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1.
Gene ; 762: 145044, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32777528

RESUMO

OBJECTIVE: Gastric cancer is the most common malignant tumor. Most patients suffering from gastric cancer die of metastasis. The role of Atrial natriuretic peptide (ANP) in inhibiting and eliminating kinds of cancer cells has been reported. Aberrant activation of Hedgehog (Hh) signaling pathway contributes to initiation and progression of various malignancies. We have previously reported that the inhibitor of Hh, cyclopamine, reduces the metastatic activity of MGC-803 via inhibiting the expression of matrix metalloproteinases (MMP)-9. It remains to be further demonstrated that ANP has the suppressive effects on invasion and metastasis in gastric cancer via Hh-mediated MMP-9 production. METHODS: Transwell, western blot, qRT-PCR were used after application of ANP on MGC-803 gastric cancer cells to determine the levels of cell migration and invasion, protein levels of MMP-9 and Hh, as well as mRNAs of MMP-9 and Hh, respectively. RESULTS: It was demonstrated that the migration and invasion were significantly lower, MMP-9 and Hh as well as their mRNAs were lower as well, in ANP-treated MGC-803 gastric cancer cells than those in control. CONCLUSIONS: The expression of MMP-9 induced by aberrant activation of Hh in MGC-803 was inhibited by ANP, which may contribute to the inhibition of cell migration and invasion. These results suggested the potential of ANP to be used in gastric cancer therapy as an inhibitor targetting Hh signaling pathway to inhibit the proliferation as well as invasion and metastasis of gastric cancer.


Assuntos
Fator Natriurético Atrial/farmacologia , Proteínas Hedgehog/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Metaloproteinase 9 da Matriz/genética , Transdução de Sinais/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 117(33): 20127-20138, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747535

RESUMO

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.


Assuntos
Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Células-Tronco Neurais/fisiologia , Anilidas/farmacologia , Animais , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Galectina 1/genética , Galectina 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Camundongos , Neoplasias Experimentais , Receptor Patched-1/genética , Piridinas/farmacologia
3.
PLoS One ; 15(7): e0225351, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735563

RESUMO

Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.


Assuntos
Barreira Hematorretiniana/metabolismo , Cílios/metabolismo , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/fisiologia , Células Endoteliais/citologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Larva/metabolismo , Mutagênese , Vasos Retinianos/citologia , Transdução de Sinais , Alcaloides de Veratrum/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Life Sci ; 257: 118053, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634424

RESUMO

AIMS: Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates in the development of various tissues and organs. However, the effect of Pax9 on atherosclerosis and VSMCs and the underlying mechanisms remain unclear. MAIN METHODS: Western blotting was performed to assess Pax9 expression in atherosclerosis and VSMCs. Pax9 siRNA and overexpression plasmid were constructed to explore the biological function. Cell proliferation assay, phalloidin staining, and Transwell assay, accompanied by the sonic hedgehog (Shh) signaling pathway antagonist, cyclopamine (5 µM) and agonist, SAG (100 nM), were used to evaluate the VSMC phenotype, proliferation, and migration, as well as explore the associated mechanisms. KEY FINDINGS: We first discovered Pax9 to be significantly increased in atherosclerotic mice and platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Pax9 knockdown inhibited the phenotypic transformation, proliferation, and migration of VSMCs, whereas the opposite effect was observed when Pax9 was overexpressed. Next, we established that Shh was activated in PDGF-BB-induced VSMCs. Moreover, Pax9 overexpression further activated Shh and exacerbated the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. These changes were effectively inhibited by treatment with the Shh signaling pathway antagonist. Consistently, Pax9 knockdown down-regulated Shh expression and inhibited the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. Treatment with the Shh signaling pathway agonist prevented these changes. SIGNIFICANCE: Pax9 regulated VSMC phenotypic transformation, proliferation, and migration via Shh, which may represent a novel target for the treatment of atherosclerosis.


Assuntos
Aterosclerose/genética , Proteínas Hedgehog/genética , Miócitos de Músculo Liso/citologia , Fator de Transcrição PAX9/genética , Animais , Aterosclerose/patologia , Becaplermina/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Cicloexilaminas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais/genética , Tiofenos/farmacologia , Alcaloides de Veratrum/farmacologia
5.
PLoS One ; 15(7): e0235922, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673370

RESUMO

We have previously established that epigenetic regulator RING1 and YY1 binding protein (RYBP) is required for the contractility of embryonic stem (ES) cell derived cardiomyocytes (CMCs), suggesting its essential role in contractility. In order to investigate the underlying molecular events of this phenotype, we compared the transcriptomic profile of the wild type and Rybp null mutant ES cells and CMCs differentiated from these cell lines. We identified genes related to ion homeostasis, cell adhesion and sarcomeric organization affected in the Rybp null mutant CMCs, by using hierarchical gene clustering and Gene Ontology analysis. We have also demonstrated that the amount of RYBP is drastically reduced in the terminally differentiated wild type CMCs whilst it is broadly expressed in the early phase of differentiation when progenitors form. We also describe that RYBP is important for the proper expression of key cardiac transcription factors including Mesp1, Shh and Mef2c. These findings identify Rybp as a gene important for both early cardiac gene transcription and consequent sarcomere formation necessary for contractility. Since impairment of sarcomeric function and contractility plays a central role in reduced cardiac pump function leading to heart failures in human, current results might be relevant to the pathophysiology of cardiomyopathies.


Assuntos
Proteínas Repressoras/genética , Sarcômeros/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência
6.
Proc Natl Acad Sci U S A ; 117(32): 19321-19327, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719137

RESUMO

Phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes under different environmental conditions, is critical for the origins and maintenance of biodiversity; however, the genetic mechanisms underlying plasticity as well as how variation in those mechanisms can drive evolutionary change remain poorly understood. Here, we examine the cichlid feeding apparatus, an icon of both prodigious evolutionary divergence and adaptive phenotypic plasticity. We first provide a tissue-level mechanism for plasticity in craniofacial shape by measuring rates of bone deposition within functionally salient elements of the feeding apparatus in fishes forced to employ alternate foraging modes. We show that levels and patterns of phenotypic plasticity are distinct among closely related cichlid species, underscoring the evolutionary potential of this trait. Next, we demonstrate that hedgehog (Hh) signaling, which has been implicated in the evolutionary divergence of cichlid feeding architecture, is associated with environmentally induced rates of bone deposition. Finally, to demonstrate that Hh levels are the cause of the plastic response and not simply the consequence of producing more bone, we use transgenic zebrafish in which Hh levels could be experimentally manipulated under different foraging conditions. Notably, we find that the ability to modulate bone deposition rates in different environments is dampened when Hh levels are reduced, whereas the sensitivity of bone deposition to different mechanical demands increases with elevated Hh levels. These data advance a mechanistic understanding of phenotypic plasticity in the teleost feeding apparatus and in doing so contribute key insights into the origins of adaptive morphological radiations.


Assuntos
Ciclídeos/metabolismo , Proteínas de Peixes/metabolismo , Proteínas Hedgehog/metabolismo , Crânio/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Proteínas de Peixes/genética , Proteínas Hedgehog/genética , Transdução de Sinais , Crânio/metabolismo
7.
Proc Natl Acad Sci U S A ; 117(27): 15712-15723, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561646

RESUMO

The mechanisms used by embryos to pattern tissues across their axes has fascinated developmental biologists since the founding of embryology. Here, using single-cell technology, we interrogate complex patterning defects and define a Hedgehog (Hh)-fibroblast growth factor (FGF) signaling axis required for anterior mesoderm lineage development during gastrulation. Single-cell transcriptome analysis of Hh-deficient mesoderm revealed selective deficits in anterior mesoderm populations, culminating in defects to anterior embryonic structures, including the pharyngeal arches, heart, and anterior somites. Transcriptional profiling of Hh-deficient mesoderm during gastrulation revealed disruptions to both transcriptional patterning of the mesoderm and FGF signaling for mesoderm migration. Mesoderm-specific Fgf4/Fgf8 double-mutants recapitulated anterior mesoderm defects and Hh-dependent GLI transcription factors modulated enhancers at FGF gene loci. Cellular migration defects during gastrulation induced by Hh pathway antagonism were mitigated by the addition of FGF4 protein. These findings implicate a multicomponent signaling hierarchy activated by Hh ligands from the embryonic node and executed by FGF signals in nascent mesoderm to control anterior mesoderm patterning.


Assuntos
Fator 4 de Crescimento de Fibroblastos/genética , Fator 8 de Crescimento de Fibroblasto/genética , Gastrulação/genética , Proteína GLI1 em Dedos de Zinco/genética , Animais , Padronização Corporal/genética , Linhagem da Célula/genética , Embrião de Galinha , Fatores de Crescimento de Fibroblastos/genética , Gástrula/crescimento & desenvolvimento , Gástrula/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Hedgehog/genética , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Transdução de Sinais/genética , Análise de Célula Única , Transcriptoma/genética
8.
Hum Genet ; 139(11): 1455-1470, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32504121

RESUMO

In humans, pathogenic variants in the DHH gene underlie cases of 46,XY gonadal dysgenesis. DHH is part of the Hedgehog family of proteins, which require extensive processing, including self-cleavage of the precursor for efficient signalling. In our work, we have assessed the effect of several human DHH pathogenic variants involved in recessive complete or partial gonadal dysgenesis, on protein processing and sub-cellular localization. We found that a subset of variants was unable to perform self-cleavage, which correlated albeit not perfectly with an altered subcellular localization of the resulting proteins. For the processing-proficient variants, we used structural modelling tools and molecular dynamic (MD) simulations to predict the potential impact of the variants on protein conformation and/or interaction with partners. Our study contributes to a better understanding of the molecular mechanisms involved in DHH dysfunction leading to 46,XY disorders of sex development.


Assuntos
Predisposição Genética para Doença/genética , Disgenesia Gonadal 46 XY/genética , Proteínas Hedgehog/genética , Mutação/genética , Linhagem Celular Tumoral , Feminino , Células HeLa , Humanos , Masculino , Simulação de Dinâmica Molecular , Conformação Proteica , Proteólise
9.
PLoS Genet ; 16(6): e1008792, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32579612

RESUMO

While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. We report a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, we identified qualitative and quantitative phenotypes in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, we found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. We further observed disruptions in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. Our findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.


Assuntos
Variações do Número de Cópias de DNA , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Transtornos do Neurodesenvolvimento/genética , Animais , Olho Composto de Artrópodes/embriologia , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Especificidade de Órgãos , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Asas de Animais/embriologia , Asas de Animais/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
10.
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
11.
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
12.
BMC Med Genet ; 21(1): 60, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209048

RESUMO

BACKGROUND: Brachydactyly type A1(BDA-1) is an autosomal dominant disorder which is caused by heterozygous pathogenic variants in a specific region of the N-terminal active fragment of Indian Hedgehog (IHH). The disorder is mainly characterized by shortening or missing of the middle phalanges. In this study, Our purpose is to identify the pathogenic variations associated with BDA-1 involved in a five-generation Chinese family. METHODS: A BDA-1 family with 8 affected and 14 unaffected family members was recruited. Whole exome sequencing (WES) was performed to identify the pathogenic variant in the proband, and which was later confirmed and segregated by Sanger sequencing. The significance of variants were assessed using several molecular and bioinformatics analysis methods. RESULTS: We uncovered a novel heterozygous missense variant c.299A > G (p.D100G) at the mutational hotspot of IHH gene following whole-exome sequencing of a Chinese family with BDA-1. The variant co-segregated with BDA-1 in the pedigree, showed 100% penetrance for phalange phenotype with variable expressivity. CONCLUSIONS: In conclusion, this study reports a five-generation Chinese family with BDA-1 due to a novel pathogenic variant (c.299A > G (p.D100G)) of IHH and expands the clinical and genetic spectrum of BDA-1.


Assuntos
Braquidactilia/genética , Proteínas Hedgehog/genética , Mutação de Sentido Incorreto , Adulto , Substituição de Aminoácidos , Ácido Aspártico/genética , Braquidactilia/diagnóstico , Braquidactilia/patologia , China , Análise Mutacional de DNA , Família , Feminino , Predisposição Genética para Doença , Glicina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma , Adulto Jovem
13.
Artigo em Inglês | MEDLINE | ID: mdl-32130032

RESUMO

We have previously demonstrated that upregulation of Sonic hedgehog (SHH) expression in allergic airway epithelia essentially contributes to the goblet cell metaplasia and mucous hypersecretion. However, the mechanism underlying the upregulation of SHH expression remains completely unknown. In cultured human airway epithelial cells, IL-4/IL-13 but not IL-5 robustly induces the mRNA and protein expression of SHH and in turn activates SHH signaling by promoting the JAK/STAT6-controlling transcription of SHH gene. Moreover, intratracheal instillation of IL-4 and/or IL-13 robustly activates STAT6 and concomitantly upregulates SHH expression in mouse airway epithelia, whereas, in Club cell 10-kDa protein (CC10)-positive airway epithelial cells of children with asthma, activated STAT6 closely correlates with the increased expression of SHH and high activity of SHH signaling. Finally, intratracheal inhibition of STAT6 by AS-1517499 significantly diminished the allergen-induced upregulation of SHH expression, goblet cell phenotypes, and airway hyperresponsiveness, in an ovalbumin- or house dust mite-induced mouse model with allergic airway inflammation,. Together, upregulation of SHH expression by IL-4/IL-13-induced JAK/STAT6 signaling contributes to allergic airway epithelial remodeling, and this study thus provides insight into how morphogen signaling is coordinated with Th2 cytokine pathways to regulate tissue remodeling in chronic airway diseases.


Assuntos
Asma/genética , Proteínas Hedgehog/genética , Interleucina-13/genética , Interleucina-4/genética , Mucosa Respiratória/imunologia , Animais , Antiasmáticos/farmacologia , Asma/induzido quimicamente , Asma/tratamento farmacológico , Asma/patologia , Linhagem Celular , Criança , Feminino , Regulação da Expressão Gênica , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/imunologia , Células Caliciformes/patologia , Proteínas Hedgehog/imunologia , Humanos , Interleucina-13/imunologia , Interleucina-13/farmacologia , Interleucina-4/imunologia , Interleucina-4/farmacologia , Interleucina-5/genética , Interleucina-5/imunologia , Janus Quinases/genética , Janus Quinases/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/administração & dosagem , Cultura Primária de Células , Pirimidinas/farmacologia , Pyroglyphidae/química , Pyroglyphidae/imunologia , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/patologia , Fator de Transcrição STAT6/antagonistas & inibidores , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/imunologia , Transdução de Sinais , Transcrição Genética , Uteroglobina/genética , Uteroglobina/imunologia
14.
Biochemistry ; 59(6): 736-741, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32013401

RESUMO

Hedgehog proteins, a family of vital cell signaling factors, are expressed in precursor form, which requires specialized autoprocessing, called cholesterolysis, for full biological activity. Cholesterolysis occurs in cis through the action of the precursor's C-terminal enzymatic domain, HhC. In this work, we describe HhC activator compounds (HACs), a novel class of noncovalent modulators that induce autoprocessing infidelity, diminishing native cholesterolysis in favor of precursor autoproteolysis, an otherwise minor and apparently nonphysiological side reaction. HAC-induced autoproteolysis generates hedgehog protein that is cholesterol free and hence signaling deficient. The most effective HAC has an AC50 of 9 µM, accelerates HhC autoproteolytic activity by 225-fold, and functions in the presence and absence of cholesterol, the native substrate. HACs join a rare class of "antagonists" that suppress native enzymatic activity by subverting mechanistic fidelity.


Assuntos
Colesterol/biossíntese , Proteínas de Drosophila/biossíntese , Proteínas Hedgehog/biossíntese , Catálise , Colesterol/genética , Proteínas de Drosophila/genética , Variação Genética/fisiologia , Proteínas Hedgehog/genética , Proteólise
15.
PLoS One ; 15(1): e0227780, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945113

RESUMO

The engraftment of human stem cell-derived cardiomyocytes (hSC-CMs) is a promising treatment for remuscularizing the heart wall post-infarction, but it is plagued by low survival of transplanted cells. We hypothesize that this low survival rate is due to continued ischemia within the infarct, and that increasing the vascularization of the scar will ameliorate the ischemia and improve hSC-CM survival and engraftment. An adenovirus expressing the vascular growth factor Sonic Hedgehog (Shh) was injected into the infarcted myocardium of rats immediately after ischemia/reperfusion, four days prior to hSC-CM injection. By two weeks post-cell injection, Shh treatment had successfully increased capillary density outside the scar, but not within the scar. In addition, there was no change in vessel size or percent vascular volume when compared to cell injection alone. Micro-computed tomography revealed that Shh failed to increase the number and size of larger vessels. It also had no effect on graft size or heart function when compared to cell engraftment alone. Our data suggests that, when combined with the engraftment of hSC-CMs, expression of Shh within the infarct scar and surrounding myocardium is unable to increase vascularization of the infarct scar, and it does not improve survival or function of hSC-CM grafts.


Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Adenoviridae/genética , Animais , Diferenciação Celular , Vasos Coronários/diagnóstico por imagem , Modelos Animais de Doenças , Vetores Genéticos/genética , Coração/diagnóstico por imagem , Proteínas Hedgehog/genética , Humanos , Masculino , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/mortalidade , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Traumatismo por Reperfusão/complicações , Taxa de Sobrevida , Transfecção , Resultado do Tratamento , Regulação para Cima , Microtomografia por Raio-X
16.
Nat Commun ; 11(1): 44, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896750

RESUMO

Sclerosing stromal tumor (SST) of the ovary is a rare type of sex cord-stromal tumor (SCST), whose genetic underpinning is currently unknown. Here, using whole-exome, targeted capture and RNA-sequencing, we report recurrent FHL2-GLI2 fusion genes in 65% (17/26) of SSTs and other GLI2 rearrangements in additional 15% (4/26) SSTs, none of which are detected in other types of SCSTs (n = 48) or common cancer types (n = 9,950). The FHL2-GLI2 fusions result in transcriptomic activation of the Sonic Hedgehog (SHH) pathway in SSTs. Expression of the FHL2-GLI2 fusion in vitro leads to the acquisition of phenotypic characteristics of SSTs, increased proliferation, migration and colony formation, and SHH pathway activation. Targeted inhibition of the SHH pathway results in reversal of these oncogenic properties, indicating its role in the pathogenesis of SSTs. Our results demonstrate that the FHL2-GLI2 fusion is likely the oncogenic driver of SSTs, defining a genotypic-phenotypic correlation in ovarian neoplasms.


Assuntos
Proteínas com Homeodomínio LIM/genética , Proteínas Musculares/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias Ovarianas/genética , Tumores do Estroma Gonadal e dos Cordões Sexuais/genética , Fatores de Transcrição/genética , Proteína Gli2 com Dedos de Zinco/genética , Adolescente , Adulto , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Pessoa de Meia-Idade , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Neoplasias Ovarianas/patologia , Esclerose , Tumores do Estroma Gonadal e dos Cordões Sexuais/patologia , Células Estromais/patologia , Sequenciamento Completo do Exoma , Adulto Jovem
17.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936485

RESUMO

Understanding of adipogenesis is important to find remedies for obesity and related disorders. In addition, it is also critical in bone disorders because there is a reciprocal relationship between adipogenesis and osteogenesis in bone micro-environment. Oxysterols are pro-osteogenic and anti-adipogenic molecules via hedgehog activation in pluripotent bone marrow stomal cells. However, no study has evaluated the role of specific oxysterols in C3H10T1/2 cells, which are a good cell model for studying osteogenesis and adipogenesis in bone-marrows. Thus, we investigated the effects of specific oxysterols on adipogenesis and expression of adipogenic transcripts in C3H10T1/2 cells. Treatment of cells with DMITro significantly induced mRNA expression of Pparγ. This induction was significantly inhibited by 25-HC. The expression of C/cepα, Fabp4 and Lpl was also inhibited by 25-HC. To determine the mechanism by which 25-HC inhibits adipogenesis, the effects of the hedgehog signalling pathway inhibitor, cyclopamine and CUR61414, were evaluated. Treatment of C3H10T1/2 cells with DMITro + cyclopamine or DMITro + CUR61414 for 96h did not modulate adipocyte differentiation; cyclopamine and CUR61414 did not reverse the inhibitory effects of 25-HC, suggesting that the canonical hedgehog signalling may not play a role in the anti-adipogenic effects of 25-HC in C3H10T1/2 cells. In addition, LXR agonist did not inhibit adipogenesis, but 25-HC strongly inhibits adipogenesis of C3H10T1/2 cells. Our observations showed that 25-HC was the most potent oxysterol in inhibiting adipogenesis and the expression of key adipogenic transcripts in C3H10T1/2 cells among the tested oxysterols, suggesting its potential application in providing an intervention in osteoporosis and obesity. We also report that the inhibitory effects of 25-HC on adipogenic differentiation in C3H10T1/2 cells are not mediated by hedgehog signaling and LXR.


Assuntos
Adipogenia/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Células-Tronco Pluripotentes/citologia , Adipogenia/genética , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Camundongos , Oxisteróis/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Fatores de Tempo
18.
Genes (Basel) ; 11(1)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31963388

RESUMO

Congenital anomalies in phalluses caused by endocrine disruptors have gained a great deal of attention due to its annual increasing rate in males. However, the endocrine-driven molecular regulatory mechanism of abnormal phallus development is complex and remains largely unknown. Here, we review the direct effect of androgen and oestrogen on molecular regulation in phalluses using the marsupial tammar wallaby, whose phallus differentiation occurs after birth. We summarize and discuss the molecular mechanisms underlying phallus differentiation mediated by sonic hedgehog (SHH) at day 50 pp and phallus elongation mediated by insulin-like growth factor 1 (IGF1) and insulin-like growth factor binding protein 3 (IGFBP3), as well as multiple phallus-regulating genes expressed after day 50 pp. We also identify hormone-responsive long non-coding RNAs (lncRNAs) that are co-expressed with their neighboring coding genes. We show that the activation of SHH and IGF1, mediated by balanced androgen receptor (AR) and estrogen receptor 1 (ESR1) signalling, initiates a complex regulatory network in males to constrain the timing of phallus differentiation and to activate the downstream genes that maintain urethral closure and phallus elongation at later stages.


Assuntos
Macropodidae/crescimento & desenvolvimento , Pênis/crescimento & desenvolvimento , Diferenciação Sexual/efeitos dos fármacos , Androgênios/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Disruptores Endócrinos , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Genitália Feminina , Genitália Masculina/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Macropodidae/metabolismo , Masculino , Pênis/metabolismo , Receptores Androgênicos/metabolismo
19.
Sci Rep ; 10(1): 1244, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988301

RESUMO

We aimed to characterise the response of locally advanced basal cell carcinoma (BCC) to systemic treatment with Vismodegib, a Hedgehog pathway inhibitor, by changes in the expression levels of Hedgehog pathway genes. Data were collected prospectively on 12 patients treated systemically for locally advanced BCC. Biopsy samples taken on admission and after treatment cessation were analysed pathologically and with the NanoString nCounter system to quantify the expression of 40 Hedgehog signaling pathway genes. Findings were compared before and after treatment, between complete and partial responders, and with localised BCC samples from 22 patients. Sixteen Hedgehog pathway genes changed significantly from before to after treatment. GAS1 was the only gene with a significantly different expression at baseline between complete responders (6 patients) and partial responders (4 patients) to Vismodegib (P = 0.014). GAS, GLIS2 and PRKACG1 showed different expression before treatment between the locally advanced and localised BCCs. The baseline expression level of GAS1 appears to be predictive of the response of locally advanced BCC to systemic Vismodegib treatment. A change in expression of many Hedgehog pathway genes, albeit expected by the known activity of Vismodegib, may nevertheless serve as an indicator of the response potential of the tumour.


Assuntos
Anilidas/uso terapêutico , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/genética , Piridinas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Progressão da Doença , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Resultado do Tratamento
20.
BMC Genomics ; 21(1): 22, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910818

RESUMO

BACKGROUND: Early sex differentiation genes of zebrafish remain an unsolved mystery due to the difficulty to distinguish the sex of juvenile zebrafish. However, aromatase inhibitors (AIs) could direct juvenile zebrafish sex differentiation to male and even induce ovary-to-testis reversal in adult zebrafish. RESULTS: In order to determine the transcriptomic changes of sex differentiation in juvenile zebrafish and early sex-reversal in adult zebrafish, we sequenced the transcriptomes of juvenile and adult zebrafish treated with AI exemestane (EM) for 32 days, when juvenile zebrafish sex differentiation finished. EM treatment in females up-regulated the expression of genes involved in estrogen metabolic process, female gamete generation and oogenesis, including gsdf, macf1a and paqr5a, while down-regulated the expression of vitellogenin (vtg) genes, including vtg6, vtg2, vtg4, and vtg7 due to the lower level of Estradiol (E2). Furthermore, EM-juveniles showed up-regulation in genes related to cell death and apoptosis, such as bcl2l16 and anax1c, while the control-juveniles exhibited up-regulation of genes involved in positive regulation of reproductive process and oocyte differentiation such as zar1 and zpcx. Moreover, EM-females showed higher enrichment than control females in genes involved in VEGF signaling pathway, glycosaminoglycan degradation, hedgehog signaling pathway, GnRH signaling pathway and steroid hormone biosynthesis. CONCLUSIONS: Our study shows anti-masculinization in EM-treated adult females but not in EM-treated juveniles. This may be responsible for the lower sex plasticity in adults than juveniles.


Assuntos
Inibidores da Aromatase/farmacologia , Diferenciação Sexual/genética , Vitelogênese/genética , Vitelogeninas/genética , Peixe-Zebra/genética , Androstadienos/farmacologia , Animais , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Hedgehog/genética , Masculino , Reprodução/genética , Diferenciação Sexual/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Vitelogênese/efeitos dos fármacos
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