Mediator Of Wnt Signaling Research Articles

Two functional forms of the Meckel-Gruber syndrome protein TMEM67 generated by proteolytic cleavage by ADAMTS9 mediate Wnt signaling and ciliogenesis.

TMEM67 mutations are the major cause of Meckel-Gruber syndrome. TMEM67 is involved in both ciliary transition zone assembly, and non-canonical Wnt signaling mediated by its extracellular domain. How TMEM67 performs these two separate functions is not known. We identify a novel cleavage motif in the extracellular domain of TMEM67 cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage regulates the abundance of two functional forms: A C-terminal portion which localizes to the ciliary transition zone regulating ciliogenesis, and a non-cleaved form which regulates Wnt signaling. By characterizing three TMEM67 ciliopathy patient variants within the cleavage motif utilizing mammalian cell culture and C. elegans, we show the cleavage motif is essential for cilia structure and function, highlighting its clinical significance. We generated a novel non-cleavable TMEM67 mouse model which develop severe ciliopathies phenocopying Tmem67 -/- mice, but in contrast, undergo normal Wnt signaling, substantiating the existence of two functional forms of TMEM67.

The emerging understanding of Frizzled receptors.

The Wnt signaling pathway is a huge network governing development and homeostasis, dysregulation of which is associated with a myriad of human diseases. The Frizzled receptor (FZD) family comprises receptors for Wnt ligands, which indispensably mediate Wnt signaling jointly with a variety of co-receptors. Studies of FZDs have revealed that 10 FZD subtypes play diverse roles in physiological processes. At the same time, dysregulation of FZDs is also responsible for various diseases, in particular human cancers. Enormous attention has been paid to the molecular understanding and targeted therapy of FZDs in the past decade. In this review, we summarize the latest research on FZD structure, function, regulation and targeted therapy, providing a basis for guiding future research in this field.

Elucidating activity for WNT signaling mediators in breast cancer and Paclitaxel chemoresistance

Breast cancer continues to be among the most common cancers found in patients worldwide with more than 300,000 new cases projected to be diagnosed in 2024 for women in the United States (Siegel et al. 2023). Metastatic breast cancer is heterogenous, which indicates both its complexity and the increased degree of chemotherapeutic intervention necessary for advanced or refractory cases. The development of chemoresistance further complicates intervention, as even previously effective therapeutics against early-stage cancers can develop resistance and generate refractory, high-grade tumors. Paclitaxel is a microtubule-stabilizing compound that remains of clinical importance due to its ongoing applications in early and metastatic disease. Studies in paclitaxel chemoresistance remain of critical consideration as even this promising therapeutic has been shown, like all compounds, to result in chemoresistance (Steding 2016). Studies have demonstrated pivotal roles for the WNT signaling pathway within tumor development, progression, and acquired chemoresistance (Zhong and Virshup 2019). The WNT signaling pathway has been implicated as a driving mechanism of chemoresistance in several cancer types, including both early and metastatic breast cancer. As WNT signaling can increase both as an immediate response to Paclitaxel treatment and as a potential mechanism of paclitaxel chemoresistance, it is important to elucidate further whether WNT signaling is actually a driving force of chemoresistance or merely a byproduct of its development. Furthermore, the widespread mechanisms and players involved in WNT signaling require further isolation and consideration to determine the most significant players in chemoresistance. Prior evidence has indicated that reductions in WNT signaling have the potential to restore sensitivity to treatment to a wide range of chemotherapeutics for some cancer types (Chikazawa 2010). These findings may be cell type specific or be a consequence of specific changes of WNT signaling mediators rather than global WNT signaling. Preliminary evidence observed in paclitaxel chemoresistant cell populations indicated a wide range of changes in WNT signaling mediators including both downregulation and upregulation of components within the same pathway. With the potential for conflicting results across cancers and these preliminary findings in mind, further investigation into the role of WNT signaling mediators in paclitaxel sensitivity and resistance remains of significant interest. In order to define actual mechanisms of action, roles for global WNT signaling as well as specific roles for key players within the pathway must be confirmed. We hypothesized that changes in global WNT signaling would significantly alter breast cancer sensitivity to paclitaxel through both canonical and noncanonical pathways via specific mediators. To begin to assess this hypothesis, breast cancer cells were treated with a global WNT inhibitor or an inhibitor targeting Beta-catenin (β-catenin). Treatment of cells with both inhibitors were found to suppress overall WNT activity to similar levels; however, the targeting of β-catenin altered paclitaxel effcacy compared to paclitaxel alone or in combination with the global inhibitor. Further analysis of specific changes following the combined treatment implicated the noncanonical pathway as playing a more substantial role in paclitaxel sensitivity. Although investigations remain ongoing, these findings indicate novel behaviors for WNT signaling in paclitaxel sensitivity and support further exploration into the complicated world of WNT signaling and its signaling mediators in chemoresistance. This research was supported in part by an Indiana Academy of Sciences Senior Research Grant (IASSG-S21-07) as well as a Marian University Faculty Research Development Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Structure-Based Ligand Discovery Targeting the Transmembrane Domain of Frizzled Receptor FZD7.

Despite the essential roles of Frizzled receptors (FZDs) in mediating Wnt signaling in embryonic development and tissue homeostasis, ligands targeting FZDs are rare. A few antibodies and peptide modulators have been developed that mainly bind to the family-conserved extracellular cysteine-rich domain of FZDs, while the canonical binding sites in the transmembrane domain (TMD) are far from sufficiently addressed. Based on the recent structures of FZDs, we explored small-molecule ligand discovery by targeting TMD. From the ChemDiv library with ∼1.6 million compounds, we identified compound F7H as an antagonist of FZD7 with an IC50 at 1.25 ± 0.38 μM. Focusing on this hit, the structural dissection study, together with computing studies such as molecular docking, molecular dynamics simulation, and free energy perturbation calculations, defined the binding pocket with key residue recognition. Our results revealed the structural basis of ligand recognition and demonstrated the feasibility of structure-guided ligand discovery for FZD7-TMD.

OTUD7B Activates Wnt Signaling Pathway through the Interaction with LEF1.

The Wnt signaling pathway plays a critical role in regulating normal cellular processes, including proliferation, differentiation, and apoptosis. Dysregulation of Wnt signaling has been implicated in various human diseases, including cancer. β-catenin and LEF1 are key mediators of Wnt signaling, and their dysregulation is a hallmark of many cancer types. In this study, we aimed to identify the deubiquitinases (DUBs) that regulate the Wnt signaling pathway through the essential component LEF1. Screening candidate DUBs from the human DUB library, we discovered that OTUD7B interacts with LEF1 and activates Wnt signaling. OTUD7B and LEF1 interact with each other through the UBA and HMG domains, respectively. Furthermore, OTUD7B promotes the nuclear localization of LEF1, leading to an increased interaction with β-catenin in the nucleus while not noticeably affecting ubiquitination on LEF1. Using qPCR array analysis, we found that OTUD7B overexpression leads to an upregulation of 75% of the tested Wnt target genes compared to the control. These findings suggest that OTUD7B may serve as a potential therapeutic target in human diseases, including cancers where Wnt signaling is frequently dysregulated.

Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-β Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia

To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-β on T cell acute lymphoblastic leukemia (T-ALL) cell lines. Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. Different concentrations of FA-2-b-β significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-β inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-β reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/β-catenin inhibitor (ICG001), the process was further intensified. Agaricus Blazei Extract FA-2-b-β inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

YAP/TEAD1 and β-catenin/LEF1 synergistically induce estrogen receptor α to promote osteogenic differentiation of bone marrow stromal cells.

Bone remodeling is vital to the maintenance of bone homeostasis and may lead to destructive skeletal diseases once the balance is disrupted. Crosstalk between Wnt and estrogen receptor (ER) signaling has been proposed in bone remodeling, but the underlying mechanism remains unclear. This study was designed to explore the effect of Wnt-ER signaling during the osteogenic differentiation of bone marrow stromal cells (BMSCs). Rat BMSCs were isolated and identified using flow cytometry and stimulated with Wnt3a. Wnt3a treatment promoted osteogenic differentiation and mineralization of the BMSCs. Meanwhile, Wnt3a enhanced the expression of ERα as well as the canonical Wnt signaling mediator β-catenin and the alternative Wnt signaling effector Yes-associated protein 1 (YAP1). Interestingly, DNA pulldown assay revealed direct binding of transcriptional enhanced associate domain 1 (TEAD1) and lymphoid enhancer binding factor 1 (LEF1), transcriptional partners of YAP1 and β-catenin, respectively, to the promoter region of ERα. In addition, inhibition of TEAD1 and LEF1 suppressed Wnt3-promoted BMSC osteogenic differentiation and blocked Wnt3a-induced ERα expression. Furthermore, an in vivo model of femoral bone defect also supported that Wnt3a facilitated bone healing in an ERα-dependent way. Together, we suggest that Wnt3a promotes the osteogenic activity of BMSCs through YAP1 and β-catenin-dependent activation of ERα, via direct binding of TEAD1 and LEF1 to the ERα promoter.

Synovial gene signatures associated with the development of rheumatoid arthritis in at risk individuals: A prospective study

ObjectiveTo identify molecular changes in synovium before arthritis development in individuals at risk of developing rheumatoid arthritis (RA). Materials and methodsWe included 67 IgM rheumatoid factor and/or anti-citrullinated protein antibody positive individuals with arthralgia but without arthritis. Synovial biopsies were collected after which individuals were prospectively followed for at least 2 years during which 17 developed arthritis. An exploratory genome-wide transcriptional profiling study was performed in 13 preselected individuals to identify transcripts associated with arthritis development (n = 6). Findings were validated using quantitative real-time PCR and immunohistochemistry in the total cohort. ResultsMicroarray-based survival analyses identified 5588 transcripts whose expression levels in synovium were significantly associated with arthritis development. Pathway analysis revealed that synovial tissue of at risk individuals who later developed arthritis display higher expression of genes involved in adaptive immune response-related pathways compared to at risk individuals who did not develop arthritis. Lower expression was observed for genes involved in extracellular matrix receptor interaction, Wnt-mediated signal transduction and lipid metabolism. Two-way hierarchical clustering analyses of a 27-gene signature separated the total at risk cohort into two groups, where pre-RA individuals preferred to cluster together. Immunohistochemistry studies revealed more podoplanin positive cells and lower lipid droplet staining in synovial tissue from pre-RA individuals. ConclusionSynovial alterations in adaptive immune response and lipid metabolism are associated with future development of arthritis. Since this data show synovial changes without overt cellular infiltration, these may be attributed to preclinical changes in resident synovial tissue cells such as fibroblasts, macrophages and tissue resident T cells.

WNT5A-RHOA Signaling Is a Driver of Tumorigenesis and Represents a Therapeutically Actionable Vulnerability in Small Cell Lung Cancer.

The p130-WNT5A-RHOA pathway drives SCLC progression and is a potential target for the development of therapeutic interventions and biomarkers to improve patient treatment.

Heterozygous variants in the DVL2 interaction region of DACT1 cause CAKUT and features of Townes–Brocks syndrome 2

Most patients with congenital anomalies of the kidney and urinary tract (CAKUT) remain genetically unexplained. In search of novel genes associated with CAKUT in humans, we applied whole-exome sequencing in a patient with kidney, anorectal, spinal, and brain anomalies, and identified a rare heterozygous missense variant in the DACT1 (dishevelled binding antagonist of beta catenin 1) gene encoding a cytoplasmic WNT signaling mediator. Our patient’s features overlapped Townes–Brocks syndrome 2 (TBS2) previously described in a family carrying a DACT1 nonsense variant as well as those of Dact1-deficient mice. Therefore, we assessed the role of DACT1 in CAKUT pathogenesis. Taken together, very rare (minor allele frequency ≤ 0.0005) non-silent DACT1 variants were detected in eight of 209 (3.8%) CAKUT families, significantly more frequently than in controls (1.7%). All seven different DACT1 missense variants, predominantly likely pathogenic and exclusively maternally inherited, were located in the interaction region with DVL2 (dishevelled segment polarity protein 2), and biochemical characterization revealed reduced binding of mutant DACT1 to DVL2. Patients carrying DACT1 variants presented with kidney agenesis, duplex or (multi)cystic (hypo)dysplastic kidneys with hydronephrosis and TBS2 features. During murine development, Dact1 was expressed in organs affected by anomalies in patients with DACT1 variants, including the kidney, anal canal, vertebrae, and brain. In a branching morphogenesis assay, tubule formation was impaired in CRISPR/Cas9-induced Dact1−/− murine inner medullary collecting duct cells. In summary, we provide evidence that heterozygous hypomorphic DACT1 variants cause CAKUT and other features of TBS2, including anomalies of the skeleton, brain, distal digestive and genital tract.

Study of the biological relevance of Wnt/β-catenin signaling pathway and β-adrenergic regulation in osteoblastic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can invade the bone site, proliferate and differentiate into cartilage and bone. Therefore, they serve as an important target for developing modalities to enhance local and systemic bone formation. It has been demonstrated that the Wnt/β-catenin signaling pathway plays a key role in the movement of MSCs toward osteoblast lineage. β-Catenin, an indispensable mediator of canonical Wnt signaling, is a transcriptional factor regulating the mesenchymal precursor's differentiation to mature osteoblasts. On the other hand, the β-adrenergic nervous system negatively impacts the osteogenic differentiation of MSCs. β-Adrenergic receptors (β-AR) are the dominant synergistic receptors on MSCs. It is established that catecholamines activate β-adrenergic receptors and inhibit osteoblastogenesis through an unknown molecular mechanism. This study evaluated the β-adrenergic system mediates inhibition of osteoblastogenesis through the inhibition of the Wnt/β-catenin signaling pathway. Hence, we have treated MSCs with a β-adrenergic agonist (Isoproterenol) and a β-adrenergic antagonist (propranolol). Next, on days 7 and 14 post-treatment, we have examined mineralization, osteogenic markers and the expression of genes related to Wnt signaling. Our findings suggested that isoproterenol (β-adrenergic agonist) inhibits the differentiation of mesenchymal stem cells to osteoblasts via stimulation of β2-adrenergic receptor and the subsequent inhibition of Wnt β-catenin signaling by lowering the expression of β-catenin. Nevertheless, the SOST expression did not exhibit a meaningful pattern to support that Isoproterenol inhibits the Wnt signaling pathway by increasing sclerostin expression.

Lead inhibits the odontogenic differentiation potential of dental pulp stem cells by affecting WNT1/β-catenin signaling and related miRNAs expression

Lead (Pb) is ubiquitous in environment that accumulates in teeth and calcified tissues from where it releases gradually with aging and adversely affects dental health. This study aimed to determine the effect of Pb exposure on odontogenic differentiation potential of isolated human dental pulp stem cells and investigate the possible underlying epigenetic factors. In the absence of Pb exposure, stem cells displayed significant odontogenic markers including elevated Alkaline Phosphatase (ALP) activity, Alizarin red staining intensity, and increased expression of odontogenic DMP1 and DSPP genes. Exposure to 60 μM Pb resulted in reduced ALP activity and calcium deposition. Also, diminished expression of RUNX2, DMP1, and DSPP, as well as Wnt signaling mediators including WNT1, and β-catenin were detected. The expression of Wnt signaling related microRNAs, miRNA-139-5p and miRNA-142-3p, on the other hand, were shown to have a significant increase. We concluded that Pb could adversely affect the odontogenic differentiation potential of dental pulp stem cell. The underlying mechanism might related to Pb-induced epigenetic dysregulation of WNT1/β-catenin pathway-related miRNAs leading to down-regulation of Wnt/β-catenin related odontogenic genes and eventually impaired odontogenic differentiation process.

Abstract 6387: Inhibition of Lymphoid Enhancer Factor1(LEF1) in acute myeloid leukemia overcomes acquired cytarabine resistance

Abstract Canonical Wnt-β-catenin signalling is frequently dysregulated in myeloid leukemia. LEF1 (Lymphoid Enhancer Factor 1), a key mediator of Wnt signalling, functions as an interacting partner with β-catenin. Previous studies have shown that LEF1 is associated with leukemic transformation and is an independent prognostic factor in normal karyotype AML. Through high throughput transcriptome profiling between the parental and acquired cytarabine resistant cell lines, we identified that LEF1 to be a potential target mediating this resistance. Here we evaluated the role, of molecular and pharmacological inhibition of LEF1 in overcoming acquired cytarabine resistance. The differential expression of LEF1 in THP1, MV4-11 and U937 parental and Ara-C resistant AML cell lines was checked by quantitative RT-PCR, immunoblot and immunofluorescence. LEF1 knock-out (KO) cells were generated using the CRISPR-Cas9 in the THP1 cytarabine resistant cell line. Lentiviral based over-expression of LEF1 was carried out in THP1 parental cell line to ensure that LEF1 promotes Ara-C resistance. For nuclear translocation of LEF1, the small molecule CHIR99021 was used in over-expressed cells.The knock-out and over-expressed cells were characterized by analysing proliferation, cell cycle and cytotoxicity assay. The effect of pharmacological inhibition of LEF1 using niclosamide was examined by in-vitro-cytotoxicity assay.Although LEF1 transcript expression was high in all the three cytarabine resistant cell lines compared to the parental cell lines, THP1-araC-R cell line showed around ≈ 500-fold change in expression and hence was used for further experiments. THP1-araC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC &amp;gt;4µM). LEF1 knock-out showed complete absence of LEF1 protein, decreased rate of proliferation and improved sensitivity to Ara-C (Ara-C IC50- 1430 µM in the untransduced vs. 387.4 µM after LEF1 KO). Over-expression of LEF1 with a fold change of ≈1000 followed by enforced nuclear translocation using the small molecule CHIR99021 in parental cell line resulted in increased resistance to Ara-C (IC50 Parental- 50.7µM vs OE CHIR treated 240.05µM). Further, niclosamide, the pharmacological inhibitor of LEF1 was able to re-sensitize the THP1 AraC -resistant cells (IC50: 1430 µM for UT vs 516.8 µM treated with niclosamide). Our results suggest that inhibition of LEF1 by molecular and pharmacological means showed considerable increase in cytarabine sensitivity in-vitro while we are testing its effect in-vivo using a transplantable AML mouse model. Citation Format: Saswati Das, Raveen Stephen Illangeswaran, Daniel Zechariah Paul Jebanesan, Rakhi Thalayatta Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi Murugan Rajamani, Martin Michaelis, Jindrich Cinatl Jr., Vikram Mathews, Shaji Ramchandran Velayudhan, Poonkuzhali Balasubramanian. Inhibition of Lymphoid Enhancer Factor1(LEF1) in acute myeloid leukemia overcomes acquired cytarabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6387.

GWAS of thyroid dysgenesis identifies a risk locus at 2q33.3 linked to regulation of Wnt signaling.

Congenital hypothyroidism due to thyroid dysgenesis (TD), presented as thyroid aplasia, hypoplasia or ectopia, is one of the most prevalent rare diseases with an isolated organ malformation. The pathogenesis of TD is largely unknown, although a genetic predisposition has been suggested. We performed a genome-wide association study (GWAS) with 142 Japanese TD cases and 8380 controls and found a significant locus at 2q33.3 (top single nucleotide polymorphism, rs9789446: P = 4.4 × 10-12), which was replicated in a German patient cohort (P = 0.0056). A subgroup analysis showed that rs9789446 confers a risk for thyroid aplasia (per allele odds ratio = 3.17) and ectopia (3.12) but not for hypoplasia. Comprehensive epigenomic characterization of the 72-kb disease-associated region revealed that it was enriched for active enhancer signatures in human thyroid. Analysis of chromosome conformation capture data showed long-range chromatin interactions of this region with promoters of two genes, FZD5 and CCNYL1, mediating Wnt signaling. Moreover, rs9789446 was found to be a thyroid-specific quantitative trait locus, adding further evidence for a cis-regulatory function of this region in thyroid tissue. Specifically, because the risk rs9789446 allele is associated with increased thyroidal expression of FDZ5 and CCNYL1 and given the recent demonstration of perturbed early thyroid development following overactivation of Wnt signaling in zebrafish embryos, an enhanced Wnt signaling in risk allele carriers provides a biologically plausible TD mechanism. In conclusion, our work found the first risk locus for TD, exemplifying that in rare diseases with relatively low biological complexity, GWAS may provide mechanistic insights even with a small sample size.

Interference with connective tissue growth factor attenuated fibroblast-to-myofibroblast transition and pulmonary fibrosis.

BackgroundThe aberrant activation and phenotype shift of resident fibroblasts in lung tissues via fibroblast-to-myofibroblast transition (FMT) is considered a pivotal step in pulmonary fibrogenesis, resulting in excessive extracellular matrix (ECM) production and deposition. However, the molecular mechanisms regulating FMT and lung fibrosis are still unclear. Connective tissue growth factor (CTGF) has been reported to be both an ECM protein and a versatile signaling molecule that is involved in multiple pathophysiological contexts, especially fibrosis. The relationship between CTGF, FMT, and lung fibrosis has not yet been well defined.MethodsIn this study, a pulmonary fibrosis (PF) rat model and FMT cell model induced by paraquat (PQ) were established to explore the relevant regulatory mechanisms in vivo and in vitro.ResultsThe results showed that the CTGF was highly activated and was a mediator of canonical Wnt signaling during FMT and PF. The inhibition of the CTGF by small-interfering ribonucleic acid decreased the expression of FMT markers, including α-smooth muscle actin, vimentin, and collagen I, inhibited the activated Wnt signaling pathway, and ameliorated lung fibrosis.ConclusionsOur findings showed that CTGF was the key effector of the FMT and fibrotic changes, and emphasized the therapeutic potential of the inhibitor or monoclonal antibody against CTGF for PF.

β-catenin regulates HIV latency and modulates HIV reactivation.

Latency is the main obstacle towards an HIV cure, with cure strategies aiming to either elicit or prevent viral reactivation. While these strategies have shown promise, they have only succeeded in modulating latency in a fraction of the latent HIV reservoir, suggesting that the mechanisms controlling HIV latency are not completely understood, and that comprehensive latency modulation will require targeting of multiple latency maintenance pathways. We show here that the transcriptional co-activator and the central mediator of canonical Wnt signaling, β-catenin, inhibits HIV transcription in CD4+ T cells via TCF-4 LTR binding sites. Further, we show that inhibiting the β-catenin pathway reactivates HIV in a primary TCM cell model of HIV latency, primary cells from cART-controlled HIV donors, and in CD4+ latent cell lines. β-catenin inhibition or activation also enhanced or inhibited the activity of several classes of HIV latency reversing agents, respectively, in these models, with significant synergy of β-catenin and each LRA class tested. In sum, we identify β-catenin as a novel regulator of HIV latency in vitro and ex vivo, adding new therapeutic targets that may be combined for comprehensive HIV latency modulation in HIV cure efforts.

Benzo(a)pyrene induces airway epithelial injury through Wnt5a-mediated non-canonical Wnt-YAP/TAZ signaling

Wnt5a is a key mediator of non-canonical Wnt signaling, and an early indicator of epithelial injury and lung dysfunction. Polycyclic aromatic hydrocarbons (PAHs) could induce acute pulmonary pathogenesis, of which the underlying mechanism remains unclear. To elucidate the potential role of Wnt5a-mediated non-canonical Wnt-YAP/TAZ signaling in the lung injury induced by short-term exposure of benzo(a)pyrene (BaP, a representative PAHs), intratracheally instilled mouse model was used and further interfered with its Wnt5a level by small molecule antagonists and agonists. Our data revealed that BaP exposure induced the lung inflammatory response and reduced the expression of Clara cell secretory protein (CC16) in a dose-dependent manner. More importantly, the activation of Wnt5a and downstream YAP/TAZ were accompanied with the enhanced release of epithelial-derived thymic stromal lymphopoietin and interleukin-33, which acted as pro-inflammatory cytokines. Functionally, inhibition of Wnt5a attenuated the BaP-induced inflammation and recuperated CC16 expression, as well as suppressed the epithelial cytokines release. Whereas promoting Wnt5a expression affected the toxic effects of BaP oppositely. Our findings together suggest that Wnt5a is a potential endogenous regulator in lung inflammation and airway epithelial injury, and Wnt5a-YAP/TAZ signaling contributes to lung dysfunction in acute exposure to BaP.

REGγ drives Lgr5+ stem cells to potentiate radiation induced intestinal regeneration.

Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), a marker of intestinal stem cells (ISCs), is considered to play key roles in tissue homoeostasis and regeneration after acute radiation injury. However, the activation of Lgr5 by integrated signaling pathways upon radiation remains poorly understood. Here, we show that irradiation of mice with whole-body depletion or conditional ablation of REGγ in Lgr5+ stem cell impairs proliferation of intestinal crypts, delaying regeneration of intestine epithelial cells. Mechanistically, REGγ enhances transcriptional activation of Lgr5 via the potentiation of both Wnt and Hippo signal pathways. TEAD4 alone or cooperates with TCF4, a transcription factor mediating Wnt signaling, to enhance the expression of Lgr5. Silencing TEAD4 drastically attenuated β-catenin/TCF4 dependent expression of Lgr5. Together, our study reveals how REGγ controls Lgr5 expression and expansion of Lgr5+ stem cells in the regeneration of intestinal epithelial cells. Thus, REGγ proteasome appears to be a potential therapeutic target for radiation-induced gastrointestinal disorders.

β-Catenin Restricts Zika Virus Internalization by Downregulating Axl.

The latest outbreak of Zika virus (ZIKV) in the Americas was associated with significant neurologic complications, including microcephaly of newborns. We evaluated mechanisms that regulate ZIKV entry into human fetal astrocytes (HFAs). Astrocytes are key players in maintaining brain homeostasis. We show that the central mediator of canonical Wnt signaling, β-catenin, regulates Axl, a receptor for ZIKV infection of HFAs, at the transcriptional level. In turn, ZIKV inhibited β-catenin, potentially as a mechanism to overcome its restriction of ZIKV internalization through regulation of Axl. This was evident with three ZIKV strains tested but not with a laboratory-adapted strain which has a large deletion in its envelope gene. Finally, we show that β-catenin-mediated Axl-dependent internalization of ZIKV may be of increased importance for brain cells, as it regulated ZIKV infection of astrocytes and human brain microvascular cells but not kidney epithelial (Vero) cells. Collectively, our studies reveal a role for β-catenin in ZIKV infection and highlight a dynamic interplay between ZIKV and β-catenin to modulate ZIKV entry into susceptible target cells. IMPORTANCE ZIKV is an emerging pathogen with sporadic outbreaks throughout the world. The most recent outbreak in North America was associated with small brains (microcephaly) in newborns. We studied the mechanism(s) that may regulate ZIKV entry into astrocytes. Astrocytes are a critical resident brain cell population with diverse functions that maintain brain homeostasis, including neurogenesis and neuronal survival. We show that three ZIKV strains (and not a heavily laboratory-adapted strain with a large deletion in its envelope gene) require Axl for internalization. Most importantly, we show that β-catenin, the central mediator of canonical Wnt signaling, negatively regulates Axl at the transcriptional level to prevent ZIKV internalization into human fetal astrocytes. To overcome this restriction, ZIKV downregulates β-catenin to facilitate Axl expression. This highlights a dynamic host-virus interaction whereby ZIKV inhibits β-catenin to promote its internalization into human fetal astrocytes through the induction of Axl.

Transcriptomic Analysis of Peripheral Monocytes upon Fingolimod Treatment in Relapsing Remitting Multiple Sclerosis Patients.

Fingolimod (FTY), a second-line oral drug approved for relapsing remitting Multiple Sclerosis (RRMS) acts in preventing lymphocyte migration outside lymph nodes; moreover, several lines of evidence suggest that it also inhibits myeloid cell activation. In this study, we investigated the transcriptional changes induced by FTY in monocytes in order to better elucidate its mechanism of action. CD14+ monocytes were collected from 24 RRMS patients sampled at baseline and after 6months of treatment and RNA profiles were obtained through next-generation sequencing. We conducted pathway and sub-paths analysis, followed by centrality analysis of cell-specific interactomes on differentially expressed genes (DEGs). We investigated also the predictive role of baseline monocyte transcription profile in influencing the response to FTY therapy. We observed a marked down-regulation effect (60 down-regulated vs. 0 up-regulated genes). Most of the down-regulated DEGs resulted related with monocyte activation and migration like IL7R, CCR7 and the Wnt signaling mediators LEF1 and TCF7. The involvement of Wnt signaling was also confirmed by subpaths analyses. Furthermore, pathway and network analyses showed an involvement of processes related to immune function and cell migration. Baseline transcriptional profile of the HLA class II gene HLA-DQA1 and HLA-DPA1 were associated with evidence of disease activity after 2years of treatment. Our data support the evidence that FTY induces major transcriptional changes in monocytes, mainly regarding genes involved in cell trafficking and immune cell activation. The baseline transcriptional levels of genes associated with antigen presenting function were associated with disease activity after 2years of FTY treatment.