Expressão e função dos receptores ativados por proteases em células odontoblastóides MDPC­-23

• Ano de defesa: 2017
• Autor(a) principal: Alvarez, Marcela Maciel Palacio [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Receptor Ativado Por Protease (Par); Células Mdpc-2; Inflamação Dentária; Complexo Pulpo-Dentinário
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=4921233; http://repositorio.unifesp.br/handle/11600/50523

INTRODUCTION. Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and thereby act as sensors for extracellular proteases. PARs activation plays important roles in the regulation of development, inflammation, immunity, and angiogenesis. PARs activation induces canonical G protein signaling and β-arrestin-dependent signalling. The aim of the current study was to investigate the expression and role of PARs in mouse odontoblast-like cells (MDPC-23). MATERIAL AND METHODS. Regulation of PARs expression in MDPC-23 cells was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) in the absence or in the presence of LPS (0,1 mg/ml). The MDPC-23 cells were also stimulated with 10-6M trypsin and PAR-2 peptide activator SLIGRL-NH2 10-4M to assess changes in gene expression of inflammatory cytokines and matrix metalloproteinases (MMPs). Antibodies against the PAR-1 and PAR-2 were used to investigate the cellular expression of these receptors using flow cytometry and confocal microscopy. Human tooth caries immunofluorescence assays and non-carious was performed to assess the presence PAR-2. Ca2+ signalling concentration–response curves were obtained using different PARs-specific agonists, thrombin for PAR-1 activation or trypsin for PAR-2. Cytoplasmic Ca2+ influx measurements were monitored through changes in the Fluo- 4 fluorescence intensity in real time using the Flex Station 3 microplate reader system. After a baseline reading for 30 s, cells were exposed to graded concentrations of PARs agonists. RESULTS AND DISCUSSION. RT-PCR and immunofluorescence reactions demonstrated the expression of PAR-1 and PAR-2 in MDPC-23 cells. Immunohistochemical studies by confocal microscopy of human teeth showed intense positive staining for the presence of PAR-2 in carious tissue compared to the control. Our results show that LPS promoted an increase in gene expression of PAR-1 and PAR-2, as well as an increase in the gene expression of IL-6 and TNF-α in MDPC-23 cells. Treatment of MDPC-23 cells with trypsin or with the PAR-2 activator peptide SLIGRL-NH2, caused a dose-dependent and time-dependent cytoplasmic Ca2+ increase. Moreover, when the PAR-2 receptor was stimulated by agonists an increase in the TLR4 gene expression was observed, as well as a decrease in the gene expression of IL-6, NF-κB and TNF-α. We also observed alteration in the gene xxi expression of MMPs mediated by PAR-2 stimulus. The stimulation of PAR-2 caused an increase in the gene expression of MMP1, MMP9 and MMP14. On the other hand, stimulation of PAR-2 promoted decreases in the expression of MMP2 and MMP13. CONCLUSION. The results presented here demonstrate for the first time that MDPC- 23 cells constitutively express PAR-1 and PAR-2 receptors, as well as indicate that these receptors are involved in the inflammatory response and dentinogenesis of odontoblastic cells MDPC-23.