Nuova ricerca

Susanna MOLINARI

Ricercatore Universitario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

Home | Curriculum(pdf) | Didattica |


Pubblicazioni

2023 - Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives [Articolo su rivista]
Imbriano, Carol; Moresi, Viviana; Belluti, Silvia; Renzini, Alessandra; Cavioli, Giorgia; Maretti, Eleonora; Molinari, Susanna
abstract

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

2023 - Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism [Articolo su rivista]
Molinari, Susanna; Imbriano, Carol; Moresi, Viviana; Renzini, Alessandra; Belluti, Silvia; Lozanoska-Ochser, Biliana; Gigli, Giuseppe; Cedola, Alessia
abstract

2023 - Linking endocannabinoid system, palmitoylethanolamide, and sarcopenia in view of therapeutic implications [Capitolo/Saggio]
Molinari, S.; Maretti, E.; Battini, R.; Leo, E.
abstract

Sarcopenia, a debilitating skeletal muscle disease closely connected with elderly, is becoming a major public health problem with the increasing of life expectancy. In the aim to found effective, targeted and side-effect-free therapies, understanding the endocannabinoid system (ECS) role in muscle homeostasis is of strategic importance. The skeletal muscle expresses all the ECS elements; in particular, a central role is played by the nuclear receptor PPARα and its main endogenous ligand, palmitoylethanolamide (PEA), an endocannabinoid-like molecule with an important anti-inflammatory effect. It is worth highlighting that in muscle the expression level of both PPARα receptor and its coactivator PGC1a, decreases with age, suggesting a causative relation between the lower PPARα function and sarcopenia. Therefore, the administration of PEA to the muscle can be a promising approach to counteract sarcopenia. In this regard, to promote the muscle targeting, innovative drug delivery systems, such as solid lipid nanoparticles, can be considered.

2023 - Nanoparticulate systems for the delivery of Palmitoylethanolamide to muscle cells [Poster]
Maretti, Eleonora; Molinari, Susanna; Gioia, Federica; Rustichelli, Cecilia; Leo, Eliana Grazia
abstract

2022 - Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia [Articolo su rivista]
Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Truzzi, Eleonora; Iannuccelli, Valentina; Leo, Eliana
abstract

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopenia

2022 - Palmitoylethanolamide-loaded Solid Lipid Nanoparticles for a therapeutic anti-inflammatory approach of Sarcopenia involving ECS [Relazione in Atti di Convegno]
Leo, Eliana Grazia; Maretti, Eleonora; Molinari, Susanna; Battini, Renata; Rustichelli, Cecilia; Iannuccelli, Valentina
abstract

2021 - Development of solid lipid nanoparticles for the delivery of an anti- inflammatory drug to muscle skeletal cells [Poster]
Maretti, Eleonora; Truzzi, Eleonora; Molinari, Susanna; Battini, Renata; Leo, Eliana Grazia
abstract

2021 - The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle [Articolo su rivista]
Rigillo, Giovanna; Basile, Valentina; Belluti, Silvia; Ronzio, Mirko; Sauta, Elisabetta; Ciarrocchi, Alessia; Latella, Lucia; Saclier, Marielle; Molinari, Susanna; Vallarola, Antonio; Messina, Graziella; Mantovani, Roberto; Dolfini, Diletta; Imbriano, Carol
abstract

2019 - PIN1: a putative molecular target to protect skeletal muscle against age-related muscle loss [Relazione in Atti di Convegno]
Brocca, Lorenza; Grosso, Martina; Pezzini, Camilla; Semeghini, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Mucci, Adele; Righi, Valeria; Lorenzo Puri, Pier; Battini, Renata; Bottinelli, Roberto; Antonietta Pellegrino, Maria; Molinari, Susanna
abstract

Aging is associated with a progressive loss in skeletal muscle mass and strength, known as sarcopenia. Sarcopenia results in a decrease in mobility and an increased risk of developing chronic metabolic disease, thus it represents a major socio-economical problem. Age-related muscle loss cannot be consistently prevented by physical therapy and a pharmacologic therapy does not exist, probably because the molecular basis of this condition is still largely unknown. Many factors such as mitochondrial dysfunction, oxidative stress, inflammation, changes in the innervation of muscle fibers probably play an important role in age-related muscle decline. PIN1 is a widely expressed Peptydyl Prolyl cis/trans isomerase, involved in post-phosphorylation control of the function of multiple target proteins. Many evidences indicate that PIN1 controls signaling pathways involved in skeletal muscle wasting. Our results indicate that skeletal muscle of Pin1 KO mice is protected against muscle loss and weakness during aging. At the molecular level, we found 1) an increased expression of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), a transcription factor that promotes mitochondrial biogenesis, in skeletal muscle of Pin1 KO mice. Coherently with their resistance to muscle loss, we also found 2) an increase in the expression of the protein synthetic signaling proteins p70 ribosomal S6 kinase (S6K) and of its phosphorylated form in aged skeletal muscle of Pin1 KO mice compared to the wild type controls, suggesting that in these mice protein synthesis is maintained efficient. We also found 3) a significant decrease of Myostatin levels in skeletal muscle of aged Pin1 KO mice. It is well known that Myostatin is upregulated sarcopenia, it activates SMAD2/3 signaling and contributes to protein degradation and muscle atrophy. The transcriptional effects of Pin1 depletion on PGC1α and S6K genes must be mediated by a transcription factor. A putative candidate to mediate these effects is represented by the transcription factor Myocyte Enhancer Factor 2C (MEF2C), a known PIN1 target. In skeletal muscle cells a specific splice variant of MEF2C, MEF2C α1, activates the increase of skeletal muscle mass by activating the expression of IGF1 and S6K. These activities are repressed by its phosphorylation, that renders it a target for the inhibitory effect of PIN1 on its protein stability and activity. Coherently with these premises, we found 4) a decrease of MEF2C protein phosphorylation levels in aged KO mice compared to the control animals. This might at least partially contribute to the increased expression of PGC1α and of S6K. Our results indicate that PIN1 could represent a valuable pharmacological target to counteract age-related muscle loss, simultaneously modulating multiple targets in a concerted way.

2018 - Alternative splicing of transcription factors genes in muscle physiology and pathology [Articolo su rivista]
Imbriano, Carol; Molinari, Susanna
abstract

Skeletal muscle formation is a multi-step process that is governed by complex networks of transcription factors. The regulation of their functions is in turn multifaceted, including several mechanisms, among them alternative splicing (AS) plays a primary role. On the other hand, altered AS has a role in the pathogenesis of numerous muscular pathologies. Despite these premises, the causal role played by the altered splicing pattern of transcripts encoding myogenic transcription factors in neuromuscular diseases has been neglected so far. In this review, we systematically investigate what has been described about the AS patterns of transcription factors both in the physiology of the skeletal muscle formation process and in neuromuscular diseases, in the hope that this may be useful in re-evaluating the potential role of altered splicing of transcription factors in such diseases.

2017 - Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy [Articolo su rivista]
Baruffaldi, Fiorenza; Montarras, Didier; Basile, Valentina; De Feo, Luca; Badodi, Sara; Ganassi, Massimo; Battini, Renata; Nicoletti, Carmine; Imbriano, Carol; Musarò, Antonio; Molinari, Susanna
abstract

Abstract: The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases.

2016 - NF-YA splice variants have different roles on muscle differentiation [Articolo su rivista]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Belluti, Silvia; Benatti, Paolo; Ricci, Laura; Artusi, Valentina; Tagliafico, Enrico; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

2016 - Role of the CCAAT-transcription factor NF-Y and its splice variants in myogenic differentiation and muscle regeneration [Poster]
Basile, Valentina; Baruffaldi, Fiorenza; Laura, Ricci; Molinari, Susanna; Imbriano, Carol
abstract

The expression of NF-YA, the DNA-binding subunit of the CCAAT-binding transcription factor NF-Y, is down-regulated in adult skeletal muscle to allow a complete terminal differentiation. The NF-YA gene encodes for two alternative splice variants, NF-YAs and NF-YAl: in human haematopoietic and mouse embryonic stem cells, the expression of NF-YAs or NF-YAl is crucial to promote proliferation or differentiation, respectively. The role of NF-YA isoforms in skeletal myogenesis and satellite cells fate has never been investigated. Here we show that both NF-YA isoforms are induced in mouse regenerating muscle and are expressed in satellite cells (SCs). The abrogation of NF-Y activity impairs both proliferation and differentiation of SCs. Forced expression of NF-YAs stimulates SCs proliferation, while NF-YAl enhances their differentiation. The same effects are observed in mouse C2C12 myoblasts, in which the two isoforms activate opposite transcriptional programs. NF-YAs upregulates genes annotated in growth factor binding, cell adhesion and extra cellular matrix Gene Onthology terms, while NF-YAl increases the transcription of genes belonging to sarcomere, muscle cell differentiation, development and muscle contraction categories. NF-YAl boosts myoblasts differentiation by up-regulating the transcription of novel NF-Y target genes, among which Mef2D, Six4 and Cdkn1C, which are known to be involved in the differentiation program. Overall, our results highlight the functional difference between NF-YA isoforms, whose modulation could be useful to improve stem cell based therapies to treat muscular dystrophy.

2015 - NF-Y splice variants differentially affect skeletal myogenesis [Esposizione]
Basile, Valentina; Baruffaldi, Fiorenza; Dolfini, Diletta; Ricci, Laura; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

The mechanisms that regulate skeletal muscle development involve the coordinated activity of transcription factors (TFs) and the precise timing of gene expression patterns. NF-Y is a heterotrimeric TF with a pioneer role in the transcriptional and epigenetic regulation of promoters containing the CCAAT-box. NF-Y activates the expression of various genes related to the cell cycle, particularly genes of the G2/M phase. NF-YA, the regulatory DNA-binding subunit of the complex, is expressed in proliferating myoblasts and down-regulated during terminal differentiation. The NF-YA gene encodes for two alternatively spliced isoforms, namely NF-YAs and NF-YAl, which are not functionally identical. Using mouse C2C12 cells, we provide evidence of a different role for NF-YA variants in the myogenic program. While NF-YAs enhances myoblasts proliferation, NF-YAl boosts their differentiation by up-regulating the transcription of novel target genes, among which Mef2D, Sixs and Cdkn1C, which are known to be involved in the differentiation program. We further demonstrate that NF-YA is expressed in resident stem cells (SCs) and the two isoforms are transcribed at different levels during SCs activation and differentiation. The inhibition of NF-Y activity impairs both proliferation and differentiation of SCs and the overexpression of the two NF-YA isoforms differentially affects their fate.

2015 - Phosphorylation and alternative splicing of MEF2C, a dual switch function in muscle regeneration [Abstract in Rivista]
Baruffaldi, Fiorenza; Badodi, Sara; De Feo, Luca; Ganassi, Massimo; Battini, Renata; Imbriano, Carol; Nicoletti, Carmine; Musarò, Antonio; Buckingham, Margaret; Montarras, Didier; Molinari, Susanna
abstract

Muscle regeneration is a multistep process that is regulated by a restricted number of transcription factors whose activity is modulated at multiple levels. However, how different layers of regulation are coordinated to promote adult myogenesis is not yet understood. Here we show that the MEF2C transcription factor controls multiple steps of muscle regeneration, including myogenic progression of satellite cells and muscle maturation of newly generated myofibers, exhibiting multiple functions that depend on alternative splicing and post-translational modifications. Inclusion of the 1 exon in Mef2c transcripts is upregulated in proliferating mouse satellite cells and in the early phases of muscle regeneration. The encoded MEF2C1 isoform stimulates expansion of primary myoblasts ex vivo and in vivo. The pro-proliferative activity of MEF2C is mediated by phosphorylation of two phosphoserines located in exon 1. Subsequent terminal differentiation and growth of newly formed myofibers are promoted by dephosphorylated MEF2C1 and MEF2C2. Our results thus reveal an important role for regulatory interactions between alternative splicing and post translational modifications of a single transcription factor in the control of the multilayered regulatory programs required for adult myogenesis.

2015 - Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition [Articolo su rivista]
Badodi, Sara; Baruffaldi, Fiorenza; Ganassi, Massimo; Battini, Renata; Molinari, Susanna
abstract

The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.

2014 - Distinct functions of alternatively spliced isoforms encoded by zebrafish mef2ca and mef2cb. Ganassi M, Badodi S, Polacchini A, Baruffaldi F, Battini R, Hughes SM, Hinits Y, Molinari S. Biochim Biophys Acta. 2014 Jul;1839(7):559-70. doi: 10.1016/j.bbagrm.2014.05.003. Epub 2014 May 17. [Articolo su rivista]
Ganassi, Massimo; Badodi, Sara; Polacchini, A; Baruffaldi, Fiorenza; Battini, Renata; Hughes, Sm; Hinits, Y; Molinari, Susanna
abstract

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4-6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4-6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4-5-6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity

2013 - A specific role for the splice variants of the transcription factor NF-Y in modulating the transcriptional activity of the myogenic program [Poster]
Basile, Valentina; Baruffaldi, Fiorenza; Belluti, Silvia; Benatti, Paolo; Mantovani, Roberto; Molinari, Susanna; Imbriano, Carol
abstract

Cell proliferation and differentiation programs are highly regulated transcriptional processes essential for myogenesis. The transcription factor NF-Y has been long considered a fundamental player of cell growth by supporting the basal transcription of various cell cycle genes. It is composed by the NF-YB/NF-YC heterodimer and NF-YA, which interacts with the other two subunits and confers the strict sequence specificity to the complex. The NF-YA gene encodes two alternatively splice transcripts (NF-YAs and NF-YAl), which differently regulate cell proliferation and differentiation, as shown in haematopoietic and mouse embryonic stem cells. NF-YAl expression is down-regulated in terminally differentiated muscle cells and in skeletal and cardiac muscle tissues. Its forced expression in muscle cells committed to differentiate impairs their exit from the cell cycle and indirectly interferes with the differentiation program. Here we show that the two NF-YA isoforms play a different role in the transcriptional activity of the myogenic program and may regulate the activity of muscle satellite cells.

2013 - IL FATTORE DI TRASCRIZIONE MEF2C AI CROCEVIA TRA PROLIFERAZIONE, SVILUPPO E DIFFERENZIAMENTO MUSCOLARE [Poster]
Ferrari, Stefano; Battini, Renata; Magli, Alessandro; Angelelli, Cecilia; Badodi, Massimo Ganassi Sara; Baruffaldi, Fiorenza; Molinari, Susanna
abstract

Il principale interesse del laboratorio è lo studio della funzione della proteina MEF2C, un fattore di trascrizione abbondante nel tessuto muscolare scheletrico dove dirige il differenziamento terminale dei precursori miogenici (mioblasti). In specifico, ci occupiamo di definire i meccanismi che regolano la funzione di MEF2C nella progressione miogenica. Abbiamo identificato un meccanismo di repressione dell'attività di MEF2C nei mioblasti proliferanti che coinvolge la fosforilazione della proteina e la sua successiva interazione fosfo-specifica con l'enzima Pin1, una peptidil-prolil cis trans isomerasi che diminuisce la stabilità di MEF2C. Questo meccanismo repressivo è importante per inibire un differenziamento prematuro dei precursori miogenici che in tal modo possono amplificarsi. La repressione Pin1-dipendente di MEF2C è attiva sia nella linea cellulare miogenica C2C12 che in cellule satelliti, vale a dire mioblasti primari di muscolo adulto, responsabili dei meccanismi di rigenerazione in caso di danno muscolare. Ci proponiamo di valutare se questo meccanismo è alterato nella distrofia di Duchenne, dove è stata osservata una diminuita capacità proliferativa delle cellule satelliti. L'attività di MEF2C è anche regolata da meccanismi di splicing alternativo del corrispondente trascritto. Nel nostro laboratorio stiamo definendo le funzioni specifiche di due isoforme di splicing di MEF2C che si differenziano per la presenza di due esoni mutualmente esclusivi: esone alpha1 e alpha 2. I nostri dati preliminari indicano che le due varianti di splicing svolgono funzioni opposte nel corso della progressione miogenica, in particolare la variante alpha1 sembra essere importante nel controllare la progressione nel ciclo delle cellule muscolari mentre la variante alpha 2 sembra essenziale per la trascrizione muscolo-specifica. Infine abbiamo osservato che in Zebrafish, Danio rerio, Mef2c svolge un ruolo importante nel regolare lo sviluppo embrionale, specificamente nella determinazione dorso-ventrale dell'embrione, inoltre la sua attività viene modulata attraverso meccanismi di splicing anche in questo organismo.

2013 - Regulation of Mef2c activity by alternative splicing in zebrafish muscle development [Poster]
Ganassi, Massimo; Badodi, Sara; Alessio, Polacchini; Baruffaldi, Fiorenza; Molinari, Susanna
abstract

Teleost fish possess two Mef2c gene paralogues: mef2ca and mef2cb. In zebrafish, Danio rerio, the Mef2cs proteins function to promote cardiomyogenic differentiation and myofibrilogenesis in nascent skeletal muscle fibers. In mouse and human, an array of MEF2C proteins are generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform in vivo. We found that the transcripts of zebrafish mef2ca and mef2cb are alternatively spliced, the resulting splice variants have differing biological activity. Among the two mef2c paralogues, mef2ca is more abundantly expressed in developing skeletal muscle and the splicing pattern of its transcript is tuned through zebrafish development. At 24 hours post fertilization (hpf), a stage when mef2ca activity is required for proper myogenesis, we found the expression of a highly active full length protein. At earlier stages mef2ca activity is attenuated by AS, indeed a high proportion of the mef2ca transcripts lack part of the transcriptional activation domain. We present evidence that the developmentally regulated AS of mef2ca transcripts is important for a correct development of the zebrafish embryos.

2012 - A POTENTIAL ROLE OF MEF2C IN THE MYOGENIC PROGRESSION BESIDE TERMINAL DIFFERENTIATION [Poster]
Baruffaldi, Fiorenza; Badodi, Sara; Ganassi, Massimo; Angelelli, Cecilia; Magli, Alessandro; Margaret Buckingham, Didier Montarras; Molinari, Susanna
abstract

MEF2C belongs to the family of Myocyte Enhancer Factor 2 transcription factors, which activate the muscle-specific gene expression program. Its activity is finely modulated at several levels but some aspects of this regulation still remains uncharacterized; for example, MEF2C is already expressed in proliferating myoblasts, but it is transcriptionally silent unless the cells are stimulated to withdraw the cell cycle and differentiate. Phosphorylation of MEF2 factors at so-called Ser/Thr-Pro motifs can modulate protein function through the induction of conformational changes by the peptidyl–prolyl cis/trans isomerase Pin1. This regulatory mechanism is based on the physical interaction between Pin1 and the Ser98 and Ser110 phosphoacceptor sites located in the alternative spliced exon α1 of MEF2C. The MEF2C/Pin1 interaction results in the repression of MEF2C stability and transcriptional activity, inhibiting muscle differentiation. We investigated the function of this regulatory mechanism in primary myogenic stem cells (SCs) combining the analysis of the dynamics of MEF2C phosphorylation with the study of the alternative splicing pattern. We demonstrated that the conditions necessary for the interaction between MEF2C and Pin1 are satisfied exclusively in proliferating SCs, where:  Pin1 expression is upregulated  MEF2C isoform containing the exon α1 specifically appears in response to activation signals  MEF2C phosphorylation on the Pin1-binding sites occurs specifically in proliferating SCs Indeed we showed that MEF2C and Pin1 can interact in the nuclei of C2C7 and SCs-derived myoblasts. Overall we provide evidence that this interaction not only would serve as a failsafe mechanism to keep silent the MEF2C-dependent transcription of muscle specific genes in proliferating SCs but we hypothesize that the expression of the MEF2Cα1 isoform phosphorylated on Ser98 and Ser110 and the interaction with Pin1 might actively contribute to promote SCs proliferation, allowing the expansion of the activated SCs pool and avoiding their premature differentiation.

2011 - Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells [Poster]
Magli, Alessandro; Ganassi, Massimo; Baruffaldi, Fiorenza; Badodi, Sara; Angelelli, Cecilia; Battini, Renata; Sal, Giannino Del; Molinari, Susanna
abstract

MEF2 (myocyte enhancer factor 2) transcription factors (MEF2A-D) are highly expressed in skeletal muscle cells, they bind to a conserved AT rich DNA sequence through their N-ter MADS and MEF2 domains and activate transcription via their C-ter transcriptional activation domains (TAD), the functional domains of MEF2C are indicated in Figure 1. MEF2 proteins interact with members of the MyoD family of basic helix–loop–helix (bHLH) proteins to establish a unique transcriptional code for skeletal muscle gene activation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2 phosphorylation and its association with other transcriptional cofactors. We show that the Pin1 isomerase, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds, interacts with phosphorylated MEF2C in muscle cells. This interaction requires two novel phospho-Ser-Pro motifs in MEF2C: Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenesis. Furthermore Pin1 modulates the skeletal muscle differentiation program because down-regulation of Pin1 markedly promotes myogenic differentiation. We suggest that Pin1 is a novel regulator of MEF2C function and muscle differentiation, it is expressed in muscle cells and a significant proportion of Pin1 in myotubes but not in myoblasts is excluded from the nucleus. We observed a reduction of phosphorylation of the Ser(98) and Ser(110) Pin1 binding sites in differentiated myocytes. Based on these results we propose a model in which, in proliferating myoblasts, Pin1, upon binding to phosphorylated nuclear MEF2C, leads to decreased levels and transcriptional activity of MEF2C. Upon induction of terminal differentiation, to establish a full activity of MEF2 proteins, a reduced Pin1-MEF2C association is required, possibly due to the relegation of Pin1 to the cytoplasm and to a reduced level of phosphorylation of Ser98 and Ser110.

2010 - PROLINE ISOMERASE PIN1 REPRESSES TERMINAL DIFFERENTIATION AND MYOCYTE ENHANCER FACTOR 2C FUNCTION IN SKELETAL MUSCLE CELLS [Articolo su rivista]
Magli, Alessandro; Angelelli, Cecilia; Ganassi, Massimo; Baruffaldi, Fiorenza; V., Matafora; Battini, Renata; A., Bachi; G., Messina; A., Rustighi; G., Del Sal; S., Ferrari; Molinari, Susanna
abstract

Reversible proline-directedphosphorylation at Ser/Thr-Pro motifs has anessential role in myogenesis, a multistep processstrictly regulated by several signalling pathwaysthat impinge on two families of myogeniceffectors, the basic Helix Loop Helix (bHLH)myogenic transcription factors and the MyocyteEnhancer Factor 2 (MEF2) proteins. Thequestion of how these signals are deciphered bythe myogenic effectors remains largelyundefined. In this study we show that thepeptidyl-prolyl isomerase Pin1, which catalyzesthe isomerization of phosphorylated Ser/Thr-Propeptide bonds to induce conformational changesof its target proteins, acts as an inhibitor ofmuscle differentiation as its knock-down inmyoblasts promotes myotube formation. Withthe aim of clarifying the mechanism of Pin1function in skeletal myogenesis, we investigatedwhether MEF2C, a critical regulator of themyogenic program that is the endpoint of severalsignalling pathways, might serve as a/the targetfor the inhibitory effects of Pin1 on muscledifferentiation. We show that Pin1 interactsselectively with phosphorylated MEF2C inskeletal muscle cells, both in vitro and in vivo.The interaction with Pin1 requires two novelcritical pSer/Thr-Pro motifs in MEF2C, Ser98and Ser110, which are phosphorylated in vivo.Overexpression of Pin1 decreases MEF2Cstability and activity, and its ability to cooperatewith MyoD to activate myogenic conversion.Collectively these findings reveal a novel role forPin1 as a regulator of muscle terminaldifferentiation and suggest that Pin1 mediatedrepression of MEF2C function could contributeto this function.

2008 - Differentiation-dependent lysine 4 acetylation enhances MEF2C binding to DNA in skeletal muscle cells [Articolo su rivista]
Angelelli, Cecilia; Magli, Alessandro; Ferrari, Daniela; Ganassi, M; Matafora, V; Parise, Flavia; Razzini, Giorgia; Bachi, A; Ferrari, Stefano; Molinari, Susanna
abstract

Myocyte enhancer factor 2 (MEF2) proteins play a key role in promoting the expression of muscle-specific genes in differentiated muscle cells. MEF2 activity is regulated by the association with several transcriptional co-factors and by post-translational modifications. In the present report, we provide evidence for a novel regulatory mechanism of MEF2C activity, which occurs at the onset of skeletal muscle differentiation and is based on Lys4 acetylation. This covalent modification results in the enhancement of MEF2C binding to DNA and chromatin. In particular, we report that the kinetic parameters of MEF2/DNA association change substantially upon induction of differentiation to give a more stable complex and that this effect is mediated by Lys4 acetylation. We also show that Lys4 acetylation plays a prominent role in the p300-dependent activation of MEF2C.

2004 - A novel complex regulates cardiac actin gene expression through interaction of Emb, a class VI POU domain protein, MEF2D, and the histone transacetylase p300 [Articolo su rivista]
Molinari, Susanna; Relaix, F; Lemonnier, M; Kirschbaum, B; Schafer, B; Buckingham, M.
abstract

Expression of the mouse cardiac actin gene depends on a distal enhancer (-7 kbp) which has been shown, in transgenic mice, to direct expression to embryonic skeletal muscle. The presence of this distal sequence is also associated with reproducible expression of cardiac actin transgenes. In differentiated skeletal muscle cells, activity of the enhancer is driven by an E box, binding MyoD family members, and by a 3' AT-rich sequence which is in the location of a DNase I-hypersensitive site. This sequence does not bind MEF2 proteins, or other known muscle transcription factors, directly. Oct1 and Emb, a class VI POU domain protein, bind to consensus sites on the DNA, and it is the binding of Emb which is important for activity. Emb binds as a major complex with MEF2D and the histone transacetylase p300. The form of Emb present in this complex and as a major form in muscle cell extracts is longer (80 kDa) than that previously described. These results demonstrate the importance of this novel complex in the transcriptional regulation of the cardiac actin gene and suggest a potential role in chromatin remodeling associated with muscle gene activation.

2004 - Low-density lipoprotein (LDL) receptor/transferrin fusion protein: in vivoproduction and functional evaluation as a potential therapeutic tool forlowering plasma LDL cholesterol. [Articolo su rivista]
Razzini, Giorgia; Parise, Flavia; Calebiro, D; Battini, Renata; Bagni, Bruno; Corazzari, Tolmino; Tarugi, Patrizia Maria; Angelelli, Cecilia; Molinari, Susanna; Falqui, L; Ferrari, Stefano
abstract

A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.

2004 - The in vivo form of the murine class VIPOU protein Emb is larger than that encoded by previously described transcripts [Articolo su rivista]
Relaix, F; Molinari, Susanna; Lemonnier, M; Schafer, B; Buckingham, M.
abstract

The class VI POU domain family member known as Emb in the mouse (rat Bm5 or human mPOU/TCFbeta1) is present in vivo as a protein migrating at about 80 kDa on western blots, considerably larger than that predicted (about 42 kDa) from previously cloned coding sequences. By RT-PCR and 5´ RACE strategies a full-length Emb sequence, Emb FL, is now identified. Shorter sequences encoding the -COOH terminal, and an -NH2 terminal isoform, EmbN, were also isolated. Comparisons of Emb coding sequences between species, including the full-length zebra fish, POU(c), are presented, together with a compilation of the multiple transcripts produced by alternative splicing and the presence of different transcriptional start and stop sites, from the Emb gene.

2001 - The nuclear localization domain of the MEF2 family of transcription factors shows member-specific features and mediates the nuclear import of histone deacetylase 4 [Articolo su rivista]
Borghi, S.; Molinari, Susanna; Razzini, G.; Parise, F.; Battini, Renata; Ferrari, Stefano
abstract

Targeting of myocyte enhancer binding factor 2 (MEF2) proteins to the nucleus depends on a C-terminal bipartite nuclear localization signal (NLS). By expression of green fluorescent protein (GFP)/MEF2 fusion proteins in transfected myoblasts, we show that MEF2C contains an additional 13 amino acids domain, located immediately upstream of the NLS, which contributes to its nuclear retention. We also show that the NLS present in MEF2 proteins is required for efficient nuclear localization of histone deacetylase 4 (HDAC4). In muscle cells, transfected HDAC4 is largely cytoplasmic or, to a lesser extent, pancellular. Co-transfection of either MEF2A or MEF2C causes HDAC4 to accumulate in the nucleus in association with MEF2. This effect strongly depends on MEF2 NLS; it also requires the specific interaction of HDAC4 with MEF2, since the isolated NLS is not sufficient for targeting HDAC4 to the nucleus and other nuclear proteins, such as NF-Y, cannot substitute MEF2. Therefore, we demonstrate that HDAC4, different from HDAC5, is mainly a cytoplasmic resident protein, requiring a trans-acting NLS for nuclear localization. The physiological implications of MEF2 carrying its own inhibitor to the nucleus are discussed.

1997 - Absence of mef2 binding to the A/T-rich element in the muscle creatine kinase (MCK) enhancer correlates with lack of early expression of the MCK gene in embryonic mammalian muscle [Articolo su rivista]
Ferrari, S.; Molinari, S.; Melchionna, R.; Angelis, M. G. C.; Battini, R.; De Angelis, L.; Kelly, R.; Cossu, G.
abstract

During skeletal muscle development, different types of muscle fibers are generated, which express different combinations of muscle-specific gene products, For example, the muscle creatine kinase gene (MCK) is highly expressed in fetal but not embryonic myotubes, We performed transient transfections of CAT reporter constructs, driven by the MCK promoter with variable lengths of 5'-flanking sequence, into primary cultures of embryonic and fetal muscle cells, Reporter activity was observed in fetal but not embryonic muscle cells, We assayed the ability of nuclear extracts prepared from embryonic and fetal muscle and C2C12 myotubes to bind specific regulatory elements in the MCK enhancer, The profile of DNA/protein complexes resulting from electrophoretic mobility shift assays was qualitatively the same with all extracts used when the oligonucleotide probes represented the MCK E-box, MHox site, CArG-box, and AP2 site, In contrast, no binding activity to the MEF2 site was observed with embryonic nuclear extract, Interestingly, MEF2 mRNAs and proteins were detected in both fetal and embryonic muscle, with the exception of the MEF2D1b isoform, which is restricted to fetal muscle, Furthermore, we found that protein phosphatase inhibitors included in the preparation of embryonic nuclear extracts or added to the medium of transfected embryonic myotubes can restore MEF2 DNA binding activity, as well as reporter activity driven by the MCK promoter and partial transcriptional activation of the endogenous MCK gene, We propose that phosphorylation of MEF2 regulates its activity and represents an important aspect of the mechanism controlling stage-specific transcription during skeletal myogenesis.

1996 - Deficits in memory and hippocampal long-term potentiation in mice with reduced calbindin D28K expression [Articolo su rivista]
Molinari, Susanna; Battini, Renata; Ferrari, Stefano; L., Pozzi; As, Killcross; Tw, Robbins; A., Jouvenceau; Jm, Billard; P., Dutar; Y., Lamour; Wa, Baker; H., Cox; Pc, Emson
abstract

The influx of calcium into the postsynaptic neuron is likely to be an important event in memory formation. Among the mechanisms that nerve cells may use to alter the Lime course or size of a spike of intracellular calcium are cytosolic calcium binding or ''buffering'' proteins. To consider the role in memory formation of one of these proteins, calbindin D-28K, which is abundant in many neurons, including the CA1 pyramidal tells of the hippocampus, transgenic mice deficient in calbindin D-28K have been created. These mice show selective impairments in spatial learning paradigms and fail to maintain long-term potentiation. These results suggest a role for calbindin D28K protein in temporally extending a neuronal calcium signal, allowing the activation of calcium-dependent intracellular signaling pathways underlying memory function.

1996 - Phosphorylation-dependent binding of MEF2 to the A/T rich element in the muscle creatine kinase enhancer correlates with lack of expression in embryonic mammalian muscle [Articolo su rivista]
Ferrari, S.; Molinari, S.; Melchionna, R.; Cusella-De Angelis, M. G.; Battini, R.; De Angelis, L.; Kelly, R.; Cossu, G.
abstract

1994 - Differential response of embryonic and fetal myoblasts to TGF-beta. A possible regulatory mechanism of skeletal histogenesis [Articolo su rivista]
CUSELLA DE ANGELIS, Mg; Molinari, Susanna; LE DONNE, A; Coletta, M; Vivarelli, E; Bouche, M; Molinaro, M; Ferrari, Stefano; Cossu, G.
abstract

Embryonic and fetal skeletal myoblasts were grown in culture in the presence of TGF beta. Under the conditions employed, TGF beta inhibited differentiation of fetal but not of embryonic myoblasts, To investigate the possible relevance of these data to skeletal muscle histogenesis in vivo, we studied the proliferation/differentiation state of mesodermal cells in the proximal region of the limb bud at the time of primary fiber formation. BrdU labeling and immunostaining for myosin heavy chains revealed that very few mesodermal cells enter the S phase of the cycle when differentiated primary fibers fist appear. However, a few hours later, many cells in S phase surround newly formed muscle fibers, suggesting that the latter may be a source of mitogens for undifferentiated myoblasts. Coculture experiments supported this hypothesis, showing that medium conditioned by fiber-containing explants can stimulate myoblast proliferation. Taken together these data suggested a possible mechanism for the regulation of muscle fiber formation. The model assumes that fibers form in the proximal region of the limb bud, where TGF beta is known to be present, and BrdU labeling experiments did not reveal cells in S phase. It is conceivable that non-dividing embryonic myoblasts (which do not respond to TGF beta) can undergo differentiation, while fetal myoblasts are inhibited by TGF beta. Once formed, primary fibers may stimulate a new wave of proliferation in fetal myoblasts, in order to expand the pool of cells needed to form secondary fibers. o test this model we developed an organ culture for limb buds which resulted in the production of myotubes with a phenotype similar to embryonic (primary) and fetal (secondary) fibers, roughly at the time when primary and secondary fibers form in vivo. When these cultures were treated with TGF beta, embryonic myotubes did form (as expected), but fetal myotubes never appeared. Conversely, when these cultures were treated with anti-TGF beta neutralizing antibodies, fetal myotubes developed earlier than in control cultures, suggesting that endogenously produced TGF beta may repress differentiation of fetal cells in vitro and, possibly, in vivo.

1994 - Specific binding to vitamin D response elements of chicken intestinal DNA-binding activity is not related to the vitamin D receptor [Articolo su rivista]
Ferrari, Stefano; Battini, Renata; Molinari, Susanna
abstract

In this report we confirm that the putative vitamin D response element (VDRE), located between -320 and -360 in the chicken calbindin-D-28k gene, is not a binding site for the vitamin D-3 receptor (VDR). In examining the ability of chicken intestinal nuclear extracts (CINE) to bind known VDREs, we observed a specific VDRE-binding activity, which is distinct from VDR. In fact, VDR-depleted CINE retains the ability to bind the rat osteocalcin VDRE. The VDRE-binding activity binds DNA with high affinity and contacts it as the same guanine residues as VDR. Its specificity in binding structural variants of the AGGTCA repeat is broader than that of VDR, as direct repeats spaced by 3, 4, and 5 base pairs are almost equally effective competitors when added to the probe in molar excess. Palindromic arrangements of the same motif are lower affinity competitors. The retinoid-X-receptors is involved in the binding complex, as incubation of CINE with antibody to retinoid-X receptor results in a quantitative supershift. Antibodies to retinoic acid receptors (RAR alpha and -beta), T-3 receptor, or chicken ovalbumin up-stream promoter-transcription factor has no apparent effect. These data suggest that species specificty is a relevant aspect of VDR/DVRE recognition, and that a novel factor(s), different from VDR, might be involved in the effect of vitamin D on gene expression.

1992 - Induction of Calbindin-D28K by 1,25-dihydroxyvitamin D3 in cultured chicken intestinal cells [Articolo su rivista]
Ferrari, Stefano; Molinari, Susanna; Battini, Renata; Cossu, G; Lamonfava, S.
abstract

Intestinal cells from chicken embryos were grown in chemically defined, serum-free medium. The majority of cultured cells exhibits an epithelial-like morphology. As demonstrated by indirect immunofluorescence, the epithelial cells, and not the contaminating fibroblasts, express Calbindin-D28K only after 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, is added to the culture medium. The highly sensitive reverse transcriptase-polymerase chain reaction shows that both Calbindin-D28K mRNA and the corresponding primary unprocessed transcripts (pre-mRNA) are dramatically increased in cultured intestinal cells treated with 1,25-dihydroxyvitamin D3, thus indicating that Calbindin-D28K is induced by the increased rate of transcription of the corresponding gene. © 1992.