Expression Of Wnt10b Research Articles

Vulpinic Acid Controls Stem Cell Fate toward Osteogenesis and Adipogenesis.

Vulpinic acid, a naturally occurring methyl ester of pulvinic acid, has been reported to exert anti-fungal, anti-cancer, and anti-oxidative effects. However, its metabolic action has not been implicated yet. Here, we show that vulpinic acid derived from a mushroom, Pulveroboletus ravenelii controls the cell fate of mesenchymal stem cells and preadipocytes by inducing the acetylation of histone H3 and α-tubulin, respectively. The treatment of 10T1/2 mesenchymal stem cells with vulpinic acid increased the expression of Wnt6, Wnt10a, and Wnt10b, which led to osteogenesis inhibiting the adipogenic lineage commitment, through the upregulation of H3 acetylation. By contrast, treatment with vulpinic acid promoted the terminal differentiation of 3T3-L1 preadipocytes into mature adipocytes. In this process, the increase in acetylated tubulin was accompanied, while acetylated H3 was not altered. As excessive generation of adipocytes occurs, the accumulation of lipid drops was not concentrated, but dispersed into a number of adipocytes. Consistently, the expressions of lipolytic genes were upregulated and inflammatory factors were downregulated in adipocytes exposed to vulpinic acid during adipogenesis. These findings reveal the multiple actions of vulpinic acid in two stages of differentiation, promoting the osteogenesis of mesenchymal stem cells and decreasing hypertrophic adipocytes, which can provide experimental evidence for the novel metabolic advantages of vulpinic acid.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis invitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway

BackgroundThe NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway.MethodsReverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/β-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo.ResultsNSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/β-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice.ConclusionIn conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.

CSIG-27. PRC2-INDEPENDENT FUNCTION OF EZH2/HP1BP3 COMPLEX IN GLIOMA STEM CELLS

Abstract Glioblastoma (GBM) displays cellular and genetical heterogeneity harboring a subpopulation of glioma stem cells (GSCs). Enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, is the core subunit of the polycomb repressor 2 (PRC2) complex, mediates gene transcriptional repression in both normal and tumor stem cells. An oncogenic role of EZH2 as a PRC2-dependent transcriptional silencer is well established; however, non-canonical functions of EZH2 are incompletely understood. Here we found a novel oncogenic mechanism for EZH2 in a PRC2-indenpend way in GSCs. Using HPLC-MS/MS and IP assay, EZH2 bound to HP1BP3 (heterochromatin protein 1 binding protein 3), a heterochromatin-related protein, with its pre-SET domain. Overexpression of H1P3B3 enhanced the proliferation, self-renewal and temozolomide (TMZ) resistance of GBM cells. Intriguingly, H1PBP3 was up-regulated in high grade gliomas with proneural (PN) subtypes and had a high predictive value on prognosis in patients with PN gliomas. Furthermore, EZH2 and HP1BP3 co-activated the expression of WNT7B by blocking the methylation of H3K9, thereby increasing TMZ resistance and tumorigenicity of glioblastoma cells. Interestingly, inhibition of WNT7B autocrine via LGK974, a specific porcupine inhibitor, effectively reversed the TMZ resistance of both GSCs and GBM glioma cells expressing HP1BP3. Hence, targeting the PRC2-independent function of EZH2 is an effective approach to enhance the efficacy of treating GBM.

Wnt signaling contributes to withdrawal symptoms from opioid receptor activation induced by morphine exposure or chronic inflammation.

Preventing and treating opioid dependence and withdrawal is a major clinical challenge, and the underlying mechanisms of opioid dependence and withdrawal remain elusive. We hypothesized that prolonged morphine exposure or chronic inflammation-induced μ-opioid receptor activity serves as a severe stress that elicits neuronal alterations and recapitulates events during development. Here, we report that Wnt signaling, which is important in developmental processes of the nervous system, plays a critical role in withdrawal symptoms from opioid receptor activation in mice. Repeated exposures of morphine or peripheral inflammation produced by intraplantar injection of complete Freund's adjuvant significantly increase the expression of Wnt5b in the primary sensory neurons in dorsal root ganglion (DRG). Accumulated Wnt5b in DRG neurons quickly transmits to the spinal cord dorsal horn (DH) after naloxone treatment. In the DH, Wnt5b, acts through the atypical Wnt-Ryk receptor and alternative Wnt-YAP/TAZ signaling pathways, contributing to the naloxone-precipitated opioid withdrawal-like behavioral symptoms and hyperalgesia. Inhibition of Wnt synthesis and blockage of Wnt signaling pathways greatly suppress the behavioral and neurochemical alterations after naloxone-precipitated withdrawal. These findings reveal a critical mechanism underlying naloxone-precipitated opioid withdrawal, suggesting that targeting Wnt5b synthesis in DRG neurons and Wnt signaling in DH may be an effective approach for prevention and treatment of opioid withdrawal syndromes, as well as the transition from acute to chronic pain.

LncRNA TMPO-AS1 serves as a ceRNA to promote osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis.

Osteosarcoma (OS) is a common kind of aggressive tumor in bone which was mostly identified in children and adolescents with extremely high risk of death. Accumulating research works have displayed that long noncoding RNAs (lncRNAs) exert an essential role in the development of multiple cancers. It has been reported that TMPO-AS1 is an oncogene in cancers; nonetheless, its molecular mechanism in OS is totally unclear. Our present study elucidated that a remarkable overexpression of TMPO-AS1 was found in OS tissues and cells. Moreover, TMPO-AS1 depletion restrained Wnt/β-catenin pathway and cell proliferation as well as facilitated cell apoptosis. Further molecular mechanism investigations showed that TMPO-AS1 can sponge to miR-199a-5p. Moreover, miR-199a-5p was at a low level at OS cells. Importantly, miR-199a-5p's overexpression was associated with the OS cells' decreased proliferation and increased apoptosis. In addition, WNT7B was confirmed as a downstream gene of miR-199a-5p. Also the WNT7B expression was reversely modulated by miR-199a-5p and positively modulated by TMPO-AS1. Rescue experiments suggested that downregulated WNT7B rescued miR-199a-5p inhibitor-mediated repression on OS progression, but the treatment of LiCl counteracted the effect of WNT7B downregulation. In a word, TMPO-AS1 serves as a competing endogenous RNA to boost osteosarcoma tumorigenesis by regulating miR-199a-5p/WNT7B axis, which provided an underlying therapeutic target for patients with OS.

Sevoflurane Inhibited Osteosarcoma Cell Proliferation And Invasion Via Targeting miR-203/WNT2B/Wnt/β-Catenin Axis.

BackgroundOsteosarcoma is one of the most common primary bone cancers with predominant occurrence in children and adolescents. This study aimed to determine the effects of sevoflurane treatment on the osteosarcoma progression and to explore the underlying molecular mechanisms.Materials and methodsThe mRNA and protein expression levels were determined by qPCR and Western blot, respectively. Osteosarcoma cell proliferation, apoptosis and invasion were determined by MTT, caspase-3 activity, colony formation and Transwell invasion assays, respectively. The interaction between miR-203 and WNT2B 3ʹ untranslated region was confirmed by luciferase reporter assay.ResultsSevoflurane treatment for 6 hrs concentration-dependently suppressed cell viability, increased caspase-3 activity and up-regulated miR-203 expression in both U2OS and MG63 cells. MiR-203 overexpression suppressed cell viability, increased caspase-3 activity and suppressed cell growth and invasion of osteosarcoma cells. In addition, miR-203 knockdown attenuated the tumor-suppressive effects of sevoflurane treatment on osteosarcoma cells. Mechanistic studies showed that miR-203 repressed the expression of WNT2B in U2OS cells, and inhibition of miR-203 attenuated the suppressive effects of sevoflurane on WNT2B expression. More importantly, WNT2B overexpression attenuated the effects of sevoflurane treatment on cell viability, caspase-3 activity, cell growth and invasion of U2OS cells. MiR-203 overexpression suppressed Wnt/β-catenin signalling. Similarly, sevoflurane suppressed the activity of Wnt/β-catenin signalling, which was partially reversed by miR-203 knockdown and WTN2B overexpression.ConclusionOur data showed the tumor-suppressive effects of sevoflurane on osteosarcoma cells, and mechanistic studies revealed that sevoflurane inhibited osteosarcoma cell proliferation and invasion partly via targeting the miR-203/WNT2B/Wnt/β-catenin axis.

Higher Plasma Sclerostin and Lower Wnt Signaling Gene Expression in White Adipose Tissue of Prediabetic South Asian Men Compared with White Caucasian Men.

BackgroundSouth Asians generally have an unfavourable metabolic phenotype compared with white Caucasians, including central obesity and insulin resistance. The Wnt protein family interacts with insulin signaling, and impaired Wnt signaling is associated with adiposity and type 2 diabetes mellitus. We aimed to investigate Wnt signaling in relation to insulin signaling in South Asians compared with white Caucasians.MethodsTen Dutch South Asian men with prediabetes and overweight or obesity and 10 matched Dutch white Caucasians were included. Blood samples were assayed for the Wnt inhibitor sclerostin. Subcutaneous white adipose tissue (WAT) and skeletal muscle biopsies were assayed for Wnt and insulin signaling gene expression with quantitative reverse transcription polymerase chain reaction (Clinicaltrials.gov NCT02291458).ResultsPlasma sclerostin was markedly higher in South Asians compared with white Caucasians (+65%, P<0.01). Additionally, expression of multiple Wnt signaling genes and key insulin signaling genes were lower in WAT in South Asians compared with white Caucasians. Moreover, in WAT in both ethnicities, Wnt signaling gene expression strongly positively correlated with insulin signaling gene expression. In skeletal muscle, WNT10B expression in South Asians was lower, but expression of other Wnt signaling and insulin signaling genes was comparable between ethnicities. Wnt and insulin signaling gene expression also positively correlated in skeletal muscle, albeit less pronounced.ConclusionSouth Asian men with overweight or obesity and prediabetes have higher plasma sclerostin and lower Wnt signaling gene expression in WAT compared with white Caucasians. We interpret that reduced Wnt signaling could contribute to impaired insulin signaling in South Asians.

CD8+ T lymphocytes enhance the anabolic effect of intermittent parathyroid hormone on cementoblasts

Root resorption is usually inflammatory in nature and has a tight link with immune system. Intermittent parathyroid hormone (iPTH) could promote cementum regeneration. The cross-talk of immune cells and cementoblasts may play an important role in the regeneration which stayed to be elucidated. In this study, a CD8+ T cells-OCCM-30 cells coculture system was established in vitro to investigate whether CD8+ T cells could enhance the anabolic effect of iPTH on cementoblasts and to find out the potential link of the effect with Wnt signal pathway. Determined by real-time PCR and Western Blot, we found an amplified cementogenesis in the OCCM-30 cells from coculture system, including increased mRNA and protein expression of Alp, Opn and Runx2, ALP activity and mineralization. We also found iPTH could increase the expression of Wnt10b in CD8+ T cells by ELISA. In addition, Wnt10b would promote the proliferation of OCCM-30 cells, while the effect on differentiation was various in different culture medium. These results demonstrated that the stimulating effect of iPTH on cementoblasts could be mediated through an interaction with CD8+ T cells, and T-cell-induced Wnt10b might be a key mechanism in the mediation.

WNT5B exerts oncogenic effects and is negatively regulated by miR-5587-3p in lung adenocarcinoma progression.

WNT5B glycoprotein belongs to the Wnt protein family. Limited investigations revealed a possible role of WNT5B in malignancies, such as triple-negative breast cancer and oral squamous cell carcinoma. However, whether WNT5B contributes to the progression of lung adenocarcinoma (LAD) remains unclear. Here, we initially determine that WNT5B is highly expressed in LAD and is positively correlated with lymph node metastasis and TNM stage. Consistently, clinical analysis reveals WNT5B as an independent prognostic biomarker in LAD. Silencing WNT5B suppresses the proliferation of LAD both in vitro and in vivo by interfering G1/S cell-cycle progression and modulating amino acid metabolism, revealing its remarkable oncogenic role in LAD. Of note, we also identified miR-5587-3p as a negative upstream regulator of WNT5B in LAD, which may help develop therapies targeting LAD patients with high WNT5B expression. Taken together, our results revealed an oncogenic role of WNT5B in LAD, which could be a prognostic biomarker and promising therapeutic target for LAD patients.

MiR-342-5p inhibits osteosarcoma cell growth, migration, invasion, and sensitivity to Doxorubicin through targeting Wnt7b

ABSTRACT Osteosarcoma (OS) accounts for 9 percent of cancer-related deaths in young people. The PI3K/Akt signaling, a well-known carcinogenic signaling pathway in human cancer, cooperates with other signaling pathways such as Wnt signaling to promote cancer progression. Wnt7b, as a transforming member of the Wnt family, could activate mTORC1 through PI3K-AKT signaling and is upregulated in OS. In the present study, we found that miR-342-5p inhibits Wnt7b expression via direct binding to Wnt7b 3′-UTR. miR-342-5p overexpression remarkably suppressed the viability and invasion while enhanced the apoptosis of OS cells; meanwhile, Wnt7b, β-catenin, c-myc, and cyclin D1 proteins were reduced while E-cadherin protein showed to be increased. Consistent with its expression pattern, Wnt7b exerted oncogenic effects on OS cells. Wnt7b could significantly attenuate the impacts of miR-342-5p. In conclusion, we demonstrated a miR-342-5p/Wnt7b axis that regulates the capacity of OS cells to proliferate and to invade through Wnt/β-catenin signaling. The miR-342-5p/Wnt7b axis might be novel targets for OS targeted therapy, which needs further in vivo and clinical investigations.

MicroRNA-19b restricts Wnt7b to the hem, which induces aspects of hippocampus development in the avian forebrain

The functions of the hippocampus are conserved between birds and mammals; however, it is not known whether similar mechanisms are responsible for its development in these two classes. In mammals, hippocampus development is known to be regulated by the hem organizer. Here, we have identified that, in birds, Wnt7b secreted from the hem is sufficient for inducing the expression of hippocampal markers. Furthermore, we have demonstrated that a microRNA, miR-19b, which is selectively excluded from the hem region, is necessary and sufficient for restricting the expression of Wnt7b to the hem. This study suggests that the role of the Wnt signal emanating from the hem is conserved between birds and mammals, and that a microRNA-based mechanism is crucial for determining the position of the hippocampus.

OS12.4 In vivo dynamics and targeting of vessel co-option in glioma

Abstract BACKGROUND Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. MATERIAL AND METHODS Here, we intravitally study preclinical syngenetic models of glioma as well as patient-derived cells transplanted orthotopically. Moreover, we profoundly confirm our preclinical results with histological studies on patient specimens. RESULTS We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. CONCLUSION Here, we show that glioma is able to employ vessel co-option, i.e. the movement of tumor cells towards and along the pre-existing vasculature. Glioma oligodendrocyte-like (OPCL) cells express Wnt7 that is necessary for vessel co-option and Wnt inhibitors significantly improve survival with temozolomide. Moreover, we demonstrated that anti-VEGF-treatment of glioma selects for Olig2/Wnt7+ cells

The role of WNT10B in normal prostate gland development and prostate cancer

WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

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

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. 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. 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. 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.

Aging of spermatogonial stem cells by Jnk-mediated glycolysis activation

Because spermatogonial stem cells (SSCs) are immortal by serial transplantation, SSC aging in intact testes is considered to be caused by a deteriorated microenvironment. Here, we report a cell-intrinsic mode of SSC aging by glycolysis activation. Using cultured SSCs, we found that aged SSCs proliferated more actively than young SSCs and showed enhanced glycolytic activity. Moreover, they remained euploid and exhibited stable androgenetic imprinting patterns with robust SSC activity despite having shortened telomeres. Aged SSCs showed increased Wnt7b expression, which was associated with decreased Polycomb complex 2 activity. Our results suggest that aberrant Wnt7b expression activated c-jun N-terminal kinase (JNK), which down-regulated mitochondria numbers by suppressing Ppargc1a Down-regulation of Ppargc1a probably decreased reactive oxygen species and enhanced glycolysis. Analyses of the Klotho-deficient aging mouse model and 2-y-old aged rats confirmed JNK hyperactivation and increased glycolysis. Therefore, not only microenvironment but also intrinsic activation of JNK-mediated glycolysis contributes to SSC aging.

Abstract 1118: FOXQ1 stimulates WNT-signaling through transcriptional activation of WNT5B to promote the epithelial to mesenchymal transition

Abstract Aberrant induction of the epithelial to mesenchymal transition (EMT) is an acknowledged mechanism of acquired metastatic competence and drug resistance in many epithelial cancer types. Both physiological and pathological forms of EMT are thought to occur through a network of transcription factors (TFs) that drive cell plasticity and the acquisition of stem-like traits. The forkhead box transcription factor, FOXQ1, is a potent driver of EMT. FOXQ1 has been shown to be specifically upregulated in the triple negative subtype of breast cancer (TNBC) where is it associated with worse clinical prognosis. However, the role of FOXQ1 within the EMT transcriptional network has not been extensively characterized. In this study, we employed chromatin immunoprecipitation and RNA sequencing in a human mammary luminal epithelial cell line with ectopic FOXQ1 (HMLE/FOXQ1), as a model of breast EMT. We identified FOXQ1 localization within the promoter regions of prominent EMT-TFs, including TWIST1, ZEB1, FOXC2 and SIX2. Further, FOXQ1 direct gene targets are enriched in signaling pathways previously associated with EMT, including the WNT-signaling pathway. Within the WNT pathway, the WNT5B signaling ligand was a direct target for transcriptional activation by FOXQ1. Gene expression analysis in a panel of breast cancer cell lines revealed both FOXQ1 and WNT5B are enriched in the basal B subtype of TNBC. FOXQ1 and WNT5B expression were also found to be upregulated and correlated downstream of TGFβ induced EMT. Disruption of WNT5B expression attenuated EMT-associated stem-like traits, including decreases in mammosphere formation and in the CD44+/CD24- population, and significantly reduced cell invasion. Analysis of breast cancer gene expression from the TCGA revealed that FOXQ1 and WNT5B expression are correlated across all breast samples (r=0.46) and differentially upregulated in ER- breast cancer. Additionally, both FOXQ1 and WNT5B expression were associated with worse recurrence-free survival. Our data supports that WNT5B is a critical FOXQ1 target for mediating EMT features and suggests that disrupting the FOXQ1-WNT5B axis could be a potential strategy to target the EMT cell population in TNBC patients. Citation Format: Allison V. Mitchell, Guojun Wu. FOXQ1 stimulates WNT-signaling through transcriptional activation of WNT5B to promote the epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1118.

Loss of FLCN inhibits canonical WNT signaling via TFE3.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/β inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/β inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression-two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/β inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/β inhibition. Finally, we found that a constitutively active β-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

Osteoclast-Released Wnt-10b Underlies Cinacalcet Related Bone Improvement in Chronic Kidney Disease.

Secondary hyperparathyroidism (SHPT) relates to high turnover bone loss and is responsible for most bone fractures among chronic kidney disease (CKD) patients. Changes in the Wingless/beta-catenin signaling (Wnt/β-catenin) pathway and Wnt inhibitors have been found to play a critical role in CKD related bone loss. A calcimimetic agent, cinacalcet, is widely used for SHPT and found to be similarly effective for parathyroidectomy clinically. A significant decrease in hip fracture rates is noted among US hemodialysis Medicare patients since 2004, which is probably related to the cinacalcet era. In our previous clinical study, it was proven that cinacalcet improved the bone mineral density (BMD) even among severe SHPT patients. In this study, the influence of cinacalcet use on bone mass among CKD mice was determined. Cinacalcet significantly reduced the cortical porosity in femoral bones of treated CKD mice. It also improved the whole-bone structural properties through increased stiffness and maximum load. Cinacalcet increased femoral bone wingless 10b (Wnt10b) expression in CKD mice. In vitro studies revealed that cinacalcet decreased osteoclast bone resorption and increased Wnt 10b release from osteoclasts. Cinacalcet increased bone mineralization when culturing the osteoblasts with cinacalcet treated osteoclast supernatant. In conclusion, cinacalcet increased bone quantity and quality in CKD mice, probably through increased bone mineralization related with osteoclast Wnt 10b secretion.