Role of histamine H4 receptor in the anti-inflammatory pathway of glucocorticoid-induced leucin zipper (GILZ) in a model of lung fibrosis.

INTRODUCTION: This study investigates the interactions between histaminergic system and glucocorticoid-induced leucin zipper (GILZ) in the inflammatory process and glucocorticoid modulation in lung fibrosis. METHODS: Wild-type (WT) and GILZ Knock-Out (KO) mice were treated with bleomycin (0.05 IU) or saline, delivered by intra-tracheal injection. After surgery, mice received a continuous infusion of JNJ7777120 (JNJ, 2 mg/kg b.wt.) or vehicle for 21 days. Lung function was studied by measuring airway resistance to air insufflation through the analysis of pressure at airway opening (PAO). Lung samples were collected to evaluate the expression of histamine H4R, Anx-A1, and p65-NF-kB, the activity of myeloperoxidase (MPO), and the production of pro-inflammatory cytokines. RESULTS: Airway fibrosis and remodeling were assessed by measuring TGF-ß production and α-SMA deposition. JNJ reduces PAO in WT but not in GILZ KO mice (from 22 ± 1 mm to 15 ± 0.5 and from 24 ± 1.5 to 19 ± 0.5 respectively), MPO activity (from 204 ± 3.13 pmol/mg to 73.88 ± 2.63 in WT and from 221 ± 4.46 pmol/mg to 107 ± 5.54 in GILZ KO), the inflammatory response, TGF-ß production, and α-SMA deposition in comparison to WT and GILZ KO vehicle groups. CONCLUSION: In conclusion, the role of H4R and GILZ in relation to glucocorticoids could pave the way for innovative therapies to counteract pulmonary fibrosis.

Anti-Inflammatory Effects of Synthetic Peptides Based on Glucocorticoid-Induced Leucine Zipper (GILZ) Protein for the Treatment of Inflammatory Bowel Diseases (IBDs).

Glucocorticoids (GCs) are commonly used to treat autoimmune and inflammatory diseases, but their clinical effects and long-term use can lead to serious side effects. New drugs that can replace GCs are needed. Glucocorticoid-induced leucine zipper (GILZ) is induced by GCs and mediates many of their anti-inflammatory effects, such as inhibiting the pro-inflammatory molecule NF-κB. The GILZ C-terminal domain (PER region) is responsible for GILZ/p65NF-κB interaction and consequent inhibition of its transcriptional activity. A set of five short peptides spanning different parts of the PER region of GILZ protein was designed, and their anti-inflammatory activity was tested, both in vitro and in vivo. We tested the biological activity of GILZ peptides in human lymphocytic and monocytic cell lines to evaluate their inhibitory effect on the NF-κB-dependent expression of pro-inflammatory cytokines. Among the tested peptides, the peptide named PEP-1 demonstrated the highest efficacy in inhibiting cell activation in vitro. Subsequently, PEP-1 was further evaluated in two in vivo experimental colitis models (chemically induced by DNBS administration and spontaneous colitis induced in IL-10 knock-out (KO) mice (to assess its effectiveness in counteracting inflammation. Results show that PEP-1 reduced disease severity in both colitis models associated with reduced NF-κB pro-inflammatory activity in colon lamina propria lymphocytes. This study explored GILZ-based 'small peptides' potential efficacy in decreasing lymphocyte activation and inflammation associated with experimental inflammatory bowel diseases (IBDs). Small peptides have several advantages over the entire protein, including higher selectivity, better stability, and bioavailability profile, and are easy to synthesize and cost-effective. Thus, identifying active GILZ peptides could represent a new class of drugs for treating IBD patients.

Cardiolipin-mediated temporal response to hydroquinone toxicity in human retinal pigmented epithelial cell line.

Hydroquinone, a potent toxic agent of cigarette smoke, damages retinal pigmented epithelial cells by triggering oxidative stress and mitochondrial dysfunction, two events causally related to the development and progression of retinal diseases. The inner mitochondrial membrane is enriched in cardiolipin, a phospholipid susceptible of oxidative modifications which determine cell-fate decision. Using ARPE-19 cell line as a model of retinal pigmented epithelium, we analyzed the potential involvement of cardiolipin in hydroquinone toxicity. Hydroquinone exposure caused an early concentration-dependent increase in mitochondrial reactive oxygen species, decrease in mitochondrial membrane potential, and rise in the rate of oxygen consumption not accompanied by changes in ATP levels. Despite mitochondrial impairment, cell viability was preserved. Hydroquinone induced cardiolipin translocation to the outer mitochondrial membrane, and an increase in the colocalization of the autophagosome adapter protein LC3 with mitochondria, indicating the induction of protective mitophagy. A prolonged hydroquinone treatment induced pyroptotic cell death by cardiolipin-mediated caspase-1 and gasdermin-D activation. Cardiolipin-specific antioxidants counteracted hydroquinone effects pointing out that cardiolipin can act as a mitochondrial "eat-me signal" or as a pyroptotic cell death trigger. Our results indicate that cardiolipin may act as a timer for the mitophagy to pyroptosis switch and propose cardiolipin-targeting compounds as promising approaches for the treatment of oxidative stress-related retinal diseases.

Optimizing therapeutic outcomes of immune checkpoint blockade by a microbial tryptophan metabolite.

BACKGROUND: Despite the great success, the therapeutic benefits of immune checkpoint inhibitors (ICIs) in cancer immunotherapy are limited by either various resistance mechanisms or ICI-associated toxic effects including gastrointestinal toxicity. Thus, novel therapeutic strategies that provide manageable side effects to existing ICIs would enhance and expand their therapeutic efficacy and application. Due to its proven role in cancer development and immune regulation, gut microbiome has gained increasing expectation as a potential armamentarium to optimize immunotherapy with ICI. However, much has to be learned to fully harness gut microbiome for clinical applicability. Here we have assessed whether microbial metabolites working at the interface between microbes and the host immune system may optimize ICI therapy. METHODS: To this purpose, we have tested indole-3-carboxaldehyde (3-IAld), a microbial tryptophan catabolite known to contribute to epithelial barrier function and immune homeostasis in the gut via the aryl hydrocarbon receptor (AhR), in different murine models of ICI-induced colitis. Epithelial barrier integrity, inflammation and changes in gut microbiome composition and function were analyzed. AhR, indoleamine 2,3-dioxygenase 1, interleukin (IL)-10 and IL-22 knockout mice were used to investigate the mechanism of 3-IAld activity. The function of the microbiome changes induced by 3-IAld was evaluated on fecal microbiome transplantation (FMT). Finally, murine tumor models were used to assess the effect of 3-IAld treatment on the antitumor activity of ICI. RESULTS: On administration to mice with ICI-induced colitis, 3-IAld protected mice from intestinal damage via a dual action on both the host and the microbes. Indeed, paralleling the activation of the host AhR/IL-22-dependent pathway, 3-IAld also affected the composition and function of the microbiota such that FMT from 3-IAld-treated mice protected against ICI-induced colitis with the contribution of butyrate-producing bacteria. Importantly, while preventing intestinal damage, 3-IAld did not impair the antitumor activity of ICI. CONCLUSIONS: This study provides a proof-of-concept demonstration that moving past bacterial phylogeny and focusing on bacterial metabolome may lead to a new class of discrete molecules, and that working at the interface between microbes and the host immune system may optimize ICI therapy.

Glucocorticoid-Induced Leucine Zipper (GILZ) in Cardiovascular Health and Disease.

Glucocorticoids (GCs) are essential in regulating functions and homeostasis in many biological systems and are extensively used to treat a variety of conditions associated with immune/inflammatory processes. GCs are among the most powerful drugs for the treatment of autoimmune and inflammatory diseases, but their long-term usage is limited by severe adverse effects. For this reason, to envision new therapies devoid of typical GC side effects, research has focused on expanding the knowledge of cellular and molecular effects of GCs. GC-induced leucine zipper (GILZ) is a GC-target protein shown to mediate several actions of GCs, including inhibition of the NF-κB and MAPK pathways. GILZ expression is not restricted to immune cells, and it has been shown to play a regulatory role in many organs and tissues, including the cardiovascular system. Research on the role of GILZ on endothelial cells has demonstrated its ability to modulate the inflammatory cascade, resulting in a downregulation of cytokines, chemokines, and cellular adhesion molecules. GILZ also has the capacity to protect myocardial cells, as its deletion makes the heart, after a deleterious stimulus, more susceptible to apoptosis, immune cell infiltration, hypertrophy, and impaired function. Despite these advances, we have only just begun to appreciate the relevance of GILZ in cardiovascular homeostasis and dysfunction. This review summarizes the current understanding of the role of GILZ in modulating biological processes relevant to cardiovascular biology.

LINE-1 transcription in round spermatids is associated with accretion of 5-carboxylcytosine in their open reading frames.

Chromatin of male and female gametes undergoes a number of reprogramming events during the transition from germ cell to embryonic developmental programs. Although the rearrangement of DNA methylation patterns occurring in the zygote has been extensively characterized, little is known about the dynamics of DNA modifications during spermatid maturation. Here, we demonstrate that the dynamics of 5-carboxylcytosine (5caC) correlate with active transcription of LINE-1 retroelements during murine spermiogenesis. We show that the open reading frames of active and evolutionary young LINE-1s are 5caC-enriched in round spermatids and 5caC is eliminated from LINE-1s and spermiogenesis-specific genes during spermatid maturation, being simultaneously retained at promoters and introns of developmental genes. Our results reveal an association of 5caC with activity of LINE-1 retrotransposons suggesting a potential direct role for this DNA modification in fine regulation of their transcription.

Glucocorticoid-induced leucine zipper regulates liver fibrosis by suppressing CCL2-mediated leukocyte recruitment.

Liver fibrosis (LF) is a dangerous clinical condition with no available treatment. Inflammation plays a critical role in LF progression. Glucocorticoid-induced leucine zipper (GILZ, encoded in mice by the Tsc22d3 gene) mimics many of the anti-inflammatory effects of glucocorticoids, but its role in LF has not been directly addressed. Here, we found that GILZ deficiency in mice was associated with elevated CCL2 production and pro-inflammatory leukocyte infiltration at the early LF stage, resulting in enhanced LF development. RNA interference-mediated in vivo silencing of the CCL2 receptor CCR2 abolished the increased leukocyte recruitment and the associated hepatic stellate cell activation in the livers of GILZ knockout mice. To highlight the clinical relevance of these findings, we found that TSC22D3 mRNA expression was significantly downregulated and was inversely correlated with that of CCL2 in the liver samples of patients with LF. Altogether, these data demonstrate a protective role of GILZ in LF and uncover the mechanism, which can be targeted therapeutically. Therefore, modulating GILZ expression and its downstream targets represents a novel avenue for pharmacological intervention for treating LF and possibly other liver inflammatory disorders.

Deficit of glucocorticoid-induced leucine zipper amplifies angiotensin-induced cardiomyocyte hypertrophy and diastolic dysfunction.

Poor prognosis in heart failure and the lack of real breakthrough strategies validate targeting myocardial remodelling and the intracellular signalling involved in this process. So far, there are no effective strategies to counteract hypertrophy, an independent predictor of heart failure progression and death. Glucocorticoid-induced leucine zipper (GILZ) is involved in inflammatory signalling, but its role in cardiac biology is unknown. Using GILZ-knockout (KO) mice and an experimental model of hypertrophy and diastolic dysfunction, we addressed the role of GILZ in adverse myocardial remodelling. Infusion of angiotensin II (Ang II) resulted in myocardial dysfunction, inflammation, apoptosis, fibrosis, capillary rarefaction and hypertrophy. Interestingly, GILZ-KO showed more evident diastolic dysfunction and aggravated hypertrophic response compared with WT after Ang II administration. Both cardiomyocyte and left ventricular hypertrophy were more pronounced in GILZ-KO mice. On the other hand, Ang II-induced inflammatory and fibrotic phenomena, cell death and reduction in microvascular density, remained invariant between the WT and KO groups. The analysis of regulators of hypertrophic response, GATA4 and FoxP3, demonstrated an up-regulation in WT mice infused with Ang II; conversely, such an increase did not occur in GILZ-KO hearts. These data on myocardial response to Ang II in mice lacking GILZ indicate that this protein is a new element that can be mechanistically involved in cardiovascular pathology.

Liver haploinsufficiency of RuvBL1 causes hepatic insulin resistance and enhances hepatocellular carcinoma progression.

RuvBL1 is an AAA+ ATPase whose expression in hepatocellular carcinoma (HCC) correlates with a poor prognosis. In vitro models suggest that targeting RuvBL1 could be an effective strategy against HCC. However, the role of RuvBL1 in the onset and progression of HCC remains unknown. To address this question, we developed a RuvBL1hep+/- mouse model and evaluated the outcome of DEN-induced liver carcinogenesis up to 12 months of progression. We found that RuvBL1 haploinsufficiency initially delayed the onset of liver cancer, due to a reduced hepatocyte turnover in RuvBL1hep+/- mice. However, RuvBL1hep+/- mice eventually developed HCC nodules that, with aging, grew larger than in the control mice. Moreover, RuvBL1hep+/- mice developed hepatic insulin resistance and impaired glucose homeostasis. We could determine that RuvBL1 regulates insulin signaling through the Akt/mTOR pathway in liver physiology in vivo as well as in normal hepatocytic and HCC cells in vitro. Whole transcriptome analysis of mice livers confirmed the major role of RuvBL1 in the regulation of hepatic glucose metabolism. Finally, RuvBL1 expression was found significantly correlated to glucose metabolism and mTOR signaling by bioinformatic analysis of human HCC sample from the publicly available TGCA database. These data uncover a role of RuvBL1 at the intersection of liver metabolism, hepatocyte proliferation and HCC development, providing a molecular rationale for its overexpression in liver cancer.

Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome.

BACKGROUND: Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. RESULTS: MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. CONCLUSIONS: Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies.

GPBAR1 Functions as Gatekeeper for Liver NKT Cells and provides Counterregulatory Signals in Mouse Models of Immune-Mediated Hepatitis.

BACKGROUND & AIMS: GPBAR1, also known as TGR5, is a G protein-coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. MATERIAL AND METHODS: Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. RESULTS: In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1-/- mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. CONCLUSION: Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity.

Glucocorticoid-Induced Leucine Zipper: A Novel Anti-inflammatory Molecule.

Glucocorticoids (GCs) are the most commonly used drugs for treatment of autoimmune and inflammatory diseases. Their efficacy is due to their ability to bind cytoplasmic receptors (glucocorticoid receptors, GR) and other cytoplasmic proteins, thus regulating gene expression. Although GCs are potent life-saving drugs, their therapeutic effects are transitory and chronic use of GCs is accompanied by serious side effects. Therefore, new drugs are needed to replace GCs. We have identified a gene, glucocorticoid-induced leucine zipper (GILZ or tsc22d3), that is rapidly and invariably induced by GCs. Human GILZ is a 135-amino acid protein that mediates many GC effects, including inhibition of the NF-κB and MAPK pathways. Similar to GCs, GILZ exerts anti-inflammatory activity in experimental disease models, including inflammatory bowel diseases and arthritis. While transgenic mice that overexpress GILZ are more resistant, GILZ knockout mice develop worse inflammatory diseases. Moreover, the anti-inflammatory effect of GCs is attenuated in GILZ-deficient mice. Importantly, in vivo delivery of recombinant GILZ protein cured colitis and facilitated resolution of lipopolysaccharide-induced inflammation without apparent toxic effects. A synthetic GILZ-derived peptide, corresponding to the GILZ region that interacts with NF-κB, was able to suppress experimental autoimmune encephalomyelitis. Collectively, these findings indicate that GILZ is an anti-inflammatory molecule that may serve as the basis for designing new therapeutic approaches to inflammatory diseases.

Selective CB2 inverse agonist JTE907 drives T cell differentiation towards a Treg cell phenotype and ameliorates inflammation in a mouse model of inflammatory bowel disease.

Cannabinoids are known to possess anti-inflammatory and immunomodulatory properties, but the mechanisms involved are not fully understood. CB2 is the cannabinoid receptor that is expressed primarily on hematopoietic cells and mediates the immunoregulatory functions of cannabinoids. In order to study the effect of JTE907, a selective/inverse agonist of CB2 with anti-inflammatory properties, on the differentiation of T cell subtypes, we used an in vitro system of Th lineage-specific differentiation of naïve CD4+ T lymphocytes isolated from the mouse spleen. The results indicate that JTE907 was able to induce the differentiation of Th0 cells into the Treg cell phenotype, which was characterized by the expression of FoxP3, TGF-ß and IL-10. P38 phosphorylation and STAT5A activation were found to mediate the signaling pathway triggered by JTE907 via the CB2 receptor in Th0 lymphocytes. In mice with DNBS-induced colitis, JTE907 treatment was able to induce an increase in the number of CD4+CD25+FoxP3+ cells in the lamina propria after 24 h of disease onset and reduce disease severity after 48 h. Further, longer JTE907 treatment resulted in less severe colitis even when administered orally, resulting in less body weight loss, reduction of the disease score, prevention of NF-κB activation, and reduction of the expression of adhesion molecules. Collectively, the results of this study indicate that specific signals delivered through the CB2 receptor can drive the immune response towards the Treg cell phenotype. Thus, ligands such as JTE907 may have use as potential therapeutic agents in autoimmune and inflammatory diseases.

Probing Internalization Effects and Biocompatibility of Ultrasmall Zirconium Metal-Organic Frameworks UiO-66 NP in U251 Glioblastoma Cancer Cells.

The synthesis of ultrasmall UiO-66 nanoparticles (NPs) with an average size of 25 nm, determined by X-ray powder diffraction and electron microscopies analysis, is reported. The NPs were stabilized in water by dialyzing the NP from the DMF used for the synthesis. DLS measurements confirmed the presence of particles of 100 nm, which are spherical aggregates of smaller particles of 20⁻30 nm size. The NP have a BET surface area of 700 m²/g with an external surface area of 300 m²/g. UiO-66_N (UiO-66 nanoparticles) were loaded with acridine orange as fluorescent probe. UV-vis spectroscopy analysis revealed no acridine loss after 48 h of agitation in simulated body fluid. The biocompatibility of UiO-66_N was evaluated in human glioblastoma (GBM) cell line U251, the most malignant (IV grade of WHO classification) among brain tumors. In U251 cells, UiO-66_N are inert since they do not alter the cell cycle, the viability, migration properties, and the expression of kinases involved in cancer cell growth. The internalization process was evident after a few hours of incubation. After 24 h, UiO-66_N@Acr (UiO-66_N loaded with acridine orange) were detectable around the nuclei of the cells. These data suggest that small UiO-66 are biocompatible NP and could represent a potential carrier for drug delivery in glioblastoma therapies.

Glucocorticoid-Induced Leucine Zipper Inhibits Interferon-Gamma Production in B Cells and Suppresses Colitis in Mice.

Glucocorticoid-induced leucine zipper (GILZ) is transcriptionally upregulated by glucocorticoids (GCs) and mediates many of the anti-inflammatory effects of GCs. Since B cell activity has been linked to cytokine production and modulation of inflammatory responses, we herein investigated the role of GILZ in B cells during colitis development. B cell-specific gilz knock-out (gilz B cKO) mice exhibited increased production of the pro-inflammatory cytokine IFN-γ in B cells, and consequently CD4+ T cell activation. Increased IFN-γ production in B cells was associated with enhanced transcriptional activity of the transcription factor activator protein-1 (AP-1) on the IFN-γ promoter. Moreover, GILZ deficiency in B cells was linked to enhanced susceptibility to experimental colitis in mice, and this was reversed by administering GILZ protein. Interestingly, we observed increased production of IFN-γ in both B and T cells infiltrating the lamina propria (LP) of gilz B cKO mice. Together, these findings indicate that GILZ controls IFN-γ production in B cells, which also affects T cell activity, and increased production of IFN-γ by B and T cells in LP is associated with predisposition to inflammatory colitis in mice.

Glucocorticoids, Sex Hormones, and Immunity.

Glucocorticoid hormones regulate essential body functions in mammals, control cell metabolism, growth, differentiation, and apoptosis. Importantly, they are potent suppressors of inflammation, and multiple immune-modulatory mechanisms involving leukocyte apoptosis, differentiation, and cytokine production have been described. Due to their potent anti-inflammatory and immune-suppressive activity, synthetic glucocorticoids (GCs) are the most prescribed drugs used for treatment of autoimmune and inflammatory diseases. It is long been noted that males and females exhibit differences in the prevalence in several autoimmune diseases (AD). This can be due to the role of sexual hormones in regulation of the immune responses, acting through their endogenous nuclear receptors to mediate gene expression and generate unique gender-specific cellular environments. Given the fact that GCs are the primary physiological anti-inflammatory hormones, and that sex hormones may also exert immune-modulatory functions, the link between GCs and sex hormones may exist. Understanding the nature of this possible crosstalk is important to unravel the reason of sexual disparity in AD and to carefully prescribe these drugs for the treatment of inflammatory diseases. In this review, we discuss similarities and differences between the effects of sex hormones and GCs on the immune system, to highlight possible axes of functional interaction.

PP242 Counteracts Glioblastoma Cell Proliferation, Migration, Invasiveness and Stemness Properties by Inhibiting mTORC2/AKT.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and is associated with poor prognosis due to its thorny localization, lack of efficacious therapies and complex biology. Among the numerous pathways driving GBM biology studied so far, PTEN/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) signaling plays a pivotal role, as it controls cell survival, proliferation and metabolism and is involved in stem cell maintenance. In front of recent and numerous evidences highlighting mTOR upregulation in GBM, all the strategies developed to inhibit this pathway have been substantially unsuccessful. Our study focused on mTOR complex 2 (mTORC2) to understand its involvement in GBM cell growth, proliferation, migration and invasiveness. We utilized an in vitro model, characterized by various genetic alterations (i.e., GL15, U257, U87MG and U118MG cell lines) in order to achieve the clonal heterogeneity observed in vivo. Additionally, being the U87MG cell line endowed with glioblastoma stem cells (GSCs), we also investigated the role of the PTEN/PI3K/AKT/mTOR pathway in this specific cell population, which is responsible for GBM relapse. We provide further insights that explain the reasons for the failure of numerous clinical trials conducted to date targeting PI3K or mTOR complex 1 (mTORC1) with rapamycin and its analogs. Additionally, we show that mTORC2 might represent a potential clinically valuable target for GBM treatment, as proliferation, migration and GSC maintenance appear to be mTORC2-dependent. In this context, we demonstrate that the novel ATP-competitive mTOR inhibitor PP242 effectively targets both mTORC1 and mTORC2 activation and counteracts cell proliferation via the induction of high autophagy levels, besides reducing cell migration, invasiveness and stemness properties.

Levels of S100B protein drive the reparative process in acute muscle injury and muscular dystrophy.

Regeneration of injured skeletal muscles relies on a tightly controlled chain of cellular and molecular events. We show that appropriate levels of S100B protein are required for timely muscle regeneration after acute injury. S100B released from damaged myofibers and infiltrating macrophages expands the myoblast population, attracts macrophages and promotes their polarization into M2 (pro-regenerative) phenotype, and modulates collagen deposition, by interacting with RAGE (receptor for advanced glycation end-products) or FGFR1 (fibroblast growth factor receptor 1) depending on the muscle repair phase and local conditions. However, persistence of high S100B levels compromises the regeneration process prolonging myoblast proliferation and macrophage infiltration, delaying M1/M2 macrophage transition, and promoting deposition of fibrotic tissue via RAGE engagement. Interestingly, S100B is released in high abundance from degenerating muscles of mdx mice, an animal model of Duchenne muscular dystrophy (DMD), and blocking S100B ameliorates histopathology. Thus, levels of S100B differentially affect skeletal muscle repair upon acute injury and in the context of muscular dystrophy, and S100B might be regarded as a potential molecular target in DMD.