丙戊酸钠通过上调Wnt/β-catenin信号通路促进培养神经元的生长(英文)

目的:丙戊酸盐(丙戊酸valproic acid,VPA)在过去数十年应用于临床治疗癫痫和偏头痛。然而,母亲怀孕早期使用VPA将大大增加子代罹患孤独症群谱障碍的易感性。鉴于Wnt/β-catenin信号通路对神经元增殖、分化、突起生长及凋亡的重要作用,本文旨在研究VPA在原代培养神经元中对Wnt/β-catenin信号通路的影响。方法:用VPA处理原代培养神经元,以生理盐水处理为对照,运用Western Blot检测Wnt/β-catenin通路相关信号分子Wnt1,Wnt2,WIF-1,Dickkopf 1及效应分子β-catenin的表达变化,同时运用免疫荧光技术观察神经元形态变化。结果:与对照组比较,VPA处理显著增加Wnt1及Wnt2的表达(P<0.05~0.01),而未增加WIF-1及Dickkopf 1的表达(P>0.05);VPA处理也导致Wnt/β-catenin通路活性上调,表现为神经元内β-catenin含量显著上升。此外,与对照组比较,VPA处理促进神经元生长,表现为神经元突起数目(P<0.05~0.01)及总长度显著增加(P<0.05~0.01);Wnt/β-catenin通路抑制剂能部分抑制VPA引起的Wnt/β-catenin通路活性上调及神经元生长。结论:VPA通过上调Wnt/β-catenin信号通路促进神经元生长,可能是VPA增加子代罹患孤独症群谱障碍易感性的原因。     

Inactive Wnt/β‐catenin pathway in conventional high‐grade osteosarcoma

Osteosarcoma is the most common malignant bone tumour, with a peak incidence in children and young adolescents, suggesting a role of rapid bone growth in its pathogenesis. The Wnt/β‐catenin pathway plays a crucial role in skeletal development and is indispensable for osteoblasts' lineage determination. Previous studies suggesting an oncogenic role for the Wnt/β‐catenin pathway in osteosarcoma were based on cytoplasmic staining of β‐catenin or the detection of one component of this pathway. However, those approaches are inappropriate to address whether the Wnt/β‐catenin pathway is functionally active. Therefore, in this study, we examined nuclear β‐catenin expression in 52 human osteosarcoma biopsies, 15 osteoblastomas (benign bone tumours), and four human osteosarcoma cell lines by immunohistochemistry. Furthermore, we modulated Wnt/β‐catenin pathway activity using a GIN (GSK3β inhibitor) and evaluated its effect on cell growth and osteogenic differentiation. Absence of nuclear β‐catenin staining was found in 90% of the biopsies and all osteosarcoma cell lines, whereas strong nuclear β‐catenin staining was observed in all osteoblastomas. Wnt‐luciferase activity was comparable to the negative control in all osteosarcoma cell lines. GIN stimulated the Wnt/β‐catenin pathway, as shown by translocation of β‐catenin into the nucleus and increased Wnt‐luciferase activity as well as mRNA expression of AXIN2, a specific downstream target gene. Stimulation of the Wnt/β‐catenin pathway by GIN significantly reduced cell proliferation in the cell lines MG‐63 and U‐2‐OS and enhanced differentiation in the cell lines HOS and SJSA‐1, as shown by an increase in alkaline phosphatase (ALP) activity and mineralization. In contrast with the oncogenic role of the Wnt/β‐catenin pathway in osteosarcoma as previous studies suggested, here we demonstrate that this pathway is inactivated in osteosarcoma. Moreover, activation of the Wnt/β‐catenin pathway inhibits cell proliferation or promotes osteogenic differentiation in osteosarcoma cell lines. Our data suggest that loss of Wnt/β‐catenin pathway activity, which is required for osteoblast differentiation, may contribute to osteosarcoma development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Wnt/β‐catenin and sonic hedgehog pathways interact in the regulation of the development of the dorsal mesenchymal protrusion

Background: The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β‐catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. Results: Tissue‐specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β‐catenin pathway‐intermediates β‐catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock‐out mouse could be attributed to reduced Wnt/β‐catenin signaling, LiCl, a pharmacological activator of this Wnt/β‐catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. Conclusions: The data presented suggest that the Wnt/β‐catenin pathway interact with the Shh pathway in the regulation of SHF/DMP‐precursor proliferation and, hence, the development of the DMP. Developmental Dynamics 245:103–113, 2016. © 2015 Wiley Periodicals, Inc.     

Loss of adenomatous polyposis coli (apc) results in an expanded ciliary marginal zone in the zebrafish eye

The distal region of neural retina (ciliary marginal zone [CMZ]) contains stem cells that produce non‐neural and neuronal progenitors. We provide a detailed gene expression analysis of the eyes of apc mutant zebrafish where the Wnt/β‐catenin pathway is constitutively active. Wnt/β‐catenin signaling leads to an expansion of the CMZ accompanied by a central shift of the retinal identity gene sox2 and the proneural gene atoh7. This suggests an important role for peripheral Wnt/β‐catenin signaling in regulating the expression and localization of neurogenic genes in the central retina. Retinal identity genes rx1 and vsx2, as well as meis1 and pax6a act upstream of Wnt/β‐catenin pathway activation. Peripheral cells that likely contain stem cells can be identified by the expression of follistatin, otx1, and axin2 and the lack of expression of myca and cyclinD1. Our results introduce the zebrafish apc mutation as a new model to study signaling pathways regulating the CMZ. Developmental Dynamics 239:2066–2077, 2010. © 2010 Wiley‐Liss, Inc.     

SOX1 inhibits breast cancer cell growth and invasion through suppressing the Wnt/β‐catenin signaling pathway

Abnormal activation of the Wnt/β‐catenin signaling pathway is common in human cancers. Several studies have demonstrated that SRY (sex‐determining region Y)‐box (SOX) family genes serve as either tumor suppressor genes or oncogenes by regulating the Wnt signaling pathway in different cancers. However, the role of SOX1 in breast cancer and the underlying mechanism is still unclear. The aim of this study was to explore the effect and mechanism of SOX1 on the breasted cancer cell growth and invasion. In this study, we established overexpressed SOX1 and investigated its function by in vitro experiments. SOX1 was down‐regulated in breast cancer tissues and cell lines. Overexpression of SOX1 inhibited cell proliferation and invasion in vitro, and it promoted cell apoptosis. Furthermore, SOX1 inhibited the expression of β‐catenin, cyclin D1, and c‐Myc in breast cancer cells. Taken together, these data suggest that SOX1 can function as a tumor suppressor partly by interfering with Wnt/β‐catenin signaling in breast cancer.     

Circular RNA ITCH mediates H2O2‐induced myocardial cell apoptosis by targeting miR‐17‐5p via wnt/β‐catenin signalling pathway

Cardiovascular disease is a severe threat health worldwide, and circRNAs have been shown to be correlated with the development of cardiovascular disease. Expression of circ‐ITCH and miR‐17a‐5p was evaluated by RT‐qPCR. Cell viability was measured using CCK‐8. Flow cytometry was applied to measure apoptosis rate. Binding between miR‐17‐5p and circ‐ITCH was detected via luciferase reporter assays. Levels of ATP in cells were examined with ATP testing. Western blot was used to evaluate apoptosis‐related proteins and proteins in Wnt/β‐catenin signalling pathway. H₂O₂ induced apoptosis of H9c2 cells and lowered cell viability as well as ATP levels and circ‐ITCH expression. After overexpression, circ‐ITCH enhanced cell viability and ATP concentration. Meanwhile, apoptosis was inhibited. MiR‐17‐5p was the target of circ‐ITCH as evidenced by luciferase report assays, with higher expression in H₂O₂‐induced H9c2 cells. Knockdown of miR‐17‐5p could promote cell viability and level of ATP and curb apoptosis and p53 and PARP expression. Moreover, overexpressed miR‐17‐5p could reverse the function of upregulated circ‐ITCH. Wnt3a, Wnt5a and β‐catenin in Wnt/β‐catenin signalling pathway were increased after H₂O₂ induction. Suppression of Wnt/β‐catenin signalling pathway could initiate the process of injury in H9c2 cells. Circ‐ITCH could protect myocardial cells from injuries caused by H₂O₂ by suppressing apoptosis while miR‐17‐5p played a reverse role, which could upregulate apoptosis and inhibit cell viability via Wnt/β‐catenin signalling pathway.     

Critical role of Frizzled1 in age‐related alterations of Wnt/β‐catenin signal in myogenic cells during differentiation

Activation of Wnt/β‐catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age‐related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age‐related alteration of Wnt/β‐catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real‐time RT‐PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC‐derived myogenic cells from aged mice and associated with activation of Wnt/β‐catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.     

Activation of canonical Wnt/β‐catenin signaling stimulates proliferation in neuromasts in the zebrafish posterior lateral line

Background: The posterior lateral line in zebrafish develops from a migrating primordium that deposits clusters of cells that differentiate into neuromasts at regular intervals along the trunk. The deposition of these neuromasts is known to be coordinated by Wnt and FGF signals that control the proliferation, migration, and organization of the primordium. However, little is known about the control of proliferation in the neuromasts following their deposition. Results: We show that pharmacological activation of the Wnt/β‐catenin signaling pathway with 1‐azakenpaullone upregulates proliferation in neuromasts post‐deposition. This results in increased size of the neuromasts and overproduction of sensory hair cells. We also show that activation of Wnt signaling returns already quiescent supporting cells to a proliferative state in mature neuromasts. Additionally, activation of Wnt signaling increases the number of supporting cells that return to the cell cycle in response to hair cell damage and the number of regenerated hair cells. Finally, we show that inhibition of Wnt signaling by overexpression of dkk1b suppresses proliferation during both differentiation and regeneration. Conclusions: These data suggest that Wnt/β‐catenin signaling is both necessary and sufficient for the control of proliferation of lateral line progenitors during development, ongoing growth of the neuromasts, and hair cell regeneration. Developmental Dynamics 242:832–846, 2013. © 2013 Wiley Periodicals, Inc.     

Twist1 contributes to cranial bone initiation and dermal condensation by maintaining wnt signaling responsiveness

Background: Specification of cranial bone and dermal fibroblast progenitors in the supraorbital arch mesenchyme is Wnt/β‐catenin signaling‐dependent. The mechanism underlying how these cells interpret instructive signaling cues and differentiate into these two lineages is unclear. Twist1 is a target of the Wnt/β‐catenin signaling pathway and is expressed in cranial bone and dermal lineages. Results: Here, we show that onset of Twist1 expression in the mouse cranial mesenchyme is dependent on ectodermal Wnts and mesenchymal β‐catenin activity. Conditional deletion of Twist1 in the supraorbital arch mesenchyme leads to cranial bone agenesis and hypoplastic dermis, as well as craniofacial malformation of eyes and palate. Twist1 is preferentially required for cranial bone lineage commitment by maintaining Wnt responsiveness. In the conditional absence of Twist1, the cranial dermis fails to condense and expand apically leading to extensive cranial dermal hypoplasia with few and undifferentiated hair follicles. Conclusions: Thus, Twist1, a target of canonical Wnt/β‐catenin signaling, also functions to maintain Wnt responsiveness and is a key effector for cranial bone fate selection and dermal condensation. Developmental Dynamics 245:144–156, 2016. © 2015 Wiley Periodicals, Inc.     

An ATF2‐based luciferase reporter to monitor non‐canonical Wnt signaling in xenopus embryos

Non‐canonical/planar cell polarity (PCP) Wnt signaling plays important roles in embryonic development and tissue homeostasis, and is implicated in human disease. Monitoring Wnt/PCP signaling relies mostly on semi‐quantitative bioassays or biochemical analysis. Here we describe a luciferase reporter assay based on an ATF2 response element, which faithfully monitors non‐canonical Wnt signaling in Xenopus embryos. The assay is simple, quantitative, and robust. It can be used to detect non‐canonical Wnt signaling changes following gain and loss of function of pathway components, including Wnt, Frizzled, Ror2, Disheveled, Rac1, MKK7, and JNK. Wnt/PCP signaling has recently been implicated in left‐right asymmetry and our reporter assay suggests that in gastrula embryos there is a right‐ward bias in Wnt/PCP signaling. We also mapped Wnt/PCP signaling in the early Xenopus embryo and find that it peaks in the dorso‐vegetal region, paralleling Wnt/β‐catenin signaling. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Serine‐arginine protein kinase 1 promotes a cancer stem cell‐like phenotype through activation of Wnt/β‐catenin signalling in NSCLC

Cancer stem cells (CSCs) are commonly associated with cancer recurrence and metastasis that occurs in up to 30–55% of non‐small‐cell lung carcinoma (NSCLC) patients. Herein, we showed that serine‐arginine protein kinase 1 (SRPK1) was highly expressed at both the mRNA and the protein levels in human NCSLC. SRPK1 was associated with the clinical features of human NSCLC, including clinical stage (p < 0.001) and T (p = 0.001), N (p = 0.007), and M (p = 0.001) classifications. Ectopic overexpression of SRPK1 promoted the acquisition of a stem cell‐like phenotype in human NSCLC cell lines cultured in vitro. Overexpression of SRPK1 increased sphere formation and the proportion of side‐population cells that exclude Hoechst dye. Conversely, SRPK1 silencing reduced the number of spheres and the proportion of side‐population cells. Mouse studies indicated that SRPK1 promoted NSCLC cell line tumour growth and SRPK1 overexpression reduced the number of tumour cells required to initiate tumourigenesis in vivo. Mechanistically, gene set enrichment analysis showed that Wnt/β‐catenin signalling correlated with SRPK1 mRNA levels and this signalling pathway was hyperactivated by ectopic SRPK1 expression in NSCLC cell lines. Immunofluorescence demonstrated that SRPK1 enhanced β‐catenin accumulation in the nuclei of NSCLC cell lines, and inhibition of β‐catenin signalling abrogated the SRPK1‐induced stem cell‐like phenotype. Together, our findings suggest that SRPK1 promotes a stem cell‐like phenotype in NSCLC via Wnt/β‐catenin signalling. Moreover, SRPK1 may represent a novel target for human NSCLC diagnosis and therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Beta‐catenin status in paediatric medulloblastomas: correlation of immunohistochemical expression with mutational status,genetic profiles,and clinical characteristics

Medulloblastoma is the most frequent malignant paediatric brain tumour. The activation of the Wnt/β‐catenin pathway occurs in 10‐15% of medulloblastomas and has been recently described as a marker for favourable patient outcome. We report a series of 72 paediatric medulloblastomas evaluated for β‐catenin protein expression, CTNNB1 mutations, and comparative genomic hybridization. Gene expression profiles were also available in a subset of 40 cases. Immunostaining of β‐catenin showed extensive nuclear staining (>50% of the tumour cells) in six cases and focal nuclear staining (<10% of cells) in three cases. The other cases either exhibited a signal strictly limited to the cytoplasm (58 cases) or were negative (five cases). CTNNB1 mutations were detected in all β‐catenin extensively nucleopositive cases. The expression profiles of these cases documented strong activation of the Wnt/β‐catenin pathway. Remarkably, five out of these six tumours showed a complete loss of chromosome 6. In contrast, cases with focal nuclear β‐catenin staining, as well as tumours with negative or cytoplasmic staining, never demonstrated CTNNB1 mutation, Wnt/β‐catenin pathway activation or chromosome 6 loss. Patients with extensive nuclear staining were significantly older at diagnosis and were in continuous complete remission after a mean follow‐up of 75.7 months (range 27.5–121.2 months) from diagnosis. All three patients with focal nuclear staining of β‐catenin died within 36 months from diagnosis. Altogether, these data confirm and extend previous observations that CTNNB1‐mutated tumours represent a distinct molecular subgroup of medulloblastomas with favourable outcome, indicating that therapy de‐escalation should be considered. International consensus on the definition criteria of this distinct medulloblastoma subgroup should be achieved. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Wnt/β‐Catenin Signaling Pathway Upregulates c‐Myc Expression to Promote Cell Proliferation of P19 Teratocarcinoma Cells

Aberrant activation of the Wnt/β‐catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/β‐catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3β specific inhibitor (SB216763) was used to activate Wnt/β‐catenin signaling. All trans‐retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real‐time PCR, and Western‐blotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c‐myc expression. The results showed that SB216763 induced the nuclear translocation of β‐catenin, upregulated the expression of c‐myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans‐RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c‐myc overexpression. C‐myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/β‐catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/β‐catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c‐myc expression. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Oncogenic KRAS is not necessary for Wnt signalling activation in APC‐associated FAP adenomas

Recent studies have suggested that APC loss alone may be insufficient to promote aberrant Wnt/β‐catenin signalling. Our aim was to comprehensively characterize Wnt signalling components in a set of APC‐associated familial adenomatous polyposis (FAP) tumours. Sixty adenomas from six FAP patients with known pathogenic APC mutations were included. Somatic APC and KRAS mutations, β‐catenin immunostaining, and qRT‐PCR of APC, MYC, AXIN2 and SFRP1 were analysed. Array‐comparative genomic hybridization (aCGH) was also assessed in 26 FAP adenomas and 24 paired adenoma–carcinoma samples. A somatic APC alteration was present in 15 adenomas (LOH in 11 and four point mutations). KRAS mutations were detected in 10% of the cases. APC mRNA was overexpressed in adenomas. MYC and AXIN2 were also overexpressed, with significant intra‐case heterogeneity. Increased cytoplasmic and/or nuclear β‐catenin staining was seen in 94% and 80% of the adenomas. β‐Catenin nuclear staining was strongly associated with MYC levels (p value 0.03) but not with KRAS mutations. Copy number aberrations were rare. However, the recurrent chromosome changes observed more frequently contained Wnt pathway genes (p value 0.012). Based on β‐catenin staining and Wnt pathway target genes alterations the Wnt pathway appears to be constitutively activated in all APC‐FAP tumours, with alterations occurring both upstream and downstream of APC. Wnt aberrations are present at both the DNA and the RNA level. Somatic profiling of APC‐FAP tumours provides new insights into the role of APC in tumourigenesis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.