RESUMEN
During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.
Asunto(s)
Cilios , Proteínas Quinasas Dependientes de AMP Cíclico , Quinasa 2 del Receptor Acoplado a Proteína-G , Proteínas Hedgehog , Transducción de Señal , Receptor Smoothened , Pez Cebra , Animales , Cilios/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Hedgehog/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Ratones , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Pez Cebra/metabolismo , Fosforilación , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Células 3T3 NIHRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias Cerebelosas , ADN (Citosina-5-)-Metiltransferasa 1 , Proteínas Hedgehog , Meduloblastoma , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Animales , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Humanos , Ratones , Línea Celular Tumoral , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Técnicas de Inactivación de Genes/métodosRESUMEN
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.
Asunto(s)
Cilios , Proteínas Hedgehog , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Receptor Smoothened , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor , Proteína Gli2 con Dedos de Zinc , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Fibroblastos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Membrana Celular/metabolismo , Ratones NoqueadosRESUMEN
In this issue of Cell Chemical Biology, Liu et al.1 report the identification of Q29, a synthetic diterpenoid that blocks covalent cholesterol modification of smoothened (SMO) and inhibits hedgehog signaling. Q29 is capable of suppressing tumor cell growth, both in vitro and in vivo, and overcoming resistance to SMO inhibitors.
Asunto(s)
Colesterol , Diterpenos , Resistencia a Antineoplásicos , Proteínas Hedgehog , Receptor Smoothened , Humanos , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Colesterol/metabolismo , Colesterol/química , Diterpenos/farmacología , Diterpenos/química , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Animales , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Transducción de Señal/efectos de los fármacosRESUMEN
Asthma is the most prevalent chronic inflammatory disease of the airways in children. The most prevalent phenotype of asthma is eosinophilic asthma, which is driven by a Th2 immune response and can be effectively managed by inhaled corticosteroid therapy. However, there are phenotypes of asthma with Th17 immune response that are insensitive to corticosteroid therapy and manifest a more severe phenotype. The treatment of this corticosteroid-insensitive asthma is currently immature and requires further attention. The objective of this study is to elucidate the regulation of the Hedgehog signaling pathway in Th17 cell differentiation in asthma. The study demonstrated that both Smo and Gli3, key components of the Hedgehog signaling pathway, were upregulated in Th17 polarization in vitro and in a Th17-dominant asthma model in vivo. Inhibiting Smo with a small molecule inhibitor or genetically knocking down Gli3 was found to suppress Th17 polarization. Smo was found to increase in Th1, Th2, Th17 and Treg polarization, while Gli3 specifically increased in Th17 polarization. ChIP-qPCR analyses indicated that Gli3 can directly interact with IL-6 in T cells, inducing STAT3 phosphorylation and promoting Th17 cell differentiation. Furthermore, the study demonstrated a correlation between elevated Gli3 expression and IL-17A and IL-6 expression in children with asthma. In conclusion, the study demonstrated that the Hedgehog signaling pathway plays an important role in the pathogenesis of asthma, as it regulates the differentiation of Th17 cells through the IL-6/STAT3 signaling. This may provide a potential therapeutic target for corticosteroid-insensitive asthma driven by Th17 cells.
Asunto(s)
Asma , Diferenciación Celular , Proteínas Hedgehog , Interleucina-6 , Factor de Transcripción STAT3 , Transducción de Señal , Células Th17 , Proteína Gli3 con Dedos de Zinc , Asma/inmunología , Asma/metabolismo , Asma/tratamiento farmacológico , Células Th17/inmunología , Factor de Transcripción STAT3/metabolismo , Animales , Interleucina-6/metabolismo , Diferenciación Celular/efectos de los fármacos , Proteínas Hedgehog/metabolismo , Humanos , Proteína Gli3 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/genética , Ratones , Niño , Masculino , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Femenino , Ratones Endogámicos BALB C , Modelos Animales de Enfermedad , Células Cultivadas , Proteínas del Tejido NerviosoRESUMEN
INTRODUCTION: Oral cancer poses a significant burden on public health in India, with higher incidence and mortality rates. Despite advancements in treatment modalities, prognosis remains poor due to factors such as localized recurrence and lymph node metastasis, potentially influenced by cancer stem cells. Among signaling pathways implicated in CSC regulation, the Hedgehog pathway plays a crucial role in oral squamous cell carcinoma (OSCC). MATERIAL & METHODS: 97 OSCC patients' tissue samples were collected and subjected to RNA isolation, cDNA synthesis and quantitative real-time PCR to analyze PTCH1 and SMO expression. Protein expression was assessed through immunohistochemistry. Clinicopathological parameters were correlated with gene and protein expression. Statistical analysis included Pearson chi-square tests, co-relation co-efficient tests, Kaplan-Meier survival analysis and ROC curve analysis. RESULTS: PTCH1 expression correlated with lymphatic permeation (p = 0.002) and tumor stage (p = 0.002), while SMO expression correlated with lymph node status (p = 0.034) and tumor stage (p = 0.021). PTCH1 gene expression correlated with lymph node status (p = 0.024). High PTCH1 gene expression was associated with shorter survival in tongue cancer patients. ROC curve analysis indicated diagnostic potential for PTCH1 and SMO gene and cytoplasmic SMO expression in distinguishing malignant tissues from adjacent normal tissues. CONCLUSION: PTCH1 and SMO play a crucial role in oral cancer progression, correlating with tumor stages and metastatic potential. Despite not directly influencing overall survival, PTCH1 expression at specific anatomical sites hints at its prognostic implications. PTCH1 and SMO exhibit diagnostic potential, suggesting their utility as molecular markers in oral cancer management and therapeutic strategies.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de la Boca , Receptor Patched-1 , Receptor Smoothened , Humanos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/diagnóstico , Femenino , Masculino , Persona de Mediana Edad , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/diagnóstico , Adulto , Anciano , Regulación Neoplásica de la Expresión Génica , Pronóstico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Estimación de Kaplan-Meier , Curva ROC , Metástasis Linfática/genética , Estadificación de NeoplasiasRESUMEN
OBJECTIVE: Apart from the role of the retinoblastoma gene, the genomic events associated with poor outcomes in patients with ophthalmic tumors are poorly understood. METHODS: We retrospectively analyzed 48 patients with six types of ophthalmic tumors. We searched for high-frequency mutated genes and susceptibility genes in these patients using combined exome and transcriptome analysis. RESULTS: We identified four clearly causative genes (TP53, PTCH1, SMO, BAP1). Susceptibility gene analysis identified hotspot genes, including RUNX1, APC, IDH2, and BRCA2, and high-frequency gene analysis identified several genes, including TP53, TTN, and MUC16. Transcriptome analysis identified 5868 differentially expressed genes, of which TOP2A and ZWINT were upregulated in all samples, while CFD, ELANE, HBA1, and HBB were downregulated. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the phosphoinositide 3-kinase (PI3K)-Akt and Transcriptional misregulation in cancer signaling pathways may be involved in ophthalmic tumorigenesis. CONCLUSIONS: TP53 is clearly involved in ophthalmic tumorigenesis, especially in basal cell carcinoma, and the PI3K-Akt signaling pathway may be an essential pathway involved in ophthalmic tumorigenesis. RUNX1, SMO, TOP2A, and ZWINT are also highly likely to be involved in ophthalmic tumorigenesis, but further functional experiments are needed to verify the mechanisms of these genes in regulating tumorigenesis.
Asunto(s)
Neoplasias del Ojo , Genómica , Mutación , Proteína p53 Supresora de Tumor , Humanos , Femenino , Masculino , Proteína p53 Supresora de Tumor/genética , Genómica/métodos , Neoplasias del Ojo/genética , Estudios Retrospectivos , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Receptor Patched-1/genética , Perfilación de la Expresión Génica , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas Supresoras de Tumor/genética , Anciano , Proteínas de Unión a Poli-ADP-Ribosa/genética , Adulto , Transducción de Señal/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Biomarcadores de Tumor/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ADN-Topoisomerasas de Tipo II , Ubiquitina Tiolesterasa , Subunidad alfa 2 del Factor de Unión al Sitio PrincipalRESUMEN
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Asunto(s)
Quinasas DyrK , Proteínas Hedgehog , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Transducción de Señal , Proteína Gli2 con Dedos de Zinc , Proteína Gli3 con Dedos de Zinc , Animales , Proteína Gli3 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/genética , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli2 con Dedos de Zinc/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proliferación Celular , Cilios/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Nucleares , Proteínas RepresorasRESUMEN
During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that ß-arrestin1 and ß-arrestin2 (ß-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that ß-arrestins are expressed in rat commissural neurons. We also found that Smo, ß-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on ß-arrestins. ß-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that ß-arrestins are required to activate Src kinase downstream of Shh. ß-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative ß-arrestins, ß-arrestin1 V53D which blocks the internalization of Smo and ß-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative ß-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that ß-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.
Asunto(s)
Orientación del Axón , Proteínas Hedgehog , beta-Arrestinas , Animales , Proteínas Hedgehog/metabolismo , Ratas , Orientación del Axón/fisiología , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Arrestinas/genética , Femenino , Axones/fisiología , Axones/metabolismo , Ratas Sprague-Dawley , Células Cultivadas , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Familia-src Quinasas/metabolismo , Masculino , Médula Espinal/metabolismo , Médula Espinal/embriología , Médula Espinal/citología , Embrión de Pollo , HumanosRESUMEN
The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 µg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.
Asunto(s)
Enterotoxinas , Proteínas Hedgehog , Transducción de Señal , Linfocitos T , Timo , Animales , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Femenino , Embarazo , Ratas , Timo/metabolismo , Timo/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Efectos Tardíos de la Exposición Prenatal/inmunología , Diferenciación Celular/efectos de los fármacos , Ratas Sprague-Dawley , MasculinoRESUMEN
Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina , Resistencia a Antineoplásicos , Proteínas Hedgehog , Transducción de Señal , Receptor Smoothened , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Receptor Smoothened/antagonistas & inhibidores , Animales , Humanos , Ratones , Resistencia a Antineoplásicos/genética , Transducción de Señal/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Elementos de Facilitación Genéticos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Sistemas CRISPR-CasRESUMEN
Daunorubicin, also known as daunomycin, is a DNAtargeting anticancer drug that is used as chemotherapy, mainly for patients with leukemia. It has also been shown to have anticancer effects in monotherapy or combination therapy in solid tumors, but at present it has not been adequately studied in colorectal cancer (CRC). In the present study, from a screening using an FDAapproved drug library, it was found that daunorubicin suppresses GLIdependent luciferase reporter activity. Daunorubicin also increased p53 levels, which contributed to both GLI1 suppression and apoptosis. The current detailed investigation showed that daunorubicin promoted the ßTrCPmediated ubiquitination and proteasomal degradation of GLI1. Moreover, a competition experiment using BODIPYcyclopamine, a wellknown Smo inhibitor, suggested that daunorubicin does not bind to Smo in HCT116 cells. Administration of daunorubicin (2 mg/kg, ip, qod, 15 days) into HCT116 xenograft mice profoundly suppressed tumor progress and the GLI1 level in tumor tissues. Taken together, the present results revealed that daunorubicin suppresses canonical Hedgehog pathways in CRC. Ultimately, the present study discloses a new mechanism of daunorubicin's anticancer effect and might provide a rationale for expanding the clinical application of daunorubicin.
Asunto(s)
Apoptosis , Neoplasias Colorrectales , Daunorrubicina , Proteína con Dedos de Zinc GLI1 , Animales , Humanos , Ratones , Antibióticos Antineoplásicos/farmacología , Antibióticos Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daunorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genéticaRESUMEN
Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.
Asunto(s)
Neuronas Dopaminérgicas , Proteínas Hedgehog , Células Madre Pluripotentes Inducidas , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson , Receptor Patched-1 , Proteína con Dedos de Zinc GLI1 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Neuronas Dopaminérgicas/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , AMP Cíclico/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Mutación/genética , Calcio/metabolismo , Diferenciación Celular/genética , Transducción de Señal/genéticaRESUMEN
The smoothened (Smo) receptor facilitates hedgehog signaling between kidney fibroblasts and tubules during acute kidney injury (AKI). Tubule-derived hedgehog is protective in AKI, but the role of fibroblast-selective Smo is unclear. Here, we report that Smo-specific ablation in fibroblasts reduced tubular cell apoptosis and inflammation, enhanced perivascular mesenchymal cell activities, and preserved kidney function after AKI. Global proteomics of these kidneys identified extracellular matrix proteins, and nidogen-1 glycoprotein in particular, as key response markers to AKI. Intriguingly, Smo was bound to nidogen-1 in cells, suggesting that loss of Smo could affect nidogen-1 accessibility. Phosphoproteomics revealed that the 'AKI protector' Wnt signaling pathway was activated in these kidneys. Mechanistically, nidogen-1 interacted with integrin ß1 to induce Wnt in tubules to mitigate AKI. Altogether, our results support that fibroblast-selective Smo dictates AKI fate through cell-matrix interactions, including nidogen-1, and offers a robust resource and path to further dissect AKI pathogenesis.
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Lesión Renal Aguda , Fibroblastos , Receptor Smoothened , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/genética , Animales , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Ratones , Fibroblastos/metabolismo , Fibroblastos/patología , Vía de Señalización Wnt , Humanos , Ratones Noqueados , Microambiente Celular , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genéticaRESUMEN
Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function), and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (Smoothened Agonist (SAG) or cyclopamine treatment, respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting oligodendrocyte precursor cells (OPCs) in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.
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Diferenciación Celular , Proteínas Hedgehog , Ratones Transgénicos , Vaina de Mielina , Células Precursoras de Oligodendrocitos , Receptor Smoothened , Animales , Proteínas Hedgehog/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Células Precursoras de Oligodendrocitos/efectos de los fármacos , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Vaina de Mielina/metabolismo , Diferenciación Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Alcaloides de Veratrum/farmacología , Ratones , Remielinización/fisiología , Remielinización/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/fisiología , Ratones Endogámicos C57BL , Transducción de Señal/fisiología , Transducción de Señal/efectos de los fármacosRESUMEN
Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. Currently, there are no effective drugs for the treatment of DN. Although several studies have reported the therapeutic potential of mesenchymal stem cells, the underlying mechanisms remain largely unknown. Here, we report that both human umbilical cord MSCs (UC-MSCs) and UC-MSC-derived exosomes (UC-MSC-exo) attenuate kidney damage, and inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis in streptozotocin-induced DN rats. Strikingly, the Hedgehog receptor, smoothened (SMO), was significantly upregulated in the kidney tissues of DN patients and rats, and positively correlated with EMT and renal fibrosis. UC-MSC and UC-MSC-exo treatment resulted in decrease of SMO expression. In vitro co-culture experiments revealed that UC-MSC-exo reduced EMT of tubular epithelial cells through inhibiting Hedgehog/SMO pathway. Collectively, UC-MSCs inhibit EMT and renal fibrosis by delivering exosomes and targeting Hedgehog/SMO signaling, suggesting that UC-MSCs and their exosomes are novel anti-fibrotic therapeutics for treating DN.
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Diabetes Mellitus , Nefropatías Diabéticas , Exosomas , Células Madre Mesenquimatosas , Humanos , Ratas , Animales , Nefropatías Diabéticas/metabolismo , Exosomas/metabolismo , Receptor Smoothened , Proteínas Hedgehog/metabolismo , Fibrosis , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Both Hedgehog and androgen signaling pathways are known to promote myelin regeneration in the central nervous system. Remarkably, the combined administration of agonists of each pathway revealed their functional cooperation towards higher regeneration in demyelination models in males. Since multiple sclerosis, the most common demyelinating disease, predominates in women, and androgen effects were reported to diverge according to sex, it seemed essential to assess the existence of such cooperation in females. Here, we developed an intranasal formulation containing the Hedgehog signaling agonist SAG, either alone or in combination with testosterone. We show that SAG promotes myelin regeneration and presumably a pro-regenerative phenotype of microglia, thus mimicking the effects previously observed in males. However, unlike in males, the combined molecules failed to cooperate in the demyelinated females, as shown by the level of functional improvement observed. Consistent with this observation, SAG administered in the absence of testosterone amplified peripheral inflammation by presumably activating NK cells and thus counteracting a testosterone-induced reduction in Th17 cells when the molecules were combined. Altogether, the data uncover a sex-dependent effect of the Hedgehog signaling agonist SAG on the peripheral innate immune system that conditions its ability to cooperate or not with androgens in the context of demyelination.
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Enfermedades Desmielinizantes , Testosterona , Animales , Femenino , Masculino , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/tratamiento farmacológico , Ratones , Testosterona/farmacología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/agonistas , Ratones Endogámicos C57BL , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Sistema Nervioso Central/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/agonistas , Vaina de Mielina/metabolismo , Modelos Animales de Enfermedad , Transducción de Señal/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/inmunología , Caracteres SexualesRESUMEN
Hedgehog (Hh) signaling is crucial in cardiovascular development and maintenance. However, the biological role of Patched1 (Ptch1), an inhibitory receptor of the Hh signaling pathway, remains elusive. In this study, a Ptch1 ortholog was characterized in Nile tilapia (Oreochromis niloticus), and its function was investigated through CRISPR/Cas9 gene knockout. When one-cell embryos were injected with CRISPR/Cas9 targeting ptch1, the mutation efficiency exceeded 70%. During 0-3 days post fertilization (dpf), no significant differences were observed between the ptch1 mutant group and the control group; at 4 dpf (0 day after hatching), about 10% of the larvae showed an angiogenesis defect and absence of blood flow; from 5 dpf, most larvae exhibited an elongated heart, large pericardial cavity, and blood leakage and coagulation, ultimately dying during the 6-8 dpf period due to the lack of blood circulation. Consistently, multiple differentially expressed genes related to angiogenesis, blood coagulation, and heart development were enriched in the ptch1 mutants. Furthermore, Smoothened (Smo) antagonist (cyclopamine) treatment of the ptch1 mutants greatly rescued the cardiovascular disorders. Collectively, our study suggests that Ptch1 is required for cardiovascular development and vascular integrity via Smo signaling, and excessive Hh signaling is detrimental to cardiovascular development.
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Cíclidos , Animales , Cíclidos/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Transducción de Señal , Técnicas de Inactivación de Genes , Mutación , Receptor Smoothened/genéticaRESUMEN
The hedgehog (Hh) signaling pathway has long been a hotspot for anti-cancer drug development due to its important role in cell proliferation and tumorigenesis. However, most clinically available Hh pathway inhibitors target the seven-transmembrane region (7TM) of smoothened (SMO), and the acquired drug resistance is an urgent problem in SMO inhibitory therapy. Here, we identify a sterol analog Q29 and show that it can inhibit the Hh pathway through binding to the cysteine-rich domain (CRD) of SMO and blocking its cholesterylation. Q29 suppresses Hh signaling-dependent cell proliferation and arrests Hh-dependent medulloblastoma growth. Q29 exhibits an additive inhibitory effect on medulloblastoma with vismodegib, a clinically used SMO-7TM inhibitor for treating basal cell carcinoma (BCC). Importantly, Q29 overcomes resistance caused by SMO mutants against SMO-7TM inhibitors and inhibits the activity of SMO oncogenic variants. Our work demonstrates that the SMO-CRD inhibitor can be a new way to treat Hh pathway-driven cancers.
Asunto(s)
Proliferación Celular , Proteínas Hedgehog , Meduloblastoma , Transducción de Señal , Receptor Smoothened , Esteroles , Receptor Smoothened/antagonistas & inhibidores , Receptor Smoothened/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Humanos , Transducción de Señal/efectos de los fármacos , Animales , Ratones , Proliferación Celular/efectos de los fármacos , Esteroles/química , Esteroles/farmacología , Esteroles/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/metabolismo , Meduloblastoma/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Estructura Molecular , Colesterol/metabolismoRESUMEN
BACKGROUND: HERC4 has been reported to have functions in several types of tumors, but its roles in ovarian cancer have not been studied yet. METHODS: Primary tissues from ovarian cancer patients and cell lines were collected for real-time PCR. Kaplan-Meier Plotter was used to predict the prognosis of ovarian cancer patients. HERC4 was overexpressed in cells by lentivirus, and CCK-8 assay was performed to evaluate cell viability. Real-time PCR and Western blot were carried out to analyze the mRNA and protein expression, respectively. Xenograft tumor models were established to analyze HERC4 function in vivo. RESULTS: Firstly, we found that HERC4 was significantly downregulated in ovarian cancer. We then found that ovarian cancer patients with high HERC4 expression had significantly higher overall survival and progression-free survival rates compared with patients with low expression. Then, HERC4 was overexpressed in ovarian cancer cells, and we found that overexpression of HERC4 significantly inhibited ovarian cancer cell growth, as well as the expression of the target protein SMO, and the key proteins in the downstream hedgehog signaling pathway. Finally, the xenograft tumor models revealed that overexpression of HERC4 significantly inhibited tumor growth in vivo. CONCLUSIONS: Overall, these results indicate that overexpression of HERC4 inhibits cell proliferation of ovarian cancer in vitro and in vivo, suggesting that HERC4 may serve as an effective target for the treatment of ovarian cancer.