PLOD2, a key factor for MRL MSC metabolism and chondroprotective properties.

BACKGROUND: Initially discovered for its ability to regenerate ear holes, the Murphy Roth Large (MRL) mouse has been the subject of multiple research studies aimed at evaluating its ability to regenerate other body tissues and at deciphering the mechanisms underlying it. These enhanced abilities to regenerate, retained during adulthood, protect the MRL mouse from degenerative diseases such as osteoarthritis (OA). Here, we hypothesized that mesenchymal stromal/stem cells (MSC) derived from the regenerative MRL mouse could be involved in their regenerative potential through the release of pro-regenerative mediators. METHOD: To address this hypothesis, we compared the secretome of MRL and BL6 MSC and identified several candidate molecules expressed at significantly higher levels by MRL MSC than by BL6 MSC. We selected one candidate, Plod2, and performed functional in vitro assays to evaluate its role on MRL MSC properties including metabolic profile, migration, and chondroprotective effects. To assess its contribution to MRL protection against OA, we used an experimental model for osteoarthritis induced by collagenase (CiOA). RESULTS: Among the candidate molecules highly expressed by MRL MSC, we focused our attention on procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2). Plod2 silencing induced a decrease in the glycolytic function of MRL MSC, resulting in the alteration of their migratory and chondroprotective abilities in vitro. In vivo, we showed that Plod2 silencing in MRL MSC significantly impaired their capacity to protect mouse from developing OA. CONCLUSION: Our results demonstrate that the chondroprotective and therapeutic properties of MRL MSC in the CiOA experimental model are in part mediated by PLOD2.

Lactate metabolism coordinates macrophage response and regeneration in zebrafish.

Rationale: Macrophages are multifunctional cells with a pivotal role on tissue development, homeostasis and regeneration. Indeed, in response to tissue injury and the ensuing regeneration process, macrophages are challenged and undergo massive metabolic adaptations and changes. However, the control of this metabolic reprogramming by macrophage microenvironment has never been deciphered in vivo. Methods: In this study, we used zebrafish model and caudal fin resection as a robust regeneration system. We explored specific changes in gene expression after tissue amputation via single-cell RNA sequencing analysis and whole-tissue transcriptomic analysis. Based on the identification of key modifications, we confirmed the role of the lactate pathway in macrophage response and fin regeneration, through the combination of chemical and genetic inhibitors of this pathway. Results: Single cell RNA sequencing revealed the upregulation of different genes associated with glycolysis and lactate metabolism in macrophages, upon fin regeneration. Hence, using chemical inhibitors of the LDH enzyme, we confirmed the role of lactate in macrophage recruitment and polarization, to promote a pro-inflammatory phenotype and enhance fin regeneration. The genetic modulation of monocarboxylate transporters illustrated a complex regulation of lactate levels, based on both intracellular and extracellular supplies. Commonly, the different sources of lactate resulted in macrophage activation with an increased expression level of inflammatory cytokines such as TNFa during the first 24 hours of regeneration. Transcriptomic analyses confirmed that lactate induced a global modification of gene expression in macrophages. Conclusion: Altogether, our findings highlight the crucial role of lactate at the onset of macrophage differentiation toward a pro-inflammatory phenotype. The deep modifications of macrophage phenotype mediated by lactate and downstream effectors play a key role to coordinate inflammatory response and tissue regeneration.

The hexosamine pathway and coat complex II promote malignant adaptation to nutrient scarcity.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.

Exploring Macrophage-Dependent Wound Regeneration During Mycobacterial Infection in Zebrafish.

The molecular and cellular mechanisms associated with tissue degradation or regeneration in an infectious context are poorly defined. Herein, we explored the role of macrophages in orchestrating either tissue regeneration or degradation in zebrafish embryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish were inoculated with different infectious doses of M. marinum prior to fin resection. While mild infection accelerated fin regeneration, moderate or severe infection delayed this process by reducing blastemal cell proliferation and impeding tissue morphogenesis. This was correlated with impaired macrophage recruitment at the wound of the larvae receiving high infectious doses. Macrophage activation characterized, in part, by a high expression level of tnfa was exacerbated in severely infected fish during the early phase of the regeneration process, leading to macrophage necrosis and their complete absence in the later phase. Our results demonstrate how a mycobacterial infection influences the macrophage response and tissue regenerative processes.

NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration.

Fish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

Pro-resolving mediator protectin D1 promotes epimorphic regeneration by controlling immune cell function in vertebrates.

BACKGROUND AND PURPOSE: Specialized pro-resolving mediators (SPMs) are a family of lipids controlling the resolution of inflammation and playing a role in many processes including organ protection and tissue repair. While SPMs are potent bioactive molecules in vivo, their role in epimorphic regeneration of organs in vertebrates has not been tested. Using the zebrafish larva as a robust regenerative vertebrate system, we studied the role of the SPM neuroprotectin/protectin D1 (PD1) during the caudal fin fold regeneration. EXPERIMENTAL APPROACH: Regeneration of the fin fold was analysed when exposed to a synthetic PD1. The effect of PD1 on immune cell recruitment and activation was further investigated using live imaging combined with fluorescent reporter lines. Using genetic and pharmacological approaches, we dissected the role of neutrophils and macrophages on driving the pro-regenerative effect of PD1. KEY RESULTS: We showed that PD1 improves fin fold regeneration. Acting in a narrow time window during regeneration, PD1 accelerates the resolution of inflammation without affecting the initial kinetic of neutrophil recruitment but instead, promotes their reverse migration potential. In addition, PD1 induces macrophage polarization switch towards non-inflammatory states in both zebrafish and mammalian system. Finally, macrophages but not neutrophils are essential for PD1-mediated regeneration. CONCLUSION AND IMPLICATIONS: These results reveal the pro-regenerative action of PD1 and its role in regulating neutrophil and macrophage response in vertebrates. These findings strongly support the development of pro-resolving mediators as natural therapeutic candidates for degenerative disorders and the use of the zebrafish as a tool to investigate pro-regenerative drugs.

ATF4-Dependent NRF2 Transcriptional Regulation Promotes Antioxidant Protection during Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress generates reactive oxygen species (ROS) that induce apoptosis if left unabated. To limit oxidative insults, the ER stress PKR-like endoplasmic reticulum Kinase (PERK) has been reported to phosphorylate and activate nuclear factor erythroid 2-related factor 2 (NRF2). Here, we uncover an alternative mechanism for PERK-mediated NRF2 regulation in human cells that does not require direct phosphorylation. We show that the activation of the PERK pathway rapidly stimulates the expression of NRF2 through activating transcription factor 4 (ATF4). In addition, NRF2 activation is late and largely driven by reactive oxygen species (ROS) generated during late protein synthesis recovery, contributing to protecting against cell death. Thus, PERK-mediated NRF2 activation encompasses a PERK-ATF4-dependent control of NRF2 expression that contributes to the NRF2 protective response engaged during ER stress-induced ROS production.

Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death.

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.

Bioinformatic screening of human ESTs for differentially expressed genes in normal and tumor tissues.

BACKGROUND: Owing to the explosion of information generated by human genomics, analysis of publicly available databases can help identify potential candidate genes relevant to the cancerous phenotype. The aim of this study was to scan for such genes by whole-genome in silico subtraction using Expressed Sequence Tag (EST) data. METHODS: Genes differentially expressed in normal versus tumor tissues were identified using a computer-based differential display strategy. Bcl-xL, an anti-apoptotic member of the Bcl-2 family, was selected for confirmation by western blot analysis. RESULTS: Our genome-wide expression analysis identified a set of genes whose differential expression may be attributed to the genetic alterations associated with tumor formation and malignant growth. We propose complete lists of genes that may serve as targets for projects seeking novel candidates for cancer diagnosis and therapy. Our validation result showed increased protein levels of Bcl-xL in two different liver cancer specimens compared to normal liver. Notably, our EST-based data mining procedure indicated that most of the changes in gene expression observed in cancer cells corresponded to gene inactivation patterns. Chromosomes and chromosomal regions most frequently associated with aberrant expression changes in cancer libraries were also determined. CONCLUSION: Through the description of several candidates (including genes encoding extracellular matrix and ribosomal components, cytoskeletal proteins, apoptotic regulators, and novel tissue-specific biomarkers), our study illustrates the utility of in silico transcriptomics to identify tumor cell signatures, tumor-related genes and chromosomal regions frequently associated with aberrant expression in cancer.