Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1β-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.

Nuclear receptor related 1 (Nurr1) is a transcription factor known to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. Reports have confirmed that defect or obliteration of Nurr1 results in neurodegeneration and motor function impairment leading to Parkinson\'s disease (PD). Studies have also indicated that Nurr1 regulates the expression of alpha-synuclein (α-SYN) and mutations in Nurr1 cause α-SYN overexpression, thereby increasing the risk of PD. Nurr1 is modulated via various pathways including Wnt signaling pathway which is known to play an important role in neurogenesis, and deregulation of it contributes to PD pathogenesis. Both Wnt/β-catenin dependent and independent pathways are implicated in the activation of Nurr1 and subsequent downregulation of α-SYN. This review highlights the interaction between Nurr1 and Wnt signaling pathways in mDA neuronal development. We further hypothesize how modulation of Wnt signaling pathway by its antagonist, secreted frizzled related proteins (sFRPs) could be a potential route to treat PD.

OBJECTIVE: One of the hallmarks of cancer stem cells (CSC) is hyperactive Wnt β-catenin signaling due to the decreased presence of Wnt antagonists such as secreted frizzled related protein 4 (SFRP4). Cysteine-rich domain (CRD) and netrin-like domain (NLD) are the two functional domains of SFRP4 having anti-tumor properties. In this study, we have explored the effectiveness of short micropeptides SC-301 (from CRD) and SC-401 (from NLD) on CSC properties, EMT, apoptosis and autophagy in ovarian CSCs enriched from PA-1 and SKOV-3 cell lines.
METHODS: Gene expression analysis, Western blot and immunocytochemistry were performed on ovarian CSCs to evaluate the inhibitory potential of micropeptides to various CSC associated oncogenic properties. Co-immunoprecipitation was performed to detect the binding of CD24 to β-catenin protein complex. CYTO-ID Autophagy Detection Kit 2.0 was used to monitor autophagic flux in peptide treated CSCs.
RESULTS: It is clearly seen that the micropeptides derived from both the domains inhibit Wnt pathway, initiate apoptosis, inhibit migration and chemosensitize CSCs. Specifically, CD24, a defining marker of ovarian CSC was suppressed by peptide treatment. Notably, interaction between CD24 and β-catenin was disrupted upon peptide treatment. SFRP4 peptide treatment also suppressed the autophagic process which is crucial for CSC survival.
CONCLUSIONS: The study demonstrated that although both peptides have inhibitory effects, SC-401 was emphatically more effective in targeting CSC properties and down regulating the Wnt β-catenin machinery.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.

Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule-mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid-a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation-80% of Pax9del/del;Wise-/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored hyaluronic acid accumulation in the palatal mesenchyme. Together, these data identify a crucial role for canonical Wnt signaling in acting downstream of Pax9 to regulate palate morphogenesis.

TIKI2 is a negative regulator of the Wnt family. Although many Wnt antagonists play important roles in renal cell carcinoma (RCC), the molecular function of TIKI2 in human RCC has not been fully elucidated. Here, we analyzed TIKI2 mRNA level in RCC specimens, the corresponding non-tumor tissues, RCC cell lines, and human proximal tubule epithelial cell line HK-2 using qPCR. We demonstrated that TIKI2 was highly expressed in RCC tissue (P < 0.05) and most RCC cell lines. In vitro, TIKI2 knockdown significantly inhibited proliferation, invasion, and clone formation ability of 769-P cells compared with controls, while ectopic TIKI2 expression enhanced A498 cell proliferation, invasion, and clone formation ability. In vivo, the average tumor volume was significantly increased in mice injected with A498-Tiki2 cells (P < 0.05). In the 769-P cell TIKI2 knockdown group, the average tumor volume was not significantly different compared to that of the control group (P = 0.08). Moreover, Wnt/β-catenin signaling was not affected by TIKI2 knockdown or overexpression. Results of the present study indicate that TIKI2 is upregulated in RCC tissues and plays an oncogenic role in RCC.

Wnt/ß-catenin signaling is crucial for maintenance of pluripotent state of embryonic stem cell (ESC). However, it is unclear how Wnt/ß-catenin signaling affects the differentiation ability of ESC, especially with regard to rostral forebrain cells. Here, using Rax, rostral forebrain marker, and Wnt/ß-catenin reporter lines, we report ratio of Rax(+) and Wnt responding tissue (Wnt(+)) patterns, which were affected by seeding number of ESC in three-dimensional culture system. Surprisingly, we found ß-catenin level and localization are heterogeneous in ESC colony by immunostaining and time-laps imaging of ß-catenin-mEGFP signals. Moreover, activation of Wnt signaling in ESC promoted expression level and nuclear localization of ß-catenin, and mRNA levels of Wnt antagonists, axin2 and dkk1, leading to upregulating Wnt/ß-catenin reporter in ESC state and Rax expression at differentiation culture day 7. Together, our results suggest that activation of Wnt signaling in ESC promotes the differentiation efficacy of rostral forebrain cells. Wnt-priming culture method may provide a useful tool for applications in the areas of basic science and molecular therapeutics for regenerative medicine.

Secreted frizzled-related proteins (SFRPs), the first identified Wnt antagonists, have been well recognized as tumor suppressors in multiple human cancers through suppressing the Wnt/β-catenin pathway. To better elucidate the mechanisms of SFRPs involved in the carcinogenesis of oral submucous fibrosis (OSF), one of the precancerous lesions of oral squamous cell carcinoma (OSCC), we investigated expression and localization of SFRP1, SFRP5, and β-catenin in normal oral epithelium, OSF, and OSCC tissues. We found that SFRP1 and SFRP5 were readily expressed in normal oral mucous tissues but gradually decreased in OSF early, moderately advanced, and advanced tissues and rarely expressed in OSCC tissues. We found the changes of SFRP1 localization and SFRP5 localization from nucleus to cytoplasm in the carcinogenesis of OSF. There is a significant association among reduced SFRP1, SFRP5, and cytoplasmic/nuclear β-catenin expression, which is correlated with higher tumor grade and stage of OSCC. We further found that SFRP1 and SFRP5 were frequently methylated in OSCC cases with betel quid chewing habit but not in normal oral mucous and different stages of OSF tissues, suggesting that methylation of SFRP1 and SFRP5 is tumor specific in the carcinogenesis of OSF. Taken together, our data demonstrated that reduced SFRP1 and SFRP5 by promoter methylation could lead to cytoplasmic/nuclear accumulation of β-catenin and tumor progression. The changes of SFRPs and β-catenin localization, as well as SFRPs\' methylation, could be useful predictors or biomarkers of OSF malignant progression and prognosis.

Activation of Wnt/β-catenin signalling is frequently observed in many types of cancer including hepatocellular carcinoma (HCC). We recently reported that cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of solid tumours and contributes to epigenetic silencing of tumour suppressors. In this study, we characterized the function of CUL4B in HCC and investigated whether CUL4B is involved in the regulation of Wnt/β-catenin signalling. CUL4B and β-catenin were frequently up-regulated and positively correlated in HCC tissues. CUL4B activated Wnt/β-catenin signalling by protecting β-catenin from GSK3-mediated degradation, achieved through CUL4B-mediated epigenetic silencing of Wnt pathway antagonists. Knockdown of CUL4B resulted in the up-regulation of Wnt signal antagonists such as DKK1 and PPP2R2B. Simultaneous knockdown of PPP2R2B partially reversed the down-regulation of β-catenin signalling caused by CUL4B depletion. Furthermore, CRL4B promoted the recruitment and/or retention of PRC2 at the promoters of Wnt antagonists and CUL4B knockdown decreased the retention of PRC2 components as well as H3K27me3. Knockdown of CUL4B reduced the proliferation, colony formation, and invasiveness of HCC cells in vitro and inhibited tumour growth in vivo, and these effects were attenuated by introduction of exogenous β-catenin or simultaneous knockdown of PPP2R2B. Conversely, ectopic expression of CUL4B enhanced the proliferation and invasiveness of HCC cells. We conclude that CUL4B can up-regulate Wnt/β-catenin signalling in human HCC through transcriptionally repressing Wnt antagonists and thus contributes to the malignancy of HCC.

The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3β) is an important regulator of the Wnt/β-catenin signaling pathway. The mechanisms underlying GSK3β regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2\'-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3β (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of β-catenin (CTNNB1) expression decreased. The p-GSK3β (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 μM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice.