Publications by authors named "Neri Mercatelli"

24 Publications

  • Page 1 of 1

Sam68 splicing regulation contributes to motor unit establishment in the postnatal skeletal muscle.

Life Sci Alliance 2020 10 4;3(10). Epub 2020 Aug 4.

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico," Rome, Italy

RNA-binding proteins orchestrate the composite life of RNA molecules and impact most physiological processes, thus underlying complex phenotypes. The RNA-binding protein Sam68 regulates differentiation processes by modulating splicing, polyadenylation, and stability of select transcripts. Herein, we found that mice display altered regulation of alternative splicing in the spinal cord of key target genes involved in synaptic functions. Analysis of the motor units revealed that Sam68 ablation impairs the establishment of neuromuscular junctions and causes progressive loss of motor neurons in the spinal cord. Importantly, alterations of neuromuscular junction morphology and properties in mice correlate with defects in muscle and motor unit integrity. muscles display defects in postnatal development, with manifest signs of atrophy. Furthermore, fast-twitch muscles in mice show structural features typical of slow-twitch muscles, suggesting alterations in the metabolic and functional properties of myofibers. Collectively, our data identify a key role for Sam68 in muscle development and suggest that proper establishment of motor units requires timely expression of synaptic splice variants.
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http://dx.doi.org/10.26508/lsa.201900637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409371PMC
October 2020

Emerging Contribution of PancRNAs in Cancer.

Cancers (Basel) 2020 Jul 24;12(8). Epub 2020 Jul 24.

Laboratory of Cellular and Molecular Neurobiology, IRCCS Fondazione Santa Lucia, 00143 Rome, Italy.

"Cancer" includes a heterogeneous group of diseases characterized by abnormal growth beyond natural boundaries. Neoplastic transformation of cells is orchestrated by multiple molecular players, including oncogenic transcription factors, epigenetic modifiers, RNA binding proteins, and coding and noncoding transcripts. The use of computational methods for global and quantitative analysis of RNA processing regulation provides new insights into the genomic and epigenomic features of the cancer transcriptome. In particular, noncoding RNAs are emerging as key molecular players in oncogenesis. Among them, the promoter-associated noncoding RNAs (pancRNAs) are noncoding transcripts acting in to regulate their host genes, including tumor suppressors and oncogenes. In this review, we will illustrate the role played by pancRNAs in cancer biology and will discuss the latest findings that connect pancRNAs with cancer risk and progression. The molecular mechanisms involved in the function of pancRNAs may open the path to novel therapeutic opportunities, thus expanding the repertoire of targets to be tested as anticancer agents in the near future.
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http://dx.doi.org/10.3390/cancers12082035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464463PMC
July 2020

Small molecule inhibition of Ewing sarcoma cell growth via targeting the long non coding RNA HULC.

Cancer Lett 2020 01 19;469:111-123. Epub 2019 Oct 19.

Laboratory of Molecular and Cellular Neurobiology, IRCCS Santa Lucia Foundation, Rome, 00143, Italy; Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro de Bosis 6, 00135, Rome, Italy. Electronic address:

Ewing sarcomas (ES) are aggressive pediatric cancers of bone and soft tissues characterized by in frame chromosomal translocations giving rise to chimeric transcription factors, such as EWS-FLI1. An emerging strategy to block EWS-FLI1 activity is represented by the small molecule YK-4-279, which binds to EWS-FLI1 and alters its transcriptional activity. The specific effectors of the anti-oncogenic activity of YK-4-279 are still largely unknown. Herein, by performing a high-throughput screening we identify the lncRNA HULC (Highly Upregulated in Liver Cancer) as a prominent target of YK-4-279 activity in ES cells. High levels of HULC correlate with ES aggressiveness, whereas HULC depletion reduces ES cell growth. Mechanistically, we find that HULC promotes the expression of TWIST1 oncogene by sponging miR-186. Downregulation of HULC upon treatment with YK-4-279 reduces the expression of TWIST1 by unleashing miR-186 and favoring its binding to TWIST1 transcripts. Notably, high levels of miR-186 and low levels of TWIST1 correlate with better prognosis in ES patients. Our results disclose a novel oncogenic regulatory circuit mediated by HULC lncRNA that is disrupted by the small molecule YK-4-279, with promising therapeutic implications for ES treatment.
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http://dx.doi.org/10.1016/j.canlet.2019.10.026DOI Listing
January 2020

The Promoter-Associated Noncoding RNA Assembles a Protein-RNA Complex to Regulate Cyclin D1 Transcription in Ewing Sarcoma.

Cancer Res 2019 07 9;79(14):3570-3582. Epub 2019 May 9.

Laboratory of Cellular and Molecular Neurobiology, Fondazione Santa Lucia, Via del Fosso di Fiorano, Rome, Italy.

Most Ewing sarcomas are characterized by the in-frame chromosomal translocation t(11;22) generating the EWS-FLI1 oncogene. EWS-FLI1 protein interacts with the RNA helicase DHX9 and affects transcription and processing of genes involved in neoplastic transformation, including (the cyclin D1 gene), which contributes to cell-cycle dysregulation in cancer. In this study, we found that expression is significantly higher in patients with Ewing sarcoma compared with other sarcomas and that the RNA, a previously uncharacterized promoter-associated noncoding (pnc) transcript, is expressed in Ewing sarcoma cells. interacted with the RNA-binding protein Sam68 and repressed expression. Notably, knockdown of Sam68 affected subcellular localization and cyclin D1 expression. Pharmacologic impairment of DHX9/EWS-FLI1 interaction promoted RNA-dependent association of Sam68 with DHX9 and recruitment of Sam68 to the promoter, thus repressing it. Conversely, mitogenic stimulation of Ewing sarcoma cells with IGF1 impaired Sam68/DHX9 interaction and positively regulated expression. These studies uncover a fine-tuned modulation of the proto-oncogene in Ewing sarcoma cells via alternative complexes formed by DHX9 with either EWS-FLI1 or -Sam68. SIGNIFICANCE: A pncRNA-based mechanism represses expression of through the formation of a protein-RNA complex and provides new therapeutic opportunities for patients with Ewing sarcoma. http://cancerres.aacrjournals.org/content/canres/79/14/3570/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-2403DOI Listing
July 2019

The early response of αB-crystallin to a single bout of aerobic exercise in mouse skeletal muscles depends upon fiber oxidative features.

Redox Biol 2019 06 3;24:101183. Epub 2019 Apr 3.

Unit of Biology and Genetics, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro de Bosis 15, 00135, Rome, Italy. Electronic address:

Besides its substantial role in eye lens, αB-crystallin (HSPB5) retains fundamental function in striated muscle during physiological or pathological modifications. In this study, we aimed to analyse the cellular and molecular factors driving the functional response of HSPB5 protein in different muscles from mice subjected to an acute bout of non-damaging endurance exercise or in C2C12 myocytes upon exposure to pro-oxidant environment, chosen as "in vivo" and "in vitro" models of a physiological stressing conditions, respectively. To this end, red (GR) and white gastrocnemius (GW), as sources of slow-oxidative and fast-glycolytic/oxidative fibers, as well as the soleus (SOL), mainly composed of slow-oxidative type fibers, were obtained from BALB/c mice, before (CTRL) and at different times (0', 15', 30' 120') following 1-h of running. Although the total level of HSPB5 protein was not affected by exercise, we found a significantly increase of phosphorylated HSPB5 (p-HSPB5) only in GR and SOL skeletal muscle with a higher amount of type I and IIA/X myofibers. The fiber-specific activation of HSPB5 was correlated to its interaction with the actin filaments, as well as to an increased level of lipid peroxidation and carbonylated proteins. The role of the pro-oxidant environment in HSPB5 response was investigated in terminally differentiated C2C12 myotubes, where most of HSPB5/pHSPB5 pool was present in the cytosolic compartment in standard culture conditions. As a result of exposure to pro-oxidizing, but not cytotoxic, HO concentration, the p-38MAPK-mediated phosphorylation of HSPB5 resulted functional to promote its interaction with the myofibrillar components, such as β-actin, desmin and filamin 1. This study provides novel information on the molecular pathway underlying the HSPB5 physiological function in skeletal muscle, confirming the contribution of the pro-oxidant environment in HSPB5 activation and interaction with substrate/client myofibrillar proteins, offering new insights for the study of myofibrillar myopathies and cardiomyopathies.
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http://dx.doi.org/10.1016/j.redox.2019.101183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6454247PMC
June 2019

The RNA binding protein Sam68 controls T helper 1 differentiation and anti-mycobacterial response through modulation of miR-29.

Cell Death Differ 2019 06 26;26(6):1169-1180. Epub 2018 Sep 26.

Laboratory of Neuroembriology, Fondazione Santa Lucia, Rome, Italy.

Polarization of naive T cells into interferon (IFN)-γ-producing T helper 1 (Th1) cells is an essential event in the inflammatory response to pathogens. Herein, we identify the RNA binding protein Sam68 as a specific modulator of Th1 differentiation. Sam68-knockout (ko) naive T cells are strongly defective in IL-12-mediated Th1 polarization and express low levels of T-bet and Eomes. Consequently, Sam68-ko Th1 cells are significantly impaired in IFN-γ production. Moreover, we found that Sam68 is required for the induction of an inflammatory Th1 response during Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, thus limiting bacterial dissemination in the lungs. Mechanistically, Sam68 directly binds to the microRNA miR-29, a negative regulator of Th1 response, and inhibits its expression during BCG infection. These findings uncover a novel post-transcriptional mechanism required for the Th1-mediated defense against intracellular pathogens and identify a new function for Sam68 in the regulation of the immune response.
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http://dx.doi.org/10.1038/s41418-018-0201-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748099PMC
June 2019

Telomere length is independently associated with age, oxidative biomarkers, and sport training in skeletal muscle of healthy adult males.

Free Radic Res 2018 Jun 13;52(6):639-647. Epub 2018 Apr 13.

a Department of Movement, Human and Health Sciences , University of Rome "Foro Italico" , Rome , Italy.

In skeletal muscle, which mainly contains postmitotic myonuclei, it has been suggested that telomere length remains roughly constant throughout adult life, or shortens in response to physiopathological conditions in muscle diseases or in the elderly. However, telomere length results from both the replicative history of a specific tissue and the exposure to environmental, DNA damage-related factors, therefore the predictive biological significance of telomere measures should combine the analysis of the various interactive factors. In the present study, we analysed any relationship between telomere length [mean and minimum terminal restriction fragment (TRF) length] chronological age, oxidative damage (4-HNE, protein carbonyls), catalase activity, and heat shock proteins expression (αB-crystallin, Hsp27, Hsp90) in semitendinous muscle biopsies of 26 healthy adult males between 20 and 50 years of age, also exploring the influence of regular exercise participation. The multiple linear regression analysis identified age, 4-HNE, catalase, and training status as significant independent variables associated with telomere length and jointly accounting for ∼30-36% of interindividual variation in mean and/or minimum TRF length. No association has been identified between telomere length and protein carbonyl, αB-crystallin, Hsp27, and Hsp90, as well as between age and the variables related to stress response. Our results showed that skeletal muscle from healthy adults displays an age-dependent telomere attrition and that oxidised environment plays an age-independent contribution, partially influenced by exercise training.
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http://dx.doi.org/10.1080/10715762.2018.1459043DOI Listing
June 2018

The p75-mediated effect of nerve growth factor in L6C5 myogenic cells.

BMC Res Notes 2017 Dec 4;10(1):686. Epub 2017 Dec 4.

Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Piazza Lauro de Bosis, 15, 00135, Rome, Italy.

Objective: During muscle development or regeneration, myocytes produce nerve growth factor (NGF) as well as its tyrosine-kinase and p75-neurotrophin (p75) receptors. It has been published that the p75 receptor could represent a key regulator of NGF-mediated myoprotective effect on satellite cells, but the precise function of NGF/p75 signaling pathway on myogenic cell proliferation, survival and differentiation remains fragmented and controversial. Here, we verified the role of NGF in the growth, survival and differentiation of p75-expressing L6C5 myogenic cells, specifically inquiring for the putative involvement of the nuclear factor κB (NFκB) and the small heat shock proteins (sHSPs) αB-crystallin and Hsp27 in these processes.

Results: Although NGF was not effective in modulating myogenic cell growth or survival in both standard or stress conditions, we demonstrated for the first time that, under serum deprivation, NGF sustained the activity of some key enzymes involved in energy metabolism. Moreover, we confirmed that NGF promotes myogenic fusion and expression of the structural protein myosin heavy chain while modulating NFκB activation and the content of sHSPs correlated with the differentiation process. We conclude that p75 is sufficient to mediate the modulation of L6C5 myogenic differentiation by NGF in term of structural, metabolic and functional changes.
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http://dx.doi.org/10.1186/s13104-017-2994-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716223PMC
December 2017

The role of αB-crystallin in skeletal and cardiac muscle tissues.

Cell Stress Chaperones 2018 07 30;23(4):491-505. Epub 2017 Nov 30.

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.

All organisms and cells respond to various stress conditions such as environmental, metabolic, or pathophysiological stress by generally upregulating, among others, the expression and/or activation of a group of proteins called heat shock proteins (HSPs). Among the HSPs, special attention has been devoted to the mutations affecting the function of the αB-crystallin (HSPB5), a small heat shock protein (sHsp) playing a critical role in the modulation of several cellular processes related to survival and stress recovery, such as protein degradation, cytoskeletal stabilization, and apoptosis. Because of the emerging role in general health and disease conditions, the main objective of this mini-review is to provide a brief account on the role of HSPB5 in mammalian muscle physiopathology. Here, we report the current known state of the regulation and localization of HSPB5 in skeletal and cardiac tissue, making also a critical summary of all human HSPB5 mutations known to be strictly associated to specific skeletal and cardiac diseases, such as desmin-related myopathies (DRM), dilated (DCM) and restrictive (RCM) cardiomyopathy. Finally, pointing to putative strategies for HSPB5-based therapy to prevent or counteract these forms of human muscular disorders.
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http://dx.doi.org/10.1007/s12192-017-0866-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045558PMC
July 2018

Physical activity in the prevention of human diseases: role of epigenetic modifications.

BMC Genomics 2017 Nov 14;18(Suppl 8):802. Epub 2017 Nov 14.

Department of Movement, Human and Health Sciences, Unit of Biology, Genetics and Biochemistry, University of Rome "Foro Italico", Rome, Italy.

Epigenetic modification refers to heritable changes in gene function that cannot be explained by alterations in the DNA sequence. The current literature clearly demonstrates that the epigenetic response is highly dynamic and influenced by different biological and environmental factors such as aging, nutrient availability and physical exercise. As such, it is well accepted that physical activity and exercise can modulate gene expression through epigenetic alternations although the type and duration of exercise eliciting specific epigenetic effects that can result in health benefits and prevent chronic diseases remains to be determined. This review highlights the most significant findings from epigenetic studies involving physical activity/exercise interventions known to benefit chronic diseases such as metabolic syndrome, diabetes, cancer, cardiovascular and neurodegenerative diseases.
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http://dx.doi.org/10.1186/s12864-017-4193-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688489PMC
November 2017

MiR-23-TrxR1 as a novel molecular axis in skeletal muscle differentiation.

Sci Rep 2017 08 3;7(1):7219. Epub 2017 Aug 3.

Unit of Biology, Genetics and Biochemistry, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.

Thioredoxin reductase 1 (TrxR1) is a selenocysteine-containing protein involved in cellular redox homeostasis which is downregulated in skeletal muscle differentiation. Here we show that TrxR1 decrease occurring during myogenesis is functionally involved in the coordination of this cellular process. Indeed, TrxR1 depletion reduces myoblasts growth by inducing an early myogenesis -related gene expression pattern which includes myogenin and Myf5 up-regulation and Cyclin D1 decrease. On the contrary, the overexpression of TrxR1 during differentiation delays myogenic process, by negatively affecting the expression of Myogenin and MyHC. Moreover, we found that miR-23a and miR-23b - whose expression was increased in the early stage of C2C12 differentiation - are involved in the regulation of TrxR1 expression through their direct binding to the 3' UTR of TrxR1 mRNA. Interestingly, the forced inhibition of miR-23a and miR-23b during C2C12 differentiation partially rescues TrxR1 levels and delays the expression of myogenic markers, suggesting the involvement of miR-23 in myogenesis via TrxR1 repression. Taken together, our results depict for the first time a novel molecular axis, which functionally acts in skeletal muscle differentiation through the modulation of TrxR1 by miR-23.
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http://dx.doi.org/10.1038/s41598-017-07575-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543121PMC
August 2017

MicroRNAs as Multifaceted Players in Glioblastoma Multiforme.

Int Rev Cell Mol Biol 2017 21;333:269-323. Epub 2017 Apr 21.

Department of Biomedicine and Prevention, University of Rome "Tor Vergata", Rome, Italy.

Glioblastoma multiforme (GBM) is the most common and inevitably lethal primary brain tumor, with a median survival rate of only 15 months from diagnosis. The current standard treatment involves maximal surgical resection flanked by radiotherapy and chemotherapy with the alkylating agent temozolomide. However, even such aggressive treatment is never curative, and recurrent tumors always arise, commonly in more aggressive, chemo- and radio-resistant forms, leading to untreatable and deadly tumors. MicroRNAs, recognized major players in cancer, are deeply involved in GBM, as shown by more than a decade of studies. In this review, we revise the main milestones of MicroRNA studies in GBM, and the latest relevant discoveries in this field. Examples are given of MicroRNAs working as "oncomiRs" or tumor suppressors, with specific connections with GBM clinical subtypes, patients' survival, and resistance to therapies. As the interaction of GBM cells with the microenvironment was proven as a key determinant of tumor growth, the role of MicroRNAs in GBM microenvironment, tumor angiogenesis, and tumor-secreted microvesicles is also reviewed. Finally, we discuss the latest findings presenting MicroRNAs as possible therapeutic targets for GBM, or their use as circulating biomarkers in diagnosis and prognosis.
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http://dx.doi.org/10.1016/bs.ircmb.2017.03.002DOI Listing
April 2018

Post-transcriptional regulation of FUS and EWS protein expression by miR-141 during neural differentiation.

Hum Mol Genet 2017 07;26(14):2732-2746

Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", 00135 Rome, Italy.

Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.
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http://dx.doi.org/10.1093/hmg/ddx160DOI Listing
July 2017

Resistance training and redox homeostasis: Correlation with age-associated genomic changes.

Redox Biol 2016 12 21;10:34-44. Epub 2016 Sep 21.

Unit of Biology, Genetics and Biochemistry, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro de Bosis 15, 00135 Rome, Italy. Electronic address:

Regular physical activity is effective as prevention and treatment for different chronic conditions related to the ageing processes. In fact, a sedentary lifestyle has been linked to a worsening of cellular ageing biomarkers such as telomere length (TL) and/or specific epigenetic changes (e.g. DNA methylation), with increase of the propensity to aging-related diseases and premature death. Extending our previous findings, we aimed to test the hypothesis that 12 weeks of low frequency, moderate intensity, explosive-type resistance training (EMRT) may attenuate age-associated genomic changes. To this aim, TL, global DNA methylation, TRF2, Ku80, SIRT1, SIRT2 and global protein acetylation, as well as other proteins involved in apoptotic pathway (Bcl-2, Bax and Caspase-3), antioxidant response (TrxR1 and MnSOD) and oxidative damage (myeloperoxidase) were evaluated before and after EMRT in whole blood or peripheral mononuclear cells (PBMCs) of elderly subjects. Our findings confirm the potential of EMRT to induce an adaptive change in the antioxidant protein systems at systemic level and suggest a putative role of resistance training in the reduction of global DNA methylation. Moreover, we observed that EMRT counteracts the telomeres' shortening in a manner that proved to be directly correlated with the amelioration of redox homeostasis and efficacy of training regime, evaluated as improvement of both muscle's power/strength and functional parameters.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040637PMC
http://dx.doi.org/10.1016/j.redox.2016.09.008DOI Listing
December 2016

Exercise-induced ROS in heat shock proteins response.

Free Radic Biol Med 2016 09 26;98:46-55. Epub 2016 Mar 26.

Unit of Biology, Genetics and Biochemistry, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro De Bosis 15, 00135 Rome, Italy. Electronic address:

Cells have evolved multiple and sophisticated stress response mechanisms aiming to prevent macromolecular (including proteins, lipids, and nucleic acids) damage and to maintain or re-establish cellular homeostasis. Heat shock proteins (HSPs) are among the most highly conserved, ubiquitous, and abundant proteins in all organisms. Originally discovered more than 50 years ago through heat shock stress, they display multiple, remarkable roles inside and outside cells under a variety of stresses, including also oxidative stress and radiation, recognizing unfolded or misfolded proteins and facilitating their restructuring. Exercise consists in a combination of physiological stresses, such as metabolic disturbances, changes in circulating levels of hormones, increased temperature, induction of mild to severe inflammatory state, increased production of reactive oxygen and nitrogen species (ROS and RNS). As a consequence, exercise is one of the main stimuli associated with a robust increase in different HSPs in several tissues, which appears to be also fundamental in facilitating the cellular remodeling processes related to the training regime. Among all factors involved in the exercise-related modulation of HSPs level, the ROS production in the contracting muscle or in other tissues represents one of the most attracting, but still under discussion, mechanism. Following exhaustive or damaging muscle exercise, major oxidative damage to proteins and lipids is likely involved in HSP expression, together with mechanically induced damage to muscle proteins and the inflammatory response occurring several days into the recovery period. Instead, the transient and reversible oxidation of proteins by physiological concentrations of ROS seems to be involved in the activation of stress response following non-damaging muscle exercise. This review aims to provide a critical update on the role of HSPs response in exercise-induced adaptation or damage in humans, focusing on experimental results where the link between redox homeostasis and HSPs expression by exercise has been addressed. Further, with the support of in vivo and in vitro studies, we discuss the putative molecular mechanisms underlying the ROS-mediated modulation of HSP expression and/or activity during exercise.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.03.028DOI Listing
September 2016

SFRR-E Young Investigator AwardeeαB-crystallin modulation after acute exercise in skeletal muscle: the role of oxidative stress and fiber composition.

Free Radic Biol Med 2014 Oct 10;75 Suppl 1:S13-4. Epub 2014 Dec 10.

University of Rome Foro Italico (Rome), Department of Movement, Human and Health Sciences, Italy.

αB-crystallin (CRYAB) is a member of the small heat shock proteins implicated in various biological functions, particularly in skeletal muscle where it is involved in adaptive remodelling processes, activation of gene transcription and stabilization of nascent proteins.In this research we analysed αB-crystallin' response in mouse gastrocnemius at 15' and 30' of recovery from an acute aerobic exercise (1hour), correlating its modulation with oxidative stress level and fiber composition, red (RG) and white gastrocnemius (WG).We found for the first time that the acute exercise lead to a short term, specific increase of phospho-αB-crystallin level (pCRYAB) in the RG, while no changes were observed in the WG. Moreover, this induction was correlated with increased level of 4-hydroxynonenal (HNE),suggesting a putative role for oxidative stress in driving CRYAB, but not hsp70 or hsp27, activity during exercise. Any increased level of αB-crystallin' protein was observed neither in RG nor in WG. These data were also supported by our in vitro experiments showing a significant enhancement of pCRYAB in H2O2-treated C2C12 myotubes.Although our results seem suggest a fiber-dependent role of CRYAB, further experiments are in progress to clarify both the molecular pathway driving CRYAB phosphorylation and its fiber-specific induction after exercise -induced oxidative stress.This work was supported by MIUR - PRIN 2012 grant.
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http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.585DOI Listing
October 2014

Alpha B-crystallin induction in skeletal muscle cells under redox imbalance is mediated by a JNK-dependent regulatory mechanism.

Free Radic Biol Med 2015 Sep 9;86:331-42. Epub 2015 Jun 9.

Unit of Biology, Genetics and Biochemistry, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Piazza Lauro De Bosis 15, Rome 00135, Italy.

The small heat shock protein α-B-crystallin (CRYAB) is critically involved in stress-related cellular processes such as differentiation, apoptosis, and redox homeostasis. The up-regulation of CRYAB plays a key role in the cytoprotective and antioxidant response, but the molecular pathway driving its expression in muscle cells during oxidative stress still remains unknown. Here we show that noncytotoxic exposure to sodium meta-arsenite (NaAsO2) inducing redox imbalance is able to increase the CRYAB content of C2C12 myoblasts in a transcription-dependent manner. Our in silico analysis revealed a genomic region upstream of the Cryab promoter containing two putative antioxidant-responsive elements motifs and one AP-1-like binding site. The redox-sensitive transcription factors Nrf2 and the AP-1 component c-Jun were found to be up-regulated in NaAsO2-treated cells, and we demonstrated a specific NaAsO2-mediated increase of c-Jun and Nrf2 binding activity to the genomic region identified, supporting their putative involvement in CRYAB regulation following a shift in redox balance. These changes also correlated with a specific phosphorylation of JNK and p38 MAPK kinases, the well-known molecular mediators of signaling pathways leading to the activation of these transcription factors. Pretreatment of C2C12 cells with the JNK inhibitor SP600125 induced a decrease in c-Jun and Nrf2 content and was able to counteract the NaAsO2-mediated increase in CRYAB expression. Thus these data show a direct role of JNK in CRYAB regulation under redox imbalance and also point to a previously unrecognized link between c-Jun and Nrf2 transcription factors and redox-induced CRYAB expression in muscle cells.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.05.035DOI Listing
September 2015

Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training.

Physiol Rep 2014 Oct 7;2(10). Epub 2014 Oct 7.

Department of Physical Performance, Norwegian School of Sport Sciences, Oslo, Norway Norwegian Olympic Sports Center, Oslo, Norway.

Reactive oxygen and nitrogen species are important signal molecules for adaptations to training. Due to the antioxidant properties of vitamin C and E, supplementation has been shown to blunt adaptations to endurance training. In this study, we investigated the effects of vitamin C and E supplementation and endurance training on adaptations in endogenous antioxidants and heat shock proteins (HSP). Thirty seven males and females were randomly assigned to receive Vitamin C and E (C + E; C: 1000 mg, E: 235 mg daily) or placebo (PLA), and underwent endurance training for 11 weeks. After 5 weeks, a subgroup conducted a high intensity interval session to investigate acute stress responses. Muscle and blood samples were obtained to investigate changes in proteins and mRNA related to the antioxidant and HSP system. The acute response to the interval session revealed no effects of C + E supplementation on NFκB activation. However, higher stress responses to exercise in C + E group was indicated by larger translocation of HSPs and a more pronounced gene expression compared to PLA. Eleven weeks of endurance training decreased muscle GPx1, HSP27 and αB-crystallin, while mnSOD, HSP70 and GSH remained unchanged, with no influence of supplementation. Plasma GSH increased in both groups, while uric acid decreased in the C + E group only. Our results showed that C + E did not affect long-term training adaptations in the antioxidant- and HSP systems. However, the greater stress responses to exercise in the C + E group might indicate that long-term adaptations occurs through different mechanisms in the two groups.
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http://dx.doi.org/10.14814/phy2.12142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254089PMC
October 2014

Platelet-rich plasma and skeletal muscle healing: a molecular analysis of the early phases of the regeneration process in an experimental animal model.

PLoS One 2014 23;9(7):e102993. Epub 2014 Jul 23.

Unit of Internal Medicine, Department of Movement, Human and Health Sciences, University of Rome "Foro Italico", Rome, Italy.

Platelet-rich plasma (PRP) has received increasing interest in applied medicine, being widely used in clinical practice with the aim of stimulating tissue healing. Despite the reported clinical success, there is still a lack of knowledge when considering the biological mechanisms at the base of the activity of PRP during the process of muscle healing. The aim of the present study was to verify whether the local delivery of PRP modulates specific molecular events involved in the early stages of the muscle regeneration process. The right flexor sublimis muscle of anesthetized Wistar rats was mechanically injured and either treated with PRP or received no treatment. At day 2 and 5 after surgery, the animals were sacrificed and the muscle samples evaluated at molecular levels. PRP treatment increased significantly the mRNA level of the pro-inflammatory cytokines IL-1β, and TGF-β1. This phenomenon induced an increased expression at mRNA and/or protein levels of several myogenic regulatory factors such as MyoD1, Myf5 and Pax7, as well as the muscular isoform of insulin-like growth factor1 (IGF-1Eb). No effect was detected with respect to VEGF-A expression. In addition, PRP application modulated the expression of miR-133a together with its known target serum response factor (SRF); increased the phosphorylation of αB-cristallin, with a significant improvement in several apoptotic parameters (NF-κB-p65 and caspase 3), indexes of augmented cell survival. The results of the present study indicates that the effect of PRP in skeletal muscle injury repair is due both to the modulation of the molecular mediators of the inflammatory and myogenic pathways, and to the control of secondary pathways such as those regulated by myomiRNAs and heat shock proteins, which contribute to proper and effective tissue regeneration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102993PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108405PMC
November 2015

NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells.

Nucleic Acids Res 2011 May 18;39(9):3892-902. Epub 2011 Jan 18.

Department of Experimental Medicine and Biochemical Sciences, University of Rome 'Tor Vergata', 00133 Rome, Italy.

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription.
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http://dx.doi.org/10.1093/nar/gkr006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089483PMC
May 2011

AlphaB-crystallin is involved in oxidative stress protection determined by VEGF in skeletal myoblasts.

Free Radic Biol Med 2010 Aug 2;49(3):374-82. Epub 2010 May 2.

Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, 00133 Rome, Italy.

Recent studies suggest that the effects of VEGF-A, the prototype VEGF ligand, may extend to a variety of cell types other than endothelial cells. The expression of VEGF-A and its main receptors, Flt-1/VEGFR-1 and KDR/Flk-1/VEGFR-2, was indeed detected in several cell types, including cardiac myocytes and regenerating myotubes. In addition to its proangiogenic activity, evidence indicates that VEGF-A can sustain skeletal muscle regeneration by enhancing the survival and migration of myogenic cells and by promoting the growth of myogenic fibers. In this study, our aim was to investigate whether VEGF could protect skeletal muscle satellite cells from apoptotic cell death triggered by reactive oxygen species and to identify the main molecular mechanisms. C2C12 mouse myoblasts, cultured in vitro in the presence of exogenous VEGF or stably transfected with a plasmid vector expressing VEGF-A, were subjected to oxidative stress and analyzed for cell growth and survival, induction of apoptosis, and molecular signaling. The results of our study demonstrated that VEGF protects C2C12 myoblasts from apoptosis induced by oxidative or hypoxic-like stress. This protection did not correlate with the modulation of the expression of VEGF receptors, but is clearly linked to the phosphorylation of the KDR/Flk-1 receptor, the activation of NF-kappaB, and/or the overexpression of the antiapoptotic protein alphaB-crystallin.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.04.027DOI Listing
August 2010

The inhibition of the highly expressed miR-221 and miR-222 impairs the growth of prostate carcinoma xenografts in mice.

PLoS One 2008 24;3(12):e4029. Epub 2008 Dec 24.

Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy.

Background: MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity.

Methodology/principal Findings: Here we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression.

Conclusions/significance: These findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004029PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2603596PMC
February 2009

Regulation of the p27(Kip1) tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation.

EMBO J 2007 Aug 12;26(15):3699-708. Epub 2007 Jul 12.

Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27(Kip1), a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27(Kip1) levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27(Kip1) protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27(Kip1).
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http://dx.doi.org/10.1038/sj.emboj.7601790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949005PMC
August 2007

miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27Kip1.

J Biol Chem 2007 Aug 14;282(32):23716-24. Epub 2007 Jun 14.

Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier, 1 00133 Rome, Italy.

MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional level and are deeply involved in the pathogenesis of several types of cancers. Here we show that miR-221 and miR-222, encoded in tandem on chromosome X, are overexpressed in the PC3 cellular model of aggressive prostate carcinoma, as compared with LNCaP and 22Rv1 cell line models of slowly growing carcinomas. In all cell lines tested, we show an inverse relationship between the expression of miR-221 and miR-222 and the cell cycle inhibitor p27(Kip1). We recognize two target sites for the microRNAs in the 3' untranslated region of p27 mRNA, and we show that miR-221/222 ectopic overexpression directly results in p27 down-regulation in LNCaP cells. In those cells, we demonstrate that the ectopic overexpression of miR-221/222 strongly affects their growth potential by inducing a G(1) to S shift in the cell cycle and is sufficient to induce a powerful enhancement of their colony-forming potential in soft agar. Consistently, miR-221 and miR-222 knock-down through antisense LNA oligonucleotides increases p27(Kip1) in PC3 cells and strongly reduces their clonogenicity in vitro. Our results suggest that miR-221/222 can be regarded as a new family of oncogenes, directly targeting the tumor suppressor p27(Kip1), and that their overexpression might be one of the factors contributing to the oncogenesis and progression of prostate carcinoma through p27(Kip1) down-regulation.
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http://dx.doi.org/10.1074/jbc.M701805200DOI Listing
August 2007