RESUMO
IWR-1-endo, a small molecule that potently inhibits the Wnt/ß-catenin signaling pathway by stabilizing the AXIN2 destruction complex, can inhibit drug efflux at the blood−brain barrier. To conduct murine cerebral microdialysis research, validated, sensitive, and reliable liquid chromatography−tandem mass spectrometry (LC-MS/MS) methods were used to determine IWR-1-endo concentration in the murine plasma and brain microdialysate. IWR-1-endo and the internal standard (ISTD) dabrafenib were extracted from murine plasma and microdialysate samples by a simple solid-phase extraction protocol performed on an Oasis HLB µElution plate. Chromatographic separation was executed on a Kinetex C18 (100A, 50 × 2.1 mm, 4 µm particle size) column with a binary gradient of water and acetonitrile, each having 0.1% formic acid, pumped at a flow rate of 0.6 mL/min. Detection by mass spectrometry was conducted in the positive selected reaction monitoring ion mode by monitoring mass transitions 410.40 > 344.10 (IWR-1-endo) and 520.40 > 307.20 (ISTD). The validated curve range of IWR-1-endo was 5−1000 ng/mL for the murine plasma method (r2 ≥ 0.99) and 0.5−500 ng/mL for the microdialysate method (r2 ≥ 0.99). The lower limit of quantification (LLOQ) was 5 ng/mL and 0.5 ng/mL for the murine plasma and microdialysate sample analysis method, respectively. Negligible matrix effects were observed in murine plasma and microdialysate samples. IWR-1-endo was extremely unstable in murine plasma. To improve the stability of IWR-1-endo, pH adjustments of 1.5 were introduced to murine plasma and microdialysate samples before sample storage and processing. With pH adjustment of 1.5 to the murine plasma and microdialysate samples, IWR-1-endo was stable across several tested conditions such as benchtop, autosampler, freeze−thaw, and long term at −80 °C. The LC-MS/MS methods were successfully applied to a murine pharmacokinetic and cerebral microdialysis study to characterize the unbound IWR-1-endo exposure in brain extracellular fluid and plasma.
Assuntos
Espectrometria de Massas em Tandem , Via de Sinalização Wnt , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Camundongos , Microdiálise , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
Porcupine is a constituent of the 19 membered Wnt family with diverse biological features such as cell differentiation, cell proliferation, cell migration, apoptosis, etc. Porcupine is a membrane-bound o-acyltransferase family protein that modulates Wnt protein through palmitoylation to allow it to depart the secretory pathway and activate cellular responses. Inhibition of Porcupine prevents palmitoylation of Wnt ligands which in turn blocks the transport of Wnt to the extracellular membrane, thus prevents the immoderate production of ß-catenin which helps to control the aberrant cell growth. Clinically, Porcupine inhibitors have shown their potential in treating majorly colorectal cancer, pancreatic cancer, hepatocellular carcinoma, head and neck cancer etc. Till date, none of the Porcupine inhibitors have been in the market and only four molecules, LGK974, ETC159, CGX1321 and RXC004 have reached the Phase I clinical trial. Present review gives a comprehensive insight on Porcupine as a novel drug target for the treatment of cancer as well as recent update on many novel heterocyclic Porcupine inhibitors with their chemical structures and pharmacology. Their physico chemical properties were also predicted using SwissADME server. Major concerns during their development have also been summarised which may throw some light for the future development of novel Porcupine inhibitors for the treatment of cancer.
Assuntos
Aciltransferases/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Aciltransferases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Desenvolvimento de Medicamentos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Proteínas de Membrana/metabolismo , Terapia de Alvo Molecular , Neoplasias/metabolismo , Pirazinas/química , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Piridinas/química , Piridinas/farmacologia , Piridinas/uso terapêuticoRESUMO
Inhibition of Wnt/ß-catenin signaling facilitates the derivation of mouse epiblast stem cells (EpiSCs), as well as dramatically promotes EpiSC self-renewal. The specific mechanism, however, is still unclear. Here, we showed that IWR1, a Wnt/ß-catenin signaling inhibitor, allowed long-term self-renewal of EpiSCs in serum medium in combination with ROCK inhibitor Y27632. Through transcriptome data analysis, we arrived at a set of candidate transcription factors induced by IWR1. Among these, Forkhead box D3 (Foxd3) was most abundant. Forced expression of Foxd3 could recapitulate the self-renewal-promoting effect of IWR1 in EpiSCs. Conversely, knockdown of Foxd3 profoundly compromised responsiveness to IWR1, causing extinction of pluripotency markers and emergence of differentiation phenotype. Foxd3 thus is necessary and sufficient to mediate self-renewal downstream of Wnt/ß-catenin signaling inhibitor. These findings highlight an important role for Foxd3 in regulating EpiSCs and will expand current understanding of the primed pluripotency.
Assuntos
Autorrenovação Celular/efeitos dos fármacos , Fatores de Transcrição Forkhead/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Wnt/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/antagonistas & inibidores , Amidas/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camadas Germinativas/citologia , Camadas Germinativas/efeitos dos fármacos , Camadas Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Piridinas/farmacologia , Proteínas Repressoras/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Recent studies suggest that RNA polymerase II (Pol II) has to be fully assembled before being imported into the nucleus, while other reports indicate a distinct mechanism to import large and small subunits. In yeast, Iwr1 binds to the holoenzyme assembled in the cytoplasm and directs its nuclear entry. However, as IWR1 is not an essential gene, Iwr1-independent pathway(s) for the nuclear import of Pol II must exist. In this paper, we investigate the transport into the nucleus of several large and small Pol II subunits in the mutants of genes involved in Pol II biogenesis. We also analyse subcellular localization in the presence of drugs that can potentially affect Pol II nuclear import. Our results show differences in the cellular distribution between large and small subunits when Pol II biogenesis was impaired. Our data suggest that, in addition to the fully assembled holoenzyme, Pol II subunits can be imported to the nucleus, either independently or as partial assemblies, through different pathways, including passive diffusion for the small subunits.
Assuntos
Núcleo Celular/enzimologia , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/genética , RNA Polimerase II/genética , Saccharomyces cerevisiae/genéticaRESUMO
Small molecule inhibitors of growth factor signaling pathways are extremely convenient reagents for investigation of embryonic development. The chemical may be introduced at a precise time, the dose can be altered over a large range and the chemical may be removed simply by replacing the medium surrounding the embryo. Because small molecule modulators are designed to target conserved features of a protein, they are usually effective across species. Ideally the chemicals offer remarkable specificity for a particular signaling pathway and exhibit negligible off-target effects. In this study we examine the use of small molecules to modulate the Wnt and Notch signaling pathways in the Xenopus embryo. We find that IWR-1 and XAV939 are effective inhibitors of the canonical Wnt signaling pathway while BIO is an excellent activator. For Notch signaling, we find that both DAPT and RO4929097 are effective inhibitors, but that RO4929097 is the more potent reagent. This report provides researchers with useful working concentrations of reagents and a small series of genetic and biological assays that may be used to characterize the role of Wnt and Notch signaling during embryonic development.
Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Receptores Notch/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Padronização Corporal/efeitos dos fármacos , Desenvolvimento Embrionário/fisiologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imidas/farmacologia , Quinolinas/farmacologia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , XenopusRESUMO
Vitamin D (VD) deficiency plays an important role in the occurrence and development of various uterine diseases. At present, most studies on the mechanism of VD in the Wnt signaling pathway focus on cancer, while there are no relevant reports on its mechanism in endometritis. This study investigated the effect of vitamin D3 (VD3) on the Wnt signaling pathway in endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS). BEECs obtained from bovine uteri were treated with VD3 (0, 50 ng/mL) and LPS (0, 10, 100 ng/mL) separately or in combination, and treated with the Wnt signaling pathway inhibitor IWR-1 to study the mechanism of action. The proliferation of BEECs was evaluated by a CCK-8 assay. qRT-PCR was used to assess the gene expression of Wnt pathway-related factors, including MYC, PCNA, LGR5, GREM1, ß-catenin, FZD7, FZD2, Wnt4 and VDR. The results showed that VD3 had no significant effect on cell proliferation (P > 0.05); LPS inhibited BEEC proliferation in a time- and dose-dependent manner, and cells treated with LPS at different concentrations for 24-48 h in combination with VD3 promoted cell proliferation to varying degrees. IWR-1 inhibited cell proliferation in a time- and concentration-dependent manner, while LPS + IWR-1 treatment also significantly promoted cell proliferation after VD3 treatment (P < 0.01). The qRT-PCR results showed that the expression of Wnt4 and PCNA genes showed different trends with different LPS concentrations for stimulation, and the expression of the MYC and GREM1 genes was only stimulated by high-dose (100 ng/mL) LPS stimulation. The expression of FZD7, LGR5, FZD2 and ß-catenin was upregulated by LPS at both concentrations. LPS + VD3 significantly downregulated the expression of the Wnt pathway-related genes MYC, PCNA, LGR5, GREM1 and ß-catenin (P < 0.001), Wnt4 and FZD2 (P < 0.01), and significantly upregulated the expression of VDR (P < 0.05). After LPS + IWR-1 treatment, the expression of the ß-catenin (P < 0.01) and LGR5 (P < 0.05) genes was significantly downregulated, while the Wnt4 (P < 0.01) and VDR (P < 0.001) genes were significantly upregulated, MYC was downregulated but without a significant difference (P > 0.05). In conclusion, VD3 treatment can mitigate the LPS-induced abnormal expression of Wnt signaling pathway genes in BEECs, showing that the Wnt pathway may be a protective pathway of VD3 against LPS-induced gene overexpression in BEECs. The results suggest that VD3 may play a regulatory role in pathways other than the Wnt signaling pathway. Whether VD3 affects the Wnt signaling pathway by affecting Wnt4 gene expression requires further study.
Assuntos
Via de Sinalização Wnt , beta Catenina , Animais , Bovinos , Proliferação de Células , Colecalciferol/farmacologia , Células Epiteliais/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
Tumor-initiating cells or cancer stem cells are a subset of cancer cells that have tumorigenic potential in human cancer. Although several markers have been proposed to distinguish tumor-initiating cells from colorectal cancer cells, little is known about how this subpopulation contributes to tumorigenesis. Here, we characterized a tumor-initiating cell subpopulation from Caco-2 colorectal cancer cells. Based on the findings that Caco-2 cell subpopulations express different cell surface markers, we were able to discriminate three main fractions, CD44-CD133-, CD44-CD133+, and CD44+CD133+ subsets, and characterized their biochemical and tumorigenic properties. Our results show that CD44+CD133+ cells possessed an unusual capacity to proliferate and could form tumors when transplanted into NSG mice. Additionally, primary tumors grown from CD44+CD133+ Caco-2 cells contained mixed populations of CD44+CD133+ and non-CD44+CD133+ Caco-2 cells, indicating that the full phenotypic heterogeneity of the parental Caco-2 cells was re-created. Notably, only the CD44+CD133+ subset of Caco-2-derived primary tumors had tumorigenic potential in NSG mice, and the tumor growth of CD44+CD133+ cells was faster in secondary xenografts than in primary transplants. Gene expression analysis revealed that the Wnt/ß-catenin pathway was over-activated in CD44+CD133+ cells, and the growth and tumorigenic potential of this subpopulation were significantly suppressed by small-molecule Wnt/ß-catenin signaling inhibitors. Our findings suggest that the CD44+CD133+ subpopulation from Caco-2 cells was highly enriched in tumorigenic cells and will be useful for investigating the mechanisms leading to human colorectal cancer development.
Assuntos
Antígeno AC133/genética , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Antígeno AC133/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Células CACO-2 , Transformação Celular Neoplásica , Humanos , Receptores de Hialuronatos/biossíntese , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , beta Catenina/biossínteseRESUMO
Wnt/ß-catenin or canonical Wnt signaling pathway regulates the self-renewal of cancer stem-like cells (CSCs) and is involved in tumor progression and chemotherapy resistance. Previously, we reported that this pathway is activated in a subset of osteosarcoma CSCs and that doxorubicin induced stemness properties in differentiated cells through Wnt/ß-catenin activation. Here, we investigated whether pharmacological Wnt/ß-catenin inhibition, using a tankyrase inhibitor (IWR-1), might constitute a strategy to target CSCs and improve chemotherapy efficacy in osteosarcoma. IWR-1 was specifically cytotoxic for osteosarcoma CSCs. IWR-1 impaired spheres' self-renewal capacity by compromising landmark steps of the canonical Wnt signaling, namely translocation of ß-catenin to the nucleus and subsequent TCF/LEF activation and expression of Wnt/ß-catenin downstream targets. IWR-1 also hampered the activity and expression of key stemness-related markers. In vitro, IWR-1 induced apoptosis of osteosarcoma spheres and combined with doxorubicin elicited synergistic cytotoxicity, reversing spheres' resistance to this drug. In vivo, IWR-1 co-administration with doxorubicin substantially decreased tumor progression, associated with specific down-regulation of TCF/LEF transcriptional activity, nuclear ß-catenin and expression of the putative CSC marker Sox2. We suggest that targeting the Wnt/ß-catenin pathway can eliminate CSCs populations in osteosarcoma. Combining conventional chemotherapy with Wnt/ß-catenin inhibition may ameliorate therapeutic outcomes, by eradicating the aggressive osteosarcoma CSCs and reducing drug resistance.
Assuntos
Imidas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Quinolinas/farmacologia , Tanquirases/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Feminino , Humanos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Tanquirases/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
AIMS: Keloid is a benign tumor that is characterized by the hyperproliferation of dermal fibroblasts and excessive deposition of extracellular matrix (ECM) especially the collagen. Aberrant activation of Wnt/ß-catenin signaling is implicated in the pathogenesis of keloid. In this study, we investigated the effects of IWR-1, a small molecule inhibitor for Wnt/ß-catenin signaling via the inhibition of tankyrase, on production of collagen and matrix metalloproteinase (MMP) in dermal fibroblasts. MAIN METHODS: We cultured human normal skin- and keloid-derived fibroblasts, then treated with IWR-1. The effects of IWR-1 on collagen and MMP production were determined by Western blot, ELISA and zymography. KEY FINDINGS: IWR-1 significantly suppressed the proliferation and migration of both the normal and keloid fibroblasts. IWR-1 also inhibited the production and secretion of type I collagen from the fibroblasts. In addition, IWR-1 significantly increased the expression of MMPs, such as MMP-1, MMP-3 and MMP-13, along with the increase of gelatinase activity. These results suggest that inhibitory effect of IWR-1 on collagen production may be related with the increased MMP activity. SIGNIFICANCE: This study provides the possible action mechanism of IWR-1 on regulation of collagen expression, on which to base further investigation for preventing skin fibrotic diseases such as keloid.
Assuntos
Colágeno/biossíntese , Fibroblastos/metabolismo , Imidas/farmacologia , Queloide/metabolismo , Quinolinas/farmacologia , Pele/metabolismo , Adulto , Idoso , Células Cultivadas , Colagenases/biossíntese , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Iwr1 is an RNA polymerase II (RNPII) interacting protein that directs nuclear import of the enzyme which has been previously assembled in the cytoplasm. Here we present genetic and molecular evidence that links Iwr1 with transcription. Our results indicate that Iwr1 interacts with RNPII during elongation and is involved in the disassembly of the enzyme from chromatin. This function is especially important in resolving problems posed by damage-arrested RNPII, as shown by the sensitivity of iwr1 mutants to genotoxic drugs and the Iwr1's genetic interactions with RNPII degradation pathway mutants. Moreover, absence of Iwr1 causes genome instability that is enhanced by defects in the DNA repair machinery.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Cromatina/genética , Cromatina/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Instabilidade Genômica/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Wnt/ß-catenin signaling pathway is a key regulator of embryonic development that was first identified in drosophila. More recent work has suggested a role for this pathway in mammalian, myogenesis but this process remains to be more completely elucidated. The current study was designed to investigate the role of the Wnt3a/ß-catenin signaling module in C2C12 cell differentiation and myotube formation. Activation of the Wnt/ß-catenin pathway in differentiating myoblasts, using lithium chloride supplementation, increased both the number and size of C2C12 myotubes. On the other hand, pharmacological inhibitors of Wnt/ß-catenin signaling (IWR-1, XAV939) resulted in significant decreases in myotube length without affecting their numbers. In separate experiments, a neutralizing antibody to Wnt3a also reduced myotube length without affecting the number of myotubes formed during differentiation. Confocal imaging studies revealed that ß-catenin was primarily localized in the cytoplasmic region of undifferentiated myoblasts and differentiation resulted in its enhanced expression in myotubes. Additionally, LiCl treatment resulted in enhanced expression of ß-catenin in pre-differentiating myoblast cultures. These data suggest that Wnt3a/ß-catenin signaling plays a critical role in C2C12 cell differentiation and provides an appropriate model for elucidating its functions in more detail.
Assuntos
Desenvolvimento Muscular , Via de Sinalização Wnt , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Cloreto de Lítio/farmacologia , Camundongos , Desenvolvimento Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Tanquirases/antagonistas & inibidores , Tanquirases/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMO
Aberrant activation of Wnt/ß-catenin signaling is frequently observed in patients with colorectal cancer (CRC) and is considered a major determinant of CRC pathogenesis. CRC pathogenesis is particularly accompanied by epithelial-mesenchymal transition (EMT) and survivin expression. Here, we investigated the potential and mechanism of a novel Wnt/ß-catenin inhibitor IWR-1 to suppress tumor metastasis in relation with EMT and survivin expression. We first determined the EMT reversal effects of IWR-1 in in vitro (HCT116 and HT29 cells) and ex vivo (specimens of CRC patients) CRC models. It was shown that IWR-1 inhibited cell proliferation and EMT even in the presence of TNF-α-induced cancer cell stimulation. IWR-1 also significantly suppressed cell migration, invasion, and matrix metalloproteinase activities of CRC cell lines. Furthermore, we showed the evidence that IWR-1 provides EMT reversal effects by directly suppressing survivin expression by the followings: 1) IWR-1 could not completely inhibit EMT in survivin-overexpressing HCT116 cells, 2) EMT reversal effects of IWR-1 were more pronounced in survivin-suppressed cells, and 3) Survivin promoter assay directly identified the survivin promoter region responsible for inhibition of survivin transcription by IWR-1. Taken altogether, our results demonstrate that IWR-1 has the potential to suppress tumor metastasis by inhibiting Wnt/ß-catenin pathway as well as survivin expression. Therefore, IWR-1 could be considered for future clinical use as a therapeutic agent to treat CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Imidas/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Quinolinas/farmacologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Microscopia de Fluorescência , Invasividade Neoplásica , Metástase Neoplásica , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Survivina , Proteínas Wnt/antagonistas & inibidores , beta Catenina/antagonistas & inibidoresRESUMO
Interactions between FGF and Wnt/ bcat signaling control development of the midbrain. The nature of this interaction and how these regulate patterning, growth and differentiation is less clear, as it has not been possible to temporally dissect the effects of one pathway relative to the other. We have employed pharmacological and genetic tools to probe the temporal and spatial roles of FGF and Wnt in controlling the specification of early midbrain neurons. We identify a ß-catenin (bcat) independent role for GSK-3 in modulating FGF activity and hence neuronal patterning. This function is complicated by an overlap with bcat-dependent regulation of FGF signaling, through the regulation of sprouty4. Additionally we reveal how attenuation of Axin protein function can promote fluctuating levels of bcat activity that are dependent on FGF activity. This highlights the complex nature of the interactions between FGF and Wnt/ bcat and reveals that they act at multiple levels to control each others activity in the midbrain.
RESUMO
Wnt signaling is an evolutionarily conserved pathway that regulates cell proliferation, differentiation and apoptosis. To investigate the possible role of Wnt signaling in the regulation of ovarian follicular development, secondary follicles were isolated and cultured in vitro in the presence or absence of its activator (LiCl or Wnt3a) or inhibitor (IWR-1). We have demonstrated that activation of ß-catenin signals by activators dramatically suppressed follicular development by increasing granulosa cell apoptosis and inhibiting follicle steroidogenesis. In contrast, inhibition of Wnt signaling by IWR-1 was observed with better developed follicles and increased steroidogenesis. Further studies have shown that the transcription factor Forkhead box O3a (Foxo3a) and its downstream target molecules were modulated by the activators or the inhibitor. These findings provide evidence that Wnt signaling might negatively regulate follicular development potentially through Foxo3a signaling components.