Cristiano Simone

p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38α/β repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38α-β in vitro, and forced activation of p38α/β in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

Physical and functional HAT/HDAC interplay regulates protein acetylation balance.

The balance between protein acetylation and deacetylation controls several physiological and pathological cellular processes, and the enzymes involved in the maintenance of this equilibrium—acetyltransferases (HATs) and deacetylases (HDACs)—have been widely studied. Presently, the evidences obtained in this field suggest that the dynamic acetylation equilibrium is mostly maintained through the physical and functional interplay between HAT and HDAC activities. This model overcomes the classical vision in which the epigenetic marks of acetylation have only an activating function whereas deacetylation marks have a repressing activity. Given the existence of several players involved in the preservation of this equilibrium, the identification of these complex networks of interacting proteins will likely foster our understanding of how cells regulate intracellular processes and respond to the extracellular environment and will offer the rationale for new therapeutic approaches based on epigenetic drugs in human diseases.

The AMPK-FoxO3A axis as a target for cancer treatment.

FoxO proteins are an evolutionarily conserved subfamily of transcription factors involved in tumor suppression, regulation of energy metabolism and development in several tissues, and are mainly regulated by phosphorylation-dependent nuclear/cytoplasmic shuttling. The transcriptional activity of FoxO3A, one of the four members of the family, is further modulated by AMPK, one of the key regulators of cellular metabolism, which basically shifts cell machinery from energy-consuming to energy-producing pathways. We recently demonstrated that the AMPK/FoxO3A energy sensor pathway is still inducible in human cancer cells in response to metabolic stress, as it becomes activated in colorectal and ovarian cancer cells in response to the inhibition of p38α. Activation of the FoxO3A transcriptional program initially induces autophagy as an attempt to retain energy to survive, whereas under persistent stress conditions it triggers autophagic cell death. In this review, we focus on the connections between AMPK and FoxO3A, describing their central role as modulators of fundamental processes such as stress resistance, cell metabolism, autophagy and cell death, and highlighting the therapeutic potential of pharmacological modulation of the AMPK-FoxO3A axis.

SWI/SNF: The crossroads where extracellular signaling pathways meet chromatin

The coordinated expression of the genome in response to extracellular cues is ensured by enzymatic cascades signaling to the nucleus. These pathways generate chromatin modifications at specific loci controlling the transcription of signal‐dependent and tissue‐specific genes. The SWI/SNF chromatin remodeling complex offers the ideal surface for integrating these signals in the execution of diverse or even opposite biological programs. J. Cell. Physiol. 207: 309–314, 2006.

pRb: master of differentiation. Coupling irreversible cell cycle withdrawal with induction of muscle-specific transcription

The protein product of the retinoblastoma (RB) gene is necessary for the completion of the muscle differentiation program and for myogenic basic helix–loop–helix-dependent transcription. In fact, in addition to induction and maintenance of permanent cell cycle withdrawal through negative regulation of E2F-responsive genes involved in proliferation, pRb also plays a positive role in the activation of muscle-specific genes. In pRb−/− myocytes, the expression of late myogenic markers is defective and myoblast fusion into myotubes occurs without irreversible cell cycle exit. This evidence demonstrates only a partial functional redundancy between pRb and its relatives p107 and pRb2/p130, as these pRb−/− multinucleated cells, which display p107 levels higher than normal myotubes, respond to mitogens with cell cycle re-entry and DNA synthesis. At the molecular level, pRb myogenic functions are mediated by cooperation with MyoD, Myocyte enhancer factor 2 (MEF2), High mobility group box protein-1 (HBP1) and histone deacetylase1, affecting chromatin configuration and tissue-specific transcription, and by post-translational modification in response to intracellular signaling cascades.

Activation of MyoD-dependent transcription by cdk9/cyclin T2.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101–161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

p38α MAPK pathway: A key factor in colorectal cancer therapy and chemoresistance

Colorectal cancer (CRC) remains one of the most common malignancies in the world. Although surgical resection combined with adjuvant therapy is effective at the early stages of the disease, resistance to conventional therapies is frequently observed in advanced stages, where treatments become ineffective. Resistance to cisplatin, irinotecan and 5-fluorouracil chemotherapy has been shown to involve mitogen-activated protein kinase (MAPK) signaling and recent studies identified p38α MAPK as a mediator of resistance to various agents in CRC patients. Studies published in the last decade showed a dual role for the p38α pathway in mammals. Its role as a negative regulator of proliferation has been reported in both normal (including cardiomyocytes, hepatocytes, fibroblasts, hematopoietic and lung cells) and cancer cells (colon, prostate, breast, lung tumor cells). This function is mediated by the negative regulation of cell cycle progression and the transduction of some apoptotic stimuli. However, despite its anti-proliferative and tumor suppressor activity in some tissues, the p38α pathway may also acquire an oncogenic role involving cancer related-processes such as cell metabolism, invasion, inflammation and angiogenesis. In this review, we summarize current knowledge about the predominant role of the p38α MAPK pathway in CRC development and chemoresistance. In our view, this might help establish the therapeutic potential of the targeted manipulation of this pathway in clinical settings.

Inhibition of p38α unveils an AMPK-FoxO3A axis linking autophagy to cancer-specific metabolism

Autophagy is an essential process for the maintenance of cellular and metabolic homeostasis. Indeed, it is required for the recovery of ATP-generating substrates in cells subjected to different types of stress insults. Thus, the activity of the autophagic machinery strongly depends on the metabolic status of the cell.1 It has been proposed that this principle applies not only to normal, but also to cancer cells,2 despite the profound differences in their metabolism. Cancer cells predominantly produce ATP through the constitutive activation of aerobic glycolysis, a process that generally relies on the stabilization and activation of the transcription factor HIF1α, which regulates the expression of glycolytic genes.3 We recently showed that p38α is required to sustain the expression of HIF1α target genes, and that its inhibition causes a rapid drop in ATP levels in colorectal cancer cells (CRCs). This acute energy need triggers AMPK-dependent nuclear accumulation of FoxO3A and subsequent activation of its transcriptional program, leading to sequential induction of autophagy, cell cycle arrest and cell death. In vivo, pharmacological blockade of p38α has both a cytostatic and cytotoxic effect on colorectal neoplasms, associated with nuclear enrichment of FoxO3A and expression of its target genes p21 and PTEN.4 Our data suggest that CRCs impaired in their glycolytic metabolism trigger autophagy as a reversible recovery mechanism and undergo cell cycle arrest; however, the persistence of the stress insults inevitably leads to cell death.

Physical interaction between pRb and cdk9/cyclinT2 complex

Cyclin-dependent kinase 9 (cdk9) is a multifunctional kinase with roles in different cellular pathways such as transcriptional elongation, differentiation and apoptosis. Cdk9/cyclin T differs functionally from other cdk/cyclin complexes that regulate cell cycle progression, but maintains structural affinity with those complexes. In addition, previous reports have demonstrated that the cdk9 complex is able to phosphorylate p56/pRb in vitro. In this report we show in vitro and in vivo interaction between cdk9/cyclinT2 and the protein product of the retinoblastoma gene (pRb) in human cell lines. The interaction involves the region composed of residues 129–195 of cdk9, cyclinT2 (1–642 aa) and the C-terminal domain of pRb (835–928 aa). We located the minimal region of cdk9 phosphorylation on the C-terminus of pRb, by identifying the residues between 793 and 834. This region contains at least three proline-directed serines (sp), S795, S807 and S811, which have been reported to be phosphorylated in vivo and which could be targeted by the cdk9 complex. These data suggest that, in logarithmically growing cells, cdk9/cyclin T2 and pRb are located in a nuclear multiprotein complex probably involved in transduction of cellular signals to the basal transcription machinery and that one of these signals could be the cdk9 phosphorylation of pRb.

A homozygous frameshift mutation in the ESCO2 gene: Evidence of intertissue and interindividual variation in Nmd efficiency

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non‐consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real‐time ESCO2 cDNA analysis on RNA isolated from CVS‐stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3–exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2‐truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents. J. Cell. Physiol. 209: 67–73, 2006.