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1.
J Ethnopharmacol ; 265: 113295, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32841701

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellariabarbata D. Don extraction (SBE), a traditional Chinese medicine, has been proved effective against various malignant disorders in clinics with tolerable side-effects when administered alone or in combination with conventional chemotherapeutic regimens. AIM OF THIS STUDY: Multi-drug resistance of cancer is attributed to existence of cancer stemness-prone cells that harbor aberrantly high activation of Sonic Hedgehog (SHH) cascade. Our previous study has demonstrated that SBE sensitized non-small cell lung cancer (NSCLC) cells to Cisplatin (DDP) treatment by downregulating SHH pathway. Yet, whether SBE could prohibit proliferation of cancer stemness-prone cells and its underlying molecular mechanisms remain to be investigated. In this article, we further investigated intervention of SBE on NSCLC cell stemness-associated phenotypes and its potential mode of action. MATERIALS AND METHODS: CCK-8 and clonal formation detection were used to measure the anti-proliferative potency of SBE against NSCLC and normal epithelial cells. Sphere formation assay and RQ-PCR were used to detect proliferation of cancer stemness cells and associated marker expression upon SBE incubation. Mechanistically, DARTS-WB and SPR were used to unveil binding target of SBE. Immunodeficient mice were implanted with patient derived tumor bulk for in vivo validation of anti-cancer effect of SBE. RESULTS: SBE selectively attenuated proliferation and stemness-like phenotypes of NSCLC cells rather than bronchial normal epithelial cells. Drug-protein interaction analysis revealed that SBE could directly bind with stem cell-specific transcription factor sex determining region Y-box 2 (SOX2) and interfere with the SOX2/SMO/GLI1 positive loop. In vivo assay using patient-derived xenografts (PDXs) model further proved that SBE diminished tumor growth and SOX2 expression in vivo. CONCLUSION: Our data indicate that SBE represses stemness-related features of NSCLC cells via targeting SOX2 and may serve as an alternative therapeutic option for clinic treatment.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Extratos Vegetais/farmacologia , Células A549 , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Receptor Smoothened/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/metabolismo
2.
Gene ; 754: 144881, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32526259

RESUMO

OBJECTIVE: This study aims to investigate the roles of Sonic hedgehog (Shh) signaling pathway in the occurrence and progression of Myelodysplastic Syndrome (MDS) and further evaluate using jervine as therapeutic strategy for MDS by inhibiting Shh pathway. METHODS: CD34+ cells from the bone marrow of 53 MDS patients were counted by flow cytometry and isolated by magnetic bead sorting. Shh, Smo, Ptch-1 and Gli-1 (involved in Shh pathway) in CD34+ cells were examined by RT-qPCR. Besides, the relationship between Shh pathway-related genes and the clinical features or prognosis of MDS were analyzed. Further, the effects of jervine on MUTZ-1 cells regarding their proliferation, apoptosis and cell cycle as well as Shh pathway-related gene and protein expression were analyzed. RESULTS: Gene expression level of Shh, Gli-1 and Smo was significantly increased in MDS patients. Herein, Smo and Gli-1 were correlated with chromosome karyotype classification and IPSS. MDS patients with high expression of Smo or Gli-1 had a poor prognosis. Jervine inhibited gene and protein expression of Shh, Smo, Ptch-1 and Gli-1. Besides, jervine suppressed the proliferation and promoted the apoptosis of MUTZ-1 cells, as well as inhibited the transition of cells from G1 to S phase. CONCLUSION: Shh signaling pathway of MDS patients is abnormally activated and participated in the occurrence and progression of MDS. Jervine intervention is a potential therapeutic strategy for MDS.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/patologia , Alcaloides de Veratrum/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Feminino , Proteínas Hedgehog/genética , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Prognóstico , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Células Tumorais Cultivadas , Adulto Jovem , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
3.
Sci Rep ; 10(1): 7285, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350360

RESUMO

Hedgehog (Hh) signaling plays a broad role in the development of many organs including bone and teeth. It is noted that sustained Hh activity in osteoblasts negatively regulates postnatal development in mice. However, it remains unknown whether Hh signaling contributes to cementum formation. In this study, to define the roles of Hh signaling in cementum formation, we analyzed two kinds of transgenic mouse models for Hh signaling activation designed by the inactivation of Suppressor of Fused (Sufu), a negative regulator of Hh signaling, (SufuOC) and a forced endogenous activation of Smo (SmoM2OC) under the control of osteocalcin (OC) promoter-driven Cre recombinase. Interestingly, cellular cementum apposition was remarkably reduced in both mutants. Consistently, matrix formation and mineralization ability were down-regulated in OCCM-30, a cementoblast cell line, following treatment with a pharmaceutical Smo agonist. In addition, reductions in Osx expression and ß-catenin activity, which are critical for cellular cementum formation, were also detected in vitro. Furthermore, the compound mutant mice designed for the stabilization of ß-catenin with both Hh-Smo signaling activation in cementoblasts revealed a complete restoration of defective cellular cementum. In addition, Wnt antagonists such as Sostdc1 and Dkk1 were also induced by Smo activation and played a role in the reduction of Osx expression and ß-catenin activity. Collectively, our data demonstrated that Hh signaling negatively regulates cementum apposition in a Wnt/ß-catenin/Osx-dependent manner.


Assuntos
Cemento Dentário/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cemento Dentário/citologia , Proteínas Hedgehog/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Transgênicos , Osteocalcina/genética , Osteocalcina/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo
4.
PLoS One ; 15(4): e0231762, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348319

RESUMO

Canine osteosarcoma (OSA), the most common canine primary bone malignancy, has a highly aggressive biologic behavior. Despite current standard of care therapies, including amputation and adjuvant chemotherapy, most dogs still succumb to metastatic disease. Further investigations into molecular mechanisms and pathways driving OSA are needed to improve therapeutic options. The Hedgehog (HH) cell-signaling pathway has demonstrated involvement in human OSA. Several studies in canine OSA have found changes in expression of some HH pathway genes and demonstrated a role for HH transcription factors. However, the role of this pathway as well as the translational value of its targeting in canine OSA are still undefined. The objectives of this study were to determine the expression of HH components directly in canine OSA tissues and to evaluate the biologic impact of HH signaling inhibition in canine OSA cells. In situ hybridization was used to detect HH family mRNA expression in archived canine OSA tissues and revealed variable expression levels of these mRNAs in canine OSA tissues. The effect of a commercially available Smoothened inhibitor, vismodegib, was studied in established canine OSA cell lines. Alterations in cellular growth as well as assessment of downstream HH targets were evaluated. Although changes in cell growth were noted following Smoothened inhibition, inconsistent decreases in target gene expression were found. While treatment with vismodegib had a negative impact on canine OSA cell growth and viability, the mechanism remains unclear. Further studies are warranted to evaluate the clinical significance of canonical HH signaling in canine OSA.


Assuntos
Anilidas/farmacologia , Neoplasias Ósseas/patologia , Proteínas Hedgehog/metabolismo , Osteossarcoma/patologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Anilidas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/veterinária , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cães , Perfilação da Expressão Gênica , Proteínas Hedgehog/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/veterinária , Piridinas/uso terapêutico , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismo
5.
Nat Commun ; 11(1): 1994, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332736

RESUMO

Gas1 and Boc/Cdon act as co-receptors in the vertebrate Hedgehog signalling pathway, but the nature of their interaction with the primary Ptch1/2 receptors remains unclear. Here we demonstrate, using primordial germ cell migration in mouse as a developmental model, that specific hetero-complexes of Ptch2/Gas1 and Ptch1/Boc mediate the process of Smo de-repression with different kinetics, through distinct modes of Hedgehog ligand reception. Moreover, Ptch2-mediated Hedgehog signalling induces the phosphorylation of Creb and Src proteins in parallel to Gli induction, identifying a previously unknown Ptch2-specific signal pathway. We propose that although Ptch1 and Ptch2 functionally overlap in the sequestration of Smo, the spatiotemporal expression of Boc and Gas1 may determine the outcome of Hedgehog signalling through compartmentalisation and modulation of Smo-downstream signalling. Our study identifies the existence of a divergent Hedgehog signal pathway mediated by Ptch2 and provides a mechanism for differential interpretation of Hedgehog signalling in the germ cell niche.


Assuntos
Quimiotaxia/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/fisiologia , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Receptor Patched-2/metabolismo , Células 3T3 , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Embrião de Mamíferos , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Inativação de Genes , Imunoglobulina G/metabolismo , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptor Patched-1/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Receptor Smoothened/metabolismo , Imagem com Lapso de Tempo , Quinases da Família src/metabolismo
6.
Phytomedicine ; 67: 153163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901891

RESUMO

BACKGROUND: Renal interstitial fibrosis is a common pathway through which chronic kidney disease progresses to end-stage renal disease. There are currently no effective drugs available to treat kidney fibrosis, so traditional medicine is likely to be a candidate. The therapeutic potential of saikosaponin B2 (SSB2), a biologically active ingredient of Radix Bupleuri, on renal fibrosis has not been reported. METHODS: A unilateral ureteral obstruction (UUO) model was conducted to induce renal interstitial fibrosis in mice. SSB2's effect was valuated by histological staining and exploring the changes in expression of relative proteins and mRNAs. A conditional medium containing sonic hedgehog variant protein stimulating normal rat kidney interstitial fibroblast cells (NRK-49F) was used in an in vitro model to determine the possible mechanism. The molecular target of SSB2 was verified using several mutation plasmids. RESULTS: SSB2 administration reduced kidney injury and alleviated interstitial fibrosis by decreasing excessive accumulation of extracellular matrix components in UUO mice. It could also reduce the expression of α-SMA, fibronectin and Gli1, a crucial molecule of the hedgehog (Hh) signaling pathway both in vivo and in vitro. In NIH-3T3 cells simulated by conditional medium containing sonic hedgehog variant protein, SSB2 showed the ability to decrease the expression of Gli1 and Ptch1 mRNA. Using a dual-luciferase reporter assay, SSB2 suppressed the Gli-luciferase reporter activity in NIH-3T3 cells, and the IC50 was 0.49 µM, but had no effect on the TNF-α/NF-κB and Wnt/ß-catenin signaling pathways, indicating the inhibition selectivity on the Hh signaling pathway. Furthermore, SSB2 failed to inhibit the Hh pathway activity evoked by ectopic expression of Gli2ΔN and Smo D473H, suggesting that SSB2 might potentially act on smoothened receptors. CONCLUSION: SSB2 could attenuate renal fibrosis and decrease fibroblast activation by inhibiting the Hh signaling pathway.


Assuntos
Proteínas Hedgehog/metabolismo , Nefropatias/tratamento farmacológico , Rim/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Animais , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Células HEK293 , Humanos , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Células NIH 3T3 , Ácido Oleanólico/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
7.
J Cancer Res Clin Oncol ; 146(2): 297-304, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31960187

RESUMO

PURPOSE: Itraconazole is an antifungal drug that has been clinically used for over 30 years. In recent years, scholars have discovered that it possesses an anticancer effect. Moreover, its mechanism has been clarified to some degree. What deserves to be mentioned is that itraconazole acting on the Hedgehog pathway has made a new progress in the treatment of cancers. While interestingly, studies have demonstrated that the Hedgehog pathway is largely activated in different cancer cells. RESULT: This review tries to highlight the effect of itraconazole on smoothened receptor (SMO) in the Hedgehog pathway, thereby reducing the glioma-associated oncogene homolog (GLI) release and finally exhibiting a range of anticancer effects, promoting apoptosis of cancer cells, and inhibiting proliferation by indirect inhibition of NF-κB pathway and inflammation, moreover, promoting the expression of cyclin-dependent kinase inhibitors, inhibiting the expression of target genes transcribed by GLI such as BCL-2 and Cyclin-D1. Besides, itraconazole increases the number of Bnip3, subsequently, inducing the dissociation of the Beclin-1/BCL-2 binding complex, as a result of ultimately promoting autophagy of cancer cells. CONCLUSION: As a new anticancer drug, whether itraconazole eventually entering clinical application requires the joint eforts of all scholars. In any case, an in-depth study on itraconazole will bring new hope for cancer patients in the near future.


Assuntos
Proteínas Hedgehog/metabolismo , Itraconazol/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Ensaios Clínicos Fase II como Assunto , Humanos , Itraconazol/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo
8.
Development ; 147(3)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31964774

RESUMO

Sonic hedgehog (Shh) signal transduction specifies ventral cell fates in the neural tube and is mediated by the Gli transcription factors that play both activator (GliA) and repressor (GliR) roles. Cilia are essential for Shh signal transduction and the ciliary phosphatidylinositol phosphatase Inpp5e is linked to Shh regulation. In the course of a forward genetic screen for recessive mouse mutants, we identified a functional null allele of inositol polyphosphate-5-phosphatase E (Inpp5e), ridge top (rdg), with expanded ventral neural cell fates at E10.5. By E12.5, Inpp5erdg/rdg embryos displayed normal neural patterning and this correction over time required Gli3, the predominant repressor in neural patterning. Inpp5erdg function largely depended on the presence of cilia and on smoothened, the obligate transducer of Shh signaling, indicating that Inpp5e functions within the cilium to regulate the pathway. These data indicate that Inpp5e plays a more complicated role in Shh signaling than previously appreciated. We propose that Inpp5e attenuates Shh signaling in the neural tube through regulation of the relative timing of GliA and GliR production, which is important in understanding how the duration of Shh signaling regulates neural tube patterning.


Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/genética , Alelos , Animais , Padronização Corporal/genética , Embrião de Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/metabolismo , Monoéster Fosfórico Hidrolases/genética , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismo
9.
Int J Mol Sci ; 21(2)2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968603

RESUMO

Bone fracture healing involves the combination of intramembranous and endochondral ossification. It is known that Indian hedgehog (Ihh) promotes chondrogenesis during fracture healing. Meanwhile, Sonic hedgehog (Shh), which is involved in ontogeny, has been reported to be involved in fracture healing, but the details had not been clarified. In this study, we demonstrated that Shh participated in fracture healing. Six-week-old Sprague-Dawley rats and Gli-CreERT2; tdTomato mice were used in this study. The right rib bones of experimental animals were fractured. The localization of Shh and Gli1 during fracture healing was examined. The localization of Gli1 progeny cells and osterix (Osx)-positive cells was similar during fracture healing. Runt-related transcription factor 2 (Runx2) and Osx, both of which are osteoblast markers, were observed on the surface of the new bone matrix and chondrocytes on day seven after fracture. Shh and Gli1 were co-localized with Runx2 and Osx. These findings suggest that Shh is involved in intramembranous and endochondral ossification during fracture healing.


Assuntos
Condrogênese/fisiologia , Consolidação da Fratura/fisiologia , Proteínas Hedgehog/metabolismo , Osteogênese/fisiologia , Animais , Osso e Ossos/fisiologia , Diferenciação Celular , Condrócitos/fisiologia , Proteínas Hedgehog/genética , Imuno-Histoquímica , Masculino , Camundongos , Osteoblastos/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
10.
Toxicol Appl Pharmacol ; 387: 114853, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31816328

RESUMO

As an intercellular signaling molecule, Hedgehog (Hh) plays a critical role in liver fibrosis/regeneration. Transcription effectors Gli1 and Gli2 are key components of the Hh signaling pathway. However, whether inhibition of Gli1/2 activity can affect liver fibrogenesis is largely unknown. In the present study, we investigated the effect of Gant61 (a Gli1/2 transcription factor inhibitor) on liver fibrosis and its possible mechanism. Wild-type and Shh-EGFP-Cre male mice were exposed to CCl4, and then treated with or without Gant61 for four weeks. The level of liver injury/fibrosis and expression levels of mRNA and protein related to the Hh ligand/pathway were assessed. In our study, CCl4 treatment induced liver injury/fibrosis and promoted activation of hepatic stellate cells (HSCs). In addition, CCl4 induced the expression of Shh ligands in and around the fibrotic lesion, accompanied by induction of mRNA and protein expression of Hh components (Smo, Gli1 and Gli2). However, administration of Gant61 decreased liver fibrosis by reduction in HSC number, down-regulation of mRNA and protein expression of Hh components (Smo, Gli1 and Gli2), and cell-cycle arrest of HSCs. Our data highlight the importance of the Shh pathway for the development of liver fibrosis, and also suggest Glis as potential therapeutic targets for the treatment of liver fibrosis.


Assuntos
Proteínas Hedgehog/metabolismo , Cirrose Hepática Experimental/tratamento farmacológico , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Tetracloreto de Carbono/toxicidade , Regulação para Baixo/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/patologia , Masculino , Camundongos , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/metabolismo
11.
Lab Invest ; 100(4): 657-664, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31758086

RESUMO

The hedgehog signaling pathway is a vital factor for embryonic development and stem cell maintenance. Dysregulation of its function results in tumor initiation and progression. The aim of this research was to establish a disease model of hedgehog-related tumorigenesis with Gorlin syndrome-derived induced pluripotent stem cells (GS-iPSCs). Induced neural progenitor cells from GS-iPSCs (GS-NPCs) show constitutive high GLI1 expression and higher sensitivity to smoothened (SMO) inhibition compared with wild-type induced neural progenitor cells (WT-NPCs). The differentiation process from iPSCs to NPCs may have similarity in gene expression to Hedgehog signal-related carcinogenesis. Therefore, GS-NPCs may be useful for screening compounds to find effective drugs to control Hedgehog signaling activity.


Assuntos
Síndrome do Nevo Basocelular , Células-Tronco Neurais , Transdução de Sinais/genética , Receptor Smoothened , Proteína GLI1 em Dedos de Zinco , Anilidas , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Humanos , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Patched-1 , Piridinas , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
12.
Biochem J ; 477(1): 121-136, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31845979

RESUMO

Vlk is a secreted tyrosine kinase that plays crucial roles during vertebrate embryonic development including skeletal formation. Genetic studies suggest that Vlk can modulate the Hedgehog signaling pathway during skeletal development. Despite its potential roles as an extracellular regulator of signaling pathways, little is known regarding the molecular functions of Vlk. Here we show that Vlk can negatively regulate the Hedgehog signaling pathway. We found that Vlk can induce lysosomal degradation of Smoothened, a crucial transmembrane signal transducer of the Hedgehog pathway, through the interaction with the extracellular domain of Smoothened (Smo-ECD). In addition, we observed that Vlk can attenuate Hedgehog signaling-induced ciliary localization of Smoothened. Furthermore, Vlk-mediated suppression of Hedgehog signaling can be diminished by tyrosine-to-phenylalanine substitutions in Smo-ECD. Taken together, these results suggest that Vlk may function as a signaling regulator in extracellular space to modulate the Hedgehog pathway.


Assuntos
Proteínas Hedgehog/metabolismo , Proteínas Tirosina Quinases/fisiologia , Proteólise , Receptor Smoothened/metabolismo , Animais , Cílios/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Células NIH 3T3
13.
Mol Pharmacol ; 97(1): 23-34, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31707356

RESUMO

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.


Assuntos
Sítios de Ligação/genética , Bioensaio/métodos , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Compostos de Boro/química , Cinamatos/farmacologia , Descoberta de Drogas/métodos , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Luciferases/química , Morfolinas/farmacologia , Nanoestruturas/química , Purinas/farmacologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Alcaloides de Veratrum/química
14.
J Orthop Res ; 38(3): 609-619, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31608494

RESUMO

We aimed to investigate whether post-traumatic osteoarthritis (PTOA) progression is appropriately represented by a PTOA mouse model using a unique climbing cage to add mechanical loading after anterior cruciate ligament (ACL) transection and to determine how Hedgehog signaling inhibition prevents PTOA progression by observing time-dependent morphological changes. This controlled laboratory study histologically compared mice with surgically-induced ACL transection (ACLT) and those with voluntary increased activity in a climbing cage from 1 week postoperatively (ACLT + climbing). We generated conditional knockout (cKO) mice with a deleted Smoothened (Smo) gene. Time-dependent histopathological, immunohistochemical, and gene expression analyses were performed. The ACLT + climbing group showed more severe cartilage defects and massive osteophyte formation than the ACLT group. Smo deletion significantly suppressed PTOA progression. The time-dependent assessment revealed cartilaginous processes of equivalent size at the posterior tibial margin in the Smo cKO and control mice at 4 weeks postoperatively. However, at 8 weeks postoperatively, mature ossifying lesions were detected in the controls but not in Smo cKO mice. In the articular cartilage, ADAMTS5 and RUNX2 expression were observed in hypertrophic chondrocytes near the defective cartilage in controls but not in Smo cKO mice. Climbing exercise after ACLT accelerated PTOA progression more severely not only through increasing joint instability induced by ACLT but also through mechanical loading force induced by climbing exercise. Hedgehog signaling inhibition attenuated PTOA progression by suppressing chondrocyte hypertrophy induced by mechanical loads, to which ACL-deficient athletes are usually exposed. Thus, Hedgehog signaling inhibition may be a therapeutic option to prevent arthritic changes in athletes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:609-619, 2020.


Assuntos
Lesões do Ligamento Cruzado Anterior/patologia , Cartilagem Articular/patologia , Proteínas Hedgehog/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Proteína ADAMTS5/metabolismo , Animais , Ligamento Cruzado Anterior/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Articulação do Joelho/patologia , Masculino , Camundongos , Camundongos Knockout , Osteoartrite/genética , Condicionamento Físico Animal , Receptor Smoothened/genética , Tíbia/fisiologia , Ferimentos e Lesões
15.
Mol Pharmacol ; 97(2): 62-71, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31591260

RESUMO

The class Frizzled (FZD) or class F of G protein-coupled receptors consists of 10 FZD paralogues and Smoothened (SMO). FZDs coordinate wingless/Int-1 signaling and SMO mediates Hedgehog signaling. Class F receptor signaling is intrinsically important for embryonic development and its dysregulation leads to diseases, including diverse forms of tumors. With regard to the importance of class F signaling in human disease, these receptors provide an attractive target for therapeutics, exemplified by the use of SMO antagonists for the treatment of basal cell carcinoma. Here, we review recent structural insights in combination with a more detailed functional understanding of class F receptor activation, G protein coupling, conformation-based functional selectivity, and mechanistic details of activating cancer mutations, which will lay the basis for further development of class F-targeting small molecules for human therapy. SIGNIFICANCE STATEMENT: Stimulated by recent insights into the activation mechanisms of class F receptors from structural and functional analysis of Frizzled and Smoothened, we aim to summarize what we know about the molecular details of ligand binding, agonist-driven conformational changes, and class F receptor activation. A better understanding of receptor activation mechanisms will allow us to engage in structure- and mechanism-driven drug discovery with the potential to develop more isoform-selective and potentially pathway-selective drugs for human therapy.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Receptores Frizzled/agonistas , Ligantes , Terapia de Alvo Molecular/métodos , Receptor Smoothened/agonistas , Animais , Descoberta de Drogas/métodos , Desenvolvimento Embrionário/fisiologia , Receptores Frizzled/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Receptor Smoothened/metabolismo , Relação Estrutura-Atividade , Proteínas Wnt/metabolismo
16.
Dev Biol ; 457(1): 128-139, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31550483

RESUMO

Hedgehog (Hh) signaling has been shown to regulate multiple developmental processes, however, it is unclear how it regulates lipid metabolism. Here, we demonstrate that Hh signaling exhibits potent activity in Drosophila fat body, which is induced by both locally expressed and midgut-derived Hh proteins. Inactivation of Hh signaling increases, whereas activation of Hh signaling decreases lipid accumulation. The major lipase Brummer (Bmm) acts downstream of Smoothened (Smo) in Hh signaling to promote lipolysis, therefore, the breakdown of triacylglycerol (TAG). We identify a critical Ci binding site in bmm promoter that is responsible to mediate Bmm expression induced by Hh signaling. Genomic mutation of the Ci binding site significantly reduces the expression of Bmm and dramatically decreases the responsiveness to Ci overexpression. Together, our findings provide a model for lipolysis to be regulated by Hh signaling, raising the possibility for Hh signaling to be involved in lipid metabolic and/or lipid storage diseases.


Assuntos
Proteínas de Drosophila/genética , Drosophila/metabolismo , Lipase/genética , Lipólise , Transdução de Sinais , Adipócitos/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Corpo Adiposo/metabolismo , Feminino , Proteínas Hedgehog/metabolismo , Larva/metabolismo , Masculino , Receptor Smoothened/metabolismo , Fatores de Transcrição/metabolismo
17.
Cell Rep ; 29(12): 4036-4052.e10, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851932

RESUMO

The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Mutação , Neurônios/patologia , Receptor Smoothened/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Apoptose , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Feminino , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Neurônios/metabolismo , Receptor Smoothened/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Rep ; 9(1): 19623, 2019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31873117

RESUMO

Growth and patterning of the cerebellum is compromised if granule cell precursors do not properly expand and migrate. During embryonic and postnatal cerebellar development, the Hedgehog pathway tightly regulates granule cell progenitors to coordinate appropriate foliation and lobule formation. Indeed, granule cells impairment or defects in the Hedgehog signaling are associated with developmental, neurodegenerative and neoplastic disorders. So far, scant and inefficient cellular models have been available to study granule cell progenitors, in vitro. Here, we validated a new culture method to grow postnatal granule cell progenitors as hedgehog-dependent neurospheres with prolonged self-renewal and ability to differentiate into granule cells, under appropriate conditions. Taking advantage of this cellular model, we provide evidence that Ptch1-KO, but not the SMO-M2 mutation, supports constitutive and cell-autonomous activity of the hedgehog pathway.


Assuntos
Diferenciação Celular , Cerebelo/metabolismo , Proteínas Hedgehog , Células-Tronco Neurais/metabolismo , Transdução de Sinais , Receptor Smoothened , Animais , Cerebelo/citologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/citologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo
19.
Med Sci Monit ; 25: 8579-8586, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31724562

RESUMO

BACKGROUND Dysregulation of the Hedgehog (Hh) pathway modulates various aspects of hematologic and solid tumors, but its effects in human Natural killer/T-cell lymphoma (NKTCL) are unclear. Moreover, no study has examined the consequences of pharmacologically inhibiting Hh signaling in NKTCL cell lines. MATERIAL AND METHODS In this study, the expression of Smoothened (Smo) and Glioma-associated oncogene 1 (Gli1) in NKTCL tissue were scrutinized. Two human NKTCL cell lines, SNK6 and SNT8, were subjected to various doses of sonidegib (a Smo inhibitor) and incubated for distinct durations. The cell apoptosis was examined by flow cytometry, CCK-8 assay was run to assess proliferation, and protein levels were quantified by Western blotting. RESULTS Both Smo and Gli1 expression were higher in NKTCL tissue than in Lymphoid Reactive Hyperplasia (LRH). Sonidegib significantly suppressed proliferation in NKTCL cells and the effect was dose-dependent. Further analysis revealed that sonidegib treatment elevated the number of apoptotic cells in a dose- and time-dependent manner. In addition, sonidegib downregulated Smo and Gli1expression in NKTCL cells. CONCLUSIONS The Hh pathway is crucial to the development of NKTCL and thus holds huge promise as a treatment for this disease.


Assuntos
Compostos de Bifenilo/farmacologia , Linfoma de Células T/metabolismo , Piridinas/farmacologia , Receptor Smoothened/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Linfoma , Piridinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco
20.
Sci Rep ; 9(1): 16057, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690747

RESUMO

We tested whether cannabinoids (CBs) potentiate alcohol-induced birth defects in mice and zebrafish, and explored the underlying pathogenic mechanisms on Sonic Hedgehog (Shh) signaling. The CBs, Δ9-THC, cannabidiol, HU-210, and CP 55,940 caused alcohol-like effects on craniofacial and brain development, phenocopying Shh mutations. Combined exposure to even low doses of alcohol with THC, HU-210, or CP 55,940 caused a greater incidence of birth defects, particularly of the eyes, than did either treatment alone. Consistent with the hypothesis that these defects are caused by deficient Shh, we found that CBs reduced Shh signaling by inhibiting Smoothened (Smo), while Shh mRNA or a CB1 receptor antagonist attenuated CB-induced birth defects. Proximity ligation experiments identified novel CB1-Smo heteromers, suggesting allosteric CB1-Smo interactions. In addition to raising concerns about the safety of cannabinoid and alcohol exposure during early embryonic development, this study establishes a novel link between two distinct signaling pathways and has widespread implications for development, as well as diseases such as addiction and cancer.


Assuntos
Canabinoides/toxicidade , Transtornos do Espectro Alcoólico Fetal/metabolismo , Proteínas Hedgehog/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Teratogênese/efeitos dos fármacos , Animais , Etanol/efeitos adversos , Etanol/farmacologia , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Camundongos , Receptor Smoothened/metabolismo
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