RESUMO
In this issue of Developmental Cell, Shiraishi et al. investigate the epigenetic changes occurring during the formation of SHH medulloblastoma and show that an epigenomic shift renders Nuclear Factor I family of transcription factors oncogenic.
Assuntos
Epigênese Genética , Proteínas Hedgehog , Meduloblastoma , Fatores de Transcrição NFI , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fatores de Transcrição NFI/metabolismo , Fatores de Transcrição NFI/genética , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , CamundongosRESUMO
Breast cancer ranks as one of the most common malignancies among women, with its prognosis and therapeutic efficacy heavily influenced by factors associated with the tumor cell biology, particularly the tumor microenvironment (TME). The diverse elements of the TME are engaged in dynamic bidirectional signaling interactions with various pathways, which together dictate the growth, invasiveness, and metastatic potential of breast cancer. The Hedgehog (Hh) signaling pathway, first identified in Drosophila, has been established as playing a critical role in human development and disease. Notably, the dysregulation of the Hh pathway is recognized as a major driver in the initiation, progression, and metastasis of breast cancer. Consequently, elucidating the mechanisms by which the Hh pathway interacts with the distinct components of the breast cancer TME is essential for comprehensively evaluating the link between Hh pathway activation and breast cancer risk. This understanding is also imperative for devising novel targeted therapeutic strategies and preventive measures against breast cancer. In this review, we delineate the current understanding of the impact of Hh pathway perturbations on the breast cancer TME, including the intricate and complex network of intersecting signaling cascades. Additionally, we focus on the therapeutic promise and clinical challenges of Hh pathway inhibitors that target the TME, providing insights into their potential clinical utility and the obstacles that must be overcome to harness their full therapeutic potential.
Assuntos
Neoplasias da Mama , Proteínas Hedgehog , Transdução de Sinais , Microambiente Tumoral , Humanos , Proteínas Hedgehog/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Animais , FemininoRESUMO
The Sonic hedgehog (Shh) signaling pathway controls embryonic development and tissue homeostasis after birth. This requires regulated solubilization of dual-lipidated, firmly plasma membrane-associated Shh precursors from producing cells. Although it is firmly established that the resistance-nodulation-division transporter Dispatched (Disp) drives this process, it is less clear how lipidated Shh solubilization from the plasma membrane is achieved. We have previously shown that Disp promotes proteolytic solubilization of Shh from its lipidated terminal peptide anchors. This process, termed shedding, converts tightly membrane-associated hydrophobic Shh precursors into delipidated soluble proteins. We show here that Disp-mediated Shh shedding is modulated by a serum factor that we identify as high-density lipoprotein (HDL). In addition to serving as a soluble sink for free membrane cholesterol, HDLs also accept the cholesterol-modified Shh peptide from Disp. The cholesteroylated Shh peptide is necessary and sufficient for Disp-mediated transfer because artificially cholesteroylated mCherry associates with HDL in a Disp-dependent manner, whereas an N-palmitoylated Shh variant lacking C-cholesterol does not. Disp-mediated Shh transfer to HDL is completed by proteolytic processing of the palmitoylated N-terminal membrane anchor. In contrast to dual-processed soluble Shh with moderate bioactivity, HDL-associated N-processed Shh is highly bioactive. We propose that the purpose of generating different soluble forms of Shh from the dual-lipidated precursor is to tune cellular responses in a tissue-type and time-specific manner.
Assuntos
Proteínas Hedgehog , Lipoproteínas HDL , Proteínas Hedgehog/metabolismo , Animais , Lipoproteínas HDL/metabolismo , Camundongos , Humanos , Membrana Celular/metabolismo , Transdução de Sinais , Colesterol/metabolismoRESUMO
Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.
Assuntos
Proteínas Culina , Proteínas Hedgehog , Complexo de Endopeptidases do Proteassoma , Transdução de Sinais , Ubiquitinação , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos , Células NIH 3T3 , Proteínas Culina/metabolismo , Proteínas Culina/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Humanos , Regulação da Expressão GênicaRESUMO
Activation of the Hedgehog (Hh) signaling pathway is often associated with the progression of various types of cancer. The purpose of study was to search for inhibitors of the Hh signaling pathway among eight compounds belonging to the group of isoxazolyl steroids. The evaluation of the effectiveness of the compounds was based on the analysis of their cytotoxicity, effect on the cell cycle, on the expression of key Hh-signaling-pathway genes (Ptch1, Smo, and Gli1) and putative target genes MMP-2 and MMP-9. Four compounds with the most pronounced cytotoxic effect were identified: compounds 1, 2 (HeLa cells) and 3, 4 (A549 cells). Compounds 1 and 2 significantly reduced the expression of the Ptch1, Smo, Gli1 genes, but had the opposite effect on MMP-2 gene expression: Compound 1 increased it, and compound 2 decreased it. Compounds 3 and 4 did not have a noticeable inhibitory effect on the expression of the Shh pathway receptors, but significantly inhibited MMP-2 and MMP-9 expression. Thus, it was shown that inhibition of the Shh signaling pathway by isoxazolyl steroids can have the opposite effect on MMPs gene expression, which is what should be taken into account in further studies of these compounds as therapeutic agents.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog , Transdução de Sinais , Esteroides , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Transdução de Sinais/efeitos dos fármacos , Esteroides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células A549 , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Linhagem Celular Tumoral , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Antineoplásicos/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/antagonistas & inibidores , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Ciclo Celular/efeitos dos fármacosRESUMO
Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.
Assuntos
Diferenciação Celular , Proteínas Associadas aos Microtúbulos , Osteoblastos , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Linhagem Celular , Cílios/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Genetic variants in the zone of polarizing activity regulatory sequence (ZRS) that induce ectopic expression of the SHH gene have been associated with different ZRS-related phenotypes. We report the first patient with a de novo variant, c.423+4916 T>C, in ZRS (previously classified as a variant of uncertain significance) that causes tibial hemimelia-polysyndactyly-triphalangeal thumb syndrome (THPTTS). A two-month-old male patient presented with bilateral preaxial polydactyly, triphalangeal thumb, and tibial agenesis and was heterozygous for the variant c.423+4916T>C (neither of his parents was a carrier). The findings obtained from the family study were sufficient to reclassify the variant from "uncertain significance" to "likely pathogenic" according to three criteria from the American College of Medical Genetics and Genomics guidelines, as follows: (1) absence of gnomAD, (2) confirmation of paternity and maternity, and (3) strong phenotype-genotype association. In ZRS-associated syndromes, a wide clinical spectrum has been observed, ranging from polydactyly to THPTTS; our patient has the most severe and rare phenotype. We did not perform functional assays. However, the c.423+4916T>C variant is flanked by three variants, which have been proven not only to cause the phenotype but also to increase the expression of SHH. Through all this data gathering, we consider the c.423+4916T>C variant to be causative of THPTTS.
Assuntos
Ectromelia , Deformidades Congênitas da Mão , Polegar , Humanos , Lactente , Masculino , Anormalidades Múltiplas/genética , Anormalidades Congênitas , Ectromelia/genética , Estudos de Associação Genética , Deformidades Congênitas da Mão/genética , Proteínas Hedgehog/genética , Disostose Mandibulofacial , Mutação , Fenótipo , Polidactilia/genética , Polegar/anormalidades , Tíbia/anormalidades , Dedos do Pé/anormalidadesRESUMO
The absence or dysfunction of primary cilia, which are non-motile protrusions on cells, leads to a group of neurodevelopment disorders called ciliopathies. In a new study, Esther Stoeckli and colleagues identify the role of primary cilium-mediated sonic hedgehog (Shh) signaling in commissural axon guidance in mice and chick embryos. We caught up with first author, Alexandre Dumoulin, and corresponding author, Esther Stoeckli, Professor at the University of Zurich, to find out more about the work.
Assuntos
Cílios , Proteínas Hedgehog , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Cílios/metabolismo , Humanos , Camundongos , Embrião de Galinha , Transdução de Sinais , História do Século XXI , Orientação de Axônios , História do Século XXRESUMO
Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Cerebelares , DNA (Citosina-5-)-Metiltransferase 1 , Proteínas Hedgehog , Meduloblastoma , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Humanos , Camundongos , Linhagem Celular Tumoral , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Técnicas de Inativação de Genes/métodosRESUMO
Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored. This study unveils a novel role for the Bâ³gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.
Assuntos
Proteínas Hedgehog , Proteína Fosfatase 2 , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco , Proteínas Hedgehog/metabolismo , Proteína Fosfatase 2/metabolismo , Humanos , Proteína GLI1 em Dedos de Zinco/metabolismo , Fosforilação , Células HEK293 , Animais , Linhagem Celular Tumoral , Proteína Gli2 com Dedos de Zinco/metabolismo , CamundongosRESUMO
Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Animais , Humanos , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Difusão , Proteínas Hedgehog/metabolismo , Morfogênese , Imagem Individual de Molécula/métodos , CamundongosRESUMO
Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC.
Assuntos
Carcinoma Hepatocelular , Proteínas Hedgehog , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Transdução de Sinais , Sunitinibe/farmacologia , Sunitinibe/uso terapêutico , Adulto , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA-Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid-restricted high-fat diet (CDA-HFD) to induce MASLD, Smo- mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo- mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo- hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD-fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence.
Assuntos
Senescência Celular , Proteínas Hedgehog , Hepatócitos , Receptor Smoothened , Animais , Hepatócitos/metabolismo , Hepatócitos/patologia , Camundongos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Humanos , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/genética , Transdução de Sinais , Camundongos Knockout , Progressão da DoençaRESUMO
Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
Assuntos
Ativinas , Diferenciação Celular , Proteínas Hedgehog , Organoides , Retina , Transdução de Sinais , Tretinoína , Humanos , Ativinas/farmacologia , Ativinas/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , Proteínas Hedgehog/metabolismo , Tretinoína/farmacologia , Retina/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Background: The liver's regenerative capacity allows it to repair itself after injury. Extracellular vesicles and particles (EVPs) in the liver's interstitial space are crucial for signal transduction, metabolism, and immune regulation. Understanding the role and mechanism of liver-derived EVPs in regeneration is significant, particularly after partial hepatectomy, where the mechanisms remain unclear. Methods: A 70% hepatectomy model was established in mice, and EVPs were isolated and characterized using electron microscopy, nanocharacterization, and Western blot analysis. Combined metabolomic and transcriptomic analyses revealed ß-sitosterol enrichment in EVPs and activation of the Hedgehog signaling pathway during regeneration. The role of ß-sitosterol in EVPs on the Hedgehog pathway and its targets were identified using qRT-PCR, Western blot analysis. The regulation of carnitine synthesis by this pathway was determined using a dual luciferase assay. The effect of a ß-sitosterol diet on liver regeneration was verified in mice. Results: After 70% hepatectomy, the liver successfully regenerated without liver failure or death. At 24 hours post-surgery, tissue staining showed transient regeneration-associated steatosis (TRAS), with increased Ki67 positivity at 48 hours. EVPs displayed a spherical lipid bilayer structure with particle sizes of 70-130 nm. CD9, CD63, and CD81 in liver-derived EVPs were confirmed. Transcriptomic and metabolomic analyses showed EVPs supplementation significantly promoted carnitine synthesis and fatty acid oxidation. Tissue staining confirmed accelerated TRAS resolution and enhanced liver regeneration with EVP supplementation. Mass spectrometry identified ß-sitosterol in EVPs, which binds to Smo protein, activating the Hedgehog pathway. This led to the nuclear transport of Gli3, stimulating Setd5 transcription and inducing carnitine synthesis, thereby accelerating fatty acid oxidation. Mice with increased ß-sitosterol intake showed faster TRAS resolution and liver regeneration compared to controls. Conclusion: Liver-derived EVPs promote regeneration after partial hepatectomy. ß-sitosterol from EVPs accelerates fatty acid oxidation and promotes liver regeneration by activating Hedgehog signaling pathway.
Assuntos
Vesículas Extracelulares , Proteínas Hedgehog , Hepatectomia , Regeneração Hepática , Fígado , Sitosteroides , Animais , Sitosteroides/farmacologia , Sitosteroides/química , Regeneração Hepática/efeitos dos fármacos , Regeneração Hepática/fisiologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/química , Camundongos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas Hedgehog/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Carnitina/farmacologia , Tamanho da PartículaRESUMO
Developmental exposure to nonylphenol (NP) results in irreversible impairments of the central nervous system (CNS). The neural precursor cell (NPC) pool located in the subgranular zone (SGZ), a substructure of the hippocampal dentate gyrus, is critical for the development of hippocampal circuits and some hippocampal functions such as learning and memory. However, the effects of developmental exposure to NP on this pool remain unclear. Thus, our aim was to clarify the impacts of developmental exposure to NP on this pool and to explore the potential mechanisms. Animal models of developmental exposure to NP were created by treating Wistar rats with NP during pregnancy and lactation. Our data showed that developmental exposure to NP decreased Sox2-and Ki67-positive cells in the SGZ of offspring. Inhibited activation of Shh signaling and decreased levels of its downstream mediators, E2F1 and cyclins, were also observed in pups developmentally exposed to NP. Moreover, we established the in vitro model in the NE-4C cells, a neural precursor cell line, to further investigate the effect of NP exposure on NPCs and the underlying mechanisms. Purmorphamine, a small purine-derived hedgehog agonist, was used to specifically modulate the Shh signaling. Consistent with the in vivo results, exposure to NP reduced cell proliferation by inhibiting the Shh signaling in NE-4C cells, and purmorphamine alleviated this reduction in cell proliferation by restoring this signaling. Altogether, our findings support the idea that developmental exposure to NP leads to inhibition of the NPC proliferation and the NPC pool depletion in the SGZ located in the dentate gyrus. Furthermore, we also provided the evidence that suppressed activation of Shh signaling may contribute to the effects of developmental exposure to NP on the NPC pool.
Assuntos
Proliferação de Células , Giro Denteado , Proteínas Hedgehog , Células-Tronco Neurais , Fenóis , Ratos Wistar , Transdução de Sinais , Animais , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Giro Denteado/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas Hedgehog/metabolismo , Fenóis/farmacologia , Fenóis/toxicidade , Feminino , Gravidez , Ratos , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Purinas/farmacologia , Morfolinas/farmacologia , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Masculino , Fatores de Transcrição SOXB1/metabolismo , Linhagem CelularRESUMO
The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and exhibit a subset of phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish.
Assuntos
Characidae , Proteínas Hedgehog , Hematopoese , Transdução de Sinais , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Hematopoese/fisiologia , Characidae/embriologia , Hipóxia/metabolismo , Evolução Biológica , Embrião não Mamífero/metabolismo , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Alcaloides de Veratrum/farmacologia , Cavernas , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Eritrócitos/metabolismoRESUMO
The mTORC1-complex is negatively regulated by TSC1 and TSC2. Activation of Hedgehog signaling is strictly dependent on communication between Smoothened and the Hedgehog-signaling effector and transcription factor, GLI2, in the primary cilium. Details about this communication are not known, and we wanted to explore this further. Here we report that in Tsc2 -/- MEFs constitutively activated mTORC1 led to mis-localization of Smoothened to the plasma membrane, combined with increased concentration of GLI2 in the cilia and reduced Hedgehog signaling, measured by reduced expression of the Hedgehog target gene, Gli1 Inhibition of mTORC1 rescued the cellular localization of Smoothened to the cilia, reduced the cilia concentration of GLI2, and restored Hedgehog signaling. Our results reveal evidence for a two-step activation process of GLI2. The first step includes GLI2 stabilization and cilium localization, whereas the second step includes communication with cilia-localized Smoothened. We found that mTORC1 inhibits the second step. This is the first demonstration that mTORC1 is involved in the regulation of Hedgehog signaling.
Assuntos
Proteínas Hedgehog , Alvo Mecanístico do Complexo 1 de Rapamicina , Transdução de Sinais , Receptor Smoothened , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor , Proteína Gli2 com Dedos de Zinco , Animais , Camundongos , Membrana Celular/metabolismo , Cílios/metabolismo , Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
BACKGROUND/AIM: Glioblastoma is the most frequent type of adult-onset malignant brain tumor and has a very poor prognosis. Glioblastoma stem cells have been shown to be one of the mechanisms by which glioblastoma acquires therapy resistance. Therefore, there is a need to establish novel therapeutic strategies useful for inhibiting this cell population. γ-Glutamylcyclotransferase (GGCT) is an enzyme involved in the synthesis and metabolism of glutathione, which is highly expressed in a wide range of cancer types, including glioblastoma, and inhibition of its expression has been reported to have antitumor effects on various cancer types. The aim of this study was to clarify the function of GGCT in glioblastoma stem cells. MATERIALS AND METHODS: We searched for pathways affected by GGCT overexpression in mouse embryonic fibroblasts NIH-3T3 by comprehensive gene expression analysis. Knockdown of GGCT and overexpression of desert hedgehog (DHH), a representative ligand of the pathway, were performed in glioblastoma stem cells derived from a mouse glioblastoma model. RESULTS: GGCT overexpression activated the hedgehog pathway. Knockdown of GGCT inhibited proliferation of glioblastoma stem cells and reduced expression of DHH and the downstream target GLI family zinc finger 1 (GLI1). DHH overexpression significantly restored the growth-suppressive effect of GGCT knockdown. CONCLUSION: High GGCT expression is important for expression of DHH and activation of the hedgehog pathway, which is required to maintain glioblastoma stem cell proliferation. Therefore, inhibition of GGCT function may be useful in suppressing stemness of glioblastoma stem cells accompanied by activation of the hedgehog pathway.
Assuntos
Proliferação de Células , Regulação para Baixo , Glioblastoma , Proteínas Hedgehog , Células-Tronco Neoplásicas , gama-Glutamilciclotransferase , Animais , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , gama-Glutamilciclotransferase/metabolismo , gama-Glutamilciclotransferase/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Transdução de SinaisRESUMO
Astrocyte diversity is greatly influenced by local environmental modulation. Here we report that the majority of astrocytes across the mouse brain possess a singular primary cilium localized to the cell soma. Comparative single-cell transcriptomics reveals that primary cilia mediate canonical SHH signaling to modulate astrocyte subtype-specific core features in synaptic regulation, intracellular transport, energy and metabolism. Independent of canonical SHH signaling, primary cilia are important regulators of astrocyte morphology and intracellular signaling balance. Dendritic spine analysis and transcriptomics reveal that perturbation of astrocytic cilia leads to disruption of neuronal development and global intercellular connectomes in the brain. Mice with primary ciliary-deficient astrocytes show behavioral deficits in sensorimotor function, sociability, learning and memory. Our results uncover a critical role for primary cilia in transmitting local cues that drive the region-specific diversification of astrocytes within the developing brain.