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1.
Mol Med Rep ; 20(4): 3326-3336, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432193

RESUMO

The aim of the present study was to determine the association between maternal metabolism and development of the fetal palate, and to suggest a potential non­invasive prenatal diagnostic method for fetal cleft palate (CP). Dexamethasone (DXM) was used to create a CP mouse model. A 9.4­Tesla (T) magnetic resonance spectroscopy (MRS) imager was used to measure an array of metabolites in the maternal serum, placental tissue, amniotic fluid and fetal palates. Multivariate statistical analysis was performed using SIMCA­P 14.1 software. Following DXM treatment, variations were detected in multiple metabolites in the female mice and their fetuses based on 9.4T MRS. It was indicated that in the experimental group during CP formation, leucine, valine, creatine, acetate and citrate levels in the palatal tissue were lower, whereas lactate, alanine, proline/inositol and glutamate­containing metabolite levels were higher, compared with the levels in the control group. In placental tissue and amniotic fluid, succinate and choline levels were lower in the experimental group. The relative concentrations of cholesterol and lipids in palatal tissues from mice treated with DXM were higher compared with the concentrations in tissues from mice in the control group, with the exception of (CH2)n lipids. In the placental tissue, the alteration in cholesterol level exhibited the opposite trend. Lipid levels for the different lipid forms varied and most of them were unsaturated lipids.


Assuntos
Fissura Palatina , Dexametasona/efeitos adversos , Embrião de Mamíferos , Desenvolvimento Embrionário/efeitos dos fármacos , Animais , Fissura Palatina/induzido quimicamente , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Dexametasona/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Espectroscopia de Ressonância Magnética , Camundongos
2.
Zhonghua Kou Qiang Yi Xue Za Zhi ; 54(5): 328-334, 2019 May 09.
Artigo em Chinês | MEDLINE | ID: mdl-31091566

RESUMO

Objective: To investigate the expression of gamma-aminobutyric acid type A receptor beta3 subunit (GABRB3) on cleft palate in C57BL/6J mice induced by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Methods: Sixty C57BL/6J pregnant mice on gestation day (GD) 10.5 were divided into two groups: one group was administered through gastric tubes one dose of 28 µg/kg TCDD (experimental group) and the other group was administered through gastric tubes one dose of 5.6 ml/kg corn oil (control group). Embryos were removed by cesarean section from pregnant mice during the palatal formation stage (GD 13.5-17.5) and the palatal tissue studied in morphological and histological observation. The relative mRNA and protein expression of GABRB3 was measured by real-time quantitative PCR and Western blotting. Localization of GABRB3 protein was measured by immunohistochemistry or immunofluorescence. Results: The incidence of cleft palate at GD17.5 was 100% in experimental group and there was no cleft palate occurred in the control group (0); elevation of palatine processes in experimental group was completed on GD15.5 which was clearly delayed by a day compared with that in control group. On GD14.5-GD17.5, the mRNA expression (0.561±0.073, 0.728±0.104, 0.782±0.137, 0.686±0.145) and protein expression (0.288±0.013, 0.404±0.017, 0.399±0.012, 0.307±0.010) in the experimental group were significantly lower than the control group mRNA expression (0.818±0.088, 0.865±0.086, 1.021±0.054, 1.163±0.179) and protein expression (0.481±0.017, 0.456±0.009, 0.474±0.016, 0.529±0.015)(P<0.05). Immunohistochemistry and immunofluorescence showed that GABRB3 was mainly expressed in the mesenchymal cells and medial edge epithelium. Conclusions: TCDD delayed palatal shelf elevation and eventually led to cleft palate may be associated with a decrease in GABRB3. GABRB3 may play an important role in the elevation and fusion phases of the palate development.


Assuntos
Fissura Palatina , Dibenzodioxinas Policloradas , Receptores de GABA-A , Animais , Cesárea , Fissura Palatina/induzido quimicamente , Fissura Palatina/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Palato , Dibenzodioxinas Policloradas/toxicidade , Gravidez , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico
3.
Dis Model Mech ; 12(2)2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30760477

RESUMO

Diverse signaling cues and attendant proteins work together during organogenesis, including craniofacial development. Lip and palate formation starts as early as the fourth week of gestation in humans or embryonic day 9.5 in mice. Disruptions in these early events may cause serious consequences, such as orofacial clefts, mainly cleft lip and/or cleft palate. Morphogenetic Wnt signaling, along with other signaling pathways and transcription regulation mechanisms, plays crucial roles during embryonic development, yet the signaling mechanisms and interactions in lip and palate formation and fusion remain poorly understood. Various Wnt signaling and related genes have been associated with orofacial clefts. This Review discusses the role of Wnt signaling and its crosstalk with cell adhesion molecules, transcription factors, epigenetic regulators and other morphogenetic signaling pathways, including the Bmp, Fgf, Tgfß, Shh and retinoic acid pathways, in orofacial clefts in humans and animal models, which may provide a better understanding of these disorders and could be applied towards prevention and treatments.


Assuntos
Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Modelos Animais de Doenças , Via de Sinalização Wnt , Animais , Moléculas de Adesão Celular/metabolismo , Humanos , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt/genética
4.
Int J Mol Sci ; 20(3)2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30708991

RESUMO

Dioxins and related compounds induce morphological abnormalities in developing animals in an aryl hydrocarbon receptor (AhR)-dependent manner. Here we review the studies in which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is used as a prototypical compound to elucidate the pathogenesis of morphological abnormalities. TCDD-induced cleft palate in fetal mice involves a delay in palatogenesis and dissociation of fused palate shelves. TCDD-induced hydronephrosis, once considered to be caused by the anatomical obstruction of the ureter, is now separated into TCDD-induced obstructive and non-obstructive hydronephrosis, which develops during fetal and neonatal periods, respectively. In the latter, a prostaglandin E2 synthesis pathway and urine concentration system are involved. TCDD-induced abnormal development of prostate involves agenesis of the ventral lobe. A suggested mechanism is that AhR activation in the urogenital sinus mesenchyme by TCDD modulates the wingless-type MMTV integration site family (WNT)/ß-catenin signaling cascade to interfere with budding from urogenital sinus epithelium. TCDD exposure to zebrafish embryos induces loss of epicardium progenitor cells and heart malformation. AHR2-dependent downregulation of Sox9b expression in cardiomyocytes is a suggested underlying mechanism. TCDD-induced craniofacial malformation in zebrafish is considered to result from the AHR2-dependent reduction in SRY-box 9b (SOX9b), probably partly via the noncoding RNA slincR, resulting in the underdevelopment of chondrocytes and cartilage.


Assuntos
Fissura Palatina/induzido quimicamente , Hidronefrose/induzido quimicamente , Dibenzodioxinas Policloradas/toxicidade , Próstata/anormalidades , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fissura Palatina/metabolismo , Dioxinas , Embrião não Mamífero/anormalidades , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Humanos , Hidronefrose/metabolismo , Masculino , Camundongos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
5.
Birth Defects Res ; 111(1): 16-25, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30402937

RESUMO

BACKGROUND: GREM1, which encodes Gremlin 1, an antagonist of bone morphogenic proteins with effects on proliferation and apoptosis, has been considered a candidate gene for nonsyndromic cleft lip with or without cleft palate (NSCL±P). In this study, we investigated potential associations of single nucleotide polymorphisms (SNP) in GREM1 and NSCL±P risk in the Brazilian population. Additionally, SNP-SNP interactions of GREM1 with previously reported rs1880646 variant in NTN1 (netrin 1), a gene also responsible for apoptotic phenotypes were verified. METHODS: Applying Taqman allelic discrimination assays, we evaluated the variants rs16969681, rs16969816, rs16969862, and rs1258763 in 325 case-parent trios and in 1,588 isolated samples in a case-control study. Allelic and genotypic analyses, as well as interaction tests assessing gene-environmental factor (GxE) and SNP-SNP interaction with rs1880646 variant in NTN1, were performed based on logistic regression analysis adjusted for the effects of gender and genomic ancestry proportions. RESULTS: The risk alleles of all SNP were undertransmitted in NSCL±P trios, though the case-control analysis confirmed only the association with rs16969862 alleles (OR: 0.78, 95% CI: 0.63-0.96, p = .02). The GxE interaction analysis revealed a significant interaction between maternal environmental contact with agrotoxics and rs16969816 (OR: 0.25, 95% CI: 0.08-0.74, p = .01), and pairwise interaction test with NTN1 rs1880646 yielded significant p values in the 1,000 permutation test for rs16969681, rs16969816, and rs16969862. CONCLUSION: The GREM1 is involved in the etiology of NSCL±P in the Brazilian population and reveal that the interaction between GREM1 and NTN1 may be related with the pathogenesis of this common craniofacial malformation.


Assuntos
Encéfalo/anormalidades , Fenda Labial/genética , Fissura Palatina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Adulto , Alelos , Encéfalo/metabolismo , Brasil/epidemiologia , Estudos de Casos e Controles , Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Família , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Padrões de Herança , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Netrina-1/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco
6.
Biomed Res Int ; 2018: 6254308, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30345304

RESUMO

Objective: We have identified a gene YOD1 encoding deubiquitinating enzyme (DUB) responsible for nonsyndromic cleft lip with or without cleft palate (NSCL/P). We aimed to determine the effects of YOD1 RNA interference (RNAi) on cell proliferation and migration, playing an important role in lip and palate formation, and to clarify whether the mechanisms involved TGF-ß3 signaling associated with NSCL/P. Methods: RNAi was applied to construct vectors expressing YOD1 small interference RNAs (siRNAs). The vectors were transfected into the human oral keratinocytes (HOK) cells. The cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The mRNA levels were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The protein levels were investigated by western blotting. Results: The proliferation of YOD1 siRNA-transfected HOK cells was remarkably inhibited. The migration rate was significantly decreased in the YOD1 siRNA-transfected HOK cells. The TGF-ß3 mRNA and protein levels were decreased significantly by siRNA-mediated knockdown of YOD1. YOD1 RNAi reduced the phosphor-Smad2/3 levels significantly. Conclusions: YOD1 RNAi may inhibit cell proliferation and migration associated with the pathogenesis of NSCL/P through TGF-ß3 signaling. The study indicates a novel role of YOD1 in regulating TGF-ß3 signaling to affect cell proliferation and migration resulting in NSCL/P.


Assuntos
Movimento Celular , Proliferação de Células , Endopeptidases/biossíntese , Queratinócitos/metabolismo , Boca/metabolismo , Interferência de RNA , Transdução de Sinais , Tioléster Hidrolases/biossíntese , Fator de Crescimento Transformador beta3/metabolismo , Fenda Labial/genética , Fenda Labial/metabolismo , Fenda Labial/patologia , Fissura Palatina/genética , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Endopeptidases/genética , Humanos , Queratinócitos/patologia , Boca/patologia , Tioléster Hidrolases/genética , Fator de Crescimento Transformador beta3/genética
7.
Int. j. morphol ; 36(3): 991-996, Sept. 2018. graf
Artigo em Inglês | LILACS | ID: biblio-954220

RESUMO

The failure of fusion of nasal and maxillary processes results in cleft lip and palate (CLP), which is one of the most common birth defects. The morphopathogenesis of this pathology is multifactorial and still largely unclear. The aim of this study was to evaluate the presence of nestin, transcriptor factor SOX3 (Sox3) and homeobox protein DLX-4 (Dlx-4) in complete unilateral (CU) and complete bilateral (CB) CLP affected facial tissue. Oral mucosa tissue samples were obtained from 17 CUCLP and 13 CBCLP patients during surgical cleft correction and 6 unaffected control subjects. Obtained tissue sections were stained with hematoxylin and eosin and by immunohistochemistry for nestin, Sox3 and Dlx-4. The intensity of staining was graded semiquantitatively. Nestin-positive structures were detected in all CUCLP and CBCLP patients' tissue samples, varying from moderate number of nestin-positive structures to numerous. Sox3 immunoreactivity was more prominent in epithelial cells in both patient groups with frequently patchy distribution. Mainly moderate number of Dlx-4-positive cells was observed in most of tissue samples. Statistically significant moderate positive correlation was found between nestin and Sox3 factors in CUCLP patient group (Spearman's rank correlation coefficient = .517, P = .034). Increase of nestinpositive structures suggests its role in the regulation of the remodeling of tissue in both CUCLP and CBCLP affected tissue. Dominance of Sox3 positivity in cleft affected epithelium indicates its possible role in (compensatory) formation of defective oral epithelium of CUCLP and CBCLP patients. The reduced expression of Dlx-4 implicates its limited regulatory role on the craniofacial development in CUCLP and CBCLP affected tissue.


La falla en la fusión de los procesos nasal y maxilar son causante de la fisura labiopalatina (FLP), que es uno de los defectos congénitos más comunes. La morfopatogenia de esta patología es multifactorial y aún poco clara. El objetivo de este estudio fue evaluar la presencia de nestina, el factor transcriptor SOX3 (Sox3) y la proteína homeobox DLX-4 (Dlx-4) en todo el tejido facial afectado por FLP bilateral unilateral (FU) y bilateral completa (FB). Se obtuvieron muestras de tejido de mucosa oral de 17 pacientes FUFLP y 13 FBFLP durante la corrección quirúrgica de la fisura y de 6 sujetos de control no afectados. Las secciones de tejido obtenidas se tiñeron con hematoxilina y eosina y mediante inmunohistoquímica para nestina, Sox3 y Dlx-4. La intensidad de la tinción fue graduada semicuantitativamente. Se detectaron estructuras positivas para nestina en todas las muestras de tejido de pacientes FUFLP y FBFLP, variando desde un número moderado a numerosas estructuras positivas para nestina. La inmunorreactividad de Sox3 fue más prominente en las células epiteliales en ambos grupos de pacientes con distribución frecuentemente irregular. Se observó un número principalmente moderado de células Dlx-4-positivas en la mayoría de las muestras de tejido. Se encontró una correlación positiva moderada estadísticamente significativa entre los factores de nestina y Sox3 en el grupo de pacientes FUFLP (coeficiente de correlación de rangos de Spearman = 0,517, P = 0,034). El aumento de estructuras positivas para nestina sugiere su papel en la regulación de la remodelación de tejido, tanto en tejido afectado por FUFLP como FBFLP. La dominancia de la positividad de Sox3 en el epitelio afectado de la fisura, indica su posible papel en la formación (compensatoria) del epitelio oral defectuoso de pacientes FUFLP y FBFLP. La expresión reducida de Dlx-4 implica su función reguladora limitada en el desarrollo craneofacial en tejido afectado por FUFLP y FBFLP.


Assuntos
Fenda Labial/metabolismo , Fissura Palatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Nestina/metabolismo , Imuno-Histoquímica
8.
Int J Mol Sci ; 19(8)2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071673

RESUMO

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.


Assuntos
Artrogripose/genética , Fissura Palatina/genética , Pé Torto Equinovaro/genética , Mutação da Fase de Leitura , Deformidades Congênitas da Mão/genética , Processamento de RNA/genética , Fator C de Crescimento do Endotélio Vascular/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Artrogripose/metabolismo , Artrogripose/patologia , Pré-Escolar , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Pé Torto Equinovaro/metabolismo , Pé Torto Equinovaro/patologia , Feminino , Deformidades Congênitas da Mão/metabolismo , Deformidades Congênitas da Mão/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Domínios Proteicos , Fator C de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
9.
J Cell Biochem ; 119(12): 9967-9973, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30155966

RESUMO

Epithelial-mesenchymal transformation of the medial edge epithelium is the most crucial process in embryonic palatal fusion. This study aimed to explore the relationship and potential mechanism between enhancer DNA methylation and mRNA expression of histone deacetylase 4 (HDAC4) during palatal fusion induced by maternal exposure to all-trans retinoic acid (ATRA). Pregnant mice were administered ATRA (70 mg/kg) by gavage at embryonic gestation day 10.5 (E10.5) to establish a cleft palate (CP) model in C57BL/6J mice. Control groups were given an equivalent volume of corn oil. Pregnant mice were dissected at E14.5 (n = 6) to obtain embryonic palates. HDAC4 enhancer DNA methylation data were obtained from a previous MethylRAD-seq. Methylation-specific polymerase chain reaction (MSP) and real-time quantitative PCR were used to quantify enhancer methylation and the mRNA expression level of HDAC4. Enhancer DNA methylation at a non-CpG site within the HDAC4 gene was hyper-methylated at E14.5 (P: 0.011, log2 FC:1.67). The MSP results indicated a similar trend, in agreement with the MethylRAD-seq results. The change in the HDAC4 expression level was negatively correlated with its enhancer DNA methylation level, at the non-CpG site, during palatal fusion induced by ATRA. Enhancer DNA methylation of HDAC4 might play an important regulatory role during palatogenesis, especially in embryonic palatal fusion at E 14.5, and may facilitate the development of novel epigenetic biomarkers in the treatment of CP.


Assuntos
Fissura Palatina/genética , Metilação de DNA , Transição Epitelial-Mesenquimal , Histona Desacetilases/genética , Tretinoína/toxicidade , Animais , Fissura Palatina/induzido quimicamente , Fissura Palatina/metabolismo , Modelos Animais de Doenças , Elementos Facilitadores Genéticos , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Exposição Materna , Camundongos , Camundongos Endogâmicos C57BL , Palato , Gravidez , RNA Mensageiro/genética , Tretinoína/administração & dosagem
10.
Cell Reprogram ; 20(4): 215-224, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29989433

RESUMO

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare monogenic disease with autosomal dominant inheritance caused by mutations in the TP63 gene, leading to progressive corneal keratinocyte loss, limbal stem cell deficiency (LSCD), and eventually blindness. Currently, there is no treatment available to cure or slow down the keratinocyte loss. Human oral mucosal epithelial stem cells (hOMESCs), which are a mixed population of keratinocyte precursor stem cells, are used as source of autologous tissue for treatment of bilateral LSCD. However, hOMESCs from EEC patients have a reduced life span due to TP63 mutations and cannot be used for autologous transplantation. Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of autologous limbal stem cell for EEC patients and can be genetically modified by genome editing technologies to correct the disease ex vivo before transplantation. In this study, we describe for the first time the generation of integration-free EEC-hiPSCs from hOMESCs of EEC patients by Sendai virus vector and episomal vector-based reprogramming. The generated hiPSC clones expressed pluripotency markers and were successfully differentiated into derivatives of the three germ layers, as well as toward corneal epithelium. These cells may be used for EEC disease modeling and open perspectives for applications in cell therapy of LSCD.


Assuntos
Biomarcadores/análise , Diferenciação Celular , Fenda Labial/patologia , Fissura Palatina/patologia , Displasia Ectodérmica/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mucosa Bucal/patologia , Células Cultivadas , Fenda Labial/genética , Fenda Labial/metabolismo , Fissura Palatina/genética , Fissura Palatina/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucosa Bucal/metabolismo , Mutação , Fenótipo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética
11.
Physiol Rep ; 6(14): e13728, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30030908

RESUMO

Active reabsorption of magnesium (Mg2+ ) in the distal convoluted tubule (DCT) of the kidney is crucial for maintaining Mg2+ homeostasis. Impaired activity of the Na+ -Cl- -cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg2+ balance. To further elucidate the possible interrelationship between NCC activity and renal Mg2+ handling, plasma Mg2+ levels and urinary excretion of sodium (Na+ ) and Mg2+ were measured in a mouse model of Gordon syndrome. In this model, DCT1-specific expression of a constitutively active mutant form of the NCC-phosphorylating kinase, SPAK (CA-SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)-sensitive Na+ reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg2+ levels in CA-SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA-SPAK mice exhibited greater NCC-dependent Na+ reabsorption together with decreased Mg2+ reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA-SPAK mice exhibited higher urinary Mg2+ excretion, while urinary Na+ excretion decreased to levels observed in control animals. Surprisingly, CA-SPAK mice had unaltered renal expression of Trpm6, encoding the Mg2+ -permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA-SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na+ reabsorption. Thus, Mg2+ reabsorption is not coupled to increased thiazide-sensitive Na+ reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg2+ excretion after prolonged HCTZ administration.


Assuntos
Artrogripose/metabolismo , Fissura Palatina/metabolismo , Pé Torto Equinovaro/metabolismo , Deformidades Congênitas da Mão/metabolismo , Magnésio/metabolismo , Reabsorção Renal , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Hidroclorotiazida/farmacologia , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
12.
Gene ; 666: 1-8, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-29733966

RESUMO

The Chinese sturgeon (Acipenser sinensis) is an anadromous fish distributed in the Yangtze River and the East China Sea. In this study, we report the novel finding of cleft palates in Chinese sturgeons derived from artificial fertilization. To explore the genetic basis of palate malformation in A. sinensis, Illumina RNA-seq technology was used to analyze the transcriptome data of farmed Chinese sturgeons with normal palates and cleft-palates. Raw reads were obtained and assembled into 808,612 unigenes, with an average length of 509.33 bp and an N50 of 574 bp. Sequence similarity analyses against four public databases (Nr, UniProt, KEGG, and COGs) found 158,642 unigenes that could be annotated. GABAergic synapses and TGF-ß signal pathways were the two most enriched pathways with high Rich Factors in the analyses of differentially expressed genes. In these two signal pathways, six genes (GABRA4, GS, GNS, S6K, PITX2, and BMP8) were found as candidate cleft-palate genes in Chinese sturgeon. These findings contribute to our understanding of cleft palate genetics in sturgeon, while simultaneously adding to our knowledge about craniofacial development.


Assuntos
Fissura Palatina/veterinária , Doenças dos Peixes/genética , Peixes/genética , Transcriptoma , Animais , Aquicultura , China , Fissura Palatina/genética , Fissura Palatina/metabolismo , Doenças dos Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Peixes/metabolismo , Perfilação da Expressão Gênica , Anotação de Sequência Molecular
13.
Biochim Biophys Acta Mol Cell Res ; 1865(7): 1002-1011, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29702134

RESUMO

The mammalian Grainyhead-like 3 (GRHL3) transcription factor is essential for epithelial development and plays a protective role against squamous cell carcinoma of the skin and of the oral cavity. A single nucleotide polymorphism (SNP) in GRHL3, rs141193530 (p.P455A), is associated with non-melanoma skin cancer in human patients. Moreover, it is known that this SNP, as well as another variant, rs41268753 (p.T454M), are associated with nonsyndromic cleft palate and that rs41268753 negatively affects GRHL3 transcriptional activity. These SNPs are located in adjacent codons of the GRHL3 gene, and the occurrence of either SNP abolishes a putative threonine-proline phosphorylation motif at T454 in the encoded protein. The role of phosphorylation in regulating mammalian GRHL function is currently unknown. In this work we show that GRHL3 is phosphorylated at several residues in a human keratinocyte cell line, among them at T454. This site is essential for the full transcriptional activity of GRHL3. The T454 residue is phosphorylated by p38 MAPK in vitro and activation of p38 signaling in cells causes an increase in GRHL3 activity. The regulation of GRHL3 function by this pathway is dependent on T454, as the substitution of T454 with methionine inhibits the activation of GRHL3. Taken together, our results show that T454 is one of the phosphorylated residues in GRHL3 in keratinocytes and this residue is important for the upregulation of GRHL3 transcriptional activity by the p38 pathway.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fissura Palatina/genética , Fissura Palatina/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Humanos , Queratinócitos/metabolismo , Fosforilação , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Treonina/análise , Treonina/genética , Fatores de Transcrição/análise , Fatores de Transcrição/genética
14.
Cleft Palate Craniofac J ; 55(8): 1072-1080, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29613838

RESUMO

OBJECTIVE: Knowledge about the stress response in children with cleft lip and/or palate (CL/P) is sparse and the association between the stress response and health-related quality of life (HRQoL) is unknown. Consequently, investigations on the influence of CL/P on the stress response alone and its association with HRQoL are of importance. The purpose was to determine whether salivary cortisol concentration in children with CL/P differs from that in children without clefts (controls) and whether there are any differences in salivary cortisol concentrations between ages, gender, and type of cleft. Furthermore, the final aim was to determine the correlation between salivary cortisol concentration and HRQoL. DESIGN: The study used a cross-sectional case-control design. PARTICIPANTS: Ninety-one 5- and 10-year-old children with CL/P and 180 age-matched controls. MAIN OUTCOME MEASURES: Salivary samples were collected on 2 mornings and 1 evening for each child. Samples were analyzed using a commercial competitive radioimmunoassay and HRQoL was assessed using the KIDSCREEN-52. RESULTS: Salivary cortisol concentrations were similar in children with CL/P and controls. There was no difference in salivary cortisol concentrations between children with different types of cleft. There was no correlation between cortisol concentration and HRQoL. CONCLUSION: Five- and 10-year-old children with corrected CL/P seemed not to be more stressed than controls, and there were no correlation to HRQoL. The HRQoL levels - were comparable to that of a European norm population.


Assuntos
Fenda Labial/metabolismo , Fenda Labial/psicologia , Fissura Palatina/metabolismo , Fissura Palatina/psicologia , Hidrocortisona/metabolismo , Saliva/química , Fatores Etários , Biomarcadores/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Masculino , Qualidade de Vida , Fatores Sexuais , Inquéritos e Questionários
15.
Mol Med Rep ; 17(4): 5396-5401, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29393476

RESUMO

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate formation in mice. This TCDD treatment, which may be considered an environmental factor in cleft palate formation, is associated with alterations in DNA methylation. However, the underlying molecular mechanisms of DNA methylation produced by TCDD in mouse embryos are poorly understood. DNA methyltransferases (DNMTs) and methyl­CpG binding domain proteins (MBDs) are thought to be closely associated with the actions of DNA methylation. Therefore, the present study tested the hypothesis that this cleft palate inducing effect of TCDD will alter the expression levels of DNMTs and various MBDs in palate tissue of fetal mice. Pregnant C57BL/6J mice were treated with either TCDD (64 µg/kg) or corn oil (control) at embryonic day 10.5 (E10.5) and fetal palates were harvested for structural and molecular analyses at E13.5, E14.5, E15.5 and E17.5. Expression levels of DNMTs and MBDs were assayed using reverse transcription­quantitative polymerase chain reaction and western blotting. The incidence of cleft palates in the TCDD group was 98.24%, whereas no cases of cleft palate were observed in the control group. Expression levels of DNMTs and MBDs were significantly increased in the TCDD group compared with the control. The results demonstrate clear alterations in DNMTs and MBDs, as induced by TCDD, and suggest that such alterations are important in cleft palate formation in fetal mice.


Assuntos
Fissura Palatina/etiologia , Fissura Palatina/patologia , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Variação Genética , Dibenzodioxinas Policloradas/efeitos adversos , Animais , Fissura Palatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Gravidez , Regulação para Cima
16.
Hum Exp Toxicol ; 37(2): 196-204, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29233047

RESUMO

RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.


Assuntos
Fissura Palatina/genética , Proteínas de Choque Térmico/genética , Palato Duro/anormalidades , Interferência de RNA , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Fissura Palatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos ICR , Morfogênese , Palato Duro/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos , Resposta a Proteínas não Dobradas , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
17.
Proc Natl Acad Sci U S A ; 114(45): E9520-E9528, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078335

RESUMO

Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Cálcio Tipo L/metabolismo , Animais , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Fissura Palatina/metabolismo , Cristalografia por Raios X/métodos , Acoplamento Excitação-Contração/fisiologia , Humanos , Hipertermia Maligna/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/metabolismo , Mutação/genética , Miotonia Congênita/metabolismo , Isoformas de Proteínas/metabolismo , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Xenopus laevis/metabolismo
18.
J Biol Chem ; 292(44): 18091-18097, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28912269

RESUMO

Glycoprotein A repetitions predominant (GARP) (encoded by the Lrrc32 gene) plays important roles in cell-surface docking and activation of TGFß. However, GARP's role in organ development in mammalian systems is unclear. To determine the function of GARP in vivo, we generated a GARP KO mouse model. Unexpectedly, the GARP KO mice died within 24 h after birth and exhibited defective palatogenesis without apparent abnormalities in other major organs. Furthermore, we observed decreased apoptosis and SMAD2 phosphorylation in the medial edge epithelial cells of the palatal shelf of GARP KO embryos at embryonic day 14.5 (E14.5), indicating a defect in the TGFß signaling pathway in the GARP-null developing palates. Of note, the failure to develop the secondary palate and concurrent reduction of SMAD phosphorylation without other defects in GARP KO mice phenocopied TGFß3 KO mice, although GARP has not been suggested previously to interact with TGFß3. We found that GARP and TGFß3 co-localize in medial edge epithelial cells at E14.5. In vitro studies confirmed that GARP and TGFß3 directly interact and that GARP is indispensable for the surface expression of membrane-associated latent TGFß3. Our findings indicate that GARP is essential for normal morphogenesis of the palate and demonstrate that GARP plays a crucial role in regulating TGFß3 signaling during embryogenesis. In conclusion, we have uncovered a novel function of GARP in positively regulating TGFß3 activation and function.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/metabolismo , Organogênese , Palato/metabolismo , Processamento de Proteína Pós-Traducional , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta3/agonistas , Animais , Animais Recém-Nascidos , Apoptose , Fissura Palatina/embriologia , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Heterozigoto , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Knockout , Palato/anormalidades , Palato/embriologia , Palato/patologia , Fosforilação , Gravidez , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta3/química , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/metabolismo
19.
PLoS One ; 12(9): e0184473, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28934221

RESUMO

BACKGROUND: Platelet-derived growth factor receptor alpha (PDGFRα) is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures. PURPOSE: To address the temporal requirement of Pdgfra in embryonic development. METHODS: We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies. RESULTS: Current study showed that (i) conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5) resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii) the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives. CONCLUSION: Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a) the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b) if mutations / sequence variations of these regulatory elements cause these anomalies.


Assuntos
Desenvolvimento Embrionário/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Parede Abdominal/anormalidades , Parede Abdominal/embriologia , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , Apoptose/fisiologia , Fenda Labial/embriologia , Fenda Labial/genética , Fenda Labial/metabolismo , Fissura Palatina/embriologia , Fissura Palatina/genética , Fissura Palatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Hérnia Umbilical/embriologia , Hérnia Umbilical/genética , Hérnia Umbilical/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Esqueleto/anormalidades , Esqueleto/embriologia , Esqueleto/metabolismo , Disrafismo Espinal/embriologia , Disrafismo Espinal/genética , Disrafismo Espinal/metabolismo , Tamoxifeno , Fatores de Tempo
20.
Mol Med Rep ; 16(5): 5915-5923, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28849085

RESUMO

The current study was designed to elucidate the mechanism of retinol binding protein 4 (RBP4) in cleft palate induced by all­trans retinoic acid (atRA). To establish a cleft palate model in C57BL/6J mice, pregnant mice were administered atRA (100 mg/kg) by gavage at the tenth embryonic stage (E10.0). Control groups were given the equivalent volume of corn oil. Pregnant mice were dissected at E12.5, E13.5 and E14.5 to obtain the embryonic palates. The expression levels of RBP4 in the embryonic palatal mesenchyme (EPM) were determined by immunohistochemistry, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Human embryonic palatal mesenchymal cells were exposed to atRA to detect the variation in RBP4 induced by atRA in vitro. Small interfering RNA was used to suppress the expression of RBP4, and a plasmid overexpressing RBP4 was used to examine upregulated expression. The cell counting kit­8 assay was used to evaluate the effect of RBP4 on cell proliferation. The expression levels of p27 and cyclin D1 were determined by RT­qPCR and western blotting, while the expression levels of extracellular signal­related kinase (ERK) 1/2 and protein kinase B (AKT) were assessed by western blotting. At E14.5, RBP4 was strongly expressed in the EPM, while it was downregulated following atRA treatment, which induced cleft palate in vivo. In vitro experiments indicated that atRA suppressed the expression of RBP4 and altered the expression of p27 and cyclin D1 to cause growth inhibition. Knockdown of RBP4 resulted in decreased expression of cyclin D1 and increased p27, and suppressed proliferation. Overexpression of RBP4 reversed the inhibitory effect of atRA and promoted proliferation via the ERK1/2 and AKT signaling pathways. These results suggested that RBP4 was involved in cleft palate induced by atRA and it can be suppressed by atRA to cause growth inhibition in the embryonic palate.


Assuntos
Fissura Palatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Plasmáticas de Ligação ao Retinol/genética , Tretinoína/farmacologia , Animais , Linhagem Celular , Proliferação de Células , Fissura Palatina/induzido quimicamente , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Óleo de Milho/administração & dosagem , Ciclina D1/genética , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Excipientes/administração & dosagem , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol/antagonistas & inibidores , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Transdução de Sinais
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