Germana Falcone

Microrna-221 and Microrna-222 Modulate Differentiation and Maturation of Skeletal Muscle Cells

Background MicroRNAs (miRNAs) are a class of small non-coding RNAs that have recently emerged as important regulators of gene expression. They negatively regulate gene expression post-transcriptionally by translational repression and target mRNA degradation. miRNAs have been shown to play crucial roles in muscle development and in regulation of muscle cell proliferation and differentiation. Methodology/Principal Findings By comparing miRNA expression profiling of proliferating myoblasts versus differentiated myotubes, a number of modulated miRNAs, not previously implicated in regulation of myogenic differentiation, were identified. Among these, miR-221 and miR-222 were strongly down-regulated upon differentiation of both primary and established myogenic cells. Conversely, miR-221 and miR-222 expression was restored in post-mitotic, terminally differentiated myotubes subjected to Src tyrosine kinase activation. By the use of specific inhibitors we provide evidence that expression of miR-221 and miR-222 is under the control of the Ras-MAPK pathway. Both in myoblasts and in myotubes, levels of the cell cycle inhibitor p27 inversely correlated with miR-221 and miR-222 expression, and indeed we show that p27 mRNA is a direct target of these miRNAs in myogenic cells. Ectopic expression of miR-221 and miR-222 in myoblasts undergoing differentiation induced a delay in withdrawal from the cell cycle and in myogenin expression, followed by inhibition of sarcomeric protein accumulation. When miR-221 and miR-222 were expressed in myotubes undergoing maturation, a profound alteration of myofibrillar organization was observed. Conclusions/Significance miR-221 and miR-222 have been found to be modulated during myogenesis and to play a role both in the progression from myoblasts to myocytes and in the achievement of the fully differentiated phenotype. Identification of miRNAs modulating muscle gene expression is crucial for the understanding of the circuits controlling skeletal muscle differentiation and maintenance.

Expression of RALT, a feedback inhibitor of ErbB receptors, is subjected to an integrated transcriptional and post-translational control.

Over-expression studies have demonstrated that RALT (receptor associated late transducer) is a feedback inhibitor of ErbB-2 mitogenic and transforming signals. In growth-arrested cells, expression of endogenous RALT is induced by mitogenic stimuli, is high throughout mid to late G1 and returns to baseline as cells move into S phase. Here, we show that physiological levels of RALT effectively suppress ErbB-2 mitogenic signals. We also investigate the regulatory mechanisms that preside to the control of RALT expression. We demonstrate that pharmacological ablation of extracellular signal-regulated kinase (ERK) activation leads to blockade of RALT expression, unlike genetic and/or pharmacological interference with the activities of PKC, Src family kinases, p38 SAPK and PI-3K. Tamoxifen-dependent activation of an inducible Raf : ER chimera was sufficient to induce RALT expression. Thus, activation of the Ras–Raf–ERK pathway is necessary and sufficient to drive RALT expression. The RALT protein is labile and was found to accumulate robustly upon pharmacological inhibition of the proteasome. We were able to detect ubiquitin-conjugated RALT species in living cells, suggesting that ubiquitinylation targets RALT for proteasome-dependent degradation. Such an integrated transcriptional and post-translational control is likely to provide RALT with the ability to fluctuate timely in order to tune ErbB signals.

Fine Regulation of RhoA and Rock Is Required for Skeletal Muscle Differentiation

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Signaling by exosomal microRNAs in cancer

A class of small non-coding RNAs, the microRNAs (miRNAs), have recently attracted great attention in cancer research since they play a central role in regulation of gene-expression and miRNA aberrant expression is found in almost all types of human cancer. The discovery of circulating miRNAs in body fluids and the finding that they are often tumor specific and can be detected early in tumorigenesis has soon led to the evaluation of their possible use as cancer biomarkers and treatment-response predictors. The evidence that tumor cells communicate via the secretion and delivery of miRNAs packed into tumor-released microvesicles has prompted to investigate miRNA contribution as signaling molecules to the establishment and maintenance of the tumor microenvironment and the metastatic niche in cancer. In this review we highlight the recent advances on the role of exosomal miRNAs as mediators of cancer cell-to-cell communication.

Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation.

SH3-containing proteins are involved in signal transduction by a number of growth factor receptors and in the organization of the cytoskeleton. The recently identified Eps8 protein, which contains an SH3 domain, is coupled functionally and physically to the EGFR and is tyrosine phosphorylated by this receptor and other receptors as well. Here, we examined the regulation of eps8 expression in response to mitogenic or differentiative signals. We show that Eps8 is expressed at low levels in resting fibroblasts, but its expression is strongly induced during activation by serum, phorbol esters and the v-src oncogene. Conversely, expression of Eps8, but not of other EGFR substrates such as Shc or Eps15, is virtually extinguished in non-proliferating, terminally differentiated murine myogenic cells. The putative role of Eps8 protein as a v-Src substrate was analysed in murine fibroblasts and in quail myogenic cells expressing a temperature-sensitive variant of the tyrosine kinase. Tyrosine phosphorylation of Eps8 was detected only at the permissive temperature. A non-myristylated, transformation-defective mutant of v-Src did not phosphorylate Eps8, whereas it phosphorylated Shc. Together, these findings indicate that Eps8 may be a critical substrate of v-Src. They further establish Eps8 as an example of a signal transducer whose expression senses the balance between growth and differentiation and might, therefore, be involved in the determination of the phenotype.

Induction of telomerase activity in v-myc-transformed avian cells

Telomerase activity is detectable in the majority of tumors or immortalized cell lines, but is repressed in most normal human somatic cells. It is generally assumed that reactivation of telomerase prevents the erosion of chromosome ends which occurs in cycling cells and, hence, hinders cellular replicative senescence. Here, we show that the expression of v-Myc oncoprotein by retroviral infection of telomerase-negative embryonal quail myoblasts and chicken neuroretina cells is sufficient for reactivating telomerase activity, earlier than telomere shortening could occur. Furthermore, the use of a conditional v-Myc-estrogen receptor protein (v-MycER) causes estrogen-dependent expression of detectable levels of telomerase activity in recently infected chick embryo fibroblasts and neuroretina cells. We conclude that the high levels of telomerase activity in v-Myc-expressing avian cells are not the mere consequence of transformation or of a differentiative block, since v-Src tyrosine kinase, which prevents terminal differentiation and promotes cell transformation, fails to induce telomerase activity.

Delineating v-Src downstream effector pathways in transformed myoblasts

In this study, we delineate the intracellular signalling pathways modulated by a conditional v-Src tyrosine kinase that lead to unrestrained proliferation and block of differentiation of primary avian myoblasts. By inhibiting Ras–MAPK kinase and phosphatidylinositol 3-kinase with different means, we find that both pathways play crucial roles in controlling v-Src-sustained growth factor and anchorage independence for proliferation. The Ras–MAPK kinase pathway also contributes to block of differentiation independently of cell proliferation since inhibition of this pathway both in proliferating and growth-arrested v-Src-transformed myoblasts induces expression of muscle-specific genes, fusion into multinucleated myotubes and assembly of specialized contractile structures. Importantly, we find that the p38 MAPK pathway is inhibited by v-Src in myoblasts and its forced activation results in growth inhibition and expression of differentiation, indicating p38 MAPK as a critical target of v-Src in growth transformation and myogenic differentiation. Furthermore, we show that downregulation of p38 MAPK activation may occur via Ras–MAPK kinase, thus highlighting a cross-regulation between the two pathways. Finally, we report that the simultaneous inhibition of MAPK kinase and calpain, combined to activation of p38 MAPK, are sufficient to reconstitute largely the differentiation potential of v-Src-transformed myoblasts.

Prolonged lifespan with enhanced exploratory behavior in mice overexpressing the oxidized nucleoside triphosphatase hMTH1

The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1‐Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1‐Tg mice express high levels of the hMTH1 hydrolase that degrades 8‐oxodGTP and 8‐oxoGTP and excludes 8‐oxoguanine from both DNA and RNA. Compared to wild‐type animals, hMTH1‐overexpressing mice have significantly lower steady‐state levels of 8‐oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age‐dependent accumulation of DNA 8‐oxoguanine that occurs in wild‐type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1‐Tg animals live significantly longer than their wild‐type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1‐Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1‐Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1‐Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.

CRISPR/Cas9-Mediated Deletion of CTG Expansions Recovers Normal Phenotype in Myogenic Cells Derived from Myotonic Dystrophy 1 Patients

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, characterized by progressive myopathy, myotonia, and multi-organ involvement. This dystrophy is an inherited autosomal dominant disease caused by a (CTG)n expansion within the 3′ untranslated region of the DMPK gene. Expression of the mutated gene results in production of toxic transcripts that aggregate as nuclear foci and sequester RNA-binding proteins, resulting in mis-splicing of several transcripts, defective translation, and microRNA dysregulation. No effective therapy is yet available for treatment of the disease. In this study, myogenic cell models were generated from myotonic dystrophy patient-derived fibroblasts. These cells exhibit typical disease-associated ribonuclear aggregates, containing CUG repeats and muscleblind-like 1 protein, and alternative splicing alterations. We exploited these cell models to develop new gene therapy strategies aimed at eliminating the toxic mutant repeats. Using the CRISPR/Cas9 gene-editing system, the repeat expansions were removed, therefore preventing nuclear foci formation and splicing alterations. Compared with the previously reported strategies of inhibition/degradation of CUG expanded transcripts by various techniques, the advantage of this approach is that affected cells can be permanently reverted to a normal phenotype.

MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells.

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protein.