Phosphorylation Of Β-catenin Research Articles

Vascular inflammaging: Endothelial CEACAM1 expression is upregulated by TNF-α via independent activation of NF-κB and β-catenin signaling.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro-inflammatory cytokine TNF-α. CEACAM1 is critical for aging-associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF-α in detail. Using wildtype (WT) and TNF-α knockout (Tnf-/-) mice, we confirmed that the aging-related upregulation of endothelial CEACAM1 critically depends on TNF-α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF-α time-dependently upregulated CEACAM1 invitro. In pharmacological experiments, we identified an early NF-κB- and a delayed β-catenin-mediated response. Involvement of β-catenin was further substantiated by siRNA-mediated knockdown of the β-catenin-targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co-immunoprecipitation analysis revealed release of β-catenin from adherens junctions by TNF-α. Finally, TNF-α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated β-catenin signaling by inhibiting its degradation via phosphorylation of GSK3β at Ser9 and by increased phosphorylation of β-catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging-related, inflammation-mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging.

TMEM182 inhibits myocardial differentiation of human iPS cells by maintaining the activated state of Wnt/β-catenin signaling through an increase in ILK expression.

Transmembrane protein 182 (TMEM182) is notably abundant in muscle and adipose tissue, but its role in the heart remains unknown. This study examined the contribution of TMEM182 in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. For this, we generated hiPSCs overexpressing TMEM182 in a doxycycline-inducible manner and induced their differentiation into cardiomyocytes. On Day 12 of differentiation, expression of the cardiomyocyte markers, TNNT2 and MYH6, was significantly decreased in TMEM182-overexpressing cells. Additionally, we found that phosphorylation of GSK-3β (Ser9) and β-catenin (Ser552) was increased during TMEM182 overexpression, suggesting activation of Wnt/β-catenin signaling. We further focused on integrin-linked kinase (ILK) as the mechanism by which TMEM182 activates Wnt/β-catenin signaling. Evaluation showed that ILK expression was increased in cells overexpressing TMEM182. These results suggest that TMEM182 maintains Wnt/β-catenin signaling in an activated state after mesoderm formation by increasing ILK expression, thereby suppressing hiPSCs differentiation into cardiomyocytes.

β-Catenin Phosphorylation at Ser552 Impairs Expression of β-Catenin-TCF/LEF Target Genes in Mouse Collecting Duct

β-Catenin Phosphorylation at Ser552 Impairs Expression of β-Catenin-TCF/LEF Target Genes in Mouse Collecting Duct

Oral inoculation of Fusobacterium nucleatum exacerbates ulcerative colitis via the secretion of virulence adhesin FadA

ABSTRACT Fusobacterium nucleatum (F. nucleatum), an anaerobic resident of the oral cavity, is increasingly recognized as a contributing factor to ulcerative colitis (UC). The adhesive properties of F. nucleatum are mediated by its key virulence protein, FadA adhesin. However, further investigations are needed to understand the pathogenic mechanisms of this oral pathogen in UC. The present study aimed to explore the role of the FadA adhesin in the colonization and invasion of oral F. nucleatum in dextran sulphate sodium (DSS)-induced colitis mice via molecular techniques. In this study, we found that oral inoculation of F. nucleatum strain carrying the FadA adhesin further exacerbated DSS-induced colitis, leading to elevated alveolar bone loss, disease severity, and mortality. Additionally, CDH1 gene knockout mice treated with DSS presented increases in body weight and alveolar bone density, as well as a reduction in disease severity. Furthermore, FadA adhesin adhered to its mucosal receptor E-cadherin, leading to the phosphorylation of β-catenin and the degradation of IκBα, the activation of the NF-κB signalling pathway and the upregulation of downstream cytokines. In conclusion, this research revealed that oral inoculation with F. nucleatum facilitates experimental colitis via the secretion of the virulence adhesin FadA. Targeting the oral pathogen F. nucleatum and its virulence factor FadA may represent a promising therapeutic approach for a portion of UC patients.

Structural and functional effects of phosphopriming and scaffolding in the kinase GSK-3β.

Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.

LIM kinase 2 activates cardiac fibroblasts and exacerbates postinfarction left ventricular remodeling via crosstalk between the canonical and non-canonical Wnt pathways

Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of β-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-β treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-β-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.

Synthetic Benzylic Diselenides and Disulfides: Potential Anticancer Activities via Modulation of the ROS-dependent Akt/β-Catenin Signaling Pathway.

The natural and synthetic organodiselenides have garnered much research attention due to their chemotherapeutic and chemopreventive activities. Herein, we describe the synthesis of a series of benzylic diselenides, which were synthesized by coupling the in situ generated disodium diselenide with the corresponding benzylic halides. The diselenides were evaluated for their anticancer activities in the highly aggressive triple-negative breast cancer cells. Preliminary anti-proliferative activities indicated 4-cyano-substituted diselenide 7 to be most potent with an IC50 value of 1.9 ± 0.3 μM. Detailed mechanistic investigations showed that diselenide 7 induces apoptosis and causes G1 phase arrest of the cell cycle. It exhibits anticancer activity by suppressing the Akt/β-catenin signaling pathway. Further control experiments with LiCl (inhibitor of GSK-3β) revealed that down-regulation of β-catenin was promoted by GSK-3β-induced phosphorylation of β-catenin and its subsequent proteasomal degradation. Moreover, the intracellular ROS was found to act as an upstream mediator for the inactivation of the Akt/β-catenin signaling pathway. The present study describing the efficient anticancer activity of a synthetic benzylic diselenide towards triple-negative breast cancer cells through the modulation of ROS-dependent Akt/β-catenin signaling pathway would certainly be helpful in the future towards the development of small-molecule organoselenium compounds for the treatment of cancer.

ADAMTS16 Suppression by MicroRNA-25 Leads to Oncogenic Properties in Renal Cell Carcinoma.

Increasing evidence indicates that microRNAs (miRNAs) play a crucial role in modulating tumor growth. This study is centered on investigating the contribution of miR-25 to the progression of Renal Cell Carcinoma (RCC). The investigators examined the expression levels of miR-25 and ADAMTS16 in RCC samples and cell lines. The association between miR-25 and ADAMTS16 was validated via a luciferase reporter assay. Cell viability, apoptosis, migration, and invasion were evaluated utilizing CCK-8 and flow cytometry techniques, while the expression levels of ADAMTS16, β-catenin, GSK-3β, and p-GSK-3β were assessed through western blot analysis. The investigation revealed elevated expression levels of miR-25 in RCC tissues. Subsequently, ADAMTS16 was identified as a target of miR-25. Increased miR-25 levels were associated with decreased expression of ADAMTS16, resulting in enhanced cell viability and diminished apoptosis. Conversely, inhibition of miR-25 led to decreased cell viability, proliferation, and migration. Additionally, the researchers observed that miR-25 triggered the phosphorylation of GSK-3β and β-catenin while leaving the total GSK-3β level unaffected. This study suggests that miR-25 regulates the expression of ADAMTS16 through the Wnt/β-catenin signaling pathway, providing new insights into the cause and potential treatment of RCC.

Disintegration of Cav-1/β-catenin complex attenuates neuronal death after ischemia-reperfusion injury by promoting β-catenin nuclear translocation.

The roles of Caveolin-1 (Cav-1) and the Wnt/β-catenin signaling pathways in cerebral ischemia-reperfusion (I/R) injury are well established. The translocation of β-catenin into the nucleus is critical for regulating neuronal apoptosis, repair, and neurogenesis within the ischemic brain. It has been reported that the scaffold domain of Caveolin-1 (Cav-1) (residues 95-98) interacts with β-catenin (residues 330-337). However, the specific contribution of the Cav-1/β-catenin complex to I/R injury remains unknown. To investigate the mechanism underlying the involvement of the Cav-1/β-catenin complex in the subcellular translocation of β-catenin and its subsequent effects on cerebral I/R injury, we treated ischemic brains with ASON (Cav-1 antisense oligodeoxynucleotides) or FTVT (a competitive peptide antagonist of the Cav-1 and β-catenin interaction). Our study demonstrated that the binding of Cav-1 to β-catenin following I/R injury prevented the nuclear accumulation of β-catenin. Treatment with ASON or FTVT after I/R injury significantly increased the levels of nuclear β-catenin. Furthermore, ASON reduced the phosphorylation of β-catenin at Ser33, Ser37, and Thr41, which contributes to its proteasomal degradation, while FTVTincreased phosphorylation at Tyr333, which is associated with its nuclear translocation. The above results indicate that the formation of the Cav-1/β-catenin complex anchors β-catenin in the cytoplasm following I/R injury. Additionally, both ASON and FTVT treatments attenuated neuronal death in ischemic brains. Our study suggests that targeting the interaction between Cav-1 and β-catenin serve as a novel therapeutic strategy to protect against neuronal damage during cerebral injury.

Increased ONECUT2 induced by Helicobacter pylori promotes gastric cancer cell stemness via an AKT-related pathway

Helicobacter pylori (HP) infection initiates and promotes gastric carcinogenesis. ONECUT2 shows promise for tumor diagnosis, prognosis, and treatment. This study explored ONECUT2’s role and the specific mechanism underlying HP infection-associated gastric carcinogenesis to suggest a basis for targeting ONECUT2 as a therapeutic strategy for gastric cancer (GC). Multidimensional data supported an association between ONECUT2, HP infection, and GC pathogenesis. HP infection upregulated ONECUT2 transcriptional activity via NFκB. In vitro and in vivo experiments demonstrated that ONECUT2 increased the stemness of GC cells. ONECUT2 was also shown to inhibit PPP2R4 transcription, resulting in reduced PP2A activity, which in turn increased AKT/β-catenin phosphorylation. AKT/β-catenin phosphorylation facilitates β-catenin translocation to the nucleus, initiating transcription of downstream stemness-associated genes in GC cells. HP infection upregulated the reduction of AKT and β-catenin phosphorylation triggered by ONECUT2 downregulation via ONECUT2 induction. Clinical survival analysis indicated that high ONECUT2 expression may indicate poor prognosis in GC. This study highlights a critical role played by ONECUT2 in promoting HP infection-associated GC by enhancing cell stemness through the PPP2R4/AKT/β-catenin signaling pathway. These findings suggest promising therapeutic strategies and potential targets for GC treatment.

DSCR1–1 attenuates osteoarthritis-associated chondrocyte injury by regulating the CREB1/ALDH2/Wnt/β-catenin axis: An in vitro and in vivo study

The progression of osteoarthritis (OA) includes the initial inflammation, subsequent degradation of the extracellular matrix (ECM), and chondrocyte apoptosis. Down syndrome candidate region 1 (DSCR1) is a stress-responsive gene and expresses in varied types of cells, including chondrocytes. Bioinformatics analysis of GSE103416 and GSE104739 datasets showed higher DSCR1 expression in the inflamed cartilage tissues and chondrocytes of OA. DSCR1 had two major isoforms, isoform 1 (DSCR1–1) and isoform 4 (DSCR1–4). We found that DSCR1–1 had a faster (in vitro) and higher expression (in vivo) response to OA compared to DSCR1–4. IL-1β-induced apoptosis, inflammation, and ECM degradation in chondrocytes were attenuated by DSCR1–1 overexpression. DSCR1–1 triggered the phosphorylation of cAMP response element-binding 1 (CREB1) at 133 serine sites by decreasing calcineurin activity. Moreover, activated CREB1 moved into the cell nucleus and combined in the promoter regions of aldehyde dehydrogenase 2 (ALDH2), thus enhancing its gene transcription. ALDH2 could recover Wnt/β-catenin signaling transduction by enhancing phosphorylation of β-catenin at 33/37 serine sites and inhibiting the migration of β-catenin protein from the cellular matrix to the nucleus. In vivo, adenoviruses (1 × 108 PFU) overexpressing DSCR1–1 were injected into the articular cavity of C57BL/6 mice with medial meniscus surgery-induced OA, and it showed that DSCR1–1 overexpression ameliorated cartilage injury. Collectively, our study demonstrates that DSCR1–1 may be a potential therapeutic target of OA.

AP collagen peptides (APCPs) promote hair growth by activating the GSK-3β/β-catenin pathway and improve hair condition.

AP collagen peptides (APCPs) are enzymatically decomposed collagen peptides that contain tri-peptides such as glycine-proline-hydroxyproline. We found that APCPs increased the proliferation of both human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). APCPs also stimulated the secretion of several growth factors, including IGFBP-6, PDGF-AB, PIGF and VEGF in hDPCs. Moreover, APCPs enhanced the phosphorylation of Akt(Ser473), GSK-3β(Ser9) and β-catenin(Ser675), indicating the activation of the GSK-3β/β-catenin signalling pathway. Exvivo culture of human hair follicles (hHFs) tissue and invivo patch assay revealed that APCPs promoted the elongation of hHFs and the induction of new hair shafts. In a mouse model, APCPs significantly promoted the transition from telogen to anagen phase and prolonged anagen phase, resulting in increased hair growth. APCPs also improved the thickness, amino acid content (cystine and methionine) and roughness of mouse hair. Taken together, these findings demonstrate that APCPs accelerate hair growth and contribute to overall hair health. Therefore, APCPs have the potential to be utilized as a food supplement and ingredient for preventing hair loss and maintaining hair health.

Isoleucyl-tRNA synthetase 2 promotes pancreatic ductal adenocarcinoma proliferation and metastasis by stabilizing β-catenin

Isoleucyl-tRNA synthetase 2 (IARS2), originally regarded as an enzyme ligating isoleucine to the corresponding tRNA, has been identified as an oncogene recently. However, its function in pancreatic ductal adenocarcinoma (PDAC) remains to be discovered. Here we explored the biological role of IARS2 in PDAC. Up-regulated IARS2 was found in PDAC tissues and cell lines. Kaplan–Meier survival analysis indicated a worse prognosis in patients with high IARS2 expression. CCK-8, EdU, and colony formation assays showed IARS2 overexpression enhanced PDAC proliferation, which was reduced by IARS2 knockdown. Meanwhile, IARS2 down-regulation inhibited PDAC metastasis by impeding epithelial–mesenchymal transition. These results were also supported by subcutaneous xenograft and metastasis assays in vivo. To figure out underlying mechanisms, differential and enrichment analyses were conducted and the WNT signaling pathway was discovered. Our results demonstrated that there was no significant relationship between the WNT signaling pathway key factor CTNNB1 and IARS2 at the transcription level. However, cycloheximide assays showed that IARS2 reduced the β-catenin degradation rate. IARS2 inhibited the phosphorylation of β-catenin at the Ser33/37 site and regulated downstream targets of WNT signaling including c-MYC, c-JUN, and MMP7. The enhancement of proliferation and metastasis caused by IARS2 could be reversed by MSAB, an agent that promotes β-catenin degradation. In summary, IARS2 facilitates PDAC proliferation and metastasis by stabilizing β-catenin, which leads to WNT/β-catenin activation. IARS2 serves as an underlying prognosis marker and a potential therapeutic target for PDAC.

Dorsomorphin inhibits AMPK, upregulates Wnt and Foxo genes and promotes the activation of dormant follicles

AMPK is a well-known energy sensor regulating cellular metabolism. Metabolic disorders such as obesity and diabetes are considered detrimental factors that reduce fecundity. Here, we show that pharmacologically induced in vitro activation (by metformin) or inhibition (by dorsomorphin) of the AMPK pathway inhibits or promotes activation of ovarian primordial follicles in cultured murine ovaries and human ovarian cortical chips. In mice, activation of primordial follicles in dorsomorphin in vitro-treated ovaries reduces AMPK activation and upregulates Wnt and FOXO genes, which, interestingly, is associated with decreased phosphorylation of β-catenin. The dorsomorphin-treated ovaries remain of high quality, with no detectable difference in reactive oxygen species production, apoptosis or mitochondrial cytochrome c oxidase activity, suggesting safe activation. Subsequent maturation of in vitro-treated follicles, using a 3D alginate cell culture system, results in mature metaphase eggs with protruding polar bodies. These findings demonstrate that the AMPK pathway can safely regulate primordial follicles by modulating Wnt and FOXO genes, and reduce β-catenin phosphorylation.

Mechanisms of vascular inflammaging: TNF-alpha mediates endothelial CEACAM1 expression by NF-KB and beta-Catenin in a biphasic manner

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Research Foundation Introduction Chronic inflammation with progressive age, also called inflammaging, is a hallmark of the pathogenesis of cardiovascular diseases. Previously we have shown increased expression of the Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) with progressive age in the vasculature of mice and humans that leads to a chronic pro-inflammatory milieu via mutual upregulation with TNF-α. Furthermore, CEACAM1 critically promotes vascular aging processes like collagen deposition, endothelial permeability as well as enhanced oxidative stress. Therefore, CEACAM1 is a main mediator of vascular inflammaging that may facilitate progress into cardiovascular diseases. In this study we identified the mechanisms that underly the upregulation of endothelial CEACAM1 expression by TNF-α in detail. Methods Wildtype (WT) and TNF-α knockout (Tnf-/-) mice of different age were used to determine the impact of TNF-α on age-dependent CEACAM1 expression in vivo. Underlying mechanisms of TNF-α-dependent CEACAM1 upregulation were further analyzed in an endothelial cell culture model using pharmacological inhibition and siRNA-mediated knockdown of signaling pathways. Activation of signaling pathways was confirmed by determining the phosphorylation status of signaling components via Western Blot and analysis of nuclear translocation of NF-κB and β-Catenin using subcellular fractionation. Results Immunohistochemical analyses of aortic sections from WT and Tnf-/- mice of different age reveal that TNF-α critically contributes to the aging-related upregulation of endothelial CEACAM1 expression in vivo. Mechanistic analyses in vitro showed that TNF-α upregulated CEACAM1 expression in a time-dependent manner. In pharmacological experiments we identified an early NF-κB- and a delayed β-Catenin-mediated response. This finding was supported by time-matched nuclear translocation of NF-κB and β-Catenin. Furthermore, siRNA-mediated knockdown of the β-Catenin-targeted transcription factor TCF4 reduced TNF-α-mediated CEACAM1 upregulation. Co-immunoprecipitation analyses show that β-Catenin is released by TNF-α-mediated adherens junctions disassembly. Furthermore, TNF-α enhanced activating phosphorylation of Akt kinase at Ser473. Akt kinase in turn increased phosphorylation of β-Catenin at Ser552, a modification reported to augment its transcriptional activity. Additionally, Akt kinase facilitated β-Catenin signaling by inhibition of its degradation via phosphorylation of GSK3β at Ser9. Conclusion Taken together, this study identified the signaling mechanisms that underly the vascular upregulation of CEACAM1 with age. Our results directly link endothelial barrier impairment, an initial step in the pathogenesis of atherosclerosis, to CEACAM1 expression. Since CEACAM1 critically promotes vascular inflammaging and age-dependent vascular alterations this further emphasizes the potential of CEACAM1 as a novel target to prevent cardiovascular diseases.

ATP6AP2, a regulator of LRP6/β-catenin protein trafficking, promotes Wnt/β-catenin signaling and bone formation in a cell type dependent manner

Wnt/β-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/β-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/β-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired β-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of β-catenin phosphorylation, but necessary for LRP6/β-catenin and N-cadherin/β-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active β-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating β-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/β-catenin signaling and trabecular bone formation.

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell-types. NDRG1 overexpression decreased β-catenin and down-regulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 that are classically induced by GSK-3β were significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression up-regulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. In fact, PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. In fact, the association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the down-regulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), that is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that up-regulate PKCα and inhibit WNT signaling.

WITHDRAWN: Recombinant osteopontin stabilizes the blood brain barrier following subarachnoid hemorrhage in rats

Background and PurposeRecombinant osteopontin (rOPN) decreases neuronal cell death and brain edema and improves the neurological scores following subarachnoid hemorrhage (SAH) in rats. The molecular mechanisms of the preservation of blood-brain barrier of rOPN following SAH in rats were examined in this study. MethodsAdult rats were divided into 3 groups: sham, vehicle, and treatment groups. Each animal in the SAH group underwent a surgical procedure to induce SAH, and the brains was harvested at 24 hours for analysis. For the mechanism part of the study two additional groups were operated and treated with Fib-14 and Wortmannin additional to rOPN. rOPN was administered via the nasal route and neurological scores as well as brain water content were evaluated at 24 hours after SAH induction. The blood brain barrier (BBB) permeability was evaluated using Evans Blue dye extravasation. The expressions of tight junction proteins claudin-3, claudin-5, occludin and the phosphorylation of β-catenin were examined using Western blot analysis. ResultsrOPN improved neurological scores and reduced brain edema at 24 hours after SAH. The BBB preservation action of rOPN is mediated in part through stabilizing β-catenin and increasing expression of tight junction proteins, claudin-3, claudin-5, and occludin. rOPN is thought to exert its effects through FAK activated PI3K-Akt pathway. ConclusionSince BBB disruption is one of the major pathophysiology of EBI after SAH, rOPN may be a promising drug to maintain BBB integrity and functions in SAH treatment.

Dependence of lymphopoiesis on efficient β-catenin degradation

Abstract Wnt/β-catenin pathway is tightly controlled by β-catenin destruction complex to regulate lineage specification and differentiation. Compared to epithelial lineage, lymphoid cells display higher rates of β-catenin degradation to achieve constitutively low levels of β-catenin. Emphasizing the reliance of lymphoid cells on β-catenin degradation, expression of degradation resistant form of β-catenin or deletion of APC or CK1 suppressed lymphoid development. Similarly, small molecules inhibiting GSK3β mediated phosphorylation or proteasomal degradation of β-catenin induced higher toxicity selectively in lymphoid cells compared to other lineages. Our interactome and CHIP-seq analysis highlighted that the unique dependency of lymphoid cells on efficient degradation of β-catenin is mediated by Ikaros zinc finger (IKZF) proteins and nucleosome remodeling and deacetylase (NuRD) complexes. Unlike activating TCF/β-catenin complexes in epithelial cells, recruitment of repressive β-catenin/IKZF/NuRD complexes to MYC superenhancer (SE) regions lead to suppression of Myc expression and induced cell death. Deletion of both IKZF1 and IKZF3 or mutation of Ikaros binding motifs within the Myc-SE subverted the β-catenin mediated toxicity. Upon loss of Ikaros factors, β-catenin interaction with TCF-factors resulted in de novo activation of epithelial specific genes. Our findings suggest that β-catenin degradation is an evolutionary requirement for Ikaros mediated protection of lymphoid identity.

β-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells

β-catenin has influential roles affecting embryonic development, tissue homeostasis, and human diseases including cancer. Cellular β-catenin levels are exquisitely controlled by a variety of regulatory mechanisms. In the course of exploring the functions of the Nek10 tyrosine kinase, we observed that deletion of Nek10 in lung adenocarcinoma cells resulted in dramatic stabilization of β-catenin, suggestive of a Nek10 role in the control of β-catenin turnover. Nek10-deficient cells exhibited diminished ability to form tumorspheres in suspension, grow in soft agar, and colonize mouse lung tissue following tail vein injection. Mechanistically, Nek10 associates with the Axin complex, responsible for β-catenin degradation, where it phosphorylates β-catenin at Tyr30, located within the regulatory region governing β-catenin turnover. In the absence of Nek10 phosphorylation, GSK3-mediated phosphorylation of β-catenin, a prerequisite for its turnover, is impaired. This represents a divergent function within the Nek family, whose other members are serine-threonine kinases involved in different elements of the centrosomal cycle, primary cilia function, and DNA damage responses.