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Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of early childhood with a poor survival rate, thus there is a requirement for improved treatment strategies. Induced pluripotent stem cells offer the ability to model disease and develop new treatment strategies. JMML is frequently associated with mutations in PTPN11. Children with Noonan syndrome, a development disorder, have an increased incidence of JMML associated with specific germline mutations in PTPN11. We undertook a proteomic assessment of myeloid cells derived from induced pluripotent stem cells obtained from Noonan syndrome patients with PTPN11 mutations, either associated or not associated with an increased incidence of JMML. We report that the proteomic perturbations induced by the leukemia-associated PTPN11 mutations are associated with TP53 and NF-Kκb signaling. We have previously shown that MYC is involved in the differential gene expression observed in Noonan syndrome patients associated with an increased incidence of JMML. Thus, we employed drugs to target these pathways and demonstrate differential effects on clonogenic hematopoietic cells derived from Noonan syndrome patients, who develop JMML and those who do not. Further, we demonstrated these small molecular inhibitors, JQ1 and CBL0137, preferentially extinguish primitive hematopoietic cells from sporadic JMML patients as opposed to cells from healthy individuals.

Characterization of pluripotent states, in which cells can both self-renew or differentiate, with the irreversible loss of pluripotency, are important research areas in developmental biology. Although microRNAs (miRNAs) have been shown to play a relevant role in cellular differentiation, the role of miRNAs integrated into gene regulatory networks and its dynamic changes during these early stages of embryonic stem cell (ESC) differentiation remain elusive. Here we describe the dynamic transcriptional regulatory circuitry of stem cells that incorporate protein-coding and miRNA genes based on miRNA array expression and quantitative sequencing of short transcripts upon the downregulation of the Estrogen Related Receptor Beta (Esrrb). The data reveals how Esrrb, a key stem cell transcription factor, regulates a specific stem cell miRNA expression program and integrates dynamic changes of feed-forward loops contributing to the early stages of cell differentiation upon its downregulation. Together these findings provide new insights on the architecture of the combined transcriptional post-transcriptional regulatory network in embryonic stem cells.

Although pluripotent cells lack circadian oscillations, this study reveals a novel function of the master circadian regulator BMAL1 in metabolism and early differentiation of embryonic stem cells., BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.

The aryl hydrocarbon receptor (AhR) is well-known for its major contributions to the cellular responses against environmental toxins and carcinogens. Notably, AhR has also emerged as a key transcription factor controlling many physiological processes including cell proliferation and apoptosis, differentiation, adhesion and migration, pluripotency and stemness. These novel functions have broadened our understanding of the signalling pathways and molecular intermediates interacting with AhR under both homeostatic and pathological conditions. Recent discoveries link AhR with the function of essential organs such as liver, skin and gonads, and with complex organismal structures including the immune and cardiovascular systems. The identification of potential endogenous ligands able to regulate AhR activity, opens the possibility of designing ad hoc molecules with pharmacological and/or therapeutic value to treat human diseases in which AhR may have a causal role. Integration of experimental data from in vitro and in vivo studies with "omic" analyses of human patients affected with cancer, immune diseases, inflammation or neurological disorders will likely contribute to validate the clinical relevance of AhR and the possible benefits of modulating its activity by pharmacologically-driven strategies. In this review, we will highlight signalling pathways involved in human diseases that could be targetable by AhR modulators and discuss the feasibility of using such molecules in therapy. The pros and cons of AhR-aimed approaches will be also mentioned.

Epigenetic mechanisms and transcription factor networks essential for differentiation of cardiac myocytes have been uncovered. However, reshaping of the epigenome of these terminally differentiated cells during fetal development, postnatal maturation, and in disease remains unknown. Here, we investigate the dynamics of the cardiac myocyte epigenome during development and in chronic heart failure. We find that prenatal development and postnatal maturation are characterized by a cooperation of active CpG methylation and histone marks at cis-regulatory and genic regions to shape the cardiac myocyte transcriptome. In contrast, pathological gene expression in terminal heart failure is accompanied by changes in active histone marks without major alterations in CpG methylation and repressive chromatin marks. Notably, cis-regulatory regions in cardiac myocytes are significantly enriched for cardiovascular disease-associated variants. This study uncovers distinct layers of epigenetic regulation not only during prenatal development and postnatal maturation but also in diseased human cardiac myocytes.

Summary Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS), whereby 40% of patients develop hypertrophic cardiomyopathy (HCM). As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC) model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ) paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies., Graphical Abstract, Highlights • Cardiomyocytes and fibroblast-like cells can be purified separately from EBs • BRAF-mutant cardiomyocytes display hypertrophy and intrinsic Ca2+-handling defects • BRAF-mutant fibroblast-like cells influence cardiomyocyte hypertrophy through TGFβ • The hypertrophic phenotype can be rescued by TGFβ or RAS/MAPK inhibition, To better understand the role of RAS/MAPK signaling in the pathogenesis of hypertrophic cardiomyopathy (HCM), Gelb and colleagues purified cardiomyocytes and fibroblast-like cells from hiPSCs derived from patients with BRAF mutations causing RASopathy-associated HCM. While mutant cardiomyocytes were pro-hypertrophic with altered Ca2+ handling, mutant fibroblast-like cells critically modulated cardiomyocyte hypertrophy through increased TGFβ signaling.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.

Planar cell polarity (PCP)-signaling and associated tissue polarization are evolutionarily conserved. A well documented feature of PCP-signaling in vertebrates is its link to centriole/cilia positioning, although the relationship of PCP and ciliogenesis is still debated. A recent report in Drosophila established that Frizzled (Fz)-PCP core signaling has an instructive input to polarized centriole positioning in non-ciliated Drosophila wing epithelia as a PCP read-out. Here, we review the impact of this observation in the context of recent descriptions of the relationship(s) of core Fz-PCP signaling and cilia/centriole positioning in epithelial and non-epithelial cells. All existing data are consistent with a model where Fz-PCP signaling functions upstream of centriole/cilia positioning, independent of ciliogenesis. The combined data sets indicate that the Fz-Dsh PCP complex is instructive for centriole/ciliary positioning via an actin-based mechanism. Thereby, centriole/cilia/centrosome positioning can be considered an evolutionarily conserved readout and common downstream effect of PCP-signaling from flies to mammals.

Background Myocarditis is inflammation of the heart muscle that can follow various viral infections. Why children only rarely develop life-threatening acute viral myocarditis (AVM), given that the causal viral infections are common, is unknown. Genetic lesions might underlie such susceptibilities. Mouse genetic studies demonstrated that interferon (IFN)-α/β immunity defects increased susceptibility to virus-induced myocarditis. Moreover, variations in human TLR3, a potent inducer of IFNs, were proposed to underlie AVM. Objectives This study sought to evaluate the hypothesis that human genetic factors may underlie AVM in previously healthy children. Methods We tested the role of TLR3-IFN immunity using human induced pluripotent stem cell-derived cardiomyocytes. We then performed whole-exome sequencing of 42 unrelated children with acute myocarditis (AM), some with proven viral causes. Results We found that TLR3- and STAT1-deficient cardiomyocytes were not more susceptible to Coxsackie virus B3 (CVB3) infection than control cells. Moreover, CVB3 did not induce IFN-α/β and IFN-α/β-stimulated genes in control cardiomyocytes. Finally, exogenous IFN-α did not substantially protect cardiomyocytes against CVB3. We did not observe a significant enrichment of rare variations in TLR3- or IFN-α/β-related genes. Surprisingly, we found that homozygous but not heterozygous rare variants in genes associated with inherited cardiomyopathies were significantly enriched in AM-AVM patients compared with healthy individuals (p = 2.22E-03) or patients with other diseases (p = 1.08E-04). Seven of 42 patients (16.7%) carried rare biallelic (homozygous or compound heterozygous) nonsynonymous or splice-site variations in 6 cardiomyopathy-associated genes (BAG3, DSP, PKP2, RYR2, SCN5A, or TNNI3). Conclusions Previously silent recessive defects of the myocardium may predispose to acute heart failure presenting as AM, notably after common viral infections in children.

Traditionally considered as a critical intermediate in the toxic and carcinogenic response to dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), the Aryl hydrocarbon/Dioxin receptor (AhR) has proven to be also an important regulator of cell physiology and organ homeostasis. AhR has become an interesting and actual area of research mainly boosted by a significant number of recent studies analyzing its contribution to the proper functioning of the immune, hepatic, cardiovascular, vascular and reproductive systems. At the cellular level, AhR establishes functional interactions with signaling pathways governing cell proliferation and cell cycle, cell morphology, cell adhesion and cell migration. Two exciting new aspects in AhR biology deal with its implication in the control of cell differentiation and its more than likely involvement in cell pluripotency and stemness. In fact, it is possible that AhR could help modulate the balance between differentiation and pluripotency in normal and transformed tumor cells. At the molecular level, AhR regulates an increasingly large array of physiologically relevant genes either by traditional transcription-dependent mechanisms or by unforeseen processes involving genomic insulators, chromatin dynamics and the transcription of mobile genetic elements. AhR is also closely related to epigenetics, not only from the point of view of target gene expression but also with respect to its own regulation by promoter methylation. It is reasonable to consider that deregulation of these many functions could have a causative role, or at least contribute to, human disease. Consequently, several laboratories have proposed that AhR could be a valuable tool as diagnostic marker and/or therapeutic target in human pathologies. An additional point of interest is the possibility of regulating AhR activity by endogenous non-toxic low weight molecules agonist or antagonist molecules that could be present or included in the diet. In this review, we will address these molecular and functional features of AhR biology within physiological and pathological contexts.

13 páginas, 12 figuras.-- et al., The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR-/-) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR- /- cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR-/- fibroblasts had reduced Racl activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Racl shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knock-down of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3-/- MEFs, as AhR-/- mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states. © 2009 by The American Society for Cell Biology., This work was supported by grants to P.M.F.-S. from the Spanish Ministry of Education and Sciences (SMES) (SAF2005-00130, SAF2008-0462), the Junta de Extremadura (2PR04A060) and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/1016, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health) and by grants to X.R.B. from the US National Cancer Institute/NIH (5R01-CA73735-11), the Spanish Ministry of Education and Science (MES) (SAF2006-01789), the Castilla-León Autonomous Government (SA053A05), and the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (RD06/0020/0001, Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health). J.M.C.-G. and A.C.R. were supported by fellowships from the Junta de Extremadura and the SMES, respectively. V. S. has been partially supported by the SMES Juan de la Cierva program and the National Institutes of Health. All Spanish funding is cosponsored by the European Union FEDER program.

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2'-deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

Hypertrophic cardiomyopathy (HCM) is a pathological disorder predominantly due to mutations in sarcomeric components. Germline mutations in BRAF cause a developmental syndrome called cardio-facio-cutaneous syndrome (CFCS), in which 40% of patients also develop HCM. Since the role of the RAS/MAPK pathway in HCM is still unclear, we generated a human induced pluripotent stem cell model for CFCS from three patients with activating BRAF T599R or Q257R mutations. In order to examine hiPSC-derived cell-type specific phenotypes and cellular interactions underpinning HCM, we generated a method to purify cardiomyocytes and non-cardiomyocytes simultaneously by cell sorting based on SIRPα and CD90 expression. Purified BRAF-mutant SIRPα+/CD90- cells were >95% cardiomyocytes, displayed cellular hypertrophy with pro-hypertrophic gene expression, and dysregulation of the RAS/MAPK pathway. BRAF-mutant cardiomyocytes also displayed intrinsic calcium handling defects, including increased calcium transient irregularity and increased stored calcium within the sarcoplasmic reticulum. In addition, purified BRAF-mutant SIRPα-/CD90+ cells, which were fibroblast-like, displayed activation of the RAS/MAPK pathway and exhibited a pro-fibrotic phenotype. Cross-culture studies revealed that BRAF-mutant fibroblast-like cells critically modulate cardiomyocyte hypertrophy through TGFβ paracrine signaling, as TGFβ inhibition prevented cardiomyocyte hypertrophy induced by BRAF-mutant fibroblast-like cells. Additionally, inhibition of RAS/MAPK signaling was capable of rescuing BRAF-mutant cardiomyocyte hypertrophy and cardiomyocyte-intrinsic calcium handling abnormalities. Thus, we show for the first time that cell autonomous and non-autonomous defects underlie RASopathy-associated HCM. As fibroblast activation has been documented previously in sarcomeric HCM, our findings suggest that cardiac fibroblasts may contribute to pathologic hypertrophy in addition to causing fibrosis in primary forms of HCM. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type ( AhR +/+) and null ( AhR –/–) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-β binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR –/– MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1 , again suggesting an AhR-regulated mechanism. Treatment of AhR +/+ MEF with transforming growth factor-β(TGF-β) downregulated AhR and, simultaneously, increased Ltbp-1 , further supporting the role of this receptor in LTBP-1 expression. AhR –/– conditioned medium had higher levels of active and total TGF-β activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-β concentrations. TGF-β did not appear to directly regulate Ltbp-1 given that addition of TGFβ neutralizing antibody or TGFβ protein to AhR –/– MEF had no effect on Ltbp-1 expression. AhR –/– MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFβ activity and cell proliferation.

Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO over-production. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a significant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor l-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca2+ concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca2+ entry could contribute to this process. Thus, neurodegeneration could diminish the induction of specific P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

Resveratrol, a natural phytoalexin, has gained much interest on the basis of its potential chemopreventive activity against human cancer. In this work, using the human breast cancer cell lines MCF-7 and MDA-MB-231, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Our results show that although resveratrol inhibited cell proliferation and viability in both cell lines, apoptosis was induced in a concentration- and cell-specific manner. In MDA-MB-231, resveratrol (up to 200 microM) lowered the expression and kinase activities of positive G1/S and G2/M cell cycle regulators and inhibited ribonucleotide reductase activity in a concentration dependent manner, without a significant effect on the low expression of tumor suppressors p21, p27, and p53. These cells died by a non-apoptotic process in the absence of a significant change in cell cycle distribution. In MCF-7, resveratrol produced a significant and transient (

The aryl hydrocarbon receptor (AHR) is a transcription factor that is highly conserved during evolution and shares important structural features with the Drosophila developmental regulators Sim and Per. Although much is known about the mechanism of AHR activation by xenobiotics, little information is available regarding its activation by endogenous stimuli in the absence of exogenous ligand. In this study, using embryonic primary fibroblasts, we have analyzed the role of proteasome inhibition on AHR transcriptional activation in the absence of xenobiotics. Proteasome inhibition markedly reduced cytosolic AHR without affecting its total cellular content. Cytosolic AHR depletion was the result of receptor translocation into the nuclear compartment, as shown by transient transfection of a green fluorescent protein-tagged AHR and by immunoblot analysis of nuclear extracts. Gel retardation experiments showed that proteasome inhibition induced transcriptionally active AHR-ARNT heterodimers able to bind to a consensus xenobiotic-responsive element. Furthermore, nuclear AHR was transcriptionally active in vivo, as shown by the induction of the endogenous target gene CYP1A2. Synchronized to AHR activation, proteasome inhibition also induced a transient increase in AHR nuclear translocator (ARNT) at the protein and mRNA levels. Since nuclear levels of AHR and ARNT are relevant for AHR transcriptional activation, our data suggest that proteasome inhibition, through a transient increase in ARNT expression, could promote AHR stabilization and accumulation into the nuclear compartment. An elevated content of nuclear AHR could favor AHR-ARNT heterodimers able to bind to xenobiotic-responsive elements and to induce gene transcription in the absence of xenobiotics. Thus, depending on the cellular context, physiologically regulated proteasome activity could participate in the control of endogenous AHR functions.

Recent studies have suggested a regulatory role for the dioxin receptor (AhR) in cell adhesion and migration. Following our previous work, we report here that the C-terminal Src kinase-binding protein (Cbp) signaling pathway controls β1 integrin activation and that this mechanism is AhR dependent. T-FGM AhR −/− fibroblasts displayed higher integrin β1 activation, revealed by the increased binding of the activation reporter 9EG7 anti-β1 mAb and of a soluble fibronectin fragment, as well as by enhanced talin-β1 association. AhR −/− fibroblasts also showed increased fibronectin secretion and impaired directional migration. Notably, interfering Cbp expression in AhR −/− fibroblasts reduced β1 integrin activation, improved cell migration and rescued wild-type cell morphology. Cbp over-expression in T-FGM AhR −/− cells enhanced the formation of inhibitory Csk–Cbp complexes which in turn reduced c-Src p-Tyr 416 activation and focal adhesion kinase (FAK) phosphorylation at the c-Src-responsive residues p-Tyr 576 and p-Tyr 577 . The c-Src target and migration-related protein Cav1 was also hypophosphorylated at p-Tyr 14 in AhR −/− cells, and such effect was rescued by down-modulating Cbp levels. Thus, AhR regulates fibroblast migration by modulating β1 integrin activation via Cbp-dependent, Src-mediated signaling.

Changes in chromatin structure are an essential mechanism for gene regulation (transcription, replication, DNA repair and recombination). ATP-dependent chromatin-remodeling enzymes have previously been implicated in maintaining and regulating chromatin structure. These enzymes are divided into several groups including the SWI/SNF, ISWI and CHD families. Although the Chromodomain Helicase DNA-binding protein (CHD) family members are involved in key cellular processes, the disrupted regulation of their expression has not been fully assessed. An impairment of chromatin remodeling activity, mediated by promoter CpG island hypermethylation of the described candidate genes, could be key in cancer pathology. Herein, we examine the DNA methylation profiles of CHD family members (CHD1-9) in different tumor types. For all CHDs, CpG island hypermethylation was only observed at the CHD5 promoter in human cancer cell lines and primary tumors, particularly gliomas and colon and breast carcinomas. RT-qPCR analyses correlated CHD5 loss of expression with hypermethylation of the promoter, and restoration of CHD5 mRNA levels upon treatment with a DNA demethylating agent. These results underpin the epigenetic inactivation of the chromating remodeling factor CHD5 as one contributor for the aberrant structural changes of chromatin throughout the genome of the cancer cell.

As our knowledge on the mechanisms that control cell function increases, more complex signaling pathways and quite intricate cross-talks among regulatory proteins are discovered. Establishing accurate interactions between cellular networks is essential for a healthy cell and different alterations in signaling are known to underline human disease. Transforming growth factor beta (TGFbeta) is an extracellular cytokine that regulates such critical cellular responses as proliferation, apoptosis, differentiation, angiogenesis and migration, and it is assumed that the latency-associated protein LTBP-1 plays a relevant role in TGFbeta targeting and activation in the extracellular matrix (ECM). The dioxin receptor (AhR) is a unique intracellular protein long studied because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Yet, a large set of studies performed in cellular systems and in vivo animal models have suggested important xenobiotic-independent functions for AhR in cell proliferation, differentiation and migration and in tissue homeostasis. Remarkably, AhR activity converges with TGFbeta-dependent signaling through LTBP-1 since cells lacking AhR expression have phenotypic alterations that can be explained, at least in part, by the coordinated regulation of both proteins. Here, we will discuss the existence of functional interactions between AhR and TGFbeta signaling. We will focus on regulatory and functional aspects by analyzing how AhR status determines TGFbeta activity and by proposing a mechanism through which LTBP-1, a novel AhR target gene, mediates such effects. We will integrate ECM proteases in the AhR-LTBP-1-TGFbeta axis and suggest a model that could help explain some in vivo phenotypes associated to AhR deficiency.

The importance of epigenetic processes in the development of cancer is clear. The study of epigenetics is therefore bound to contribute to the improvement of human health. Aberrations in DNA methylation, post-translational modifications of histones, chromatin remodeling and microRNAs patterns are the main epigenetic alterations, and these are associated with tumorigenesis. Epigenetic technologies in cancer studies are helping increase the number of cancer candidate genes and allow us to examine changes in 5-methylcytosine DNA and histone modifications at a genome-wide level. In fact, all the various cellular pathways contributing to the neoplastic phenotype are affected by epigenetic genes in cancer. They are being explored as biomarkers in clinical use for early detection of disease, tumor classification and response to treatment with classical chemotherapy agents, target compounds and epigenetic drugs. Encouraging results have been obtained with histone deacetylase and DNA methyltransferase inhibitors, leading the US Food and Drug Administration to approve several of them for the treatment of hematological malignancies and lymphoproliferative disorders, such as myelodysplastic syndrome and cutaneous lymphoma. However, many tasks remains to be done, such as the clinical validation of epigenetic biomarkers to allow the accurate prediction of the outcome of cancer patients and their potential chemosensitivity to current pharmacological treatments.

In mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR), high levels of latent transforming growth factor-beta (TGF-beta)-binding protein-1 (LTBP-1) correlated with increased TGF-beta1 activity, an observation suggesting that LTBP-1 could contribute to maintain TGF-beta1 levels. Here, using small interfering RNAs (siRNA), we have first analyzed if LTBP-1 expression affected TGF-beta1 activity in MEF cells. We have then determined how LTBP-1 levels could alter the activity of extracellular proteases known to activate TGF-beta1, and finally, whether protease inhibition could reduce TGF-beta1 activation. LTBP-1 inhibition by siRNA in AhR-/- MEF decreased the amount of active TGF-beta1 and reduced plasminogen activators (PA)/plasmin and elastase activities and thrombospondin-1 (TSP-1) expression, without significantly affecting their mRNA levels. On the contrary, LTBP-1 siRNA restored matrix metalloproteinase-2 (MMP-2) activity in AhR-/- MEF. Interestingly, whereas a TGF-beta1 neutralizing antibody mimicked many of the LTBP-1 siRNA effects on extracellular proteases, addition of recombinant TGF-beta1 protein increased proteases activity over basal levels in AhR-/- MEF. These proteases contributed to TGF-beta activation since their specific inhibitors reduced active TGF-beta levels in these cells. These results suggest that LTBP-1 contributes to TGF-beta1 activation in MEF, possibly by influencing the activities of PA/plasmin, elastase, TSP-1, and MMP-2. TGF-beta1, on the other hand, could be also involved in maintaining the activity of these extracellular proteases. Thus, LTBP-1 appears to play a role in TGF-beta1 activation through a process involving extracellular protease activities, which, in turn, could be affected by TGF-beta1 levels.

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERaalpha)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kappaB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kappaB, were both inhibited by RES. The patterns for NF-kappaB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kappaB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kappaB and calpain protease activity. Therefore, Bcl-2 and NF-kappaB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells.

11 páginas, 9 figuras.-- PMID: 15946950 [PubMed].-- et al., Although the dioxin receptor, the aryl hydrocarbon receptor (AhR), is considered a major regulator of xenobiotic-induced carcinogenesis, its role in tumor formation in the absence of xenobiotics is still largely unknown. Trying to address this question, we have produced immortalized cell lines from wild-type (T-FGM-AhR+/+) and mutant (T-FGM-AhR-/-) mouse mammary fibroblasts by stable co-transfection with the simian virus 40 (SV-40) large T antigen and proto-oncogenic c-H-Ras. Both cell lines had a myofibroblast phenotype and similar proliferation, doubling time, SV-40 and c-H-Ras expression and activity, and cell cycle distribution. AhR+/+ and AhR-/- cells were also equally able to support growth factor- and anchorage-independent proliferation. However, the ability of T-FGM-AhR-/- to induce subcutaneous tumors (leimyosarcomas) in NOD/SCID-immunodeficient mice was close to 4-fold lower than T-FGM-AhR+/+. In culture, T-FGM-AhR-/- had diminished migration in collagen-I and decreased lamellipodia formation. VEGFR-1/Flt-1, a VEGF receptor that regulates cell migration and blood vessel formation, was also down-regulated in AhR-/- cells. Signaling through the ERK-FAK-PKB/AKT-Rac-1 pathway, which contributes to cell motility and invasion, was also significantly inhibited in T-FGM-AhR-/-. Thus, the lower tumorigenic potential of T-FGM-AhR-/- could result from a compromised adaptability of these cells to the in vivo microenvironment, possibly because of an impaired ability to migrate and to respond to angiogenesis., This work has been funded by Grant SAF2002-00034 from the Spanish Ministry of Science and Technology and from the Junta de Extremadura (2PR01A092) (to P. M. F.-S.). Work in the I. S. G. laboratory was supported by grants from the MCyT (BIO2000-0453-01, SAF2003-01103, and FIT-010000-2004-157), Junta de Castilla y León (CSI06/13), ADE Castilla y León (04/04/SA/0001), FIS (PI020138, G03/179, and G03/136) and the USAL-CIBASA project.

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming.