Wingless-type MMTV Integration Site Research Articles

Taurine Activates BMP-2/Wnt3a-Mediated Osteoblast Differentiation and Mineralization via Akt and MAPK Signaling

We aimed to elucidate the preventive effects of taurine against osteopenia in ovariectomized (OVX) rats and the mechanisms by which taurine regulates osteoblastogenesis in vitro and in vivo. The effects of the taurine on human osteoblast MG-63 cell differentiation and osteoblastogenesis effect in OVX rat were examined Konkuk University in 2018 by evaluating osteoblast differentiation, and expression of osteoblast-specific factors by western blotting analysis. Taurine supplementation significantly improved alkaline phosphatase (ALP) activity and mineralization in a concentration-dependent manner. Further, taurine induced the expression of osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), small mothers against decapentaplegic 1/5/8 (SMAD1/5/8), wingless-type MMTV integration site family member 3A (Wnt3a), and collagen type 1 (COL-1) via mitogen-activated protein kinase (MAPK) and serine/threonine protein kinase (Akt). Moreover, the RUNX2 activity of the taurine-treated group was enhanced by protein-protein interactions such as Wnt3a-induced p-AKT/RUNX2 and BMP-mediated SMADs/MAPK/RUNX2 interactions. Our in vitro and in vivo results suggested that taurine can be considered as a potential therapeutic candidate agent for preventing bone loss in postmenopausal osteoporosis.

Muscle Cell Morphogenesis, Structure, Development and Differentiation Processes Are Significantly Regulated during Human Ovarian Granulosa Cells In Vitro Cultivation.

Granulosa cells (GCs) have many functions and are fundamental for both folliculogenesis and oogenesis, releasing hormones and communicating directly with the oocyte. Long-term in vitro cultures of GCs show significant stem-like characteristics. In the current study, RNA of human ovarian granulosa cells was collected at 1, 7, 15 and 30 days of long-term in vitro culture. Understanding the process of differentiation of GCs towards different cell lineages, as well as the molecular pathways underlying these mechanisms, is fundamental to revealing other possible stemness markers of this type of cell. Identifying new markers of GC plasticity may help to understand the aetiology and recurrence of a wide variety of diseases and health conditions and reveal possible clinical applications of the ovarian tissue cells, affecting not only the reproductive ability but also sex hormone production. Granulosa cells were the subject of this study, as they are readily available as remnant material leftover after in vitro fertilisation procedures and exhibit significant stem-like characteristics in culture. The change in gene expression was investigated through a range of molecular and bioinformatic analyses. Expression microarrays were used, allowing the identification of groups of genes typical of specific cellular pathways. This candidate gene study focused on ontological groups associated with muscle cell morphogenesis, structure, development and differentiation, namely, “muscle cell development”, “muscle cell differentiation”, “muscle contraction”, “muscle organ development”, “muscle organ morphogenesis”, “muscle structure development”, “muscle system process” and “muscle tissue development”. The results showed that the 10 most upregulated genes were keratin 19, oxytocin receptor, connective tissue growth factor, nexilin, myosin light chain kinase, cysteine and glycine-rich protein 3, caveolin 1, actin, activating transcription factor 3 and tropomyosin, while the 10 most downregulated consisted of epiregulin, prostaglandin-endoperoxide synthase 2, transforming growth factor, interleukin, collagen, 5-hydroxytryptmine, interleukin 4, phosphodiesterase, wingless-type MMTV integration site family and SRY-box 9. Moreover, ultrastructural observations showing heterogeneity of granulosa cell population are presented in the study. At least two morphologically different subpopulations were identified: large, light coloured and small, darker cells. The expression of genes belonging to the mentioned ontological groups suggest the potential ability of GCs to differentiate and proliferate toward muscle lineage, showing possible application in muscle regeneration and the treatment of different diseases.

Shh-Mediated Increase in β-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of β-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that β-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-mycwas found to bind the β-catenin promoter, and the increase in β-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of β-catenin. We propose that in culture, Shh signaling regulates β-catenin expression through N-mycand results in increased CGNP proliferation.

Integrated miRNA-mRNA network revealing the key molecular characteristics of ossification of the posterior longitudinal ligament

Background:Ossification of the posterior longitudinal ligament (OPLL) refers to an ectopic ossification disease originating from the posterior longitudinal ligament of the spine. Pressing on the spinal cord or nerve roots can cause limb sensory and motor disorders, significantly reducing the patient's quality of life. At present, the pathogenesis of OPLL is still unclear. The purpose of this study is to integrate microRNA (miRNA)-mRNA biological information data to further analyze the important molecules in the pathogenesis of OPLL, so as to provide targets for future OPLL molecular therapy.Methods:miRNA and mRNA expression profiles of GSE69787 were downloaded from Gene Expression Omnibus database and analyzed by edge R package. Funrich software was used to predict the target genes and transcription factors of de-miRNA. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) were carried out based on CLUEGO plug-in in Cytoscape. Using data collected from a search tool for the retrieval of interacting genes online database, a protein-protein interaction (PPI) network was constructed using Cytoscape. The hub gene selection and module analysis of PPI network were carried out by cytoHubba and molecular complex detection, plug-ins of Cytoscape software respectively.Results:A total of 346 genes, including 247 up-regulated genes and 99 down-regulated genes were selected as DEGs. SP1 was identified as an upstream transcription factor of de-miRNAs. Notably, gene ontology enrichment analysis shows that up- and down-regulated DEGs are mainly involved in BP, such as skeletal structure morphogenesis, skeletal system development, and animal organ morphogenesis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that only WNT signaling pathway was associated with osteogenic differentiation. Lymphoid enhancer binding factor 1 and wingless-type MMTV integration site family member 2 Wingless-Type MMTV Integration site family member 2 were identified as hub genes, miR-520d-3p, miR-4782-3p, miR-6766-3p, and miR-199b-5p were identified as key miRNAs. In addition, 2 important network modules were obtained from PPI network.Conclusions:In this study, we established a potential miRNA-mRNA regulatory network associated with OPLL, revealing the key molecular mechanism of OPLL and providing targets for future treatment or prevent its occurrence.

Identification of candidate lncRNAs and circRNAs regulating WNT3/β-catenin signaling in essential hypertension.

Mounting evidence suggests that noncoding RNAs (ncRNAs) contribute to the pathogenesis of cardiovascular diseases. However, their role in essential hypertension (EH) is still unclear. We therefore identified differentially expressed long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in EH patients from a high-risk population group and constructed a competing endogenous RNA regulatory network that predicts interactions of potential diagnostic and therapeutic relevance between specific lncRNA/circRNA-microRNA-mRNA triplets. Our analysis identified two lncRNAs, transmembrane protein 183A pseudogene (LOC646616) and leucine aminopeptidase 3 pseudogene 2 (LAP3P2), and two circRNAs, hsa_circ_0039388 and hsa_circ_0038648, that are highly co-expressed with both wingless-type MMTV integration site family member 3 (WNT3) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) mRNAs and also share common microRNA binding sites with these two transcripts. We also confirmed that a mutually regulated network composed of LOC646616/microRNA-637/WNT3 controls WNT3 expression and influences viability and invasive properties in human arterial smooth muscle cells in vitro. These findings highlight a novel ncRNA-based regulatory mechanism potentially driving WNT/β-catenin activation in EH, and suggest that the identified ncRNAs may represent useful biomarkers and therapeutic targets for this condition.

A novel Sprouty4-ERK1/2-Wnt/β-catenin regulatory loop in marrow stromal progenitor cells controls osteogenic and adipogenic differentiation

BackgroundSprouty (SPRY) proteins play critical roles in controlling cell proliferation, differentiation, and survival by inhibiting receptor tyrosine kinase (RTK)-mediated extracellular signal-regulated kinase (ERK) signaling. Recent studies have demonstrated that SPRY4 negatively regulates angiogenesis and tumor growth. However, whether SPRY4 regulates osteogenic and/or adipogenic differentiation of mesenchymal stem cells remains to be explored. ResultsIn this study, we investigated the expression pattern of Spry4 and found that its expression was regulated during the differentiation of mouse marrow stromal progenitor cells and increased in the metaphysis of ovariectomized mice. In vitro loss-of-function and gain-of-function studies demonstrated that SPRY4 inhibited osteogenic differentiation and stimulated adipogenic differentiation of progenitor cells. In vivo experiments showed that silencing of Spry4 in the marrow of C57BL/6 mice blocked fat accumulation and promoted osteoblast differentiation in ovariectomized mice. Mechanistic investigations revealed the inhibitory effect of SPRY4 on canonical wingless-type MMTV integration site (Wnt) signaling and ERK pathway. ERK1/2 was shown to interact with low-density lipoprotein receptor-related protein 6 (LRP6) and activate the canonical Wnt signaling pathway. Inactivation of Wnt signaling attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by Spry4 small interfering RNA (siRNA). Finally, promoter study revealed that β-catenin transcriptionally inhibited the expression of Spry4. ConclusionsOur study for the first time suggests that a novel SPRY4-ERK1/2-Wnt/β-catenin regulatory loop exists in marrow stromal progenitor cells and plays a key role in cell fate determination. It also highlights the potential of SPRY4 as a novel therapeutic target for the treatment of metabolic bone disorders such as osteoporosis.

Up-regulated miR-374a-3p relieves lipopolysaccharides induced injury in CHON-001 cells via regulating Wingless-type MMTV integration site family member 5B

BackgroundOsteoarthritis (OA) is a frequent and incurable joint disease, inducing significant pain and seriously threatening to human health. It has been reported that microRNAs (miRNAs) play crucial roles on cancers and inflammatory diseases via cooperating with genes. However, the effect of miR-374a-3p/Wingless-type MMTV integration site family, member 5B (WNT5B) pair in OA remains to be explored. MethodsGSE105027 and GSE55457 datasets were obtained to reveal the expression of miR-374a-3p and WNT5B in OA cartilages using log-scale. The OA cell model was established by lipopolysaccharides (LPS) stimulation in CHON-001 cells and the functional role of miR-374a-3p on OA was investigated by analyzing cell proliferation, cell apoptosis and the expression of apoptosis-related proteins (Bcl-2, Bax and Bim). Through bioinformatics prediction, WNT5B, the target gene of miR-374a-3p, was predicted and the association between miR-374a-3p and WNT5B was further explored by luciferase reporter assay. Functional experiments in vitro were conducted to assess whether WNT5B was involved in the regulation of miR-374a-3p to LPS-stimulated CHON-001. Finally, the expression of JNK/ERK/MAPK pathway-related proteins was detected to explore the underlying molecular mechanism. ResultsThe data set showed that miR-374a-3p was decreased in OA cartilages and the consistent expressional pattern was observed in LPS-stimulated CHON-001 cells.Overexpression of miR-374a-3p significantly alleviated LPS-induced damage in CHON-001 cells, whereas miR-374a-3p inhibitor aggravated LPS-stimulated injury. Further experiments demonstrated that WNT5B was a target of miR-374a-3p and its expression was decreased by miR-374a-3p. WNT5B expression was increased in OA cartilages. Silencing WNT5B prevented CHON-001 cells from LPS-induced damage. Down-regulation of WNT5B strengthened the protective effect of miR-374a-3p on LPS-stimulated CHON-001 cells. Moreover, miR-374a-3p cooperated with WNT5B to affect cell behaviors of LPS-stimulated CHON-001 cells via mediating the JNK/ERK/MAPK pathway. ConclusionThese results indicated that overexpression of miR-374a-3p protects CHON-001 cells against LPS challenge by modulating WNT5B and inhibiting the JNK/ERK/MAPK pathway.

Role of Astrocytic Dysfunction in the Pathogenesis of Parkinson's Disease Animal Models from a Molecular Signaling Perspective.

Despite the fact that astrocytes are the most abundant glial cells, critical for brain function, few studies have dealt with their possible role in neurodegenerative diseases like Parkinson's disease (PD). This article explores relevant evidence on the involvement of astrocytes in experimental PD neurodegeneration from a molecular signaling perspective. For a long time, astrocytic proliferation was merely considered a byproduct of neuroinflammation, but by the time being, it is clear that astrocytic dysfunction plays a far more important role in PD pathophysiology. Indeed, ongoing experimental evidence suggests the importance of astrocytes and dopaminergic neurons' cross-linking signaling pathways. The Wnt-1 (wingless-type MMTV integration site family, member 1) pathway regulates several processes including neuron survival, synapse plasticity, and neurogenesis. In PD animal models, Frizzled (Fzd) neuronal receptors' activation by the Wnt-1 normally released by astrocytes following injuries leads to β-catenin-dependent gene expression, favoring neuron survival and viability. The transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor also participates in experimental PD genesis. Activation of astrocyte TRPV1 receptors by noxious stimuli results in reduced inflammatory response and increased ciliary neurotrophic factor (CNTF) synthesis, which enhances neuronal survival and differentiation. Another major pathway involves IκB kinase (IKK) downregulation by ARL6ip5 (ADP-ribosylation-like factor 6 interacting protein 5, encoded by the cell differentiation-associated, JWA, gene). Typically, IKK releases the proinflammatory NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) molecule from its inhibitor. Therefore, by downregulating NF-κB inhibitor, ARL6ip5 promotes an anti-inflammatory response. The evidence provided by neurotoxin-induced PD animal models guarantees further research on the neuroprotective potential of normalizing astrocyte function in PD.

Maternal Inulin Supplementation Alters Hepatic DNA Methylation Profile and Improves Glucose Metabolism in Offspring Mice.

ScopeAs a prebiotic, inulin may have a protective effect on glucose metabolism. However, the mechanism of inulin treatment on glucose intolerance in offspring exposed to a maternal high-fat (HF) diet is still not clear. Here, we examined the hepatic DNA methylation profile to determine how maternal inulin supplementation modified glucose metabolism in offspring mice.ProceduresFemale mice were fed a HF diet, control diet (CON), or a HF diet with inulin supplementation (HF-inulin) during gestation and lactation. Upon weaning, pup livers were obtained. A hepatic genome DNA methylation array was performed.ResultsPups exposed to a maternal HF diet exhibited glucose intolerance and insulin resistance. Maternal inulin treatment moderated glucose metabolism. A DNA methylation array identified differentially methylated regions associated with 970 annotated genes from pups exposed to a HF diet in response to maternal inulin treatment. In particular, the wingless-type MMTV integration site family member 5A (Wnt5a) gene was hypermethylated, and the phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 alpha (Pik3c2a), phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (Pik3c2b), and phosphoinositide-3-kinase regulatory subunit 2 (Pik3r2) genes were hypomethylated in inulin-treated pups. Consistently, hepatic Wnt5a gene expression was reduced and Pik3c2a, Pik3c2b, and Pik3r2 gene expression were increased in the inulin group.ConclusionMaternal inulin treatment improved glucose intolerance by changing DNA methylation and gene expression of Wnt5a and Pi3k in mice exposed to a maternal HF diet.

Caveolin-1 Promotes Chemoresistance of Gastric Cancer Cells to Cisplatin by Activating WNT/β-Catenin Pathway

Drug resistance is a major challenge for chemotherapy in treating human gastric cancer (GC), as the underlying molecular mechanism of chemoresistance in GC remains unknown. Caveolin-1 (Cav-1) is a scaffold protein of plasma membrane caveolae that acts as a tumor modulator by interacting with several cell signals. In this research, we showed that the survival rate of GC cells to cisplatin (CDDP) increased in the presence of Cav-1. Moreover, Cav-1 overexpression inhibited cisplatin-induced apoptosis and improved the survival rate of GC cells. Cav-1 overexpression and knock-down experiments indicated that Cav-1 expression stimulated wingless-type MMTV integration site (WNTs) pathway through the phosphorylation of LRP6 and dephosphorylation of β-catenin. Cav-1 was positively associated with the increase of WNT downstream target gene Met, which led to the activation of HER2 signaling. Moreover, our results demonstrated that the expression of Cav-1 and Met were positively associated with the resistance of GC cells to cisplatin. Collectively, Cav-1 enhances the cisplatin-resistance of GC cells by activating the WNT signaling pathway and Met-HER2 crosstalk. Understanding the role of Cav-1 in the chemoresistance of GC would help to develop novel therapies for a better treatment outcome of GC patients.

Enhancing Activity of Pleurotus sajor-caju (Fr.) Sing β-1,3-Glucanoligosaccharide (Ps-GOS) on Proliferation, Differentiation, and Mineralization of MC3T3-E1 Cells through the Involvement of BMP-2/Runx2/MAPK/Wnt/β-Catenin Signaling Pathway.

Osteoporosis is a leading world health problem that results from an imbalance between bone formation and bone resorption. β-glucans has been extensively reported to exhibit a wide range of biological activities, including antiosteoporosis both in vitro and in vivo. However, the molecular mechanisms responsible for β-glucan-mediated bone formation in osteoblasts have not yet been investigated. The oyster mushroom Pleurotus sajor-caju produces abundant amounts of an insoluble β-glucan, which is rendered soluble by enzymatic degradation using Hevea glucanase to generate low-molecular-weight glucanoligosaccharide (Ps-GOS). This study aimed to investigate the osteogenic enhancing activity and underlining molecular mechanism of Ps-GOS on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells. In this study, it was demonstrated for the first time that low concentrations of Ps-GOS could promote cell proliferation and division after 48 h of treatment. In addition, Ps-GOS upregulated the mRNA and protein expression level of bone morphogenetic protein-2 (BMP-2) and runt-related transcription factor-2 (Runx2), which are both involved in BMP signaling pathway, accompanied by increased alkaline phosphatase (ALP) activity and mineralization. Ps-GOS also upregulated the expression of osteogenesis related genes including ALP, collagen type 1 (COL1), and osteocalcin (OCN). Moreover, our novel findings suggest that Ps-GOS may exert its effects through the mitogen-activated protein kinase (MAPK) and wingless-type MMTV integration site (Wnt)/β-catenin signaling pathways.

Analysis of Wnt7B and BMP4 expression patterns in congenital pulmonary airway malformation.

Congenital pulmonary airway malformation (CPAM) is a rare disorder characterized by aberrant overgrowth of terminal bronchioles. The objective of this study was to describe wingless-type MMTV integration site family 7B (Wnt7B) and bone morphogenetic protein 4 (BMP4) expression patterns in human CPAM lesions and to explore the possible roles of Wnt7B and BMP4 in the pathogenesis of CPAM. Fifteen tissue samples from patients with CPAM were obtained from the Pathology Department of Shengjing Hospital of China Medical University. Samples representing CPAM lesions and adjacent normal lung tissues were collected and Wnt7B and BMP4 expression was detected through immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. IHC revealed that Wnt7B immunopositive cells were only detected in epithelial cells, whereas BMP4 immunopositive cells were detected in epithelial and mesenchymal cells. Expression of Wnt7B and BMP4 immunopositive cells was higher in CPAM lesions than that in adjacent normal lung tissue. qRT-PCR and Western blotting showed that Wnt7B and BMP4 mRNA and protein expression were significantly higher in CPAM lesions than in adjacent normal lung tissue (P < .05). Overall, the level of BMP4 was higher than that of Wnt7B. Increased expression of Wnt7B and BMP4 appear to be related to the pathogenesis of CPAM and abnormal pulmonary development. Upregulation of Wnt7B and BMP4 could play an important role in the development of the bronchial-alveolar structures that characterize CPAM.

Epithelial Wnt10a Is Essential for Tooth Root Furcation Morphogenesis

WNT10A (Wingless-type MMTV integration site family, member 10A) plays a crucial role in tooth development, and patients with biallelic WNT10A mutation and mice lacking Wnt10a show taurodontism. However, whether epithelial or mesenchymal WNT10A controls the initiation of the root furcation formation remains unclear, and the functional significance of WNT10A in regulating root morphogenesis has not been clarified. Here, we investigated how Wnt10a affects tooth root development by generating different tissue-specific Wnt10a conditional knockout mice. Wnt10a knockout in the whole tissue (EIIa-Cre;Wnt10aflox/flox) and in dental epithelium (K14-Cre;Wnt10aflox/flox) led to an absence of or apically located root furcation in molars of mice, a phenotype that resembled taurodontism. An RNAscope analysis showed that the dynamic epithelial and mesenchymal Wnt10a expression pattern occurred during root development. Immunofluorescent staining of E-cadherin and EdU revealed decreased epithelial cell proliferation at the cervical region of the molar in K14-Cre;Wnt10aflox/flox mice at postnatal day 0 (PN0), just before the initiation of root morphogenesis. Interestingly, we found increased pulpal mesenchymal cell proliferation in the presumptive root furcating region of the molar in K14-Cre;Wnt10aflox/flox mice at PN4 and PN7. RNA-seq indicated that among the Wnt ligands with high endogenous expression levels in molars, Wnt4 was increased after epithelial knockout of Wnt10a. The RNAscope assay confirmed that the expression of Wnt4 and Axin2 in the dental papilla of the presumptive root furcating region, where dental pulp overgrowth occurred, was increased in K14-Cre;Wnt10aflox/flox molars. Furthermore, after suppression of the elevated Wnt4 level in K14-Cre;Wnt10aflox/flox molars by Wnt4 shRNA adenovirus and kidney capsule grafts, the root furcation defect was partially rescued. Taken together, our study provides the first in vivo evidence that epithelial Wnt10a guides root furcation formation and plays a crucial role in controlling the organized proliferation of adjacent mesenchymal cells by regulating proper Wnt4 expression during root furcation morphogenesis.

Pirfenidone inhibits epithelial-mesenchymal transition in keloid keratinocytes.

Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-β1; 10ng/mL), with or without pirfenidone (400μg/mL), for an additional 24h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000μg/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000μg/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-β1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-β1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400μg/mL) in both normal and keloid keratinocytes. For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.

RIPK4 suppresses the TGF-β1 signaling pathway in HaCaT cells.

Receptor-interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor-β 1 (TGF-β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF-β signaling during epidermal homeostasis-related events and suppression of RIPK4 expression by TGF-β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor-κB (NF-κB), protein kinase C (PKC), wingless-type MMTV integration site family (Wnt), and (mitogen-activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad-mediated TGF-β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad-mediated TGF-β1 signaling events through suppression of TGF-β1-induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3-Smad4 interaction, nuclear localization, and TGF-β1-induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound-scratch assay demonstrated that RIPK4 suppressed TGF-β1-mediated wound healing through blocking TGF-β1-induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF-β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF-β1 signaling.

Melatonin Attenuates β-Glycerophosphate-Induced Calcification of Vascular Smooth Muscle Cells via a Wnt1/β-Catenin Signaling Pathway.

Background Melatonin has been demonstrated to protect against calcification in cyclosporine nephrotoxicity. The wingless-type MMTV integration site family member 1 (Wnt1)/β-catenin pathway is associated with cardiovascular calcification. This study aimed to explore whether melatonin could attenuate VSMC calcification through regulating the Wnt1/β-catenin signaling pathway. Methods The effects of melatonin on vascular calcification were investigated in vascular smooth muscle cells (VSMCs). Calcium deposits were visualized by Alizarin Red Staining. Calcium content and alkaline phosphatase (ALP) activity were used to evaluate osteogenic differentiation. Western blots were used to measure the expression of runt-related transcription factor 2 (Runx2), α-smooth muscle actin (α-SMA), and cleaved caspase-3. Results Melatonin markedly ameliorated calcium deposition and ALP activity. Runx2 and cleaved caspase-3 were found to be reduced and α-SMA was found to be increased by melatonin, together with a decrease in apoptosis. Immunofluorescence assay revealed a lower Runx2 protein level in the melatonin group. Melatonin treatment significantly decreased the expression of Wnt1 and β-catenin. Treatment with lithium chloride or transglutaminase 2 abrogated the protective effects of melatonin. Conclusion Melatonin can attenuate β-GP-induced VSMC calcification through the suppression of Wnt1/β-catenin system.

Polymorphisms and endometriosis: a systematic review and meta-analyses.

Endometriosis is an estrogen-dependent gynecological disorder that affects at least 10% of women of reproductive age. It may lead to infertility and non-specific symptoms such as chronic pelvic pain. Endometriosis screening and diagnosis are difficult and time-consuming. Late diagnosis (with a delay ranging from 3.3 to 10.7years) is a major problem and may contribute to disease progression and a worse response to treatment once initiated. Efficient screening tests might reduce this diagnostic delay. As endometriosis is presumed to be a complex disease with several genetic and non-genetic pathogenic factors, many researchers have sought to identify polymorphisms that predispose to this condition. We performed a systematic review and meta-analysis of the most regularly reported polymorphisms in order to identify those that might predispose to endometriosis and might thus be of value in screening. The MEDLINE database was searched for English-language publications on DNA polymorphisms in endometriosis, with no date restriction. The PubTator text mining tool was used to extract gene names from the selected publications' abstracts. We only selected polymorphisms reported by at least three studies, having applied strict inclusion and exclusion criteria to their control populations. No stratification based on ethnicity was performed. All steps were carried out according to PRISMA guidelines. The initial selection of 395 publications cited 242 different genes. Sixty-two genes (corresponding to 265 different polymorphisms) were cited at least in three publications. After the application of our other selection criteria (an original case-control study of endometriosis, a reported association between endometriosis and at least one polymorphism, data on women of reproductive age and a diagnosis of endometriosis in the cases established by surgery and/or MRI and confirmed by histology), 28 polymorphisms were eligible for meta-analysis. Only five of the 28 polymorphisms were found to be significantly associated with endometriosis: interferon gamma (IFNG) (CA) repeat, glutathione S-transferase mu 1 (GSTM1) null genotype, glutathione S-transferase pi 1 (GSTP1) rs1695 and wingless-type MMTV integration site family member 4 (WNT4) rs16826658 and rs2235529. Six others showed a significant trend towards an association: progesterone receptor (PGR) PROGINS, interCellular adhesion molecule 1 (ICAM1) rs1799969, aryl-hydrocarbon receptor repressor (AHRR) rs2292596, cytochrome family 17 subfamily A polypeptide 1 (CYP17A1) rs743572, CYP2C19 rs4244285 and peroxisome proliferator-activated receptor gamma (PPARG) rs1801282), and 12 showed a significant trend towards the lack of an association: tumor necrosis factor (TNF) rs1799964, interleukin 6 (IL6) rs1800796, transforming growth factor beta 1 (TGFB1) rs1800469, estrogen receptor 1 (ESR1) rs2234693, PGR rs10895068, FSH receptor (FSHR) rs6166, ICAM1 rs5498, CYP1A1 rs4646903, CYP19A1 rs10046, tumor protein 53 (TP53) rs1042522, X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) rs25487 and serpin peptidase inhibitor clade E member 1 (SERPINE1) rs1799889; however, for the 18 polymorphisms identified in the latter two groups, further studies of the potential association with the endometriosis risk are needed. The remaining five of the 28 polymorphisms were not associated with endometriosis: glutathione S-transferase theta 1 (GSTT1) null genotype, vascular endothelial growth factor alpha (VEGFA) rs699947, rs833061, rs2010963 and rs3025039. By carefully taking account of how the control populations were defined, we identified polymorphisms that might be candidates for use in endometriosis screening and polymorphisms not associated with endometriosis. This might constitute the first step towards identifying polymorphism combinations that predispose to endometriosis (IFNG (CA) repeat, GSTM1 null genotype, GSTP1 rs1695, WNT4 rs16826658 and WNT4 rs2235529) in a large cohort of patients with well-defined inclusion criteria. In turn, these results might improve the diagnosis of endometriosis in primary care. Lastly, our present findings may enable a better understanding of endometriosis and improve the management of patients with this disease.

GPX2 silencing relieves epithelial-mesenchymal transition, invasion, and metastasis in pancreatic cancer by downregulating Wnt pathway.

Glutathione peroxidase 2 (GPX2) participates in many cancers including pancreatic cancer (PC), and overexpression of GPX2 promotes tumor growth. Herein, we identified the role of GPX2 in epithelial-mesenchymal transformation (EMT), invasion, and metastasis in PC. Bioinformatics prediction was applied to select PC-related genes. The regulatory function of GPX2 in PC was explored by treatment with short hairpin RNA against GPX2 or LiCl (activator of wingless-type MMTV integration site [Wnt] pathway) in PC cells. GPX2 level in PC tissues, the levels of GPX2, β-catenin, Vimentin, Snail, epithelial-cadherin (E-cadherin), matrix metalloproteinase 2 (MMP2), MMP9, and Wnt2 in cells were determined. Subsequently, cell proliferation, invasion, and metastasis were assayed. Bioinformatics analysis revealed that GPX2 was involved in PC development mediated by the Wnt pathway. GPX2 was highly expressed in PC tissues. GPX2 silencing downregulated levels of β-catenin, Vimentin, Snail, MMP2, MMP9, and Wnt2 but upregulated levels of E-cadherin. It was confirmed that GPX2 silencing suppressed PC cell proliferation, metastasis, and invasion. Furthermore, the trend of EMT and invasion and metastasis of PC induced by the LiCl-activated Wnt pathway was reversed when the GPX2 was silenced. GPX2 silencing could inhibit the Wnt pathway, subsequently suppress PC development.

Knockout of TLR4 promotes fracture healing by activating Wnt/β-catenin signaling pathway

ObjectivesThe aim of this study was to investigate the effect of Toll like receptor 4 (TLR4) on fracture healing. MethodsThe open tibial fracture models in TLR4 knockout (TLR4−/−) and wild type (WT) C57BL-6 J mice were established. The radiological examination, tartrate-resistant acid phosphatase (TRAP) staining, Micro-CT scan and biological torsion test were performed on 7, 14 and 21 days after operation. Enzyme Linked Immunosorbent Assay (ELISA) kit was used to detect the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β) and interleukin 6 (IL-6). Western blotting was used to detect the expression of β-catenin, Wingless-type MMTV integration site family, member 4 and 5B (Wnt4 and Wnt5B), proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-2 (BMP-2) of the callus tissue obtained from mice. ResultsTLR4 knockout promoted fracture healing, reduced the number of osteoclasts, increased bone callus volume (BV) and callus mineralized volume fraction (BV/TV%) (P < 0.05), increased the maximum torque and torsional stiffness of callus (P < 0.05), reduced TNF-α, IL-1β and IL-6 expression (P < 0.01), and increased the expression levels of β-catenin, Wnt4, Wnt5B, PCNA and BMP-2 (P < 0.01). ConclusionTLR4 knockout reduced inflammatory and promoted fracture healing by activating Wnt/β-catenin signaling pathway.

Wingless-type MMTV integration site family member 5a: a novel biomarker regulated in type 2 diabetes mellitus and diabetic kidney disease

ObjectivesType 2 diabetes mellitus (T2DM) is sustained by insulin resistance (IR) and reduced β-cell mass, which is largely due to insulin secretory dysfunction. Wnt5a protein is essential to islet formation and β-cell migration in the development of pancreas in vertebrates. Levels of the Wnt5a protein antagonist plasma secreted frizzled-related protein 5 (Sfrp5) were elevated in patients with T2DM. However, the association between Wnt5a, T2DM patients and diabetic kidney disease (DKD) is unknown. We aim to investigate the circulating Wnt5a levels in in different clinical stages of T2DM and evaluate its correlation of duration of diabetes mellitus chronic complication.MethodsA total of 329 participants (187 males, 142 females; age range 40 to 80 years) were enrolled in this study. Serum Wnt5a levels were measured by an enzyme-linked immunosorbent assay (ELISA). The demographic and clinical parameters evaluated in subjects with new onset T2DM, onset T2DM after treatment and DKD at different clinical phases.ResultsWnt5a was significantly down-regulated in newly diagnosed T2DM patients and gradually increased after 3 months of treatment. Interesting, serum wnt5a was gradually increased in patients with long-term diabetes and kidney disease compared to patients with T2DM and onset DKD.ConclusionsWe speculated that the Wnt5a protein might regulate islet function and be involved in the onset of diabetes as a protective factor. It may be one of the inflammatory factors adversely involved in the progression of diabetic nephropathy.