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1.
J Biol Chem ; 298(9): 102295, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35872015

RESUMO

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.


Assuntos
Amelogênese , Regulação da Expressão Gênica , Proteína HMGN2 , Proteínas de Homeodomínio , Fator 1 de Ligação ao Facilitador Linfoide , Fatores de Transcrição , Transcrição Gênica , Amelogênese/genética , Amelogenina/genética , Animais , Cromatina/metabolismo , Proteína HMGN2/genética , Proteína HMGN2/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Proteína Homeobox PITX2
2.
Hum Mol Genet ; 23(1): 194-208, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23975681

RESUMO

Patients with Axenfeld-Rieger Syndrome (ARS) present various dental abnormalities, including hypodontia, and enamel hypoplasia. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in early tooth development. However, because Pitx2 is also a major regulator of later stages of tooth development, especially during amelogenesis, it is unclear how mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel) expression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is controlled by chromatin-associated factor Hmgn2, and that Hmgn2 prevents Pitx2 from efficiently binding to and activating the Amel promoter. Consistent with a physiological significance to this interaction, we show that K14-Hmgn2 transgenic mice display a severe loss of Amel expression on the labial side of the lower incisors, as well as enamel hypoplasia-consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carry PITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in a novel mouse model of ARS.


Assuntos
Amelogenina/metabolismo , Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/patologia , Proteína HMGN2/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Incisivo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Amelogenina/genética , Animais , Segmento Anterior do Olho/patologia , Linhagem Celular , Esmalte Dentário/metabolismo , Esmalte Dentário/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Anormalidades do Olho/genética , Oftalmopatias Hereditárias , Regulação da Expressão Gênica , Proteína HMGN2/genética , Humanos , Incisivo/patologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Regiões Promotoras Genéticas , Proteína Homeobox PITX2
3.
Organogenesis ; 17(3-4): 136-149, 2021 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-34845978

RESUMO

Cells with osteogenic potential are believed to be an ideal source for bone tissue bioengineering. Large bone defects require temporary substitution of the damaged parts. In this respect, the transplantation of bone cells cultured on osteogenic substrates has been investigated. To use the natural bone matrix, one approach is the so-called demineralized bone matrix (DBM). In this study, we evaluated the interaction of human fetal osteoblasts (hFOB 1.19 cells, a human fetal osteoblastic cell line) with DBM fragments. No additional bone differentiation inducer was used other than the DBM itself. The samples were processed, had adhesion pattern evaluated and analyzed by light microscopy (cytochemical and immunocytochemical analysis) and electron microscopy (scanning and transmission). The adhesion pattern of hFOB cells on DBM was similar to what was observed on the cell culture plate. Morphological analysis showed that the hFOB cells had emitted filopodia and cellular projections on both controls and DBM. On DBM, the adhered cells emitted prolongations and migrated into the matrix. The monolayer growth pattern was observed as well as the accumulation of filamentous and reticulate extracellular materials when hFOB cells were cultured on the DBM surface. EDS analysis revealed the deposition of calcium on DBM. Immunocytochemical data showed that the hFOB cells were able to secrete extracellular matrix molecules such as fibronectin and laminin on DBM. Our data indicate that DBM successfully stimulates the osteoblastic phenotype of osteoblast-like cells and corroborate with the fact that DBM is a considerable natural matrix that promotes fractured-bone healing.


Assuntos
Matriz Óssea , Osteoblastos , Matriz Óssea/química , Osso e Ossos , Diferenciação Celular , Humanos , Osteogênese
4.
Exp Hematol ; 36(3): 265-72, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18179859

RESUMO

OBJECTIVE: alpha-Hemoglobin stabilizing protein (AHSP) binds alpha-hemoglobin (Hb), avoiding its precipitation and its pro-oxidant activity. In the presence of betaHb, the alphaHb-AHSP complex is dismembered and betaHb displaces AHSP to generate the quaternary structure of Hb. The relationship between Hb formation and alterations in AHSP expression, which may affect human erythropoiesis, has not yet been described in human cells. Hence, in this study, we examined the effects of AHSP knockdown in hemin-induced K562 and erythropoietin-induced CD34(+) cells with particular reference to cellular aspects and gene expression. MATERIALS AND METHODS: Short-hairpin RNA expression vectors aimed at the AHSP mRNA target sequence were cloned and transfected into K562 and CD34(+) cells. K562 and CD34(+) cells were stimulated to erythroid differentiation. Cells were examined in terms of gene expression using quantitative real-time polymerase chain reaction; reactive oxygen species (ROS) production, apoptosis, and Hb production through flow cytometry assays; and immunofluorescence assays for globin chains. RESULTS: RNA interference-mediated knockdown of AHSP expression resulted in considerable alphaHb precipitation, as well as in a significant decrease in HbF formation. AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. CONCLUSION: These findings strengthen the hypothesis that AHSP stabilizes the alphaHb chain, avoiding its precipitation and its ability to generate ROS, which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.


Assuntos
Antígenos CD34/efeitos dos fármacos , Proteínas Sanguíneas/efeitos dos fármacos , Eritropoetina/farmacologia , Globinas/efeitos dos fármacos , Hemina/farmacologia , Hemoglobinas/biossíntese , Chaperonas Moleculares/efeitos dos fármacos , Antígenos CD34/biossíntese , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/biossíntese , Proteínas Sanguíneas/genética , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Clonagem Molecular , Células Precursoras Eritroides/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Globinas/metabolismo , Humanos , Células K562 , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Interferência de RNA , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
5.
J Exp Med ; 212(11): 1931-46, 2015 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-26438360

RESUMO

Bone surfaces attract hematopoietic and nonhematopoietic cells, such as osteoclasts (OCs) and osteoblasts (OBs), and are targeted by bone metastatic cancers. However, the mechanisms guiding cells toward bone surfaces are essentially unknown. Here, we show that the Gαi protein-coupled receptor (GPCR) EBI2 is expressed in mouse monocyte/OC precursors (OCPs) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) is secreted abundantly by OBs. Using in vitro time-lapse microscopy and intravital two-photon microscopy, we show that EBI2 enhances the development of large OCs by promoting OCP motility, thus facilitating cell-cell interactions and fusion in vitro and in vivo. EBI2 is also necessary and sufficient for guiding OCPs toward bone surfaces. Interestingly, OCPs also secrete 7α,25-OHC, which promotes autocrine EBI2 signaling and reduces OCP migration toward bone surfaces in vivo. Defective EBI2 signaling led to increased bone mass in male mice and protected female mice from age- and estrogen deficiency-induced osteoporosis. This study identifies a novel pathway involved in OCP homing to the bone surface that may have significant therapeutic potential.


Assuntos
Densidade Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Osteoclastos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/fisiologia , Células-Tronco/efeitos dos fármacos , Animais , Osso e Ossos/citologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/fisiologia , Transdução de Sinais , Células-Tronco/fisiologia
6.
Appl Immunohistochem Mol Morphol ; 22(3): 188-99, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23702648

RESUMO

We performed immunohistochemistry for macrophage colony-stimulating factor 1 receptor (also known as c-fms proto-oncogene product) on tissue microarrays of human nontumor lung, pulmonary squamous cell carcinomas (SCC) and adenocarcinomas (ADC), and breast and ovarian carcinomas using a commercially available anti-cFMS antibody. The specificity of the antibody was validated by Western blot and mass spectrometry analysis. Staining of cFMS was restricted to stromal fibroblasts in pulmonary SCC and ADC specimens and was not identified in tumor epithelium or epithelium and stromal cells of nontumor lung. Evaluation of pulmonary SCC (n=63) and ADC (n=71) specimens revealed stromal fibroblast cFMS staining in 60% (38 of 63) and 35% (25 of 71) of the tumor samples, respectively. A similar pattern of stromal fibroblast cFMS staining was observed in breast (n=21) and ovarian (n=50) carcinomas. It was reported that glucocorticoids induced cFMS expression in breast carcinomas and choriocarcinomas. To investigate whether stromal cFMS expression in lung cancers was associated with glucocorticoid signaling, glucocorticoid receptor protein distribution was evaluated in lung tissue microarrays by immunohistochemistry. Stromal fibroblast glucocorticoid receptor staining was only observed in 18% (2 of 11) of pulmonary SCC and 6% (1 of 17) of ADC specimens, suggesting that cFMS expression may not be directly mediated by glucocorticoids in stromal fibroblasts of lung cancers. The tumor stromal cell expression of cFMS in certain tumor types (lung, ovarian, and breast) suggests the potential for more diverse tumor therapeutic options and presents an attractive target for drug development.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Neoplasias Ovarianas/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Adenocarcinoma/patologia , Idoso , Neoplasias da Mama/patologia , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Imuno-Histoquímica , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Terapia de Alvo Molecular , Neoplasias Ovarianas/patologia , Proto-Oncogene Mas , Análise Serial de Tecidos
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