Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
Mais filtros












Intervalo de ano de publicação
1.
J Oral Biosci ; 66(1): 151-159, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38030062

RESUMO

OBJECTIVES: This study aimed to clarify the molecular mechanism underlying the higher invasion and metastasis abilities of LMF4 cells than those of HSC-3 cells by comparing the expression levels of the tumor suppressor factor, cell adhesion molecule 1 (CADM1). METHODS: We explored 1) whether CADM1 expression level was downregulated in LMF4 cells compared with HSC-3 cells, 2) whether CADM1 expression knockdown increased the expression levels of matrix metalloproteinases (MMPs), 3) the exact cellular signaling pathways responsible for increased MMP expression after knockdown of CADM1 expression, and 4) whether disruption of CADM1-dependent HSC-3 cell adhesion increased the migratory and invasive activities of HSC-3 cells. RESULTS: CADM1 expression was lower in the LMF4 than in the HSC-3 cells. The knockdown of CADM1 increased the expression of MMP-2 and MMP-9 in HSC-3 cells. In addition, the upregulation of MMP-2 expression after CADM1 knockdown was abrogated by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 and the phosphoinositide 3-kinase (PI3K) inhibitor LY294002. The upregulation of MMP-9 expression after the knockdown of CADM1 was abrogated by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the p38 MAP kinase (MAPK) inhibitor SB203580 and LY294002. Anti-CADM1 neutralizing antibody evoked migratory and invasive abilities of HSC-3 cells. CONCLUSION: The disruption of CADM1-dependent cell-cell adhesion in human oral squamous cell carcinoma cells resulted in tumor progression, possibly through an increase in MMP-2 expression in a MEK/PI3K-dependent manner and an increase in MMP-9 expression in a JNK/p38 MAPK/PI3K-dependent manner.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adesão Celular/genética , Neoplasias Bucais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Molécula 1 de Adesão Celular/genética , Molécula 1 de Adesão Celular/metabolismo
2.
J Oral Biosci ; 65(1): 97-103, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36584898

RESUMO

OBJECTIVES: Temporomandibular joint osteoarthritis (TMJ-OA) is a multifactorial disease caused by inflammation and oxidative stress. It has been hypothesized that mechanical stress-induced injury of TMJ tissues induces the generation of reactive oxygen species (ROS), such as hydroxyl radical (OH∙), in the synovial fluid (SF). In general, the overproduction of ROS contributes to synovial inflammation and dysfunction of the subchondral bone in OA. However, the mechanism by which ROS-injured synoviocytes recruit inflammatory cells to TMJ-OA lesions remains unclear. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of chemoattractant molecules. The phosphorylation levels of intracellular signaling molecules were evaluated using western blot analysis. RESULTS: Hydrogen peroxide (H2O2) treatment significantly promoted mRNA expression of neutrophil chemoattractant CXCL15/Lungkine in a dose-dependent manner (100-500 µM) in fibroblast-like synoviocytes (FLSs) derived from mouse TMJ. H2O2 (500 µM) significantly upregulated the phosphorylation of extracellular signal-regulated kinase (ERK)1 and ERK2 in FLSs. Intriguingly, the mitogen-activated protein (MAP)/ERK kinase (MEK) inhibitor U0126 (10 µM) nullified H2O2-induced increase in CXCL15/Lungkine mRNA expression. Additionally, H2O2 (500 µM) administration significantly upregulated OH∙ production in FLSs, as assessed by live-cell permeant fluorescent probe targeted against OH∙ under fluorescence microscopy. Furthermore, the ROS inhibitor N-acetyl-l-cysteine (5 mM) partially but significantly reversed H2O2-mediated phosphorylation of ERK1/2. CONCLUSIONS: H2O2-induced oxidative stress promoted the expression of CXCL15/Lungkine mRNA in a MEK/ERK-dependent manner in mouse TMJ-derived FLSs, suggesting that FLSs recruit neutrophils to TMJ-OA lesions through the production of CXCL15/Lungkine and exacerbate the local inflammatory response.


Assuntos
Osteoartrite , Sinoviócitos , Animais , Camundongos , Fatores Quimiotáticos/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Peróxido de Hidrogênio/efeitos adversos , Peróxido de Hidrogênio/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/patologia
3.
Int J Mol Sci ; 21(22)2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207747

RESUMO

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase-substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resposta ao Choque Frio/fisiologia , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteômica
4.
Exp Ther Med ; 20(3): 1967-1974, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32782506

RESUMO

Osteoarthritis (OA)-related fibrosis is a possible cause of temporomandibular joint (TMJ) stiffness. However, the molecular mechanisms underlying the fibrogenic activity in fibroblast-like synoviocytes (FLSs) remain to be clarified. The present study examined the effects of receptor tyrosine kinase (RTK) ligands, such as fibroblast growth factor (FGF)-1 and epidermal growth factor (EGF), on myofibroblastic differentiation of the FLS cell line FLS1, which is derived from the mouse TMJ. The present study revealed that both FGF-1 and EGF dose-dependently suppressed the expression of the myofibroblast (MF) markers, including α-smooth muscle actin (α-SMA) and type I collagen, in FLS1 cells. Additionally, both FGF-1 and EGF activated extracellular signal-regulated kinase (ERK) in FLS1 cells. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 abrogated the FGF-1- and EGF-mediated suppression of MF marker expression. On the other hand, inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, also suppressed the expression of MF markers in FLS1 cells. Importantly, U0126 abrogated the inflammatory cytokine-mediated suppression of MF marker expression. Interestingly, RTK ligands and inflammatory cytokines additively suppressed the expression of type I collagen. These results suggested that RTK ligands and inflammatory cytokines cooperatively inhibited the fibrogenic activity in FLSs derived from the TMJ in a MEK/ERK-dependent manner. The present findings partially clarify the molecular mechanisms underlying the development of OA-related fibrosis in the TMJ and may aid in identifying therapeutic targets for this condition. Additionally, FGF-1 and EGF could be therapeutically utilized to prevent OA-related fibrosis around the inflammatory TMJ.

5.
Oncol Lett ; 20(1): 474-482, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32565972

RESUMO

Squamous cell carcinoma (SCC) is the most frequent cancer that develops in the oral cavity. Epithelial-mesenchymal transition (EMT) is known to play an important role in the process of metastasis of SCC cells. In our previous study, we demonstrated that TGF-ß1 induced EMT in the human oral SCC (hOSCC) cell line HSC-4. We also found that Slug plays an important role in suppressing E-cadherin expression and promotion of the migratory activity of HSC-4 cells. However, we also demonstrated that Slug does not participate in upregulation of N-cadherin expression, suggesting that EMT-related transcription factors other than Slug also play an important role in the process. In the present study, we aimed to elucidate how the transcription factor Sox9 affects the TGF-ß1-induced upregulation of N-cadherin expression in HSC-4 cells. We found that TGF-ß1 upregulated Sox9 expression in HSC-4 cells. In addition, Sox9 siRNA significantly abrogated the TGF-ß1-induced upregulation of N-cadherin expression and inhibited the TGF-ß1-promoted migratory activity in HSC-4 cells. We also demonstrated that TGF-ß1 upregulated the phosphorylation status of Sox9 and then promoted nuclear translocation of Sox9 from the cytoplasm, possibly resulting in an increase in N-cadherin expression. The cyclic AMP-dependent protein kinase A inhibitor H-89, which is known to suppress phosphorylation of Sox9, significantly abrogated the TGF-ß1-induced upregulation of N-cadherin expression. These results suggested that TGF-ß1 induced N-cadherin expression by upregulating Sox9 expression and promoting its nuclear translocation, which results in EMT progression in hOSCC cells.

6.
Int J Mol Med ; 42(3): 1484-1494, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29901090

RESUMO

Mechanosensitive (MS) neurons in the periodontal ligament (PDL) pass information to the trigeminal ganglion when excited by mechanical stimulation of the tooth. During occlusal tooth trauma of PDL tissues, MS neurons are injured, resulting in atrophic neurites and eventual degeneration of MS neurons. Nerve growth factor (NGF), a neurotrophic factor, serves important roles in the regeneration of injured sensory neurons. In the present study, the effect of pro­inflammatory cytokines, including interleukin 1ß (IL­1ß) and tumor necrosis factor α (TNF­α), on transforming growth factor ß1 (TGF­ß1)­induced NGF expression was evaluated in rat PDL­derived SCDC2 cells. It was observed that TGF­ß1 promoted NGF expression via Smad2/3 and p38 mitogen­activated protein kinase (MAPK) activation. IL­1ß and TNF­α suppressed the TGF­ß1­induced activation of Smad2/3 and p38 MAPK, resulting in the abrogation of NGF expression. NGF secreted by TGF­ß1­treated SCDC2 cells promoted neurite extension and the expression of tyrosine hydroxylase, a rate­limiting enzyme in dopamine synthesis in rat pheochromocytoma PC12 cells. These results suggested that pro­inflammatory cytokines suppressed the TGF­ß­mediated expression of NGF in PDL­derived fibroblasts through the inactivation of TGF­ß­induced Smad2/3 and p38 MAPK signaling, possibly resulting in the disturbance of the regeneration of injured PDL neurons.


Assuntos
Fibroblastos/metabolismo , Interleucina-1beta/farmacologia , Fator de Crescimento Neural/metabolismo , Ligamento Periodontal/citologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Fibroblastos/efeitos dos fármacos , Humanos , Fator de Crescimento Neural/genética , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Células PC12 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo
7.
Biosci Rep ; 38(2)2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29535277

RESUMO

Many inflammatory cells are known to be home to inflamed temporomandibular joint (TMJ) tissues by stimulation with cytokines and chemokines produced by inflammatory lesions in the TMJ. However, how the inflammatory cells affect the progression of inflammation in TMJ synovial tissues after their homing to inflamed TMJ site is still uncertain. Here, we isolated and cultured TMJ synoviocyte-like cells (TMJSCs) from murine TMJ tissues. We demonstrated that interleukin 1ß (IL-1ß) up-regulated expression of monocyte chemoattractant protein 1 (MCP-1) in TMJSCs. In addition, we found that IL-1ß-treated TMJSCs strongly promoted migratory activity of mouse monocyte/macrophage RAW264.7 cells through secretion of MCP-1. On the other hand, IL-1ß up-regulated expression levels of intracellular adhesion molecule 1 (ICAM-1), a leukocyte adhesion ligand in TMJSCs. In addition, IL-1ß promoted cell-cell adhesion between TMJSCs and RAW264.7 cells. Intriguingly, we also found that cell-cell interactions mediated through soluble factors other than IL-1ß and cell-cell adhesion molecules between IL-1ß-stimulated TMJSCs and RAW264.7 cells synergistically augmented secretion of MCP-1 from these cells. Therefore, these results suggested that the IL-1ß-induced recruitment of monocyte/macrophage lineage cells to inflamed synovial membranes in TMJ was further augmented by the cell-cell interaction-induced secretion of MCP-1 from the inflammation site, possibly resulting in prolonged inflammatory responses in TMJ synovial tissue.


Assuntos
Comunicação Celular/imunologia , Quimiocina CCL2/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Sinoviócitos/imunologia , Articulação Temporomandibular/imunologia , Animais , Inflamação/imunologia , Inflamação/patologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Monócitos/patologia , Células RAW 264.7 , Sinoviócitos/patologia , Articulação Temporomandibular/patologia
8.
Mol Med Rep ; 17(3): 3448-3454, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29257332

RESUMO

Surface pre-reacted glass­ionomer (S­PRG)-containing dental materials, including composite and coating resins have been used for the restoration and/or prevention of dental cavities. S­PRG is known to have the ability to release aluminum, boron, fluorine, silicon, and strontium ions. Aluminum ions are known to be inhibitors whereas boron, fluorine, silicon, and strontium ions are known to be promoters of mineralization, via osteoblasts. However, it remains to be clarified how an aqueous eluate obtained from S­PRG containing these ions affects the ability of mesenchymal stem cells (MSCs), which are known to be present in dental pulp and bone marrow, to differentiate into osteogenic cell types. The present study demonstrated that 200­ to 1,000­fold­diluted aqueous eluates obtained from S­PRG significantly upregulated the mRNA expression level of the osteogenic differentiation marker alkaline phosphatase in human MSCs (hMSCs) without exhibiting the cytotoxic effect. In addition, the 500­ to 1,000­fold­diluted aqueous eluates obtained from S­PRG significantly and clearly promoted mineralization of the extracellular matrix of hMSCs. It was additionally demonstrated that hMSCs cultured on the cured resin composites containing S­PRG fillers exhibited osteogenic differentiation in direct correlation with the weight percent of S­PRG fillers. These results strongly suggested that aqueous eluates of S­PRG fillers promoted hard tissue formation by hMSCs, implicating that resins containing S­PRG may act as a useful biomaterial to cover accidental exposure of dental pulp.


Assuntos
Resinas Acrílicas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Dióxido de Silício/farmacologia , Resinas Acrílicas/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Materiais Dentários/química , Matriz Extracelular/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , RNA Mensageiro/metabolismo , Dióxido de Silício/química , Regulação para Cima/efeitos dos fármacos , Água/química
9.
Exp Cell Res ; 358(2): 411-420, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28712928

RESUMO

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.


Assuntos
Medula Óssea/metabolismo , Comunicação Celular , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Anti-Inflamatórios/farmacologia , Diferenciação Celular/imunologia , Hipóxia Celular , Células Cultivadas , Técnicas de Cocultura , Macrófagos/imunologia , Camundongos , Molécula 1 de Adesão de Célula Vascular/metabolismo
10.
Int J Mol Med ; 39(4): 799-808, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28259960

RESUMO

Malocclusion caused by abnormal jaw development or muscle overuse during mastication results in abnormal mechanical stress to the tissues surrounding the temporomandibular joint (TMJ). Excessive mechanical stress against soft and hard tissues around the TMJ is involved in the pathogenesis of inflammatory diseases, including osteoarthritis (OA). OA-related fibrosis is a possible cause of joint stiffness in OA. However, cellular and molecular mechanisms underlying fibrosis around the TMJ remain to be clarified. Here, we established a cell line of fibroblast­like synoviocytes (FLSs) derived from the mouse TMJ. Then, we examined whether the Rho­associated coiled­coil forming kinase (ROCK)/actin/myocardin-related transcription factor (MRTF) gene regulatory axis positively regulates the myofibroblast (MF) differentiation status of FLSs. We found that i) FLSs extensively expressed the MF markers α­smooth muscle actin (α­SMA) and type I collagen; and ii) an inhibitor against the actin­polymerizing agent ROCK, Y­27632; iii) an actin-depolymerizing agent cytochalasin B; iv) an inhibitor of the MRTF/serum response factor­regulated transcription, CCG­100602, clearly suppressed the mRNA levels of α­SMA and type I collagen in FLSs; and v) an MF differentiation attenuator fibroblast growth factor­1 suppressed filamentous actin formation and clearly suppressed the mRNA levels of α-SMA and type I collagen in FLSs. These results strongly suggest that the ROCK/actin/MRTF axis promotes the fibrogenic activity of synoviocytes around the TMJ. Our findings partially clarify the molecular mechanisms underlying the emergence of TMJ­OA and may aid in identifying drug targets for treating this condition at the molecular level.


Assuntos
Actinas/metabolismo , Osteoartrite/metabolismo , Transdução de Sinais , Sinoviócitos/metabolismo , Articulação Temporomandibular/metabolismo , Transativadores/metabolismo , Animais , Feminino , Má Oclusão/metabolismo , Má Oclusão/patologia , Camundongos , Osteoartrite/patologia , Estresse Mecânico , Sinoviócitos/patologia , Articulação Temporomandibular/patologia , Quinases Associadas a rho
11.
Oncol Rep ; 37(2): 713-720, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28035402

RESUMO

Squamous cell carcinoma is the most common cancer in the oral cavity. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) promotes the epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (hOSCC) cells; however, it remains to be clarified whether the TGF-ß superfamily member bone morphogenetic protein (BMP) affects this process in hOSCC cells. Here, we examined the independent and collective effects of TGF-ß1 and BMP-2 on EMT and mesenchymal­epithelial transition (MET) in a panel of four hOSCC cell lines. Notably, we found that HSC-4 cells were the most responsive to BMP-2 stimulation, which resulted in the upregulation of Smad1/5/9 target genes such as the MET inducers ID1 and cytokeratin 9 (CK9). Furthermore, BMP-2 downregulated the mesenchymal marker N-cadherin and the EMT inducer Snail, but upregulated epithelial CK9 expression, indicating that BMP-2 prefers to induce MET rather than EMT. Moreover, TGF-ß1 dampened BMP-2-induced epithelial gene expression by inhibiting Smad1/5/9 expression and phosphorylation. Functional analysis revealed that TGF-ß1 and BMP-2 significantly enhanced HSC-4 cell migration and proliferation, respectively. Collectively, these data suggest that TGF-ß positively regulates hOSCC invasion in the primary tumor, whereas BMP-2 facilitates cancer cell colonization at secondary metastatic sites. Thus, the invasive and metastatic characteristics of hOSCC appear to be reciprocally regulated by BMP and TGF-ß.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Bucais/patologia , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Smad/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismo , Células Tumorais Cultivadas
12.
Int J Mol Med ; 38(1): 139-47, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27176567

RESUMO

Bisphosphonates (BPs) are analogues of pyrophosphate that are known to prevent bone resorption by inhibiting osteoclast activity. Nitrogen-containing BPs, such as zoledronic acid (ZA), are widely used in the treatment of osteoporosis and bone metastasis. However, despite having benefits, ZA has been reported to induce BP-related osteonecrosis of the jaw (BRONJ) in cancer patients. The molecular pathological mechanisms responsible for the development of BRONJ, including necrotic bone exposure after tooth extraction, remain to be elucidated. In this study, we examined the effects of ZA on the transforming growth factor-ß (TGF­ß)-induced myofibroblast (MF) differentiation of human gingival fibroblasts (hGFs) and the migratory activity of hGFs, which are important for wound closure by fibrous tissue formation. The ZA maximum concentration in serum (Cmax) was found to be approximately 1.47 µM, which clinically, is found after the intravenous administration of 4 mg ZA, and ZA at this dose is considered appropriate for the treatment of cancer bone metastasis or bone diseases, such as Erdheim-Chester disease. At Cmax, ZA significantly suppressed i) the TGF­ß-induced promotion of cell viability, ii) the TGF­ß-induced expression of MF markers such as α-smooth muscle actin (α-SMA) and type I collagen, iii) the TGF­ß-induced migratory activity of hGFs and iv) the expression level of TGF­ß type I receptor on the surfaces of hGFs, as well as the TGF­ß-induced phosphorylation of Smad2/3. Thus, ZA suppresses TGF­ß-induced fibrous tissue formation by hGFs, possibly through the inhibition of Smad­dependent signal transduction. Our findings partly elucidate the molecular mechanisms underlying BRONJ and may prove to be beneficial to the identification of drug targets for the treatment of this symptom at the molecular level.


Assuntos
Difosfonatos/farmacologia , Fibroblastos/patologia , Gengiva/patologia , Imidazóis/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Ácido Zoledrônico
13.
J Biochem ; 159(6): 631-40, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26861993

RESUMO

Molecular mechanism underlying the invasion of oral cancer cells remains to be clarified. We previously demonstrated that transforming growth factor-ß1 (TGF-ß1) induces the expression of mesenchymal markers in human oral squamous cell carcinoma HSC-4 cells. Intriguingly, the expression of the epithelial-mesenchymal transition-related transcription factor Slug was also significantly upregulated upon TGF-ß1 stimulation. However, the mechanism by which Slug transduces the TGF-ß1-induced signal to enhance the invasiveness of HSC-4 cells is poorly understood. Proteomic analysis revealed that the expression of matrix metalloproteinase (MMP)-10 was upregulated in TGF-ß1-stimulated cells. Additionally, a Boyden chamber assay revealed that the TGF-ß1-induced increase in invasiveness of HSC-4 cells was significantly inhibited by MMP-10 small interfering RNA (siRNA). Intriguingly, Slug siRNA suppressed TGF-ß1-induced expression of MMP-10. These results suggest that TGF-ß1 induces invasion in HSC-4 cells through the upregulation of MMP-10 expression in a Slug-dependent manner. On the other hand, Slug siRNA suppressed TGF-ß1-induced Wnt-5b expression. Wnt-5b significantly induced MMP-10 expression, whereas Wnt-5b siRNA suppressed the TGF-ß1-induced increase in invasiveness, suggesting that TGF-ß1-induced expression of MMP-10 and the resulting upregulation of invasiveness are mediated by Wnt-5b. Overall, these results suggest that TGF-ß1 stimulates HSC-4 cell invasion through the Slug/Wnt-5b/MMP-10 signalling axis.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Metaloproteinase 10 da Matriz/metabolismo , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Wnt/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/patologia , Invasividade Neoplásica
14.
J Appl Oral Sci ; 23(1): 49-55, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25760267

RESUMO

OBJECTIVE: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. MATERIAL AND METHODS: Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS: Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. CONCLUSION: The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues.


Assuntos
Proteínas do Esmalte Dentário/farmacologia , Células Epiteliais/efeitos dos fármacos , Periodonto/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Gengiva/citologia , Gengiva/efeitos dos fármacos , Regeneração Tecidual Guiada Periodontal/métodos , Humanos , Camundongos , Periodontite/tratamento farmacológico , Valores de Referência , Reprodutibilidade dos Testes , Coloração pela Prata , Fatores de Tempo
15.
J. appl. oral sci ; 23(1): 49-55, Jan-Feb/2015. tab, graf
Artigo em Inglês | LILACS, BBO | ID: lil-741590

RESUMO

Objective Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration. However, the effects of EMD on gingival epithelial cells during regeneration of periodontal tissues are unclear. In this in vitro study, we purified ameloblastin from EMD and investigated its biological effects on epithelial cells. Material and Methods Bioactive fractions were purified from EMD by reversed-phase high-performance liquid chromatography using hydrophobic support with a C18 column. The mouse gingival epithelial cell line GE-1 and human oral squamous cell carcinoma line SCC-25 were treated with purified EMD fraction, and cell survival was assessed with a WST-1 assay. To identify the proteins in bioactive fractions of EMD, we used proteome analysis with two-dimensional gel electrophoresis followed by identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Results Purified fractions from EMD suppressed proliferation of GE-1 and SCC-25. LC-MS/MS revealed that ameloblastin in EMD is the component responsible for inhibiting epithelial cell proliferation. The inhibitory effect of ameloblastin on the proliferation of GE-1 and SCC-25 was confirmed using recombinant protein. Conclusion The inhibitory effects of EMD on epithelial cell proliferation are caused by the biological activities of ameloblastin, which suggests that ameloblastin is involved in regulating epithelial downgrowth in periodontal tissues. .


Assuntos
Humanos , Animais , Camundongos , Proteínas do Esmalte Dentário/farmacologia , Células Epiteliais/efeitos dos fármacos , Periodonto/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Gengiva/citologia , Gengiva/efeitos dos fármacos , Regeneração Tecidual Guiada Periodontal/métodos , Periodontite/tratamento farmacológico , Valores de Referência , Reprodutibilidade dos Testes , Coloração pela Prata , Fatores de Tempo
16.
Insect Biochem Mol Biol ; 55: 61-9, 2014 12.
Artigo em Inglês | MEDLINE | ID: mdl-25460512

RESUMO

The laccase in the pupal cuticle of the silkworm, Bombyx mori, is thought to accumulate as an inactive precursor that can be activated stage-dependently. In this study we isolated an 81-kDa laccase from cuticular extract of B. mori that was prepared by digestion of the pupal cuticles with α-chymotrypsin. The mass spectrometric analysis of the purified protein indicates that this 81-kDa laccase is a product of the Bombyx laccase2 gene. The purified 81-kDa laccase (α-chymotrypsin-solubilized Bombyx laccase2: Bm-clac2) has an N-terminal sequence of RNPADS that corresponds to Arg146 to Ser151 of the deduced protein sequence of Bmlaccase2 cDNA, indicating that Bm-clac2 lacks the N-terminal part upstream from residue Arg146. Bm-clac2 shows enzymatic activity, but its specific activity is increased around 17-fold after treatment with trypsin, which involves cleavage of peptide bonds at the C-terminal region. We also found that the activity of Bm-clac2 is increased in the presence of isopropanol. In previous reports, proteolytic processing has been hypothesized as a system for laccase activation in vivo, but the present result implies that this type of processing is not the only way to convert Bm-clac2 to the high-activity enzyme.


Assuntos
Bombyx/enzimologia , Lacase/metabolismo , Muda , Sequência de Aminoácidos , Animais , Bombyx/genética , Quimotripsina , Eletroforese em Gel de Poliacrilamida , Lacase/genética , Lacase/isolamento & purificação , Espectrometria de Massas , Dados de Sequência Molecular , Pupa/enzimologia
17.
Cell Physiol Biochem ; 32(4): 899-914, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24217646

RESUMO

BACKGROUND/AIMS: Remodeling of fibrous and vascular tissues in the periodontal ligament (PDL) around the tooth root was observed during tooth movement by orthodontic force application. We previously demonstrated that a single cell-derived culture (SCDC) of primarily cultured PDL fibroblasts, called SCDC2, has an endothelial progenitor cell (EPC)-like character and can form endothelial cell (EC) marker-positive blood vessel-like structures. However, the types of molecular mechanisms that control the in vivo kinetic properties and the differentiation of the PDL-derived EPC-like cells into myofibroblasts (MFs), which are known to expand fibrous tissues, require clarification. METHODS: Using specific mitogen activated protein kinase (MAPK) inhibitors, we examined how epidermal growth factor (EGF)-mediated MAPK signals affected the proliferation, migration, and MF differentiation of these cells. RESULTS: EGF induced SCDC2 cell proliferation in MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)- and c-Jun N-terminal kinase (JNK)-dependent manners. In addition, EGF suppressed the expression of MF differentiation markers in these cells in a MEK/ERK-dependent manner, and, moreover, stimulated the cell migration in a MEK/ERK-dependent manner. CONCLUSION: EGF regulates fibrous tissue remodeling in PDLs through MEK/ERK- and JNK-mediated signals by affecting the proliferation, migration, and MF differentiation of the PDL-derived EPC-like cells.


Assuntos
Células Endoteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ligamento Periodontal/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , Imunofluorescência , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Ratos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
18.
J Biochem ; 153(3): 303-15, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23248240

RESUMO

We investigated whether transforming growth factor (TGF)-ß1 promoted epithelial-mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-ß target genes expression were most clearly upregulated following TGF-ß1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-ß1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-ß1 stimulation. Interestingly, E-cadherin and ß-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-ß1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-ß1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-ß1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3ß1-targeted proteins were upregulated in TGF-ß1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and ß1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-ß1-induced cell migration. These results suggest that the EMT and integrin α3ß1/FAK pathway-mediated migration of TGF-ß1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.


Assuntos
Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Integrina alfa3beta1/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfa3beta1/genética , Microscopia Confocal , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad7/genética , Proteína Smad7/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética
19.
Int J Biol Sci ; 8(7): 1062-74, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22949889

RESUMO

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-ß (TGF-ß) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-ß signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-ß1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-ß1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-ß-induced Smad-dependent signaling, suppressed the TGF-ß1-induced growth inhibition and SMC markers expression, but did not the TGF-ß1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-ß1-induced downregulation of EC marker expression. In addition, the TGF-ß1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-ß1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-ß1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.


Assuntos
Células Endoteliais/citologia , Miócitos de Músculo Liso/metabolismo , Ligamento Periodontal/citologia , Proteínas Smad/metabolismo , Células-Tronco/citologia , Fator de Crescimento Transformador beta/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Imunofluorescência , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos
20.
J Plant Res ; 125(2): 311-6, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22246111

RESUMO

Early interactions between invading penetration hyphae of the pathogenic fungus Magnaporthe oryzae and rice cells occur at the apoplast, the free diffusional space outside the plasma membrane of leaves. After initial colonization, intercellular hyphae are again in intimate contact with the rice apoplast. While several studies have looked at proteomics in rice-Magnaporthe interactions, none have focused on apoplast localized proteins. We adjusted a protocol for intercellular washing fluids (IWF) to rice leaves infected with Magnaporthe oryzae for proteomic analysis. In our IWF extract, we identified several proteins associated with compatible or incompatible pathogen interactions. Three DUF26 domain proteins were identified as changing in abundance 12 h after inoculation, confirming DUF26 domain-containing proteins are among early, pathogen stress-responsive proteins induced by infection with Magnaporthe oryzae. A Magnaporthe cyclophilin, previously identified as a virulence factor was also identified in the intercellular washing fluid.


Assuntos
Líquido Extracelular/metabolismo , Proteínas Fúngicas/metabolismo , Magnaporthe/fisiologia , Oryza/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Eletroforese em Gel Bidimensional , Interações Hospedeiro-Patógeno , Espectrometria de Massas , Oryza/microbiologia , Oryza/fisiologia , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteoma , Proteômica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...