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1.
Molecules ; 26(22)2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34833848

RESUMO

Dentin matrix protein 1 (DMP1) contains a large number of acidic domains, multiple phosphorylation sites, a functional arginine-glycine-aspartate (RGD) motif, and a DNA binding domain, and has been shown to play essential regulatory function in dentin and bone mineralization. DMP1 could also orchestrate bone matrix formation, but the ability of DMP1 on Ti to human mesenchymal stem cell (hMSC) conversion to osteoblasts has not been studied. There is importance to test if the DMP1 coated Ti surface would promote cell migration and attachment to the metal surface and promote the differentiation of the attached stem cells to an osteogenic lineage. This study aimed to study the human mesenchymal stem cells (hMSCs) attachment and proliferation on DMP1 coated titanium (Ti) disks compared to non-coated disks, and to assess possible osteoblastic differentiation of attached hMSCs. Sixty-eight Ti disks were divided into two groups. Group 1 disks were coated with dentin matrix protein 1 and group 2 disks served as control. Assessment with light microscopy was used to verify hMSC attachment and proliferation. Cell viability was confirmed through fluorescence microscopy and mitochondrial dehydrogenase activity. Real-time polymerase chain reaction analysis was done to study the gene expression. The proliferation assay showed significantly greater cell proliferation with DMP1 coated disks compared to the control group (p-value < 0.001). Cell vitality analysis showed a greater density of live cells on DMP1 coated disks compared to the control group. Alkaline phosphatase staining revealed higher enzyme activity on DMP1 coated disks and showed itself to be significantly higher than the control group (p-value < 0.001). von Kossa staining revealed higher positive areas for mineralized deposits on DMP1 coated disks than the control group (p-value < 0.05). Gene expression analysis confirmed upregulation of runt-related transcription factor 2, osteoprotegerin, osteocalcin, osteopontin, and alkaline phosphatase on DMP1 coated disks (p-value < 0.001). The dentin matrix protein promoted the adhesion, proliferation, facilitation differentiation of hMSC, and mineralized matrix formation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/farmacologia , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Fosfoproteínas/farmacologia , Titânio/farmacologia , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Propriedades de Superfície
2.
Connect Tissue Res ; 59(sup1): 6-12, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29745808

RESUMO

Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.


Assuntos
Calcificação Fisiológica , Cálcio/metabolismo , Diferenciação Celular , Odontoblastos/metabolismo , Osteoblastos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Cálcio/farmacologia , Linhagem Celular , Camundongos , Odontoblastos/citologia , Osteoblastos/citologia
3.
Connect Tissue Res ; 59(sup1): 13-19, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29745814

RESUMO

In search for bone and dentin extracellular matrix (ECM) proteins, transforming growth factor beta receptor II interacting protein 1 (TRIP-1) was identified as a novel protein synthesized by osteoblasts and odontoblasts and exported to the ECM. TRIP-1 is a WD-40 (WD is Tryptophan-Aspartic acid dipeptide) protein that has been well recognized for its physiological role in the endoplasmic reticulum (ER). In the ER, TRIP-1 functions as an essential subunit of eukaryotic elongation initiation factor 3 and is involved in the protein translational machinery. Recently, we reported that TRIP-1 is localized in the ECM of bone and dentin. In this study, we demonstrate that varying concentrations of TRIP-1 can participate in the nucleation of calcium phosphate polymorphs. Nucleation studies performed with high calcium and phosphate concentration demonstrated that recombinant TRIP-1 could orchestrate the formation of hydroxyapatite crystals. Nucleation experiments performed on demineralized and deproteinized dentin wafer under physiological conditions and subsequent transmission electron microscope analysis of the deposits at the end of 7 and 14 days showed that TRIP-1 promoted the deposition of calcium phosphate mineral aggregates in the gap-overlap region of type I collagen. Taken together, we provide mechanistic insight into the role of this intracellular protein in matrix mineralization.


Assuntos
Colágeno Tipo I/química , Durapatita/química , Fator de Iniciação 3 em Eucariotos/química , Proteínas da Matriz Extracelular/química , Colágeno Tipo I/metabolismo , Durapatita/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
4.
J Mech Behav Biomed Mater ; 81: 26-38, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29477893

RESUMO

Titanium (Ti) is widely used in biomedical devices due to its recognized biocompatibility. However, implant failures and subsequent clinical side effects are still recurrent. In this context, improvements can be achieved by designing biomaterials where the bulk and the surface of Ti are independently tailored. The conjugation of biomolecules onto the Ti surface can improve its bioactivity, thus accelerating the osteointegration process. Ti was modified with TiO2, two different spacers, 3-(4-aminophenyl) propionic acid (APPA) or 3-mercaptopropionic acid (MPA) and dentin matrix protein 1 (DMP1) peptides. X-ray photoelectron spectroscopy analysis revealed the presence of carbon and nitrogen for all samples, indicating a success in the functionalization process. Furthermore, DMP1 peptides showed an improved coverage area for the samples with APPA and MPA spacers. Biological tests indicated that the peptides could modulate cell affinity, proliferation, and differentiation. Enhanced results were observed in the presence of MPA. Moreover, the immobilization of DMP1 peptides through the spacers led to the formation of calcium phosphate minerals with a Ca/P ratio near to that of hydroxyapatite. Corrosion and tribocorrosion results indicated an increased resistance to corrosion and lower mass loss in the functionalized materials, showing that this new type of functional material has attractive properties for biomaterials application.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Teste de Materiais , Osteogênese/efeitos dos fármacos , Peptídeos/química , Titânio/química , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Corrosão , Eletroquímica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Propriedades de Superfície
5.
Sci Rep ; 6: 37885, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27883077

RESUMO

Transforming growth factor beta receptor II interacting protein 1 (TRIP-1), a predominantly intracellular protein is localized in the ECM of bone. TRIP-1 lacks a signal peptide, therefore, in this study, we provide evidence that intracellular TRIP-1 can be packaged and exported to the ECM via exosomes. Overexpression of TRIP-1 in MC3T3-E1 cells resulted in increased matrix mineralization during differentiation and knockdown resulted in reduced effects. In vivo function of TRIP-1 was studied by an implantation assay performed using TRIP-1 overexpressing and knockdown cells cultured in a 3-dimmensional scaffold. After 4 weeks, the subcutaneous tissues from TRIP-1 overexpressing cells showed higher calcium and phosphate deposits, arranged collagen fibrils and increased expression of Runx2 and alkaline phosphatase. Nucleation studies on demineralized and deproteinized dentin wafer is a powerful tool to determine the functional role of noncollagenous proteins in matrix mineralization. Using this system, we provide evidence that TRIP-1 binds to Type-I collagen and can promote mineralization. Surface plasmon resonance analysis demonstrated that TRIP-1 binds to collagen with KD = 48 µM. SEM and TEM analysis showed that TRIP-1 promoted the nucleation and growth of calcium phosphate mineral aggregates. Taken together, we provide mechanistic insights of this intracellular protein in matrix mineralization.


Assuntos
Calcificação Fisiológica/fisiologia , Fatores de Iniciação em Eucariotos/metabolismo , Matriz Extracelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoblastos/metabolismo , Animais , Fosfatos de Cálcio/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Colágeno Tipo I/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Exossomos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Nus , Osteoblastos/fisiologia , Osteogênese/genética , Ratos , Crânio/citologia
6.
Connect Tissue Res ; 55 Suppl 1: 121-4, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25158195

RESUMO

Dentin matrix protein 1 (DMP1) is a key regulator of biomineralization within the extracellular matrix (ECM) of bone and plays a role in regulating osteogenic gene expression. Osteocalcin (OCN) is one of the most abundantly expressed non-collagenous proteins by osteoblasts. In the present study, we generated a mouse model (OC-DMP1) that overexpresses full-length DMP1 utilizing the mouse OCN promoter. Expression of genes encoding osteogenic transcription factors and ECM proteins during early post-natal development in male OC-DMP1 and wild type (WT) mice was evaluated in femurs and calvaria. Bones were dissected from n = 4 animals at 15, 30, 60 and 90-d of age. Total RNA was isolated, reverse transcribed, and real-time PCR analysis was performed. Results confirmed a difference (p < 0.05) in osteogenic gene expression between OC-DMP1 and WT mice at the specified time points. Additionally, distinctive osteogenic gene expression profiles for calvaria and femur, representing intramembranous and endochondral bone formation, were identified. These data suggest that bone-specific DMP1 overexpression changes the pattern in osteogenic gene expression pattern thereby influencing bone development. This animal model presented here provides new opportunities for analysis of in vivo roles of DMP1 in bone.


Assuntos
Osso e Ossos/metabolismo , Proteínas da Matriz Extracelular/genética , Osteogênese/genética , Fosfoproteínas/genética , Animais , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteogênese/fisiologia , Ratos , Fatores de Transcrição/metabolismo
7.
Biomed Res Int ; 2013: 182965, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23841055

RESUMO

NUMB is a multifunctional protein implicated to function in self-renewal and differentiation of progenitors in several tissues. To characterize the transcripts and to analyze the expression pattern of NUMB in odontogenesis, we isolated 2 full-length clones for NUMB from mouse dental pulp mRNA. One novel sequence contained 200 bp insertion in the phosphotyrosine binding domain (PTB). Confocal microscopy analysis showed strong NUMB expression in human dental pulp stem cells (hDPSC) and preameloblasts. Western blot analysis indicated that NUMB isoforms were differentially expressed in various dental tissues. Immunohistochemical analysis showed that in postnatal mouse tooth germs, NUMB was differentially expressed in the preameloblasts, odontoblasts, cervical loop region, and in the dental pulp stem cells during development. Interestingly, overexpression of NUMB in HAT-7, a preameloblast cell line, had dramatic antagonizing effects on the protein expression level of activated Notch 1. Further analysis of the Notch signaling pathway showed that NUMB significantly downregulates sonic hedgehog (Shh) expression in preameloblasts. Therefore, we propose that NUMB maintains ameloblast progenitor phenotype at the cervical loop by downregulating the activated Notch1 protein and thereby inhibiting the mRNA expression of Shh.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Odontogênese , RNA Mensageiro/genética , Animais , Diferenciação Celular/genética , Linhagem Celular , Polpa Dentária/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Odontoblastos/metabolismo , RNA Mensageiro/isolamento & purificação , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais
8.
J Biol Chem ; 288(22): 16098-109, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23589294

RESUMO

Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/química , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/farmacologia , Estrutura Terciária de Proteína , Sialoglicoproteínas/genética , Sialoglicoproteínas/farmacologia
9.
J Biol Chem ; 288(12): 8585-8595, 2013 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-23362283

RESUMO

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca(2+). This calcium flux triggered the activation of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca(2+) depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.


Assuntos
Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Proteínas da Matriz Extracelular/fisiologia , Células-Tronco Mesenquimais/enzimologia , Fosfoproteínas/fisiologia , Sialoglicoproteínas/fisiologia , Proteína Smad1/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Antígenos de Diferenciação/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteogênese , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Smad Reguladas por Receptor/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
J Periodontol ; 84(3): 389-95, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22612367

RESUMO

BACKGROUND: Periodontitis can ultimately result in tooth loss. Many natural and synthetic materials have been tried to achieve periodontal regeneration, but the results remain variable and unpredictable. We hypothesized that exogenous treatment with dentin matrix protein 1 (DMP1) activates specific genes and results in phenotypic and functional changes in human periodontal ligament stem cells (hPDLSCs). METHODS: hPDLSCs were isolated from extracted teeth and cultured in the presence or absence of DMP1. Quantitative polymerase chain reactions were performed to analyze the expression of several genes involved in periodontal regeneration. hPDLSCs were also processed for immunocytochemical and Western blot analysis using phosphorylated extracellular signal-regulated kinase (pERK) and ERK antibodies. Alkaline phosphatase and von Kossa staining were performed to characterize the differentiation of hPDLSCs into osteoblasts. Field emission scanning electron microscopic analysis of the treated and control cell cultures were also performed. RESULTS: Treatment with DMP1 resulted in the upregulation of genes, such as matrix metalloproteinase-2, alkaline phosphatase, and transforming growth factor ß1. Activation of ERK mitogen-activated protein kinase signaling pathway and translocation of pERK from the cytoplasm to the nucleus was observed. Overall, DMP1-treated cells showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation when compared with untreated controls. CONCLUSION: DMP1 can orchestrate a coordinated expression of genes and phenotypic changes in hPDLSCs by activation of the ERK signaling pathway, which may provide a valuable strategy for tissue engineering approaches in periodontal regeneration.


Assuntos
Proteínas da Matriz Extracelular/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Fosfoproteínas/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Ligamento Periodontal/citologia , Ligamento Periodontal/enzimologia , Fosfoproteínas/farmacologia , Fosforilação , Transporte Proteico , Proteínas Recombinantes , Regeneração/genética , Células-Tronco/efeitos dos fármacos
11.
Cell Adh Migr ; 6(4): 307-11, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22588498

RESUMO

Cell adhesion to DPP substrate is an integrin-mediated event and involves integrin binding, clustering, assembly of focal adhesion complexes and cytoskeletal organization. Cells perceive the DPP substrate through the integrin receptor αvß1 and bind the actin cytoskeleton to the membrane via focal adhesion sites. The cells respond to this proteinaceous rigid substrate by activating the mechano-chemical signaling events leading to cell spreading and formation of focal adhesions. Focal adhesions, which are sites of integrin binding to the extracellular matrix, form in the leading edge during cell migration. These sites are dynamic and the supramolecular assemblies contain structural and signaling components regulating cell functions. In our study, we present a scenario that integrins utilize the actin network to permit activation of the mitogen-activated kinase modules to transduce signals through the cytoplasm to the nucleus in the presence of DPP. We specifically demonstrate that ERK-mediated transcriptional events impinge on activation of transcription factors leading to cell differentiation.


Assuntos
Adesão Celular , Matriz Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfoproteínas/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Movimento Celular , Citoesqueleto/metabolismo , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos
12.
Histochem Cell Biol ; 138(1): 113-25, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22527697

RESUMO

Glucose-regulated protein 78 (GRP-78) is one of the many endoplasmic reticulum chaperone proteins that have been shown to possess multifunctional roles. We have previously demonstrated that GRP-78 functions as a receptor for dentin matrix protein 1 (DMP1) and is required for DMP1-mediated calcium release; that it is a secreted protein and can bind to type I collagen and DMP1 extracellularly and aid in the nucleation of calcium phosphate. We provide evidence in this study that tyrosine phosphorylation is required for DMP1/GRP-78-mediated calcium release in mesenchymal cells. We further demonstrate that GRP-78 is localized in the nucleus of mesenchymal cells and that the cell surface GRP-78 is not associated with the G-protein Gαq in mesenchymal cells. Results from this study show that during development of mineralized tissues, increased expression of GRP-78 can be observed in condensing cartilage and mesenchymal cells of the alveolar bone, endochondral bone and dental pulp. Additionally, we show that GRP-78 is present in the mineralizing matrices of teeth, bone and in the extracellular matrix of differentiating human marrow stromal cells and dental pulp stem cells. Collectively, our observations provide a new perspective on GRP-78 with respect to mineralized matrix formation.


Assuntos
Proteínas de Choque Térmico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Calcificação Fisiológica , Diferenciação Celular , Polpa Dentária/metabolismo , Chaperona BiP do Retículo Endoplasmático , Matriz Extracelular/metabolismo , Proteínas de Choque Térmico/análise , Humanos , Camundongos , Especificidade de Órgãos , Fosforilação
13.
J Histochem Cytochem ; 60(4): 323-37, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22260994

RESUMO

Transforming growth factor beta receptor II (TGFßR-II) interacting protein 1 (TRIP-1) is a WD-40 protein that binds to the cytoplasmic domain of the TGF-ß type II receptor in a kinase-dependent manner. To investigate the role of TRIP-1 in mineralized tissues, we examined its pattern of expression in cartilage, bone, and teeth and analyzed the relationship between TRIP-1 overexpression and mineralized matrix formation. Results demonstrate that TRIP-1 was predominantly expressed by osteoblasts, odontoblasts, and chondrocytes in these tissues. Interestingly, TRIP-1 was also localized in the extracellular matrix of bone and at the mineralization front in dentin, suggesting that TRIP-1 is secreted by nonclassical secretory mechanisms, as it is devoid of a signal peptide. In vitro nucleation studies demonstrate a role for TRIP-1 in nucleating calcium phosphate polymorphs. Overexpression of TRIP-1 favored osteoblast differentiation of undifferentiated mesenchymal cells with an increase in mineralized matrix formation. These data indicate an unexpected role for TRIP-1 during development of bone, teeth, and cartilage.


Assuntos
Osso e Ossos/metabolismo , Calcificação Fisiológica , Fator de Iniciação 3 em Eucariotos/metabolismo , Dente/metabolismo , Linhagem Celular , Humanos , Imuno-Histoquímica
14.
J Biol Chem ; 287(8): 5211-24, 2012 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-22134916

RESUMO

Dentin phosphoprotein (DPP), a major noncollagenous protein of the dentin matrix, is a highly acidic protein that binds Ca(2+) avidly and is thus linked to matrix mineralization. Here, we demonstrate that the RGD domain in DPP can bind to integrins on the cell surface of undifferentiated mesenchymal stem cells and pulp cells. This coupling generates intracellular signals that are channeled along cytoskeletal filaments and activate the non-receptor tyrosine kinase focal adhesion kinase, which plays a key role in signaling at sites of cellular adhesion. The putative focal adhesion kinase autophosphorylation site Tyr(397) is phosphorylated during focal adhesion assembly induced by DPP on the substrate. We further demonstrate that these intracellular signals propagate through the cytoplasm and activate anchorage-dependent ERK signaling. Activated ERK translocates to the nucleus and phosphorylates the transcription factor ELK-1, which in turn coordinates the expression of downstream target genes such as DMP1 and dentin sialoprotein (DSP). These studies suggest a novel paradigm demonstrating that extracellular DPP can induce intracellular signaling that can be propagated to the nucleus and thus alter gene activities.


Assuntos
Proteínas da Matriz Extracelular/farmacologia , Integrinas/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Fosfoproteínas/farmacologia , Sialoglicoproteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Polpa Dentária/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Camundongos , Minerais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Odontoblastos/metabolismo , Paxilina/metabolismo , Fosfoproteínas/química , Estrutura Terciária de Proteína , Sialoglicoproteínas/química , Proteínas Elk-1 do Domínio ets/metabolismo
15.
Cells Tissues Organs ; 194(2-4): 255-60, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21546758

RESUMO

DMP1 has been shown to play many roles in osteogenesis. We recently demonstrated that calcium-mediated stress kinase activation by DMP1 leads to osteoblast differentiation. In this study we demonstrate that DMP1 can also activate the extracellular signal-regulated kinase (ERK)-MAPK pathway. This activation was mediated through the RGD integrin-binding domain in DMP1. Further, we demonstrate that Runx2, an essential transcription factor, is stimulated by the ERK-MAPK pathway.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/citologia , Osteoblastos/enzimologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/química , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oligopeptídeos/metabolismo , Osteoblastos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
16.
J Biol Chem ; 286(11): 8729-39, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21239500

RESUMO

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.


Assuntos
Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Osteoblastos/metabolismo , Crânio/metabolismo , Animais , Fosfatos de Cálcio/metabolismo , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Endocitose/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Choque Térmico/genética , Camundongos , Osteoblastos/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Ratos , Crânio/citologia
17.
J Biol Chem ; 285(47): 36339-51, 2010 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-20841352

RESUMO

Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.


Assuntos
Cálcio/metabolismo , Diferenciação Celular , Proteínas da Matriz Extracelular/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Células Cultivadas , Chaperona BiP do Retículo Endoplasmático , Proteínas da Matriz Extracelular/genética , Imunofluorescência , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/genética , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Crânio/citologia , Crânio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
J Histochem Cytochem ; 57(3): 227-37, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19001636

RESUMO

Formation of bone and dentin are classical examples of matrix-mediated mineralization. The mineral phase is essentially the same in these two tissues and primarily consists of a carbonated hydroxyapatite, but the difference lies in the crystal size and shape. There are three components that are necessary for proper mineralization, namely the proper synthesis and secretion of the non-collagenous proteins (NCPs), self-assembly of the collagenous matrix, and delivery of calcium and phosphate ions to the extracellular matrix. Three major NCPs present in the dentin matrix are dentin matrix protein 1 (DMP1), dentin phosphophorin (DPP), and dentin sialoprotein (DSP). In this study, we show the temporal and spatial localization of these NCPs and correlate their expression with the presence of collagenous matrix and calcified deposits in developing mouse incisors and molars. DMP1, an acidic protein, is present predominantly at the mineralization front and in the nucleus of undifferentiated preodontoblast cells. DPP, the major NCP, is present in large amounts at the mineralization front and might function to regulate the size of the growing hydroxyapatite crystals. For the first time, we report the localization of DPP in the nucleus of preodontoblast cells, suggesting a signaling function during the odontoblast differentiation process. DSP is localized predominantly in the dentinal tubules at the site of peritubular dentin, which is highly mineralized in nature. Thus, the precise localization of DMP1, DPP, and DSP in the dentin tissue suggests that a concerted effort between several NCPs is necessary for dentin formation.


Assuntos
Proteínas da Matriz Extracelular/biossíntese , Precursores de Proteínas/biossíntese , Dente/metabolismo , Animais , Animais Recém-Nascidos , Calcificação Fisiológica , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Fosfoproteínas , Sialoglicoproteínas , Dente/embriologia , Dente/crescimento & desenvolvimento
19.
J Biol Chem ; 283(44): 29658-70, 2008 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-18757373

RESUMO

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant K(D)=85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Proteínas de Choque Térmico/fisiologia , Chaperonas Moleculares/fisiologia , Fosfoproteínas/fisiologia , Sequência de Aminoácidos , Animais , Desenvolvimento Ósseo , Endocitose , Proteínas da Matriz Extracelular/química , Proteínas de Choque Térmico/metabolismo , Humanos , Microscopia Confocal , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/química , Ratos , Proteínas Recombinantes/química , Dente/embriologia
20.
J Biol Chem ; 282(21): 15357-65, 2007 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-17369251

RESUMO

Formation of calcified tissues is a well regulated process. In dentin, the odontoblasts synthesize several biomolecules that function as nucleators or inhibitors of mineralization. To identify genes that are odontoblast-specific, a subtractive hybridization technique was employed that resulted in the identification of a previously undescribed novel gene synthesized by the odontoblasts. Based on the nomenclature in our laboratory, this gene has been named dentin matrix protein 4 (DMP4). The protein encoded by mouse DMP4 cDNA contained 579 amino acids, including a 26-amino acid signal peptide. Analysis of the protein sequence demonstrated the presence of a Greek key calcium-binding domain and one conserved domain of unknown function in all the species examined thus far. Calcium binding property was confirmed by (45)Ca binding assays and the corresponding change in conformation by far-ultraviolet circular dichroism. Northern analysis demonstrated high expression levels of a single 3-kb mRNA transcript in tooth, whereas low expression levels were detected in other tissues. In situ hybridization analysis showed high expression levels of DMP4 in odontoblasts and low levels in osteoblasts and ameloblasts during tooth development. Gain and loss of function experiments demonstrated that DMP4 had the potential to differentiate mesenchymal precursor cells into functional odontoblast-like cells.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Odontoblastos/metabolismo , Ameloblastos/citologia , Ameloblastos/metabolismo , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Transformada , Dicroísmo Circular , DNA Complementar/genética , Proteínas da Matriz Extracelular/genética , Células-Tronco Mesenquimais/citologia , Camundongos , Odontoblastos/citologia , Especificidade de Órgãos/fisiologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Ligação Proteica/genética , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Dente/citologia , Dente/metabolismo
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