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1.
FASEB J ; 37(4): e22861, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36929047

RESUMO

Enamel is formed by the repetitive secretion of a tooth-specific extracellular matrix and its decomposition. Calcification of the enamel matrix via hydroxyapatite (HAP) maturation requires pH cycling to be tightly regulated through the neutralization of protons released during HAP synthesis. We found that Gpr115, which responds to changes in extracellular pH, plays an important role in enamel formation. Gpr115-deficient mice show partial enamel hypomineralization, suggesting that other pH-responsive molecules may be involved. In this study, we focused on the role of Gpr111/Adgrf2, a duplicate gene of Gpr115, in tooth development. Gpr111 was highly expressed in mature ameloblasts. Gpr111-KO mice showed enamel hypomineralization. Dysplasia of enamel rods and high carbon content seen in Gpr111-deficient mice suggested the presence of residual enamel matrices in enamel. Depletion of Gpr111 in dental epithelial cells induced the expression of ameloblast-specific protease, kallikrein-related peptidase 4 (Klk4), suggesting that Gpr111 may act as a suppressor of Klk4 expression. Moreover, reduction of extracellular pH to 6.8 suppressed the expression of Gpr111, while the converse increased Klk4 expression. Such induction of Klk4 was synergistically enhanced by Gpr111 knockdown, suggesting that proper enamel mineralization may be linked to the modulation of Klk4 expression by Gpr111. Furthermore, our in vitro suppression of Gpr111 and Gpr115 expression indicated that their suppressive effect on calcification was additive. These results suggest that both Gpr111 and Gpr115 respond to extracellular pH, contribute to the expression of proteolytic enzymes, and regulate the pH cycle, thereby playing important roles in enamel formation.


Assuntos
Hipomineralização do Esmalte Dentário , Receptores Acoplados a Proteínas G , Animais , Camundongos , Ameloblastos/metabolismo , Hipomineralização do Esmalte Dentário/genética , Hipomineralização do Esmalte Dentário/metabolismo , Células Epiteliais/metabolismo , Concentração de Íons de Hidrogênio , Calicreínas/metabolismo , Receptores Acoplados a Proteínas G/genética
2.
Endocr J ; 69(4): 399-406, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-34853196

RESUMO

This study aimed to reveal the relationship between quality of life (QOL) and sleep quality in patients with type 1 diabetes mellitus (T1DM). Overall, 202 patients with T1DM were registered in our study, and 192 were eligible for analysis. Baseline characteristics and laboratory values were determined. Patients completed the Japanese versions of the Pittsburgh Sleep Quality Index (PSQI) and Diabetes Therapy-Related QOL (DTR-QOL) questionnaires. We investigated the relationship between the global PSQI and DTR-QOL total scores by using linear regression analysis. In univariate regression analysis, DTR-QOL total scores were associated with body mass index, alcohol consumption, hypertension, hemoglobin A1c (HbA1c), and global PSQI score (all p-value <0.05) but not with sleep duration. When the association between PSQI subscales and DTR-QOL total scores was examined, DTR-QOL total scores were significantly related to subjective sleep quality and daytime dysfunction. In a multivariate regression analysis, the global PSQI score was negatively related to DTR-QOL total scores. Patients with an HbA1c concentration ≥8.0% had significantly lower DTR-QOL total scores. We revealed a relationship between QOL and sleep quality in T1DM patients and showed that the relationship between QOL and PSQI subscales in T1DM patients may be different from that in patients with type 2 diabetes mellitus. Assessing and managing sleep quality may be necessary for patients with diabetes to improve QOL.


Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Transtornos do Sono-Vigília , Estudos Transversais , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Hemoglobinas Glicadas , Humanos , Japão , Qualidade de Vida , Sono , Qualidade do Sono , Transtornos do Sono-Vigília/complicações , Transtornos do Sono-Vigília/etiologia , Inquéritos e Questionários
3.
J Biol Chem ; 295(45): 15328-15341, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32868297

RESUMO

Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.


Assuntos
Ameloblastos/metabolismo , Esmalte Dentário/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Ratos , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética
4.
Biochem Biophys Res Commun ; 581: 89-95, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34662808

RESUMO

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.


Assuntos
Ameloblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Fatores de Transcrição Kruppel-Like/genética , Dente Molar/metabolismo , Odontoblastos/metabolismo , Odontogênese/genética , Ameloblastos/citologia , Amelogenina/genética , Amelogenina/metabolismo , Animais , Animais Recém-Nascidos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dente Molar/citologia , Dente Molar/crescimento & desenvolvimento , Odontoblastos/citologia , Regiões Promotoras Genéticas , Proteoglicanas/genética , Proteoglicanas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo
5.
J Biol Chem ; 294(10): 3406-3418, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30504223

RESUMO

The development of ectodermal organs, such as teeth, requires epithelial-mesenchymal interactions. Basic helix-loop-helix (bHLH) transcription factors regulate various aspects of tissue development, and we have previously identified a bHLH transcription factor, AmeloD, from a tooth germ cDNA library. Here, we provide both in vitro and in vivo evidence that AmeloD is important in tooth development. We created AmeloD-knockout (KO) mice to identify the in vivo functions of AmeloD that are critical for tooth morphogenesis. We found that AmeloD-KO mice developed enamel hypoplasia and small teeth because of increased expression of E-cadherin in inner enamel epithelial (IEE) cells, and it may cause inhibition of the cell migration. We used the CLDE dental epithelial cell line to conduct further mechanistic analyses to determine whether AmeloD overexpression in CLDE cells suppresses E-cadherin expression and promotes cell migration. Knockout of epiprofin (Epfn), another transcription factor required for tooth morphogenesis and development, and analysis of AmeloD expression and deletion revealed that AmeloD also contributed to multiple tooth formation in Epfn-KO mice by promoting the invasion of dental epithelial cells into the mesenchymal region. Thus, AmeloD appears to play an important role in tooth morphogenesis by modulating E-cadherin and dental epithelial-mesenchymal interactions. These findings provide detailed insights into the mechanism of ectodermal organ development.


Assuntos
Movimento Celular , Células Epiteliais/citologia , Dente/citologia , Fatores Genéricos de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Camundongos , Dente/metabolismo
6.
Microvasc Res ; 129: 103986, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32017943

RESUMO

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.


Assuntos
Inibidores da Angiogênese/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Corioide/irrigação sanguínea , Neovascularização de Coroide/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Neovascularização Retiniana/prevenção & controle , Vasos Retinianos/efeitos dos fármacos , Degeneração Macular Exsudativa/prevenção & controle , Animais , Proteínas de Ligação ao Cálcio/síntese química , Proteínas de Ligação ao Cálcio/genética , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/síntese química , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Degeneração Macular Exsudativa/genética , Degeneração Macular Exsudativa/metabolismo , Degeneração Macular Exsudativa/patologia
7.
Int J Cancer ; 145(8): 2157-2169, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30924128

RESUMO

Glioblastoma (GBM) is pathologically characterized by highly malignant neoplastic cells, focal necrosis and aberrant blood vessels composed of disorganized endothelial cells and pericytes. The recent cancer microarray database revealed upregulation of fibulin-7 (Fbln7), a member of the fibulin family, but provided no information on the tissue localization or biological function. In the present study, we demonstrated that Fbln7 is markedly overexpressed by the GBM tissue among astrocytic tumors, and immunolocalized mainly to endothelial cells and pericytes of the glomeruloid and hypertrophied microvessels. The production of Fbln7 by endothelial cells and pericytes was confirmed in cultured human umbilical vein endothelial cells (HUVEC) and human brain vascular pericytes (HBVP) and vascular endothelial growth factor (VEGF) stimulated the Fbln7 expression in HUVEC. Fbln7 bound to angiopoietin-1, but not angiopoietin-2 or Tie2 receptor, through interaction between the N-terminal portions of Fbln7 and angiopoietin-1, and it blocked phosphorylation of Tie2 receptor in HUVEC. In a coculture assay using HUVEC and HBVP, multilayered and irregular-shaped tube-like structures of HUVEC were induced by treatment with a high concentration of VEGF. This was accompanied by Fbln7 overproduction by HUVEC and angiopoietin-1 expression by HBVP. The production of aberrant VEGF-induced tube-like structures was attenuated by treatment with antibody or synthetic peptides specific to the Fbln7 N-terminal domain or knockdown of Fbln7. These data demonstrate that Fbln7 is overexpressed by endothelial cells and pericytes of the abnormal microvessels in GBM, and suggest that Fbln7 may contribute to the aberrant vessel formation by modulation of the angiopoietin-1/angiopoietin-2-Tie2 axis.


Assuntos
Angiopoietina-1/genética , Neoplasias Encefálicas/genética , Proteínas de Ligação ao Cálcio/genética , Glioblastoma/genética , Neovascularização Patológica/genética , Angiopoietina-1/metabolismo , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Técnicas de Cocultura , Regulação Neoplásica da Expressão Gênica , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Neovascularização Patológica/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Pericitos/citologia , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Ligação Proteica , Fator A de Crescimento do Endotélio Vascular/farmacologia
8.
Biochem Biophys Res Commun ; 518(4): 672-677, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31470976

RESUMO

Link protein is encoded by the Hapln1 gene and is a prototypical protein found in the cartilage matrix. It acts as an important component of the endochondral skeleton during early development. To study its transcriptional regulation, promoter fragments derived from the link protein gene were coupled to the ß-galactosidase reporter and used to study in vivo transgene expression in mice. In day 15.5 mouse embryos, a link promoter fragment spanning -1020 to +40 nucleotides demonstrated highly specific ß-galactosidase staining of skeletal structures, including the appendicular and axial cartilaginous tissues. Two shorter promoter fragments, spanning -690 to +40 and -315 to +40 nucleotides, demonstrated limb- and genitalia-specific expression resembling that of homeodomain-regulated tissues. Bioinformatic analysis revealed a highly conserved, Hox-like binding site (HLBS) at approximately -220 bp of the promoter, shared by both constructs, which contained the Hox-core consensus sequence TAATTA. Electromobility shift assays demonstrated binding of Hox-B4 recombinant protein to the HLBS, which was eliminated with nucleotide substitutions within the core-binding element. Co-transfection analysis of the HLBS demonstrated a 22-fold transcriptional activation by HoxA9 expression, which was ablated with a substitution within the core HLBS element. Together these findings establish promoter regions within the link protein gene that are important for in vivo expression and identify the potential role of homeodomain-containing proteins in controlling cartilage and limb gene expression.


Assuntos
Cartilagem/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/genética , Proteoglicanas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Cartilagem/embriologia , Proteínas da Matriz Extracelular/metabolismo , Extremidades/embriologia , Genitália/embriologia , Genitália/metabolismo , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Transgênicos , Proteoglicanas/metabolismo , Homologia de Sequência do Ácido Nucleico , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
9.
FASEB J ; 32(9): 4889-4898, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29634368

RESUMO

Fibulin-7 (Fbln7) has been identified as the latest member of the fibulin family of secreted glycoproteins in developing teeth, functioning as a cell adhesion molecule and interacting with other matrix proteins, receptors, and growth factors. More recently, we have shown that the C-terminal Fbln7 fragment (Fbln7-C) has antiangiogenic activity in vitro. Fbln7 is also expressed in immune-privileged tissues, such as eye and placenta, but its functional significance is unknown. In the current study, we show that human monocytes adhere to both full-length Fbln7 (Fbln7-FL) and Fbln7-C, in part, via integrins α5ß1 and α2ß1. Morphologic studies and surface expression analyses of CD14, mannose receptor (CD206), major histocompatibility complex II, and CD11b receptors revealed that both Fbln7-FL and Fbln7-C inhibit M-CSF-induced monocyte differentiation. Fbln7-C had significantly greater negative effects on cell spreading and stress fiber formation, including the production of IL-6 and metalloproteinase-1/-9 compared with Fbln7-FL. Furthermore, in an LPS-induced systemic inflammation model, Fbln7-C and Fbln7-FL reduced the infiltration of immune cells, such as neutrophils and macrophages, to the inflamed peritoneum. Thus, these results suggest that Fbln7 and Fbln7-C could modulate the activity of immune cells and have therapeutic potential for inflammatory diseases.-Sarangi, P. P., Chakraborty, P., Dash, S. P., Ikeuchi, T., de Vega, S., Ambatipudi, K., Wahl, L., Yamada, Y. Cell adhesion protein fibulin-7 and its C-terminal fragment negatively regulate monocyte and macrophage migration and functions in vitro and in vivo.


Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular/fisiologia , Macrófagos/metabolismo , Monócitos/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Lectinas Tipo C/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptores de Superfície Celular/metabolismo
10.
PLoS Genet ; 12(7): e1006156, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27441836

RESUMO

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.


Assuntos
Cálcio/metabolismo , Colágeno Tipo I/biossíntese , Canais Iônicos/genética , Osteogênese Imperfeita/genética , Adulto , Sinalização do Cálcio , Colágeno Tipo I/metabolismo , Consanguinidade , Análise Mutacional de DNA , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Feminino , Genes Recessivos , Estudos de Associação Genética , Predisposição Genética para Doença , Homeostase , Humanos , Lactente , Masculino , Linhagem , Processamento de Proteína Pós-Traducional
11.
J Biol Chem ; 292(33): 13531-13540, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28673966

RESUMO

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.


Assuntos
Fosfatase Alcalina/metabolismo , Esmalte Dentário/metabolismo , Indução Enzimática , Subunidade 1 do Complexo Mediador/metabolismo , Receptor Notch1/agonistas , Transdução de Sinais , Calcificação de Dente , Fosfatase Alcalina/química , Animais , Linhagem Celular Transformada , Esmalte Dentário/ultraestrutura , Genes Reporter , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Imunoprecipitação , Subunidade 1 do Complexo Mediador/antagonistas & inibidores , Subunidade 1 do Complexo Mediador/genética , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Regiões Promotoras Genéticas , Multimerização Proteica , Proteólise , Interferência de RNA , Receptor Notch1/metabolismo , Elementos de Resposta
12.
J Cell Sci ; 129(5): 1018-30, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26759176

RESUMO

Pannexin 3 (Panx3) and connexin 43 (Cx43; also known as GJA1) are two major gap junction proteins expressed in osteoblasts. Here, we studied their functional relationships in skeletal formation by generating Panx3(-/-) and Panx3(-/-);Cx43(-/-) mice and comparing their skeletal phenotypes with Cx43(-/-) mice. Panx3(-/-) mice displayed defects in endochondral and intramembranous ossification, resulting in severe dwarfism and reduced bone density. The skeletal abnormalities of Panx3(-/-);Cx43(-/-) mice were similar to those in Panx3(-/-) mice. The gross appearance of newborn Cx43(-/-) skeletons showed no obvious abnormalities, except for less mineralization of the skull. In Panx3(-/-) mice, proliferation of chondrocytes and osteoblasts increased and differentiation of these cells was inhibited. Panx3 promoted expression of osteogenic proteins such as ALP and Ocn (also known as ALPL and BGLAP, respectively), as well as Cx43, by regulating Osx (also known as SP7) expression. Panx3 was induced in the early differentiation stage and reduced during the maturation stage of osteoblasts, when Cx43 expression increased in order to promote mineralization. Furthermore, only Panx3 functioned as an endoplasmic reticulum (ER) Ca(2+) channel to promote differentiation, and it could rescue mineralization defects in Cx43(-/-) calvarial cells. Our findings reveal that Panx3 and Cx43 have distinct functions in skeletal formation.


Assuntos
Conexina 43/fisiologia , Conexinas/fisiologia , Osteogênese , Animais , Proliferação de Células , Condrócitos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Transdução de Sinais , Crânio/citologia , Crânio/crescimento & desenvolvimento , Crânio/metabolismo , Tíbia/citologia , Tíbia/crescimento & desenvolvimento , Tíbia/metabolismo
13.
PLoS Genet ; 10(8): e1004468, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25166858

RESUMO

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/ßcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.


Assuntos
Padronização Corporal/genética , Proteínas de Ligação a DNA/biossíntese , Extremidades/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição/biossíntese , Animais , Proteínas de Ligação a DNA/genética , Ectoderma , Embrião de Mamíferos , Desenvolvimento Embrionário , Extremidades/embriologia , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas Wnt/biossíntese , Proteínas Wnt/genética
14.
J Cell Sci ; 127(Pt 24): 5261-72, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25344255

RESUMO

The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development.


Assuntos
Diferenciação Celular , Epiderme/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Sp/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Epiderme/crescimento & desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/patologia , Humanos , Queratinócitos/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/deficiência , Camundongos , Modelos Biológicos , Morfogênese/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Receptores Notch/metabolismo , Proteína do Retinoblastoma/metabolismo
15.
Biopolymers ; 106(2): 184-195, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26491858

RESUMO

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin ß1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

16.
J Biol Chem ; 289(5): 2839-51, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24338011

RESUMO

Canonical Wnt signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. Here, we show that Panx3 (pannexin 3) plays a key role in this transition by inhibiting the proliferation and promoting the cell cycle exit. Using primary calvarial cells and explants, C3H10T1/2 cells, and C2C12 cells, we found that Panx3 expression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We also found that the Panx3 hemichannel inhibited cell growth by promoting ß-catenin degradation through GSK3ß activation. Additionally, the Panx3 hemichannel inhibited cyclin D1 transcription and Rb phosphorylation through reduced cAMP/PKA/CREB signaling. Furthermore, the Panx3 endoplasmic reticulum Ca(2+) channel induced the transcription and phosphorylation of p21, through the calmodulin/Smad pathway, and resulted in the cell cycle exit. Our results reveal that Panx3 is a new regulator that promotes the switch from proliferation to differentiation of osteoprogenitors via multiple Panx3 signaling pathways.


Assuntos
Conexinas/metabolismo , Osteócitos/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt/fisiologia , Quinases Ativadas por p21/metabolismo , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Conexinas/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Crânio/citologia , beta Catenina/metabolismo
17.
Am J Pathol ; 184(6): 1683-94, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24720953

RESUMO

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-ß1-induced Smad2 phosphorylation, and inhibitors of TGF-ß1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-ß1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-ß/Smad signaling.


Assuntos
Envelhecimento/metabolismo , Proliferação de Células , Matriz Extracelular/metabolismo , Mesângio Glomerular/metabolismo , Laminina/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Animais , Matriz Extracelular/genética , Matriz Extracelular/patologia , Mesângio Glomerular/patologia , Glomerulonefrite/genética , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Laminina/genética , Camundongos , Camundongos Knockout , Fosforilação/genética , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/patologia , Transdução de Sinais/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
18.
FASEB J ; 28(3): 1386-97, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24344332

RESUMO

Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Membrana/fisiologia , Neuritos , Transdução de Sinais , Animais , Sequência de Bases , Primers do DNA , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Arch Biochem Biophys ; 545: 148-53, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24480309

RESUMO

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Animais , Proteínas de Ligação ao Cálcio/química , Adesão Celular , Proteína Substrato Associada a Crk/metabolismo , Ativação Enzimática , Adesões Focais/metabolismo , Adesões Focais/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Integrinas/metabolismo , Camundongos , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fibras de Estresse/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Connect Tissue Res ; 55 Suppl 1: 38-42, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25158178

RESUMO

In enamel formation, the deposition of minerals as crystallites starts when the mineralization front first forms at the start of the secretory stage. During maturation, the enamel layer accumulates significant amounts of new mineral as the crystallites grow in volume. Inversely related to mineral gain is loss of protein and water from the forming enamel. Both ameloblastin (Ambn) and enamelin are essential components for formation of a functional enamel layer. The aim of this study was to quantify the proportion of mineral and non-mineral material present in developing enamel relative to Ambn concentration using Ambn mutant mice mated with others overexpressing full-length Ambn from the mouse amelogenin promoter at lower (+), similar (++) or higher (+++) concentration than normal. Mandibular incisors (age: 7 weeks, n = 8) were imaged by micro-computed tomography and the enamel was analyzed from the apical region to the incisal edge in sequential 1.0 mm volumes of interest. Mineral density was determined using a series of hydroxyapatite (HA) phantoms to calibrate enamel density measurements. At the site where the mandibular incisor emerged into the oral cavity, the enamel volume, mineral weight, and mineral density were reduced when Tg Ambn was expressed at lower or higher levels than normal. While in wild-type the % mineral was >95%, it was negligible in Ambn-/-, 22.3% in Ambn-/-, Tg(+), 75.4% in Ambn-/-, Tg(++), and 45.2% in Ambn-/-, Tg(+++). These results document that the deposition of mineral and removal of non-mineral components are both very sensitive to expressed Ambn concentrations.


Assuntos
Amelogênese/genética , Amelogenina/ultraestrutura , Esmalte Dentário/ultraestrutura , Amelogenina/genética , Animais , Densidade Óssea , Incisivo/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microtomografia por Raio-X
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