RESUMO
Intracerebral hemorrhage (ICH) is a significant public health matter that has no effective treatment. ICH-induced destruction of the blood-brain barrier (BBB) leads to neurological deterioration. Astrocytic sonic hedgehog (SHH) alleviates brain injury by maintaining the integrity of the BBB after ICH. Silent information regulator 1 (SIRT1) is neuroprotective in several central nervous system diseases via BBB regulation. It is also a possible influential factor of the SHH signaling pathway. Nevertheless, the role of SIRT1 on BBB and the underlying pathological process associated with the SHH signaling pathway after ICH remain unclear. We established an intracerebral hemorrhagic mouse model by collagenase injection. SRT1720 (a selective agonist of SIRT1) was used to evaluate the effect of SIRT1 on BBB integrity after ICH. SIRT1 expression was reduced in the mouse brain after ICH. SRT1720 attenuated neurobehavioral impairments and brain edema of ICH mouse. After ICH induction, SRT1720 improved BBB integrity and tight junction expressions in the mouse brain. The SHH signaling pathway-related factors smoothened and glioma-associated oncogene homolog-1 were increased with the intervention of SRT1720, while cyclopamine (a specific inhibitor of the SHH signaling pathway) reversed these effects. These findings suggest that SIRT1 protects from ICH by altering BBB permeability and tight junction expression levels. This process is associated with the SHH signaling pathway, suggesting that SIRT1 may be a potential therapeutic target for ICH.
Assuntos
Barreira Hematoencefálica , Hemorragia Cerebral , Compostos Heterocíclicos de 4 ou mais Anéis , Sirtuína 1 , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Sirtuína 1/metabolismo , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/tratamento farmacológico , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Masculino , Camundongos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/agonistas , Edema Encefálico/tratamento farmacológico , Edema Encefálico/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Doenças Desmielinizantes , Testosterona , Animais , Feminino , Masculino , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/tratamento farmacológico , Camundongos , Testosterona/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/agonistas , Camundongos Endogâmicos C57BL , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/agonistas , Bainha de Mielina/metabolismo , Modelos Animais de Doenças , Transdução de Sinais/efeitos dos fármacos , Sistema Imunitário/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/imunologia , Caracteres SexuaisRESUMO
The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.
Assuntos
Proteínas Hedgehog , Humanos , Colesterol/metabolismo , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Ligantes , Proteínas Oncogênicas , EsteróisRESUMO
Embryonic stem cells (ESCs) have been shown to have an ability to form a large number of functional endothelial cells in vitro, but generating organ-specific endothelial cells remains a challenge. Sonic hedgehog (SHH) pathway is one of the crucial developmental pathways that control differentiation of many embryonic cell types such as neuroectodermal, primitive gut tube and developing limb buds; SHH pathway is important for functioning of adult cell of skin, bone, liver as well as it regulates haematopoiesis. Misregulation of SHH pathway leads to cancers such as hepatic, pancreatic, basal cell carcinoma, medulloblastoma, etc. However, its role in differentiation of human ESCs into endothelial cells has not been completely elucidated. Here, we examined the role of SHH signalling pathway in endothelial differentiation of hESCs by growing them in the presence of an SHH agonist (purmorphamine) and an SHH antagonist (SANT-1) for a period of 6 days. Interestingly, we found that activation of SHH pathway led to a higher expression of set of transcription factors such as BRACHYURY, GATA2 and RUNX1, thus favouring hemogenic endothelium; whereas inhibition of SHH pathway led to a reduced expression of set of markers such as RUNX1 and BRACHURY, and an increased expression of set of markers - NFATC1, c-KIT, GATA4, CD31 & CD34, thus favouring endocardiogenic endothelium. The results of this study have revealed the previously unreported deterministic role of SHH pathway in specification of endothelial cells differentiated from human ESCs into hemogenic vs. endocardiogenic lineage; this finding could have major implications for clinical applications.
Assuntos
Padronização Corporal , Diferenciação Celular , Proteínas Hedgehog/metabolismo , Hemangioblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Transdução de Sinais , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Padronização Corporal/genética , Diferenciação Celular/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Mesoderma/metabolismo , Modelos Biológicos , Transdução de Sinais/genéticaRESUMO
The G-protein-coupled receptor 126 (GPR126) may play an important role in tumor development, although its role remains poorly understood. We found that GPR126 had higher expression in most colorectal cancer cell lines than in normal colon epithelial cell lines, and higher expression levels in colorectal cancer tissues than in normal adjacent colon tissues. GPR126 knockdown induced by shRNA inhibited cell viability and colony formation in HT-29, HCT116, and LoVo cells, decreased BrdU incorporation into newly synthesized proliferating HT-29 cells, led to an arrest of cell cycle progression at the G1 phase in HCT-116 and HT-29 cells, and suppressed tumorigenesis of HT-29, HCT116, and LoVo cells in nude mouse xenograft models. GPR126 knockdown engendered decreased transcription and translation of histone deacetylase 2 (HDAC2), previously implicated in the activation of GLI1 and GLI2 in the Hedgehog signaling pathway. Ectopic expression of HDAC2 in GPR126-silenced cells restored cell viability and proliferation, GLI2 luciferase reporter activity, partially recovered GLI2 expression, and reduced the cell cycle arrest. HDAC2 regulated GLI2 expression and, along with GLI2, it bound to the PTCH1 promoter, as evidenced by a chip assay with HT-29 cells. Purmorphamine, a hedgehog agonist, largely restored the cell viability and expression of GLI2 proteins in GPR126-silenced HT-29 cells, whereas GANT61, a hedgehog inhibitor, further enhanced the GPR126 knockdown-induced inhibitory effects. Our findings demonstrate that GPR126 regulates colorectal cancer cell proliferation by mediating the expression of HDAC2 and GLI2, therefore it may represent a suitable therapeutic target for colorectal cancer treatment.
Assuntos
Proliferação de Células/fisiologia , Neoplasias Colorretais/metabolismo , Histona Desacetilase 2/metabolismo , Proteínas Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Colo/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , DNA/biossíntese , Fase G1 , Técnicas de Silenciamento de Genes , Células HT29 , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Xenoenxertos , Humanos , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Nus , Morfolinas/farmacologia , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Receptor Patched-1/metabolismo , Purinas/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs in vivo was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) (P < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist (P < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor (P < 0.05). In vivo study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling (P < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.
Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Sinoviócitos/metabolismo , Artrite Reumatoide/patologia , Biomarcadores , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Imuno-Histoquímica , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Pessoa de Meia-Idade , Alcaloides de Veratrum/farmacologiaRESUMO
ZNF521 is a transcription co-factor with recognized regulatory functions in haematopoietic, osteo-adipogenic and neural progenitor cells. Among its diverse activities, ZNF521 has been implicated in the regulation of medulloblastoma (MB) cells, where the Hedgehog (HH) pathway, has a key role in the development of normal cerebellum and of a substantial fraction of MBs. Here a functional cross-talk is shown for ZNF521 with the HH pathway, where it interacts with GLI1 and GLI2, the major HH transcriptional effectors and enhances the activity of HH signalling. In particular, ZNF521 cooperates with GLI1 and GLI2 in the transcriptional activation of GLI (glioma-associated transcription factor)-responsive promoters. This synergism is dependent on the presence of the N-terminal, NuRD-binding motif in ZNF521, and is sensitive to HDAC (histone deacetylase) and GLI inhibitors. Taken together, these results highlight the role of ZNF521, and its interaction with the NuRD complex, in determining the HH response at the level of transcription. This may be of particular relevance in HH-driven diseases, especially regarding the MBs belonging to the SHH (sonic HH) subgroup where a high expression of ZNF521 is correlated with that of HH pathway components.
Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Linhagem Celular , Neoplasias Cerebelares/genética , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Meduloblastoma/genética , Camundongos , Família Multigênica , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica , Regulação para Cima , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3-5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results-combined with signalling studies and molecular dynamics simulations-delineate the structural basis for PTCH1-SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.
Assuntos
Membrana Celular/química , Proteínas Hedgehog/agonistas , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Esteróis/farmacologia , Animais , Sítios de Ligação , Técnicas Biossensoriais , Domínio Catalítico/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Colesterol/farmacologia , Proteínas Hedgehog/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Receptor Patched-1/antagonistas & inibidores , Receptor Patched-1/metabolismo , Conformação Proteica , Estabilidade Proteica , Anticorpos de Cadeia Única/imunologia , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/química , Esteróis/química , Esteróis/metabolismo , Proteínas de Xenopus/químicaRESUMO
Fracture healing is a complex process of many coordinated biological pathways. This system can go awry resulting in nonunion, which leads to significant patient morbidity. The Hedgehog (Hh) signaling pathway is upregulated in fracture healing. We hypothesized that the Hh signaling pathway can be pharmacologically modulated to positively affect fracture healing. Diaphyseal femur fractures were created in elderly mice (18 months, C57BL/6 females), which have a blunted and delayed healing response compared to younger mice, and were stabilized with intramedullary pins. To activate the Hh pathway we targeted the receptor Smoothened using an agonist (Hh-Ag1.5 [Hh-Ag]) and compared this to a vehicle control. Expression of Hh target genes were significantly increased in the fracture callus of the agonist group compared to controls, indicating pathway activation. Expression of osteogenic and chondrogenic-related genes was greatly upregulated in fracture callus versus intact femora, although Hh agonist treatment did not consistently enhance this response. Blindly graded, radiographic callus healing scores were significantly higher in the Hh-Ag groups at post operative day (POD) 14, indicating earlier callus bridging. On microCT, Hh-Ag treatment led to greater callus volume (+40%) and bone volume (+25%) at POD21. By day 14, callus vascularity, as assessed by 3D microCT angiography vessel volume, was 85% greater in the Hh-Ag group. Finally, mechanical strength of the calluses in the Hh-Ag groups was significantly greater than in the control groups at POD21. In conclusion, systemic administration of a Hh agonist appears to improve the osseous and vascular healing responses in a mouse fracture healing-impaired model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
Assuntos
Consolidação da Fratura/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Fatores Etários , Animais , Calo Ósseo/diagnóstico por imagem , Calo Ósseo/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Fraturas do Fêmur/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Liver is the second most transplanted organ according to United network for organ sharing. Due to shortage of compatible donors, surgical difficulties, immunological hindrance, and high postoperative cost, stem cell therapy is an attractive substitute of liver transplant for millions of patients suffering from hepatic failure. Due to several technical limitations such as viral integration, inefficient differentiation, and adult phenotypes and epigenetic memory of fibroblasts, induced pluripotent stem cells, mesenchymal stem cells, or induced hepatocyte may not present a great clinical substitute for liver transplant. We pioneered a novel technology for robust expansion of quiescent liver stem cells (LSCs) from mice via utilizing of Hedgehog agonist HhAg1.5 for 3 weeks. These expanded LSCs retained stem-like properties after multiple passaging and differentiated to hepatocytes and cholangiocytes. Grafting of ex vivo expanded LSCs in Fah-/- Rag2-/- Il2rg-/- knockout mice, significantly increased life span compared to control group (P < 0.001). Thus in this study, we provide a promising viable substitute for primary hepatocytes for regenerative medicine and for life-threatening metabolic liver diseases.
Assuntos
Células-Tronco Adultas/citologia , Proteínas Hedgehog/agonistas , Falência Hepática/terapia , Fígado/citologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antígeno AC133/imunologia , Células-Tronco Adultas/imunologia , Animais , Sistema Biliar/citologia , Diferenciação Celular , Hepatócitos/citologia , Antígenos Comuns de Leucócito/imunologia , Fígado/imunologia , Longevidade , Camundongos , Camundongos Knockout , Medicina RegenerativaRESUMO
Early loss of up to 50% of cells is common for in vitro chondrogenesis of mesenchymal stromal cells (MSC) in pellet culture, reducing the efficacy and the tissue yield for cartilage engineering. Enhanced proliferation could compensate for this unwanted effect, but relevant signaling pathways remain largely unknown. The aim of this study was to identify the contribution of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and hedgehog (HH) signaling toward cell proliferation during chondrogenesis and investigate whether a further mitogenic stimulation is possible and promising. Human MSC were subjected to chondrogenesis in the presence or absence of pathway inhibitors or activators up to Day 14 or from Days 14 to 28, before proliferation, DNA and proteoglycan content were quantified. [3H]-thymidine incorporation revealed arrest of proliferation on Day 3, after which cell division was reinitiated. Although BMP signaling was essential for proliferation throughout chondrogenesis, IGF signaling was relevant only up to Day 14. In contrast, FGF and HH signaling drove proliferation only from Day 14 onward. Early BMP4, IGF-1, or FGF18 treatment neither prevented early cell loss nor allowed further mitogenic stimulation. However, application of the HH-agonist purmorphamine from Day 14 increased proliferation 1.44-fold (p < 0.05) and late BMP4-application enhanced the DNA and proteoglycan content, with significant effects on tissue yield. Conclusively, a differential and phase-dependent contribution of the four pathways toward proliferation was uncovered and BMP4 treatment was promising to enhance tissue yield. Culture forms less prone to size limitations by nutrient/oxygen gradients and a focus on early apoptosis prevention may be considered as the next steps to further enhance chondrocyte formation from MSC.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Condrogênese/genética , Células-Tronco Mesenquimais/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Morfogenética Óssea 4/genética , Cartilagem/efeitos dos fármacos , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/genética , Humanos , Fator de Crescimento Insulin-Like I/agonistas , Fator de Crescimento Insulin-Like I/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Morfolinas/farmacologia , Purinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: The present study was been designed to investigate the role and pharmacological potential of hedgehog in oestrogen-deficient rat heart. METHODS: Oestrogen deficiency was produced in female Wistar rats by the surgical removal of both ovaries and these animals were used four weeks later. Isolated rat heart was subjected to 30 min ischaemia followed by 120 min of reperfusion (I/R). The heart was subjected to pharmacological preconditioning with the hedgehog agonist purmorphamine (1µM) and GDC-0449, a hedgehog antagonist, in the last episode of reperfusion before I/R. Myocardial infarction was assessed in terms of the increase in lactate dehydrogenase (LDH), creatinine kinase-MB (CK-MB), myeloperoxidase (MPO) level and infarct size (triphenyltetrazolium chloride staining). Immunohistochemistry analysis was done for the assessment of tumour necrosis factor (TNF)-α level in cardiac tissue. eNOS expression was estimated by rt-PCR. RESULTS: Pharmacological preconditioning with purmorphamine significantly attenuated I/R-induced myocardial infarction, TNF-α, MPO level and release of LDH and CK-MB compared to the I/R control group. However, GDC-0449 prevented the ameliorative preconditioning effect of estradiol. CONCLUSION: It may be concluded that the hedgehog agonist purmorphamine prevents the ovariectomised heart from ischaemic reperfusion injury.
Assuntos
Cardiotônicos/uso terapêutico , Coração/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Morfolinas/uso terapêutico , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Purinas/uso terapêutico , Animais , Feminino , Coração/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ovariectomia , Ratos WistarRESUMO
The cerebellum undergoes rapid growth during the third trimester and is vulnerable to injury and deficient growth in infants born prematurely. Factors associated with preterm cerebellar hypoplasia include chronic lung disease and postnatal glucocorticoid administration. We modeled chronic hypoxemia and glucocorticoid administration in neonatal mice to study whole cerebellar and cell type-specific effects of dual exposure. Chronic neonatal hypoxia resulted in permanent cerebellar hypoplasia. This was compounded by administration of prednisolone as shown by greater volume loss and Purkinje cell death. In the setting of hypoxia and prednisolone, administration of a small molecule Smoothened-Hedgehog agonist (SAG) preserved cerebellar volume and protected against Purkinje cell death. Such protective effects were observed even when SAG was given as a one-time dose after dual insult. To model complex injury and determine cell type-specific roles for the hypoxia inducible factor (HIF) pathway, we performed conditional knockout of von Hippel Lindau (VHL) to hyperactivate HIF1α in cerebellar granule neuron precursors (CGNP) or Purkinje cells. Surprisingly, HIF activation in either cell type resulted in no cerebellar deficit. However, in mice administered prednisolone, HIF overactivation in CGNPs resulted in significant cerebellar hypoplasia, whereas HIF overactivation in Purkinje cells caused cell death. Together, these findings indicate that HIF primes both cell types for injury via glucocorticoids, and that hypoxia/HIF + postnatal glucocorticoid administration act on distinct cellular pathways to cause cerebellar injury. They further suggest that SAG is neuroprotective in the setting of complex neonatal cerebellar injury.
Assuntos
Anti-Inflamatórios/uso terapêutico , Cerebelo/anormalidades , Cicloexilaminas/uso terapêutico , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Tiofenos/uso terapêutico , Aminoácidos Dicarboxílicos/farmacologia , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Cerebelo/efeitos dos fármacos , Deficiências do Desenvolvimento/etiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glucocorticoides/farmacologia , Hipóxia Encefálica/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/etiologia , Prednisolona/uso terapêutico , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
Normally, hepatic progenitor cells (HPCs) are activated and differentiate into hepatocytes or bile ductular cells to repair liver damage during liver injury. However, it remains controversial whether the abnormal differentiation of HPCs occurs under abnormal conditions. Lipopolysaccharide (LPS), a component of the microenvironment, promotes liver fibrosis. In the present study, HPCs promoted liver fibrosis in rats following carbon tetrachloride (CCl4) treatment. Meanwhile, the LPS level in the portal vein was elevated and played a primary role in the fate of HPCs. In vitro, LPS inhibited the hepatobiliary differentiation of HPCs. Concurrently, HPCs co-cultured with LPS for 2 weeks showed a tendency to differentiate into myofibroblasts (MFs). Thus, we conclude that LPS promotes the aberrant differentiation of HPCs into MFs as a third type of descendant. This study provides insight into a novel differentiation fate of HPCs in their microenvironment, and could thus lead to the development of HPCs for treatment methods in liver fibrosis.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Proteínas Hedgehog/genética , Lipopolissacarídeos/farmacologia , Cirrose Hepática/genética , Transplante de Células-Tronco , Células-Tronco/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Tetracloreto de Carbono , Diferenciação Celular , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Lipopolissacarídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/agonistas , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/agonistas , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
BACKGROUND AND PURPOSE: Because of the limitation in treatment window of the r-tPA (recombinant tissue-type plasminogen activator), the development of delayed treatment for stroke is needed. In this study, we examined the efficacy of delayed poststroke treatment (post 3-8 days) of the sonic hedgehog pathway agonist on functional recovery and the underlying mechanisms. METHODS: We evaluated functional recovery at 1 month after stroke using locomotion analysis and Barnes maze test for cognitive function. We used a genetically inducible neural stem cell-specific reporter mouse line (nestin-CreERT2-R26R-YFP) to label and track their proliferation, survival, and differentiation in ischemic brain. Brain tissue damage, angiogenesis, and cerebral blood flow recovery was evaluated using magnetic resonance imaging techniques and immunostaining. RESULTS: Our results show that delayed treatment of sonic hedgehog pathway agonist in stroke mice results in enhanced functional recovery both in locomotor function and in cognitive function at 1 month after stroke. Furthermore, using the Nestincre-ERT2-YFP mice, we showed that poststroke sonic hedgehog pathway agonist treatment increased surviving newly born cells derived from both subventricular zone and subgranular zone neural stem cells, total surviving DCX+ (Doublecortin) neuroblast cells, and neurons (NeuN+/YFP+) in the ischemic brain. Sonic hedgehog pathway agonist treatment also improved the brain tissue repair in ischemic region supported by our T2-weighted magnetic resonance imaging, cerebral blood flow map by arterial spin labeling, and immunohistochemistry (α-smooth muscle actin and CD31 immunostaining). CONCLUSIONS: These data confirm an important role for the hedgehog pathway in poststroke brain repair and functional recovery, suggesting a prolonged treatment window for potential treatment strategy to modulate sonic hedgehog pathway after stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Comportamento Animal , Modelos Animais de Doenças , Proteína Duplacortina , Proteínas Hedgehog/administração & dosagem , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Nestina , Células-Tronco NeuraisRESUMO
Myocardial infarction (MI) is caused by the occlusion of a coronary artery due to underlying atherosclerosis complicated by localized thrombosis. The blockage of blood flow leads to cardiomyocyte (CM) death in the infarcted area. Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury; however, some pathways active during embryogenesis and silent during adult life are recruited in response to tissue injury. One such example is hedgehog (Hh) signaling. Hh is involved in the embryonic development of the heart and coronary vascular system. Pathological conditions including ischemia activate Hh signaling in adult tissues. This review highlights the involvement of Hh signaling in ischemic tissue regeneration with a particular emphasis on heart regeneration and discusses its potential role as a therapeutic agent.
Assuntos
Coração/fisiologia , Proteínas Hedgehog/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/terapia , Regeneração , Transdução de Sinais , Animais , Terapia Baseada em Transplante de Células e Tecidos/métodos , Técnicas de Reprogramação Celular/métodos , Descoberta de Drogas , Coração/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Humanos , Terapia de Alvo Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Regeneração/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Granule neuron precursors (GNPs) proliferate under the influence of Sonic hedgehog (Shh) that is secreted by Purkinje neurons during early postnatal cerebellar development. To investigate microRNA (miRNA) function in this developmental process, we conditionally deleted the Dicer1 gene under the activity of human glial fibrillary acidic protein (hGFAP) promoter. We report that Dicer1-ablated GNPs display decreased proliferation and survival at early postnatal stages and that the proliferation defect of mutant GNPs cannot be rescued by treatment of an Shh agonist in vitro as assayed by 5-bromo-2'-deoxyuridine (BrdU) pulse labeling and Shh target gene expression detection. Further analysis reveals that the expression of distinct cell cycle regulator genes including cell cycle inhibitor, CDKN1a (p21), selectively increases in Dicer1-ablated GNPs. Subsequently, we demonstrate that miR-17-5p exhibits high expression level in the developing cerebellum and that transfection of a synthetic miR-17-5p mimic downregulates p21 protein expression in GNPs and promotes proliferation of GNPs in culture. Therefore, Dicer1 ablation impairs Shh-induced GNP proliferation by disrupting the expression of distinct cell cycle regulator genes that are targets of miR-17â¼92 cluster members. This study establishes a molecular link between miRNAs and cell cycle progression in the proliferating GNPs during normal cerebellar development and may facilitate miRNA application in treating medulloblastoma.
Assuntos
Proliferação de Células/fisiologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , RNA Helicases DEAD-box/deficiência , Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/metabolismo , Ribonuclease III/deficiência , Animais , Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Cerebelo/patologia , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Hedgehog/agonistas , Humanos , Camundongos Transgênicos , MicroRNAs/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Regiões Promotoras Genéticas , Ribonuclease III/genéticaRESUMO
We studied the effect of an activator (ShTh) and an inhibitor (cyclopamine) of the Hedgehog signaling pathway on proliferation of human glioma cell lines U87-MG and U251-MG and cultured human astrocytes. The Hedgehog signaling pathway is activated in glioma cells, but not in cultured human astrocytes. Experiments with Shh and cyclopamine can serve as an additional criterion for assessing activity of Hedgehog signaling in known cell lines and primary cultured cells.
Assuntos
Proliferação de Células , Glioblastoma/metabolismo , Transdução de Sinais , Astrócitos/fisiologia , Linhagem Celular Tumoral , Glioblastoma/patologia , Proteínas Hedgehog/agonistas , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Alcaloides de Veratrum/farmacologiaRESUMO
Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showed that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures.
Assuntos
Cicloexilaminas/administração & dosagem , Consolidação da Fratura/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Tiofenos/administração & dosagem , Animais , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Modelos Animais de Doenças , Consolidação da Fratura/fisiologia , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/tratamento farmacológico , Fraturas da Tíbia/patologia , Microtomografia por Raio-XRESUMO
BACKGROUND/AIMS: Lipid accumulation, inflammatory responses and oxidative stress have been implicated in the pathology of alcoholic liver disease (ALD). Targeting inhibition of these features may provide a promising therapeutic strategy for ALD. Baicalin, a flavonoid isolated from Scutellaria baicalensis Georgi, has been shown to exert a hepatoprotective effect. However, its effects on ALD remain obscure. This study was aimed to investigate the effects of baicalin on alcohol-induced liver injury and its related mechanisms. METHODS: For in vivo experiments, rats were supplied intragastrical administration of alcohol continuously for 4 or 8 weeks, and then received baicalin treatment in the latter 4 weeks in the presence / absence of alcohol intake. Liver histology and function, inflammatory cytokines, oxidative mediators, and the components of the Sonic hedgehog pathway were evaluated. For in vitro experiments, alcohol-stimulated human normal liver cells LO2 were used. RESULTS: Baicalin treatment significantly alleviated alcoholic liver injury, improved liver function impaired by alcohol, and inhibited hepatocytes apoptosis. In addition, baicalin decreased the expression levels of proinflammatory cytokines TNF-α, IL-1ß, IL-6) and malonyldialdehyde (MDA), and increased the activities of antioxidant enzymes SOD and GSH-Px. Furthermore, baicalin modulated the activation of Sonic hedgehog (Shh) pathway. Administration of baicalin upregulated the expression of sonic hedgehog (Shh), patched (Ptc), Smoothened (Smo), and Glioblastoma-1(Gli-1). Blockade of the Shh pathway in cyclopamine abolished the effects of baicalin in vitro. CONCLUSION: Both in vivo and in vitro experimental results indicate that baicalin exerts hepatoprotective roles in alcohol-induced liver injury through inhibiting oxidative stress, inflammatory response, and the regulation of the Shh pathway.