The antifibrotic potential of IMT504: modulation of GLAST + Wnt1 + bone marrow stromal progenitors and hepatic microenvironment.

BACKGROUND: The immunomodulatory oligodeoxynucleotide (ODN) IMT504 might harbor antifibrotic properties within the liver. METHODS: Fibrosis models were induced in mice through thioacetamide (TAA) administration and bile-duct ligation. Cre-loxP mice were utilized to identify GLAST + Wnt1 + bone marrow stromal progenitors (BMSPs) and to examine their contribution with cells in the liver. In vivo and in vitro assays; flow-cytometry, immunohistochemistry, and qPCR were conducted. RESULTS: IMT504 demonstrated significant inhibition of liver fibrogenesis progression and reversal of established fibrosis. Early responses to IMT504 involved the suppression of profibrogenic and proinflammatory markers, coupled with an augmentation of hepatocyte proliferation. Additionally, this ODN stimulated the proliferation and mobilization of GLAST + Wnt1 + BMSPs, likely amplifying their contribution with endothelial- and hepatocytes-like cells. Moreover, IMT504 significantly modulated the expression levels of Wnt ligands and signaling pathway/target genes specifically within GLAST + Wnt1 + BMSPs, with minimal impact on other BMSPs. Intriguingly, both IMT504 and conditioned media from IMT504-pre-treated GLAST + Wnt1 + BMSPs shifted the phenotype of fibrotic macrophages, hepatic stellate cells, and hepatocytes, consistent with the potent antifibrotic effects observed. CONCLUSION: In summary, our findings identify IMT504 as a promising candidate molecule with potent antifibrotic properties, operating through both direct and indirect mechanisms, including the activation of GLAST + Wnt1 + BMSPs.

Vestigial-dependent induction contributes to robust patterning but is not essential for wing-fate recruitment in Drosophila.

Cell recruitment is a process by which a differentiated cell induces neighboring cells to adopt its same cell fate. In Drosophila, cells expressing the protein encoded by the wing selector gene, vestigial (vg), drive a feed-forward recruitment signal that expands the Vg pattern as a wave front. However, previous studies on Vg pattern formation do not reveal these dynamics. Here, we use live imaging to show that multiple cells at the periphery of the wing disc simultaneously activate a fluorescent reporter of the recruitment signal, suggesting that cells may be recruited without the need for their contact neighbors be recruited in advance. In support of this observation, when Vg expression is inhibited either at the dorsal-ventral boundary or away from it, the activation of the recruitment signal still occurs at a distance, suggesting that Vg expression is not absolutely required to send or propagate the recruitment signal. However, the strength and extent of the recruitment signal is clearly compromised. We conclude that a feed-forward, contact-dependent cell recruitment process is not essential for Vg patterning, but it is necessary for robustness. Overall, our findings reveal a previously unidentified role of cell recruitment as a robustness-conferring cell differentiation mechanism.

A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the vestigial expression pattern.

Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. Here we investigate the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. We find that the Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, we find that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, we provide direct evidence that cell recruitment is initiated between early and mid third-instar larval development. Altogether, our work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.

A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos.

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

Nuclear localization of ß-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia.

OBJECTIVES: To evaluate the localization of ß-catenin in oral dysplastic cells, the expression of target genes upregulated in oral dysplasia, and the role of Wnt ligands in these events. MATERIALS AND METHODS: Subcellular localization of total and non-phosphorylated (transcriptionally active) ß-catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes (DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited with the inhibitor of porcupine, C59. Wnt3a and ß-catenin were evaluated in biopsies by tissue immunofluorescence. RESULTS: Immunofluorescence and fractionation experiments showed augmented nuclear ß-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of ß-catenin and Tcf/Lef-dependent transcription in OKF6 and primary oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased Wnt3a secretion, nuclear ß-catenin and the expression of survivin and cyclin D1 at both mRNA and protein levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples, since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear ß-catenin in oral dysplasia, but not in healthy mucosa biopsies. CONCLUSION: These data indicate that secretion of Wnt ligands is critical for ß-catenin nuclear localization and expression of target genes in oral dysplasia.

Wnt1 and SFRP1 as potential prognostic factors and therapeutic targets in cutaneous squamous cell carcinoma.

The Wnt signaling pathway plays a key role in insurgence and progression of many different forms of cancer. Some crucial components of the Wnt pathway have been proposed to be novel targets for cancer therapy. To date, the Wnt signaling pathway has not been studied in cutaneous squamous cell carcinoma (CSCC). This study was designed to investigate the expression of Wnt1 and SFRP1 from the Wnt pathway in CSCC. Tissue samples were obtained from 35 patients with CSCC and 30 controls admitted to the Xinjiang Uygur Autonomous Region People's Hospital at Urumchi City, China. Gene and protein expressions of Wnt1 and SFRP1 were quantified by immunohistochemistry and western blotting. Wnt1 expression was significantly higher (P < 0.05) in CSCC samples than in normal skin cells of the control subjects; in contrast, SFRP1 expression was significantly lower in CSCC tissues than that in tissues of control subjects (P < 0.05). Moreover, Wnt1 expression (P < 0.05) was found to be correlated with histopathological differentiation in CSCC, and negatively correlated with SFRP1 expression in CSCC (rs = -0.473, P = 0.015). Therefore, we concluded that Wnt1 and SFRP1 play important roles in the development of CSCC and could be potent markers for diagnosis, prevention, and therapy of CSCC.

Wnt1-induced MAFK expression promotes osteosarcoma cell proliferation.

Osteosarcoma is one of the most common primary bone tumors in children and young adults. In this study, we investigated the role of musculoaponeurotic fibrosarcoma oncogene homolog K (MAFK) in osteosarcoma cell proliferation in vitro and the possible pathways that contributed to MAFK-related osteosarcoma development. We first reported that MAFK was expressed at low levels in an osteosarcoma cell line. Furthermore, a significant correlation between MAFK and the Wnt signaling pathway was observed in osteosarcoma by using a gene microarray assay. We found that expression of MAFK could be induced by Wnt1 in a dose-dependent manner. Furthermore, Wnt1-induced MAFK expression caused a significant increase of cell viability, whereas a Wnt pathway inhibitor, IWR-1-endo, abolished Wnt1-induced effects on MAFK. Finally, cell cycle analysis showed that enhanced cell proliferation might be attributed to re-distribution of the cell cycle. Together, our results suggested that Wnt1-induced MAFK expression promoted cell proliferation in MG63 cells, and that the role of MAFK in osteosarcoma might be closely linked to the Wnt signaling pathway.

Phylogeography of the sand dune ant Mycetophylax simplex along the Brazilian Atlantic Forest coast: remarkably low mtDNA diversity and shallow population structure.

BACKGROUND: During past glacial periods, many species of forest-dwelling animals experienced range contractions. In contrast, species living outside such moist habitats appear to have reacted to Quaternary changes in different ways. The Atlantic Forest represents an excellent opportunity to test phylogeographic hypotheses, because it has a wide range of vegetation types, including unforested habitats covered predominantly by herbaceous and shrubby plants, which are strongly influenced by the harsh environment with strong wind and high insolation. Here, we investigated the distribution of genetic diversity in the endemic sand dune ant Mycetophylax simplex across its known range along the Brazilian coast, with the aim of contributing to the understanding of alternative phylogeographic patterns. We used partial sequences of the mitochondrial gene cytochrome oxidase I and nuclear gene wingless from 108 specimens and 51 specimens, respectively, to assess the phylogeography and demographic history of this species. To achieve this we performed different methods of phylogenetic and standard population genetic analyses. RESULTS: The observed genetic diversity distribution and historical demographic profile suggests that the history of M. simplex does not match the scenario suggested for other Atlantic Forest species. Instead, it underwent demographic changes and range expansions during glacial periods. Our results show that M. simplex presents a shallow phylogeographic structure with isolation by distance among the studied populations, living in an almost panmictic population. Our coalescence approach indicates that the species maintained a stable population size until roughly 75,000 years ago, when it underwent a gradual demographic expansion that were coincident with the low sea-level during the Quaternary. Such demographic events were likely triggered by the expansion of the shorelines during the lowering of the sea level. CONCLUSIONS: Our data suggest that over evolutionary time M. simplex did not undergo dramatic range fragmentation, but rather it likely persisted in largely interconnected populations. Furthermore, we add an important framework about how both glacial and interglacial events could positively affect the distribution and diversification of species. The growing number of contrasting phylogeographic patterns within and among species and regions have shown that Quaternary events influenced the distribution of species in more ways than first supposed.

The Wnt1 ligand/Frizzled 3 receptor system plays a regulatory role in the achievement of the 'in vitro' capacitation and subsequent 'in vitro' acrosome exocytosis of porcine spermatozoa.

The aim of this work was to determine the existence of a functional Wnt/ß-catenin signaling pathway in boar spermatozoa, which would be linked with the already well-known GSK-3 signaling pathway. This was first confirmed by detecting the presence of the specific Frizzled 3 receptor in these cells. Furthermore, this signaling pathway was activated in boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome exocytosis (IVAE) by incubating cells with separate concentrations of Wnt1 ligand, the Wnt/ß-catenin signaling pathway-specific effector. Incubation with the Wnt1 ligand increased the rhythm of the time-dependent reduction in sperm viability during the achievement of both IVC and IVAE. This finding was concomitant with an increase in the percentage of spermatozoa with altered membrane fluidity and permeability determined through both merocyanine-540 and YO-PRO-1 stains. While the Wnt1 ligand did not affect total sperm motility during the achievement of the IVC, it induced a fast and transient increase in the overall motility patterns in spermatozoa subjected to IVAE. This IVAE-linked action was related to a decrease in the percentage of cells with high mitochondrial membrane potential and an increase in the percentage of cells with high intracellular Fluo-3-marked Ca(2+) content. In conclusion, our results suggest that the Wnt1 ligand-modulated Wnt/ß-catenin signaling pathway plays a relevant role in the modulation of both IVC and subsequent, progesterone-induced IVAE. Furthermore, our data indicate that the transduction pathways by which the Wnt1 ligand acts on IVC and IVAE are different, and that the Wnt/ß-catenin pathway is independent from GSK-3 activity in the achievement of IVC.

Identificação e validação de marcadores moleculares das vias de sinalização WNT, PI3K e processo EMT para risco de progressão de carcinoma ductal in situ de mama / Identification and validation of molecular markers of WNT and PI3K signaling pathway and EMT process for the risk of progression of ductal carcinoma in situ of the breast

O carcinoma ductal da mama é o tipo histológico mais freqüente, sendo que o in situ (CDIS) representa uma neoplasia com incidência crescente, devido aos métodos de detecção precoce de lesões mamárias não palpáveis. Essa neoplasia da mama inclui um grupo heterogêneo de tumores pré-invasivos, com potencial maligno distinto, que podem progredir rapidamente para a forma invasiva, ou não apresentarem evolução, após um longo período da doença. Um estudo prévio (CASTRO et al. 2008) aplicou o conceito de divergência molecular em grupos de lesões que mimetizam a progressão do câncer de mama [epitélio normal (EN), carcinoma ductal in situ (CDIS) puro, componente in situ de lesão que coexiste com invasão (CDIS-CDI) e do carcinoma ductal invasivo (CDI)], utilizando as metodologias de microdissecção a laser e cDNA microarray. O padrão de expressão gênica do grupo de células epiteliais tumorais do componente in situ do CDIS-CDI é semelhante ao grupo de células tumorais do CDI e diferente do grupo de células CDIS puro, cujos aspectos morfológicos são semelhantes ao componente in situ do CDIS-CDI. Isso sugeriu que as modificações moleculares das células do componente in situ do CDIS-CDI, já estejam presentes antes da manifestação morfológica de invasão e que os genes diferentemente expressos entre os dois grupos de células de lesões pré-invasivas, sejam potenciais preditores de risco de progressão do CDIS puro. Assim, nós avaliamos 28 genes das vias de sinalização WNT, PI3K e processo EMT, previamente selecionados do estudo anterior (CASTRO et al. 2008) através de RT-PCR quantitativo (RT-qPCR), em células tumorais capturadas de amostras congeladas do CDIS puro e do componente in situ do CDIS-CDI. Esse trabalho confirmou a diferença de expressão em células epiteliais entre os dois grupos de lesões pré-invasivas, em 14 (70%) de 20 genes avaliados, que apresentaram dados confiáveis nos ensaios de TLDA, sendo que 13 genes apresentaram maior expressão em CDIS...

Among breast tumors, ductal breast carcinoma is the most common histologic type. Ductal carcinoma in situ (DCIS) has increasing incidence, mainly due to early detection methods of non-palpable breast lesions. DCIS includes a heterogeneous group of pre-invasive tumors, with distinct malignant potential, which can either rapidly progress to the invasive form, or show no progression after a long period of surveillance. In a previous study from the group (CASTRO et al. 2008) based on the expression pattern of epithelial cells, using laser capture microdissection and cDNA microarray, the concept of molecular divergence was applied to groups of breast lesions which mimic the progression of breast cancer [cells from normal epithelium, cells from pure ductal carcinoma in situ (DCIS), cells from in situ component that coexists with invasive lesion (DCIS-IDC) and cells from invasive ductal carcinoma (IDC)]. The gene expression pattern of the cells from the in situ component of DCIS-IDC is more similar to the group of IDC tumor cells other than to the group of pure DCIS of cells, which presents higher similarity in terms of morphological features to the in situ component of DCIS-IDC. This suggests that the molecular changes of the cells from the in situ component of DCIS-IDC are already present before morphological manifestations of invasion and that the genes differentially expressed between the two groups of cells of pre-invasive lesions, are potential predictors of risk of progression of pure DCIS. Thus, we evaluated 28 previously selected genes of WNT signaling pathway, PI3K and EMT process (CASTRO et al. 2008) by quantitative RT-PCR (RT-qPCR) in tumor cells captured from in situ lesions of pure DCIS and DCIS-IDC from frozen samples. This work confirmed the difference in expression of epithelial cells between the two groups of preinvasive lesions, in 14 (70%) of the 20 genes evaluated, by reliable TLDA assays. Most genes (13 genes) showed higher expression in pure...

A broad expression profile of the GMR-GAL4 driver in Drosophila melanogaster.

The GAL4/UAS binary system has been widely used in Drosophila melanogaster for ectopic expression of transgenes in a tissue-specific manner. The GMR-GAL4 driver, which expresses the yeast transcription factor GAL4 under the control of glass multiple reporter (GMR) promoter elements, has been commonly utilized to express target transgenes, specifically in the developing eye. However, we have observed abnormal wing phenotypes; this is a result of the activity of critical wing developing genes, e.g., components of the Notch or Wg pathway, that are up- or down-regulated under the control of the GMR-GAL4 driver. X-gal staining confirmed that UAS-LacZ is expressed in third-instar larva wing imaginal discs, as well as in eye discs, when driven by the GMR-GAL4 driver. Furthermore, we found that GMR-GAL4 also drives UAS-LacZ expression in other tissues, such as brain, trachea, and leg discs. These results indicate that GMR-GAL4 has a broad expression profile, rather than the eye-specific pattern described previously, and that one should be careful when using it as a tool for targeted gene expression.

Effect of different resistance-training regimens on the WNT-signaling pathway.

The purpose of the present study was to evaluate the effects of 8 weeks of strength and power training on the expression of genes related to the canonical WNT pathway and ß-catenin protein levels in physically active men. Twenty-five subjects (27.4 ± 4.6 years) were balanced based on their relative maximum strength in the squat exercise (squat 1RM/body mass) and randomly assigned to strength training (ST) (n = 10), power training (PT) (n = 10), and control (C) (n = 5) groups. The ST and the PT groups performed high and low intensity squats, respectively, thrice a week, for 8 weeks. Muscle biopsies from the vastus lateralis muscle were collected before and after the training period. Relative strength and power increased similarly in both ST and PT groups (P < 0.001). Fiber cross-sectional area also increased similarly in both ST and PT groups. Gene expression and ß-catenin protein expression levels were assessed by real-time PCR and Western blot. Certain genes were up-regulated in the ST group (WNT1: 6.4-fold, P < 0.0001; SFRP1: 3.3-fold, P < 0.0001 and LEF1: 7.3-fold, P < 0.0001) and also in the PT group (WNT1: 24.9-fold, P < 0.0001; SFRP1: 2.7-fold, P < 0.0001; LEF1: 34.1-fold, P < 0.0001 and Cyclin D1: 7.7-fold, P < 0.001). In addition, the expression of key WNT pathway genes was substantially more responsive to PT than to ST (WNT1: P < 0.0001; LEF1: P < 0.0001 and Cyclin D1: P < 0.001). Finally, the total ß-catenin protein content increased only in the PT group (P < 0.05). Our data indicate that a PT regimen triggers greater responses in key elements of the WNT pathway.

Wnt/beta-catenin signalling pathway following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

The Wnt/beta-catenin signaling pathway plays an important role in development, tissue homeostasis, and regeneration. Inappropriate activation of the Wnt pathway is linked to a wide range of human cancers. The purpose of this study was to characterize the Wnt/beta-catenin signaling pathway as depicted by the expression of Wnt1, Frizzled-1, Wnt5a, Frizzled-5 and beta-catenin during 4NQO-induced rat tongue carcinogenesis by immunohistochemistry. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4NQO solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, an overexpression of Wnt5a was noticed when compared to control group (p<0.05). The Wnt1 showed significant differences (p<0.05) in pre-neoplastic lesions at 12 weeks following carcinogen exposure. In well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO, Wnt1 was expressed in the majority of the dysplasic cells and tumor cells. This was statistically significant (p<0.05). No significant differences (p>0.05) were found in expression of Frizzled-1, Frizzled-5 or beta-catenin following oral carcinogenesis. Taken together, our results support the belief that expression of Wnt1 and Wnt5a is related to malignant transformation and conversion of oral mucosa.

Molecular analysis of the Wnt-1 proto-oncogene in Ambystoma mexicanum (axolotl) embryos.

To analyze Wnt-1 expression during neurulation in urodele embryos, we have isolated a Wnt-1 cDNA clone, Awnt-1, from an Ambystoma mexicanum (axolotl) neurula-stage cDNA library. Awnt-1 codes for a protein of 369 amino acids rich in cysteine residues, is preceded by a hydrophobic leader peptide sequence and contains four possible sites for N-linked glycosylation. The temporal expression profile of Awnt-1 was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Awnt-1 expression in the axolotl embryo is biphasic. Awnt-1 transcripts are found in early blastulae until gastrulation, are barely detectable during gastrulation, and are present again from neurulation until late embryogenesis. Transcripts are present before the midblastula transition, indicating that they might be of maternal origin. To localize Awnt-1 expression in embryos during the first phase of expression, early gastrulae were dissected by cutting along the animal-vegetal and future dorso-ventral axes and analyzed by RT-PCR. At the early gastrula stage Awnt-1 transcripts appear to be located in the future ventral region of the embryo. Hatching larvae no longer express Awnt-1. PCR reactions performed using cDNA library-phage DNA templates derived from whole neurulae versus embryos with the neuroectoderm removed suggest that, in the neurula, Awnt-1 transcripts are located in the neuroectoderm. This suggest that, as is the case for Wnt-1 in other vertebrates, Awnt-1 may be involved in neurogenesis. These results suggest that Wnt-1 has earlier roles in development than has been considered until now.