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1.
J Orofac Orthop ; 81(1): 10-21, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31591651

RESUMO

PURPOSE: Orthodontic treatment is based on the principle of force application to teeth and subsequently to the surrounding tissues and periodontal cells. Sequestosome 1 (SQSTM1) is a well-known marker for autophagy, which is an important cellular mechanism of adaptation to stress. The aim of this study was to analyze whether biomechanical loading conditions regulate SQSTM1 in periodontal cells and tissues, thereby providing further information on the role of autophagy in orthodontic tooth movement. METHODS: Periodontal ligament (PDL) fibroblasts were exposed to cyclic tensile strain of low magnitude (3%, CTSL), and the regulation of autophagy-associated targets was determined with an array-based approach. SQSTM1 was selected for further biomechanical loading experiments with dynamic and static tensile strain and assessed via real-time polymerase chain reaction (RT-PCR) and immunoblotting. Signaling pathways involved in SQSTM1 activation were analyzed by using specific inhibitors, including an autophagy inhibitor. Finally, SQSTM1 expression was analyzed in gingival biopsies and histological sections of rats in presence and absence of orthodontic forces. RESULTS: Multiple autophagy-associated targets were regulated by CTSL in PDL fibroblasts. All biomechanical loading conditions tested increased the SQSTM1 expression significantly. Stimulatory effects of CTSL on SQSTM1 expression were diminished by inhibition of the c­Jun N­terminal kinase (JNK) pathway and of autophagy. Increased SQSTM1 levels after CTSL were confirmed by immunoblotting. Orthodontic force application also led to significantly elevated SQTSM1 levels in the gingiva and PDL of treated animals as compared to control. CONCLUSIONS: Our in vitro and in vivo findings provide evidence of a role of SQSTM1 and thereby autophagy in orthodontic tooth movement.


Assuntos
Autofagia , Dente , Animais , Fenômenos Biomecânicos , Ligamento Periodontal , Ratos , Estresse Mecânico , Técnicas de Movimentação Dentária
2.
Clin Oral Investig ; 22(8): 2933-2941, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29442188

RESUMO

OBJECTIVES: Damage-regulated autophagy modulator (DRAM) 1 is a p53 target gene with possible involvement in oral inflammation and infection. This study sought to examine the presence and regulation of DRAM1 in periodontal diseases. MATERIAL AND METHODS: In vitro, human periodontal ligament fibroblasts were exposed to interleukin (IL)-1ß and Fusobacterium nucleatum for up to 2 days. The DRAM1 synthesis and its regulation were analyzed by real-time PCR, immunocytochemistry, and ELISA. Expressions of other autophagy-associated genes were also studied by real-time PCR. In vivo, synthesis of DRAM1 in gingival biopsies from rats and patients with and without periodontal disease was examined by real-time PCR and immunohistochemistry. For statistics, ANOVA and post-hoc tests were applied (p < 0.05). RESULTS: In vitro, DRAM1 was significantly upregulated by IL-1ß and F. nucleatum over 2 days and a wide range of concentrations. Additionally, increased DRAM1 protein levels in response to both stimulants were observed. Autophagy-associated genes ATG3, BAK1, HDAC6, and IRGM were also upregulated under inflammatory or infectious conditions. In vivo, the DRAM1 gene expression was significantly enhanced in rat gingival biopsies with induced periodontitis as compared to control. Significantly increased DRAM1 levels were also detected in human gingival biopsies from sites of periodontitis as compared to healthy sites. CONCLUSION: Our data provide novel evidence that DRAM1 is increased under inflammatory and infectious conditions in periodontal cells and tissues, suggesting a pivotal role of DRAM1 in oral inflammation and infection. CLINICAL RELEVANCE: DRAM1 might be a promising target in future diagnostic and treatment strategies for periodontitis.


Assuntos
Fibroblastos/efeitos dos fármacos , Fusobacterium nucleatum , Proteínas de Membrana/biossíntese , Adolescente , Animais , Autofagia , Biópsia , Criança , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Interleucina-1beta/farmacologia , Ligamento Periodontal/citologia , Periodontite/microbiologia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
3.
Clin Oral Investig ; 20(7): 1781-9, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26620730

RESUMO

OBJECTIVES: Different studies suggest that inflammation as well as hypoxia leads to an increase of p53 protein levels. However, the implication of p53 during oral inflammatory processes is still unknown. The aim of this study was therefore to investigate the effect of hypoxia and inflammation on p53 regulation in human periodontium in vitro and in vivo. MATERIALS AND METHODS: Under hypoxic and normoxic conditions, human primary periodontal ligament (PDL) fibroblasts (n = 9) were stimulated with lipopolysaccharides (LPS) from Porphyromonas gingivalis (P.g.), a periodontal pathogenic bacterium. After different time points, cell viability was tested; p53 gene expression, protein synthesis, and activation were measured using quantitative RT-PCR, immunoblotting, and immunofluorescence. Moreover, healthy and inflamed periodontal tissues were obtained from 12 donors to analyze p53 protein in oral inflammatory diseases by immunohistochemistry. RESULTS: LPS-P.g. and hypoxia initially induced a significant upregulation of p53 mRNA expression and p53 protein levels. Nuclear translocation of p53 after inflammatory stimulation supported these findings. Hypoxia first enhanced p53 levels, but after 24 h of incubation, protein levels decreased, which was accompanied by an improvement of PDL cell viability. Immunohistochemistry revealed an elevation of p53 immunoreactivity in accordance to the progression of periodontal inflammation. CONCLUSIONS: Our data indicate that p53 plays a pivotal role in PDL cell homeostasis and seems to be upregulated in oral inflammatory diseases. CLINICAL RELEVANCE: Upregulation of p53 may promote the destruction of periodontal integrity. A possible relationship with carcinogenesis may be discussed.


Assuntos
Fibroblastos/metabolismo , Ligamento Periodontal/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sobrevivência Celular , Imunofluorescência , Humanos , Hipóxia , Immunoblotting , Imuno-Histoquímica , Inflamação , Lipopolissacarídeos , Ligamento Periodontal/citologia , Porphyromonas gingivalis , Reação em Cadeia da Polimerase em Tempo Real
4.
Mediators Inflamm ; 2015: 438085, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25861162

RESUMO

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1ß, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1ß, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1ß, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Hipóxia/complicações , NF-kappa B/fisiologia , Doenças Periodontais/etiologia , Ligamento Periodontal/metabolismo , Porphyromonas gingivalis/patogenicidade , Humanos , Interleucina-1beta/genética , Lipopolissacarídeos/farmacologia , Metaloproteinase 1 da Matriz/genética , Ligamento Periodontal/citologia , Fator A de Crescimento do Endotélio Vascular/fisiologia
5.
Mediators Inflamm ; 2014: 986264, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25374447

RESUMO

Oxidative stress is characterized by an accumulation of reactive oxygen species (ROS) and plays a key role in the progression of inflammatory diseases. We hypothesize that hypoxic and inflammatory events induce oxidative stress in the periodontal ligament (PDL) by activating NOX4. Human primary PDL fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (LPS-PG), a periodontal pathogen bacterium under normoxic and hypoxic conditions. By quantitative PCR, immunoblot, immunostaining, and a specific ROS assay we determined the amount of NOX4, ROS, and several redox systems. Healthy and inflamed periodontal tissues were collected to evaluate NOX4 and redox systems by immunohistochemistry. We found significantly increased NOX4 levels after hypoxic or inflammatory stimulation in PDL cells (P < 0.001) which was even more pronounced after combination of the stimuli. This was accompanied by a significant upregulation of ROS and catalase (P < 0.001). However, prolonged incubation with both stimuli induced a reduction of catalase indicating a collapse of the protective machinery favoring ROS increase and the progression of inflammatory oral diseases. Analysis of inflamed tissues confirmed our hypothesis. In conclusion, we demonstrated that the interplay of NOX4 and redox systems is crucial for ROS formation which plays a pivotal role during oral diseases.


Assuntos
Lipopolissacarídeos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Ligamento Periodontal/metabolismo , Periodontite/etiologia , Porphyromonas gingivalis/patogenicidade , Catalase/metabolismo , Hipóxia Celular , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Imuno-Histoquímica , Lipopolissacarídeos/isolamento & purificação , NADPH Oxidase 4 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Periodontite/metabolismo , Periodontite/microbiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo
6.
Arch Oral Biol ; 55(3): 215-22, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20116783

RESUMO

OBJECTIVE: To characterize in vivo the role of IGF-I and its signalling, as an early reaction in the mechanotransduction process and to analyse changes of the local expression related to the magnitude of the applied force. MATERIALS AND METHODS: Forces of 0.1N, 0.25 N and 0.5 N were applied to move the right upper first molars of 12 anaesthetized rats mesially. These forces were kept constant for 4h. The untreated contralateral side served as a control. Paraffin-embedded sections of the resected jaws were prepared for immunohistochemistry to localize insulin-like growth factor-I (IGF-I), its receptor (IGF-IR), and insulin receptor substrate 1 (IRS1). Histomorphometric analysis was performed to count the percentage of immunoreactive cells in different parts of the periodontal ligament. RESULTS: IGF-I, IGF-IR and IRS1 positive cells were observed in the periodontal tissues of the control and loaded teeth. In the experimental group, the number of IGF-I-, IGF-IR- and IRS1-positive cells increased significantly on the tension side and decreased on the compression side. CONCLUSIONS: These data indicate a close relationship between mechanical loading of the PDL and the autocrine/paracrine expression of IGF components as an early step in the mechanotransduction process leading in the long term to an organized remodelling of the alveolar bone.


Assuntos
Proteínas Substratos do Receptor de Insulina/análise , Fator de Crescimento Insulin-Like I/análise , Ligamento Periodontal/citologia , Receptor IGF Tipo 1/análise , Técnicas de Movimentação Dentária , Processo Alveolar/citologia , Animais , Fenômenos Biomecânicos , Cemento Dentário/citologia , Fibroblastos/citologia , Masculino , Mecanotransdução Celular/fisiologia , Dente Molar/fisiologia , Fios Ortodônticos , Osteoblastos/citologia , Osteoclastos/citologia , Pressão , Ratos , Ratos Wistar , Estresse Mecânico , Fatores de Tempo , Torque , Transdutores
7.
J Periodontal Res ; 45(2): 246-54, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19778325

RESUMO

BACKGROUND AND OBJECTIVE: Cementum and bone are rather similar hard tissues, and osteocytes and cementocytes, together with their canalicular network, share many morphological and cell biological characteristics. However, there is no clear evidence that cementocytes have a function in tissue homeostasis of cementum comparable to that of osteocytes in bone. Recent studies have established an important role for the secreted glycoprotein sclerostin, the product of the SOST gene, as an osteocyte-derived signal to control bone remodelling. In this study, we investigated the expression of sclerostin in cementocytes in vivo as well as the expression of SOST and sclerostin in periodontal ligament cell cultures following induction of mineralization. MATERIAL AND METHOD: Immunolocalization of sclerostin was performed in decalcified histological sections of mouse and human teeth and alveolar bone. Additionally, periodontal ligament cells from human donors were cultured in osteogenic conditions, namely in the presence of dexamethasone, ascorbic acid and beta-glycerophosphate, for up to 3 wk. The induction of calcified nodules was visualized by von Kossa stain. SOST mRNA was detected by real-time PCR, and the presence of sclerostin was verified using immunohistochemistry and western blots. RESULTS: Expression of sclerostin was demonstrated in osteocytes of mouse and human alveolar bone. Distinct immunolocalization in the cementocytes was shown. In periodontal ligament cultures, following mineralization treatment, increasing levels of SOST mRNA as well as of sclerostin protein could be verified. CONCLUSION: The identification of SOST/sclerostin in cementocytes and mineralizing periodontal ligament cells adds to our understanding of the biology of the periodontium, but the functional meaning of these findings can only be unravelled after additional in vitro and in vivo studies.


Assuntos
Proteínas Morfogenéticas Ósseas/análise , Cemento Dentário/citologia , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Idoso , Processo Alveolar/citologia , Animais , Ácido Ascórbico/farmacologia , Western Blotting , Calcificação Fisiológica/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Criança , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Marcadores Genéticos , Glicerofosfatos/farmacologia , Glicoproteínas , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Pessoa de Meia-Idade , Osteócitos/citologia , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Reação em Cadeia da Polimerase , Adulto Jovem
8.
Cell Tissue Res ; 325(3): 469-79, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16670921

RESUMO

Periodontal ligament (PDL) cells exhibit several osteoblastic traits and are parathyroid hormone (PTH)-responsive providing evidence for a role of these cells in dental hard-tissue repair. To examine the hypothesis that PDL cells respond to PTH stimulation with changes in proliferation and apoptotic signaling through independent but convergent signaling pathways, PDL cells were cultured from human bicuspids obtained from six patients. PDL cells at different states of maturation were challenged with PTH(1-34) intermittently for 0, 1, or 24 h/cycle or exposed continuously. Specific inhibitors to protein kinases A and C (PKA, PKC) and the mitogen-activated protein kinase cascade (MAPK) were employed. At harvest, the cell number, BrdU incorporation, and DNA fragmentation were determined by means of cell counting and immunoassays. Intermittent PTH(1-34) caused a significant increase in cell number in confluent cells as opposed to a reduction in pre-confluent cells. In confluent cells, the effect resulted from a significant increase in proliferation, whereas DNA fragmentation was reduced when PTH(1-34) was administered for 1 h/cycle but increased after PTH(1-34) for 24 h/cycle. Inhibition of PKC inhibited PTH(1-34)-induced proliferation but enhanced apoptosis. Inhibition of PKA enhanced proliferation and DNA fragmentation. Similar results were obtained in less mature cells, although, in the presence of the PKA inhibitor, the PTH(1-34)-induced changes were more pronounced than in confluent cells. In the presence of the MAPK inhibitor, all of the parameters examined were reduced significantly in both maturation states. Thus, PTH(1-34) mediates proliferative and apoptotic signaling in human PDL cells in a maturation-state-dependent manner via PKC-dependent and PKA-dependent pathways.


Assuntos
Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Ligamento Periodontal/fisiologia , Proteína Quinase C/metabolismo , Adolescente , Dente Pré-Molar/citologia , Contagem de Células , Células Cultivadas , Criança , Humanos , Imunoensaio , Técnicas In Vitro , Modelos Biológicos , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Ligamento Periodontal/citologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
9.
Osteoarthritis Cartilage ; 14(10): 1023-32, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16731008

RESUMO

OBJECTIVES: Physical therapies are commonly used for limiting joint inflammation. To gain insight into their mechanisms of actions for optimal usage, we examined persistence of mechanical signals generated by cyclic tensile strain (CTS) in chondrocytes, in vitro. We hypothesized that mechanical signals induce anti-inflammatory and anabolic responses that are sustained over extended periods. METHODS: Articular chondrocytes obtained from rats were subjected to CTS for various time intervals followed by a period of rest, in the presence of interleukin-1beta (IL-1beta). The induction for cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-9, MMP-13 and aggrecan was analyzed by real-time polymerase chain reaction (PCR), Western blot analysis and immunofluorescence. RESULTS: Exposure of chondrocytes to constant CTS (3% CTS at 0.25 Hz) for 4-24 h blocked more than 90% (P<0.05) of the IL-1beta-induced transcriptional activation of proinflammatory genes, like iNOS, COX-2, MMP-9 and MMP-13, and abrogated inhibition of aggrecan synthesis. CTS exposure for 4, 8, 12, 16, or 20 h followed by a rest for 20, 16, 12, 8 or 4h, respectively, revealed that 8h of CTS optimally blocked (P<0.05) IL-1beta-induced proinflammatory gene induction for ensuing 16 h. However, CTS for 8h was not sufficient to inhibit iNOS expression for ensuing 28 or 40 h. CONCLUSIONS: Data suggest that constant application of CTS blocks IL-1beta-induced proinflammatory genes at transcriptional level. The signals generated by CTS are sustained after its removal, and their persistence depends upon the length of CTS exposure. Furthermore, the sustained effects of mechanical signals are also reflected in their ability to induce aggrecan synthesis. These findings, once extrapolated to human chondrocytes, may provide insight in obtaining optimal sustained effects of physical therapies in the management of arthritic joints.


Assuntos
Agrecanas/biossíntese , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico Sintase/biossíntese , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Interleucina-1beta/farmacologia , Ratos , Estresse Mecânico
10.
Osteoarthritis Cartilage ; 14(3): 264-72, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16290189

RESUMO

OBJECTIVE: We sought to determine the molecular basis for the anticatabolic effects of mechanical signals on fibrocartilage cells by studying the expression of a variety of matrix metalloproteinases (MMPs). Furthermore, we examined whether the effects of biomechanical strain on MMP gene expression are sustained. METHODS: Fibrochondrocytes from temporomandibular joint (TMJ) discs were exposed to dynamic tensile strain for various time intervals in the presence of interleukin (IL)-1beta. The regulation of the messenger RNA (mRNA) expression and synthesis of MMPs and tissue inhibitors of MMPs (TIMPs) were examined by end-point and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Western blot analysis. RESULTS: Fibrochondrocytes expressed mRNA for MMP-2, -3, -7, -8, -9, -11, -13, -14, -16, -17, and -19 as well as TIMP-1, -2, and -3, IL-1beta induced a significant (P<0.05) upregulation of mRNA for MMP-3, -7, -8, -9, -13, -16, -17, and -19. The IL-1beta-stimulated upregulation of these MMPs was significantly (P<0.05) abrogated by dynamic tensile strain. However, MMP-2, -11, -14, and TIMPs were not affected by either IL-1beta or tensile strain. Biomechanical strain also inhibited the IL-1beta-stimulated protein synthesis of MMP-3, -7, -8, -9, -13, -16, and -17. Application of mechanical strain for various time intervals during a 24-h incubation with IL-1beta showed that the suppressive effects of mechanical signals are sustained. CONCLUSIONS: The data provide evidence that biomechanical signals can downregulate the catabolic activity of fibrocartilage cells in an inflammatory environment by inhibiting the expression of a variety of MMPs. Furthermore, the matrix-protective effects of biomechanical signals are sustained even in an inflammatory environment.


Assuntos
Condrócitos/enzimologia , Fibrocartilagem/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloproteinases da Matriz/biossíntese , Mecanotransdução Celular/fisiologia , Animais , Western Blotting , Células Cultivadas , Condrócitos/metabolismo , Regulação para Baixo/fisiologia , Fibrocartilagem/citologia , Fibrocartilagem/metabolismo , Interleucina-1/farmacologia , Metaloproteinases da Matriz/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estresse Mecânico , Articulação Temporomandibular/citologia , Articulação Temporomandibular/enzimologia , Articulação Temporomandibular/metabolismo , Inibidores Teciduais de Metaloproteinases/biossíntese , Inibidores Teciduais de Metaloproteinases/genética
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