Wnt signaling pathway plays an important role in both the regulation of host innate immunity and the nervous system. In this study, two key genes in the Wnt signaling pathway, β-catenin and GSK-3β, were first characterized from Procambarus clarkii, and significantly upregulated in hemocytes during Spiroplasma eriocheiris infection. At the cellular level, overexpression of Pcβ-catenin in Drosophila S2 cells significantly increased the cell viability and reactive oxygen species (ROS) production, decreased the cell necrosis and intracellular S. eriocheiris replication, while PcGSK-3β overexpression exerted an opposite effect. The Co-IP results revealed that PcGSK-3β could interact with Pcβ-catenin. Further, co-transfection of PcGSK-3β and Pcβ-catenin into S2 cells markedly reduced the cell survival and ROS level upon S. eriocheiris infection. At the individual level, knockdown of Pcβ-catenin significantly induced the apoptosis of hemocytes and increased the mortality of the crayfish following S. eriocheiris infection. Conversely, PcGSK-3β deficiency significantly elevated the ROS level in hemocytes thereby enhancing the resistance of P. clarkii to S. eriocheiris infection. In conclusion, this study has proved the regulation mechanism of Wnt signaling pathway in response to S. eriocheiris infection, which may contribute to our understanding of innate immunity in invertebrates.

In multicellular organisms, Wnt proteins govern stem and progenitor cell renewal and differentiation to regulate embryonic development, adult tissue homeostasis and tissue regeneration. Defects in canonical Wnt signalling, which is transduced intracellularly by β-catenin, have been associated with developmental disorders, degenerative diseases and cancers. Although a simple model describing Wnt-β-catenin signalling is widely used to introduce this pathway and has largely remained unchanged over the past 30 years, in this Review we discuss recent studies that have provided important new insights into the mechanisms of Wnt production, receptor activation and intracellular signalling that advance our understanding of the molecular mechanisms that underlie this important cell-cell communication system. In addition, we review the recent development of molecules capable of activating the Wnt-β-catenin pathway with selectivity in vitro and in vivo that is enabling new lines of study to pave the way for the development of Wnt therapies for the treatment of human diseases.

WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation, promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases casein kinase 1α (CSNK1A1) and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain E3 ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigated the mechanism underlying this requirement. In HAP1 cells lacking CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was smaller than the reduction in WNT target gene transcription. To test whether the reduction in WNT signaling caused by HUWE1 loss resulted from reduced CTNNB1 alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells did not change CTNNB1 abundance but still reduced WNT signaling, demonstrating that another mechanism was at play. Genetic interaction and overexpression analyses revealed that the reduction in WNT signaling caused by HUWE1 loss required not only GSK3A or GSK3B, but also APC and AXIN1. Therefore, in HAP1 cells lacking CSNK1A1, a residual destruction complex containing APC, AXIN1 and GSK3A or GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation, and HUWE1 enhances WNT signaling by antagonizing this activity. Regulation of WNT signaling by HUWE1 also requires its ubiquitin ligase activity. We conclude that HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that antagonizes destruction complex-mediated CTNNB1 degradation and another that is independent of changes in CTNNB1 abundance. Coordinated regulation of CTNNB1 abundance and a second signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.

Eupalinolide B (EB), a primary bioactive compound isolated from Eupatorium lindleyanum DC., has exhibited various pharmacological properties, such as antitumor, anti-inflammatory, and notably, neuroprotective effects in neurodegenerative diseases. However, the in-depth studies on the antidepressant potential of EB and its underlying mechanisms are still lacking. Herein, we investigated the therapeutic effects of EB on corticosterone (CORT)-induced neurotoxicity in PC12 cells and its antidepressant-like effects in rats subjected to chronic unpredictable mild stress (CUMS). In particular, we focused on the molecular mechanisms related to modulating the GSK-3β/β-catenin pathway. Our findings revealed that EB promoted cell proliferation while decreasing apoptosis and oxidative stress in CORT-induced PC12 cells. In vivo, EB alleviated the depressive-like behaviors in CUMS rats, as assayed by the sucrose preference test, open field test, and forced swim test. Additionally, EB attenuated the hippocampal pathological damage and increased Ki67- and doublecortin-positive cell numbers in hippocampal dentate gyrus, thus restoring hippocampal neurogenesis in CUMS rats. The binding of EB to GSK-3β was confirmed using molecular docking and cellular thermal shift assays. Overexpression of GSK-3β diminished the therapeutic effects of EB on CORT-induced PC12 cells, further indicating that GSK-3β is the target of EB. Mechanistically, EB hindered GSK-3β activity and thus activated β-catenin signaling in both CORT-induced PC12 cells and CUMS rat hippocampus, as demonstrated by increased p-GSK-3β (Ser9), reduced p-β-catenin, and elevated β-catenin expression. Collectively, this study offers new insights into the antidepressant mechanisms of EB, highlighting its potential as a candidate for depression treatment.

Evolutionarily, Wnt/β-catenin signaling is well-conserved and supports several key cell-biological processes (e.g. adhesion and proliferation). Its crucial component, β-catenin, has been described in several organisms, however, its identification and characterization are notably lacking in annelid earthworms. Here, we report a novel β-catenin homologue from the earthworm Eisenia andrei, termed Ea-β-catenin. The full-length 3253 nt Ea-β-catenin mRNA includes an open reading frame of 2499 nt encoding a putative protein with 833 amino acid residues that comprise 11 classical armadillo-repeat regions. Phylogenetic analysis indicates that Ea-β-catenin shows strong homology with Lophotrochozoan β-catenins. Ubiquitous, but variable expressions of Ea-β-catenin were observed in distinct earthworm tissues. During embryogenesis, Ea-β-catenin mRNA gradually increased from the E1 to E4 developmental stages. Regeneration experiments revealed an inverse correlation between Ea-β-catenin mRNA levels and the rate of EdU+/PY489-β-catenin+ proliferating cells during the second week of the posterior blastema formation. In vitro exposures to poly(I:C) and zymosan significantly increased Ea-β-catenin mRNA levels, while small molecule Wnt-pathway modulators such as LiCl or iCRT14 increased or decreased Ea-β-catenin mRNA expression, and nuclear translocation of PY489-β-catenin, respectively. These novel results pave the way for follow-up studies aimed at characterizing additional members of the Wnt/β-catenin pathway that may be involved in embryonic and/or postembryonic development, as well as innate immunity in earthworms.

The homeobox-containing transcription factor PAX6 is a key regulator of eye development. Pathogenic heterozygous PAX6 variants lead to variable ocular phenotypes, most commonly haploinsufficiency-induced aniridia. Missense variants are typically associated with milder ocular conditions, although variants in the DNA-binding paired domain which alter target binding lead to severe ocular phenotypes including bilateral microphthalmia, similar to SOX2-anophthalmia syndrome. However, the variant-specific pathway disruption resulting in phenotypic heterogeneity is not well understood. To investigate pathogenic mechanisms of PAX6 variants, transcriptomic and chromatin accessibility analysis was performed on hiPSC derived 3D optic cup-like organoids generated from patients with variants (i) PAX6N124K displaying combined microphthalmia, aniridia and optic nerve coloboma, and (ii) PAX6R261X exhibiting typical aniridia. Total RNA sequencing analysis revealed downregulation of SOX2 in missense PAX6N124K cups compared to both wildtype and PAX6R261X haploinsufficient aniridia controls, along with Notch signalling components and markers of proliferation and differentiation. Transcription factor binding motifs of Notch-related genes were also found to be differentially bound in PAX6N124K cups through ATACseq footprinting analysis. Our analysis of PAX6-related oculopathies using in vitro models reveals disruption to DNA binding perturbs SOX2 and Notch signalling, contributing to severe ocular phenotypes in patients with missense changes in the paired domain. This work reveals a previously unestablished role for PAX6 in SOX2 and Notch signalling regulation during early oculogenesis, as well as illuminating disease mechanisms underlying variant-specific ocular phenotypes and genotype-phenotype correlations. These novel insights can influence clinical care, and provide valuable data on potential therapeutic targets, which can guide future translational research.

The Wnt signaling pathway is critically involved in orchestrating cellular functions such as proliferation, migration, survival, and cell fate determination during development. Given its pivotal role in cellular communication, aberrant Wnt signaling has been extensively linked to the pathogenesis of various diseases. This review offers an in-depth analysis of the Wnt pathway, detailing its signal transduction mechanisms and principal components. Furthermore, the complex network of interactions between Wnt cascades and other key signaling pathways, such as Notch, Hedgehog, TGF-β, FGF, and NF-κB, is explored. Genetic mutations affecting the Wnt pathway play a pivotal role in disease progression, with particular emphasis on Wnt signaling's involvement in cancer stem cell biology and the tumor microenvironment. Additionally, this review underscores the diverse mechanisms through which Wnt signaling contributes to diseases such as cardiovascular conditions, neurodegenerative disorders, metabolic syndromes, autoimmune diseases, and cancer. Finally, a comprehensive overview of the therapeutic progress targeting Wnt signaling was given, and the latest progress in disease treatment targeting key components of the Wnt signaling pathway was summarized in detail, including Wnt ligands/receptors, β-catenin destruction complexes, and β-catenin/TCF transcription complexes. The development of small molecule inhibitors, monoclonal antibodies, and combination therapy strategies was emphasized, while the current potential therapeutic challenges were summarized. This aims to enhance the current understanding of this key pathway.

Cardiac fibroblasts (CFs) are the essential cell type for heart morphogenesis and homeostasis. In addition to maintaining the structural integrity of the heart tissue, muscle fibroblasts are involved in complex signaling cascades that regulate cardiomyocyte proliferation, migration, and maturation. While CFs serve as the primary source of extracellular matrix proteins (ECM), tissue repair, and paracrine signaling, they are also responsible for adverse pathological changes associated with cardiovascular disease. Following activation, fibroblasts produce excessive ECM components that ultimately lead to fibrosis and cardiac dysfunction. Decades of research have led to a much deeper understanding of the role of CFs in cardiogenesis. Recent studies using the single-cell genomic approach have focused on advancing the role of CFs in cellular interactions, and the mechanistic implications involved during cardiovascular development and disease. Arguably, the unique role of fibroblasts in development, tissue repair, and disease progression categorizes them into the friend or foe category. This brief review summarizes the current understanding of cardiac fibroblast biology and discusses the key findings in the context of development and pathophysiological conditions.

Piezo1, which maintains the integrity and function of the intestinal epithelial barrier, is essential for colonic epithelial homeostasis. However, whether and how Piezo1 regulates colon stem cell fate remains unclear. Here, we show that Piezo1 inhibition promotes colon stem cell proliferation. Mechanistically, stearoyl-CoA 9-desaturase 1 (SCD1) is downstream of Piezo1 to affect colon stem cell stemness by acting on the Wnt-β-catenin pathway. For mice, the altered colon stem cell stemness after Piezo1 knockdown and activation was accompanied by a reprogrammed fatty acid (FA) metabolism in colon crypts. Notably, we found that GsMTX4 protects injured colon stem cell stemness in mouse and human colitis organoids. Our results elucidated the role of Piezo1 in regulating normal and postinjury colon stem cell fates through SCD1-Wnt-β-catenin and the SCD1-mediated FA desaturation process. These results provide fresh perspectives on the mechanical factors regulating colon stem cell fate and therapeutic strategies for related intestinal diseases.

Heterozygous pathogenic variants in AXIN2 are associated with oligodontia-colorectal cancer syndrome (ODCRCS), a disorder characterized by oligodontia, colorectal cancer, and in some cases, sparse hair and eyebrows. We have identified four individuals with one of two de novo , heterozygous variants (NM_004655.4:c.196G>A, p.(Glu66Lys) and c.199G>A, p.(Gly67Arg)) in AXIN2 whose presentations expand the phenotype of AXIN2-related disorders. In addition to ODCRCS features, these individuals have global developmental delay, microcephaly, and limb, ophthalmologic, and renal abnormalities. Structural modeling of these variants suggests that they disrupt AXIN2 binding to tankyrase, which regulates AXIN2 levels through PARsylation and subsequent proteasomal degradation. To test whether these variants produce a phenotype in vivo , we utilized an innovative prime editing N1 screen to phenotype heterozygous (p.E66K) mouse embryos, which were perinatal lethal with short palate and skeletal abnormalities, contrary to published viable Axin2 null mouse models. Modeling of the p.E66K variant in the Drosophila wing revealed gain-of-function activity compared to reference AXIN2. However, the variant showed loss-of-function activity in the fly eye compared to reference AXIN2, suggesting that the mechanism by which p.E66K affects AXIN2 function is cell context-dependent. Together, our studies in humans, mice, and flies demonstrate that specific variants in the tankyrase-binding domain of AXIN2 are pathogenic, leading to phenotypic expansion with context-dependent effects on AXIN2 function and WNT signaling. Moreover, the modeling strategies used to demonstrate variant pathogenicity may be beneficial for the resolution of other de novo heterozygous variants of uncertain significance associated with congenital anomalies in humans.

Testis-specific transcript 10 (Tex10) is highly expressed in the testis, embryonic stem cells (ESCs), and primordial germ cells (PGCs). We previously generated a Tex10 knockout mouse model demonstrating its critical roles in ESC pluripotency and preimplantation development. Here, using conditional knockout mice and dTAG-degron ESCs, we show Tex10 is required for spermatogenesis and ESC-to-PGCLC differentiation. Specifically, Tex10-null spermatocytes arrest at metaphase I, compromising round spermatid formation. Tex10 depletion and overexpression compromise and enhance ESC-to-PGCLC differentiation, respectively. Mechanistically, bulk and single-cell RNA sequencing reveals that Tex10 depletion downregulates genes involved in pluripotency, PGC development, and spermatogenesis while upregulating genes promoting somatic programs. Chromatin occupancy study reveals that Tex10 binds to H3K4me3-marked promoters of Psmd3 and Psmd7, negative regulators of Wnt signaling, and activates their expression, thereby restraining Wnt signaling. Our study identifies Tex10 as a previously unappreciated factor in spermatogenesis and PGC development, offering potential therapeutic insights for treating male infertility.

The emergence of multicellularity in animals marks a pivotal evolutionary event, which was likely enabled by molecular innovations in the way cells adhere and communicate with one another. β-Catenin is significant to this transition due to its dual role as both a structural component in the cadherin-catenin complex and as a transcriptional coactivator involved in the Wnt/β-catenin signaling pathway. However, our knowledge of how this protein functions in ctenophores, one of the earliest diverging metazoans, is limited.

To study β-catenin function in the ctenophore Mnemiopsis leidyi, we generated affinity-purified polyclonal antibodies targeting Mlβ-catenin. We then used this tool to observe β-catenin protein localization in developing Mnemiopsis embryos. In this article, we provide evidence of consistent β-catenin protein enrichment at cell-cell interfaces in Mnemiopsis embryos. Additionally, we found β-catenin enrichment in some nuclei, particularly restricted to the oral pole around the time of gastrulation. The Mlβ-catenin affinity-purified antibodies now provide us with a powerful reagent to study the ancestral functions of β-catenin in cell adhesion and transcriptional regulation.

The localization pattern of embryonic Mlβ-catenin suggests that this protein had an ancestral role in cell adhesion and may have a nuclear function as well.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline and loss of neuronal integrity. Emerging evidence suggests that RhoA, Rho-associated coiled-coil kinase (ROCK), and their downstream effector molecule glycogen synthase 3β (GSK3β) interact within a complex signaling pathway (RhoA/ROCK/GSK3β) that plays a crucial role in the pathogenesis of AD. RhoA, a small GTPase, along with its downstream effector, ROCK, regulates various cellular processes, including actin cytoskeleton dynamics, apoptosis, and synaptic plasticity. GSK3β, a serine/threonine kinase, plays a key role in neuronal function and AD pathology, including the regulation of tau phosphorylation and amyloid-beta cleavage. Overactive GSK3β has been closely linked to tau hyperphosphorylation, neurodegeneration, and the progression of AD. Thus, GSK3β has been considered as a promising therapeutic target for treating AD and mitigating cognitive impairment. However, clinical trials of GSK3β in AD have faced considerable challenges due to the complexity of the specific neuronal inhibition of GSK3β. In this review, we summarize the literature regarding the relationship of RhoA/ROCK and GSK3β signaling pathways in AD pathogenesis. We further discuss recent findings of the sTREM2-transgelin-2 (TG2) axis as a potential mediator of this complex pathway and provide our review on a novel targeting strategy for AD.

Cornus officinalis Sieb. et Zucc has significant neuroprotective activity and has been widely studied for its potential to improve cognitive function. Our team's previous research has found that loganin isolated from Cornus officinalis has an antidepressant effect. Depression is a mental disorder accompanied by dysfunction of Connexin43 (Cx43)-formed astrocytic gap junctions. However, the precise mechanisms of loganin involved remain uncertain.

We aimed to examine the mechanism by which loganin produces its antidepressant properties.

Using a chronic unpredictable stress (CUS) model of depression in rats, the study evaluated the behavioral responses to treatment with loganin, fluoxetine, and their combination. Biochemical analyses were conducted to measure the expression and phosphorylation status of Cx43, β-catenin, GSK-3β in the brain. In vitro experiments were also performed how loganin protects the gap junctions in astrocytes that have been exposed to corticosterone.

After four weeks of loganin treatment, rats exposed to CUS showed a decrease in depressive-like behaviors. When combined with fluoxetine, the antidepressant-like effects were observed faster than with either treatment alone. Loganin significantly increased Cx43 expression in the prefrontal cortex and ventral hippocampus, reversed Cx43 mimetic peptide Gap26-induced depressive-like behaviors, decreased Cx43 phosphorylation at Ser368, increased β-catenin levels, and promoted GSK-3β phosphorylation at Ser9. In vitro, loganin prevented corticosterone-induced damage to gap junctions between astrocytes, an effect that was negated by XAV-939 (β-catenin inhibitor).

These results implied that loganin could exert antidepressant-like effects by improving the gap junctions of astrocytes in the prefrontal cortex and hippocampus, acting through the GSK-3β/β-catenin signaling pathway. The combination of loganin with fluoxetine may provide a faster onset of antidepressant action compared to either treatment alone, highlighting the potential of loganin as a natural adjunct therapy for depression.

Autism spectrum disorder (ASD) is a neurodevelopmental disability condition arising from a combination of genetic and environmental factors. Despite the blood-brain barrier (BBB) serving as a crucial gatekeeper, conveying environmental influences into the brain parenchyma, the contributions of BBB in ASD pathogenesis remain largely uncharted. Here we report that SHANK3, an ASD-risk gene, expresses in the BBB-forming brain endothelial cells (BECs) and regulates tight junctional (TJ) integrity essential for BBB's barrier function. Endothelium-specific Shank3 (eShank3) knockout (KO) neonatal mice exhibit male-specific BBB-hyperpermeability, reduced neuronal excitability, and impaired ultra-sonic communications. Although BBB permeability is restored during adult age, the male mutant mice display reduced neuronal excitability and impaired sociability. Further analysis reveals that the BBB-hyperpermeability is attributed to the β-Catenin imbalance triggered by eShank3-KO. These findings highlight a pathogenic mechanism stemming from the ASD-risk Shank3, emphasizing the significance of neonatal BECs in the BBB as a potential therapeutic target for ASD.

The incidence and mortality rates of colorectal cancer (CRC) are alarmingly high, and the scientific community is consistently engaged in developing newer therapeutic options for cancer cure or prevention. The fluoropyrimidine drug, 5-fluorouracil (5FU), remains the first line of treatment against CRC; nevertheless, relapses frequently occur since the cells gain resistance over time through various mechanisms. Studies have highlighted the significance of combinatorial treatment of a Wnt signaling inhibitor and 5FU as a better treatment strategy to overcome 5FU resistance. Small molecules that specifically target and disrupt β-catenin-TCF interaction, a crucial step of the Wnt signaling, are promising in CRC treatment. In this study, we investigated the synergistic cytotoxic activity of menadione with 5FU as the former has previously been shown to downregulate Wnt signaling in CRC cells. Docking and experimental results suggest that the drug combination interfered with key protein-protein interactions in the β-catenin-TCF complex, exerted synergistic anti-cancerous effects in CRC cells, and downregulated the expression of Wnt signaling proteins. Taken together, our data suggest that the simultaneous binding of 5FU and menadione to β-catenin can block Wnt signaling by disrupting β-catenin-TCF interaction and inhibit the proliferation of CRC cells.

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 in vitro. 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.

Cancer sparks if the components of the cellular signaling network are aberrantly activated, leading to uncontrolled cell growth and proliferation. One of the most important players of this highly regulated network is the Wnt/β-catenin signaling, with a significant role in human health and disease. The critical co-receptor of this pathway, LRP6, is overexpressed in various cancer types and is a target for therapy. Therefore, understanding the details of the LRP6 structural activation mechanism is of tremendous importance. This research intended to compare the structural-dynamics features of the E3E4 functional domain of LRP6 induced by the activator Wnt3a and the inhibitor, Dkk1_C, compared with the receptor behavior in the apo-state. Using molecular docking, molecular dynamics simulation, and G_MMPBSA calculation, we characterized overlapping binding regions of Wnt3a and Dkk1_C on E3E4. Despite their overall similar interacting regions, Dkk1_C and Wnt induce remarkably different inter-blades hydrogen bonds, structural-dynamics behavior, and conformational energy landscape in E3E4. According to our findings, Dkk1_C stabilized the interaction. between BP3 blades 2-3, 3-4, and 4-5 and BP4 blades 1-6, 1-2, 2-3, and 3-4, aligned with apo-state. However, on the other hand, Wnt distinguishably destabilized the hydrogen bond networks of these blades. Our DCCM analysis also depicted a similar correlation pattern of apo and Dkk1-bound states, and dramatic differences in Wnt-bound state, with a specific enhancement of correlated movements in EGF4. These data provide atomistic-level clues of how natural regulators of Wnt signaling manipulate LRP6 dynamics and, therefore, guide the structure-based design of efficient artificial inhibitors/activators for the pathway.

The Neural Crest cells are multipotent progenitor cells formed at the neural plate border that differentiate and give rise to a wide range of cell types and organs. Directional migration of NC cells and their correct positioning at target sites are essential during embryonic development, and defects in these processes results in congenital diseases. The NC migration begins with the epithelial-mesenchymal transition and extracellular matrix remodeling. The main cellular mechanisms that sustain this migration include contact inhibition of locomotion, co-attraction, chemotaxis and mechanical cues from the surrounding environment, all regulated by proteins that orchestrate cell polarity and motility. In this review we highlight the molecular mechanisms involved in neural crest cell migration and polarity, focusing on the role of small GTPases, Heterotrimeric G proteins and planar cell polarity complex. Here, we also discuss different congenital diseases caused by altered NC cell migration.

Fortilin, a 172-amino acid polypeptide, is a multifunctional protein that interacts with various protein molecules to regulate their functions. Although fortilin has been shown to interact with cytoskeleton proteins such as tubulin and actin, its interactions with the components of adherens junctions remained unknown. Using co-immunoprecipitation western blot analyses, the proximity ligation assay, microscale thermophoresis, and biolayer interferometry, we here show that fortilin specifically interacts with CTNNA3 (α-T-catenin), but not with CTNNA1, CTNNA2, or CTNNB. The silencing of fortilin using small interfering RNA (siRNAfortilin) promotes the proteasome-mediated degradation of CTNNA3 in 293T cells. Using both fortilin-deficient THP1 cells and 293T cells that overexpress wild-type (WT), phospho-null (5A), and phospho-mimetic (5D) CTNNA3s, we also show that the absence of fortilin accelerates the phosphorylation of CTNNA3, leading to its ubiquitination and proteasome-mediated degradation. Further, the silencing of CTNNA3 using siRNACTNNA3 causes 293T cells to undergo apoptosis. These data suggest that fortilin guards the cells against apoptosis by positively regulating the pro-survival molecule CTNNA3 by protecting it against phosphorylation, ubiquitination, and proteasome-mediated degradation.

Osteoarthritis (OA) is a debilitating chronic joint disease affecting large populations of patients, especially the elderly. The pathological mechanisms of OA are currently unknown. Multiple risk factors are involved in OA development. Among these risk factors, alterations of mechanical loading in the joint leading to changes in biological signaling pathways have been known as a key event in OA development. The importance of AMPK-β-catenin-Runx2 signaling in the initiation and progression of OA has been recognized in recent years. In this review, we discuss the recent progress in understanding the role of this signaling pathway and the underlying interaction mechanisms during OA development. We also discuss the drug development aiming to target this signaling pathway for OA treatment.

Hepatocellular carcinoma (HCC), considered as one of the most common and lethal cancers worldwide, has drawn significant attention from researchers.Extensively studied diverse cancers, the function of LIMA1 in tumorigenesis and cancer progression remains ambiguous.. Moreover, the role of LIMA1 in HCC remains controversial.

The expression difference of LIMA1 in hepatocellular carcinoma, which was verified by TMT quantitative proteomics, immunohistochemistry, western blot, and the TCGA database, has been investigated in this study. Demonstrated by using transwell, cck8, sphere formation, and other experiments, the effects of LIMA1 on the migration, proliferation, stemness, and other aspects of hepatocellular carcinoma were significant. Moreover, the effect of LIMA1 on the wnt-βcatenin/Hippo pathway was revealed by using RNA sequencing and western blot, and the relationship between LIMA1 and βcatenin was verified by using COIP. Finally, the effect of m6a modification on LIMA1 was further verified using Western blotting, actinomycin D and MeRip experiments.

In HCC tissues and several HCC cell lines, LIMA1 was expressed at a relatively high level.LIMA1 positively regulated the invasion, migration, proliferation and stemness of hepatocellular carcinoma, and silencing of LIMA1 inhibited the tumorigenic ability of HCC cells in nude mice. Moreover, it was shown that LIMA1 can have an impact on the wnt-β-catenin/Hippo pathway. And silencing β-catenin suppressed the invasion, migration, proliferation and stemness of hepatocellular carcinoma cells mediated by LIMA1. Finally, it was further verified that the activation of LIMA1 in hepatocellular carcinoma cells is due to m6-methyladenosine methylation that is dependent on METTL3.

In HCC, LIMA1 functions as a tumor promoter and engages with the WNT-β-catenin and Hippo signaling pathways,, affecting the characteristics of tumor cells. LIMA1 expression is regulated by METTL3-mediated m6A modification, leading to its high expression in HCC. Our research presents a hopeful objective for the detection and therapy of HCC.

Osteosarcoma, the most prevalent primary bone malignancy in children and adolescents, exhibits high heterogeneity. The CGREF1 gene encodes a novel 301 amino acid classical secreted protein that contains the presumed N-terminal signaling peptide and EF hand motif. However, its role in osteosarcoma remains unclear.

Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized for bioinformatics analysis. Western blot and immunohistochemistry (IHC) techniques were employed to detect the expression of relevant proteins. siRNA, lentivirus, and plasmid technologies were applied to modulate gene expression. The downstream pathway of CGREF1 was identified through RNA sequencing analysis. Cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry, wound healing assay, and Transwell assay were conducted for in vitro functional experiments. In vivo experiments involved subcutaneous tumor formation in nude mice.

Our analysis of public databases and clinical samples revealed that CGREF1 is highly expressed in osteosarcoma and is associated with poor prognosis. Knockdown of CGREF1 impeded cell cycle progression and suppressed the proliferation of osteosarcoma cells. Conversely, upregulation of CGREF1 exhibited an opposing pattern. The RNA-seq data from 143B cells was subjected to analysis, revealing that the differentially expressed genes were predominantly enriched in the Wnt signaling pathway. Further experimental results demonstrated that CGREF1 affects activation of the Wnt pathway by regulating GSK3/β-catenin signaling, thereby affecting proliferation ability of osteosarcoma cells. Finally, experiments using subcutaneous transplanted tumor models in nude mice showed that CGREF1 knockdown inhibited tumor growth in vivo by inhibiting the Wnt/β-catenin signaling pathway.

The expression of CGREF1 was significantly upregulated in osteosarcoma and correlated with unfavorable prognosis. CGREF1 exerted a regulatory effect on the proliferation of osteosarcoma cells both in vitro and in vivo through modulation of the wnt/β-catenin signaling pathway. In the future, targeting CGREF1 could potentially offer a novel therapeutic strategy for treating osteosarcoma.

AKT, or protein kinase B, is a central node of the PI3K signaling pathway that is pivotal for a range of normal cellular physiologies that also underlie several pathological conditions, including inflammatory and autoimmune diseases, overgrowth syndromes, and neoplastic transformation. These pathologies, notably cancer, arise if either the activity of AKT or its positive or negative upstream or downstream regulators or effectors goes unchecked, superimposed on by its intersection with a slew of other pathways. Targeting the PI3K/AKT pathway is, therefore, a prudent countermeasure. AKT inhibitors have been tested in many clinical trials, primarily in combination with other drugs. While some have recently garnered attention for their favorable profile, concern over resistance and off-target effects have continued to hinder their widespread adoption in the clinic, mandating a discussion on alternative modes of targeting. In this review, we discuss isoform-centric targeting that may be more effective and less toxic than traditional pan-AKT inhibitors and its significance for disease prevention and treatment, including immunotherapy. We also touch on the emerging mutant- or allele-selective covalent allosteric AKT inhibitors (CAAIs), as well as indirect, novel AKT-targeting approaches, and end with a briefing on the ongoing quest for more reliable biomarkers predicting sensitivity and response to AKT inhibitors, and their current state of affairs.

Missense mutations in exon 3 of CTNNB1, the gene encoding β-catenin, are associated with poor outcomes in endometrial carcinomas (EC). Clinically, CTNNB1 mutation status has been difficult to use as a predictive biomarker as β-catenin oncogenic activity is modified by other factors, and these determinants are unknown. Here we reveal that CD73 restrains the oncogenic activity of exon 3 β-catenin mutants, and its loss associates with recurrence. Using 7 patient-specific mutants, with genetic deletion or ectopic expression of CD73, we show that CD73 loss increases β-catenin-TCF/LEF transcriptional activity. In cells lacking CD73, membrane levels of mutant β-catenin decreased which corresponded with increased levels of nuclear and chromatin-bound mutant β-catenin. These results suggest CD73 sequesters mutant β-catenin to the membrane to limit its oncogenic activity. Adenosine A1 receptor deletion phenocopied increased β-catenin-TCF/LEF activity seen with NT5E deletion, suggesting that the effect of CD73 loss on mutant β-catenin is mediated via attenuation of adenosine receptor signaling. RNA-seq analyses revealed that NT5E deletion alone drives pro-tumor Wnt/β-catenin gene expression and, with CD73 loss, β-catenin mutants dysregulate zinc-finger and non-coding RNA gene expression. We identify CD73 as a novel regulator of oncogenic β-catenin and help explain variability in patient outcomes in CTNNB1 mutant EC.

Morphogen gradients instruct cells to pattern tissues. Although the mechanisms by which morphogens transduce chemical signals have been extensively studied, the roles and regulation of the physical communication between morphogen-receiver cells remain unclear. Here, we show that the Wnt/β-catenin-morphogen gradient, which patterns the embryonic anterior-posterior (AP) axis, generates intercellular tension gradients along the AP axis by controlling membrane cadherin levels in zebrafish embryos. This "mechano-gradient" is used for the cell competition-driven correction of noisy morphogen gradients. Naturally and artificially generated unfit cells, producing noisy Wnt/β-catenin gradients, induce local deformation of the mechano-gradients that activate mechanosensitive calcium channels in the neighboring fit cells, which then secrete annexin A1a to kill unfit cells. Thus, chemo-mechanical interconversion-mediated competitive communication between the morphogen-receiver cells ensures precise tissue patterning.

Establishment of the 3 primordial germ layers (ectoderm, endoderm, and mesoderm) during early animal development represents an essential prerequisite for the emergence of properly patterned embryos. β-catenin is an ancient protein that is known to play essential roles in this process. However, these roles have chiefly been established through inhibition of β-catenin translation or function at the time of fertilization. Comprehensive analyses reporting the totality of functions played by nuclear β-catenin during early embryogenesis of a given animal, i.e., at different developmental stages and in different germ layers, are thus still lacking. In this study, we used an inducible, conditional knockdown system in the sea urchin to characterize all possible requirements of β-catenin for germ layer establishment and patterning. By blocking β-catenin protein production starting at 7 different time points of early development, between fertilization and 12 h post fertilization, we established a clear correlation between the position of a germ layer along the primary embryonic axis (the animal-vegetal axis) and its dependence on nuclear β-catenin activity. For example, in the vegetal hemisphere, we determined that the 3 germ layers (skeletogenic mesoderm, non-skeletogenic mesoderm, and endoderm) require distinct and highly specific durations of β-catenin production for their respective specification, with the most vegetal germ layer, the skeletogenic mesoderm, requiring the shortest duration. Likewise, for the 2 animal territories (ectoderm and anterior neuroectoderm), we established that their restriction, along the animal-vegetal axis, relies on different durations of β-catenin production and that the longest duration is required for the most animal territory, the anterior neuroectoderm. Moreover, we found that 2 of the vegetal germ layers, the non-skeletogenic mesoderm and the endoderm, further require a prolonged period of nuclear β-catenin activity after their specification to maintain their respective germ layer identities through time. Finally, we determined that restriction of the anterior neuroectoderm territory depends on at least 2 nuclear β-catenin-dependent inputs and a nuclear β-catenin-independent mechanism. Taken together, this work is the first to comprehensively define the spatiotemporal requirements of β-catenin during the early embryogenesis of a single animal, the sea urchin Paracentrotus lividus, thereby providing new experimental evidence for a better understanding of the roles played by this evolutionary conserved protein during animal development.

Tumor progression of colorectal cancer (CRC) seriously affects patient prognosis. For CRC patients with advanced-stage disease, it is still necessary to continuously explore more effective targeted therapeutic drugs. Circular RNAs (circRNAs) are involved in the regulation of tumor biology. We screened circAURKA, which was significantly highly expressed in CRC by previous high-throughput RNA sequencing. In vitro experiments were performed to investigate the effect of the circRNA on the proliferation and metastasis of HCT116 and SW480 cells. In addition, we used the EdU assay, Transwell assay, nude mouse xenograft tumor model and nude mouse tail vein metastasis model to examine the effect of circAURKA on the proliferation and metastasis of CRC. Mechanistically, fluorescent in situ hybridization (FISH), RNA pull-down, RNA immunoprecipitation (RIP), protein coimmunoprecipitation (co-IP) experiments and animal models were performed to confirm the underlying mechanisms of circAURKA. CircAURKA was significantly highly expressed in CRC tissues and colorectal cells and mainly present in the cytoplasm. The circRNA promoted the proliferation and metastasis of CRC cells in vitro and in vivo. In terms of the molecular mechanism, circAURKA inhibited the degradation of the CTNNB1 protein by promoting the interaction between ACLY and the CTNNB1 protein, thereby promoting the proliferation and metastasis of CRC cells. In addition, circAURKA stability was regulated by m6A methylation modification. This study revealed that circAURKA promoted the proliferation and metastasis of CRC by inhibiting CTNNB1 protein degradation, providing a basis for the development of targeted drugs to control CRC progression.

Wnt signaling is essential for both the development and homeostasis of diverse cellular lineages, including hematopoietic stem cells. Organism-wide, Wnt signals are tightly regulated, as overactivation of the pathway can lead to tumorigenesis. Although numerous Wnt ligands and Frizzled (Fzd) receptors exist, how particular Wnt/Fzd pairings are established and how their signals are regulated is poorly understood. We have previously identified the requirements of the cognate pairing of Wnt9a and Fzd9b for early hematopoietic stem cell proliferation. However, the specific signals governing activation, but equally important, the molecular mechanisms required to turn the signal 'off,' are unknown. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) is specifically required to ubiquitinate the third intracellular loop of Fzd9b at K437, targeting it for lysosomal degradation. In contrast to other ubiquitin ligases described to regulate the cell surface availability of multiple Fzds broadly, our data indicate that Trip12 is selective for Fzd9b. We further demonstrate that this occurs through ubiquitination at K437 of Fzd9b in the third intracellular loop, ultimately leading to a decrease in Fzd9b receptor availability and in Wnt9a/Fzd9b signaling that impacts hematopoietic stem cell proliferation in zebrafish. Our results point to specific mechanisms driving the availability of different Fzd receptors. Determining how particular Fzd abundance is regulated at the membrane will be critical to developing specific therapies for human intervention.

We report the generation and characterization of the K5: CAT bigenic mouse in which the constitutively activated form of β-catenin (ΔN89 β-catenin) is conditionally expressed in cytokeratin-5 (K5) positive epidermal keratinocytes. Following short-term doxycycline intake during the telogen resting phase, the adult K5: CAT bigenic develops enlarged pilosebaceous units that expand deep into the dermis, an expansion usually observed during the anagen growth phase. Prolonged doxycycline treatment results in significant thickening and folding of the K5: CAT epidermis. During this persistent induction period, there is clear evidence of increased keratinocyte proliferation, particularly in the epidermal basal cell layer and the outer root sheath of the hair follicle. This unscheduled increase in cellular proliferation likely explains the decrease in hair density observed in the K5: CAT mouse following persistent doxycycline intake. Numerous hyperplastic endometrioid cysts, which display cornification toward their lumens, are also observed during this treatment period. Remarkably, de-induction of ΔN89 β-catenin expression through doxycycline withdrawal results in a marked reversal of the skin phenotype, suggesting that these morphological changes are dependent on continued signaling by β-catenin and/or its downstream molecular mediators. Joining a small group of mouse models for conditional β-catenin signaling, our K5: CAT mouse model will be particularly useful in identifying those molecular mediators of β-catenin that are responsible for initiating and maintaining these phenotypic responses in the K5: CAT skin. Such studies are predicted to shed more light on β-catenin signaling in epidermal epithelial morphogenesis, hair follicle cycling, and hair growth pathologies.

Casein kinase 1ε (CK1ε) and axis inhibitor 1 (AXIN1) are crucial components of the β-catenin destruction complex in canonical Wnt signaling. CK1ε has been shown to interact with AXIN1, but its physiological function and role in tumorigenesis remain unknown. In this study, we found that CK1δ/ε inhibitors significantly enhanced AXIN1 protein level in colorectal cancer (CRC) cells through targeting CK1ε. Mechanistically, CK1ε promoted AXIN1 degradation by the ubiquitin-proteasome pathway by promoting the interaction of E3 ubiquitin-protein ligase SIAH1 with AXIN1. Genetic or pharmacological inhibition of CK1ε and knockdown of SIAH1 downregulated the expression of Wnt/β-catenin-dependent genes, suppressed the viability of CRC cells, and restrained tumorigenesis and progression of CRC in vitro and in vivo. In summary, our results demonstrate that CK1ε exerted its oncogenic role in CRC occurrence and progression by regulating the stability of AXIN1. These findings reveal a novel mechanism by which CK1ε regulates the Wnt/β-catenin signaling pathway and highlight the therapeutic potential of targeting the CK1ε/SIAH1 axis in CRC.

Despite significant progress in cancer prevention, screening, and treatment, the still limited number of therapeutic options is an obstacle towards increasing the cancer cure rate. In recent years, many efforts were put forth to develop therapeutics that selectively target different components of the oncogenic Wnt/β-catenin signaling pathway. These include small molecule inhibitors, antibodies, and more recently, gene-based approaches. Although some of them showed promising outcomes in clinical trials, the Wnt/β-catenin pathway is still not targeted in routine clinical practice for cancer management. As for most anticancer treatments, a critical limitation to the use of Wnt/β-catenin inhibitors is their therapeutic index, i.e., the difficulty of combining effective anticancer activity with acceptable toxicity. Protecting healthy tissues from the effects of Wnt/β-catenin inhibitors is a major issue due to the vital role of the Wnt/β-catenin signaling pathway in adult tissue homeostasis and regeneration. In this review, we provide an up-to-date summary of clinical trials on Wnt/β-catenin pathway inhibitors, examine their anti-tumor activity and associated adverse events, and explore strategies under development to improve the benefit/risk profile of this therapeutic approach.

Protein homeostasis is the basis of normal life activities, and the proteasome family plays an extremely important function in this process. The proteasome 20S is a concentric circle structure with two α rings and two β rings overlapped. The proteasome 20S can perform both ATP-dependent and non-ATP-dependent ubiquitination proteasome degradation by binding to various subunits (such as 19S, 11S, and 200 PA), which is performed by its active subunit β1, β2, and β5. The proteasome can degrade misfolded, excess proteins to maintain homeostasis. At the same time, it can be utilized by tumors to degrade over-proliferate and unwanted proteins to support their growth. Proteasomes can affect the development of tumors from several aspects including tumor signaling pathways such as NF-κB and p53, cell cycle, immune regulation, and drug resistance. Proteasome-encoding genes have been found to be overexpressed in a variety of tumors, providing a potential novel target for cancer therapy. In addition, proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib have been put into clinical application as the first-line treatment of multiple myeloma. More and more studies have shown that it also has different therapeutic effects in other tumors such as hepatocellular carcinoma, non-small cell lung cancer, glioblastoma, and neuroblastoma. However, proteasome inhibitors are not much effective due to their tolerance and singleness in other tumors. Therefore, further studies on their mechanisms of action and drug interactions are needed to investigate their therapeutic potential.

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 downregulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3β was significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression upregulated 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. 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. 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 downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which 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 upregulate PKCα and inhibit WNT signaling.

Diabetes represents a major public health concern with a considerable impact on human life and healthcare expenditures. It is now well established that diabetes is characterized by a severe skeletal muscle pathology that limits functional capacity and quality of life. Increasing evidence indicates that diabetes is also one of the most prevalent disorders characterized by impaired skeletal muscle regeneration, yet underlying mechanisms and therapeutic treatments remain poorly established. In this review, we describe the cellular and molecular alterations currently known to occur during skeletal muscle regeneration in people with diabetes and animal models of diabetes, including its associated comorbidities, e.g., obesity, hyperinsulinemia, and insulin resistance. We describe the role of myogenic and non-myogenic cell types on muscle regeneration in conditions with or without diabetes. Therapies for skeletal muscle regeneration and gaps in our knowledge are also discussed, while proposing future directions for the field.

Adult neurogenesis in the dentate gyrus (DG) is impaired during Alzheimer's disease (AD) progression. Curcumin has been reported to reduce cell apoptosis and stimulate neurogenesis. This study aimed to investigate the influence of curcumin on adult neurogenesis in AD mice and its potential mechanism. Two-month-old male C57BL/6J mice were injected with soluble β-amyloid (Aβ1-42) using lateral ventricle stereolocalization to establish AD models. An immunofluorescence assay, including bromodeoxyuridine (BrdU), doublecortin (DCX), and neuron-specific nuclear antigen (NeuN), was used to detect hippocampal neurogenesis. Western blot and an enzyme-linked immunosorbent assay (ELISA) were used to test the expression of related proteins and the secretion of brain-derived neurotrophic factor (BDNF). A Morris water maze was used to detect the cognitive function of the mice. Our results showed that curcumin administration (100 mg/kg) rescued the impaired neurogenesis of Aβ1-42 mice, shown as enhanced BrdU+/DCX+ and BrdU+/NeuN+ cells in DG. In addition, curcumin regulated the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) -mediated glycogen synthase kinase-3β (GSK3β) /Wingless/Integrated (Wnt)/β-catenin pathway and cyclic adenosine monophosphate response element-binding protein (CREB)/BDNF in Aβ1-42 mice. Inhibiting Wnt/β-catenin and depriving BDNF could reverse both the upregulated neurogenesis and cognitive function of curcumin-treated Aβ1-42 mice. In conclusion, our study indicates that curcumin, through targeting PI3K/Akt, regulates GSK3β/Wnt/β-catenin and CREB/BDNF pathways, improving the adult neurogenesis of AD mice.

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent. The whole protein and its C-terminal domain can be degraded directly by the 20S proteasome in vitro. Partial depletion of PA28γ revealed that this regulatory subunit contributes to H1 degradation within the cell. Our study shows that histone H1 protein levels are under tight regulation to prevent its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome, a process enhanced by the regulatory subunit PA28γ.

Colorectal cancer (CRC) is a leading cause of cancer deaths worldwide. Over the past three decades, extensive efforts have sought to elucidate the genomic landscape of CRC. These studies reveal that CRC is highly heterogeneous at the molecular level, with different subtypes characterised by distinct somatic mutational profiles, epigenetic aberrations and transcriptomic signatures. This review summarises our current understanding of the genomic and epigenomic alterations implicated in CRC development and progression. Particular focus is given to how characterisation of CRC genomes is leading to more personalised approaches to diagnosis and treatment.

WNT/β-catenin signaling is mediated by the transcriptional coactivator β-catenin (CTNNB1). CTNNB1 abundance is regulated by phosphorylation and proteasomal degradation promoted by a destruction complex composed of the scaffold proteins APC and AXIN1 or AXIN2, and the kinases CSNK1A1 and GSK3A or GSK3B. Loss of CSNK1A1 increases CTNNB1 abundance, resulting in hyperactive WNT signaling. Previously, we demonstrated that the HECT domain ubiquitin ligase HUWE1 is necessary for hyperactive WNT signaling in HAP1 haploid human cells lacking CSNK1A1. Here, we investigate the mechanism underlying this requirement. In the absence of CSNK1A1, GSK3A/GSK3B still phosphorylated a fraction of CTNNB1, promoting its degradation. HUWE1 loss enhanced GSK3A/GSK3B-dependent CTNNB1 phosphorylation, further reducing CTNNB1 abundance. However, the reduction in CTNNB1 caused by HUWE1 loss was disproportionately smaller than the reduction in WNT target gene transcription. To test if the reduction in WNT signaling resulted from reduced CTNNB1 abundance alone, we engineered the endogenous CTNNB1 locus in HAP1 cells to encode a CTNNB1 variant insensitive to destruction complex-mediated phosphorylation and degradation. HUWE1 loss in these cells reduced WNT signaling with no change in CTNNB1 abundance. Genetic interaction and overexpression analyses revealed that the effects of HUWE1 on WNT signaling were not only mediated by GSK3A/GSK3B, but also by APC and AXIN1. Regulation of WNT signaling by HUWE1 required its ubiquitin ligase activity. These results suggest that in cells lacking CSNK1A1, a destruction complex containing APC, AXIN1 and GSK3A/GSK3B downregulates WNT signaling by phosphorylating and targeting CTNNB1 for degradation. HUWE1 enhances WNT signaling by antagonizing this activity. Therefore, HUWE1 enhances WNT/CTNNB1 signaling through two mechanisms, one that regulates CTNNB1 abundance and another that is independent of CTNNB1 stability. Coordinated regulation of CTNNB1 abundance and an independent signaling step by HUWE1 would be an efficient way to control WNT signaling output, enabling sensitive and robust activation of the pathway.