Wnt Gene Expression Research Articles

Combinatorial Wnt signaling landscape during brachiopod anteroposterior patterning

BackgroundWnt signaling pathways play crucial roles in animal development. They establish embryonic axes, specify cell fates, and regulate tissue morphogenesis from the early embryo to organogenesis. It is becoming increasingly recognized that these distinct developmental outcomes depend upon dynamic interactions between multiple ligands, receptors, antagonists, and other pathway modulators, consolidating the view that a combinatorial “code” controls the output of Wnt signaling. However, due to the lack of comprehensive analyses of Wnt components in several animal groups, it remains unclear if specific combinations always give rise to specific outcomes, and if these combinatorial patterns are conserved throughout evolution.ResultsIn this work, we investigate the combinatorial expression of Wnt signaling components during the axial patterning of the brachiopod Terebratalia transversa. We find that T. transversa has a conserved repertoire of ligands, receptors, and antagonists. These genes are expressed throughout embryogenesis but undergo significant upregulation during axial elongation. At this stage, Frizzled domains occupy broad regions across the body while Wnt domains are narrower and distributed in partially overlapping patches; antagonists are mostly restricted to the anterior end. Based on their combinatorial expression, we identify a series of unique transcriptional subregions along the anteroposterior axis that coincide with the different morphological subdivisions of the brachiopod larval body. When comparing these data across the animal phylogeny, we find that the expression of Frizzled genes is relatively conserved, whereas the expression of Wnt genes is more variable.ConclusionsOur results suggest that the differential activation of Wnt signaling pathways may play a role in regionalizing the anteroposterior axis of brachiopod larvae. More generally, our analyses suggest that changes in the receptor context of Wnt ligands may act as a mechanism for the evolution and diversification of the metazoan body axis.

Transcriptomic Analysis of Pubertal and Adult Virgin Mouse Mammary Epithelial and Stromal Cell Populations

Conflicting data exist as to how mammary epithelial cell proliferation changes during the reproductive cycle. To study the effect of endogenous hormone fluctuations on gene expression in the mouse mammary gland, we performed bulk RNAseq analyses of epithelial and stromal cell populations that were isolated either during puberty or at different stages of the adult virgin estrous cycle. Our data confirm prior findings that proliferative changes do not occur in every mouse in every cycle. We also show that during the estrous cycle the main gene expression changes occur in adipocytes and fibroblasts. Finally, we present a comprehensive overview of the Wnt gene expression landscape in different mammary gland cell types in pubertal and adult mice. This work contributes to understanding the effects of physiological hormone fluctuations and locally produced signaling molecules on gene expression changes in the mammary gland during the reproductive cycle and should be a useful resource for future studies investigating gene expression patterns in different cell types across different developmental timepoints.

Assessment of single and combined administration of ubiquinone and lactoferrin on histopathology, ultrastructure, oxidative stress, and WNT4 expression gene induced by thioacetamide on hepatorenal system of adult male rats

BackgroundHepatorenal syndrome is a life-threatening medical complication of liver cirrhosis. Hepatic cirrhosis is commonly accompanied by rapid failure of renal functions. Thioacetamide (TAA) is a potent hepatotoxin and a class 2-type carcinogen. Ubiquinone (Coq10) and lactoferrin (LF) are potent antioxidants with antifibrotic and antiinflammatory effects. However, whether Coq10 and LF reduce the hepatorenal injury induced by TAA remains unclear. Here, we investigated the potential protective effect of both/or Coq10 and LF in ameliorating TAA-induced hepatorenal injury and the role of WNT4 gene expression in detecting TAA-induced renal injury in rats. Seventy healthy and mature male Sprague Dawley rats, weighting (200 g ± 20 g) and aging (4–6) weeks were randomly divided into seven groups (n = 10): control, Coq10, LF, TAA, TAA + Coq10, TAA + LF, and TAA + Coq10 + LF. The hepatorenal injury was induced through intraperitoneal (i.p.) injection of TAA (150 mg/kg/twice/weekly) for nine weeks. Coq10 (10 mg/kg/day) and LF (200 mg/kg/day) were orally administered for nine weeks.ResultsTAA induced marked hepatorenal damage, evident by the significant increase in the alanine aminotransferase (ALT), aspartate transaminase (AST), serum creatinine (SCr) activities, and the blood urea nitrogen (BUN) level. Besides, the significant increases in concentrations of malondialdehyde (MDA) and nitric oxide (NOx) together with significant decreases in the activities of catalase (CAT) and superoxide dismutase (SOD). The histopathological analysis of the TAA group showed obvious fibrosis, steatosis, and inflammation of the hepatic parenchyma as well as severe glomerular and tubular damage of the renal parenchyma. In addition, TAA induced marked ultrastructural alterations and up-regulation in the expression of the WNT4 gene in the kidney. Meanwhile, the biochemical, histopathological, and ultrastructural alterations were significantly decreased with significant down-regulation in the expression of WNT4 in the groups exposed to TAA and treated with Coq10 and LF.ConclusionOur data suggested that Coq10 and LF could have protective effects on TAA hepatorenal damage, through improving the hepatic and renal functions, reduction of oxidative stress, structural and ultrastructural alterations, besides down-regulation in the expression of WNT4.

P148 CCN3-derived peptides as anti-fibrotic therapies for systemic sclerosis

Abstract Background/Aims Effective therapy for skin fibrosis in scleroderma (systemic sclerosis: SSc) remains an unmet clinical challenge. Persistently activated M2 macrophages are believed to stimulate myofibroblasts in the disease microenvironment creating a pro-fibrotic niche in the skin. Expression of the anti-fibrotic matricellular protein CCN3 is reduced in animal models of fibrosis. We have identified a small peptide based on an amino acid sequence in CCN3, BLR-200, which mimics the anti-fibrotic activity of CCN3, and propose that BLR-200 could be used to treat patients in whom lack of CCN3 activity is permissive for fibrosis. However, whether there is lack of CCN3 activity in SSc and whether BLR-200 has effective anti-fibrotic activity in SSc are unknown. Methods We used ELISAs (R&D Systems) to detect CCN3 and the M2 macrophage marker CD206 in the serum of scleroderma patients with early stage diffuse subset disease (within two years of disease onset), those with late-stage diffuse subset disease (over five years duration) as well as healthy controls (all n = 20). The bleomycin-induced mouse model of skin scleroderma, in which bleomycin is injected subcutaneously every day for 21 days, was used to assess the antifibrotic ability of BLR200 in vivo. Results In scleroderma serum, CCN3 was significantly reduced, relative to matched healthy controls and patients with inactive disease, in early onset scleroderma patients with active disease that show elevated CD206 levels (p < 0.01). In the mouse model of human SSc, compared to injection with a scrambled control peptide and relative to control mice treated with phosphate buffered saline, injection with BLR-200 prevented bleomycin-induced changes in collagen deposition, myofibroblast differentiation and skin thickness as measured by Trichrome stain, indirect immunofluorescence analysis with an anti-a-smooth muscle actin antibody, and morphometric analysis, respectively (N = 8 mice per group, p < 0.05). Similarly, RNA sequencing and real-time polymerase chain reaction analysis revealed that BLR-200 significantly impaired the ability of bleomycin to induce expression of fibrogenic genes such as: integrin alpha 11, CCN2, CCN1, Smad3, wnt4, YAP1 and tenascin-C (all N = 5, p < 0.05). Spatial transcriptomics analysis revealed that BLR-200 impaired bleomycin-induced alterations in skin cell populations, including the activation and expansion of epithelial and reticular fibroblast niches and the induction of Wnt, hippo, focal adhesion and actin cytoskeleton gene expression cluster, all of which are known to be activators of fibrosis and myofibroblast activation and persistence. Conclusion CCN3 expression is reduced in serum of scleroderma patients with active skin disease that show M2 macrophage activation as visualized by elevated CD206 levels. Since BLR-200 has antifibrotic activity in the bleomycin model of skin scleroderma, our data are consistent with the hypothesis that BLR-200 could be used to block fibrosis progression in early onset diffuse scleroderma patients who possess both low CCN3 and high CD206 in serum. Disclosure J. Nguyen: None. K. Zestranjyan: None. S. Xu: None. B.L. Riser: Corporate appointments; CEO of BLR Bio. R.J. Stratton: None. A. Leask: None.

Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL).

Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level.

Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature

PurposeOvarian tissue cryopreservation is vital for fertility preservation, yet its effect on ovarian tissue follicle survival and transcriptomic signature requires further investigation. This study delves into the effects of vitrification on tissue morphology, function, and transcriptomic changes, helping to find possibilities for vitrification protocol improvements.MethodsOvarian cortex from 19 bovine animals were used to conduct pre- and post-vitrification culture followed by histological assessment, immunohistochemistry, and TUNEL assay. Follicles’ functionality was assessed for viability and growth within the tissue and in isolated cultures. RNA-sequencing of ovarian tissue was used to explore the transcriptomic alterations caused by vitrification.ResultsFollicle density, cell proliferation, and DNA damage in ovarian stroma were unaffected by vitrification. However, vitrified cultured tissue exhibited reduced follicle density of primordial/primary and antral follicles, while freshly cultured tissue manifested reduction of antral follicles. Increased stromal cell proliferation and DNA damage occurred in both groups post-culture. Isolated follicles from vitrified tissue exhibited similar viability to fresh follicles until day 4, after which the survival dropped. RNA-sequencing revealed minor effects of vitrification on transcriptomic signatures, while culture induced significant gene expression changes in both groups. The altered expression of WNT and hormonal regulation pathway genes post-vitrification suggests the molecular targets for vitrification protocol refinement.ConclusionVitrification minimally affects tissue morphology, follicle density, and transcriptomic signature post-thawing. However, culture revealed notable changes in vitrified tissue samples, including reduced follicle density, decreased isolated follicle survival, and alteration in WNT signalling and ovarian hormonal regulation pathways, highlighted them as possible limitations of the current vitrification protocol.

Investigation of the effect of belinostat on MCF-7 breast cancer stem cells via the Wnt, Notch, and Hedgehog signaling pathway.

To evaluate belinostat's (PXD101) activity on MCF-7 breast cancer stem cells (CSCs) via Wnt, Notch, and Hedgehog. This research study was carried out at the Department of Medical Biology, Necmettin Erbakan University, Konya, Turkey, from June 2017 to July 2019. The effect of PXD101 on MCF-7 cell viability was determined by cell proliferation kit (XTT). Following belinostat treatment, CD44+/CD24- MCF-7 CSCs were isolated by FACS. Ribonucleic acid isolation and copy-deoxyribonucleic acid synthesis were carried out using HEK-293 cells, MCF-7 cells, and MCF-7 CSCs. Expression changes of metastasis-related genes, Wnt, Hedgehog, Notch, and stem cell markers were analysed by quantitative polymerase chain reaction. The IC50 in MCF-7 cancer cells was 5 μM for 48 hours. The FACS analysis indicated that 2% of the MCF-7 cancer cells were CSCs. Following belinostat treatment, the MCF-7 cell count decreased by 44%, and the MCF-7 CD44+/CD24- CSC count decreased by 66%. Belinostat treatment reduced the expression of metastasis, Wnt, Notch, Hedgehog, and stem cell marker genes. Belinostat has a potential effect on the differentiation and self-renewal of breast CSCs.

Prenatal Exposure to COVID-19 mRNA Vaccine BNT162b2 Induces Autism-Like Behaviors in Male Neonatal Rats: Insights into WNT and BDNF Signaling Perturbations

The COVID-19 pandemic catalyzed the swift development and distribution of mRNA vaccines, including BNT162b2, to address the disease. Concerns have arisen about the potential neurodevelopmental implications of these vaccines, especially in susceptible groups such as pregnant women and their offspring. This study aimed to investigate the gene expression of WNT, brain-derived neurotrophic factor (BDNF) levels, specific cytokines, m-TOR expression, neuropathology, and autism-related neurobehavioral outcomes in a rat model. Pregnant rats received the COVID-19 mRNA BNT162b2 vaccine during gestation. Subsequent evaluations on male and female offspring included autism-like behaviors, neuronal counts, and motor performance. Molecular techniques were applied to quantify WNT and m-TOR gene expressions, BDNF levels, and specific cytokines in brain tissue samples. The findings were then contextualized within the extant literature to identify potential mechanisms. Our findings reveal that the mRNA BNT162b2 vaccine significantly alters WNT gene expression and BDNF levels in both male and female rats, suggesting a profound impact on key neurodevelopmental pathways. Notably, male rats exhibited pronounced autism-like behaviors, characterized by a marked reduction in social interaction and repetitive patterns of behavior. Furthermore, there was a substantial decrease in neuronal counts in critical brain regions, indicating potential neurodegeneration or altered neurodevelopment. Male rats also demonstrated impaired motor performance, evidenced by reduced coordination and agility. Our research provides insights into the effects of the COVID-19 mRNA BNT162b2 vaccine on WNT gene expression, BDNF levels, and certain neurodevelopmental markers in a rat model. More extensive studies are needed to confirm these observations in humans and to explore the exact mechanisms. A comprehensive understanding of the risks and rewards of COVID-19 vaccination, especially during pregnancy, remains essential.

Molecular and cellular regulation on sexual fate and gonadal development in hermaphrodite yellowfin seabream Acanthopagrus latus

Yellowfin seabream (Acanthopagrus latus) is a hermaphroditic fish with male prematurity, which is also an economically important mariculture species in coastal areas of China. Although the production yield has been increasing in recent years, artificial breeding of this species remains imperfect due to the lack understanding of its sex differentiation mechanism. In this study, we conducted transcriptomic sequencing to screen for sex-related genes in different stage of the testis, ovotestis, ovary, hypothalamus, and pituitary tissues of yellowfin seabream. During gonad development (from intersex to mature stage), Gnrh3, Kiss2, and Dbh were expressed in the hypothalamus and Lhb and Fshb were expressed in the pituitary. Sex-related genes such as Hsd11b1, Foxl2l, Amh, and Gsdf were among the top 10 downregulated genes in the intersex testis compared with the mature testis. The expression of Hsd11b, Wnt, and Sox families genes differed significantly in the sex change process among functional mature gonads. In addition, treatment with estradiol resulted in feminizing of the ovotestis. Inhibition of androgens or estrogens promoted ovarian or testicular steps of ovotestis development. In summary, we systematically evaluated gene expression profiles during yellowfin seabream gonad development from intersex to functional male or female. Our results confirmed that regulation by the hypothalamus-pituitary-gonad (HPG) axis and the promotion of sex steroid hormones acted on this process, and they provide a reference for further studies of the involvement of the HPG axis in sex change and of sex induction methods in yellowfin seabream.

Wnt1 gene expression in the heart left ventricle as a response to the various durations of the intensive exercise: An experimental study.

Objective. Myocardial fibrosis is a devastating condition causing millions of deaths yearly. Several factors, such as aging, cause myocardial fibrosis. The Wnt/β-catenin pathway is one of the critical intracellular signaling for the development of cardiac fibrosis. Molecular and cellular mechanism of myocardial fibrosis induced by intensive exercise is not well-understood. The current study evaluates the effects of short- and long-term intensive exercise on the Wnt1 gene expression in a heart left ventricle in an animal model. Methods. Twenty-one male Wistar rats (mean weight 250±50 g) were divided into three groups (n=7): 1) control group (C); 2) short-term regular intensive exercise group (S-RIE, high-intensity exercise for one month six days weekly for 60 min with speed of 35 m/min), and 3) long-term regular intensive exercise group (L-RIE, high-intensity exercise for six months six days daily for 60 min with speed of 35 m/min). The heart left ventricle was isolated at the end of the experiment, and the relative gene expression of the Wnt1 gene was measured by the Real-Time PCR. Results. The L-RIE group showed a significant increase in the Wnt1 expression compared to the S-RIE and the control group. Although no difference was observed in the Wnt1 mRNA level in the S-RIE group compared to the control group, Wnt1 mRNA level increased in the L-RIE group compared to the S-RIE group. Conclusion. The exercise duration was of a great importance in the Wnt1 gene expression. Regular intensive exercise may be involved in the formation of the myocardial fibrosis by increasing the expression of the Wnt1 gene.

Knock-in of a 3’ UTR Stop Cassette into the Wnt4 locus increases mRNA expression and leads to ovarian cyst formation

Wnt4 signaling is critical for mammalian female sex determination, in female reproductive organ development, in follicular and oocyte maturation, and in steroid hormone production. When Wnt4 function is impaired, female embryos undergo partial female to male sex-reversal. This phenotype is associated with the expression of a set of somatic genes that are typical for the male differentiation pathways such as those of the Leydig cells. Given the roles of the 3`untranslated region (3`UTR) in control of gene expression, we addressed whether a knock-in of a stop cassette to 3`END of the Wnt4 gene would impact female reproductive system development or function. The 3`UTRstop cassette indeed affected Wnt4 gene expression in vivo so that the respective mRNA was upregulated in the ovaries of a three month-old female. The homozygous Wnt4 3`UTRstop mice were noted to be leaner than their wild type (WT) littermate controls. Analysis of the ovarian follicular count at the age of three months revealed increased pre-antral but reduced ovarian corpus luteum follicular counts. Furthermore, two out of five of the homozygous female Wnt4 3`UTRstop mice had ovarian cysts, not noted in WT controls. RT-qPCR and in situ hybridization analysis depicted changes in the expression of a panel of genes which encode enzymes that mediate the synthesis of female steroid hormones or their receptors due to the Wnt4 3`UTRstop knock-in. Thus, female mice which had the homozygous construct exhibited elevated ovarian Wnt4 mRNA expression and the corresponding knock-in was associated with changes in ovarian development and folliculogenesis. Our data reinforce the conclusion that deregulated Wnt4 expression impacts female sex organogenesis, ovary development and function, and that the Wnt4 3`UTRstop knock-in mouse provides a model to explore in more detail the roles of Wnt4 signaling in the process.

WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

Basal-like breast cancer (BBC) and glioblastoma multiforme (GBM) are aggressive cancers associated with poor prognosis. BBC and GBM have stem cell-like gene expression signatures, which are in part driven by forkhead box O (FOXO) transcription factors. To gain further insight into the impact of FOXO1 in BBC, we treated BT549 cells with AS1842856 and performed RNA sequencing. AS1842856 binds to unphosphorylated FOXO1 and inhibits its ability to directly bind to DNA. Gene Set Enrichment Analysis indicated that a set of WNT pathway target genes, including lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 7 (TCF7), were robustly induced after AS1842856 treatment. These same genes were also induced in GBM cell lines U87MG, LN18, LN229, A172, and DBTRG upon AS1842856 treatment. By contrast, follow-up RNA interference (RNAi) targeting of FOXO1 led to reduced LEF1 and TCF7 gene expression in BT549 and U87MG cells. In agreement with RNAi experiments, CRISPR Cas9-mediated FOXO1 disruption reduced the expression of canonical WNT genes LEF1 and TCF7 in U87MG cells. The loss of TCF7 gene expression in FOXO1 disruption mutants was restored by exogenous expression of the DNA-binding-deficient FOXO1-H215R. Therefore, FOXO1 induces TCF7 in a DNA-binding-independent manner, similar to other published FOXO1-activated genes such as TCF4 and hes family bHLH transcription factor 1. Our work demonstrates that FOXO1 promotes canonical WNT gene expression in examined BBC and GBM cells, similar to results found in Drosophila melanogaster, T-cell development, and murine acute myeloid leukemia models.

Identification of Potential Therapeutic Targets for Plasmablastic Lymphoma Through Gene Expression Analysis: Insights into RAS and Wnt Signaling Pathways

Plasmablastic lymphoma (PBL) is a rare and aggressive B-cell lymphoma with overlapping characteristics with diffuse large B-cell lymphoma (DLBCL) and multiple myeloma. Hyperactive Wnt signaling derails homeostasis and promotes oncogenesis and chemoresistance in DLBCL and multiple myeloma. Evidence suggests active cross-talk between the Wnt and RAS pathways impacting metastasis in solid cancers in which combined targeted therapies show effective results. Recent genomic studies in PBL demonstrated a high frequency of mutations linked with the RAS signaling pathway. However, the role of RAS and Wnt signaling pathway molecule expression in PBL remained unknown. We examined the expression of Wnt and RAS pathway-related genes in a well-curated cohort of PBL. Because activated B cells are considered immediate precursors of plasmablasts in B cell development, we compared this data with activated B-cell type DLBCL (ABC-DLBCL) patients, employing NanoString transcriptome analysis (770 genes). Hierarchical clustering revealed distinctive differential gene expression between PBL and ABC-DLBCL. Gene set enrichment analysis labeled the RAS signaling pathway as the most enriched (37 genes) in PBL, including upregulating critical genes, such as NRAS, RAF1, SHC1, and SOS1. Wnt pathway genes were also enriched (n = 22) by gene set enrichment analysis. Molecules linked with Wnt signaling activation, such as ligands or targets (FZD3, FZD7, c-MYC, WNT5A, WNT5B, and WNT10B), were elevated in PBL. Our data also showed that, unlike ABC-DLBCL, the deranged Wnt signaling activity in PBL was not linked with hyperactive nuclear factor κB and B-cell receptor signaling. In divergence, Wnt signaling inhibitors (CXXC4, SFRP2, and DKK1) also showed overexpression in PBL. The high expression of RAS signaling molecules reported may indicate linkage with gain–in-function RAS mutations. In addition, high expression of Wnt and RAS signaling molecules may pave pathways to explore benefiting from combined targeted therapies, as reported in solid cancer, to improve prognosis in PBL patients.

Экспрессия генов семейства WNT у больных множественной миеломой с различным ответом на противоопухолевую терапию

Aim. To compare the expression levels of the WNT family genes in mesenchymal stromal cells (MSC) of the bone marrow (BM) hematopoietic niche in multiple myeloma (MM) patients vs. healthy donors.
 Materials & Methods. The study enrolled 12 MM patients aged 49–71 years (the median age 61 years) after standard induction bortezomib therapy. The treatment efficacy was assessed in accordance with the criteria of International Myeloma Working Group (IMWG). Patients were stratified in groups with complete and partial response (CPR; group 1, n = 9) and no response (group 2, n = 3). Besides, a group of primary untreated patients was formed (n = 2). The control group included healthy donors of BM (n = 3). The levels of the WNT and CTNNB1 gene expression were assessed by real-time PCR on cDNA isolated from MSC.
 Results. In the group of 2 primary patients, two genes (WNT2B and WNT9B) considerably differed in the degree of expression. In non-responders (n = 3), the WNT2B expression could not be determined, whereas the WNT15 expression appeared to be increased. In group CPR (n = 9), mRNA level of the WNT5A gene increased after therapy, whereas the WNT3A gene expression returned to the normal level. The WNT7B gene transcription level did not differ in the control and comparison groups. In group CPR, a significant expression increase in the β-catenin-coding CTNNB1 gene was detected.
 Conclusion. The differences identified in the expression of the WNT2B, WNT9B, and CTNNB1 genes suggest the possibility of their use as prognostic molecular markers in MM.

Oncogenic and Receptor-Mediated Wnt Signaling Influence the Sensitivity of Colonic Cells to Butyrate.

Deregulated Wnt signaling is responsible for most cases of colorectal cancer (CRC). Dietary fiber is protective against CRC and this activity is likely mediated by butyrate, a breakdown product of dietary fiber that hyperactivates Wnt signaling, repressing CRC proliferation and inducing apoptosis. Receptor-mediated Wnt signaling and oncogenic Wnt signaling, which is typically initiated by mutation in more downstream elements of the pathway, activate non-overlapping patterns of gene expression. Receptor-mediated signaling is associated with a poor prognosis for CRC while oncogenic signaling is associated with a relatively good prognosis. We have compared the expression of genes differentially expressed in receptor-mediated vs. oncogenic Wnt signaling to microarray data generated in our laboratory. Most importantly we evaluated these gene expression patterns comparing the early stage colon microadenoma line LT97 with the metastatic CRC cell line SW620. LT97 cells exhibit a gene expression pattern more strongly associated with that observed with oncogenic Wnt signaling, while SW620 cells exhibit a gene expression pattern moderately associated with that observed with receptor-mediated Wnt signaling. Given that SW620 cells are more advanced and malignant compared to LT97 cells, these findings are generally consistent with the better prognosis observed with tumors exhibiting a more oncogenic Wnt gene expression pattern. Importantly, LT97 cells are more sensitive to the effects of butyrate on proliferation and apoptosis that are CRC cells. We further examine these gene expression patterns in butyrate-resistant vs. butyrate-sensitive CRC cells. Based upon all of these observations, we hypothesize that colonic neoplastic cells exhibiting a more oncogenic as compared to receptor-mediated Wnt signaling gene expression pattern would be more sensitive to the effects of butyrate, and, hence, fiber, than are those cells exhibiting a more receptor-mediated Wnt signaling pattern of expression. Diet-derived butyrate may affect the differential patient outcomes resulting from the two types of Wnt signaling. We further posit that development of butyrate resistance and concomitant changes in Wnt signaling patterns, including associations with CBP and p300, disrupts the association between the two major types of Wnt signaling (receptor-mediated and oncogenic) and neoplastic progression/prognosis. Ideas about testing the hypothesis and therapeutic implications are briefly considered.

Berberine increases stromal production of Wnt molecules and activates Lgr5+ stem cells to promote epithelial restitution in experimental colitis

BackgroundInflammatory bowel diseases (IBDs) are characterized by sustained inflammation and/or ulcers along the lower digestive tract, and have complications such as colorectal cancer and inflammation in other organs. The current treatments for IBDs, which affect 0.3% of the global population, mainly target immune cells and inflammatory cytokines with a success rate of less than 40%.ResultsHere we show that berberine, a natural plant product, is more effective than the frontline drug sulfasalazine in treating DSS (dextran sulfate sodium)-induced colitis in mice, and that berberine not only suppresses macrophage and granulocyte activation but also promotes epithelial restitution by activating Lgr5+ intestinal stem cells (ISCs). Mechanistically, berberine increases the expression of Wnt genes in resident mesenchymal stromal cells, an ISC niche, and inhibiting Wnt secretion diminishes the therapeutic effects of berberine. We further show that berberine controls the expression of many circadian rhythm genes in stromal cells, which in turn regulate the expression of Wnt molecules.ConclusionsOur findings suggest that berberine acts on the resident stromal cells and ISCs to promote epithelial repair in experimental colitis and that Wnt-β-Catenin signaling may be a potential target for colitis treatment.

Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor β1 (TGFβ) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors.β-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin-positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.

A Novel Single-Nucleotide Polymorphism in WNT4 Promoter Affects Its Transcription and Response to FSH in Chicken Follicles.

The signaling pathway of the wingless-type mouse mammary tumor virus integration site (Wnt) plays an important role in ovarian and follicular development. In our previous study, WNT4 was shown to be involved in the selection and development of chicken follicles by upregulating the expression of follicle-stimulating hormone receptors (FSHR), stimulating the proliferation of follicular granulosa cells, and increasing the secretion of steroidal hormones. FSH also stimulates the expression of WNT4. To further explore the molecular mechanism by which FSH upregulates WNT4 and characterize the cis-elements regulating WNT4 transcription, in this study, we determined the critical regulatory regions affecting chicken WNT4 transcription. We then identified a single-nucleotide polymorphism (SNP) in this region, and finally analyzed the associations of the SNP with chicken production traits. The results showed that the 5′ regulatory region from −3354 to −2689 of WNT4 had the strongest activity and greatest response to FSH stimulation, and we identified one SNP site in this segment, −3015 (G > C), as affecting the binding of NFAT5 (nuclear factor of activated T cells 5) and respones to FSH stimulation. When G was replaced with C at this site, it eliminated the NFAT5 binding. The mRNA level of WNT4 in small yellow follicles of chickens with genotype GG was significantly higher than that of the other two genotypes. Moreover, this locus was found to be significantly associated with comb length in hens. Individuals with the genotype CC had longer combs. Collectively, these data suggested that SNP−3015 (G > C) is involved in the regulation of WNT4 gene expression by responding FSH and affecting the binding of NFAT5 and that it is associated with chicken comb length. The current results provide a reference for further revealing the response mechanism between WNT and FSH.

Comprehensive Genome-Wide Analysis of Wnt Gene Family and Expression Profiling during Limb Regeneration in Portunus trituberculatus

Wnt genes encode a family of secretory glycoproteins that are involved in various stages of organ development through regulation of cell proliferation, differentiation, and growth. Recently, Wnt genes have been shown to play an important role in regeneration processes. However, there have been no previous genome-wide analyses of the Wnt gene family members in crab species. In this study, a total of 13 Wnt family genes were identified from the genome of the swimming crab Portunus trituberculatus and classified into three main groups based on the conserved domain, protein sequence, and motifs. Chromosome location analysis showed that tandem duplication may have resulted in the expansion of the PtWnt gene family. RNA-seq results indicated that most PtWnt genes had a higher Fragments Per Kilobase of transcript sequences per Million base pairs (FPKM) value in the regenerating limb bud muscle than in the normal limb muscle, and the genes were enriched in a number of pathways that had biological functions underlying limb regeneration such as the cell surface receptor signaling pathway, the hippo signaling pathway, receptor binding, and basal cell carcinoma. Moreover, quantitative real-time PCR (RT-qPCR) analysis confirmed that the expression levels of nine PtWnts except for PtWnt1, PtWnt2, and PtWnt6 had a consistently increasing trend during limb regeneration. PtWnt1, PtWnt2, and PtWnt6 exhibited significantly up- or down-regulated expression at different limb regeneration stages. These results provide valuable information for further evolutionary and functional characterization of PtWnt genes and indicate the complexity and specialized mechanism of the Wnt signaling pathway regulating regeneration in crustaceans.