Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma.

Giant congenital naevi are pigmented childhood lesions that frequently lead to melanoma, the most aggressive skin cancer. The mechanisms underlying this malignancy are largely unknown, and there are no effective therapies. Here we describe a mouse model for giant congenital naevi and show that naevi and melanoma prominently express Sox10, a transcription factor crucial for the formation of melanocytes from the neural crest. Strikingly, Sox10 haploinsufficiency counteracts Nras(Q61K)-driven congenital naevus and melanoma formation without affecting the physiological functions of neural crest derivatives in the skin. Moreover, Sox10 is also crucial for the maintenance of neoplastic cells in vivo. In human patients, virtually all congenital naevi and melanomas are SOX10 positive. Furthermore, SOX10 silencing in human melanoma cells suppresses neural crest stem cell properties, counteracts proliferation and cell survival, and completely abolishes in vivo tumour formation. Thus, SOX10 represents a promising target for the treatment of congenital naevi and melanoma in human patients.

Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

JNK3 is abundant in insulin-secreting cells and protects against cytokine-induced apoptosis.

AIMS/HYPOTHESIS: In insulin-secreting cells, activation of the c-Jun NH(2)-terminal kinase (JNK) pathway triggers apoptosis. Whereas JNK1 and JNK2 are ubiquitously produced, JNK3 has been described exclusively in neurons. This report aims to characterise the expression and role in apoptosis of the three JNK isoforms in insulin-secreting cells exposed to cytokines. METHODS: Sections of human and mouse pancreases were used for immunohistochemistry studies with isoform-specific anti-JNK antibodies. Human, pig, mouse and rat pancreatic islets were isolated by enzymatic digestion and RNA or protein extracts were prepared. RNA and protein levels were determined by quantitative RT-PCR and western blotting respectively, using JNK-isoform-specific primers and isoform-specific antibodies; activities of the three JNK isoforms were determined by kinase assays following quantitative immunoprecipitation/depletion of JNK3. JNK silencing was performed with small interfering RNAs and apoptotic rates were determined in INS-1E cells by scoring cells displaying pycnotic nuclei. RESULTS: JNK3 and JNK2 mRNAs are the predominant isoforms expressed in human pancreatic islets. JNK3 is nuclear while JNK2 is also cytoplasmic. In INS-1E cells, JNK3 knockdown increases c-Jun levels and caspase-3 cleavage and sensitises cells to cytokine-induced apoptosis; in contrast, JNK1 or JNK2 knockdown is protective. CONCLUSIONS/INTERPRETATION: In insulin-secreting cells, JNK3 plays an active role in preserving pancreatic beta cell mass from cytokine attacks. The specific localisation of JNK3 in the nucleus, its recruitment by cytokines, and its effects on key transcription factors such as c-Jun, indicate that JNK3 is certainly an important player in the transcriptional control of genes expressed in insulin-secreting cells.

Genome-wide analysis of SREBP1 activity around the clock reveals its combined dependency on nutrient and circadian signals.

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1-/- mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved.

Combinatorial effects of splice variants modulate function of Aiolos

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.

Influence of a fat overload on lipogenic regulators in metabolic syndrome patients

Several epidemiological studies have related an increase of lipids in the postprandial state to an individual risk for the development of CVD, possibly due to the increased plasma levels of TAG and fatty acids (FA) through enzymes of FA metabolism. The interaction between nutrition and the human genome determines gene expression and metabolic response. The aim of the present study was to evaluate the influence of a fat overload on the gene mRNA levels of lipogenic regulators in peripheral blood mononuclear cells (PBMC) from patients with the metabolic syndrome. The study included twenty-one patients with criteria for the metabolic syndrome who underwent a fat overload. Measurements were made before and after the fat overload of anthropometric and biochemical variables and also the gene mRNA levels of lipogenic factors. The main results were that the fat overload led to an increased mRNA levels of sterol regulatory element binding protein-1 (SREBP1), retinoid X receptor α (RXRα) and liver X receptor α (LXRα) in PBMC, and this increase was associated with the FA synthase (FASN) mRNA levels. We also found that TAG levels correlated with FASN mRNA levels. In addition, there was a positive correlation of SREBP1 with RXRα and of LXRα with the plasma lipoperoxide concentration. The fat overload led to an increase in regulators of lipogenesis in PBMC from patients with the metabolic syndrome.

Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter.

Peroxisome proliferator-activated receptor (PPARs) are members of the nuclear receptor superfamily. For transcriptional activation of their target genes, PPARs heterodimerize with the retinoid-X receptor (RXR). The convergence of the PPAR and RXR signaling pathways has been shown to have an important function in lipid metabolism. The promoter of the gene encoding the acyl-coenzyme-A oxidase (ACO), the rate-limiting enzyme in peroxisomal beta-oxidation of fatty acids, is a target site of PPAR action. In this study, we examined the role and the contribution of both cis-and trans-acting factors in the transcriptional regulation of this gene using transient transfections in insect cells. We identified several functional cis-acting elements present in the promoter of the ACO gene and established that PPAR-dependent as well as PPAR-independent mechanisms can activate the ACO promoter in these cells. We show that the PPAR/RXR heterodimer exerts its effect through two response elements within the ACO promoter, in synergy with the transcription factor Sp1 via five Sp1-binding sites. Furthermore, this functional interaction also occurs when Sp1 is co-expressed with PPAR or RXR alone, indicating that activation can occur independently of PPAR/RXR heterodimers.

Comparative genomics of ParaHox clusters of teleost fishes: gene cluster breakup and the retention of gene sets following whole genome duplications.

BACKGROUND: The evolutionary lineage leading to the teleost fish underwent a whole genome duplication termed FSGD or 3R in addition to two prior genome duplications that took place earlier during vertebrate evolution (termed 1R and 2R). Resulting from the FSGD, additional copies of genes are present in fish, compared to tetrapods whose lineage did not experience the 3R genome duplication. Interestingly, we find that ParaHox genes do not differ in number in extant teleost fishes despite their additional genome duplication from the genomic situation in mammals, but they are distributed over twice as many paralogous regions in fish genomes. RESULTS: We determined the DNA sequence of the entire ParaHox C1 paralogon in the East African cichlid fish Astatotilapia burtoni, and compared it to orthologous regions in other vertebrate genomes as well as to the paralogous vertebrate ParaHox D paralogons. Evolutionary relationships among genes from these four chromosomal regions were studied with several phylogenetic algorithms. We provide evidence that the genes of the ParaHox C paralogous cluster are duplicated in teleosts, just as it had been shown previously for the D paralogon genes. Overall, however, synteny and cluster integrity seems to be less conserved in ParaHox gene clusters than in Hox gene clusters. Comparative analyses of non-coding sequences uncovered conserved, possibly co-regulatory elements, which are likely to contain promoter motives of the genes belonging to the ParaHox paralogons. CONCLUSION: There seems to be strong stabilizing selection for gene order as well as gene orientation in the ParaHox C paralogon, since with a few exceptions, only the lengths of the introns and intergenic regions differ between the distantly related species examined. The high degree of evolutionary conservation of this gene cluster's architecture in particular - but possibly clusters of genes more generally - might be linked to the presence of promoter, enhancer or inhibitor motifs that serve to regulate more than just one gene. Therefore, deletions, inversions or relocations of individual genes could destroy the regulation of the clustered genes in this region. The existence of such a regulation network might explain the evolutionary conservation of gene order and orientation over the course of hundreds of millions of years of vertebrate evolution. Another possible explanation for the highly conserved gene order might be the existence of a regulator not located immediately next to its corresponding gene but further away since a relocation or inversion would possibly interrupt this interaction. Different ParaHox clusters were found to have experienced differential gene loss in teleosts. Yet the complete set of these homeobox genes was maintained, albeit distributed over almost twice the number of chromosomes. Selection due to dosage effects and/or stoichiometric disturbance might act more strongly to maintain a modal number of homeobox genes (and possibly transcription factors more generally) per genome, yet permit the accumulation of other (non regulatory) genes associated with these homeobox gene clusters.

VEGF gene expression in adult human thymus fat: a correlative study with hypoxic induced factor and cyclooxygenase-2.

It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to adipogenesis and angiogenesis.

Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

Molecular evidence for a functional ecdysone signaling system in Brugia malayi.

BACKGROUND: Filarial nematodes, including Brugia malayi, the causative agent of lymphatic filariasis, undergo molting in both arthropod and mammalian hosts to complete their life cycles. An understanding of how these parasites cross developmental checkpoints may reveal potential targets for intervention. Pharmacological evidence suggests that ecdysteroids play a role in parasitic nematode molting and fertility although their specific function remains unknown. In insects, ecdysone triggers molting through the activation of the ecdysone receptor: a heterodimer of EcR (ecdysone receptor) and USP (Ultraspiracle). METHODS AND FINDINGS: We report the cloning and characterization of a B. malayi EcR homologue (Bma-EcR). Bma-EcR dimerizes with insect and nematode USP/RXRs and binds to DNA encoding a canonical ecdysone response element (EcRE). In support of the existence of an active ecdysone receptor in Brugia we also cloned a Brugia rxr (retinoid X receptor) homolog (Bma-RXR) and demonstrate that Bma-EcR and Bma-RXR interact to form an active heterodimer using a mammalian two-hybrid activation assay. The Bma-EcR ligand-binding domain (LBD) exhibits ligand-dependent transactivation via a GAL4 fusion protein combined with a chimeric RXR in mammalian cells treated with Ponasterone-A or a synthetic ecdysone agonist. Furthermore, we demonstrate specific up-regulation of reporter gene activity in transgenic B. malayi embryos transfected with a luciferase construct controlled by an EcRE engineered in a B. malayi promoter, in the presence of 20-hydroxy-ecdysone. CONCLUSIONS: Our study identifies and characterizes the two components (Bma-EcR and Bma-RXR) necessary for constituting a functional ecdysteroid receptor in B. malayi. Importantly, the ligand binding domain of BmaEcR is shown to be capable of responding to ecdysteroid ligands, and conversely, ecdysteroids can activate transcription of genes downstream of an EcRE in live B. malayi embryos. These results together confirm that an ecdysone signaling system operates in B. malayi and strongly suggest that Bma-EcR plays a central role in it. Furthermore, our study proposes that existing compounds targeting the insect ecdysone signaling pathway should be considered as potential pharmacological agents against filarial parasites.

The effect of standard and high-fluence corneal cross-linking (CXL) on cornea and limbus.

PURPOSE: When treating peripheral ectatic disease-like pellucid marginal degeneration (PMD), corneal cross-linking with UV-A and riboflavin (CXL) must be applied eccentrically to the periphery of the lower cornea, partly irradiating the corneal limbus. Here, we investigated the effect of standard and double-standard fluence corneal cross-linking with riboflavin and UV-A (CXL) on cornea and corneal limbus in the rabbit eye in vivo. METHODS: Epithelium-off CXL was performed in male New Zealand White rabbits with two irradiation diameters (7 mm central cornea, 13 mm cornea and limbus), using standard fluence (5.4 J/cm(2)) and double-standard fluence (10.8 J/cm(2)) settings. Controls were subjected to epithelial removal and riboflavin instillation, but were not irradiated with UV-A. Following CXL, animals were examined daily until complete closure of the epithelium, and at 7, 14, 21, and 28 days. Animals were killed and a corneoscleral button was excised and processed for light microscopy and immunohistochemistry. RESULTS: For both irradiation diameters and fluences tested, no signs of endothelial damage or limbal vessel thrombosis were observed, and time to re-epithelialization was similar to untreated controls. Histological and immunohistochemical analysis revealed no differences in the p63 putative stem cell marker expression pattern. CONCLUSIONS: Even when using fluence twice as high as the one used in current clinical CXL settings, circumferential UV-A irradiation of the corneal limbus does not alter the regenerative capacity of the limbal epithelial cells, and the expression pattern of the putative stem cell marker p63 remains unchanged. This suggests that eccentric CXL may be performed safely in PMD.