Transcriptomic analysis and validation reveal the pathogenesis and a novel biomarker of acute exacerbation of chronic obstructive pulmonary disease.

BACKGROUND: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the main factor that leads to the deterioration of the disease. Currently, the diagnosis of AECOPD mainly relies on clinical manifestations, good predictors or biomarkers are lacking. We aim to reveal specific biomarkers and potential pathogenesis of AECOPD and provide a research basis for the diagnosis and treatment. METHODS: Four patients with AECOPD, four patients with stable COPD, and five control subjects were enrolled for RNA sequencing and KEGG analysis. The mRNA level of target genes was verified by quantitative real-time PCR (qPCR) with an expanded sample size (30 patients with AECOPD, 27 patients with stable COPD, and 35 control subjects). ELISA and immunofluorescence were used to identify the target proteins. Furthermore, the expression and function of WNT/ß-catenin signaling pathway were assessed in animal models of COPD. RESULTS: RNA sequencing showed that 54 genes were up-regulated and 111 genes were down-regulated in the AECOPD. Differentially expressed genes were mainly enriched in WNT signaling pathway, et al. QPCR revealed that multi-genes of the WNT/ß-catenin signaling were significantly down-regulated in AECOPD (P < 0.05), and ß-catenin protein was significantly decreased in plasma of AECOPD and stable COPD (P < 0.01), while phosphorylated ß-catenin was significantly up-regulated in peripheral blood mononuclear cells of AECOPD (P < 0.05). Similarly, WNT ligands, WNT receptors, and downstream signaling molecules were down-regulated, with an increased phosphorylated ß-catenin protein in animal models of COPD. Activation of WNT/ß-catenin signaling pathway by lithium chloride reduced the expression of phosphorylated ß-catenin and ameliorated the COPD-like airway inflammation in mice. CONCLUSION: WNT/ß-catenin signaling pathway is down-regulated in AECOPD patients and in animal models of COPD. Increased expression of phosphorylated ß-catenin in the blood might be a potential biomarker of AECOPD. Activation of WNT/ß-catenin pathway may also represent a therapeutic target for AECOPD.

Curcumin regulates EZH2/Wnt/ß-Catenin pathway in the mandible and femur of ovariectomized osteoporosis rats.

Osteoporosis (OP) behaves in different manners in different parts of the skeleton. This study aims to investigate the effects of curcumin on bone mass of the mandibular and femur from ovariectomized OP rats and to validate whether enhancer of zeste homolog 2 (EZH2)/Wnt/ß-Catenin pathway is involved in this process. Curcumin was administered intragastrically into ovariectomized rats for 12 weeks. The bone parameters and the morphology of the trabecular bone of the left mandible and left femur were assessed by micro-computed tomography assay. Morphological changes of the left mandible and left femur were evaluated by hematoxylin and eosin staining. The mRNA levels of EZH2, ß-Catenin, and Runx2 in the right mandible and right femur were examined by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to assess EZH2 expression. Both the mandible and femur exhibited OP-like changes in ovariectomized rats, while the mandible bone resorption was less than the femur bone resorption. Curcumin intragastric administration improved bone microstructure and promoted bone formation in the mandible and femur. Curcumin inhibited EZH2 mRNA level and induced that of ß-Catenin and Runx2 in the mandible and femur. Collectively, curcumin exerts protective effects against OP, possibly by regulating the EZH2/Wnt/ß-Catenin pathway.

FZD6 triggers Wnt-signalling driven by WNT10BIVS1 expression and highlights new targets in T-cell acute lymphoblastic leukemia.

Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. In our previous work, we described a recurrent rearrangement involving the WNT10B locus (WNT10BR ), characterized by the expression of WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analyzed an Italian cohort of patients (n = 20) and detected a high incidence (13/20) of WNT10BIVS1 expression. To address genes involved in WNT10B molecular response, we have designed a Wnt-targeted RNA sequencing panel. Identifying Wnt agonists and antagonists, it results that the expression of FZD6, LRP5, and PROM1 genes stands out in WNT10BIVS1 positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as leukemic cell models, which are characterized by the expression of WNT10BIVS1 , we have observed that WNT10B drives major Wnt activation to the FZD6 receptor complex through receipt of ligand. Additionally, short hairpin RNAs (shRNAs)-mediated gene silencing and small molecule-mediated inhibition of WNTs secretion have been observed to interfere with the WNT10B/FZD6 interaction. We have therefore identified that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as a new target in the T-ALL treatment strategy.

Human Amniotic Epithelial Cells Promote the Proliferation of Human Corneal Endothelial Cells by Regulating Telomerase Activity via the Wnt/ß-catenin Pathway.

PURPOSE: Human amniotic epithelial cells (HAECs) have regenerative properties and low immunogenicity, which have enabled their use without immune rejection in regenerative medicine applications, such as wound repair, corneal surgery and burn repair. The aim of this study was to explore the potential role of HAECs in the proliferation of human corneal endothelial cells (HCEnCs) and the possible mechanism of regulation. METHODS: HAECs and HCEnCs were isolated from donated tissue samples and were cultured; the collected HAEC culture medium (HAEC-Me) was added to the human corneal endothelium medium (CEM) to establish the HAEC-CM system. HCEnCs were cultured in CEM, 20%HAEC-Me, 20% HAEC-CM, 20% HAEC-CM supplemented with a GSK-3ß inhibitor TWS119 or CEM supplemented with TWS119. Then, cell proliferation, apoptosis, cell cycle progression, telomerase activity, and Wnt/ß-catenin pathway-related protein levels were assessed. RESULTS: We found that the HCEnCs cultured in the 20% HAEC-CM had increased proliferative capacity, telomerase activity and ß-catenin and Tcf4 expression levels, and they had a decrease in the rate of apoptosis and α-SMA expression when they were compared with the HCEnCs cultured in the 20% HAEC-Me. After GSK-3ß was inhibited by TWS119, HCEnCs cultured in CEM or 20% HAEC-CM had an increased proliferative capacity, telomerase activity, ß-catenin/Tcf4 expression and a decreased α-SMA expression, and they had a decreased apoptotic rate. CONCLUSIONS: These data indicate that the human amniotic epithelial cells microenvironment can promote the proliferation of human corneal endothelial cells, which may be related to regulating telomerase activity and epithelial-to-mesenchymal transition (EMT) via the Wnt/ß-catenin pathway.

Apical ectodermal ridge regulates three principal axes of the developing limb.

Understanding limb development not only gives insights into the outgrowth and differentiation of the limb, but also has clinical relevance. Limb development begins with two paired limb buds (forelimb and hindlimb buds), which are initially undifferentiated mesenchymal cells tipped with a thickening of the ectoderm, termed the apical ectodermal ridge (AER). As a transitional embryonic structure, the AER undergoes four stages and contributes to multiple axes of limb development through the coordination of signalling centres, feedback loops, and other cell activities by secretory signalling and the activation of gene expression. Within the scope of proximodistal patterning, it is understood that while fibroblast growth factors (FGFs) function sequentially over time as primary components of the AER signalling process, there is still no consensus on models that would explain proximodistal patterning itself. In anteroposterior patterning, the AER has a dual-direction regulation by which it promotes the sonic hedgehog (Shh) gene expression in the zone of polarizing activity (ZPA) for proliferation, and inhibits Shh expression in the anterior mesenchyme. In dorsoventral patterning, the AER activates Engrailed-1 (En1) expression, and thus represses Wnt family member 7a (Wnt7a) expression in the ventral ectoderm by the expression of Fgfs, Sp6/8, and bone morphogenetic protein (Bmp) genes. The AER also plays a vital role in shaping the individual digits, since levels of Fgf4/8 and Bmps expressed in the AER affect digit patterning by controlling apoptosis. In summary, the knowledge of crosstalk within AER among the three main axes is essential to understand limb growth and pattern formation, as the development of its areas proceeds simultaneously.

Wingless ligands and beta-catenin expression in the rat endometrium: The role of Wnt3 and Wnt7a/beta-catenin pathway at the embryo-uterine interface.

Wnt/beta-catenin signaling may play an essential role in endometrial decidualization, placentation, and the establishment of pregnancy. We investigate here the possible roles, immunolocalizations, and synthesis of the Wnt3, Wnt7a, and beta-catenin proteins in the rat endometrium during the estrous cycle and early postimplantation period. Wnt3 and Wnt7a had a similar localization and dynamic expression relative to the endometrial stages. Wnt7a immunostaining was not limited only to the luminal epithelial cells, but also to strong stainings in the stromal and endothelial cells. Wnt3, Wnt7a, and beta-catenin were highly synthesized and colocalized at the trophoblast-decidual interface; and were more obvious in the primary decidual zone, the GTCs, and the ectoplacental cone. Beta-catenin was strongly localized at the borders of the mature decidual cells; however, Wnt3 and Wnt7a immunolocalizations were decreased in those cells. As such, the immunolocalization of Wnt3, Wnt7a, and beta-catenin shifted with decidualization and placentation. The expression level of Wnt3, Wnt7a, and beta-catenin messenger RNAs increased in early pregnancy, and especially between Days 8.5 and 9.5. The dramatic changes in the expression of Wnt3, Wnt7a, and beta-catenin observed during the early days of pregnancy and the estrous cycle may indicate their roles in decidualization, stromal cell proliferation, and trophoblast invasion.

Aberrant expression of Wnt/ß-catenin signaling pathway genes in aggressive malignant gastric gastrointestinal stromal tumors.

INTRODUCTION: Recent reports on gene expression profiling (GEP) show several genes associated with malignant progression of GIST. However, genes associated with malignant transformation have not been clarified. Here, we aimed to reveal distinct genes in aggressive malignant GIST, using comprehensive gene expression analysis. MATERIALS AND METHODS: We investigated GEP obtained by microarrays for 43 gastric GISTs, which mostly harbored KIT and PDGFRA mutations and integrated clinicopathological risk information. RT-PCR and immunohistochemistry were performed for FZD7, a receptor of Wnt ligands. RESULTS: GEP divided 43 gastric GISTs into two clusters. A cluster included seven of eight high-risk GISTs (88%) in modified NIH classification and was defined as high-risk cluster; the other cluster was defined as low-risk cluster. The number of probes with over 3-fold changes between the two clusters was 1,177, in which probes corresponding to 16 oncogenes were included. Genes involved in the Wnt signaling pathway were the most abundant among the 16 oncogenes. Focusing on 73 Wnt signaling pathway genes of the 21,578 probes, 12 upregulated and 5 downregulated genes were found in the high-risk cluster. Major cascade genes promoting the Wnt/ß-catenin signaling pathway, including WNT11, FZD family, and DVL2, were upregulated in the high-risk cluster. SNAI1, SNAI2, and BIRC5, which are activated by this pathway and increase cell proliferation, were also upregulated. These gene expression alterations were consistent in the positive direction of this pathway. GISTs in high-risk cluster strongly expressed FZD7. CONCLUSION: Wnt/ß-catenin signaling pathway may play an important role in malignant transformation of indolent GIST.

WNT7A Overexpression Inhibits Growth and Migration of Hepatocellular Carcinoma via the ß-Catenin Independent Pathway.

Background/Aims. Hepatocellular carcinoma (HCC) is the lethal digestive cancer and the second leading cause of cancer death in men worldwide. Wnt7a, a 39Kd secreted glycoprotein composed of 349 amino acids, was reported to be related to various diseases. However, its role in HCC has not been studied yet. In this study, using gene expression data and clinical information obtained from the Oncomine and KMplot database, we acknowledged that WNT7A was underexpressed in HCC cancer tissue compared with normal tissue, and WNT7A underexpression was correlated with the decreased survival rate of HCC patients. The function of Wnt7a in cell viability, apoptosis, and migration was evaluated by biological behavior assay and molecular analysis. The findings revealed that WNT7A overexpression significantly restrained cell viability and migration while enhancing apoptosis. In addition, WNT7A overexpression promoted cell apoptosis by strengthening Caspase-3 activity and inhibited migration by downregulating EMT transcriptional factor Snail. Furthermore, the expression level of SKP2 was significantly downregulating in the WNT7A overexpression group. In conclusion, this study illustrated that overexpression of WNT7A inhibited cell viability and migration, which was likely attributed to the regulation of SKP2/P21.

Neuronal network remodeling and Wnt pathway dysregulation in the intra-hippocampal kainate mouse model of temporal lobe epilepsy.

Mouse models of mesial temporal lobe epilepsy recapitulate aspects of human epilepsy, which is characterized by neuronal network remodeling in the hippocampal dentate gyrus. Observational studies suggest that this remodeling is associated with altered Wnt pathway signaling, although this has not been experimentally examined. We used the well-characterized mouse intrahippocampal kainate model of temporal lobe epilepsy to examine associations between hippocampal neurogenesis and altered Wnt signaling after seizure induction. Tissue was analyzed using immunohistochemistry and confocal microscopy, and gene expression analysis was performed by RT-qPCR on RNA extracted from anatomically micro-dissected dentate gyri. Seizures increased neurogenesis and dendritic arborization of newborn hippocampal dentate granule cells in peri-ictal regions, and decreased neurogenesis in the ictal zone, 2-weeks after kainate injection. Interestingly, administration of the novel canonical Wnt pathway inhibitor XAV939 daily for 2-weeks after kainate injection further increased dendritic arborization in peri-ictal regions after seizure, without an effect on baseline neurogenesis in control animals. Transcriptome analysis of dentate gyri demonstrated significant canonical Wnt gene dysregulation in kainate-injected mice across all regions for Wnt3, 5a and 9a. Intriguingly, certain Wnt genes demonstrated differential patterns of dysregulation between the ictal and peri-ictal zones, most notably Wnt5B, 7B and DKK-1. Together, these results demonstrate regional variation in Wnt pathway dysregulation early after seizure induction, and surprisingly, suggest that some Wnt-mediated effects might actually temper aberrant neurogenesis after seizures. The Wnt pathway may therefore provide suitable targets for novel therapies that prevent network remodeling and the development of epileptic foci in high-risk patients.

Wnt signaling regulates trans-differentiation of stem cell like type 2 alveolar epithelial cells to type 1 epithelial cells.

BACKGROUND: Type 2 alveolar epithelial cells (AT2s) behave as stem cells and show clonal proliferation upon alveolar injury followed by trans-differentiation (TD) into Type 1 alveolar epithelial cells (AT1s). In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process. METHODS: AT2 cells can be isolated from human lungs and cultured in vitro where they undergo TD into AT1s. In the present study we identified signaling pathways involved in the physiological AT2-to-AT1 TD process using Affymetrix microarray, qRT-PCR, fluorescence microscopy, and an in vitro lung aggregate culture. RESULTS: Affymetrix microarray revealed Wnt signaling to play a crucial role in the TD process. Wnt7a was identified as a ligand regulating the AT1 marker, Aquaporin 5 (AQP5). Artificial Neural Network (ANN) analysis of the Affymetrix data exposed ITGAV: Integrin alpha V (ITGAV), thrombospondin 1 (THBS1) and epithelial membrane protein 2 (EMP2) as Wnt signaling targets. CONCLUSIONS: Wnt signaling targets that can serve as potential alveolar epithelial repair targets in future therapies of the gas exchange surface after injury. As ITGAV is significantly increases during TD and is regulated by Wnt signaling, ITGAV might be a potential target to speed up the alveolar healing process.

Fatty degeneration and wnt10b expression in the supraspinatus muscle after surgical repair of torn rotator cuff tendon.

PURPOSE: In the torn rotator cuff muscles, decreased expression of wnt10b prior to elevation of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) has previously been reported. The purpose of this study is to elucidate the expression profiles of these adipogenesis-related genes after rotator cuff detachment and reattachment in a rabbit model. METHODS: We investigated gene expression profiles of PPARγ, C/EBPα, and wnt10b in different parts of rabbit supraspinatus (SSP) muscle after tendon detachment (n = 6 for each time point). In addition, we assessed expression of the same genes after SSP reattachment with different intervals from initial detachment (n = 6). Fatty degeneration of the SSP muscle was examined by Oil red-O staining. Gene expression profiles were examined by quantitative real-time polymerase chain reaction. RESULTS: After SSP detachment, Oil red-O-positive oil deposits increased after 3 weeks. In the SSP reattachment model, numerous Oil red-O-positive cells were present at 5-week reattachment, following 2- and 3-week detachment. PPARγ and C/EBPα messenger ribonucleic acid expression exhibited a significant increase at 2 and 3 weeks after SSP detachment and remained increased at 5-week reattachment after 2- and 3-week detachment. A decreased expression of wnt10b was observed from 1 week after SSP detachment. Expression of wnt10b was recovered not in the central area of the SSP muscle but in the periphery after reattachment. Adipogenic change was not observed when SSP tendon was reattached after 1-week detachment. CONCLUSIONS: These results may suggest that once the adipogenic transcription factors, PPARγ and C/EBPα, were elevated, repair surgery after rotator cuff tear could not prevent the emergence of fat in the SSP muscle.

Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation.

OBJECTIVE: Exostosin-1 (Ext1) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/ß-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/ß-catenin signaling during chondrogenesis remains unexplored. METHOD: Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan, and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by ß-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with Wnt activators CHIR99021 and recombinant WNT3A. RESULTS: Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/ß-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/ß-catenin signaling. Wnt/ß-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. CONCLUSIONS: EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/ß-catenin signaling. Wnt/ß-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/ß-catenin signaling during chondrogenesis.

IQGAP1 promotes pancreatic cancer progression and epithelial-mesenchymal transition (EMT) through Wnt/ß-catenin signaling.

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that participates in several cellular functions, including cytoskeletal regulation, cell adhesion, gene transcription and cell polarization. IQGAP1 has been implicated in the tumorigenesis and progression of several human cancers. However, the role of IQGAP1 in pancreatic ductal adenocarcinoma (PDAC) is still unknown. We found that IQGAP1 expression was an independent prognostic factor for PDAC. IQGAP1 upregulation significantly promoted cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), whereas IQGAP1 downregulation impaired its oncogenic functions. Overexpression of IQGAP1 increased the protein level of Dishevelled2 (DVL2) and enhanced canonical Wnt signaling as evidenced by increased DVL2 level, ß-catenin transcriptional activity, ß-catenin nuclear translocation and expression of the direct target genes of ß-catenin (cyclin D1 and c-myc). In contrast, knockdown of IQGAP1 decreased the level of DVL2 and attenuated Wnt/ß-catenin signaling. In vivo results revealed that IQGAP1 promoted tumor growth and metastasis. Co-immunoprecipitation studies demonstrated that IQGAP1 interacted with both DVL2 and ß-catenin. Moreover, knockdown of DVL2 reversed IQGAP1-induced EMT. Our findings thus confirmed that IQGAP1 could be used as a potential target for PDAC treatment.

Loss of Wnt Secretion by Macrophages Promotes Hepatobiliary Injury after Administration of 3,5-Diethoxycarbonyl-1, 4-Dihydrocollidine Diet.

Exposure of mice to a diet containing 3,5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) induces porphyrin accumulation, cholestasis, immune response, and hepatobiliary damage mimicking hepatic porphyria and sclerosing cholangitis. Although ß-catenin signaling promotes hepatocyte proliferation, and macrophages are a source of Wnts, the role of macrophage-derived Wnts in modulating hepatobiliary injury/repair remains unresolved. We investigated the effect of macrophage-specific deletion of Wntless, a cargo protein critical for cellular Wnt secretion, by feeding macrophage-Wntless-knockout (Mac-KO) and wild-type littermates a DDC diet for 14 days. DDC exposure induced Wnt11 up-regulation in macrophages. Mac-KO mice on DDC showed increased serum alkaline phosphatase, aspartate aminotransferase, direct bilirubin, and histologic evidence of more cell death, inflammation, and ductular reaction. There was impaired hepatocyte proliferation evidenced by Ki-67 immunostaining, which was associated with decreased hepatocyte ß-catenin activation and cyclin-D1 in Mac-KO. Mac-KO also showed increased CD45, F4/80, and neutrophil infiltration after DDC diet, along with increased expression of several proinflammatory cytokines and chemokines. Gene expression analyses of bone marrow-derived macrophages from Mac-KO mice and F4/80+ macrophages isolated from DDC-fed Mac-KO livers showed proinflammatory M1 polarization. In conclusion, this study shows that a lack of macrophage Wnt secretion leads to more DDC-induced hepatic injury due to impaired hepatocyte proliferation and increased M1 macrophages, which promotes immune-mediated cell injury.

MiR-148a suppressed cell invasion and migration via targeting WNT10b and modulating ß-catenin signaling in cisplatin-resistant colorectal cancer cells.

Cancer stem cells (CSCs) are suggested to be responsible for high recurrence rate and metastasis of colorectal cancer (CRC). Identifying novel targets that can suppress CSCs proliferation and metastasis may provide novel approach to combat against CRC. In the present study, we examined the role of miR-148a in cisplatin-resistant CRC cells with enhanced stem cell marker expression and explored the underlying mechanisms. In this study, we used cisplatin to selectively enrich cisplatin-resistant CRC cells from SW480 cell line, and these selected cisplatin-resistant SW480 cells were with significantly enhanced expression of stem cell markers and increased chemoresistance. MicroRNA (miRNA) array and qRT-PCR assay identified the down-regulation of miR-148a in cisplatin-resistant SW480 cells. Overexpression of miR-148a suppressed expression of stem cell markers, inhibited sphere formation, invasion and migration, induced apoptosis, and reduced chemo-resistance in cisplatin-resistant SW480 cells. Bioinformatics prediction and luciferase reporter assay revealed that WNT10b was a downstream target of miR-148a, and overexpression of miR-148a suppressed WNT10b expression and ß-catenin signaling activities. Enforced expression WNT10b attenuated the effects of miR-148a on cisplatin-resistant SW480 cells sphere formation, invasion and migration. Further study showed that overexpression of miR-148a also suppressed in vivo tumor growth, and WNT10b expression and ß-catenin signaling activities in tumor tissues were suppressed by miR-148a overexpression. In the clinical samples, miR-184a was found to be down-regulated in CRC tissues, down-regulation of miR-148a predicted poor prognosis in CRC patients. In conclusion, our study for the first time enriched the cisplatin-resistant CRC cells with enhanced stem cell marker expression from sphere-forming and chemo-resistant SW480-derived tumor xenografts in immune-deficient mice, and miR-148a suppressed the expression of stem cell markers, increased chemo-sensitivity, cell invasion and migration at least partly via regulating WNT10b and ß-catenin signaling pathway.

Glucocorticoids suppress Wnt16 expression in osteoblasts in vitro and in vivo.

Glucocorticoid-induced osteoporosis is a frequent complication of systemic glucocorticoid (GC) therapy and mainly characterized by suppressed osteoblast activity. Wnt16 derived from osteogenic cells is a key determinant of bone mass. Here, we assessed whether GC suppress bone formation via inhibiting Wnt16 expression. GC treatment with dexamethasone (DEX) decreased Wnt16 mRNA levels in murine bone marrow stromal cells (mBMSCs) time- and dose-dependently. Similarly, Wnt16 expression was also suppressed after DEX treatment in calvarial organ cultures. Consistently, mice receiving GC-containing slow-release prednisolone pellets showed lower skeletal Wnt16 mRNA levels and bone mineral density than placebo-treated mice. The suppression of Wnt16 by GCs was GC-receptor-dependent as co-treatment of mBMSCs with DEX and the GR antagonist RU-486 abrogated the GC-mediated suppression of Wnt16. Likewise, DEX failed to suppress Wnt16 expression in GR knockout-mBMSCs. In addition, Wnt16 mRNA levels were unaltered in bone tissue of GC-treated GR dimerization-defective GR dim mice, suggesting that GCs suppress Wnt16 via direct DNA-binding mechanisms. Consistently, DEX treatment reduced Wnt16 promoter activity in MC3T3-E1 cells. Finally, recombinant Wnt16 restored DEX-induced suppression of bone formation in mouse calvaria. Thus, this study identifies Wnt16 as a novel target of GC action in GC-induced suppression of bone formation.

BATF regulates the expression of Nfil3, Wnt10a and miR155hg for efficient induction of antibody class switch recombination in mice.

BATF functions in T cells and B cells to control the host response to antigen and promote the production of class switched immunoglobulins. In this study, we demonstrate that BATF expression increases rapidly, and transiently, following B cell stimulation and use an inducible murine model of BATF deletion to show that this induction is necessary, and sufficient, for immunoglobulin (Ig) class switch recombination (CSR). We examine two genes (Nfil3 and miR155gh) that are positively regulated, and one gene (Wnt10a) that is negatively regulated by BATF during CSR. These genes play essential roles in CSR and each impacts the expression and/or function of the others. Our observations allow these targets of BATF regulation to be positioned in a network upstream of the activation of germline transcripts (GLT) from the IgH locus and of transcriptional activation of Aicda - the gene encoding the enzyme directing Ig gene rearrangements. This work extends the knowledge of the molecular control of CSR and, importantly, positions the induction and function of BATF as an early event in this process.

Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/ε.

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazole-based CK1 inhibitors, we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a plausible binding mode of IWP-2 in the ATP binding pocket of CK1δ which was confirmed by X-ray analysis. In vitro kinase assays demonstrated IWPs to be ATP-competitive inhibitors of wtCK1δ. IWPs also strongly inhibited the gatekeeper mutant M82FCK1δ. When profiled in a panel of 320 kinases, IWP-2 specifically inhibited CK1δ. IWP-2 and IWP-4 also inhibited the viability of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWP-derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but also might influence CK1δ/ε-related pathways.

Spatial and Single-Cell Transcriptional Profiling Identifies Functionally Distinct Human Dermal Fibroblast Subpopulations.

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.