Equisetum arvense standardized dried extract hinders age-related osteosarcopenia.

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ's anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

Grafted Sertoli Cells Exert Immunomodulatory Non-Immunosuppressive Effects in Preclinical Models of Infection and Cancer.

The Sertoli cells (SeCs) of the seminiferous tubules secrete a multitude of immunoregulatory and trophic factors to provide immune protection and assist in the orderly development of germ cells. Grafts of naked or encapsulated SeCs have been proved to represent an interesting therapeutic option in a plethora of experimental models of diseases. However, whether SeCs have immunosuppressive or immunomodulatory effects, which is imperative for their clinical translatability, has not been demonstrated. We directly assessed the immunopotential of intraperitoneally grafted microencapsulated porcine SeCs (MC-SeCs) in murine models of fungal infection (Aspergillus fumigatus or Candida albicans) or cancer (Lewis lung carcinoma/LLC or B16 melanoma cells). We found that MC-SeCs (i) provide antifungal resistance with minimum inflammatory pathology through the activation of the tolerogenic aryl hydrocarbon receptor/indoleamine 2,3-dioxygenase pathway; (ii) do not affect tumor growth in vivo; and (iii) reduce the LLC cell metastatic cancer spread associated with restricted Vegfr2 expression in primary tumors. Our results point to the fine immunoregulation of SeCs in the relative absence of overt immunosuppression in both infection and cancer conditions, providing additional support for the potential therapeutic use of SeC grafts in human patients.

KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK.

The imbalance in osteoblast (OB)-dependent bone formation in favor of osteoclast (OC)-dependent bone resorption is the main cause of loss of tissue mineral mass during bone remodeling leading to osteoporosis conditions. Thus, the suppression of OC activity together with the improvement in the OB activity has been proposed as an effective therapy for maintaining bone mass during aging. We tested the new dietary product, KYMASIN UP containing standardized Withania somnifera, Silybum marianum and Trigonella foenum-graecum herbal extracts or the single extracts in in vitro models mimicking osteoclastogenesis (i.e., RAW 264.7 cells treated with RANKL, receptor activator of nuclear factor kappa-Β ligand) and OB differentiation (i.e., C2C12 myoblasts treated with BMP2, bone morphogenetic protein 2). We found that the dietary product reduces RANKL-dependent TRAP (tartrate-resistant acid phosphatase)-positive cells (i.e., OCs) formation and TRAP activity, and down-regulates osteoclastogenic markers by reducing Src (non-receptor tyrosine kinase) and p38 MAPK (mitogen-activated protein kinase) activation. Withania somnifera appears as the main extract responsible for the anti-osteoclastogenic effect of the product. Moreover, KYMASIN UP maintains a physiological release of the soluble decoy receptor for RANKL, OPG (osteoprotegerin), in osteoporotic conditions and increases calcium mineralization in C2C12-derived OBs. Interestingly, KYMASIN UP induces differentiation in human primary OB-like cells derived from osteoporotic subjects. Based on our results, KYMASIN UP or Withania somnifera-based dietary supplements might be suggested to reverse the age-related functional decline of bone tissue by re-balancing the activity of OBs and OCs, thus improving the quality of life in the elderly and reducing social and health-care costs.

Sertoli Cells Improve Myogenic Differentiation, Reduce Fibrogenic Markers, and Induce Utrophin Expression in Human DMD Myoblasts.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene translating in lack of functional dystrophin and resulting in susceptibility of myofibers to rupture during contraction. Inflammation and fibrosis are critical hallmarks of DMD muscles, which undergo progressive degeneration leading to loss of independent ambulation in childhood and death by early adulthood. We reported that intraperitoneal injection of microencapsulated Sertoli cells (SeC) in dystrophic mice translates into recovery of muscle morphology and performance thanks to anti-inflammatory effects and induction of the dystrophin paralogue, utrophin at the muscle level, opening new avenues in the treatment of DMD. The aim of this study is to obtain information about the direct effects of SeC on myoblasts/myotubes, as a necessary step in view of a translational application of SeC-based approaches to DMD. We show that (i) SeC-derived factors stimulate cell proliferation in the early phase of differentiation in C2C12, and human healthy and DMD myoblasts; (ii) SeC delay the expression of differentiation markers in the early phase nevertheless stimulating terminal differentiation in DMD myoblasts; (iii) SeC restrain the fibrogenic potential of fibroblasts, and inhibit myoblast-myofibroblast transdifferentiation; and, (iv) SeC provide functional replacement of dystrophin in preformed DMD myotubes regardless of the mutation by inducing heregulin ß1/ErbB2/ERK1/2-dependent utrophin expression. Altogether, these results show that SeC are endowed with promyogenic and antifibrotic effects on dystrophic myoblasts, further supporting their potential use in the treatment of DMD patients. Our data also suggest that SeC-based approaches might be useful in improving the early phase of muscle regeneration, during which myoblasts have to adequately proliferate to replace the damaged muscle mass.

Identification of Withania somnifera-Silybum marianum-Trigonella foenum-graecum Formulation as a Nutritional Supplement to Contrast Muscle Atrophy and Sarcopenia.

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The imbalance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania somnifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs.

Targeting RAGE prevents muscle wasting and prolongs survival in cancer cachexia.

BACKGROUND: Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer. METHODS: By using murine models consisting in the injection of colon 26 murine adenocarcinoma (C26-ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager-/- (RAGE-null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager-/- mice treated with the RAGE ligand, S100B (S100 calcium-binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell- or masses-conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager-/- mice were injected with TNFα/IFNγ or S100B in a tumour-free environment. RESULTS: We demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour-derived cachexia-inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen-activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)-dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour-derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour-derived pro-cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia-inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival. CONCLUSIONS: RAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome.

Reductive stress in striated muscle cells.

Reductive stress is defined as a condition of sustained increase in cellular glutathione/glutathione disulfide and NADH/NAD+ ratios. Reductive stress is emerging as an important pathophysiological event in several diseased states, being as detrimental as is oxidative stress. Occurrence of reductive stress has been documented in several cardiomyopathies and is an important pathophysiological factor particularly in coronary artery disease and myocardial infarction. Excess activation of the transcription factor, Nrf2-the master regulator of the antioxidant response-, consequent in most cases to defective autophagy, can lead to reductive stress. In addition, hyperglycemia-induced activation of the polyol pathway can lead to increased NADH/NAD+ ratio, which might translate into increased levels of hydrogen sulfide-via enhanced activity of cystathionine ß-synthase-that would fuel reductive stress through inhibition of mitochondrial complex I. Reductive stress may be either a potential weapon against cancer priming tumor cells to apoptosis or a cancer's ally promoting tumor cell proliferation and making tumor cells resistant to reactive oxygen species-inducing drugs. In non-cancer pathological states reductive stress is definitely harmful paradoxically leading to reactive oxygen species overproduction via excess NADPH oxidase 4 activity. In face of the documented occurrence of reductive stress in several heart diseases, there is much less information about the occurrence and effects of reductive stress in skeletal muscle tissue. In the present review we describe relevant results emerged from studies of reductive stress in the heart and review skeletal muscle conditions in which reductive stress has been experimentally documented and those in which reductive stress might have an as yet unrecognized pathophysiological role. Establishing whether reductive stress has a (patho)physiological role in skeletal muscle will hopefully contribute to answer the question whether antioxidant supplementation to the general population, athletes, and a large cohort of patients (e.g. heart, sarcopenic, dystrophic, myopathic, cancer, and bronco-pulmonary patients) is harmless or detrimental.

Report and Abstracts of the 17th Meeting of IIM, the Interuniversity Institute of Myology:Virtual meeting, October 16-18, 2020.

In 2020, due to the COVID-19 pandemic, the annual meeting of the Interuniversity Institute of Myology (IIM), took place on a virtual platform. Attendees were scientists and clinicians, as well as pharmaceutical companies and patient organization representatives from Italy, several European countries, Canada and USA. Four internationally renowned Keynote speakers presented recent advances on muscle stem cells regulation, skeletal muscle regeneration, quantitative biology approaches, and metabolic regulation of muscle homeostasis. Novel, unpublished data by young trainees were presented as oral communications or posters, in five scientific sessions and two poster sessions. On October 15, 2020, selected young trainees participated to the High Training Course on "Advanced Myology", organized together with the University of Perugia, Italy. The course, on a virtual platform, showcased lectures on muscle development and regulation of muscle gene expression by international speakers, and roundtables discussions on "Single cell analysis of skeletal muscle" and "Skeletal muscle stem cell in healthy muscle and disease". The Young IIM Committee, composed by young trainee winners of awards in the past IIM Meeting editions, was directly involved in the selection of keynote speakers, the organization of scientific sessions and roundtables discussions tailored to the interests of their peers. A broad audience of Italian, European and North American participants contributed to the different initiatives. The meeting was characterized by a friendly and inclusive atmosphere, facilitating lively and stimulating discussions on emerging areas of muscle research. The meeting stimulated scientific cross-fertilization fostering novel ideas and scientific collaborations aimed at better understanding muscle normal physiology and the mechanisms underlaying muscle diseases, with the ultimate goal of developing better therapeutic strategies. The meeting was a success, and the number of meeting attendees was the highest of all IIM Meeting editions. Despite the current difficulties imposed by the COVID-19 pandemic, we are confident that the IIM community will continue to grow and deliver significant contributions to the understanding of muscle development and function, the pathogenesis of muscular diseases and the development of novel therapeutic approaches. Here, abstracts of the meeting illustrate the new results on basic, translational, and clinical research, confirming that our field is strong and healthy.

S100 proteins in obesity: liaisons dangereuses.

Obesity is an endemic pathophysiological condition and a comorbidity associated with hypercholesterolemia, hypertension, cardiovascular disease, type 2 diabetes mellitus, and cancer. The adipose tissue of obese subjects shows hypertrophic adipocytes, adipocyte hyperplasia, and chronic low-grade inflammation. S100 proteins are Ca2+-binding proteins exclusively expressed in vertebrates in a cell-specific manner. They have been implicated in the regulation of a variety of functions acting as intracellular Ca2+ sensors transducing the Ca2+ signal and extracellular factors affecting cellular activity via ligation of a battery of membrane receptors. Certain S100 proteins, namely S100A4, the S100A8/S100A9 heterodimer and S100B, have been implicated in the pathophysiology of obesity-promoting macrophage-based inflammation via toll-like receptor 4 and/or receptor for advanced glycation end-products ligation. Also, serum levels of S100A4, S100A8/S100A9, S100A12, and S100B correlate with insulin resistance/type 2 diabetes, metabolic risk score, and fat cell size. Yet, secreted S100B appears to exert neurotrophic effects on sympathetic fibers in brown adipose tissue contributing to the larger sympathetic innervation of this latter relative to white adipose tissue. In the present review we first briefly introduce S100 proteins and then critically examine their role(s) in adipose tissue and obesity.

Cellular and molecular mechanisms of sarcopenia: the S100B perspective.

Primary sarcopenia is a condition of reduced skeletal muscle mass and strength, reduced agility, and increased fatigability and risk of bone fractures characteristic of aged, otherwise healthy people. The pathogenesis of primary sarcopenia is not completely understood. Herein, we review the essentials of the cellular and molecular mechanisms of skeletal mass maintenance; the alterations of myofiber metabolism and deranged properties of muscle satellite cells (the adult stem cells of skeletal muscles) that underpin the pathophysiology of primary sarcopenia; the role of the Ca2+ -sensor protein, S100B, as an intracellular factor and an extracellular signal regulating cell functions; and the functional role of S100B in muscle tissue. Lastly, building on recent results pointing to S100B as to a molecular determinant of myoblast-brown adipocyte transition, we propose S100B as a transducer of the deleterious effects of accumulation of reactive oxygen species in myoblasts and, potentially, myofibers concurring to the pathophysiology of sarcopenia.

RAGE in the pathophysiology of skeletal muscle.

Emerging evidence suggests that the signalling of the Receptor for Advanced Glycation End products (RAGE) is critical for skeletal muscle physiology controlling both the activity of muscle precursors during skeletal muscle development and the correct time of muscle regeneration after acute injury. On the other hand, the aberrant re-expression/activity of RAGE in adult skeletal muscle is a hallmark of muscle wasting that occurs in response to ageing, genetic disorders, inflammatory conditions, cancer, and metabolic alterations. In this review, we discuss the mechanisms of action and the ligands of RAGE involved in myoblast differentiation, muscle regeneration, and muscle pathological conditions. We highlight potential therapeutic strategies for targeting RAGE to improve skeletal muscle function.

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.

S100A6 protein: functional roles.

S100A6 protein belongs to the A group of the S100 protein family of Ca2+-binding proteins. It is expressed in a limited number of cell types in adult normal tissues and in several tumor cell types. As an intracellular protein, S100A6 has been implicated in the regulation of several cellular functions, such as proliferation, apoptosis, the cytoskeleton dynamics, and the cellular response to different stress factors. S100A6 can be secreted/released by certain cell types which points to extracellular effects of the protein. RAGE (receptor for advanced glycation endproducts) and integrin ß1 transduce some extracellular S100A6's effects. Dosage of serum S100A6 might aid in diagnosis in oncology.

Artesunate induces ROS- and p38 MAPK-mediated apoptosis and counteracts tumor growth in vivo in embryonal rhabdomyosarcoma cells.

Rhabdomyosarcoma represents about 50% of soft-tissue sarcomas and 10% of malignant solid tumors in childhood. Embryonal rhabdomyosarcoma (ERMS) is the most frequent subtype, suggested to have an origin in muscle precursor cells that fail to exit the cell cycle and terminally differentiate mainly because of overexpression of the transcription factor, PAX7, which sustains proliferation, migration and invasiveness in ERMS cells. Artesunate (ARS) is a semi-synthetic derivative of artemisinin (ART), a natural compound well known as an antimalarial drug. However, ART and its derivatives have been found efficacious even as anticancer drugs that induce cell cycle arrest and/or apoptosis in several kinds of cancer. Here, we show that ARS dose-dependently induces DNA damage and apoptosis in ERMS cell lines. Production of reactive oxygen species (ROS) and activation of p38 MAPK have a central role in triggering ARS-mediated apoptosis in ERMS cells; indeed either the antioxidant, N-acetylcysteine or the p38 MAPK inhibitor, SB203580, protects ERMS cells from ARS-induced apoptosis. Moreover, ARS treatment in ERMS cells ROS-dependently induces the expression of the myo-miRs, miR-133a and miR-206, which are down-regulated in RMS, and reduces PAX7 protein levels. Finally, ARS upregulates the expression of the adhesion molecules, NCAM and integrin ß1, and reduces migration and invasiveness of ERMS cells in vitro, and ARS treatment reduces of about 50% the growth of ERMS xenografts in vivo. Our results are the first evidence of efficacy of ART derivatives in restraining ERMS growth in vivo, and suggest ARS as a potential candidate for therapeutic treatment of ERMS.

Defective RAGE activity in embryonal rhabdomyosarcoma cells results in high PAX7 levels that sustain migration and invasiveness.

Rhabdomyosarcoma is a muscle-derived malignant tumor mainly affecting children. The most frequent variant, embryonal rhabdomyosarcoma (ERMS) is characterized by overexpression of the transcription factor, PAX7 which prevents ERMS cells from exiting the cell cycle and terminally differentiating. However, a role for PAX7 in the invasive properties of ERMS cells has not been investigated in detail thus far. Here we show that ectopic expression of receptor for advanced glycation end-products (RAGE) in human ERMS cells results in the activation of a RAGE/myogenin axis which downregulates PAX7 by transcriptional and post-translational mechanisms, as in normal myoblasts, and reduces metastasis formation. High PAX7 sustains migration and invasiveness in ERMS cells by upregulating EPHA3 and EFNA1 and downregulating NCAM1 thus decreasing the neural cell adhesion molecule (NCAM)/polysialylated-NCAM ratio. Microarray gene expression analysis shows that compared with the RAGE(-ve) TE671/WT cells and similarly to primary human myoblasts, TE671/RAGE cells show upregulation of genes involved in muscle differentiation and cell adhesion, and downregulation of cell migration related and major histocompatibility complex class I genes. Our data reveal a link between PAX7 and metastasis occurrence in ERMSs, and support a role for the RAGE/myogenin axis in metastasis suppression. Thus, low RAGE expression in ERMS primary tumors may be predictive of metastatic behavior.

RAGE signaling deficiency in rhabdomyosarcoma cells causes upregulation of PAX7 and uncontrolled proliferation.

Embryonal rhabdomyosarcomas (ERMSs) show elevated levels of PAX7, a transcription factor that marks quiescent adult muscle stem (satellite) cells and is important for proliferation and survival of activated satellite cells and whose timely repression is required for myogenic differentiation. However, the mechanism of PAX7 accumulation in ERMSs and whether high PAX7 causes uncontrolled proliferation in ERMS remains to be elucidated. The receptor for advanced glycation end-products (RAGE, encoded by AGER) transduces a myogenic and anti-proliferative signal in myoblasts, and stable transfection of the ERMS cell line TE671, which does not express RAGE, with AGER results in reduced proliferation and formation of tumor masses in vivo, and enhanced apoptosis and myogenic differentiation. Herein, we show that RAGE expression is low or absent in human ERMSs. We also show that in ERMS cells (1) PAX7 accumulates owing to absent or low RAGE signaling; (2) elevated PAX7 levels reduce RAGE expression and levels of MyoD and myogenin, muscle-specific transcription factors required for myoblast proliferation arrest and differentiation, respectively; (3) PAX7 supports myoblast proliferation by reducing the levels of MyoD, primarily by promoting its degradation; and (4), when ectopically expressed in ERMS cells, that RAGE upregulates myogenin which upregulates MyoD and downregulates PAX7, with consequent inhibition of proliferation and stimulation of differentiation. Thus, failure to express RAGE and, hence, MyoD and myogenin above a critical level in ERMS cells might result in deregulated PAX7 expression leading to uncontrolled proliferation and, potentially, to rhabdomyosarcomagenesis.

Phosphocaveolin-1 enforces tumor growth and chemoresistance in rhabdomyosarcoma.

Caveolin-1 (Cav-1) can ambiguously behave as either tumor suppressor or oncogene depending on its phosphorylation state and the type of cancer. In this study we show that Cav-1 was phosphorylated on tyrosine 14 (pCav-1) by Src-kinase family members in various human cell lines and primary mouse cultures of rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma affecting childhood. Cav-1 overexpression in the human embryonal RD or alveolar RH30 cells yielded increased pCav-1 levels and reinforced the phosphorylation state of either ERK or AKT kinase, respectively, in turn enhancing in vitro cell proliferation, migration, invasiveness and chemoresistance. In contrast, reducing the pCav-1 levels by administration of a Src-kinase inhibitor or through targeted Cav-1 silencing counteracted the malignant in vitro phenotype of RMS cells. Consistent with these results, xenotransplantation of Cav-1 overexpressing RD cells into nude mice resulted in substantial tumor growth in comparison to control cells. Taken together, these data point to pCav-1 as an important and therapeutically valuable target for overcoming the progression and multidrug resistance of RMS.

Hypoxia promotes danger-mediated inflammation via receptor for advanced glycation end products in cystic fibrosis.

RATIONALE: Hypoxia regulates the inflammatory-antiinflammatory balance by the receptor for advanced glycation end products (RAGE), a versatile sensor of damage-associated molecular patterns. The multiligand nature of RAGE places this receptor in the midst of chronic inflammatory diseases. OBJECTIVES: To characterize the impact of the hypoxia-RAGE pathway on pathogenic airway inflammation preventing effective pathogen clearance in cystic fibrosis (CF) and elucidate the potential role of this danger signal in pathogenesis and therapy of lung inflammation. METHODS: We used in vivo and in vitro models to study the impact of hypoxia on RAGE expression and activity in human and murine CF, the nature of the RAGE ligand, and the impact of RAGE on lung inflammation and antimicrobial resistance in fungal and bacterial pneumonia. MEASUREMENTS AND MAIN RESULTS: Sustained expression of RAGE and its ligand S100B was observed in murine lung and human epithelial cells and exerted a proximal role in promoting inflammation in murine and human CF, as revealed by functional studies and analysis of the genetic variability of AGER in patients with CF. Both hypoxia and infections contributed to the sustained activation of the S100B-RAGE pathway, being RAGE up-regulated by hypoxia and S100B by infection by Toll-like receptors. Inhibiting the RAGE pathway in vivo with soluble (s) RAGE reduced pathogen load and inflammation in experimental CF, whereas sRAGE production was defective in patients with CF. CONCLUSIONS: A causal link between hyperactivation of RAGE and inflammation in CF has been observed, such that targeting pathogenic inflammation alleviated inflammation in CF and measurement of sRAGE levels could be a useful biomarker for RAGE-dependent inflammation in patients with CF.

HuR and miR-1192 regulate myogenesis by modulating the translation of HMGB1 mRNA.

Upon muscle injury, the high mobility group box 1 (HMGB1) protein is upregulated and secreted to initiate reparative responses. Here we show that HMGB1 controls myogenesis both in vitro and in vivo during development and after adult muscle injury. HMGB1 expression in muscle cells is regulated at the translational level: the miRNA miR-1192 inhibits HMGB1 translation and the RNA-binding protein HuR promotes it. HuR binds to a cis-element, HuR binding sites (HuRBS), located in the 3'UTR of the HMGB1 transcript, and at the same time miR-1192 is recruited to an adjacent seed element. The binding of HuR to the HuRBS prevents the recruitment of Argonaute 2 (Ago2), overriding miR-1192-mediated translation inhibition. Depleting HuR reduces myoblast fusion and silencing miR-1192 re-establishes the fusion potential of HuR-depleted cells. We propose that HuR promotes the commitment of myoblasts to myogenesis by enhancing the translation of HMGB1 and suppressing the translation inhibition mediated by miR-1192.

Muscular dystrophies share pathogenetic mechanisms with muscle sarcomas.

Several lines of recent evidence have opened a new debate on the mechanisms underlying the genesis of rhabdomyosarcoma, a pediatric soft tissue tumor with a widespread expression of muscle-specific markers. In particular, it is increasingly evident that the loss of skeletal muscle integrity observed in some mouse models of muscular dystrophy can favor rhabdomyosarcoma formation. This is especially true in old age. Here, we review these experimental findings and focus on the main molecular and cellular events that can dictate the tumorigenic process in dystrophic muscle, such as the loss of structural or regulatory proteins with tumor suppressor activity, the impaired DNA damage response due to oxidative stress, the chronic inflammation and the conflicting signals arising within the degenerated muscle niche.