Expression Of Wnt Signaling Proteins Research Articles

Combinatorial Effects of 5-Fluorouracil and Menadione on Wnt/β-Catenin Pathway in Human Colorectal Cancer Cells.

The incidence and mortality rates of colorectal cancer (CRC) are alarmingly high, and the scientific community is consistently engaged in developing newer therapeutic options for cancer cure or prevention. The fluoropyrimidine drug, 5-fluorouracil (5FU), remains the first line of treatment against CRC; nevertheless, relapses frequently occur since the cells gain resistance over time through various mechanisms. Studies have highlighted the significance of combinatorial treatment of a Wnt signaling inhibitor and 5FU as a better treatment strategy to overcome 5FU resistance. Small molecules that specifically target and disrupt β-catenin-TCF interaction, a crucial step of the Wnt signaling, are promising in CRC treatment. In this study, we investigated the synergistic cytotoxic activity of menadione with 5FU as the former has previously been shown to downregulate Wnt signaling in CRC cells. Docking and experimental results suggest that the drug combination interfered with key protein-protein interactions in the β-catenin-TCF complex, exerted synergistic anti-cancerous effects in CRC cells, and downregulated the expression of Wnt signaling proteins. Taken together, our data suggest that the simultaneous binding of 5FU and menadione to β-catenin can block Wnt signaling by disrupting β-catenin-TCF interaction and inhibit the proliferation of CRC cells.

Expression of Wnt signaling proteins in rare congenital bladder disorders

Introduction and AimsCongenital bladder anomalies are rare and are a leading cause of end stage renal failure in children. The Wnt signaling pathway, important during embryonic development, has been implicated in the pathogenesis of these conditions through regulation of gene expression, including essential transcription factors. We investigated the expression of four Wnt transcriptional targets, namely, Pygopus 1 (Pygo1), FRA1, TCF7L1 and Connexin 43 (Cx43) in three rare congenital bladder disorders: bladder exstrophy (BE), neurogenic bladder (NGB) and posterior urethral valves (PUV). MethodsBladder tissue samples were collected from patients at the Great Ormond Street Hospital for Sick Children, London, UK, with control (normally-functioning bladder, N=9), BE (N=15), NGB (N=6) and PUV (N=5). Histological analysis was performed using the van Gieson stain to differentiate smooth muscle (SM) and connective tissue (CT) compartments. An unbiased, automated, semi-quantitative immunofluorescence analysis was performed to measure the labelling intensity of four Wnt-related proteins in tissue from these four groups. Results and DiscussionThere was a significant (p<0.05) increase in the expression of Pygo1 in the smooth muscle of all anomalies examined and also in the connective tissue in PUV compared to control. Cx43 also showed overexpression in the smooth muscle across all conditions; however, there was a reduced expression in NGB and an increase in PUV in connective tissue. TCF7L1 showed a significant decrease in both tissue compartments for NGB, whereas FRA1 expression remained unchanged across all anomalies. We also measured colocalization of Wnt-related proteins. TCF7L1 exhibited increased colocalization with Pygo1 and FRA1 in exstrophy compared to control. These results suggest a complex dysregulation of the Wnt pathway in congenital bladder disorders. ConclusionWnt signaling-related proteins show dysregulation in congenital bladder disorders compared to control tissue. Understanding these mechanisms should help towards non-invasive early diagnosis, drug target discovery and development of treatment strategies for these conditions.Summary tableSummary of Wnt-related protein expression and colocalization in bladder exstrophy (BE), neurogenic bladder (NGB) and posterior urethral valves (PUV) disorder compared to normally-functioning control bladder. The table shows the levels of expression as significantly up (↑) down (↓) or no change (-) of Pygopus 1 (Pygo1), Connexin 43 (Cx43), FRA1 and TCF7L1 expression in smooth muscle and connective tissue compartments in BE, NGB and PUV relative to control bladder tissue samples. The table also shows colocalization of six protein pairs of Pygo1 and FRA1; Pygo1 and Cx43; Pygo1 and TCF7L1; FRA1 and Cx43; FRA1 and TCF7L1 and Cx43 and TCF7L1 in bladder disorders compared to control.Summary tableProtein Expression (Smooth muscle)Pygo1Cx43FRA1TCF7L1BE↑↑--NGB↑↑-↓PUV↑↑--Protein Expression (Connective tissue)BE----NGB-↓-↓PUV↑↑--ColocalizationPygo1/FRA1Pygo1/Cx43Pygo1/TCF7L1FRA1/Cx43FRA1/TCF7L1Cx43/TCF7L1Exstrophy--↑-↑-NGB------PUV------

Combination of quercetin and 2-methoxyestradiol inhibits epithelial-mesenchymal transition in PC-3 cell line via Wnt signaling pathway.

Aim:We have previously reported that quercetin (Qu) regulates epithelial–mesenchymal transition (EMT) by modulating Wnt signaling components. In this study, we investigated the synergistic effect of Qu and 2-methoxyestradiol (2-ME) and the role of Wnt signaling components in regulating EMT in PC-3 cells.Materials & methods:EMT was induced by treating PC-3 cells with TGF-β, followed by evaluation of expression of EMT markers and Wnt signaling proteins in naive, induced and after exposing induced cells to Qu and 2-ME at both gene and protein level by real-time PCR (RT-PCR) and western blot, respectively.Results:Qu and 2-ME synergistically downregulated mesenchymal markers with simultaneous upregulation of epithelial markers. Wnt signaling proteins expression was also downregulated by Qu and 2-ME in TGF-β-induced EMT in PC-3 cells.Conclusion:Thus, combination therapy of Qu and 2-ME could be a new promising therapeutic approach for the treatment of prostate cancer.

Alcohol-induced Wnt signaling inhibition during bone fracture healing is normalized by intermittent parathyroid hormone treatment.

Nearly half of orthopaedic trauma patients are intoxicated at the time of injury, and excess alcohol consumption increases the risk for fracture nonunion. Previous studies show alcohol disrupts fracture associated Wnt signaling required for normal bone fracture repair. Intermittent parathyroid hormone (PTH) promotes bone growth through canonical Wnt signaling, however, no studies have investigated the effect of PTH on alcohol‐inhibited bone fracture repair. Male C57BL/6 mice received two‐3 day alcohol binges separated by 4 days before receiving a mid‐shaft tibia fracture. Postoperatively, mice received PTH daily until euthanasia. Wnt/β‐catenin signaling was analyzed at 9 days post‐fracture. As previously observed, acute alcohol exposure resulted in a >2‐fold decrease in total and the active form of β‐catenin and a 2‐fold increase in inactive β‐catenin within the fracture callus. Intermittent PTH abrogated the effect of alcohol on β‐catenin within the fracture callus. Upstream of β‐catenin, alcohol‐treated animals had a 2‐fold decrease in total LRP6, the Wnt co‐receptor, which was restored with PTH treatment. Alcohol nor PTH had any significant effect on GSK‐3β. These data show that intermittent PTH following a tibia fracture restores normal expression of Wnt signaling proteins within the fracture callus of alcohol‐treated mice.

Pharmacologic Inhibition of Porcupine, Disheveled, and β-Catenin in Wnt Signaling Pathway Ameliorates Diabetic Peripheral Neuropathy in Rats

Wnt signaling pathway has been investigated extensively for its diverse metabolic and pain-modulating mechanisms; recently its involvement has been postulated in the development of neuropathic pain. However, there are no reports as yet on the involvement of Wnt signaling pathway in one of the most debilitating neurovascular complication of diabetes, namely, diabetic peripheral neuropathy (DPN). Thus, in the present study, involvement of Wnt signaling was investigated in DPN using Wnt signaling inhibitors namely LGK974 (porcupine inhibitor), NSC668036 (disheveled inhibitor), and PNU74654 (β-catenin inhibitor). Diabetes was induced by a single intraperitoneal injection of streptozotocin (50 mg/kg) to male Sprague–Dawley rats. Diabetic rats after 6 weeks of diabetes induction showed increased expression of Wnt signaling proteins in the spinal cord (L4–L6 lumbar segment), dorsal root ganglions and sciatic nerves. Subsequent increase in inflammation, endoplasmic reticulum stress and loss of intraepidermal nerve fiber density was also observed, leading to neurobehavioral and nerve functional deficits in diabetic rats. Intrathecal administration of Wnt signaling inhibitors (each at doses of 10 and 30 µmol/L) in diabetic rats showed improvement in pain-associated behaviors (heat, cold, and mechanical hyperalgesia) and nerve functions (motor, sensory nerve conduction velocities, and nerve blood flow) by decreasing the expression of Wnt pathway proteins, inflammatory marker, matrix metalloproteinase 2, endoplasmic reticulum stress marker, glucose-regulated protein 78, and improving intraepidermal nerve fiber density. All these results signify the neuroprotective potential of Wnt signaling inhibitors in DPN. PerspectiveThis study emphasizes the involvement of Wnt signaling pathway in DPN. Blockade of this pathway using Wnt inhibitors provided neuroprotection in experimental DPN in rats. This study may provide a basis for exploring the therapeutic potential of Wnt inhibitors in DPN patients.

Abstract 5175: γ-Mangostin, a natural xanthone derivative targets Wnt signaling pathway in colon cancer cells

Abstract Background: Colorectal cancer is the third most common cancer in incidence and cause of death in the United States. The current treatment modalities include chemotherapy, radiation, and surgery. Many people are genetically predisposed for colon cancer through mutations in genes such as adenomatous polyposis coli (APC). While the absence of APC causes aberrant Wnt/Beta catenin signaling, the APC mutations are found in more than 80% of colorectal tumors. γ-Mangostin is a major bioactive compound present in Mangosteen (Garcinia mangostana) which possess significant anti-cancer activity. Herein, we investigated the effects of γ -Mangostin on colon cancer growth and elucidated its mechanistic action through Wnt signaling pathway. Methods: HCT116, SW480 and RKO cell lines were used in the study. The effects of γ-Mangostin on cell proliferation were assessed by hexosaminidase and clonogenicity assays. Effects of γ-Mangostin on apoptosis were evaluated by cell cycle and western blot analysis. Moreover the effect of γ-Mangostin on colonosphere formation was also evaluated. Furthermore, the effect of γ-Mangostin on Wnt signaling proteins was evaluated by western blot analysis. The in vivo anti-cancer effect of γ -Mangostin was investigated on the HCT116 subcutaneous tumor xenograft model implanted in five-week-old male athymic nude mice. Further, the effect of γ -Mangostin was assessed by the specific marker expression in tissue samples by western blot analysis and immunohistochemistry. Results: γ-Mangostin treatment resulted in a dose and time dependent inhibition of proliferation and colony formation in all the three cell lines. Treatment also induced colon cancer cells to undergo G0/G1 and S-phase arrest. Apoptosis was confirmed by increased levels of Bax/Bcl2 ratio, coupled with a reduction in cyclin D1. γ -Mangostin significantly reduced the number and size of colonospheres. Moreover, γ-Mangostin treatment decreased the expression of Wnt signaling proteins, which suggest that γ-Mangostin inhibits the colon cancer growth through Wnt signaling pathway. To determine the effect of γ-Mangostin on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered with 5mg/Kg γ-Mangostin intraperitoneally for 21 days. γ-Mangostin treatment significantly reduced the tumor growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses revealed significant decrease in the expression of Wnt signaling proteins. Conclusion: Together, these data suggest that γ-Mangostin inhibits colon cancer growth through Wnt signaling pathway. γ-Mangostin may be a potential therapeutic agent for colon cancer. Citation Format: Balaji Krishnamachary, Dharmalingam Subramaniam, Thomas Attard, Seth Septer, Shrikant Anant. γ-Mangostin, a natural xanthone derivative targets Wnt signaling pathway in colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5175. doi:10.1158/1538-7445.AM2017-5175

A novel role for the Wnt inhibitor APCDD1 in adipocyte differentiation: Implications for diet-induced obesity

Impaired adipogenic differentiation during diet-induced obesity (DIO) promotes adipocyte hypertrophy and inflammation, thereby contributing to metabolic disease. Adenomatosis polyposis coli down-regulated 1 (APCDD1) has recently been identified as an inhibitor of Wnt signaling, a key regulator of adipogenic differentiation. Here we report a novel role for APCDD1 in adipogenic differentiation via repression of Wnt signaling and an epigenetic linkage between miR-130 and APCDD1 in DIO. APCDD1 expression was significantly up-regulated in mature adipocytes compared with undifferentiated preadipocytes in both human and mouse subcutaneous adipose tissues. siRNA-based silencing of APCDD1 in 3T3-L1 preadipocytes markedly increased the expression of Wnt signaling proteins (Wnt3a, Wnt5a, Wnt10b, LRP5, and β-catenin) and inhibited the expression of adipocyte differentiation markers (CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)) and lipid droplet accumulation, whereas adenovirus-mediated overexpression of APCDD1 enhanced adipogenic differentiation. Notably, DIO mice exhibited reduced APCDD1 expression and increased Wnt expression in both subcutaneous and visceral adipose tissues and impaired adipogenic differentiation in vitro Mechanistically, we found that miR-130, whose expression is up-regulated in adipose tissues of DIO mice, could directly target the 3'-untranslated region of the APCDD1 gene. Furthermore, transfection of an miR-130 inhibitor in preadipocytes enhanced, whereas an miR-130 mimic blunted, adipogenic differentiation, suggesting that miR-130 contributes to impaired adipogenic differentiation during DIO by repressing APCDD1 expression. Finally, human subcutaneous adipose tissues isolated from obese individuals exhibited reduced expression of APCDD1, C/EBPα, and PPARγ compared with those from non-obese subjects. Taken together, these novel findings suggest that APCDD1 positively regulates adipogenic differentiation and that its down-regulation by miR-130 during DIO may contribute to impaired adipogenic differentiation and obesity-related metabolic disease.

Tu1998 - Wnt Signaling Protein Expression Based Pridiction of Recurrent Free Survival in Esophageal Cancer

Tu1998 - Wnt Signaling Protein Expression Based Pridiction of Recurrent Free Survival in Esophageal Cancer

Extracellular vesicles mediate signaling between the aqueous humor producing and draining cells in the ocular system.

PurposeCanonical Wnt signaling is associated with glaucoma pathogenesis and intraocular pressure (IOP) regulation. Our goal was to gain insight into the influence of non-pigmented ciliary epithelium (NPCE)-derived exosomes on Wnt signaling by trabecular meshwork (TM) cells. The potential impact of exosomes on Wnt signaling in the ocular drainage system remains poorly understood.MethodsExosomes isolated from media collected from cultured NPCE cells by differential ultracentrifugation were characterized by dynamic light scattering (DLS), tunable resistive pulse sensing (TRPS), and nanoparticle tracking analysis (NTA), sucrose density gradient migration and transmission electron microscopy (TEM). The cellular target specificity of the NPCE-derived exosomes was investigated by confocal microscopy-based monitoring of the uptake of DiD-labeled exosomes over time, as compared to uptake by various cell lines. Changes in Wnt protein levels in TM cells induced by NPCE exosomes were evaluated by Western blot.ResultsExosomes derived from NPCE cells were purified and detected as small rounded 50–140 nm membrane vesicles, as defined by DLS, NTA, TRPS and TEM. Western blot analysis indicated that the nanovesicles were positive for classic exosome markers, including Tsg101 and Alix. Isolated nanoparticles were found in sucrose density fractions typical of exosomes (1.118–1.188 g/mL sucrose). Using confocal microscopy, we demonstrated time-dependent specific accumulation of the NPCE-derived exosomes in NTM cells. Other cell lines investigated hardly revealed any exosome uptake. We further showed that exosomes induced changes in Wnt signaling protein expression in the TM cells. Western blot analysis further revealed decreased phosphorylation of GKS3β and reduced β-catenin levels. Finally, we found that treatment of NTM cells with exosomes resulted in a greater than 2-fold decrease in the level of β-catenin in the cytosolic fraction. In contrast, no remarkable difference in the amount of β-catenin in the nuclear fraction was noted, relative to the control.ConclusionsThe data suggest that NPCE cells release exosome-like vesicles and that these nanoparticles affect canonical Wnt signaling in TM cells. These findings may have therapeutic relevance since canonical Wnt pathway is involved in intra-ocular pressure regulation. Further understanding of NPCE-derived exosome-responsive signaling pathways may reveal new targets for pharmacological intervention within the drainage system as a target for glaucoma therapy.

Interactions between canonical Wnt signaling pathway and MAPK pathway regulate differentiation, maturation and function of dendritic cells

Antigen-presenting dendritic cells interpret environmental signals to orchestrate local and systemic immune responses. In this study, the roles of Wnt proteins and their signaling pathway members in the maturation and function of monocyte-derived DCs were investigated. The present study showed higher expression of β-catenin, as well as pGSK-3β in DCs than those in monocytes. Wnt3a, Wnt5a and inhibition of GSK-3β promoted differentiation of DCs, but inhibited maturation of DCs. GSK-3β induced DCs maturation with unconventional phenotypes. Together with β-catenin silence, these treatment lead to reduced secretion of cytokines and chemokines except for IL-10 in comparison with LPS treatment, and significantly promoted proliferation of T cells. Wnt3a and inhibition of GSK-3β increased expression of MAPK signalings (p-ERK, p-p38, p-JNK). However, inhibition of MAPK signalings in turn differently regulated Wnt signaling proteins expression. These data suggest that Wnt pathway regulates DCs differentiation, maturation and function with interaction of MAPK signaling pathways.

Prognostic impact of the expression of Wnt-signaling proteins in cervical carcinoma FIGO stage I-IV treated with radiotherapy or chemoradiotherapy.

Wnt signaling proteins were assessed in patients with primary cervical carcinomas who received chemoradiation. The associations between three Wnt signaling proteins and prognosis were assessed. Specimens from 122 patients with cervical carcinomas (FIGO stage I-IV) were immunohistochemically (IHC) analyzed for β-catenin, APC and axin protein expression. Associations between these Wnt-protein expressions, clinicopathological factors, and clinical outcome data were examined.Positive IHC staining for the β-catenin protein (cell-membranes, cytoplasm and nuclei) was recorded in 88%, 58% and 5%, respectively. There was a strong association between β-catenin staining of the cell-membranes and prediction of recurrences and prognosis (p = 0. 002). Tumors with > 5% of nuclear β-catenin staining were associated with inferior cancer-specific survival (p = 0.048) compared with no staining. The overall recurrence rate was significantly higher in the group with increased nuclear staining (67%) compared with the group with no staining (33%). Nuclear APC staining of high intensity was associated with a significantly worse cancer-specific survival and increased overall recurrence rate compared to tumors with weak staining. Distant recurrences were recorded in 29% of cases with intense staining and in 14% of cases with low staining.The Wnt signaling pathway seems to be of importance in the process of cervical oncogenesis. A predictive and prognostic value was found for β-catenin, where strong cell-membrane staining was favorable, and > 5% positive nuclear staining was associated with poorer cancer-specific survival and overall recurrence rate. Nuclear APC staining intensity was also associated with a less favorable prognosis.

Abstract B49: Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells

Abstract Neuroblastoma (NB) is a pediatric malignancy derived from the sympaticoadrenal lineage of neural crest. It is the most commonly occurring extra-cranial nervous system malignancy of infants and children under the age of 5. Long-term survival is less than 40% in patients with high risk NB and over half of the survivors relapse. Emerging evidence supports the notion that there exists within tumors a small population of cells exhibiting stem-like properties, termed cancer stem-like cells (CSC), that is responsible for clinical relapse; therefore, investigating CSC targets may lead to improved treatments. Recent studies have demonstrated that deubiquitinating enzymes (DUBs) of the ubiquitin proteasome system are important players in cancer-associated pathways. These enzymes could be potential effective therapeutic targets. Previous work has identified expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1), a DUB, in the CSC pool of neuroblastoma cells. Our studies are focused on the potential pathways and mechanism by which UCHL1 is acting in the CSC pool. We performed in vitro analyses on NB cell lines SK-N-BE(2), SK-N-AS, and SK-N-DZ. These cells differ in their MYCN amplification and P53 status. The CSC pool was enriched using the sphere formation assay. The small molecule inhibitor, LDN-57444, was used to impair UCHL1 activity. Furthermore, site-directed mutagenesis was used to generate the C90S mutant, which also blocks UCHL1 activity. The effect of blocking UCHL1 activity on cellular proliferation and CSC self-renewal was determined by the MTS assay and sphere formation assay respectively. We further confirmed the effect of impairing UCHL1 activity on the CSC population using flow cytometry by assessing the CD133 and side population. The soft agar assay was used to study the anchorage independent growth of UCHL1 impaired cells. Droplet digital PCR was used to investigate the effect of impaired UCHL1 activity on genes in the Wnt signaling pathway. Immunoprecipitation assays were used to confirm presence of C90S mutant in transfected cells and to determine potential interaction between UCHL1 and Wnt signaling proteins such as β-catenin. UCHL1 inhibition with LDN-57444 demonstrated a dose-dependent decrease in cellular proliferation and impaired sphere formation capability (read-out for self-renewal). We observed differential gene expression levels of MYC and MYCN in cells with UCHL1 inhibited activity via LDN-57444 treatment. C90S mutant transfected cells also demonstrated impaired self-renewal. C90S mutant cells showed decreased gene expression of Wnt signaling proteins and differential expression of MYC and MYCN. Immunoprecipitation studies and CD133 and side population analyses for assessing the CSC population are ongoing. Our data indicates that UCHL1 is potentially an important player in the regulation of CSC self-renewal. Our results also suggest that UCHL1 may mediate self-renewal through the MYC family of transcription factors. The MYC family has been shown to be promising targets in NB. Reduced MYC or MYCN gene expression in NB leads to cell cycle arrest and/or apoptosis. Further investigation of the effect of UCHL1 impaired activity on Wnt signaling will clarify whether UCHL1 is affecting MYC transcription through this pathway. Our studies indicated that UCHL1 might potentially be an effective therapeutic target for the CSC population to supplement current treatments. Citation Format: Judy C. Chang, Jaclyn Y. Hung. Role of ubiquitin carboxyl-terminal esterase L1 (UCHL1) on neuroblastoma cancer stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B49.

Expression of Shh and Wnt Signaling Pathway Proteins in Eyelid Sebaceous Gland Carcinoma: Clinicopathologic Study

Activation or dysregulation of the Sonic hedgehog (Shh) and Wnt signaling pathways is suggested to lead to the development of many human malignancies. In this study, we investigated Shh and Wnt signaling protein expression in eyelid sebaceous gland carcinoma, and analyzed their correlation with clinical characteristics of the tumor. Patients who underwent surgical resection of eyelid sebaceous gland carcinoma from 1999 to 2011 were recruited for the study. Immunohistochemical stainings of Shh signaling proteins (Shh, Gli-1, Gli-2, Gli-3, and ABCG2) and Wnt signaling proteins (Wnt, glycogen synthase kinase 3β, β-catenin, lipoprotein receptor-related protein [LRP], and c-Myc) were conducted. Thirty-seven cases of eyelid sebaceous gland carcinoma were included in this study. Twenty-nine patients showed no metastasis, and eight patients showed lymph node or distant metastasis. Shh, ABCG2, and Wnt proteins were more highly expressed in the group with metastasis than in the group without metastasis (P = 0.031, P = 0.015, and P = 0.001, respectively; χ(2) test). Patients showing high ABCG2 expression, Wnt, or LRP expression developed metastasis more commonly than those with low ABCG2, or without Wnt or LRP expression (log rank test, P = 0.019, 0.001, and 0.000, respectively). The group with metastasis showed higher expression levels of Shh, ABCG2, and Wnt than did the group without metastasis, and patients with strong ABCG2 expression, Wnt, or LRP expression showed higher cumulative incidence of lymph node or distant metastasis, implying that activation of the Shh and Wnt signaling pathway is associated with aggressive behavior of the tumor.

Wnt Signaling in the Pathogenesis of Multiple Sclerosis-Associated Chronic Pain

Many multiple sclerosis (MS) patients develop chronic pain, but the underlying pathological mechanism is unknown. Mice with experimental autoimmune encephalomyelitis (EAE) have been widely used to model MS-related neurological complications, including CNS demyelination, neuroinflammation and motor impairments. Similar to MS patients, EAE mice also develop chronic pain. We are interested in elucidating the potential involvement of Wnt signaling in the pathogenesis of chronic pain in EAE mice. In this study, we characterized the expression of Wnt signaling proteins in the spinal cord dorsal horn (SCDH) of EAE mice, by immunoblotting and immunostaining. The EAE model was created by immunization of adult mice (C57BL/6, 10 weeks) with myelin oligodendrocyte glycoprotein (MOG) 35-55. Robust mechanical hyperalgesia and allodynia were developed in both fore- and hindpaws of the EAE mice. Wnt3a, a prototypical Wnt ligand for the canonical pathway, was significantly increased in the SCDH of the EAE mice. Another key protein in the canonical pathway, ß-catenin, was also significantly up-regulated. In addition, Wnt5a, a prototypic Wnt ligand for the non-canonical pathway, and its receptor (co-receptor) Ror2 were also up-regulated in the SCDH of the EAE mice. We further found that Wnt5a antagonist Box5 and β-catenin inhibitor indomethacin attenuated mechanical allodynia in the EAE mice. Our data collectively suggest that Wnt signaling pathways are up-regulated in the SCDH of the EAE mice and that aberrant activation of Wnt signaling contributes to the development of EAE-related chronic pain.

Ethanol Affects Differentiation‐Related Pathways and Suppresses Wnt Signaling Protein Expression in Human Neural Stem Cells

Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder. NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single-cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin-coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt-receptor complex proteins (p-LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked-2 (NKD-2), and downstream Wnt proteins (β-catenin, Tyr-p-GSK3β, Ser-p-GSK3β) were analyzed by Western blot. Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF-β signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p-LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser-p-GSK3β and β-catenin. Expression of NKD-2 and DVL3 remained unchanged and the expression of active Tyr-p-GSK3β increased significantly. EtOH can significantly alter neural differentiation pathway-related gene expression and suppress Wnt signaling proteins in differentiating human NSC.

WNT-pathway activation in IBD-associated colorectal carcinogenesis: potential biomarkers for colonic surveillance.

Objectives: The Wnt-pathway dominates the sporadic carcinogenesis whereas p53 plays a pivotal role in the colitis-associated counterpart. The expression of Wnt-signaling proteins and p53 during colitis-associated carcinogenesis was determined. Methods: A tissue microarray was constructed with colonic samples from 5 groups of patients: controls (C, n=10), IBD without neoplasia (IBD, n=12), non-dysplastic IBD with neoplasia elsewhere in the colon (IBD-NE, n=12), dysplastic lesion in IBD (IBD-DYS, n=12), and IBD-associated colorectal cancer (IBD-CRC, n=10). Immunohistochemistry was performed for β-catenin, cyclin D1 and p53. p53 sequence analysis was performed in some cases. Results: Nuclear β-catenin expression was found in 0%, 0%, 50%, 55% and 100% of the patients in the C-, IBD-, IBD-NE-, IBD-DYS- and IBD-CRC-groups, respectively. Non-dysplastic IBD mucosa with neoplasia detected elsewhere showed nuclear expression in 50% of the cases compared to 0% in IBD mucosa without neoplasia (p=0.02). Cyclin D1 staining had similar expression patterns. Overexpression of p53 was only detected in the IBD-DYS (66.7%) and IBD-CRC groups (50%). Conclusion: In contrast to previous findings, our results suggest activation of the Wnt-pathway in the early phase of colitis-associated carcinogenesis. Furthermore, as Wnt activation was observed in 50% of the IBD-NE cases, nuclear β-catenin may facilitate detection of neoplasia.

WNT-Pathway Activation in IBD-Associated Colorectal Carcinogenesis: Potential Biomarkers for Colonic Surveillance

Objectives: The Wnt-pathway dominates the sporadic carcinogenesis whereas p53 plays a pivotal role in the colitis-associated counterpart. The expression of Wnt-signaling proteins and p53 during colitis-associated carcinogenesis was determined.Methods: A tissue microarray was constructed with colonic samples from 5 groups of patients: controls (C, n=10), IBD without neoplasia (IBD, n=12), non-dysplastic IBD with neoplasia elsewhere in the colon (IBD-NE, n=12), dysplastic lesion in IBD (IBD-DYS, n=12), and IBD-associated colorectal cancer (IBD-CRC, n=10). Immunohistochemistry was performed for β-catenin, cyclin D1 and p53. p53 sequence analysis was performed in some cases.Results: Nuclear β-catenin expression was found in 0%, 0%, 50%, 55% and 100% of the patients in the C-, IBD-, IBD-NE-, IBD-DYS- and IBD-CRC-groups, respectively. Non-dysplastic IBD mucosa with neoplasia detected elsewhere showed nuclear expression in 50% of the cases compared to 0% in IBD mucosa without neoplasia (p=0.02). Cyclin D1 staining had similar expression patterns. Overexpression of p53 was only detected in the IBD-DYS (66.7%) and IBD-CRC groups (50%).Conclusion: In contrast to previous findings, our results suggest activation of the Wnt-pathway in the early phase of colitis-associated carcinogenesis. Furthermore, as Wnt activation was observed in 50% of the IBD-NE cases, nuclear β-catenin may facilitate detection of neoplasia.

Uterine leiomyomas are characterized by abnormal expression of Wnt signaling proteins and disordered collagen deposition

Objective: The pathogenesis of fibroids is not well understood. Microarray analysis of fibroid tissue identified that some genes are significantly overexpressed, including Delta-like protein (DLK) and Frizzled protein (FZD) (Tsibris, et al. 2002). We postulate that smooth muscle cells of leiomyoma are abnormally differentiated cells derived from uterine stem cells. In addition, the abnormal differentiation leads to the secretion of disordered collagen. Design: Prospective analysis of surgical specimens using immuohistochemistry (IHC), immunoblot and Gomori staining. Materials/Methods: We performed immunohistochemisty using DLK antibody (an imprinted gene involved in cell differentiation), FZD antibody (a key gene in the Wnt cell architecture signaling pathway) and Gomori stain for collagen on paraffin embedded slides of fibroids and myometrium. FZD expression was confirmed by immunoblot. Results: FZD expression was observed in the nucleus of fibroid cells. A halo effect surrounding the stained area was noted. In myometrial cells FZD was seen in the nucleus and cytoplasm. Immunoblotting demonstrated that FZD was upregulated in fibroids compared to myometrium. However, immunohistochemistry showed only nuclear expression of DLK in fibroids, while in myometrium expression was observed in the nucleus and cytoplasm. Gomori staining demonstrated disarrayed collagen with numerous clear spaces and scattered, non-aligned smooth muscle cells in leiomyoma tissue. In contrast, closely packed collagen and aligned smooth muscle fibers were observed in myometrium. Conclusions: These studies suggest that the phenotype of fibroid smooth muscle cells is different from that of the myometrial cells. These altered cells secrete poorly formed collagen, further indicating that fibroids may be a disorder of differentiation. Supported by: NICHD/NIH Intramural Research Training Award Fellowship to Cara Prupas and Chantal Mayers.

Phosphorylation of Pax2 by the c-Jun N-terminal Kinase and Enhanced Pax2-dependent Transcription Activation

The Pax gene family encodes DNA-binding proteins that can both activate and repress transcription of specific target genes during embryonic development. Pax proteins are required for pattern formation and cell differentiation in a broad spectrum of developing tissues. Consistent with its expression in the intermediate mesoderm, the optic cup and stalk, and the otic vesicle, Pax2, a member of the Pax2/5/8 subfamily, is essential for the development of the renal epithelia, the optic cup, and the inner ear. In addition to a DNA binding domain, the Pax2 protein contains a carboxyl-terminal transactivation domain rich in serine, threonine, and tyrosine. In this report, we demonstrate that the Pax2 transactivation domain is phosphorylated by the c-Jun N-terminal kinase, but not the ERK1/2 or p38 MAP kinases and that phosphorylation is coincident with increased transactivation of a Pax2-dependent reporter gene. Activation of JNK by either upstream kinase MEKK1 or DLK or by expression of Wnt signaling proteins significantly enhances Pax2 phosphorylation in cells. In vitro kinase assays using immunoprecipitated JNK or constitutively active, recombinant JNK show phosphorylation of GST-Pax2 fusion proteins. In transfected cells, phosphorylation of Pax2 correlates with increased transactivation of a Pax2-dependent reporter gene, suggesting that serine/threonine phosphorylation of the transactivation domain is important for Pax2 activity. Pax2 can form a complex with the JNK scaffolding protein JIP1, and this interaction is enhanced by activation of the JNK signaling module with the upstream kinase DLK. The data demonstrate that Pax2 is a new target for the JNK signaling module and point to a novel mechanism for mediating Pax-dependent transcription regulation.