Wnt Pathway Extracellular Components and Their Essential Roles in Bone Homeostasis

The Wnt pathway is involved in several processes essential for bone development and homeostasis. For proper functioning, the Wnt pathway is tightly regulated by numerous extracellular elements that act by both activating and inhibiting the pathway at different moments. This review aims to describe, summarize and update the findings regarding the extracellular modulators of the Wnt pathway, including co-receptors, ligands and inhibitors, in relation to bone homeostasis, with an emphasis on the animal models generated, the diseases associated with each gene and the bone processes in which each member is involved. The precise knowledge of all these elements will help us to identify possible targets that can be used as a therapeutic target for the treatment of bone diseases such as osteoporosis.

H3K27ac-activated EGFR-AS1 promotes cell growth in cervical cancer through ACTN4-mediated WNT pathway

Background Recently, extensive studies unveiled that lncRNAs exert critical function in the development and progression of cervical cancer (CC). EGFR-AS1 is a novel lncRNA which has not been well-explored in CC. Aims Our study aimed to research the function and molecular mechanism of EGFR-AS1 in CC cells. qRT-PCR analysis was performed to detect gene expression. Colony formation, EdU, flow cytometry, TUNEL, western blot and transwell assays were performed to assess the effect of EGFR-AS1 on CC cell growth. The regulatory mechanism of EGFR-AS1 was dug out through mechanism experiments. Results EGFR-AS1 was notably overexpressed in CC cell lines. Loss-of-functional experiments revealed that EGFR-AS1 promoted CC cell proliferation, migration and invasion, and suppressed cell apoptosis. Mechanistically, up-regulation of EGFR-AS1 was attributed to the activation of H3K27 acetylation (H3K27ac). Further, EGFR-AS1 was revealed to function as miR-2355-5p sponge. Additionally, miR-2355-5p was down-regulated in CC cells and ACTN4 was identified as a target gene of miR-2355-5p. Ultimately, overexpressed ACTN4 could reserve the suppressive role of EGFR-AS1 silencing in CC cell growth. Last but not least, EGFR-AS1 facilitated CC cell growth via ACTN4-mediated WNT pathway. Conclusions H3K27ac-activated EGFR-AS1 sponged miR-2355-5p and promoted CC cell growth through ACTN4-mediated WNT pathway.

Claudin-18 expression under hypoxia in neonatel lungs of brochopulmonary dysplasia model rats

Background: Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar and microvascular development. Claudin-18 is the only known lung-specific tight junction protein affecting alveolar epithelium development and transdifferentiation. Objective: To explore the changes in claudin-18 expression, alveolar epithelial cell (AEC) marker proteins, the canonical Wnt pathway, and their possible regulatory relationships in a hyperoxia-induced BPD rat model. Methods: The BPD neonatal rat model was established by exposure to hyperoxia (85%). Hematoxylin and eosin (HE) staining was used to confirm the establishment of the BPD model. The mRNA levels were assessed using quantitative real-time polymerase chain reaction, while protein expression levels were determined using western blotting, immunohistochemical staining, and immunofluorescence . Results: As confirmed by HE staining, the BPD neonatal rat model was successfully established. Compared with the air group, claudin-18 and claudin-4 expression decreased in the hyperoxia group. The expression of β-catenin of the Wnt signaling decreased, whereas that of p-GSK-3β increased. Expression of the AEC Ⅱ marker SFTPC decreased initially and then increased, whereas that of the AEC Ⅰ marker Podoplanin increased on day 14 (P < 0.05). Conclusions: Claudin-18 downregulation during hyperoxia may affect lung development and maturation, which may result in hyperoxia-induced BPD. Additionally, claudin-18 is associated with the canonical Wnt pathway and alveolar epithelial transdifferentiation.

Nuclear Receptor Interacting Protein-2 Mediates the Stabilization and Activation of β-Catenin During Podocyte Injury

Objective: Activation of β-catenin causes podocyte injury and proteinuria, but how β-catenin signalling is regulated during podocyte injury remains elusive. Nuclear receptor interacting protein 2 (NRIP2) modulates the Wnt pathway in colorectal cancer-initiating cells, but the role of NRIP2 in podocyte injury has not yet been investigated. We aimed to examine the interaction between NRIP2 and β-catenin signalling.Materials and Methods: Knockdown or overexpression of NRIP2 and β-catenin and chemical treatments were performed in cultured human podocytes. Immunoprecipitation, immunoblotting and immunofluorescence assays were used to assess protein interactions and expression. Data from the GEO dataset and kidney tissues from patients with focal segmental glomerulosclerosis (FSGS) and surgical nephrectomy were examined. An adriamycin (ADR) nephropathy model was established in NRIP2 knockout mice.Results: NRIP2 knockdown accelerated β-catenin degradation, which was reversed by MG132; specifically, NRIP2 bound β-catenin and stabilized it to prevent its degradation through the ubiquitin proteasomal pathway. Overexpression of NRIP2 led to β-catenin activation and Snail1 induction, and these effects were attenuated by β-catenin knockdown. NRIP2 knockdown blocked ADR-stimulated β-catenin activation. In ADR mice, genetic knockout of Nrip2 ameliorated podocyte injury and loss, glomerulosclerosis, and proteinuria by inhibiting β-catenin activation. Moreover, NRIP2 was significantly upregulated in podocytes of FSGS patients and colocalized with nuclear β-catenin.Conclusion: These results established NRIP2 as a stabilizer of β-catenin activation through the ubiquitin proteasomal pathway in podocyte injury.

LIM homeobox-2 suppresses hallmarks of adult and pediatric liver cancers by inactivating MAPK/ERK and Wnt/beta-catenin pathways

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, apoptosis), molecular approaches (phospho-kinase arrays, RNA-seq), bioinformatics and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 down-regulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors and low patients’ survival. Forced expression of LHX2 reduced the proliferation, migration and survival of hepatoma cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g. TLE/Groucho) and MAPK/ERK (e.g. DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

pop-1/TCF, ref-2/ZIC and T-box factors regulate the development of anterior cells in the C. elegans embryo

Patterning of the anterior-posterior axis is fundamental to animal development. The Wnt pathway plays a major role in this process by activating the expression of posterior genes in animals from worms to humans. This observation raises the question of whether the Wnt pathway or other regulators control the expression of the many anterior-expressed genes. We found that the expression of five anterior-specific genes in Caenorhabditis elegans embryos depends on the Wnt pathway effectors pop-1/TCF and sys-1/β-catenin. We focused further on one of these anterior genes, ref-2/ZIC, a conserved transcription factor expressed in multiple anterior lineages. Live imaging of ref-2 mutant embryos identified defects in cell division timing and position in anterior lineages. Cis-regulatory dissection identified three ref-2 transcriptional enhancers, one of which is necessary and sufficient for anterior-specific expression. This enhancer is activated by the T-box transcription factors TBX-37 and TBX-38, and surprisingly, concatemerized TBX-37/38 binding sites are sufficient to drive anterior-biased expression alone, despite the broad expression of TBX-37 and TBX-38. Taken together, our results highlight the diverse mechanisms used to regulate anterior expression patterns in the embryo.

Thyroid Hormone Nuclear Receptor TRα1 and Canonical WNT Pathway Cross-Regulation in Normal Intestine and Cancer

A pivotal role of thyroid hormones and their nuclear receptors in intestinal development and homeostasis have been described, whereas their involvement in intestinal carcinogenesis is still controversial. In this perspective article we briefly summarize the recent advances in this field and present new data regarding their functional interaction with one of the most important signaling pathway, such as WNT, regulating intestinal development and carcinogenesis. These complex interactions unveil new concepts and will surely be of importance for translational research.

Exosome Component 4 Promotes Epithelial Ovarian Cancer Cell Proliferation, Migration, and Invasion via the Wnt Pathway

BackgroundOf gynecologic malignancies, ovarian cancer is the leading cause of death, mainly due to the lack of sensitive tumor markers, which means it almost always presents at an advanced stage. Exosome Component 4 (EXOSC4) is involved in RNA degradation, but its role in epithelial ovarian cancer (EOC) is unclear.MethodsThe expression levels of EXOSC4 in EOC and normal ovarian tissue specimens were determined by immunohistochemical staining. The overall survival (OS) and progression-free survival (PFS) of patients with EOC were evaluated after patients were classified into high and low EXOSC4 expression groups, and the Cox regression model was established to identify independent predictors of patient prognosis. The effects of EXOSC4 on proliferation, colony formation, migration, and invasion were examined in the SKOV-3 and HO8910 cell lines by lentivirus-mediated shRNA knockdown. Flow cytometry was used to detect cell cycle changes. The mRNA levels of cyclin D1, CDK4, and c-myc were detected by RT-PCR. The protein expression levels of β-catenin, cyclin D1, CDK4, c-myc, vimentin, N-cadherin, and E-cadherin were assessed by western blot. Wnt/β-catenin activation was measured by TCF/LEF reporter assay.ResultsEXOSC4 was significantly elevated in EOC tissues and cell lines. High EXOSC4 expression was correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage and pathological grade, and identified as an independent predictor of shorter OS and PFS. EXOSC4 knockdown suppressed proliferation, migration, and invasion in EOC cell lines. Cells were arrested at G0/G1 phase after EXOSC4 knockdown. The mRNA levels of cyclin D1, CDK4, and c-myc were decreased. β-catenin, cyclin D1, CDK4, c-myc, vimentin, and N-cadherin protein expression levels were reduced, while those of E-cadherin was increased. Wnt/β-catenin activity was suppressed after the EXOSC4 knockdown.ConclusionsEXOSC4 is involved in EOC. Knockdown of EXOSC4 can inhibit the proliferation, migration, and invasion ability of EOC by suppressing the Wnt pathway. EXOSC4 is expected to be a novel biomarker and molecular target in EOC.

WNT11, a new gene associated with early-onset osteoporosis, is required for osteoblastogenesis

Monogenic early-onset osteoporosis (EOOP) is a rare disease defined by low bone mineral density (BMD) that results in increased risk of fracture in children and young adults. Although several causative genes have been identified, some of the EOOP causation remains unresolved. Whole-exome sequencing revealed a de novo heterozygous loss-of-function mutation in WNT11 (NM_004626.2:c.677_678dup p.Leu227Glyfs*22) in a 4-year-old boy with low BMD and fractures. We identified two heterozygous WNT11 missense variants (NM_004626.2:c.217G &gt; A p.Ala73Thr) and (NM_004626.2:c.865G &gt; A p.Val289Met) in a 51-year-old woman and in a 61-year-old woman respectively, both with bone fragility. U2OS cells with heterozygous WNT11 mutation (NM_004626.2:c.690_721delfs*40) generated by CRISPR-Cas9 showed reduced cell proliferation (30%) and osteoblast differentiation (80%) as compared with wild-type U2OS cells. The expression of genes in the Wnt canonical and non-canonical pathways was inhibited in these mutant cells, but recombinant WNT11 treatment rescued the expression of Wnt pathway target genes. Furthermore, the expression of RSPO2, a WNT11 target involved in bone cell differentiation, and its receptor LGR5, was decreased in WNT11 mutant cells. Treatment with WNT5A and WNT11 recombinant proteins reversed LGR5 expression, but WNT3A recombinant protein treatment had no effect on LGR5 expression in mutant cells. Moreover, treatment with recombinant RSPO2 but not WNT11 or WNT3A activated the canonical pathway in mutant cells. In conclusion, we have identified WNT11 as a new gene responsible for EOOP, with loss-of-function variant inhibiting bone formation via Wnt canonical and non-canonical pathways. WNT11 may activate Wnt signaling by inducing the RSPO2–LGR5 complex via the non-canonical Wnt pathway.

The Influence of BCL6 on the WNT Pathway in Glioblastoma Therapy Resistance

<p>Glioblastoma is a devastating disease with a median survival of 18 months from diagnosis and a 5 years survival rate of only 10%. The gold standard of treatment for glioblastoma is surgical resection followed by chemotherapeutic treatment with Temozolomide, a DNA alkylating agent, and irradiation around the remaining tumour margins. These treatments are both designed to create DNA damage to the cancerous cells, causing the cell cycle to halt, and result in apoptosis. This treatment does extend patients life for a few months, however glioblastoma cells quickly become resistant to therapy, and disease is always fatal. The anti-apoptotic protein BCL6, confers resistance to apoptosis in response to DNA damage and has been shown to be upregulated in Glioblastoma in response to DNA damaging chemotherapy and irradiation. This upregulation has been hypothesised to increase resistance to these therapies. By minimizing resistance to the standard therapies, the outlook for sufferers of glioblastoma could be greatly improved. Dysregulation of the WNT pathway has also been shown to be very important in carcinogenesis of glioblastoma and is responsible for the diffuse nature of the tumour which makes total resection nearly impossible. An RNA-seq screen was carried out on a glioblastoma cell line in which BCL6 was inhibited using the small molecule inhibitor FX1. This resulted in a change in expression of a large number of WNT related genes. This indicates that there is a link between BCL6 and the WNT pathway. Changes in expression in the WNT genes DKK1, WNT5a and WNT5b were validated. Experiments were carried out to investigate the effects of chemotherapy and BCL6 inhibition on both the canonical and non-canonical WNT pathways. It was found that BCL6 has an influence of the level of activity of the canonical WNT pathway. It also influences migration, the cell cycle, and clonogenicity. Understanding this link between WNT and BCL6 could be crucial in finding an effective treatment for glioblastoma.</p>