2 generation is required for TGF-β1-induced myofibroblast activation. Pharmacological inhibition of ADAM10 reduces sEphrin-B2 levels in bronchoalveolar lavage and prevents lung fibrosis in mice. Consistent with the mouse data, ADAM10–sEphrin-B2 signaling is upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. These results uncover a new molecular mechanism of tissue fibrogenesis and identify sEphrin-B2, its receptors EphB3 and EphB4 and ADAM10 as potential therapeutic targets in the treatment of fibrotic diseases.

OP0209 Soluble Ephrin-B2 Ectodomain Contributes to the Pathogenesis of Systemic Sclerosis

Background Fibrotic diseases including systemic sclerosis (SSc) or scleroderma are characterized by fibroblast differentiation into myofibroblasts and extracellular matrix deposition. The mechanisms driving fibroblast activation are not fully known, and the identification of new profibrotic mediators will hopefully lead to the development of new rational anti- fibrotic treatments. Microarray studies show that ephrin-B2 is overexpressed in SSc fibroblasts, suggesting that this cell- membrane-anchored ligand may be a novel profibrotic mediator. Objectives To identify the role of ephrin B2 in skin and lung fibrosis. Methods Mice were injected subcutaneously with recombinant mouse ephrin-B2/Fc (100μg/kg/mouse) daily for two weeks and assessed for the development of skin fibrosis. Fibroblast-specific ephrin-B2 knockout (KO) mice were generated and assessed for the development of lung and skin fibrosis induced by intratracheal or subcutaneous injection of bleomycin respectively, by histology, hydroxyproline levels, q-PCR and western blot. Ephrin-B2 levels were determined by western blot and ELISA in broncoalveolar lavage (BAL) fluids from these mice, and from humans with SSc. Cultured mouse and human skin fibroblasts were treated with recombinant ephrin-B2-Fc (0.1-5 mg/mL) and the expression of profibrotic genes was assessed by qPCR and western blot. 96-Multiwell Insert Systems (BD Biosciences) were used to measure the chemotaxis of mouse skin fibroblasts from mice treated with either PBS or bleomycin for 7 days. Fibroblasts in these chemotaxis experiments were transfected with 20nM siRNA (Dharmacon) targeting EphB2, EphB3 or EphB4 receptors. Non-targeting siRNA was used as a control. Results Treatment of normal human skin fibroblasts with recombinant ephrin-B2 in vitro induced myofibroblast differentiation, as indicated by increased stress fiber formation, increased expression of α-SMA and collagen type I. Mice treated sub-cutaneously with recombinant mouse ephrin-B2/Fc exhibited significant skin fibrosis indicated by increased dermal thickness, collagen deposition, hydroxyproline content and α-SMA-expressing myofibroblasts. Fibroblast-specific ephrin-B2 KO mice were significantly protected from bleomycin-induced lung and skin fibrosis, as indicated by significant reductions in hydroxyproline and TGF-b levels. Q-PCR and immunohistochemical analyses showed that ephrin-B2 expression was elevated in SSc fibroblasts and skin sections from IPF patients respectively. Soluble ephrin-B2 levels determined by ELISA and western blot were increased in BAL from SSc patients and bleomycin-challenged mice. Soluble ephrin-B2 induced fibroblast chemotaxis (through EphB3/EphB4 but not EphB2 receptors), suggesting that pathological shedding of soluble ephrin-B2 might account for the profibrotic effect of this ligand in lung and skin fibrosis. Conclusions Our study identifies ephrin-B2 as a novel mediator of fibrogenesis, and suggests that targeting ephrin-B2 ligand or its binding receptors EphB3/EphB4 could potentially be a new therapeutic strategy in SSc. Disclosure of Interest None declared