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1.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
2.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(4): 472-479, 2020 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-32691553

RESUMO

Objective: To screen the key odontogenic genes in mice and verify the odontogenic inducing effect on amniotic epithelial cells (WISH). Methods: The spatially and temporally different expression of bone morphogenetic proteins 4 (BMP4), fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), lymphoid enhancer factor 1 (LEF1) proteins and their genes expression in the early odontogenesis stage (embryo day 10.5 (E10.5)、E11.5、E14.5) in fetal mice were detected by immunohistochemistry staining and quantitative real-time PCR (RT-qPCR). According to the results, we screened the probable key odontogenic genes. Then adding osteogenic inducing solution to induce non-odontogenic epithelium cells, WISH. After 3 weeks culture of non-odontogenic epithelial WISH for osteogenic induction, the epithelial-mesenchymal transformation cap ability was evaluated by using Alizarin (ALZ) red staining and RT-qPCR on the alkaline phosphatase ( ALP) mRNA expression level. Using germ layer recombination experiment to observe and verify whether the screened genes can induce non-odontogenic epithelium cells acquire odontogenesis ability. The recombined tissue grafts containing key genes were transplanted beneath the renal capsule of mice. Results: The results of immunohistochemistry staining and RT-qPCR showed that on E10.5 BMP4 protein and gene were differently expressed in the first and second branchial arch epithelium, which synchronized the odontogenic capability transferring from epithelium to mesenchyme from E10.5-E14.5. Though the expression of FGF8 protein and gene existed such difference in the first and second branchial arch epithelium, there was no synchronization in transfer. The expression of LEF1 and SHH proteins and genes had neither difference nor synchronization. So far, we considered the BMP4 was the probable key odontogenic gene. Through 3 weeks' osteogenic induction, ALZ red stained positively and calcium nodules were observed in WISH, and the expression level of ALP mRNA increased. In the germ layer recombination experiment, exogenous BMP4 protein enabled the second branchial arch mesenchyme forming tooth-like structures after recombined with the second branchial arch epithelium or WISH. Conclusions: The proteins and genes of BMP4, FGF8, SHH and LEF1 are spatially and temporally differently expressed in the early tooth development stage in mice. The protein and gene of BMP4 are differently expressed between the first and second branchial arch epithelium and enables the non-odontogenic epithelium acquiring odontogenic ability. BMP4 is the possible key odontogenic gene.


Assuntos
Proteína Morfogenética Óssea 4 , Células Epiteliais , Regulação da Expressão Gênica no Desenvolvimento , Odontogênese , Dente , Animais , Proteína Morfogenética Óssea 4/genética , Células Epiteliais/citologia , Mesoderma/metabolismo , Camundongos , Odontogênese/genética , Dente/metabolismo
3.
Nat Cell Biol ; 22(6): 651-662, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393886

RESUMO

BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
4.
Pediatr Pulmonol ; 55(3): 765-770, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31962011

RESUMO

BACKGROUND: Congenital pulmonary airway malformation (CPAM) is a rare disorder characterized by aberrant overgrowth of terminal bronchioles. The objective of this study was to describe wingless-type MMTV integration site family 7B (Wnt7B) and bone morphogenetic protein 4 (BMP4) expression patterns in human CPAM lesions and to explore the possible roles of Wnt7B and BMP4 in the pathogenesis of CPAM. METHODS: Fifteen tissue samples from patients with CPAM were obtained from the Pathology Department of Shengjing Hospital of China Medical University. Samples representing CPAM lesions and adjacent normal lung tissues were collected and Wnt7B and BMP4 expression was detected through immunohistochemical (IHC) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. RESULTS: IHC revealed that Wnt7B immunopositive cells were only detected in epithelial cells, whereas BMP4 immunopositive cells were detected in epithelial and mesenchymal cells. Expression of Wnt7B and BMP4 immunopositive cells was higher in CPAM lesions than that in adjacent normal lung tissue. qRT-PCR and Western blotting showed that Wnt7B and BMP4 mRNA and protein expression were significantly higher in CPAM lesions than in adjacent normal lung tissue (P < .05). Overall, the level of BMP4 was higher than that of Wnt7B. CONCLUSIONS: Increased expression of Wnt7B and BMP4 appear to be related to the pathogenesis of CPAM and abnormal pulmonary development. Upregulation of Wnt7B and BMP4 could play an important role in the development of the bronchial-alveolar structures that characterize CPAM.


Assuntos
Proteína Morfogenética Óssea 4/genética , Malformação Adenomatoide Cística Congênita do Pulmão/genética , Proteínas Wnt/genética , Proteína Morfogenética Óssea 4/metabolismo , Malformação Adenomatoide Cística Congênita do Pulmão/metabolismo , Humanos , Pulmão/metabolismo , Proteínas Wnt/metabolismo
5.
Cancer Res ; 80(6): 1304-1315, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941699

RESUMO

Metastasis is the major cause of death in patients with cancer; with no therapeutic cure, treatments remain largely palliative. As such, new targets and therapeutic strategies are urgently required. Here, we show that bone morphogenetic protein-4 (BMP4) blocks metastasis in animal models of breast cancer and predicts improved survival in patients. In preclinical models of spontaneous metastasis, BMP4 acted as an autocrine mediator to modulate a range of known metastasis-regulating genes, including Smad7, via activation of canonical BMP-SMAD signaling. Restored BMP4 expression or therapeutically administered BMP4 protein, blocked metastasis and increased survival by sensitizing cancer cells to anoikis, thereby reducing the number of circulating tumor cells. Gene silencing of Bmp4 or its downstream mediator Smad7, reversed this phenotype. Administration of recombinant BMP4 markedly reduced spontaneous metastasis to lung and bone. Elevated levels of BMP4 and SMAD7 were prognostic for improved recurrence-free survival and overall survival in patients with breast cancer, indicating the importance of canonical BMP4 signaling in the suppression of metastasis and highlighting new avenues for therapy against metastatic disease. SIGNIFICANCE: Targeting the BMP4-SMAD7 signaling axis presents a novel therapeutic strategy to combat metastatic breast cancer, a disease that has had no reduction in patient mortality over 20 years. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/6/1304/F1.large.jpg.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Neoplasias Ósseas/genética , Neoplasias da Mama/patologia , Proteína Smad7/metabolismo , Animais , Comunicação Autócrina , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/uso terapêutico , Neoplasias Ósseas/prevenção & controle , Neoplasias Ósseas/secundário , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Linhagem Celular Tumoral/transplante , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Animais/patologia , Mastectomia , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Prognóstico , Proteínas Recombinantes/uso terapêutico , Transdução de Sinais/genética , Proteína Smad4/metabolismo , Proteína Smad7/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cancer Lett ; 469: 390-398, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31730904

RESUMO

Metastasis is a major lethal cause of esophageal squamous cell carcinoma (ESCC) and confers a poor prognosis. Previous studies demonstrated that serpin family E member 2 (SERPINE2) is involved in tumor metastasis. However, the function and mechanism of SERPINE2 in ESCC metastasis remains unclear. In this study, we found that SERPINE2 was increased in ESCC and associated with tumor metastasis. SERPINE2 knockdown inhibited tumor cell invasion and lymph node and lung metastasis by inducing epithelial-mesenchymal transition (EMT). We identified a total of 410 differentially expressed genes in SERPINE2-knockdown cells by RNA-Seq analysis. Among them, bone morphogenetic protein 4 (BMP4) was significantly downregulated. Conversely, BMP4 was increased in SERPINE2-overexpressing cells. Inhibiting BMP4 could attenuate SERPINE2-induced migration and invasion. Moreover, SERPINE2 was positively correlated with clinical stage, tumor invasion depth and lymph node metastasis in ESCC patients. These findings suggest that SERPINE2 promotes tumor metastasis by activating BMP4 and could serve as a potential therapeutic target for clinical intervention in ESCC.


Assuntos
Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 4/genética , Carcinoma de Células Escamosas do Esôfago/genética , Serpina E2/genética , Movimento Celular , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica
7.
Turk Neurosurg ; 30(3): 371-376, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31452178

RESUMO

AIM: To investigate the effects of different therapeutic radiation doses on the prevalence of neural tube defects (NTDs) in chick embryos and bone morphogenetic protein (BMP) 4 and BMP7 expression levels. MATERIAL AND METHODS: The chick embryos (n=143) were derived from fertile, specific pathogen-free eggs of domestic fowl. The presence of NTDs was analyzed using a stereomicroscope, and BMP4 and BMP7 expression levels were assessed by immunohistochemical staining. The chick embryos were divided into five groups: control (no radiation exposure) (n=23), exposure to thorax computerized tomography (CT) (n=30); exposure to abdominopelvic CT (n=30), exposure to cranium CT (n=30), and exposure to brain perfusion CT (n=30). RESULTS: The prevalence of NTDs and BMP4 and BMP7 expression levels in the different groups were compared. In the cranium CT dose group, both the NTD prevalence (20%, p=0.002) and BMP7 (p=0.031) expression levels were significantly higher than those in the other groups. However, none of the medical doses of irradiation altered BMP4 expression levels (p=0.242). No NTDs were detected in the thorax CT and abdominopelvic CT groups. CONCLUSION: Exposure to irradiation at cranium CT doses may induce the development of NTDs and increase BMP7 expression. Dose radiation exposure using thorax CT and abdominopelvic CT protocols does not appear to induce NTDs.


Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 7/biossíntese , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/radioterapia , Doses de Radiação , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Embrião de Galinha , Expressão Gênica , Tubo Neural/diagnóstico por imagem , Tubo Neural/efeitos da radiação , Defeitos do Tubo Neural/diagnóstico por imagem , Defeitos do Tubo Neural/genética
8.
Congenit Anom (Kyoto) ; 60(1): 15-21, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30714224

RESUMO

The external genitalia are appendage organs outgrowing from the posterior body trunk. Murine genital tubercle (GT), anlage of external genitalia, initiates its outgrowth from embryonic day (E) 10.5 as a bud structure. Several growth factors such as fibroblast growth factor (FGF), Wnt and Sonic hedgehog (Shh) are essential for the GT outgrowth. However, the mechanisms of initiation of GT outgrowth are poorly understood. We previously identified bone morphogenetic protein (Bmp) signaling as a negative regulator for GT outgrowth. We show here novel aspects of Bmp4 functions for GT outgrowth. We identified the Bmp4 was already expressed in cloaca region at E9.5, before GT outgrowth. To analyze the function of Bmp4 at early stage for the initiation of GT outgrowth, we utilized the Hoxa3-Cre driver and Bmp4 flox/flox mouse lines. Hoxa3 Cre/+ ; Bmp4 flox/flox mutant mice showed the hypoplasia of GT with reduced expression of outgrowth promoting genes such as Wnt5a, Hoxd13 and p63, whereas Shh expression was not affected. Formation of distal urethral epithelium (DUE) marked by the Fgf8 expression is essential for controlling mesenchymal genes expression in GT and subsequent its outgrowth. Furthermore, Fgf8 expression was dramatically reduced in such mutant mice indicating the defective DUE formation. Hence, current results indicate that Bmp4 is an essential growth factor for the initiation of GT outgrowth independent of Shh signaling. Thus, Bmp4 positively regulates for the formation of DUE. The current study provides new insights into the function of Bmp signaling at early stage for the initiation of GT outgrowth.


Assuntos
Proteína Morfogenética Óssea 4/genética , Genitália/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Animais , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genitália/metabolismo , Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Mesoderma/metabolismo , Camundongos , Transdução de Sinais/genética , Fatores de Transcrição/genética
9.
Aging (Albany NY) ; 11(23): 11520-11540, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831718

RESUMO

Liver has numerous critical metabolic functions including lipid metabolism, which is usually dysregulated in obesity, the metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD). Increasing evidence indicates bone morphogenetic proteins (BMPs) play an important role in adipogenesis and thermogenic balance in adipogenic progenitors and adipose tissue. However, the direct impact of BMPs on hepatic steatosis and possible association with NAFLD are poorly understood. Here, we found that BMP4 was up-regulated in oleic acid-induced steatosis and during the development of high fat diet (HFD)-induced NAFLD. Exogenous BMP4 reduced lipid accumulation and up-regulated the genes involved in lipid synthesis, storage and breakdown in hepatocytes. Exogenous BMP4 inhibited hepatic steatosis, reduced serum triglyceride levels and body weight, and alleviated progression of NAFLD in vivo. Mechanistically, BMP4 overexpression in hepatocytes down-regulated most components of the mTORC1 signaling axis. Collectively, these findings strongly suggest that BMP4 may play an essential role in regulating hepatic lipid metabolism and the molecular pathogenesis of NAFLD. Manipulating BMP4 and/or mTORC1 signaling axis may lead to the development of novel therapeutics for obesity, metabolic syndrome, and NAFLD.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Hepatócitos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/farmacologia , Células Cultivadas , Fígado Gorduroso , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Humanos , Metabolismo dos Lipídeos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Regulação para Cima
10.
Dis Model Mech ; 12(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848143

RESUMO

Idiopathic scoliosis (IS) is the most common type of musculoskeletal defect affecting children worldwide, and is classified by age of onset, location and degree of spine curvature. Although rare, IS with onset during infancy is the more severe and rapidly progressive form of the disease, associated with increased mortality due to significant respiratory compromise. The pathophysiology of IS, in particular for infantile IS, remains elusive. Here, we demonstrate the role of PRMT5 in the infantile IS phenotype in mouse. Conditional genetic ablation of PRMT5 in osteochondral progenitors results in impaired terminal hypertrophic chondrocyte differentiation and asymmetric defects of endochondral bone formation in the perinatal spine. Analysis of these several markers of endochondral ossification revealed increased type X collagen (COLX) and Ihh expression, coupled with a dramatic reduction in Mmp13 and RUNX2 expression, in the vertebral growth plate and in regions of the intervertebral disc in the Prmt5 conditional mutant mice. We also demonstrate that PRMT5 has a continuous role in the intervertebral disc and vertebral growth plate in adult mice. Altogether, our results establish PRMT5 as a critical promoter of terminal hypertrophic chondrocyte differentiation and endochondral bone formation during spine development and homeostasis.This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteína Morfogenética Óssea 4/genética , Condrócitos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Escoliose/genética , Alelos , Animais , Proteína Morfogenética Óssea 4/metabolismo , Cartilagem/metabolismo , Diferenciação Celular , Proliferação de Células , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cruzamentos Genéticos , Deleção de Genes , Proteínas Hedgehog/metabolismo , Homeostase , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Mutação , Osteogênese/genética , Fenótipo , Regiões Promotoras Genéticas , Células-Tronco/citologia
12.
PLoS Biol ; 17(10): e3000498, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613879

RESUMO

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.


Assuntos
Proteína Morfogenética Óssea 4/genética , Gastrulação/genética , Mesoderma/metabolismo , Proteína Nodal/genética , Transdução de Sinais , Proteínas Wnt/genética , Benzotiazóis/farmacologia , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Gástrula/citologia , Gástrula/efeitos dos fármacos , Gástrula/metabolismo , Gastrulação/efeitos dos fármacos , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Modelos Biológicos , Modelos Estatísticos , Proteína Nodal/deficiência , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteínas Wnt/metabolismo
13.
PLoS Biol ; 17(10): e3000081, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31634368

RESUMO

In vitro models of postimplantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon bone morphogenetic protein 4 (BMP4) treatment of geometrically confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line-associated heterogeneity was found to be attributable to endogenous Nodal expression, with up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues.


Assuntos
Proteína Morfogenética Óssea 4/farmacologia , Linhagem da Célula/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento , Proteína Nodal/genética , Células-Tronco Pluripotentes/efeitos dos fármacos , Fenômenos Biomecânicos , Padronização Corporal/genética , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/genética , Gastrulação/efeitos dos fármacos , Gastrulação/genética , Perfilação da Expressão Gênica , Heterogeneidade Genética , Ensaios de Triagem em Larga Escala , Humanos , Modelos Biológicos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Proteína Nodal/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Propriedades de Superfície
14.
Differentiation ; 109: 28-33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31494396

RESUMO

Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to pro-atherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. RESULTS: Comparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22-alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4-6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. CONCLUSION: Our data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.


Assuntos
Aterosclerose/patologia , Desdiferenciação Celular , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Animais , Aterosclerose/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Transdução de Sinais
15.
Sci Signal ; 12(598)2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31506384

RESUMO

Inflammation alters bone marrow hematopoiesis to favor the production of innate immune effector cells at the expense of lymphoid cells and erythrocytes. Furthermore, proinflammatory cytokines inhibit steady-state erythropoiesis, which leads to the development of anemia in diseases with chronic inflammation. Acute anemia or hypoxic stress induces stress erythropoiesis, which generates a wave of new erythrocytes to maintain erythroid homeostasis until steady-state erythropoiesis can resume. Although hypoxia-dependent signaling is a key component of stress erythropoiesis, we found that inflammation also induced stress erythropoiesis in the absence of hypoxia. Using a mouse model of sterile inflammation, we demonstrated that signaling through Toll-like receptors (TLRs) paradoxically increased the phagocytosis of erythrocytes (erythrophagocytosis) by macrophages in the spleen, which enabled expression of the heme-responsive gene encoding the transcription factor SPI-C. Increased amounts of SPI-C coupled with TLR signaling promoted the expression of Gdf15 and Bmp4, both of which encode ligands that initiate the expansion of stress erythroid progenitors (SEPs) in the spleen. Furthermore, despite their inhibition of steady-state erythropoiesis in the bone marrow, the proinflammatory cytokines TNF-α and IL-1ß promoted the expansion and differentiation of SEPs in the spleen. These data suggest that inflammatory signals induce stress erythropoiesis to maintain erythroid homeostasis when inflammation inhibits steady-state erythropoiesis.


Assuntos
Proteínas de Ligação a DNA/imunologia , Eritropoese/imunologia , Heme/imunologia , Inflamação/imunologia , Estresse Fisiológico/imunologia , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/imunologia , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Células Precursoras Eritroides/imunologia , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/imunologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Heme/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/genética , Fagocitose/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Estresse Fisiológico/genética
16.
Growth Factors ; 37(3-4): 153-163, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500477

RESUMO

The objective of this study was to test the association of the variation in a 360 bp region in exon 2 of the ovine bone morphogenetic protein 4 (BMP4) gene with growth performance (birth weight, pre-weaning average daily gain, weaning weight, post-weaning average daily gain and marketing weight) and body conformational traits (height at withers, height at hips, body length, heart girth, thigh circumference, body mass index, skeletal muscle index, body index and relative body index) in 242 Barki lambs using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). Two variants (A and B) and three genotypes (AA, AB and BB) were detected. The BMP4 genotype significantly affected (p < .05 or p < .01) post-weaning daily gain, marketing weight, height at hips, thigh circumference, body mass index and skeletal muscle index. The results provided valuable information indicating selection for the BMP4 genotype might increase growth and muscularity in Barki lambs.


Assuntos
Composição Corporal/genética , Tamanho Corporal/genética , Proteína Morfogenética Óssea 4/genética , Ovinos/crescimento & desenvolvimento , Ovinos/genética , Animais , Sequência de Bases , Éxons/genética , Feminino , Variação Genética/genética , Genótipo , Masculino , Fenótipo , Polimorfismo Conformacional de Fita Simples/genética , Análise de Sequência de DNA , Ganho de Peso/genética
17.
Sci Rep ; 9(1): 11724, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409851

RESUMO

Bone morphogenetic protein (BMP) signaling and Notch signaling play important roles in tumorigenesis in various organs and tissues, including the breast. BMP-4 enhanced epithelial mesenchymal transition (EMT) and stem cell properties in both mammary epithelial cell line and breast carcinoma cell line. BMP-4 increased the expression of EMT biomarkers, such as fibronectin, laminin, N-cadherin, and Slug. BMP-4 also activated Notch signaling in these cells and increased the sphere forming efficiency of the non-transformed mammary epithelial cell line MCF-10A. In addition, BMP-4 upregulated the sphere forming efficiency, colony formation efficiency, and the expression of cancer stem cell markers, such as Nanog and CD44, in the breast carcinoma cell line MDA-MB-231. Inhibition of Notch signaling downregulated EMT and stem cell properties induced by BMP-4. Down-regulation of Smad4 using siRNA impaired the BMP-4-induced activation of Notch signaling, as well as the BMP-4-mediated EMT. These results suggest that EMT and stem cell properties are increased in mammary epithelial cells and breast cancer cells through the activation of Notch signaling in a Smad4-dependent manner in response to BMP-4.


Assuntos
Proteína Morfogenética Óssea 4/genética , Transição Epitelial-Mesenquimal/genética , Neoplasias/etiologia , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Proteína Smad4/genética , Proteína Smad4/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Oncol Rep ; 42(3): 1035-1046, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322275

RESUMO

Bone marrow­derived mesenchymal stem cells (MSCs), are the basic cellular components that make up the bone marrow microenvironment (BMM). In acute myeloid leukemia (AML), the morphology and function of MSCs changes in accordance with the transformation of the BMM. Moreover, the transformation of MSCs into osteoblasts is determined through the bone morphogenetic protein (BMP) pathway, ultimately leading to an altered expression of the downstream adhesion molecule, connective tissue growth factor (CTGF). In this study, we aimed to explore the interaction of possible pathways in AML­derived mesenchymal stem cells (AML­MSCs) co­cultured with the K562 and K562­ADM cell lines. AML­MSCs were co­cultured with K562/K562­ADM cells, and the interactions between the cells were verified by morphological detection, peroxidase staining (POX), reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and fluorescence in situ hybridization (FISH). The proliferation of K562/K562­ADM cells under co­culture conditions was detected by flow cytometry. The expression levels of BMP4 and CTGF were examined by RT­qPCR and western blot (WB) analysis. The detection of interleukin (IL)­6 and IL­32 was also determined by enzyme linked immunosorbent assay (ELISA). In the co­culture system, the K562­ADM cells underwent fusiform transformation. The occurrence of this transformation was associated with an increased expression of CTGF due to the dysregulation of the BMP pathway. The AML­MSCs promoted the proliferation of the K562­ADM cell, but inhibited that of the K562 cells. These findings were confirmed by changes in the expression of the soluble cytokines, IL­6 and IL­32. On the whole, the findings of this study demonstrate that AML­MSCs regulate the expression of CTGF through the BMP pathway. In addition, they affect cytokine production, induce spindle­shaped transformation, and increase drug resistance in the K562­ADM cells. Thus, the morphological transformation through the BMP pathway provides us with a novel target with which to circumvent tumor occurrence, development, drug resistance, invasion and metastasis.


Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Proteína Morfogenética Óssea 4/genética , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Criança , Fator de Crescimento do Tecido Conjuntivo/genética , Feminino , Seguimentos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Microambiente Tumoral , Adulto Jovem
19.
Dev Biol ; 455(2): 382-392, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31315026

RESUMO

Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice.


Assuntos
Desenvolvimento Embrionário , Células Germinativas , Receptores Estrogênicos/fisiologia , Animais , Proteína Morfogenética Óssea 4/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ectoderma/embriologia , Elementos Facilitadores Genéticos , Camundongos , Camundongos Knockout , Análise Serial de Proteínas
20.
J Trace Elem Med Biol ; 55: 15-19, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31345353

RESUMO

The trace element strontium has a significant impact on cartilage metabolism. However, the direct effects of strontium on alkaline phosphatase (ALP), a marker of bone growth, and bone morphogenetic protein-4 (BMP-4), which plays a key role in the regulation of bone and cartilage development, are not entirely clear. In order to understand the mechanisms involved in these processes, the chondrocytes were isolated from Wistar rat articular cartilage by enzymatic digestion and cultured under standard conditions. They were then treated with strontium at 0.5, 1.0, 2.0, 5.0, 20.0 and 100.0 µg/mL for 72 h. The mRNA abundance and protein expression levels of ALP and BMP-4 were measured using real-time polymerase chain reaction (real-time PCR) and Western blot analysis. The results showed that the levels of expression of ALP and BMP-4 in chondrocytes increased as the concentration of strontium increased relative to the control group, and the difference became significant at 1.0 µg/mL strontium (P<0.05). These results indicated that strontium could be involved in cartilage development via regulating ALP and BMP-4 expression.


Assuntos
Fosfatase Alcalina/genética , Proteína Morfogenética Óssea 4/genética , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Estrôncio/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Western Blotting , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Masculino , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real
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