Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.224
Filtrar
1.
Nat Chem Biol ; 15(10): 975-982, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548691

RESUMO

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.


Assuntos
Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Animais , Colesterol/química , Regulação da Expressão Gênica , Células HEK293 , Proteínas Hedgehog/química , Proteínas Hedgehog/genética , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Receptor Patched-1/química , Ligação Proteica , Conformação Proteica , Anticorpos de Domínio Único
2.
Nat Commun ; 10(1): 2863, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253779

RESUMO

Cancer stem cells (CSCs) represent a major source of treatment resistance and tumor progression. However, regulation of CSCs stemness is not entirely understood. Here, we report that TSPAN8 expression is upregulated in breast CSCs, promotes the expression of the stemness gene NANOG, OCT4, and ALDHA1, and correlates with therapeutic resistance. Mechanistically, TSPAN8 interacts with PTCH1 and inhibits the degradation of the SHH/PTCH1 complex through recruitment of deubiquitinating enzyme ATXN3. This results in the translocation of SMO to cilia, downstream gene expression, resistance of CSCs to chemotherapeutic agents, and enhances tumor formation in mice. Accordingly, expression levels of TSPAN8, PTCH1, SHH, and ATXN3 are positively correlated in human breast cancer specimens, and high TSPAN8 and ATXN3 expression levels correlate with poor prognosis. These findings reveal a molecular basis of TSPAN8-enhanced Sonic Hedgehog signaling and highlight a role for TSPAN8 in promoting cancer stemness.


Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/fisiologia , Tetraspaninas/metabolismo , Animais , Neoplasias da Mama , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Regulação para Cima
3.
Cell Mol Life Sci ; 76(20): 3939-3952, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201464

RESUMO

Articular cartilage is formed at the end of epiphyses in the synovial joint cavity and permanently contributes to the smooth movement of synovial joints. Most skeletal elements develop from transient cartilage by a biological process known as endochondral ossification. Accumulating evidence indicates that articular and growth plate cartilage are derived from different cell sources and that different molecules and signaling pathways regulate these two kinds of cartilage. As the first sign of joint development, the interzone emerges at the presumptive joint site within a pre-cartilage tissue. After that, joint cavitation occurs in the center of the interzone, and the cells in the interzone and its surroundings gradually form articular cartilage and the synovial joint. During joint development, the interzone cells continuously migrate out to the epiphyseal cartilage and the surrounding cells influx into the joint region. These complicated phenomena are regulated by various molecules and signaling pathways, including GDF5, Wnt, IHH, PTHrP, BMP, TGF-ß, and FGF. Here, we summarize current literature and discuss the molecular mechanisms underlying joint formation and articular development.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Regulação da Expressão Gênica , Cápsula Articular/metabolismo , Via de Sinalização Wnt , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular , Condrócitos/citologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Cápsula Articular/citologia , Cápsula Articular/crescimento & desenvolvimento , Osteogênese/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
4.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31072004

RESUMO

Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.


Assuntos
Família 26 do Citocromo P450/genética , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Ácido Retinoico 4 Hidroxilase/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Embrião de Mamíferos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Organogênese/genética , Transdução de Sinais/genética , Dente/crescimento & desenvolvimento , Dente/metabolismo , Tretinoína/metabolismo
5.
Anticancer Res ; 39(5): 2351-2360, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092427

RESUMO

BACKGROUND/AIM: Hepatic hemangiomas (HH) can show an aggressive course with significant complications. Prognostic markers that identify an aggressive course are entirely absent. Since we have showed that Hedgehog signaling is overexpressed in aggressive hemangiomas of the skin. Here, we hypothesize that it is also altered in aggressive HH. MATERIALS AND METHODS: Immunohistological staining for GLUT1 and quantitative PCR was performed in seven specimens with aggressive HH. For comparison, we included specimens of kaposiform hemangioendothelioma (KHE), skin hemangioma and normal liver tissue. RESULTS: Overexpression of the Hedgehog signaling components SHH and GLI2 and its target gene FOXA2 in HH were similar to those found in aggressive skin hemangioma and KHE, their expression being significantly higher than in mild skin hemangioma. High expression levels of SHH and FOXA2 positively correlated with HH, but not with normal liver tissue. CONCLUSION: Hedgehog signaling is up-regulated in aggressive HH. This finding may lead to a biomarker allowing early intervention.


Assuntos
Proteínas Hedgehog/genética , Hemangioma/genética , Fator 3-beta Nuclear de Hepatócito/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Proteína Gli2 com Dedos de Zinco/genética , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Transportador de Glucose Tipo 1/genética , Hemangioendotelioma/genética , Hemangioendotelioma/patologia , Hemangioma/patologia , Humanos , Lactente , Recém-Nascido , Síndrome de Kasabach-Merritt/genética , Síndrome de Kasabach-Merritt/patologia , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/patologia , Transdução de Sinais/genética
6.
Hum Genet ; 138(7): 703-713, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139930

RESUMO

Neural tube defects (NTD) result from complex mechanisms between genes, nutrition and environment. The identification of genetic predictors by genome exome sequencing and their influence on genome methylation need further consideration. Gene variants related to 1-CM metabolism (1-CM) could influence the methylation of genes involved in neural tube embryogenesis through impaired synthesis of S-adenosyl methionine. We performed exome sequencing of 6116 genes referenced in OMIM and NTD risk and genome-wide methylation in 23 NTD cases. We replicated the most significant associations in 81 other cases. The analysis of exome sequencing identified one gene of 1-CM, LRP2, and one gene of Sonic Hedgehog (SHH), GLI3, in the 23 NTD cases. The analysis restricted to genes of 1-CM and neural tube embryogenesis identified five gene predictors of 1-CM (LRP2, rs137983840; MMAA, rs148142853; TCN2, rs35838082; FPGS, rs41306702; BHMT, rs763726268) and two of SHH (GLI3, rs35364414; MKS1, rs151023718). We replicated the association of TCN2, BHMT and GLI3 with NTD risk in the 81 cases. We found a significant hemimethylation of CFAP46 that may influence SHH activation in one case, who carried risk alleles in BHMT, LRP2, MMAA and GLI3. In conclusion, we identified new candidate genes and rare variants that highlight an interacting influence of genes involved in SHH and 1-CM in the puzzle of genetic components of NTD risk.


Assuntos
Biomarcadores/metabolismo , Carbono/metabolismo , Exoma , Proteínas Hedgehog/genética , Defeitos do Tubo Neural/genética , Vitamina B 12/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Humanos , Masculino , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Transdução de Sinais , Sequenciamento Completo do Exoma , Adulto Jovem
7.
Cell Mol Life Sci ; 76(19): 3875-3889, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30980110

RESUMO

Re-directing mesenchymal stromal cell (MSC) chondrogenesis towards a non-hypertrophic articular chondrocyte-(AC)-like phenotype is important for improving articular cartilage neogenesis to enhance clinical cartilage repair strategies. This study is the first to demonstrate that high levels of non-canonical WNT5A followed by WNT11 and LEF1 discriminated MSC chondrogenesis from AC re-differentiation. Moreover, ß-catenin seemed incompletely silenced in differentiating MSCs, which altogether suggested a role for WNT signaling in hypertrophic MSC differentiation. WNT inhibition with the small molecule IWP-2 supported MSC chondrogenesis according to elevated proteoglycan deposition and reduced the characteristic upregulation of BMP4, BMP7 and their target ID1, as well as IHH and its target GLI1 observed during endochondral differentiation. Along with the pro-hypertrophic transcription factor MEF2C, multiple hypertrophic downstream targets including IBSP and alkaline phosphatase activity were reduced by IWP-2, demonstrating that WNT activity drives BMP and hedgehog upregulation, and MSC hypertrophy. WNT inhibition almost matched the strong anti-hypertrophic capacity of pulsed parathyroid hormone-related protein application, and both outperformed suppression of BMP signaling with dorsomorphin, which also reduced cartilage matrix deposition. Yet, hypertrophic marker expression under IWP-2 remained above AC level, and in vivo mineralization and ectopic bone formation were reduced but not eliminated. Overall, the strong anti-hypertrophic effects of IWP-2 involved inhibition but not silencing of pro-hypertrophic BMP and IHH pathways, and more advanced silencing of WNT activity as well as combined application of IHH or BMP antagonists should next be considered to install articular cartilage neogenesis from human MSCs.


Assuntos
Condrogênese , Células-Tronco Mesenquimais/fisiologia , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomineralização/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Regulação da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Hipertrofia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos SCID , Pessoa de Meia-Idade , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo , Adulto Jovem
8.
Molecules ; 24(8)2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018591

RESUMO

Sonic hedgehog (Shh) signaling plays a tumor-promoting role in many epithelial cancers. Cancer cells produce soluble a Shh that signals to distant stromal cells that express the receptor Patched (Ptc). These receiving cells respond by producing other soluble factors that promote cancer cell growth, generating a positive feedback loop. To interfere with reinforced Shh signaling, we examined the potential of defined heparin and heparan sulfate (HS) polysaccharides to block Shh solubilization and Ptc receptor binding. We confirm in vitro and in vivo that proteolytic cleavage of the N-terminal Cardin-Weintraub (CW) amino acid motif is a prerequisite for Shh solubilization and function. Consistent with the established binding of soluble heparin or HS to the Shh CW target motif, both polysaccharides impaired proteolytic Shh processing and release from source cells. We also show that HS and heparin bind to, and block, another set of basic amino acids required for unimpaired Shh binding to Ptc receptors on receiving cells. Both modes of Shh activity downregulation depend more on HS size and overall charge than on specific HS sulfation modifications. We conclude that heparin oligosaccharide interference in the physiological roles of HS in Shh release and reception may be used to expand the field of investigation to pharmaceutical intervention of tumor-promoting Shh functions.


Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas Hedgehog/química , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Receptor Patched-1/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular Tumoral , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Heparina/química , Heparitina Sulfato/química , Humanos , Modelos Moleculares , Receptor Patched-1/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Solubilidade , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
9.
Nat Commun ; 10(1): 1929, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028265

RESUMO

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.


Assuntos
Células Endoteliais/patologia , Proteínas Hedgehog/genética , Prolapso da Valva Mitral/patologia , Valva Mitral/patologia , Células-Tronco Pluripotentes/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos , Endocárdio/metabolismo , Endocárdio/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Transcrição GATA5/genética , Fator de Transcrição GATA5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Valva Mitral/metabolismo , Prolapso da Valva Mitral/genética , Prolapso da Valva Mitral/metabolismo , Prolapso da Valva Mitral/terapia , Modelos Biológicos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Cultura Primária de Células , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteína Wnt3A/farmacologia
10.
J Appl Genet ; 60(2): 199-207, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30997664

RESUMO

Sonic Hedgehog (Shh) regulates many key developmental processes during vertebrate limb development, fat formation, and skeletal tissue regeneration. Current whole genome sequencing data have identified a copy number variation mapping to bovine Sonic Hedgehog gene (SHH-CNV). The object of this study was to characterize the SHH-CNV distributions in 648 individuals from 11 Chinese cattle populations and further to investigate the associations of the copy number changes with gene expression and cattle growth traits. The SHH-CNV showed a high variance within Chinese indigenous yellow cattle. Compared to yak and dairy cattle, the beef cattle like Luxi and Xianan breed had significantly higher median copy numbers, suggesting the diversity of SHH-CNV in beef cattle selections. The negative correlation of SHH-CNV with SHH transcriptional level in adult adipose tissue (P < 0.01) indicated the dosage effects of SHH-CNV related to bovine fat formation. Association analysis of SHH-CNV and body size traits was conducted in five breeds. The results revealed that the copy number gain type of SHH-CNV exhibited significantly better chest depth in 24 months old Qinchuan cattle, and better body weight, body length, and chest girth in 18 months old Nanyang cattle, whereas the normal copy number had superior chest girth and body weight in adult Jinnan cattle (P < 0.05 or P < 0.01). In summary, this research uncovered meaningful effects of SHH-CNV on gene expression and cattle phenotypic traits, indicating its potential applications for genetic improvement of beef cattle.


Assuntos
Cruzamento , Variações do Número de Cópias de DNA/genética , Proteínas Hedgehog/genética , Locos de Características Quantitativas/genética , Animais , Composição Corporal/genética , Tamanho Corporal , Peso Corporal/genética , Bovinos , China , Regulação da Expressão Gênica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Carne Vermelha
11.
J Mol Histol ; 50(3): 217-227, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30895425

RESUMO

Sjögren's syndrome or radiotherapy for head and neck cancer leads to the irreversible hypofunction of salivary gland (SG). The stem/progenitor cell-based regenerative strategy has been proven to be the most promising approach to repair the function of SG. The molecular mechanisms that regulate SG morphogenesis, especially during lumen formation, provide valuable hints for establishment of such regenerative strategies. It has been demonstrated that numerous growth factors particularly belonging to SHH, BMP, and FGF signaling pathway are involved in the regulation of lumen formation and have shown protective effects on the SG from irradiation in mouse models. However, it remains elusive whether the expression pattern and function of these signaling molecules are conserved in humans. In this study, we examined the expression patterns of the molecules critical for SHH, BMP, and FGF signaling cascades from the canalicular stage to the terminal bud stage, the key stages for lumen formation, in human SG and compared them with the expression data observed in mice. Our results manifested that genes involved in SHH signaling pathway showed identical expression patterns, while genes involved in BMP as well as FGF pathway exhibited similar but distinct expression patterns in humans to those in the mouse. We concluded that the expression patterns of genes involved in SHH, BMP, and FGF pathways in the development of human SG exhibit high similarity to that in the development of mouse SG during lumen formation, suggesting that the molecular mechanism regulating the morphogenesis of SG during lumen formation may be conserved in mice and humans. Our results will have an implication in the future establishment of stem-cell based approaches for the repair of SG function.


Assuntos
Fatores de Crescimento de Fibroblastos/genética , Proteínas Hedgehog/genética , Morfogênese/genética , Glândulas Salivares/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Hibridização In Situ , Camundongos , Glândulas Salivares/crescimento & desenvolvimento , Transdução de Sinais/genética , Células-Tronco/metabolismo
12.
Methods Mol Biol ; 1922: 151-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838573

RESUMO

Whole-mount in situ hybridization (WMISH) is a commonly used technique for visualizing the expression profile of mRNAs in embryos. Unlike traditional in situ hybridization techniques, which require thin tissue sections, the WMISH technique allows gene expression patterns to be assessed over the entire embryo and structure. Here, we describe the detailed procedural steps of WMISH, including probe production, embryo fixation and staining, and post-hybridization signal detection. Using this protocol, we visualized highly specific expression patterns of Sonic hedgehog and Bmp4 mRNAs in E12.5 mouse embryos.


Assuntos
Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , RNA Mensageiro/análise , Animais , Proteína Morfogenética Óssea 4/genética , Digoxigenina , Desenvolvimento Embrionário/genética , Proteínas Hedgehog/genética , Camundongos , Sondas RNA
13.
Int J Mol Sci ; 20(6)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884865

RESUMO

The Hedgehog (Hh) pathway has regulatory roles in maintaining and restoring lingual taste organs, the papillae and taste buds, and taste sensation. Taste buds and taste nerve responses are eliminated if Hh signaling is genetically suppressed or pharmacologically inhibited, but regeneration can occur if signaling is reactivated within the lingual epithelium. Whereas Hh pathway disruption alters taste sensation, tactile and cold responses remain intact, indicating that Hh signaling is modality-specific in regulation of tongue sensation. However, although Hh regulation is essential in taste, the basic biology of pathway controls is not fully understood. With recent demonstrations that sonic hedgehog (Shh) is within both taste buds and the innervating ganglion neurons/nerve fibers, it is compelling to consider Hh signaling throughout the tongue and taste organ cell and tissue compartments. Distinctive signaling centers and niches are reviewed in taste papilla epithelium, taste buds, basal lamina, fibroblasts and lamellipodia, lingual nerves, and sensory ganglia. Several new roles for the innervation in lingual Hh signaling are proposed. Hh signaling within the lingual epithelium and an intact innervation each is necessary, but only together are sufficient to sustain and restore taste buds. Importantly, patients who use Hh pathway inhibiting drugs confront an altered chemosensory world with loss of taste buds and taste responses, intact lingual touch and cold sensation, and taste recovery after drug discontinuation.


Assuntos
Epitélio/metabolismo , Proteínas Hedgehog/genética , Percepção Gustatória/genética , Paladar/genética , Proteínas Hedgehog/metabolismo , Humanos , Sensação/genética , Sensação/fisiologia , Transdução de Sinais/genética , Células Estromais/metabolismo , Paladar/fisiologia , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologia , Percepção Gustatória/fisiologia , Língua/inervação , Língua/fisiologia
14.
Curr Top Dev Biol ; 132: 417-450, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30797516

RESUMO

Distinct classes of neurons arise at different positions along the dorsal-ventral axis of the spinal cord leading to spinal neurons being segregated along this axis according to their physiological properties and functions. Thus, the neurons associated with motor control are generally located in, or adjacent to, the ventral horn whereas the interneurons (INs) that mediate sensory activities are present within the dorsal horn. Here, we review classic and recent studies examining the developmental mechanisms that establish the dorsal-ventral axis in the embryonic spinal cord. Intriguingly, while the cellular organization of the dorsal and ventral halves of the spinal cord looks superficially similar during early development, the underlying molecular mechanisms that establish dorsal vs ventral patterning are markedly distinct. For example, the ventral spinal cord is patterned by the actions of a single growth factor, sonic hedgehog (Shh) acting as a morphogen, i.e., concentration-dependent signal. Recent studies have shed light on the mechanisms by which the spatial and temporal gradient of Shh is transduced by cells to elicit the generation of different classes of ventral INs, and motor neurons (MNs). In contrast, the dorsal spinal cord is patterned by the action of multiple factors, most notably by members of the bone morphogenetic protein (BMP) and Wnt families. While less is known about dorsal patterning, recent studies have suggested that the BMPs do not act as morphogens to specify dorsal IN identities as previously proposed, rather each BMP has signal-specific activities. Finally, we consider the promise that elucidation of these mechanisms holds for neural repair.


Assuntos
Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/metabolismo , Medula Espinal/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Interneurônios/citologia , Interneurônios/metabolismo , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Neurônios/citologia , Transdução de Sinais/genética , Medula Espinal/citologia , Medula Espinal/embriologia , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
15.
Int J Oncol ; 54(4): 1306-1316, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720134

RESUMO

Trans­3,4',5­trihydroxystilbene (resveratrol) is a naturally occurring polyphenolic phytoalexin with marked anticancer activities, and is mainly found in grapes, berries and peanuts. However, due to a low bioavailability, it has not progressed to clinical practice for cancer treatment. Therefore, the aims of the present study were to examine the anticancer activities of the resveratrol derivative, triacetyl resveratrol (TCRV), in pancreatic cancer cells. Apoptosis was measured by fluorescence­activated cell sorting and terminal deoxynucleotidyl transferase (TdT)­mediated dUTP nick­end labeling assays. Gene expression was measured by reverse transcription­quantitative polymerase chain reaction. TCRV inhibited colony formation and induced apoptosis through caspase­3 activation in human pancreatic cancer AsPC­1 and PANC­1 cells, whereas it exerted no effect on human pancreatic normal ductal epithelial cells (HPNE). TCRV inhibited epithelial­mesenchymal transition (EMT) by upregulating the expression of E­cadherin and suppressing the expression of N­cadherin and the transcription factors, Snail, Slug and Zeb1. TCRV inhibited Zeb1 3'UTR­luciferase activity through the upregulation of microRNA (miR)­200 family members. The inhibitory effects of TCRV on pancreatic cancer cell migration and invasion were counteracted by anti­miR­200 family members. The inhibitory effects of TCRV on EMT and the induction of apoptosis were exerted through the suppression of the sonic hedgehog (Shh) pathway, and through the modulation of cyclin D1 and Bcl­2 expression. The hyperactivation of the Shh pathway by either Shh protein or Gli1 overexpression abrogated the biological effects of TCRV. Taken together, the results of this study demonstrate that TCRV inhibits pancreatic cancer growth and EMT by targeting the Shh pathway and its downstream signaling mediators. TCRV inhibited EMT through the upregulation of miR­200 family members. Since TCRV effectively inhibited the growth of human pancreatic cancer cells by modulating the Shh pathway, without affecting the growth of HPNE cells, our findings suggest the possible use of TCRV as a promising candidate for the treatment and/or prevention of pancreatic cancer.


Assuntos
MicroRNAs/genética , Neoplasias Pancreáticas/genética , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Resveratrol/análogos & derivados
16.
Clin Sci (Lond) ; 133(3): 483-495, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30705106

RESUMO

Activation of hedgehog (Hh) signaling contributes to the progression of Barrett's esophagus (BE), which increases the risk of esophageal adenocarcinoma. Recent clinical studies revealed that proton-pump inhibitors (PPIs) but not H2 receptor antagonists (H2RAs) were associated with a decreased risk of esophageal adenocarcinoma. We would like to know whether PPIs interfere with BE progression during BE treatment. Here, we explored the role of omeprazole on Hh signaling and expression of two crucial biomarkers of BE, SOX9 and CDX2. We demonstrated that bile acids elevated expression of Hh pathway target genes, such as GLI1 and PTCH1, and induced SOX9 and CDX2 up-regulation in both CP-A and CP-B cells. Omeprazole, but not famotidine, down-regulated these genes induced by bile acids. In addition, omeprazole-induced down-regulation of SOX9 and CDX2 was mediated by Hh signaling. To explore the mechanisms by which omeprazole inhibits Hh signaling, we performed luciferase assay but did not find any effects of omeprazole on the activity of GLI1 promoter, the critical transcription factor of Hh signaling. Therefore, we used miRNA sequencing and a bioinformatics tool in our study to identify the differently expressed miRNAs in BE organoids treated with or without omeprazole, and we identified miR-2116-3p was involved in omeprazole-mediated inhibition of Hh signaling and subsequent down-regulation of SOX9 and CDX2. Collectively, our data indicate omeprazole inhibits Hh signaling and subsequent SOX9 and CDX2 expression via up-regulating miR-2116-3p. We have demonstrated a novel acid-independent mechanism of omeprazole that might yield valuable insight into clinical management of BE progression, irrespective of acid reflux symptoms.


Assuntos
Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Esôfago de Barrett/metabolismo , Fator de Transcrição CDX2/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas Hedgehog/metabolismo , Omeprazol/farmacologia , Fatores de Transcrição SOX9/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Esôfago de Barrett/tratamento farmacológico , Esôfago de Barrett/genética , Fator de Transcrição CDX2/genética , Linhagem Celular Tumoral , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Fatores de Transcrição SOX9/genética , Transdução de Sinais/efeitos dos fármacos
17.
Mol Genet Genomic Med ; 7(4): e00569, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30729724

RESUMO

BACKGROUND: Mutations in mediator of RNA polymerase II transcription subunit 12 homolog (MED12, OMIM 300188) cause X-linked intellectual disability (XLID) disorders including FG, Lujan, and Ohdo syndromes. The Gli3-dependent Sonic Hedgehog (SHH) signaling pathway has been implicated in the original FG syndrome and Lujan syndrome. How are SHH-signaling defects related to the complex clinical phenotype of MED12-associated XLID syndromes are not fully understood. METHODS: Quantitative RT-PCR was used to study expression levels of three SHH-signaling genes in lymophoblast cell lines carrying four MED12 mutations from four unrelated XLID families. Genotype and phenotype correlation studies were performed on these mutations. RESULTS: Three newly identified and one novel MED12 mutations in six affected males from four unrelated XLID families were studied. Three mutations (c.2692A>G; p.N898D, c.3640C>T; p.R1214C, and c.3884G>A; p.R1295H) are located in the LS domain and one (c.617G>A; p.R206Q) is in the L domain of MED12. These mutations involve highly conserved amino acid residues and segregate with ID and related congenital malformations in respective probands families. Patients with the LS-domain mutations share many features of FG syndrome and some features of Lujan syndrome. The patient with the L-domain mutation presented with ID and predominant neuropsychiatric features but little dysmorphic features of either FG or Lujan syndrome. Transcript levels of three Gli3-dependent SHH-signaling genes, CREB5, BMP4, and NEUROG2, were determined by quantitative RT-PCR and found to be significantly elevated in lymphoblasts from patients with three mutations in the MED12-LS domain. CONCLUSIONS: These results support a critical role of MED12 in regulating Gli3-dependent SHH signaling and in developing ID and related congenital malformations in XLID syndromes. Differences in the expression profile of SHH-signaling genes potentially contribute to variability in clinical phenotypes in patients with MED12-related XLID disorders.


Assuntos
Anormalidades Craniofaciais/genética , Complexo Mediador/genética , Retardo Mental Ligado ao Cromossomo X/genética , Mutação de Sentido Incorreto , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Anormalidades Craniofaciais/patologia , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/genética , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Complexo Mediador/química , Complexo Mediador/metabolismo , Retardo Mental Ligado ao Cromossomo X/patologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Domínios Proteicos , Transdução de Sinais
18.
J Biol Chem ; 294(14): 5549-5561, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30782847

RESUMO

In Sonic hedgehog (SHH) signaling, GLI family zinc finger (GLI)-mediated diverse gene transcription outcomes are strictly regulated and are important for SHH function in both development and disease. However, how the GLI factors differentially regulate transcription in response to variable SHH activities is incompletely understood. Here, using a newly generated, tagged Gli3 knock-in mouse (Gli3TAP ), we performed proteomic analyses and identified the chromatin-associated SAFB-like transcription modulator (SLTM) as a GLI-interacting protein that context-dependently regulates GLI activities. Using immunoprecipitation and immunoblotting, RT-quantitative PCR, and ChIP assays, we show that SLTM interacts with all three GLI proteins and that its cellular levels are regulated by SHH. We also found that SLTM enhances GLI3 binding to chromatin and increases GLI3 repressor (GLI3R) form protein levels. In a GLI3-dependent manner, SLTM promoted the formation of a repressive chromatin environment and functioned as a GLI3 co-repressor. In the absence of GLI3 or in the presence of low GLI3 levels, SLTM co-activated GLI activator (GLIA)-mediated target gene activation and cell differentiation. Moreover, in vivo Sltm deletion generated through CRISPR/Cas9-mediated gene editing caused perinatal lethality and SHH-related abnormal ventral neural tube phenotypes. We conclude that SLTM regulates GLI factor binding to chromatin and contributes to the transcriptional outcomes of SHH signaling via a novel molecular mechanism.


Assuntos
Proteínas Hedgehog/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Transdução de Sinais , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Sistemas CRISPR-Cas , Cromatina , Edição de Genes , Técnicas de Introdução de Genes , Proteínas Hedgehog/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteômica , Proteína Gli3 com Dedos de Zinco/genética
19.
Toxicol Appl Pharmacol ; 365: 1-8, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30610878

RESUMO

Lung cancer is a leading lethal disease with a 5-year survival rate of only 16%. Inadequate potent anti-cancer drugs appear to be a bottleneck in the treatment of lung cancer; hence, how to develop effective anti-lung cancer therapeutics is an urgent problem. In this study, we aim to explore a novel compound with potent anti-lung cancer effect and study its anti-cancer mechanisms. We found that triptonide at very low concentrations of 5-10 nM caused a marked suppression of cell proliferation and colony formation of lung cancer cells. More interestingly, triptonide also robustly inhibited the lung cancer cell formation of tumor spheres, and reduced the stemness and tumorigenicity of the sphere-forming cells. In vivo studies showed that administration of triptonide significantly inhibited the tumor growth with low toxicity. Molecular mechanistic studies revealed that triptonide significantly decreased expression of the Gli1 at both mRNA and protein levels by repressing Gli1 gene promoter activity. Additionally, triptonide reduced the levels of cancer stem cell key signaling protein sonic hedgehog (Shh), but increased the amount of Ptch1, a protein binding to SMO to diminish the Shh signal transduction, thus inhibition of the Shh-Gli1 signaling pathway. Together, our findings show that triptonide effectively inhibits lung cancer cell growth, stemness, and tumorigenicity, and support the notion that triptonide is a new Shh-Gli1 signaling inhibitor and a novel anti-lung cancer drug candidate for further developing effective lung cancer therapeutics.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Células A549 , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Proteínas Hedgehog/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor Patched-1/metabolismo , Fenótipo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/genética
20.
PLoS One ; 14(1): e0211333, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30695055

RESUMO

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.


Assuntos
Neoplasias Ósseas/genética , Condrossarcoma/genética , Perfilação da Expressão Gênica/métodos , Proteínas Nucleares/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Animais , Neoplasias Ósseas/metabolismo , Condrossarcoma/metabolismo , Imunoprecipitação da Cromatina , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Camundongos , Proteínas Nucleares/química , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína GLI1 em Dedos de Zinco/química , Proteína Gli2 com Dedos de Zinco/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA