Nuclear Β-catenin Protein Levels Research Articles

MiR-575/RIPK4 axis modulates cell cycle progression and proliferation by inactivating the Wnt/β-catenin signaling pathway through inhibiting RUNX1 in colon cancer.

Receptor interacting protein serine/threonine kinase 4 (RIPK4) is widely involved in human cancer development. Nevertheless, its role in colon cancer (COAD) has not been elucidated till now. Our research aimed at exploring the function and underlying molecular mechanism of RIPK4 in COAD progression. Through bioinformatic analyses and RT-qPCR, RIPK4 was discovered to be increased in COAD cells and tissues, and its high level predicted poor prognosis. Loss-of-function assays revealed that RIPK4 silencing suppressed COAD cell growth, induced cell cycle arrest, and enhanced cell apoptosis. In vivo experiments also proved that tumor growth was inhibited by silencing of RIPK4. Luciferase reporter assay validated that RIPK4 was targeted and negatively regulated by miR-575. Western blotting demonstrated that Wnt3a, phosphorylated (p)-GSK-3β, and cytoplasmic and nuclear β-catenin protein levels, β-catenin nuclear translocation, and Cyclin D1, CDK4, Cyclin E, and c-Myc protein levels were reduced by RIPK4 knockdown, which however was reversed by treatment with LiCl, the Wnt/β-catenin pathway activator. LiCl also offset the influence of RIPK4 knockdown on COAD cell growth, cell cycle process, and apoptosis. Finally, RIPK4 downregulation reduced RUNX1 level, which was upregulated in COAD and its high level predicted poor prognosis. RIPK4 is positively associated with RUNX1 in COAD. Overexpressing RUNX1 antagonized the suppression of RIPK4 knockdown on RUNX1, Wnt3a, p-GSK-3β, cytoplasmic β-catenin, nuclear β-catenin, Cyclin D1, CDK4, Cyclin E, and c-Myc levels. Collectively, miR-575/RIPK4 axis repressed COAD progression via inactivating the Wnt/β-catenin pathway through downregulating RUNX1.

Immunity-related long noncoding RNA WDFY3-AS2 inhibited cell proliferation and metastasis through Wnt/β-catenin signaling in oral squamous cell carcinoma

ObjectiveLong noncoding RNA WDFY3-AS2 has been shown to play dual roles in the modulation of cancer progression. This study aimed at clarifying the biological role of WDFY3-AS2 as well as the association between WDFY3-AS2 expression, β-catenin expression, and OSCC immunity in oral squamous cell carcinoma (OSCC). DesignBioinformatics analyses, CCK8, EdU, wound healing, transwell, RT-qPCR, western blot, immunofluorescence, in situ hybridization, and immunohistochemistry assays were adopted for exploring the role of WDFY3-AS2 in OSCC. ResultsBioinformatics analyses showed that WDFY3-AS2 conferred a poor prognosis for OSCC patients. Further analyses identified WDFY3-AS2 as an independent prognostic indicator for OSCC. Moreover, silencing WDFY3-AS2 inhibits OSCC cell proliferation, migration and invasion. Gene set enrichment analysis indicated that WDFY3-AS2 participated in the regulation of Wnt signaling. In addition, WDFY3-AS2 expression was positively associated with β-catenin mRNA levels, the key component of Wnt signaling. Interestingly, WDFY3-AS2 knockdown inhibited β-catenin expression and nuclear translocation, thus suppressing OSCC progression through Wnt signaling. Furthermore, WDFY3-AS2 expression correlated with an immunosuppressive phenotype in the tumor immune microenvironment. In situ hybridization and immunohistochemistry verified that WDFY3-AS2 was positively associated with total and nuclear β-catenin protein levels and negatively associated with CD4 expression. ConclusionsThis study demonstrates that the immunity-associated WDFY3-AS2 augments OSCC proliferation and metastasis through Wnt/β-catenin signaling and may serve as a novel treatment target and a new prognostic factor for OSCC.

IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers.

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.

Calcium-sensing receptor mediates interleukin-1β-induced collagen expression in mouse collecting duct cells.

The mechanisms that underlie the profibrotic effect of interleukin (IL)-1β are complicated and not fully understood. Recent evidence has suggested the involvement of the calcium-sensing receptor (CaSR) in tubular injury. Therefore, the current study aimed to investigate whether CaSR mediates IL-1β-induced collagen expression in cultured mouse inner medullary collecting duct cells (mIMCD3) and to determine the possible downstream signaling effector. The results showed that IL-1β significantly upregulated the expression of type I and III collagens in a concentration- and time-dependent manner. Moreover, CaSR was expressed in mIMCD3 cells, and its expression was increased by increasing the concentrations and times of IL-1β treatment. Selective inhibitors (Calhex231 or NPS2143) or the siRNA of CaSR attenuated the enhanced expression of type I and III collagens. Furthermore, IL-1β increased nuclear β-catenin protein levels and decreased cytoplasmic β-catenin expression in cells. In contrast, blockage of CaSR by the pharmacological antagonists or siRNA could partially attenuate such changes in the IL-1β-induced nuclear translocation of β-catenin. DKK1, an inhibitor of β-catenin nuclear translocation, further inhibited the expression of type I and III collagens in cells treated with IL-1β plus CaSR antagonist. In summary, these data demonstrated that IL-1β-induced collagen I and III expressions in collecting duct cells might be partially mediated by CaSR and the downstream nuclear translocation of β-catenin.

Regular exercise and creatine supplementation prevent chronic mild stress-induced decrease in hippocampal neurogenesis via Wnt/GSK3β/β-catenin pathway.

[Purpose]Chronic stress can lead to mood-related psychomotor behaviors such as despair. Decreased hippocampal neurogenesis has been observed in patients with depression and in animal models of depression. Exercise enhances the population of the new born cells in the dentate gyrus (DG). A few studies have demonstrated that creatine has antidepressant effects in humans. However, the mechanism underpinning these effects is poorly understood. Therefore, we examined whether regular exercise and/or creatine was closely associated with the activity of the Wnt/GSK3β/β-catenin pathway in the hippocampal DG.[Methods]Mice were subjected to 4 weeks of chronic mild stress starting a week prior to the start of a 4-week protocol of treadmill running and creatine supplementation. Tail suspension (TST) and forced swimming tests (FST) were carried out 2 days after the final treadmill running session. Immunohistochemical and western blot analyses were conducted to evaluate hippocampal neurogenesis, GSK3β activity, and nuclear β-catenin protein levels in the DG. Furthermore, Wnt signaling antagonism in the DG using stereotaxic injection was performed.[Results]Chronic mild stress-induced increase in immobility in the TST and FST were restored by treadmill running and/or creatine supplementation. The number of Ki-67+ and doublecortin (DCX)+ cells were decreased by chronic stress, and this decline was reversed by the exercise and supplement regimen, along with the changes in GSK3β activity and nuclear β-catenin protein levels in the DG. Local antagonism of DG Wnt signaling caused an increase in immobility even 5 days after injection with C59.[Conclusion]Regular exercise combined with creatine supplementation had a greater effect on hippocampal neurogenesis via the Wnt/GSK3β/β-catenin pathway activation compared with each treatment in chronic mild stress-induced behavioral depression.

Loss of the vitamin D receptor in human breast and prostate cancers strongly induces cell apoptosis through downregulation of Wnt/\u03b2-catenin signaling

Vitamin D co-regulates cell proliferation, differentiation and apoptosis in numerous tissues, including cancers. The known anti-proliferative and pro-apoptotic actions of the active metabolite of vitamin D, 1,25-dihydroxy-vitamin D [1,25(OH)2D] are mediated through binding to the vitamin D receptor (VDR). Here, we report on the unexpected finding that stable knockdown of VDR expression in the human breast and prostate cancer cell lines, MDA-MB-231 and PC3, strongly induces cell apoptosis and inhibits cell proliferation in vitro. Implantation of these VDR knockdown cells into the mammary fat pad (MDA-MB-231), subcutaneously (PC3) or intra-tibially (both cell lines) in immune-incompetent nude mice resulted in reduced tumor growth associated with increased apoptosis and reduced cell proliferation compared with controls. These growth-retarding effects of VDR knockdown occur in the presence and absence of vitamin D and are independent of whether cells were grown in bone or soft tissues. Transcriptome analysis of VDR knockdown and non-target control cell lines demonstrated that loss of the VDR was associated with significant attenuation in the Wnt/β-catenin signaling pathway. In particular, cytoplasmic and nuclear β-catenin protein levels were reduced with a corresponding downregulation of downstream genes such as Axin2, Cyclin D1, interleukin-6 (IL-6), and IL-8. Stabilization of β-catenin using the GSK-3β inhibitor BIO partly reversed the growth-retarding effects of VDR knockdown. Our results indicate that the unliganded VDR possesses hitherto unknown functions to promote breast and prostate cancer growth, which appear to be operational not only within but also outside the bone environment. These novel functions contrast with the known anti-proliferative nuclear actions of the liganded VDR and may represent targets for new diagnostic and therapeutic approaches in breast and prostate cancer.

Abstract 2101: Ormeloxifene suppresses the growth of prostate tumor via inhibition of β-catenin induced AR signaling

Abstract Background: Prostate cancer (PrCa) first manifests as an androgen-dependent disease and can be treated with androgen-deprivation therapy. Despite the initial success of androgen ablation therapy, resistance to anti-androgen therapy displays by progression to hormone refractory (androgen-independent) advanced stage PrCa which is primary cause of patient’s death. Main underlying cause for the onset of hormone refractory cancer is ligand independent activation of AR signaling in PrCa cells. It has been shown that β-catenin acts as a non-androgen activator of AR which enhances AR transactivation in PrCa cells. Thus, identification of agents with excellent pharmacokinetics and pharmacodynamics parameters that can inhibit ligand independent activation of AR signaling might be highly useful for the treatment of advanced stage PrCa. Herein, we identified a synthetic molecule, ormeloxifene (ORM), which efficiently represses β-catenin mediated ligand independent activation of AR signaling, thus, inhibits growth and metastatic features of PrCa cells. Methods: Androgen-refractory but AR positive PrCa cell (C4-2) was used as an in vitro and in vivo model systems. Effect of ORM on AR and PSA protein levels was determined by Western blot analysis. Effect of ORM treatment was analyzed on AR and PSA luciferase activities by transiently transfecting the C4-2 cells by AR and PSA luciferase plasmids. Renilla construct was used as an internal control. C4-2 cells nuclear and cytoplasmic lysates were prepared using Active Motif kit. Immunoprecipitation analysis was performed to determine if ORM inhibits physical interaction of β-catenin with AR. Therapeutic efficacy of ORM was evaluated in cell lines and PrCa xenograft mouse models. Results: ORM dose-dependently (10, 15 and 20 µM) inhibited the protein levels of AR and its downstream target protein PSA. ORM (10 and 20μM) treatment also inhibited AR transactivation as determined by decreased promoter activities of AR and its target gene PSA. ORM (10 and 20μM) treatment inhibited protein levels of nuclear β-catenin and physical interaction of β-catenin with AR in PrCa cells. ORM administration dose- dependently (intra-peritoneal; 100 and/or 500µg/mouse; thrice/week) significantly (P<0.01) inhibited growth of C4-2 cells derived xenograft tumors in athymic nude mice. ORM treatment significantly (P<0.01) inhibited the expressions of nuclear AR and β-catenin expressions in xenograft tumor tissues. These ORM treated mice did not show any apparent toxicity in our study. Conclusion: Our study demonstrates that ORM is a potent inhibitor of β-catenin-mediated activation of AR signaling. Based on its safety profile, ORM might be an ideal candidate for repurposing to treat advanced stage PrCa alone or in combination with other therapies. Citation Format: Aditya Ganju, Bilal Bin Hafeez, Mohammed Sikander, Vivek Kumar Kashyap, Murali Mohan Yallapu, Subhash C. Chauhan, Meena Jaggi. Ormeloxifene suppresses the growth of prostate tumor via inhibition of β-catenin induced AR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2017-2101

The activation of Wnt signaling by a STAT6-dependent macrophage phenotype promotes mucosal repair in murine IBD.

The complete repair of the mucosa constitutes a key goal in inflammatory bowel disease (IBD) treatment. The Wnt signaling pathway mediates mucosal repair and M2 macrophages that coordinate efficient healing have been related to Wnt ligand expression. Signal transducer and activator of transcription 6 (STAT6) mediates M2 polarization in vitro and we hypothesize that a STAT6-dependent macrophage phenotype mediates mucosal repair in acute murine colitis by activating the Wnt signaling pathway. Our results reveal an impaired mucosal expression of M2 macrophage-associated genes and delayed wound healing in STAT6(-/-) mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). These mice also exhibited decreased mucosal expression of Wnt2b, Wnt7b, and Wnt10a, diminished protein levels of nuclear β-catenin that is mainly located in crypts adjacent to damage, and reduced mRNA expression of two Wnt/β-catenin target molecules Lgr5 and c-Myc when compared with wild-type (WT) mice. Murine peritoneal macrophages treated with interleukin-4 (IL-4) and polarized toward an M2a phenotype overexpressed Wnt2b, Wnt7b, and Wnt10a in a STAT6-dependent manner. Administration of a Wnt agonist as well as transfer of properly polarized M2a macrophages to STAT6(-/-) mice activated the Wnt signaling pathway in the damaged mucosa and accelerated wound healing. Our results demonstrate that a STAT6-dependent macrophage phenotype promotes mucosal repair in TNBS-treated mice through activation of the Wnt signaling pathway.

Abstract 270: IκB Kinase β Signaling in Adipose Progenitor Cells Promotes Obesity and Metabolic Disorders

Objective: Obesity is associated with a state of chronic low-grade inflammation that is a major contributor to diabetes and cardiovascular diseases. IκB kinase β (IKKβ), a central coordinator of inflammation through activation of NF-κB, has been implicated in the pathogenesis of obesity-associated metabolic disorders. However, the role of IKKβ in adipose tissue development and metabolism remains elusive. We have recently revealed a potential role of IKKβ in the regulation of adipocyte differentiation. This study aims to investigate the impact of adipose progenitor cell IKKβ deficiency in diet-induced obesity and metabolic disorders. Approach and Results: To determine the role of IKKβ in adipose tissue development, we selectively deleted IKKβ in the white adipose lineage. This was attained by using a PDGFRβ-Cre/IKKβ-loxP system. Cell lineage analysis confirmed that PDGFRβ-Cre is active in primary adipose stromal vascular (SV) cells and PDGFRβ-positive SV cells had higher adipogenic potential than did PDGFRβ-negative cells. PDGFRβ-Cre-mediated IKKβ deletion diminished the ability of SV cells to differentiate into adipocytes. By analyzing mice that selectively lacked IKKβ in the white adipose lineage, we found that deficiency of IKKβ protected mice from high-fat diet-induced obesity. Moreover, IKKβ deficient mice had decreased plasma proinflammatory cytokine levels and enhanced insulin sensitivity. Mechanistically, IKKβ deficiency inhibited proteasome-mediated β-catenin ubiquitination and degradation in adipose SV cells, causing an accumulation of nuclear β-catenin protein levels and impaired adipocyte differentiation. Consistently, CRISPR-mediated IKKβ deletion in murine 3T3-L1 preadipocytes also increased nuclear β-catenin protein levels and blocked 3T3-L1 cell differentiation. Conclusions: Our results establish IKKβ as an important regulator of adipogenesis and adipose tissue development. Overnutrition-mediated IKKβ activation may serve as an initial signal that triggers adipose progenitor cell differentiation in response to consumption of a high-fat diet. Inhibition of IKKβ may present as a novel therapeutic approach to combat obesity and diabetes.

HSP105 recruits protein phosphatase 2A to dephosphorylate β-catenin.

The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study.

Nuclear translocation of β-catenin mediates the parathyroid hormone-induced endothelial-to-mesenchymal transition in human renal glomerular endothelial cells.

Emerging evidence shows that increased parathyroid hormone (PTH) accelerates endothelial injury and subsequent organ fibrosis. Although the underlying mechanisms remain largely unknown, the endothelial-to-mesenchymal transition (EndMT) has recently been demonstrated to be a crucial event during fibrotic disorders. Therefore, the present study aimed to investigate whether elevated PTH could induce EndMT in primary human renal glomerular endothelial cells (GECs) and to determine the possible underlying signaling pathway. The expression of EndMT-related markers was determined by real-time PCR, Western blotting, and confocal microscopy. The results showed that PTH receptor (PTHR) was expressed in GECs and its expression was decreased by increasing concentration of PTH. Moreover, PTH significantly inhibited the expression of endothelial marker CD31 and increased the expression of mesenchymal markers FSP1 and α-SMA in concentration- and time-dependent manners. Confocal microscopy revealed an increasing overlap of CD31 with FSP1 in some GECs after PTH treatment. The expression of type I collagen was upregulated by PTH. Furthermore, PTH enhanced the nuclear β-catenin protein levels, and decreased cytoplasmic β-catenin expression in GECs was observed. In contrast, DKK1, an inhibitor of β-catenin nuclear translocation, attenuated such changes in EndMT-related markers induced by PTH. In summary, these data demonstrated that elevated PTH-induced EndMT in human GECs might be partially mediated by the nuclear translocation of β-catenin.

Β-catenin regulates NF-κB activity and inflammatory cytokine expression in bronchial epithelial cells treated with lipopolysaccharide.

In the present study, we demonstrate that lipopolysaccharide (LPS) induces the expression of inflammatory cytokines, including interleukin (IL)-6, IL-8, IL-1β, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 in BEAS-2B human bronchial epithelial cells in a dose- and time-dependent manner. This increase was accompanied by an increased activity of nuclear factor (NF)‑κB. When the expression of β-catenin was analyzed following treatment with LPS, the mRNA level was unaltered; however, the β-catenin protein levels increased with a decrease in phosphorylation at the serine 33/37 residues. Nuclear β-catenin protein levels also increased along with the reporter activity of a β-catenin-responsive TOPFlash vector. To elucidate the regulatory role of β-catenin in the LPS-induced inflammatory response of bronchial epithelial cells, β-catenin production was knocked down using siRNA. Our results revealed that β-catenin protein levels and TOPFlash vector reporter activity were reduced to basal levels by siRNA transfection. In this experimental condition, NF-κB activity, measured by enzyme-linked immunosorbent assay (ELISA), electrophoretic mobility shift assay (EMSA) and an NF-κB responsive reporter assay, was reduced to basal levels. Similarly, LPS-induced inflammatory cytokine expression was reduced almost to basal levels following transfection with β-catenin siRNA. These results demonstrate that β-catenin positively regulates NF-κB activity, as well as the expression of inflammatory cytokines in the inflammatory response of LPS-treated bronchial epithelial cells.

Abstract 055: β-Catenin Mediates an Interplay between Genetic Background and Growth-Regulating Effects of Estrogen in the Healthy Heart

In diseased hearts, estrogen (E2) has been shown to exert anti-hypertrophic actions. Little is known about the role of E2 in the healthy heart. Our initial aim was to characterize structurally and molecularly the effects of E2 in the healthy mouse heart. Two-month-old female C57Bl/6J mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet ( n = 17-18/group). The three-month physiological dose of E2 led to a higher relative heart weight compared with CON ( P < 0.001). We also confirmed increased cardiomyocyte cross-sectional area by E2 ( P < 0.01). No activation of the fetal gene program and no fibrosis were observed. Transcriptome analysis revealed induction of growth-related pathways by E2, such as the Wnt signaling pathway ( n = 5/group; adjusted P < 0.05). To further confirm activation of Wnt/β-catenin signaling, we verified increased nuclear β-catenin protein levels by E2 compared with CON ( P < 0.01) and hypothesized that β-catenin mediates the actions of E2. Cardiac deletion of β-catenin blunted the E2 effects on cardiac growth ( n = 13/group). Surprisingly, in wild-type littermates with the background C57Bl/6N, E2 decreased the relative heart weight and cardiomyocyte cross-sectional area compared with CON ( n = 7-11/group; P < 0.001). This was underlain by decreased nuclear β-catenin protein levels by E2 compared with CON ( P < 0.001). Furthermore, E2 increased glycogen synthase kinase 3β (GSK3β) phosphorylation and the endosomal/autophagosomal markers Rab5, Rab7 and LC3-II in C57Bl/6J but not C57Bl/6N mice. Assessing a polymorphism linked to Snap29 , we confirmed higher Snap29 protein levels in C57Bl/6J than C57Bl/6N mice ( P < 0.01). This could lead to distinct regulation of endosomes and could be the potential cause of the strain difference. In conclusion, E2 regulates cardiac growth through β-catenin in a strain-specific manner. Collectively, we identified a molecular mechanism that demonstrates a divergent response of mouse sub-strains to E2.

Nuclear Translocation of β-catenin is Essential for Glioma Cell Survival

Identification of molecular pathways that are essential for cancer cell survival is vital for understanding the underlying biology, as well as to design effective cancer therapeutics. β-catenin, a multifunctional oncogenic protein, participates in cell development. Its multifaceted functions primarily lie to the subcellular distribution. The present study demonstrated that β-catenin accumulated in the nucleus to a greater extent in high-grade gliomas compared with low-grade gliomas. In addition, nuclear localization correlated with a worse prognosis for patients, as determined by immunohistochemical analysis of 74 glioma samples. Nuclear expression of β-catenin was down-regulated in LN229 and U87 glioma cells by a small molecule inhibitor of β-catenin/TCF4 signaling, demonstrating strongly inhibited β-catenin/TCF4 transcriptional activity and STAT3 luciferase activity, as well as decreased mRNA and protein levels of nuclear β-catenin, TCF4, EGFR, AKT1, AKT2 and STAT3. Furthermore, repressed nuclear translocation of β-catenin resulted in inhibition of proliferation and invasiveness, and also induced apoptosis of glioma cells. Similar results were also observed in vivo; intratumoral injection of such small molecule inhibitor downregulated expression of nuclear β-catenin, TCF4, and components of the EGFR pathway, and also delayed tumor growth in nude mice harboring subcutaneous U87 xenografts. Results from the present study provided evidence that nuclear accumulation of β-catenin participated in malignant progression of gliomas and implicated poor prognosis, highlighting it as a potential therapeutic target for gliomas.

Bone morphogenetic protein-2-induced Wnt/beta-catenin signaling pathway activation through enhanced low-density-lipoprotein receptor-related protein 5 catabolic activity contributes to hypertrophy in osteoarthritic chondrocytes

IntroductionEvents normally taking place in the terminal chondrocyte differentiation in the growth plate are also observed during osteoarthritis (OA) development, suggesting that molecules, such as Wnts and bone morphogenetic proteins (BMPs) regulating chondrocyte activity in the growth plate, may play a key role in osteoarthritis pathogenesis. The aim of the study was to investigate the possible cross-talk between BMP-2 and Wnt/β-catenin pathways in OA progression.MethodsLow-density-lipoprotein receptor-related protein 5 (LRP-5) and 6, BMP-2, -4, and -7, bone morphogenetic protein receptor-IA and IB (BMPR-IA and BMPR-IA), lymphoid enhancer factor-1 (LEF-1), and transcription factor 4 (TCF-4) expression levels were investigated in normal and osteoarthritic chondrocytes. LRP-5, β-catenin (phospho and active form), matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, as well as collagen X (COL10A1) expression levels were evaluated after LRP-5 silencing in BMP-2-treated chondrocytes. The investigation of Smad1/5/8 binding to LRP-5 promoter was assessed with chromatin immunoprecipitation (ChIP). Furthermore, we evaluated the effect of experimental activation of the Wnt/β-catenin pathway with LiCl and LEF-1 silencing, in LiCl-treated chondrocytes, on matrix metalloproteinases (MMPs) 7, 9, 13, 14, ADAMTS-4, 5, and collagen X (COL10A1) expression, as well as possible interactions between LEF-1 and MMPs and COL10A1 promoters by using a ChIP assay.ResultsLRP-5, BMP-2, BMP-4, BMPR-IA, and LEF-1 mRNA and protein expression levels were found to be significantly upregulated in osteoarthritic chondrocytes compared with normal. We showed that treatment of cultured chondrocytes with BMP-2 resulted in increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter. LRP-5 silencing reduced nuclear β-catenin protein levels, MMPs and collagen X expression, whereas increased phospho-β-catenin protein levels in BMP-2-treated chondrocyte. Furthermore, we demonstrated that activation of the Wnt/β-catenin signaling pathway by LiCl and LEF-1 downregulation by using siRNA regulates MMP-9, 13, 14, ADAMTS-5, and COL10A1 expression, evidenced by the observed strong binding of LEF-1 to MMP-9, 13, 14, ADAMTS-5 and COL10A promoters.ConclusionsOur findings suggest, for the first time to our knowledge, that BMP-2-induced Wnt/β-catenin signaling activation through LRP-5 may contribute to chondrocyte hypertrophy and cartilage degradation in osteoarthritis.

Axin2 controls bone remodeling through the β-catenin–BMP signaling pathway in adult mice

To investigate the role of Wnt-beta-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and 12-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear beta-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the beta-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3beta, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2(fx/fx);Bmp4(fx/fx) mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the beta-catenin-BMP2/4-Osx signaling pathway in osteoblasts.

Wdr5 Is Essential for Osteoblast Differentiation

Wdr5 is developmentally expressed in osteoblasts and accelerates osteoblast differentiation in vitro and in vivo. To address whether Wdr5 is essential for osteoblast differentiation, plasmid-based small interfering RNAs were used to stably suppress endogenous Wdr5 protein levels in MC3T3-E1 cells. Reduction of endogenous Wdr5 levels markedly inhibited osteoblast differentiation, evidenced by a significant decrease in alkaline phosphatase activity, Runx-2 and osteocalcin mRNAs, and absence of mineralized matrix formation. Wdr5 suppression also resulted in a reduction of histone H3 lysine 4 trimethylation, confirming its critical role in this modification. Because Wdr5 overexpression enhances canonical Wnt signaling in osteoblasts in vivo, the effects of Wdr5 silencing on this pathway were examined. The expression of the canonical Wnt target gene, c-myc, was decreased, whereas that of sfrp2, which is repressed by Wnt signaling, was increased with Wdr5 knockdown. Although only a minimal increase in apoptosis was observed, the antiapoptotic effect of Wnt signaling was also impaired with Wdr5 silencing. The expression of canonical Wnts was significantly decreased with Wdr5 knockdown, resulting in a decrease in nuclear beta-catenin protein levels. Activation of the canonical Wnt signaling pathway did not overcome the effects of Wdr5 knockdown on the expression of Wnt target genes. Chromatin immunoprecipitation demonstrated that Wdr5 is present on the Wnt1 promoter and on canonical Wnt response elements of the c-myc and Runx-2 promoters. These studies demonstrate that Wdr5 suppression interferes with the canonical Wnt signaling pathway at multiple stages and that optimal Wdr5 levels are required for induction of the osteoblast phenotype.