Systemic Collagen VII Replacement Therapy for Advanced Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin blistering disease associated with progressive multiorgan fibrosis. RDEB is caused by biallelic mutations in COL7A1 encoding the extracellular matrix protein collagen VII (C7), which is necessary for epidermal‒dermal adherence. C7 is not simply a structural protein but also has multiple functions, including the regulation of TGFß bioavailability and the inhibition of skin scarring. Intravenous (IV) administration of recombinant C7 (rC7) rescues C7-deficient mice from neonatal lethality. However, the effect on established RDEB has not been determined. In this study, we used small and large adult RDEB animal models to investigate the disease-modulating abilities of IV rC7 on established RDEB. In adult RDEB mice, rC7 accumulated at the basement membrane zone in multiple organs after a single infusion. Fortnightly IV injections of rC7 for 7 weeks in adult RDEB mice reduced fibrosis of skin and eye. The fibrosis-delaying effect was associated with a reduction of TGFß signaling. IV rC7 in adult RDEB dogs incorporated in the dermal‒epidermal junction of skin and improved disease by promoting wound healing and reducing dermal‒epidermal separation. In both species, IV C7 was well-tolerated. These preclinical studies suggest that repeated IV administration of rC7 is an option for systemic treatment of established adult RDEB.

Increased abundance of Cbl E3 ligases alters PDGFR signaling in recessive dystrophic epidermolysis bullosa.

In recessive dystrophic epidermolysis bullosa (RDEB), loss of collagen VII, the main component of anchoring fibrils critical for epidermal-dermal cohesion, affects several intracellular signaling pathways and leads to impaired wound healing and fibrosis. In skin fibroblasts, wound healing is also affected by platelet-derived growth factor receptor (PDGFR) signaling. To study a potential effect of loss of collagen VII on PDGFR signaling we performed unbiased disease phosphoproteomics. Whereas RDEB fibroblasts exhibited an overall weaker response to PDGF, Cbl E3 ubiquitin ligases, negative regulators of growth factor signaling, were stronger phosphorylated. This increase in phosphorylation was linked to higher Cbl mRNA and protein levels due to increased TGFß signaling in RDEB. In turn, increased Cbl levels led to increased PDGFR ubiquitination, internalization, and degradation negatively affecting MAPK and AKT downstream signaling pathways. Thus, our results indicate that elevated TGFß signaling leads to an attenuated response to growth factors, which contributes to impaired dermal wound healing in RDEB.

Pro-inflammatory immunity supports fibrosis advancement in epidermolysis bullosa: intervention with Ang-(1-7).

Recessive dystrophic epidermolysis bullosa (RDEB), a genetic skin blistering disease, is a paradigmatic condition of tissue fragility-driven multi-organ fibrosis. Here, longitudinal analyses of the tissue proteome through the course of naturally developing disease in RDEB mice revealed that increased pro-inflammatory immunity associates with fibrosis evolution. Mechanistically, this fibrosis is a consequence of altered extracellular matrix organization rather than that of increased abundance of major structural proteins. In a humanized system of disease progression, we targeted inflammatory cell fibroblast communication with Ang-(1-7)-an anti-inflammatory heptapeptide of the renin-angiotensin system, which reduced the fibrosis-evoking aptitude of RDEB cells. In vivo, systemic administration of Ang-(1-7) efficiently attenuated progression of multi-organ fibrosis and increased survival of RDEB mice. Collectively, our study shows that selective down-modulation of pro-inflammatory immunity may mitigate injury-induced fibrosis. Furthermore, together with published data, our data highlight molecular diversity among fibrotic conditions. Both findings have direct implications for the design of therapies addressing skin fragility and fibrosis.

Mouse models for dominant dystrophic epidermolysis bullosa carrying common human point mutations recapitulate the human disease.

Heterozygous missense mutations in the human COL7A1 gene - coding for collagen VII - lead to the rare, dominantly inherited skin disorder dominant dystrophic epidermolysis bullosa (DDEB), which is characterised by skin fragility, blistering, scarring and nail dystrophy. To better understand the pathophysiology of DDEB and develop more effective treatments, suitable mouse models for DDEB are required but to date none have existed. We identified the two most common COL7A1 mutations in DDEB patients (p.G2034R and p.G2043R) and used CRISPR-Cas9 to introduce the corresponding mutations into mouse Col7a1 (p.G2028R and p.G2037R). Dominant inheritance of either of these two alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of epidermolysis bullosa, which frequently show early lethality and severe disease, these mouse models, which to our knowledge are the first for DDEB, show no reduction in growth and survival, and - together with a relatively mild phenotype - represent a practically and ethically tractable tool for better understanding and treating epidermolysis bullosa. This article has an associated First Person interview with the first author of the paper.

Raft-like lipid microdomains drive autophagy initiation via AMBRA1-ERLIN1 molecular association within MAMs.

Mitochondria-associated membranes (MAMs) are essential communication subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. We previously demonstrated that, upon macroautophagy/autophagy induction, AMBRA1 is recruited to the BECN1 complex and relocalizes to MAMs, where it regulates autophagy by interacting with raft-like components. ERLIN1 is an endoplasmic reticulum lipid raft protein of the prohibitin family. However, little is known about its association with the MAM interface and its involvement in autophagic initiation. In this study, we investigated ERLIN1 association with MAM raft-like microdomains and its interaction with AMBRA1 in the regulation of the autophagic process. We show that ERLIN1 interacts with AMBRA1 at MAM raft-like microdomains, which represents an essential condition for autophagosome formation upon nutrient starvation, as demonstrated by knocking down ERLIN1 gene expression. Moreover, this interaction depends on the "integrity" of key molecules, such as ganglioside GD3 and MFN2. Indeed, knocking down ST8SIA1/GD3-synthase or MFN2 expression impairs AMBRA1-ERLIN1 interaction at the MAM level and hinders autophagy. In conclusion, AMBRA1-ERLIN1 interaction within MAM raft-like microdomains appears to be pivotal in promoting the formation of autophagosomes.Abbreviations: ACSL4/ACS4: acyl-CoA synthetase long chain family member 4; ACTB/ß-actin: actin beta; AMBRA1: autophagy and beclin 1 regulator 1; ATG14: autophagy related 14; BECN1: beclin 1; CANX: calnexin; Cy5: cyanine 5; ECL: enhanced chemiluminescence; ER: endoplasmic reticulum; ERLIN1/KE04: ER lipid raft associated 1; FB1: fumonisin B1; FE: FRET efficiency; FRET: Förster/fluorescence resonance energy transfer; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GD3: aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)ceramide; HBSS: Hanks' balanced salt solution; HRP: horseradish peroxidase; LMNB1: lamin B1; mAb: monoclonal antibody; MAMs: mitochondria-associated membranes; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MYC/cMyc: proto-oncogene, bHLH transcription factor; P4HB: prolyl 4-hydroxylase subunit beta; pAb: polyclonal antibody; PE: phycoerythrin; SCAP/SREBP: SREBF chaperone; SD: standard deviation; ST8SIA1: ST8 alpha-N-acetyl-neuraminide alpha-2,8 sialyltransferase 1; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; TUBB/beta-tubulin: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VDAC1/porin: voltage dependent anion channel 1.

Proteomic Profiling of Fibroblasts Isolated from Chronic Wounds Identifies Disease-Relevant Signaling Pathways.

Chronic skin wounds accompany many prevalent age-related diseases and are a major cause of morbidity and mortality. Both keratinocytes and fibroblasts contribute to the pathomechanisms in chronic skin wounds. Dysregulated pathways in the epidermis have been extensively studied, but little is known of the influence of dermal fibroblasts on chronic wounding. We isolated fibroblasts from chronic wounds, propagated them in vitro, and analyzed them using proteomic profiling in combination with functional characterization of the proteomic changes. Chronic wound-associated fibroblasts exhibit a unique proteome profile characteristic of lysosomal dysfunction and dysregulated TGFß signaling. They display a decreased propensity for cell proliferation and migration, combined with an enhanced ability to contract the extracellular matrix. With these properties, chronic wound-associated fibroblasts actively contribute to pathological inabilities to close wounds and represent potential targets for pharmacological interference for changing cellular phenotypes.

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling.

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

Generation of rabbit polyclonal human and murine collagen VII monospecific antibodies: A useful tool for dystrophic epidermolysis bullosa therapy studies.

High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.

Influenza A Virus Induces Autophagosomal Targeting of Ribosomal Proteins.

Seasonal epidemics of influenza A virus are a major cause of severe illness and are of high socio-economic relevance. For the design of effective antiviral therapies, a detailed knowledge of pathways perturbed by virus infection is critical. We performed comprehensive expression and organellar proteomics experiments to study the cellular consequences of influenza A virus infection using three human epithelial cell lines derived from human lung carcinomas: A549, Calu-1 and NCI-H1299. As a common response, the type I interferon pathway was up-regulated upon infection. Interestingly, influenza A virus infection led to numerous cell line-specific responses affecting both protein abundance as well as subcellular localization. In A549 cells, the vesicular compartment appeared expanded after virus infection. The composition of autophagsomes was altered by targeting of ribosomes, viral mRNA and proteins to these double membrane vesicles. Thus, autophagy may support viral protein translation by promoting the clustering of the respective molecular machinery in autophagosomes in a cell line-dependent manner.

Impaired lymphoid extracellular matrix impedes antibacterial immunity in epidermolysis bullosa.

Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB), a skin fragility disorder that, unexpectedly, manifests also with elevated colonization of commensal bacteria and frequent wound infections. Here, we describe an unprecedented systemic function of collagen VII as a member of a unique innate immune-supporting multiprotein complex in spleen and lymph nodes. In this complex, collagen VII specifically binds and sequesters the innate immune activator cochlin in the lumen of lymphoid conduits. In genetic mouse models, loss of collagen VII increased bacterial colonization by diminishing levels of circulating cochlin LCCL domain. Intraperitoneal injection of collagen VII, which restored cochlin in the spleen, but not in the skin, reactivated peripheral innate immune cells via cochlin and reduced bacterial skin colonization. Systemic administration of the cochlin LCCL domain was alone sufficient to diminish bacterial supercolonization of RDEB mouse skin. Human validation demonstrated that RDEB patients displayed lower levels of systemic cochlin LCCL domain with subsequently impaired macrophage response in infected wounds. This study identifies an intrinsic innate immune dysfunction in RDEB and uncovers a unique role of the lymphoid extracellular matrix in systemic defense against bacteria.

Identification of tissue damage, extracellular matrix remodeling and bacterial challenge as common mechanisms associated with high-risk cutaneous squamous cell carcinomas.

In this study we used a genetic extracellular matrix (ECM) disease to identify mechanisms associated with aggressive behavior of cutaneous squamous cell carcinoma (cSCC). cSCC is one of the most common malignancies and usually has a good prognosis. However, some cSCCs recur or metastasize and cause significant morbidity and mortality. Known factors that are associated with aggressiveness of cSCCs include tumor grading, size, localization and microinvasive behavior. To investigate molecular mechanisms that influence biologic behavior we used global proteomic and histologic analyses of formalin-fixed paraffin-embedded tissue of primary human cSCCs. We compared three groups: non-recurring, non-metastasizing low-risk sporadic cSCCs; metastasizing sporadic cSCCs; and cSCCs from patients with recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a genetic skin blistering and ECM disease caused by collagen VII deficiency. Patients commonly suffer from high-risk early onset cSCCs that frequently metastasize. The results indicate that different processes are associated with formation of RDEB cSCCs compared to sporadic cSCCs. Sporadic cSCCs show signs of UV damage, whereas RDEB cSCCs have higher mutational rates and display tissue damage, inflammation and subsequent remodeling of the dermal ECM as tumor initiating factors. Interestingly the two high-risk groups - high-risk metastasizing sporadic cSCCs and RDEB cSCCs - are both associated with tissue damage and ECM remodeling in gene-ontology enrichment and Search Tool for the Retrieval of Interacting Genes/Proteins analyses. In situ histologic analyses validate these results. The high-risk cSCCs also show signatures of enhanced bacterial challenge. Histologic analyses confirm correlation of bacterial colonization with worse prognosis. Collectively, this unbiased study - performed directly on human patient material - reveals that common microenvironmental alterations linked to ECM remodeling and increased bacterial challenges are denominators of high-risk cSCCs. The proteins identified here could serve as potential diagnostic markers and therapeutic targets in high-risk cSCCs.

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy.

Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy.

Respiratory status determines the effect of emodin on cell viability.

The anthraquinone emodin has been shown to have antineoplastic properties and a wealth of unconnected effects have been linked to its use, most of which are likely secondary outcomes of the drug treatment. The primary activity of emodin on cells has remained unknown. In the present study we demonstrate dramatic and extensive effects of emodin on the redox state of cells and on mitochondrial homeostasis, irrespectively of the cell type and organism, ranging from the yeast Saccharomyces cerevisiae to human cell lines and primary cells. Emodin binds to redox-active enzymes and its effectiveness depends on the oxidative and respiratory status of cells. We show that cells with efficient respiratory metabolism are less susceptible to emodin, whereas cells under glycolytic metabolism are more vulnerable to the compound. Our findings indicate that emodin acts in a similar way as known uncouplers of the mitochondrial electron transport chain and causes oxidative stress that particularly disturbs cancer cells.

Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition.

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC-PCP-SILAC, we analyzed protein-protein interactions of hyperactive BRAFV600E and wild-type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K-RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

Inhibition of ß-catenin signaling by phenobarbital in hepatoma cells in vitro.

The antiepileptic drug phenobarbital (PB) exerts hepatic effect based on indirect activation of the constitutive androstane receptor (CAR) via inhibition of the epidermal growth factor receptor (EGFR) and the kinase Src. It has furthermore been observed that in mice PB suppresses the growth of hepatocellular carcinoma with overactive signaling through the oncogenic Wnt/ß-catenin pathway, thus suggesting an interference of PB with ß-catenin signaling. The present work was aimed to characterize effects of PB on ß-catenin signaling at different cellular levels and to elucidate molecular details of the interaction of PB and ß-catenin in an in vitro system of mouse hepatoma cells. PB efficiently inhibited signaling through ß-catenin. This phenomenon was in-depth characterized at the levels of ß-catenin protein accumulation and transcriptional activity. Mechanistic analyses revealed that the effect of PB on ß-catenin signaling was independent of the activation of CAR and also independent of the cytosolic multi-protein complex responsible for physiological post-translation control of the ß-catenin pathway via initiation of ß-catenin degradation. Instead, evidence is provided that PB diminishes ß-catenin protein production by inhibition of protein synthesis via signal transduction through EGFR and Src. The proposed mechanism is well in agreement with previously published activities of PB at the EGFR and Src-mediated regulation of ß-catenin mRNA translation. Inhibition of ß-catenin signaling by PB through the proposed mechanism might explain the inhibitory effect of PB on the growth of specific sub-populations of mouse liver tumors. In conclusion, the present data comprehensively characterize the effect of PB on ß-catenin signaling in mouse hepatoma cells in vitro and provides mechanistic insight into the molecular processes underlying the observed effect.

Axitinib and sorafenib are potent in tyrosine kinase inhibitor resistant chronic myeloid leukemia cells.

BACKGROUND: Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl. Bcr-Abl tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), revolutionized CML therapy. Nevertheless, about 20 % of CMLs display primary or acquired TKI resistance. TKI resistance can be either caused by mutations within the Bcr-Abl kinase domain or by aberrant signaling by its effectors, e.g. Lyn or Gab2. Bcr-Abl mutations are frequently observed in TKI resistance and can only in some cases be overcome by second line TKIs. In addition, we have previously shown that the formation of Gab2 complexes can be regulated by Bcr-Abl and that Gab2 signaling counteracts the efficacy of four distinct Bcr-Abl inhibitors. Therefore, TKI resistance still represents a challenge for disease management and alternative therapies are urgently needed. FINDINGS: Using different CML cell lines and models, we identified the clinically approved TKIs sorafenib (SF) and axitinib (AX) as drugs overcoming the resistance mediated by the Bcr Abl(T315I) mutant as well as the one mediated by Gab2 and Lyn(Y508F). In addition, we demonstrated that AX mainly affects the Bcr-Abl/Grb2/Gab2 axis, whereas SF seems to act independently of the fusion kinase and most likely by blocking signaling pathways up- and downstream of Gab2. CONCLUSION: We demonstrate that SF and AX show potency in various and mechanistically distinct scenarios of TKI resistance, including Bcr-Abl(T315I) as well as Lyn- and Gab2-mediated resistances. Our data invites for further evaluation und consideration of these inhibitors in the treatment of TKI resistant CML.

AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation.

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

AMBRA1 interplay with cullin E3 ubiquitin ligases regulates autophagy dynamics.

Autophagy maintains cellular homeostasis by degrading harmful or unnecessary intracellular components. How the autophagy response is induced rapidly and transiently remains largely unknown. We report that the E3 ubiquitin ligases Cullin-5 and Cullin-4 regulate the onset and termination of autophagy, respectively, by dynamically interacting with AMBRA1, a regulator of autophagy. Under normal conditions, Cullin-4 binding to AMBRA1 limits its protein abundance. Autophagy stimuli promote AMBRA1 stabilization by causing ULK1-dependent Cullin-4 release. Notably, Cullin-4/AMBRA1 dissociation is transient, and the re-established interaction triggers AMBRA1 degradation, terminating the autophagy response. Moreover, Cullin-4 inhibits the interaction between AMBRA1 and another Cullin E3 ligase. Indeed, upon Cullin-4 dissociation, AMBRA1 binds and inhibits Cullin-5, thus promoting the accumulation of the mTOR inhibitor DEPTOR. Through DEPTOR stabilization, AMBRA1 establishes a feedback loop that ensures the rapid onset of autophagy by enhancing mTOR inactivation. Our findings show that Cullin-mediated degradation of autophagy regulators temporally controls the autophagy response.