Activation Of Wnt Pathway Research Articles

NID1 promotes laryngeal cancer stemness via activating WNT pathway.

Laryngeal cancer (LCA) is one of the most common head and neck squamous cell carcinoma with poor outcome. LCA stem cells are the main reason for LCA therapy resistance and relapse. Understanding the molecular mechanisms of the self-renew of LCA stem cells is critical to develop now targets and strategies for LCA therapy. Q-PCR and western blotting assays were used to determine NID1 level in LCA tissues and normal laryngeal tissues. MTT, colony formation assay, apoptosis assay and animal model were used to investigate the effect of NID1 on radiotherapy resistance. Side population assay and sphere formation assay were used to determine the role of LCA in the self-renew of LCA stem cells. NID1 was upregulated in LCA tissues, particularly in LCA tissues derived from relapsed patients, and associated with had poor outcome. NID1 knockdown suppressed radiotherapy resistance and the self-renew of LCA stem cells, while NID1 overexpression promoted radiotherapy resistance and the self-renew of LCA stem cells. Further analysis showed that NID1 promotes radiotherapy resistance and the self-renew of LCA stem cells via activating WNT pathway. Moreover, NID1 level was positively correlated with nuclear β-Catenin level in LCA tissues. Our results show that NID1 promotes radiotherapy resistance and the self-renew of LCA stem cells via activating WNT pathway, providing a novel potential target for LCA treatment.

Reconstructing signaling history of single cells with imaging-based molecular recording.

The intensity and duration of biological signals encode information that allows a few pathways to regulate a wide array of cellular behaviors. Despite the central importance of signaling in biomedical research, our ability to quantify it in individual cells over time remains limited. Here, we introduce INSCRIBE, an approach for reconstructing signaling history in single cells using endpoint fluorescence images. By regulating a CRISPR base editor, INSCRIBE generates mutations in genomic target sequences, at a rate proportional to signaling activity. The number of edits is then recovered through a novel ratiometric readout strategy, from images of two fluorescence channels. We engineered human cell lines for recording WNT and BMP pathway activity, and demonstrated that INSCRIBE faithfully recovers both the intensity and duration of signaling. Further, we used INSCRIBE to study the variability of cellular response to WNT and BMP stimulation, and test whether the magnitude of response is a stable, heritable trait. We found a persistent memory in the BMP pathway. Progeny of cells with higher BMP response levels are likely to respond more strongly to a second BMP stimulation, up to 3 weeks later. Together, our results establish a scalable platform for genetic recording and in situ readout of signaling history in single cells, advancing quantitative analysis of cell-cell communication during development and disease.

Extraembryonic mesoderm cells derived from human embryonic stem cells rely on Wnt pathway activation.

Extraembryonic mesoderm cells (EXMCs) are involved in the development of multiple embryonic lineages and umbilical cord formation, where they subsequently develop into mesenchymal stem cells (MSCs). Although EXMCs can be generated from human naïve embryonic stem cells (ESCs), it is unclear whether human primed ESCs (hpESCs) can differentiate into EXMCs that subsequently produce MSCs. The present report described a three-dimensional differentiation protocol to induce hpESCs into EXMCs by activating the Wnt pathway using CHIR99021. Single-cell transcriptome and immunostaining analyses revealed that the EXMC characteristics were similar to those of post-implantation embryonic EXMCs. Cell sorting was used to purify and expand the EXMCs. Importantly, these EXMCs secreted extracellular matrix proteins, including COL3A1 and differentiated into MSCs. Inconsistent with other MSC types, these MSCs exhibited a strong differentiation potential for chondrogenic and osteogenic cells and lacked adipocyte differentiation. Together, these findings provided a protocol to generate EXMCs and subsequent MSCs from hpESCs.

The soluble factor milieu in idiopathic pulmonary fibrosis dysregulates epithelial differentiation.

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-β alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-β were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-β alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-β had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-β. Although both IPFc and TGF-β increased extracellular matrix gene expression, only TGF-β increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-β. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-β. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF.

A novel mutation in the BTB domain impairs transcriptional repression function of KCTD1 leading to syndromic microtia

Microtia is a common birth defect affecting the external ears and encompasses a spectrum of congenital anomalies of the auricle. For some of the microtia-associated syndromes, the additional abnormalities are not easily observed or with variable expressivity. Identifying pathogenic mutations through genetic testing is of great help in recognizing these highly heterogeneous syndromes in clinical practice. We reported a novel de novo KCTD1 mutation in a Chinese patient with congenital microtia. It expands the mutational spectrum of KCTD1 and provide an additional scalp‐ear‐nipple patient with typical and atypical clinical presentations. The identified mutation in the BTB domain impairs the suppressive activity of the AP-2 transcription factor family and may impact on maintaining the finely tuned activity of WNT pathway, which directs stem cell development in ectoderm patterning and craniofacial development. Due to the variable expressive clinical phenotypes of syndromic microtia, genetic molecular testing could be of great help in the definite diagnosis.

Stimulating Wnt signaling reveals context-dependent genetic effects on gene regulation in primary human neural progenitors.

Gene regulatory effects have been difficult to detect at many non-coding loci associated with brain-related traits, likely because some genetic variants have distinct functions in specific contexts. To explore context-dependent gene regulation, we measured chromatin accessibility and gene expression after activation of the canonical Wnt pathway in primary human neural progenitors (n = 82 donors). We found that TCF/LEF motifs and brain-structure-associated and neuropsychiatric-disorder-associated variants were enriched within Wnt-responsive regulatory elements. Genetically influenced regulatory elements were enriched in genomic regions under positive selection along the human lineage. Wnt pathway stimulation increased detection of genetically influenced regulatory elements/genes by 66%/53% and enabled identification of 397 regulatory elements primed to regulate gene expression. Stimulation also increased identification of shared genetic effects on molecular and complex brain traits by up to 70%, suggesting that genetic variant function during neurodevelopmental patterning can lead to differences in adult brain and behavioral traits.

Cuproptosis in microsatellite stable colon cancer cells affects the cytotoxicity of CD8+T through the WNT signaling pathway

The microsatellite stable (MSS) colon cancer (CC) has long been considered resistant to immunotherapy. Cuproptosis, as a novel form of cell death, may interact with tumor immunity. This project focused on the impact of cuproptosis on the cytotoxicity of CD8+T in MSS CC, aiming to provide effective clues for improving the treatment strategy of MSS CC. The study developed an MSS CC cuproptosis model using 50 nM elesclomol and 1 μM CuCl2. Cuproptotic SW480 cells were directly co-cultured with CD8+ T cells. Cuproptosis levels were assessed via intracellular copper ion detection, Western blot, and confocal laser scanning microscopy. CCK-8, Hochest/PI staining, CFSE cell proliferation assay, LDH cytotoxicity detection, and ELISA were used to evaluate CD8+ T cell immune activity and cytotoxicity. Transcriptome sequencing and bioinformatics analysis identified regulated signals in cuproptotic SW480 cells. A rescue experiment utilized a WNT pathway activator (BML-284). PD-L1 expression in cells/membranes was analyzed using qRT-PCR, Western blot, and flow cytometry. NSG mice were immunoreconstituted, and the effects of cuproptosis on immune infiltration and cancer progression in MSS CC mice were assessed using ELISA and immunohistochemistry (IHC). Treatment with 50 nM elesclomol and 1 μM CuCl2 significantly increased cuproptosis in SW480 cells. Co-culture with CD8+ T cells enhanced their cytotoxicity. Sequencing revealed cuproptosis-mediated modulation of immune and inflammatory pathways, including WNT signaling. Rescue experiments showed downregulation of WNT signaling in cuproptotic SW480 cells. Indirectly, CD8+ T cell immune function was enhanced by reducing PD-L1 expression. In mice, cuproptosis resulted in increased infiltration of CD8+ T cells in tumor tissue, leading to delayed cancer progression compared to the control group. Cuproptosis in MSS CC cells enhances the cytotoxicity of CD8+ T cells, which may be achieved through downregulation of the WNT signaling pathway and decreased expression of PD-L1. In the future, drugs that can induce cuproptosis may be a promising approach to improve MSS CC immunotherapy.

The overexpression of R-spondin 3 affects hair morphogenesis and hair development along with the formation and maturation of the hair follicle stem cells.

Mice hair follicles (HFs) are a valuable model for studying various aspects of hair biology, including morphogenesis, development, and regeneration due to their easily observable phenotype and genetic manipulability. The initiation and progression of hair follicle morphogenesis, as well as the hair follicle cycle, are regulated by various signaling pathways, of which the main role is played by the Wingless-type MMTV integration site family (Wnt) and the Bone Morphogenic Protein (BMP). During the hair follicle cycle, the BMP pathway maintains hair follicle stem cells (HFSCs) in a dormant state while the Wnt pathway activates them for hair growth. Given the pivotal role of the Wnt pathway in hair biology and HFSCs regulation, we investigated the influence of the Wnt modulator - R-spondin 3 (Rspo3), in these processes. For this purpose, we developed a transgenic mice model with the overexpression of Rspo3 (Rspo3GOF) in the whole ectoderm and its derivatives, starting from early morphogenesis. Rspo3GOF mice exhibited a distinct phenotype with sparse hair and visible bald areas, caused by reduced proliferation and increased apoptosis of hair matrix progenitor cells, which resulted in a premature anagen-to-catagen transition with a shortened growth phase and decreased overall length of all hair types. In addition, Rspo3GOF promoted induction of auchene and awl, canonical Wnt-dependent hair type during morphogenesis, but the overall hair amount remained reduced. We also discovered a delay in the pre-bulge formation during morphogenesis and prolonged immaturity of the HFSC population in the bulge region postnatally, which further impaired proper hair regeneration throughout the mice's lifespan. Our data supported that Rspo3 function observed in our model works in HFSCs' formation of pre-bulge during morphogenesis via enhancing activation of the canonical Wnt pathway, whereas in contrast, in the postnatal immature bulge, activation of canonical Wnt signaling was attenuated. In vitro studies on keratinocytes revealed changes in proliferation, migration, and colony formation, highlighting the inhibitory effect of constitutive overexpression of Rspo3 on these cellular processes. Our research provides novel insights into the role of Rspo3 in the regulation of hair morphogenesis and development, along with the formation and maturation of the HFSCs, which affect hair regeneration.

Cadherin adhesion complexes direct cell aggregation in the epithelial transition of Wnt-induced nephron progenitor cells.

In the developing mammalian kidney, nephron formation is initiated by a subset of nephron progenitor cells (NPCs). Wnt input activates a β-catenin (Ctnnb1)-driven, transcriptional nephrogenic program and the mesenchymal to epithelial transition (MET) of NPCs. Using an in vitro mouse NPC culture model, we observed that activation of the Wnt pathway results in the aggregation of induced NPCs, which is an initiating step in the MET program. Genetic removal showed aggregation was dependent on β-catenin. Modulating extracellular Ca2+ levels showed cell-cell contacts were Ca2+ dependent, suggesting a role for cadherin (Cdh)-directed cell adhesion. Molecular analysis identified Cdh2, Cdh4 and Cdh11 in NPCs, and the β-catenin directed upregulation of Cdh3 and Cdh4 accompanying the MET of induced NPCs. Mutational analysis of β-catenin supported a role for a Lef/Tcf-β-catenin-mediated transcriptional response in the cell aggregation process. Genetic removal of all four cadherins, and independent removal of α-catenin or of β-catenin-α-catenin interactions, abolished aggregation, but not the inductive response to Wnt pathway activation. These findings, and data in an accompanying article highlight the role of β-catenin in linking transcriptional programs to the morphogenesis of NPCs in mammalian nephrogenesis.

Comprehensive analysis of transcription factors involved in odontoblast differentiation mechanism.

Primary cultured odontoblasts rapidly lose their tissue-specific phenotype. To identify transcription factors (TF) that are important for the maintenance of the odontoblast phenotype, primary cultures of C57BL/6J mouse dental mesenchymal cells (DMC) were isolated, and expression of TF and odontoblast marker genes in cells immediately after isolation and 2days after culture were comprehensively evaluated and compared using RNA-sequencing (RNA-seq). The expression of odontoblast markers in mouse dental mesenchymal cells decreased rapidly after isolation. In addition, the expression of Hedgehog-related, Notch-related, and immediate- early gene (IEG)-related transcription factors significantly decreased. Forced expression of these genes in lentiviral vectors, together with fibroblast growth factor 4 (FGF4), fibroblast growth factor 9 (FGF9), and the Wnt pathway activator CHIR99021, significantly induced the expression of odontogenic marker genes. These results indicate, for the first time, that Notch signaling and early genes may be important for maintaining odontoblast cultures. Furthermore, simultaneous stimulation of FGF, Wnt, Hedgehog, Notch pathways, and IEG transcription factors cooperatively promoted the maintenance of the odontoblast phenotype. These results suggest that the Hedgehog and Notch signaling pathways may play an important role in maintaining odontoblast phenotypes, in addition to FGF and Wnt signaling.

Pilose antler extracts promotes hair growth in androgenetic alopecia mice by activating hair follicle stem cells via the AKT and Wnt pathways.

Background: Angrogenetic alopecia (AGA) is one of the most prevalent hair loss disorders worldwide. The hair follicle stem cell (HFSC) is closely related to the formation of hair follicle (HF) structure and HF self-renewal. The activation of HFSC in AGA is critical for hair growth. Pilose antler has been reported to have hair growth-promoting activity, but the mechanism of action on AGA and HFSC has not been reported. Methods: We previously extracted an active component from the pilose antler known as PAEs. In this study, we conducted experiments using AGA mice and HFSC. The effects of PAEs on hair growth in AGA mice were firstly detected, and then the mechanisms of PAEs for AGA were predicted by integrating network pharmacology and de novo transcriptomics data of pilose antler. Finally, biological experiments were used to validate the molecular mechanism of PAEs in treating AGA both in vivo and in vitro. Results: It was found that PAEs promoted hair regrowth by accelerating the activation of anagen, delaying the anagen-catagen transition. It also alleviated the morphological changes, such as hair shortening, thinning, miniaturization, and HF number reduction, and regulated the hair regeneration process of four subtypes of hair. We further found that PAEs could promote the proliferation of HFSC, outer root sheath (ORS) cells, and hair bulb cells in AGA mice. We then integrated network pharmacology and pilose antler transcriptomics data to predict that the mechanism of PAEs treatment in AGA mice is closely related to the PI3K-AKT/Wnt-β-Catenin pathways. Subsequently, it was also verified that PAEs could activate both pathways in the skin of AGA mice. In addition, we found that PAEs perhaps increased the number of blood vessels around dermal papilla (DP) in experiments in vivo. Meanwhile, the PAEs stimulated the HFSC proliferation in vitro and activated the AKT and Wnt pathways. However, the proliferative activity of HFSC was inhibited after blocking the Wnt pathway and AKT activity. Conclusion: This study suggests that the hair growth-promoting effect of PAEs in AGA mice may be closely related to the stimulation of the AKT and Wnt pathways, which in turn activates the proliferation of HFSC.

P-312 Transcriptional profiling of SSEA-1+ endometrial epithelial cells highlights their role in endometrial regeneration, remodelling and homeostasis

Abstract Study question Will the transcriptional profile of SSEA-1+ endometrial epithelial cells (EECs) explain their functional role, which can be explored in an in vitro endometrial organoid model? Summary answer SSEA-1+ EECs demonstrate differentially expressed genes (DEGs) and associated functional pathways expected from a basal stem/progenitor cell (SPC) population, agreeing with the in vitro experiments. What is known already Endometrial SPCs are postulated to reside in the basalis and to be responsible for the full phenotypic and functional restoration of the endometrial functionalis layer. SSEA-1+ EECs assume the postulated basalis SPC niche. Previous studies demonstrated isolated SSEA-1+ EECs to have a higher capacity to generate organoids in 3D matrix and display growth like that observed after endometrial denudation. They have lower steroid hormone expression and higher telomerase activity with longer telomere lengths, all suggesting a SPC phenotype. SSEA-1+ EECs co-express nuclear SRY-box transcription factor 9 (nSOX9) and nuclear b-catenin, suggesting an activated Wnt pathway. Study design, size, duration Endometrial samples were collected from eight pre-menopausal women attending the Liverpool Women’s Hospital for benign gynaecological procedures, aged 30-47 years. These patients were having regular menstrual cycles and had not been on any hormonal therapy for at least three months prior to enrolment. Three further endometrial samples were obtained for 3D EEC organoid culture studies. Participants/materials, setting, methods Magnetic-activated cell sorting was used to isolate SSEA-1 enriched/depleted EECs from freshly harvested endometrial samples. Microarray analysis was performed using Agilent human arrays. DEGs and pathway enrichment analysis were explored using R-studio and Ingenuity Pathway Analysis (IPA). Internal and external validation used RT-qPCR, dual-immunofluorescence, and comparison against publicly available endometrial single cell spatial transcriptomic data. Human EEC organoids models were employed to assess hormonal regulation of SSEA-1 expression using immunohistochemistry. Main results and the role of chance SSEA-1+ EECs have a distinct transcriptional profile, with 1,059 DEGs compared with SSEA-1- EECs. Pathway analysis highlighted their role in endometrial regeneration, adhesion, remodelling, and neovascularisation, enriching for pathways such as ‘extracellular matrix structural constituent’, ‘epithelial to mesenchymal transition’, ‘angiogenesis’, ‘TNFα signalling via NFKB’ and ‘Notch signalling’. IPA’s biological functions demonstrated their involvement in tissue homeostasis, tumour suppression and their more quiescent SPC phenotype, with activation of ‘organismal death’ and inhibition of ‘cell movement/migration’ and ‘cell movement/migration of tumour cell lines’. The activation of canonical pathways such as ‘PTEN signalling’ and ‘endocannabinoid cancer inhibition’ demonstrated their fine equilibrium to drive proliferation during regeneration, whilst providing protection from hyperplastic/carcinogenic transformation. Ten selected DEGs were internally validated using RT-qPCR, demonstrating concordance. Dual-immunofluorescence of gene products of DEGs MMP7, MMP26, C11orf52 and CD47 confirmed co-localisation alongside SSEA-1 in an external biological cohort of endometrial samples. External in silico validation using the Garcia-Alonso et al endometrial single-cell transcriptomics dataset, mapped significantly upregulated DEGs MMP7 and MMP26 to mainly SOX9+ and SOX9+/LGR5+ EECs. EEC organoids exposed to hormones (including oestradiol) demonstrated an induction of proliferation and higher proportion of organoids expressing SSEA-1 (46.8% vs 67%) simulating endometrial glandular regeneration process. Limitations, reasons for caution Freshly isolated/sorted EECs were obtained from women at different menstrual cycle phases. Our organoid model system only contained EECs without the stromal component, thus, was not suitable to assess progestogenic response of EECs that occur in vivo. Wider implications of the findings This study provides novel information on human SSEA-1+ EECs, suggesting their pivotal role in endometrial regeneration. Since endometrial epithelial SPCs make a vital contribution to endometrial repair and pregnancy establishment, characterising of these cells in health will allow the development of targeted novel therapies on aberrant SPCs within gynaecological disease. Trial registration number Not applicable

Physiological regulation of neuronal Wnt activity is essential for TDP-43 localization and function

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease.

Altered glia-neuron communication in Alzheimer’s Disease affects WNT, p53, and NFkB Signaling determined by snRNA-seq

BackgroundAlzheimer’s disease is the most common cause of dementia and is characterized by amyloid-β plaques, tau neurofibrillary tangles, and neuronal loss. Although neuronal loss is a primary hallmark of Alzheimer’s disease, it is known that non-neuronal cell populations are ultimately responsible for maintaining brain homeostasis and neuronal health through neuron-glia and glial cell crosstalk. Many signaling pathways have been proposed to be dysregulated in Alzheimer’s disease, including WNT, TGFβ, p53, mTOR, NFkB, and Pi3k/Akt signaling. Here, we predict altered cell-cell communication between glia and neurons.MethodsUsing public snRNA-sequencing data generated from postmortem human prefrontal cortex, we predicted altered cell-cell communication between glia (astrocytes, microglia, oligodendrocytes, and oligodendrocyte progenitor cells) and neurons (excitatory and inhibitory). We confirmed interactions in a second and third independent orthogonal dataset. We determined cell-type-specificity using Jaccard Similarity Index and investigated the downstream effects of altered interactions in inhibitory neurons through gene expression and transcription factor activity analyses of signaling mediators. Finally, we determined changes in pathway activity in inhibitory neurons.ResultsCell-cell communication between glia and neurons is altered in Alzheimer’s disease in a cell-type-specific manner. As expected, ligands are more cell-type-specific than receptors and targets. We identified ligand-receptor pairs in three independent datasets and found involvement of the Alzheimer’s disease risk genes APP and APOE across datasets. Most of the signaling mediators of these interactions were not significantly differentially expressed, however, the mediators that are also transcription factors had differential activity between AD and control. Namely, MYC and TP53, which are associated with WNT and p53 signaling, respectively, had decreased TF activity in Alzheimer’s disease, along with decreased WNT and p53 pathway activity in inhibitory neurons. Additionally, inhibitory neurons had both increased NFkB signaling pathway activity and increased TF activity of NFIL3, an NFkB signaling-associated transcription factor.ConclusionsCell-cell communication between glia and neurons in Alzheimer’s disease is altered in a cell-type-specific manner involving Alzheimer’s disease risk genes. Signaling mediators had altered transcription factor activity suggesting altered glia-neuron interactions may dysregulate signaling pathways including WNT, p53, and NFkB in inhibitory neurons.

A gene edited pig model for studying LGR5+ stem cells: implications for future applications in tissue regeneration and biomedical research.

Recent advancements in genome editing techniques, notably CRISPR-Cas9 and TALENs, have marked a transformative era in biomedical research, significantly enhancing our understanding of disease mechanisms and helping develop novel therapies. These technologies have been instrumental in creating precise animal models for use in stem cell research and regenerative medicine. For instance, we have developed a transgenic pig model to enable the investigation of LGR5-expressing cells. The model was designed to induce the expression of H2B-GFP under the regulatory control of the LGR5 promoter via CRISPR/Cas9-mediated gene knock-in. Notably, advancements in stem cell research have identified distinct subpopulations of LGR5-expressing cells within adult human, mouse, and pig tissues. LGR5, a leucine-rich repeat-containing G protein-coupled receptor, enhances WNT signaling and these LGR5+ subpopulations demonstrate varied roles and anatomical distributions, underscoring the necessity for suitable translational models. This transgenic pig model facilitates the tracking of LGR5-expressing cells and has provided valuable insights into the roles of these cells across different tissues and species. For instance, in pulmonary tissue, Lgr5+ cells in mice are predominantly located in alveolar compartments, driving alveolar differentiation of epithelial progenitors via Wnt pathway activation. In contrast, in pigs and humans, these cells are situated in a unique sub-basal position adjacent to the airway epithelium. In fetal stages a pattern of LGR5 expression during lung bud tip formation is evident in humans and pigs but is lacking in mice. Species differences with respect to LGR5 expression have also been observed in the skin, intestines, and cochlea further reinforcing the need for careful selection of appropriate translational animal models. This paper discusses the potential utility of the LGR5+ pig model in exploring the role of LGR5+ cells in tissue development and regeneration with the goal of translating these findings into human and animal clinical applications.

Increasing GSH-Px Activity and Activating Wnt Pathway Promote Fine Wool Growth in FGF5-Edited Sheep.

Fibroblast growth factor 5 (FGF5) plays key roles in promoting the transition from the anagen to catagen during the hair follicle cycle. The sheep serves as an excellent model for studying hair growth and is frequently utilized in various research processes related to human skin diseases. We used the CRISPR/Cas9 system to generate four FGF5-edited Dorper sheep and only low levels of FGF5 were detected in the edited sheep. The density of fine wool in GE sheep was markedly increased, and the proportion of fine wool with a diameter of 14.4-20.0 μm was significantly higher. The proliferation signal in the skin of gene-edited (GE) sheep was stronger than in wild-type (WT) sheep. FGF5 editing decreased cortisol concentration in the skin, further activated the activity of antioxidant enzymes such as Glutathione peroxidase (GSH-Px), and regulated the expression of Wnt signaling pathways containing Wnt agonists (Rspondins, Rspos) and antagonists (Notum) in hair regeneration. We suggest that FGF5 not only mediates the activation of antioxidant pathways by cortisol, which constitutes a highly coordinated microenvironment in hair follicle cells, but also influences key signals of the Wnt pathway to regulate secondary hair follicle (SHF) development. Overall, our findings here demonstrate that FGF5 plays a significant role in regulating SHF growth in sheep and potentially serves as a molecular marker of fine wool growth in sheep breeding.

Trefoil factor 1 suppresses stemness and enhances chemosensitivity of pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies, partly due to resistance to conventional chemotherapy. The chemoresistance of malignant tumors is associated with epithelial-mesenchymal transition (EMT) and the stemness of cancer cells. The aim of this study is to investigate the availability and functional mechanisms of trefoil factor family 1 (TFF1), a tumor-suppressive protein in pancreatic carcinogenesis, to treat pancreatic cancer. To investigate the role of endogenous TFF1 in human and mice, specimens of human pancreatic cancer and genetically engineered mouse model of pancreatic cancer (KPC/TFF1KO; Pdx1-Cre/LSL-KRASG12D/LSL-p53R172H/TFF1-/-) were analyzed by immunohistochemistry (IHC). To explore the efficacy of extracellular administration of TFF1, recombinant and chemically synthesized TFF1 were administered to pancreatic cancer cell lines, a xenograft mouse model and a transgenic mouse model. The deficiency of TFF1 was associated with increased EMT of cancer cells in mouse models of pancreatic cancer, KPC. The expression of TFF1 in cancer cells was associated with better survival rate of the patients who underwent chemotherapy, and loss of TFF1 deteriorated the benefit of gemcitabine in KPC mice. Extracellular administration of TFF1 inhibited gemcitabine-induced EMT, Wnt pathway activation and cancer stemness, eventually increased apoptosis of pancreatic cancer cells invitro. Invivo, combined treatment of gemcitabine and subcutaneous administration of TFF1 arrested tumor growth in xenograft mouse model and resulted in the better survival of KPC mice by inhibiting EMT and cancer stemness. These results indicate that TFF1 can contribute to establishing a novel strategy to treat pancreatic cancer patients by enhancing chemosensitivity.

Molecular characteristics of non-human papillomavirus driven cervical adenocarcinoma and adenosquamous carcinoma.

e17532 Background: Cervical cancer is the fourth most common malignancy in women worldwide. Although infection from human papillomavirus (HPV) has been the major cause of cervical cancer, about 3-8% of cervical cancer cases are HPV-negative. Clinical data showed that patients with HPV-negative cervical cancer had significantly lower DFS than those with HPV-positive cervical cancer. The detection of HPV DNA tends to be lower in adenosquamous carcinoma (AC/ASC) than in squamous cell carcinoma. Because the mechanism of HPV-negative cervical cancer development is unclear, this study aims to find the pattern of differential gene expression in HPV-negative AC/ASC and verify the underlying potential mechanism. Methods: In this study, we aimed to exam the differential gene expression profiling by exome-capture RNA sequencing on formalin-fixed, paraffin-embedded tumor tissue of prospectively enrolled cervical AC/ASC patients (HPV-negative: HPV-positive: 1:1) and validate the selected biomarkers on the remaining (unmatched; n = 110) prospective cohort and the 274 retrospective cohort patients. Subsequently, dysregulated differentially expressed genes specifically existed in HPV-negative cervical AC/ASC tissues and HPV-negative cell lines were validated by Western blotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining (IHC staining). Results: Exome-capture RNA sequencing on HPV-negative and age/grade matched HPV-positive AC/ASC (10:6) for discovery initially. We found differentially activated WNT pathway from HPV positive/negative AC/ASC in the prospective cohort. In the preliminary results of 28 prospective cohort patients, SOX17 and MMP7 highly expressed genes in HPV-negative (n = 13) then the HPV-positive (n = 11) cervical AC/ASC by IHC staining (p = 0.1029 and p = 0.0306, respectively). Moreover, plasma MMP7 levels were upregulated in HPV-negative AC/ASC (n = 10) compared with HPV-positive AC/ASC (n = 17) (p = 0.0086) by ELISA analysis in the prospective cohort. Conclusions: Further studies and more analyses will be continued to investigate the effects of changes in the expression of SOX17 and MMP7 related mechanisms, and targeting WNT/SOX17/MMP7 pathways specifically associated with HPV-negative AC/ASC to provide new insights into the early screening and effective treatment of HPV-negative AC/ASC.

Semaphorin Receptors Antagonize Wnt Signaling Through Beta-Catenin Degradation.

Precise control of morphogen signaling levels is essential for proper development. An outstanding question is: what mechanisms ensure proper morphogen activity and correct cellular responses? Previous work has identified Semaphorin (SEMA) receptors, Neuropilins (NRPs) and Plexins (PLXNs), as positive regulators of the Hedgehog (HH) signaling pathway. Here, we provide evidence that NRPs and PLXNs antagonize Wnt signaling in both fibroblasts and epithelial cells. Further, Nrp1/2 deletion in fibroblasts results in elevated baseline Wnt pathway activity and increased maximal responses to Wnt stimulation. Notably, and in contrast to HH signaling, SEMA receptor-mediated Wnt antagonism is independent of primary cilia. Mechanistically, PLXNs and NRPs act downstream of Dishevelled (DVL) to destabilize β-catenin (CTNNB1) in a proteosome-dependent manner. Further, NRPs, but not PLXNs, act in a GSK3b/CK1-dependent fashion to antagonize Wnt signaling, suggesting distinct repressive mechanisms for these SEMA receptors. Overall, this study identifies SEMA receptors as novel Wnt pathway antagonists that may also play larger roles integrating signals from multiple inputs.

Evolutionary and functional analyses of LRP5 in archaic and extant modern humans

BackgroundThe human lineage has undergone a postcranial skeleton gracilization (i.e. lower bone mass and strength relative to body size) compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could also be genetically influenced.ResultsWe have analyzed the LRP5 gene, which is known to be associated with high bone mineral density conditions, from an evolutionary and functional point of view. Taking advantage of the published genomes of archaic Homo populations, our results suggest that this gene has a complex evolutionary history both between archaic and living humans and within living human populations. In particular, we identified the presence of different selective pressures in archaics and extant modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000 Genomes Project. Furthermore, we observed a very limited evidence of archaic introgression in this gene (only at three haplotypes of East Asian ancestry out of the 1000 Genomes), compatible with a general erasing of the fingerprint of archaic introgression due to functional differences in archaics compared to extant modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing bone mineral density. In particular, four of five archaic missense mutations affecting the first β-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation.ConclusionsIn summary, these data suggest a genetic component contributing to the understanding of skeletal differences between extant modern humans and archaic Homo populations.