ABSTRACT
The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.
Subject(s)
Breast Neoplasms , Triple Negative Breast Neoplasms , Humans , Female , Animals , Breast Neoplasms/metabolism , Resveratrol , Hedgehogs/genetics , Hedgehogs/metabolism , DNA Methylation , Epigenesis, Genetic , Triple Negative Breast Neoplasms/genetics , Wnt Signaling Pathway , DNA/metabolism , Cell Line, Tumor , Gene Expression Regulation, NeoplasticABSTRACT
Breast cancer is a complex and diverse condition that disrupts multiple signaling pathways essential for cell proliferation, survival, and differentiation. Recently, the significant involvement of long-chain non-coding RNAs (lncRNAs) in controlling key signaling pathways associated with breast cancer development has been discovered. This review aims to explore the interaction between lncRNAs and various pathways, including the AKT/PI3K/mTOR, Wnt/ß-catenin, Notch, DNA damage response, TGF-ß, Hedgehog, and NF-κB signaling pathways, to gain a comprehensive understanding of their roles in breast cancer. The AKT/PI3K/mTOR pathway regulates cell growth, survival, and metabolic function. Recent data suggests that specific lncRNAs can influence the functioning of this pathway, acting as either oncogenes or tumor suppressors. Dysregulation of this pathway is commonly observed in breast cancer cases. Moreover, breast cancer development has been associated with other pathways such as Wnt/ß-catenin, Notch, TGF-ß, Hedgehog, and NF-κB. Emerging studies have identified lncRNAs that modulate breast cancer's growth, progression, and metastasis by interacting with these pathways. To advance the development of innovative diagnostic tools and targeted treatment options, it is crucial to comprehend the intricate relationship between lncRNAs and vital signaling pathways in breast cancer. By fully harnessing the therapeutic potential of lncRNAs, there is a possibility of developing more effective and personalized therapy choices for breast cancer patients. Further investigation is necessary to comprehensively understand the role of lncRNAs within breast cancer signaling pathways and fully exploit their therapeutic potential.
Subject(s)
Breast Neoplasms , RNA, Long Noncoding , Humans , Female , Animals , Breast Neoplasms/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , beta Catenin/metabolism , Proto-Oncogene Proteins c-akt/metabolism , NF-kappa B/metabolism , Hedgehogs/genetics , Hedgehogs/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Transforming Growth Factor beta/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Gene Expression Regulation, Neoplastic/geneticsABSTRACT
Neural stem cells (NSCs) in the adult hippocampus are composed of multiple subpopulations. However, their origin and functional heterogeneity are still unclear. Here, we found that the contribution of murine Wnt-responsive (Axin2+) and Hedgehog-responsive (Gli1+) embryonic neural progenitors to adult NSCs started from early and late postnatal stages, respectively. Axin2+ adult NSCs were intended to actively proliferate, whereas Gli1+ adult NSCs were relatively quiescent and responsive to external stimuli. Moreover, Gli1+ NSC-derived adult-born neurons exhibited more complex dendritic arborization and connectivity than Axin2+ NSC-derived ones. Importantly, genetic cell ablation analysis identified that Axin2+ and Gli1+ adult NSCs were involved in hippocampus-dependent learning, but only Axin2+ adult NSCs were engaged in buffering stress responses and depressive behavior. Together, our study not only defined the heterogeneous multiple origins of adult NSCs but also advanced the concept that different subpopulations of adult NSCs may function differently.
Subject(s)
Adult Stem Cells , Neural Stem Cells , Mice , Animals , Hedgehogs/metabolism , Zinc Finger Protein GLI1/genetics , Neural Stem Cells/metabolism , Hippocampus/metabolism , Neurons/metabolism , Adult Stem Cells/metabolism , Neurogenesis/physiologyABSTRACT
Hepatic fibrosis is one of the major worldwide health concerns which requires tremendous research due to the limited outcomes of the current therapies. The present study was designed to assess, for the first time, the potential therapeutic effect of rupatadine (RUP) in diethylnitrosamine (DEN)-induced liver fibrosis and to explore its possible mechanistic actions. For the induction of hepatic fibrosis, rats were treated with DEN (100 mg/kg, i.p.) once weekly for 6 consecutive weeks, and on the 6th week, RUP (4 mg/kg/day, p.o.) was administered for 4 weeks. Treatment with RUP ameliorated changes in body weights, liver indices, liver function enzymes, and histopathological alterations induced by DEN. Besides, RUP amended oxidative stress, which led to the inhibition of PAF/NF-κB p65-induced inflammation, and, subsequently, prevention of TGF-ß1 elevation and HSCs activation as indicated by reduced α-SMA expression and collagen deposition. Moreover, RUP exerted significant anti-fibrotic and anti-angiogenic effects by suppressing Hh and HIF-1α/VEGF signaling pathways. Our results highlight, for the first time, a promising anti-fibrotic potential of RUP in rat liver. The molecular mechanisms underlying this effect involve the attenuation of PAF/NF-κB p65/TGF-ß1 and Hh pathways and, subsequently, the pathological angiogenesis (HIF-1α/VEGF).
Subject(s)
NF-kappa B , Transforming Growth Factor beta1 , Rats , Animals , NF-kappa B/metabolism , Transforming Growth Factor beta1/metabolism , Hedgehogs/metabolism , Vascular Endothelial Growth Factor A , Liver Cirrhosis/chemically induced , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Liver/metabolismABSTRACT
Normal lung development critically depends on HH (Hedgehog) and PDGF (platelet-derived growth factor) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH signaling and PDGF signaling and their impact on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of Pdgfa (platelet-derived growth factor subunit A) and Pdgfra (platelet-derived growth factor receptor alpha) knockouts during secondary alveolar septation. Using a dual signaling reporter, Gli1lZ;PdgfraEGFP, we show that HH and PDGF pathway intermediates are concurrently expressed during alveolar septal myofibroblast accumulation, suggesting pathway convergence in the generation of lung myofibroblasts. Consistent with this hypothesis, HH inhibition reduces Pdgfra expression and diminishes the number of Pdgfra-positive and Pdgfra-lineage cells in postnatal lungs. Bulk RNA sequencing data of Pdgfra-expressing cells from Postnatal Day 8 (P8) lungs show that HH inhibition alters the expression not only of well-established HH targets but also of several putative PDGF target genes. This, together with the presence of Gli-binding sites in PDGF target genes, suggests HH input into PDGF signaling. We identified these HH/PDGF targets in several postnatal lung mesenchymal cell populations, including myofibroblasts, using single-cell transcriptomic analysis. Collectively, our data indicate that HH signaling and PDGF signaling intersect to support myofibroblast/fibroblast function during secondary alveolar septum formation. Moreover, they provide a molecular foundation relevant to perinatal lung diseases associated with impaired alveolarization.
Subject(s)
Hedgehogs , Lung , Pregnancy , Female , Animals , Mice , Hedgehogs/metabolism , Lung/metabolism , Platelet-Derived Growth Factor/metabolism , Myofibroblasts/metabolism , Fibroblasts/metabolism , Receptor, Platelet-Derived Growth Factor alpha/metabolismABSTRACT
Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.
Subject(s)
Hedgehogs , Wnt Signaling Pathway , Humans , Mice , Animals , Hedgehogs/metabolism , Gene Expression Regulation , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Cell Differentiation/genetics , Proteins/metabolism , Chondrocytes/metabolismABSTRACT
BACKGROUND AND AIMS: It remains unknown how patients with liver failure maintain essential albumin levels. Here, we delineate a hierarchical transcription regulatory network that ensures albumin expression under different disease conditions. APPROACH AND RESULTS: We examined albumin levels in liver tissues and serum in 157 patients, including 84 with HCC, 38 decompensated cirrhosis, and 35 acute liver failure. Even in patients with liver failure, the average serum albumin concentrations were 30.55 g/L. In healthy subjects and patients with chronic liver diseases, albumin was expressed in hepatocytes. In patients with massive hepatocyte loss, albumin was expressed in liver progenitor cells (LPCs). The albumin gene (ALB) core promoter possesses a TATA box and nucleosome-free area, which allows constitutive RNA polymerase II binding and transcription initiation. Chromatin immunoprecipitation assays revealed that hepatocyte nuclear factor 4 alpha (HNF4α), CCAAT/enhancer-binding protein alpha (C/EBPα), and forkhead box A2 (FOXA2) bound to the ALB enhancer. Knockdown of either of these factors reduced albumin expression in hepatocytes. FOXA2 acts as a pioneer factor to support HNF4α and C/EBPα. In hepatocytes lacking HNF4α and C/EBPα expression, FOXA2 synergized with retinoic acid receptor (RAR) to maintain albumin transcription. RAR nuclear translocation was induced by retinoic acids released by activated HSCs. In patients with massive hepatocyte loss, LPCs expressed HNF4α and FOXA2. RNA sequencing and quantitative PCR analyses revealed that lack of HNF4α and C/EBPα in hepatocytes increased hedgehog ligand biosynthesis. Hedgehog up-regulates FOXA2 expression through glioblastoma family zinc finger 2 binding to the FOXA2 promoter in both hepatocytes and LPCs. CONCLUSIONS: A hierarchical regulatory network formed by master and pioneer transcription factors ensures essential albumin expression in various pathophysiological conditions.
Subject(s)
Carcinoma, Hepatocellular , Liver Failure , Liver Neoplasms , Humans , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Hedgehogs/metabolism , Liver Neoplasms/metabolism , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Hepatocytes/metabolism , Liver/metabolism , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Albumins , Liver Failure/metabolismABSTRACT
The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.
Subject(s)
Anti-Bacterial Agents/history , Arthrodermataceae/metabolism , Hedgehogs/metabolism , Hedgehogs/microbiology , Methicillin Resistance/genetics , Methicillin-Resistant Staphylococcus aureus/genetics , Selection, Genetic/genetics , Animals , Anti-Bacterial Agents/metabolism , Arthrodermataceae/genetics , Denmark , Europe , Evolution, Molecular , Geographic Mapping , History, 20th Century , Humans , Methicillin-Resistant Staphylococcus aureus/metabolism , New Zealand , One Health , Penicillins/biosynthesis , Phylogeny , beta-Lactams/metabolismABSTRACT
The hedgehog (Hh) pathway is essential for animal development, but aberrant activation promotes cancer growth. In this study, we show that GIPC3, a PDZ domain-containing protein with putative adaptor protein function, positively modulates Hh target gene expression in normal fibroblasts and melanoma cells and supports melanoma tumor growth. Using overexpression and epistasis studies, we show that Gipc3 potentiates Hh transcriptional output and that it modulates GLI-dependent transcription independently of Sufu. Whereas we find that GIPC3 protein does not interact with Hh pathway components, Ingenuity Pathway Analyses of GIPC3-interacting proteins identified by coimmunoprecipitation and mass spectrometry show an association with cancer pathogenesis. Subsequent interrogation of The Cancer Genome Atlas and the Human Protein Atlas databases reveals GIPC3 upregulation in many cancers. Using expression screens in selected groups of GIPC3-upregulated cancers with reported Hh pathway activation, we find a significant positive correlation of GIPC3 expression with Hh pathway components GLI1, GLI2, and GPR161 in melanoma lines. Consistently, GIPC3 knockdown in melanoma lines significantly reduces GLI1 and GLI2 expression, cell viability, colony formation, and allograft tumor growth. Our findings highlight previously unidentified roles of GIPC3 in potentiating Hh response and melanoma tumorigenesis and suggest that GIPC3 modulation on Hh signaling may be targeted to reduce melanoma growth.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Melanoma/metabolism , Skin Neoplasms/metabolism , Adaptor Proteins, Signal Transducing/genetics , Allografts , Animals , Carcinogenesis , Cell Growth Processes , Gene Expression Regulation, Neoplastic , Hedgehogs/metabolism , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein Gli2/genetics , Zinc Finger Protein Gli2/metabolismABSTRACT
Disruption of the transcriptional activity of the Hippo pathway members YAP1 and TAZ has become a major target for cancer treatment. However, detailed analysis of the effectiveness and networks affected by YAP1/TAZ transcriptional targeting is limited. In this study, we utilize TEAD inhibitor, an inhibitor of the binding of YAP1 and TAZ with their main transcriptional target TEAD in a mouse model of basal cell carcinoma, to unveil the consequences of YAP1/TAZ transcriptional blockage in cancer cells. Both TEAD inhibitor and YAP1/TAZ knockdown lead to reduced proliferation and increased differentiation of mouse basal cell carcinoma driven by oncogenic hedgehog-smoothened (SmoM2) activity. Although TEAD-transcriptional networks were essential to inactivate differentiation, this inactivation was found to be indirect and potentially mediated through the repression of KLF4 by SNAI2. By comparing the transcriptional effects of TEAD inhibition with those caused by YAP1/TAZ depletion, we determined YAP1/TAZâTEADâindependent effects in cancer cells that impact STAT3 and NF-κB. Our results reveal the gene networks affected by targeting YAP1/TAZâTEAD in basal cell carcinoma tumors and expose the potential pitfalls for targeting TEAD transcription in cancer.
Subject(s)
Carcinoma, Basal Cell/metabolism , Hedgehogs/metabolism , TEA Domain Transcription Factors/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins/metabolism , YAP-Signaling Proteins/metabolism , Animals , Carcinogenesis , Carcinoma, Basal Cell/genetics , Cell Differentiation , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Hippo Signaling Pathway , Humans , Kruppel-Like Factor 4/metabolism , Mice , NF-kappa B/metabolism , STAT3 Transcription Factor/metabolism , Snail Family Transcription Factors/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins/genetics , YAP-Signaling Proteins/geneticsABSTRACT
We examined several aspects of African hedgehog adenovirus (AhAdv-1) that was isolated from an African pygmy hedgehog, including: replication kinetics of, virus-induced cytopathic effect (CPE), activation status of mitogen-activated protein kinase (MAPK) signaling pathways, and possible roles of these signaling pathways in virus replication and virus-induced CPE in MDCK cells. AhAdv-1 efficiently replicated and induced CPE in infected cells and caused accumulation of cleaved caspase-3 at 24 h post-infection (p.i.), suggesting apoptosis induction. Analysis of several intracellular signal transduction pathways, which are involved in apoptosis, showed activation of p38 MAPK, Akt and ERK1/2 pathways at 3 h p.i., and upregulation of phosphorylated SAPK/JNK at 24 h p.i. Although p38 MAPK inhibitor and SAPK/JNK inhibitor suppressed activation of the respective pathways in infected cells, they did not inhibit virus-induced CPE. Treatment of infected cells with inhibitor of the Akt pathway, the p38 pathway, the SAPK/JNK pathway or the ERK pathway revealed that inhibitors of p38 pathway inhibited viral replication by real-time PCR and TCID50 assay in infected MDCK cells, suggesting that AhAdv-1 uses p38 pathway for multiplication in infected cells.
Subject(s)
Adenoviridae , JNK Mitogen-Activated Protein Kinases , MAP Kinase Signaling System , Virus Replication , Adenoviridae/genetics , Animals , Apoptosis , Dogs , Hedgehogs/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Madin Darby Canine Kidney Cells , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Bioluminescence has been recognized as an important means for inter- and intra-species communication. A growing number of reports of red fluorescence occurring in keratinaceous materials have become available. The fluorophore(s) in these cases were shown to be, or suspected to be, free base porphyrins. The red fluorescence found in the downs of bustards was associated with inter-species signaling in mate selection. First reported in 1925, we confirm that spines of the European hedgehog (Erinaceus europaeus) when irradiated with UV (365-395 nm) light display red fluorescence localized in the light-colored sections of their proximal ends. Using reflectance fluorescence spectroscopy, we confirmed that the fluorophores responsible for the emission are free-base porphyrins, as suspected in the original report. Base-induced degradation of the spine matrix and subsequent HPLC, UV-vis, and ESI+ mass spectrometry analysis revealed the presence of a mixture of coproporphyrin III and uroporphyrin III as predominant porphyrins and a minor fraction of protoporphyrin IX. Investigation of the spine microbiome uncovered the abundant presence of bacteria known to secrete and/or interconvert porphyrins and that are not present on the non-fluorescing quills of the North American porcupine (Erethizon dorsatum). Given this circumstantial evidence, we propose the porphyrins could originate from commensal bacteria. Furthermore, we hypothesize that the fluorescence may be incidental and of no biological function for the hedgehog.
Subject(s)
Fluorescence , Hedgehogs/metabolism , Hedgehogs/microbiology , Porphyrins/metabolism , Spine , Animals , Hedgehogs/anatomy & histologyABSTRACT
Afrotheria is a clade of African-origin species with striking dissimilarities in appearance and habitat. In this study, we compared whole proteome sequences of six Afrotherian species to obtain a broad viewpoint of their underlying molecular make-up, to recognize potentially unique proteomic signatures. We find that 62% of the proteomes studied here, predominantly involved in metabolism, are orthologous, while the number of homologous proteins between individual species is as high as 99.5%. Further, we find that among Afrotheria, L. africana has several orphan proteins with 112 proteins showing < 30% sequence identity with their homologues. Rigorous sequence searches and complementary approaches were employed to annotate 156 uncharacterized protein sequences and 28 species-specific proteins. For 122 proteins we predicted potential functional roles, 43 of which we associated with protein- and nucleic-acid binding roles. Further, we analysed domain content and variations in their combinations within Afrotheria and identified 141 unique functional domain architectures, highlighting proteins with potential for specialized functions. Finally, we discuss the potential relevance of highly represented protein families such as MAGE-B2, olfactory receptor and ribosomal proteins in L. africana and E. edwardii, respectively. Taken together, our study reports the first comparative study of the Afrotherian proteomes and highlights salient molecular features.
Subject(s)
Eutheria/classification , Eutheria/genetics , Animals , Conserved Sequence , Databases, Protein , Elephants/classification , Elephants/genetics , Elephants/metabolism , Eutheria/metabolism , Evolution, Molecular , Hedgehogs/classification , Hedgehogs/genetics , Hedgehogs/metabolism , Molecular Sequence Annotation , Moles/classification , Moles/genetics , Moles/metabolism , Phylogeny , Protein Domains , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Proteome/genetics , Proteomics , Shrews/classification , Shrews/genetics , Shrews/metabolism , Species Specificity , Trichechus manatus/classification , Trichechus manatus/genetics , Trichechus manatus/metabolismABSTRACT
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.
Subject(s)
Organoids/pathology , Organoids/physiology , Regeneration , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/physiopathology , Urinary Bladder/pathology , Urinary Bladder/physiology , Adult , Animals , Bone Morphogenetic Proteins/metabolism , Female , Hedgehogs/metabolism , Hepatocyte Nuclear Factor 3-alpha/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Organoids/physiopathology , Single-Cell Analysis , Stem Cells/cytology , Stem Cells/pathology , Stem Cells/physiology , Transcriptome , Urinary Bladder/cytology , Urinary Tract Infections/metabolism , Urinary Tract Infections/pathologyABSTRACT
The present study assessed the effects of microRNA1 (miR1) on the development of osteoarthritis using human tissues and a Col2a1CreERT2/GFPfl/flRFPmiR1 mouse model of osteoarthritis. Human cartilage tissues (n=20) were collected for reverse transcriptionquantitative polymerase chain reaction (RTqPCR), histological analysis and immunohistochemistry experiments. A transgenic mouse model of osteoarthritis was established by subjecting Col2a1CreERT2/GFPfl/flRFPmiR1 transgenic mice to anterior cruciate ligament transection (ACLT). Mice were subjected to radiography and in vivo fluorescence molecular tomography (FMT), while mouse tissues were collected for histological analysis, RTqPCR and Safranin O staining. It was found that the miR1 level was downregulated, whereas the levels of Indian hedgehog (Ihh), as well as those of its downstream genes were upregulated in human osteoarthritic cartilage. In the transgenic mice, treatment with tamoxifen induced miR1, as well as collagen, type II (Col2a1) and Aggrecan (Acan) expression; however, it decreased Ihh, gliomaassociated oncogene homolog (Gli)1, Gli2, Gli3, smoothened homolog (Smo), matrix metalloproteinase (MMP)13 and collagen type X (Col10) expression. Safranin O staining revealed cartilage surface damage in the nontamoxifen + ACLT group, compared with that in the tamoxifen + ACLT group. Histologically, an intact cartilage surface and less fibrosis were observed in the tamoxifen + ACLT group. Immunohistochemistry revealed that the protein expression of Ihh, Col10, and MMP13 was significantly higher in the joint tissues of the nontamoxifen + ACLT group than in those of the tamoxifen + ACLT group. However, Col2a1 expression was lower in the joint tissues of the nontamoxifen + ACLT group than in those of the tamoxifen + ACLT group. The results of RTqPCR and FMT further confirmed these findings. On the whole, the findings of the present study demonstrate that miR1 expression protects against osteoarthritisinduced cartilage damage and gene expression by inhibiting Ihh signaling.
Subject(s)
Collagen Type II/metabolism , Hedgehog Proteins/metabolism , MicroRNAs/metabolism , Osteoarthritis/metabolism , Osteoarthritis/pathology , Animals , Collagen Type II/genetics , Hedgehog Proteins/genetics , Hedgehogs/genetics , Hedgehogs/metabolism , Immunohistochemistry , Mice , Mice, Transgenic , MicroRNAs/genetics , Osteoarthritis/geneticsABSTRACT
The aim of the study was to identify ultradian (intraday) and infradian (multi-day) rhythms in the dynamics of testosterone concentration in the blood serum of white-breasted hedgehogs. Blood sampling was performed from the femoral veins of 12 male hedgehogs. We found ultradian rhythms of testosterone on both sampling dates-March 7-8 (a day length of 11 hours and 15 minutes) and May 10-11 (a day length of 16 hours). An 8-hour rhythm of testosterone concentration has been established. The acrophases were at the same times in both photoperiods and thus independent of sunset times. The study of the infradian rhythms of testosterone was daily carried out on May 22-June 3, at 07:40 to 08:50 and from June 27 to July 7, at 16:15-16:50. It revealed an infradian rhythm of the testosterone concentration with a period of 4-days in both the morning and the evening sampling. According to our previous investigation, the infradian rhythms of testosterone among individual hedgehogs, rodents and primates have the same period. That indicates the common mechanisms for their formation. In case of experimental studies, the phase of ultradian and infradian biorhythms will need to be taken into account because the testosterone concentration in acrophase is 2-4 times higher than in bathyphase.
Subject(s)
Circadian Rhythm , Hedgehogs/metabolism , Testosterone/blood , Animals , Hedgehogs/physiology , Male , PhotoperiodABSTRACT
Spinal cord patterning is orchestrated by multiple cell signalling pathways. Neural progenitors are maintained by Notch signalling, whereas ventral neural fates are specified by Hedgehog (Hh) signalling. However, how dynamic interactions between Notch and Hh signalling drive the precise pattern formation is still unknown. We applied the PHRESH (PHotoconvertible REporter of Signalling History) technique to analyse cell signalling dynamics in vivo during zebrafish spinal cord development. This approach reveals that Notch and Hh signalling display similar spatiotemporal kinetics throughout spinal cord patterning. Notch signalling functions upstream to control Hh response of neural progenitor cells. Using gain- and loss-of-function tools, we demonstrate that this regulation occurs not at the level of upstream regulators or primary cilia, but rather at the level of Gli transcription factors. Our results indicate that Notch signalling maintains Hh responsiveness of neural progenitors via a Gli-dependent mechanism in the spinal cord.
Subject(s)
Body Patterning , Hedgehogs/metabolism , Receptors, Notch/metabolism , Signal Transduction , Spinal Cord/embryology , Zinc Finger Protein GLI1/metabolism , Animals , Gene Expression Regulation, Developmental , ZebrafishABSTRACT
Obesity is a risk factor for numerous metabolic disorders. In this study, we investigated the effects of the isothiocyanates sulforaphane (SA) and sulforaphene (SE) on adipogenesis in 3T3-L1 adipocytes. SE, a compound that is abundant in radish, inhibited adipogenesis by suppressing the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ, 69.2 ± 2.4%, P < 0.05) and CCAAT/enhancer-binding protein α (C/EBPα, 36.1 ± 3.1%, P < 0.05), thereby reducing fat accumulation in 3T3-L1 adipocytes (45.6 ± 2.7%, P < 0.05); SA was less effective. SE exerted these activities through the activation of the Hedgehog (Hh) signaling pathway by restoring Smo ((2.1 ± 0.2)-fold, P < 0.05) and Gli1 ((2.8 ± 0.1)-fold, P < 0.05) expression, which was suppressed by adipogenic signals. These effects of SE were abrogated by treatment with the Hh inhibitor vismodegib. Thus, SE inhibits adipocyte differentiation via Hh signaling and may be an effective natural agent for preventing adipocyte hyperplasia and obesity.
Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Anti-Obesity Agents/pharmacology , Hedgehogs/metabolism , Isothiocyanates/pharmacology , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/metabolism , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Hedgehogs/genetics , Mice , PPAR gamma/genetics , PPAR gamma/metabolism , Signal Transduction/drug effectsABSTRACT
Understanding how patterning influences cell behaviors to generate three dimensional morphologies is a central goal of developmental biology. Additionally, comparing these regulatory mechanisms among morphologically diverse tissues allows for rigorous testing of evolutionary hypotheses. Zebrafish skin is endowed with a coat of precisely patterned bony scales. We use in-toto live imaging during scale development and manipulations of cell signaling activity to elucidate core features of scale patterning and morphogenesis. These analyses show that scale development requires the concerted activity of Wnt/ß-catenin, Ectodysplasin (Eda) and Fibroblast growth factor (Fgf) signaling. This regulatory module coordinates Hedgehog (HH) dependent collective cell migration during epidermal invagination, a cell behavior not previously implicated in skin appendage morphogenesis. Our analyses demonstrate the utility of zebrafish scale development as a tractable system in which to elucidate mechanisms of developmental patterning and morphogenesis, and suggest a single, ancient origin of skin appendage patterning mechanisms in vertebrates.