Neuroglobin (Ngb) has been demonstrated by our lab and others to be neuroprotective against neurological disorders including stroke. However, the roles of Ngb in neurogenesis remain elusive. Neurogenesis can occur in adulthood and can be induced by pathological conditions in the brain such as stroke, and significantly contributes to functional recovery, thus enhancing endogenous neurogenesis may be a promising therapeutic strategy for neurodegenerative diseases. In this study we aimed to investigate the roles of Ngb in neurogenesis using Lentivirus overexpressing Ngb (Lv-Ngb). We show that Ngb overexpression promoted the proliferation of neural progenitor cells (NPC) marked by increased neurosphere number and size. Ngb overexpression also enhanced neuronal differentiation of cultured NPC under differentiation conditions. Moreover, subventricular injection of Lv-Ngb in mice after middle cerebral artery occlusion (MCAO) increased PSA-NCAM positive neuroblastoma cells and Tuj1 positive immature neurons, suggesting that Ngb overexpression promotes neurogenesis in mice brain after stroke. We further show that the pro-neurogenesis effect of Ngb overexpression might be mediated through Dvl1 up-regulation, and subsequent activation of Wnt signaling, indicated by increased nuclear localization of beta-catenin. These results suggest that Ngb may play an important role in promoting neurogenesis in neurodegenerative diseases such as stroke, which may eventually benefit the development of stroke therapeutics targeting neurogenesis through Ngb upregulation.

Several developmental pathways contribute to processes that regulate tissue growth and organ size. The Hippo pathway has emerged as one such critical regulator. However, how Hippo signaling is integrated with other pathways to coordinate these processes remains unclear. Here, we show that the Hippo pathway restricts Wnt/beta-Catenin signaling by promoting an interaction between TAZ and DVL in the cytoplasm. TAZ inhibits the CK1delta/epsilon-mediated phosphorylation of DVL, thereby inhibiting Wnt/beta-Catenin signaling. Abrogation of TAZ levels or Hippo signaling enhances Wnt3A-stimulated DVL phosphorylation, nuclear beta-Catenin, and Wnt target gene expression. Mice lacking Taz develop polycystic kidneys with enhanced cytoplasmic and nuclear beta-Catenin. Moreover, in Drosophila, Hippo signaling modulates Wg target gene expression. These results uncover a cytoplasmic function of TAZ in regulating Wnt signaling and highlight the role of the Hippo pathway in coordinating morphogenetic signaling with growth control.

The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators.

Wnt/β-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs) are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/β-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/β-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC) to upregulate Wnt/β-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/β-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding β-catenin) through binding to its 3' untranslated region (3' UTR). Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/β-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/β-catenin signaling, which help to understand the complex regulation of Wnt/β-catenin signaling.

The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirement for Armadillo (Arm, Drosophila beta-catenin) and Pangolin (Pan, Drosophila TCF) in the Wnt/Wingless(Wg)-induced transcriptional response of Drosophila Kc cells. Using somatic genetics, we demonstrate that both Arm and Pan are absolutely required for mediating activation and repression of target genes. Furthermore, by means of STARR-sequencing we identified Wnt/Wg-responsive enhancer elements and found that all responsive enhancers depend on Pan. Together, our results confirm the dogma of canonical Wnt/Wg signaling and argue against the existence of distal pathway branches in this system.

Mineralization of bone is a dynamic process, involving a complex interplay between cells, secreted macromolecules, signaling pathways, and enzymatic reactions; the dysregulation of bone mineralization may lead to serious skeletal disorders, including hypophosphatemic rickets, osteoporosis, and rheumatoid arthritis. Very few studies have reported the role of osteocytes - the most abundant bone cells in the skeletal system and the major orchestrators of bone remodeling in bone mineralization, which is owed to their nature of being deeply embedded in the mineralized bone matrix. The Wnt/β-catenin signaling pathway is actively involved in various life processes including osteogenesis; however, the role of Wnt/β-catenin signaling in the terminal mineralization of bone, especially in the regulation of osteocytes, is largely unknown. This research demonstrates that during the terminal mineralization process, the Wnt/β-catenin pathway is downregulated, and when Wnt/β-catenin signaling is activated in osteocytes, dendrite development is suppressed and the expression of dentin matrix protein 1 (DMP1) is inhibited. Aberrant activation of Wnt/β-catenin signaling in osteocytes leads to the spontaneous deposition of extra-large mineralized nodules on the surface of collagen fibrils. The altered mineral crystal structure and decreased bonding force between minerals and the organic matrix indicate the inferior integration of minerals and collagen. In conclusion, Wnt/β-catenin signaling plays a critical role in the terminal differentiation of osteocytes and as such, targeting Wnt/β-catenin signaling in osteocytes may serve as a potential therapeutic approach for the management of bone-related diseases.

Cellular nucleic acid binding protein (CNBP) is a small and highly conserved protein with nucleic acid chaperone activity that binds single-stranded nucleic acids. Data collected so far suggests that CNBP is required for proper craniofacial development. Despite the advances achieved in the last decade, the identity of the molecular targets of CNBP responsible for its role in rostral head development remains elusive.

Teneurins (Tens) are a highly conserved family of proteins necessary for cell-cell adhesion. Tens can be cleaved, and some of their proteolytic products, such as the teneurin c-terminal associated-peptide (TCAP) and the intracellular domain (ICD), have been demonstrated to be biologically active. Although Tens are considered critical for central nervous system development, they have also been demonstrated to play important roles in adult tissues, suggesting a potential link between their deregulation and various pathological processes, including neurodegeneration and cancer. However, knowledge regarding how Ten expression is modulated is almost absent. Relevantly, the functions of Tens resemble several of the effects of canonical and non-canonical Wnt pathway activation, including the effects of the Wnt pathways on neuronal development and function as well as their pivotal roles during carcinogenesis. Accordingly, in this initial study, we decided to evaluate whether Wnt signaling can modulate the expression of Tens. Remarkably, in the present work, we used a specific inhibitor of porcupine, the key enzyme for Wnt ligand secretion, to not only demonstrate the involvement of Wnt signaling in regulating Ten-3 expression for the first time but also reveal that Wnt3a, a canonical Wnt ligand, increases the expression of Ten-3 through a mechanism dependent on the secretion and activity of the non-canonical ligand Wnt5a. Although our work raises several new questions, our findings seem to demonstrate the upregulation of Ten-3 by Wnt signaling and also suggest that Ten-3 modulation is possible because of crosstalk between the canonical and non-canonical Wnt pathways.

Cholangiocarcinoma (CCA) is a refractory malignancy derived from bile duct epithelial cells. This study aimed to explore the role and molecular mechanisms of action of sevoflurane in CCA.

Histone deacetylase (HDAC) inhibitors promote differentiation through post-translational modifications of histones. BML-281, an HDAC6 inhibitor, has been known to prevent tumors, acute dextran sodium sulfate-associated colitis, and lung injury. However, the neurogenic differentiation effect of BML-281 is poorly understood. In this study, we investigated the effect of BML-281 on neuroblastoma SH-SY5Y cell differentiation into mature neurons by immunocytochemistry (ICC), reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR), and western blotting analysis. We found that the cells treated with BML-281 showed neurite outgrowth and morphological changes into mature neurons under a microscope. It was confirmed that the gene expression of neuronal markers (NEFL, MAP2, Tuj1, NEFH, and NEFM) was increased with certain concentrations of BML-281. Similarly, the protein expression of neuronal markers (NeuN, Synaptophysin, Tuj1, and NFH) was upregulated with BML-281 compared to untreated cells. Following treatment with BML-281, the expression of Wnt5α increased, and downstream pathways were activated. Interestingly, both Wnt/Ca2+ and Wnt/PCP pathways activated and regulated PKC, Cdc42, RhoA, Rac1/2/3, and p-JNK. Therefore, BML-281 induces the differentiation of SH-SY5Y cells into mature neurons by activating the non-canonical Wnt signaling pathway. From these results, we concluded that BML-281 might be a novel drug to differentiation into neuronal cells through the regulation of Wnt signaling pathway to reduce the neuronal cell death.

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.

A family of signal transduction pathways known as wingless type (Wnt) signaling pathways is essential to developmental processes like cell division and proliferation. Mutation in Wnt signaling results in a variety of diseases, including cancers of the breast, colon, and skin, metabolic disease, and neurodegenerative disease; thus, the Wnt signaling pathways have been attractive targets for disease treatment. However, the complicatedness and large involveness of the pathway often hampers pinpointing the specific targets of the metabolic process. In our current study, we investigated the differential metabolic regulation by the overexpression of the Wnt signaling pathway in a timely-resolved manner by applying high-throughput and un-targeted metabolite profiling. We have detected and annotated 321 metabolite peaks from a total of 36 human embryonic kidney (HEK) 293 cells using GC-TOF MS and LC-Orbitrap MS. The un-targeted metabolomic analysis identified the radical reprogramming of a range of central carbon/nitrogen metabolism pathways, including glycolysis, TCA cycle, and glutaminolysis, and fatty acid pathways. The investigation, combined with targeted mRNA profiles, elucidated an explicit understanding of activated fatty acid metabolism (β-oxidation and biosynthesis). The findings proposed detailed mechanistic biochemical dynamics in response to Wnt-driven metabolic changes, which may help design precise therapeutic targets for Wnt-related diseases.

Progressive fibrosis of the dermal tissues is a challenging complication of radiotherapy whose underlying mechanism is not fully understood, and there are few available treatments. The canonical Wnt/β-catenin signaling pathway plays an important role in fibrosis as well as in the epithelial-to-mesenchymal transition (EMT). We investigated whether inhibition of Wnt/β-catenin signaling with sLRP6E1E2, a molecule that binds to extracellular Wnt ligands, ameliorated radiation-induced fibrosis both in vitro and in vivo. Radiation with a single dose of 2 Gy not only facilitated fibrosis in cultured human dermal fibroblasts via activation of the Wnt/β-catenin pathway but also initiated EMT in cultured keratinocytes, developing collagen-producing mesenchymal cells. sLRP6E1E2-expressing adenovirus treatment exerted anti-fibrotic activity in irradiated cultured dermal fibroblasts and keratinocytes. In a mouse model, a single fraction of 15 Gy was delivered to the dorsal skins of 36 mice randomized into three groups: those receiving PBS, those receiving control adenovirus, and those receiving decoy Wnt receptor-expressing adenovirus (dE1-k35/sLRP6E1E2). The mice were observed for 16 weeks, and excessive deposition of type I collagen was suppressed by sLRP6E1E2-expressing adenovirus treatment. These results demonstrate that the modulation of the Wnt/β-catenin pathway has the potential to decrease the severity of radiation-induced dermal fibrosis.

The TGF-β and Wnt signaling pathways are interrelated in many cell types and tissues, and control cell functions in coordination. Here, we report that SMAD4, a downstream effector of the TGF-β signaling pathway, induces FZD4, a receptor of the Wnt signaling pathway, establishing a novel route of communication between these two pathways in granulosa cells (GCs). We found that SMAD4 is a strong inducer of FZD4, not only initiating FZD4 transcription but also activating FZD4-dependent Wnt signaling and GC apoptosis. Furthermore, we identified the direct and indirect mechanisms by which SMAD4 promotes expression of FZD4 in GCs. First, SMAD4 functions as a transcription factor to directly bind to the FZD4 promoter region to increase its transcriptional activity. Second, SMAD4 promotes production of SDNOR, a novel lncRNA that acts as a sponge for miR-29c, providing another mean to block miR-29c from degenerating FZD4 mRNA. Overall, our findings not only reveal a new channel of crosstalk between the TGF-β and Wnt signaling pathways, SMAD4-FZD4 axis, but also provide new insights into the regulatory network of GC apoptosis and follicular atresia. These RNA molecules, such as miR-29c and lnc-SDNOR, represent potential targets for treatment of reproductive diseases and improvement of female fertility.

The WNT signaling pathway plays a crucial role in oviduct/fallopian development. However, the specific physiological processes regulated by the WNT pathway in the fallopian/oviduct function remain obscure. Benefiting from the Lgr4 knockout mouse model, we report the regulation of oviduct epithelial secretion by LGR4. Specifically, the loss of Lgr4 altered the mouse oviduct size and weight, severely reduced the number of oviductal epithelial cells, and ultimately impaired the epithelial secretion. These alterations were mediated by a failure of CTNNB1 protein accumulation in the oviductal epithelial cytoplasm, by the modulation of WNT pathways, and subsequently by a profound change of the gene expression profile of epithelial cells. In addition, selective activation of the WNT pathway triggered the expression of steroidogenic genes, like Cyp11a1 and 3β-Hsd1, through the activation of the transcriptional factor NR5A2 in an oviduct primary cell culture system. As demonstrated, the LGR4 protein modulates a WNT-NR5A2 signaling cascade facilitating epithelial secretory cell maturation and steroidogenesis to safeguard oviduct development and function in mice.

Wingless-type protein (Wnt)/β-catenin pathway alterations in non-small cell lung cancer (NSCLC) are associated with poor prognosis and resistance. In 598 stage III-IV NSCLC patients receiving platinum-based chemotherapy at the MD Anderson Cancer Center (MDACC), we correlated survival with 441 host single-nucleotide polymorphisms (SNPs) in 50 Wnt pathway genes. We then assessed the most significant SNPs in 240 Mayo Clinic patients receiving platinum-based chemotherapy for advanced NSCLC, 127 MDACC patients receiving platinum-based adjuvant chemotherapy and 340 early stage MDACC patients undergoing surgery alone (cohorts 2-4). In multivariate analysis, survival correlates with SNPs for AXIN2 (rs11868547 and rs4541111, of which rs11868547 was assessed in cohorts 2-4), Wnt-5B (rs12819505), CXXC4 (rs4413407) and WIF-1 (rs10878232). Median survival was 19.7, 15.6 and 10.7 months for patients with 1, 2 and 3-5 unfavorable genotypes, respectively (P=3.8 × 10(-9)). Survival tree analysis classified patients into two groups (median survival time 11.3 vs 17.3 months, P=4.7 × 10(-8)). None of the SNPs achieved significance in cohorts 2-4; however, there was a trend in the same direction as cohort 1 for 3 of the SNPs. Using online databases, we found rs10878232 displayed expression quantitative trait loci correlation with the expression of LEMD3, a neighboring gene previously associated with NSCLC survival. In conclusion, results from cohort 1 provide further evidence for an important role for Wnt in NSCLC. Investigation of Wnt inhibitors in advanced NSCLC would be reasonable. Lack of an SNP association with outcome in cohorts 2-4 could be due to low statistical power, impact of patient heterogeneity or false-positive observations in cohort 1.

The Wnt signaling pathway plays key roles in development, adult tissue homeostasis and stem cell maintenance. Further understanding of the function of Wnt signaling in specific cell types could benefit from lentiviral vectors expressing reporters for the Wnt pathway or vectors interfering with signaling.

Human BCL7 gene family consists of BCL7A, BCL7B, and BCL7C. A number of clinical studies have reported that BCL7 family is involved in cancer incidence, progression, and development. Among them, BCL7B, located on chromosome 7q11.23, is one of the deleted genes in patients with Williams-Beuren syndrome. Although several studies have suggested that malignant diseases occurring in patients with Williams-Beuren syndrome are associated with aberrations in BCL7B, little is known regarding the function of this gene at the cellular level. In this study, we focused on bcl-7, which is the only homolog of BCL7 gene family in Caenorhabditis elegans, and analyzed bcl-7 deletion mutants. As a result, we found that bcl-7 is required for the asymmetric differentiation of epithelial seam cells, which have self-renewal properties as stem cells and divide asymmetrically through the WNT pathway. Distal tip cell development, which is regulated by the WNT pathway in Caenorhabditis elegans, was also affected in bcl-7-knockout mutants. Interestingly, bcl-7 mutants exhibited nuclear enlargement, reminiscent of the anaplastic features of malignant cells. Furthermore, in KATOIII human gastric cancer cells, BCL7B knockdown induced nuclear enlargement, promoted the multinuclei phenotype and suppressed cell death. In addition, this study showed that BCL7B negatively regulates the Wnt-signaling pathway and positively regulates the apoptotic pathway. Taken together, our data indicate that BCL7B/BCL-7 has some roles in maintaining the structure of nuclei and is involved in the modulation of multiple pathways, including Wnt and apoptosis. This study may implicate a risk of malignancies with BCL7B-deficiency, such as Williams-Beuren syndrome.

The Wnt signaling pathway plays a crucial role in neurodevelopment and in regulating the function and structure of the adult nervous system. Schizophrenia (SCZ) and bipolar disorder (BD) are severe mental disorders with evidence of subtle neurodevelopmental, structural and functional neuronal abnormalities. We aimed to elucidate the role of aberrant regulation of the Wnt system in these disorders by evaluating plasma levels of secreted Wnt modulators in patients (SCZ = 551 and BD = 246) and healthy controls (HCs = 639) using enzyme immune-assay. We also investigated the expression of 141 Wnt-related genes in whole blood in a subsample (SCZ = 338, BD = 241, and HCs = 263) using microarray analysis. Both SCZ and BD had dysregulated mRNA expression of Wnt-related genes favoring attenuated canonical (beta-catenin-dependent) signaling, and there were also indices of enhanced non-canonical Wnt signaling. In particular, FZD7, which may activate all Wnt pathways, but favors non-canonical signaling, and NFATc3, a downstream transcription factor and readout of the non-canonical Wnt/Ca2+ pathway, were significantly increased in SCZ and BD (p < 3 × 10-4). Furthermore, patients had lower plasma levels of soluble dickkopf 1 and sclerostin (p < 0.01) compared with HC. Our findings suggest that SCZ and BD are characterized by abnormal Wnt gene expression and plasma protein levels, and we propose that drugs targeting the Wnt pathway may have a role in the treatment of severe mental disorders.

In this study, we investigated the development of the Wnt signaling pathway in vitamin D (VitD) to improve systemic lupus erythematosus in mice to breakthrough clinical treatment approaches.