RESUMO
BACKGROUND AND OBJECTIVE: Matrix metalloproteinase (MMP)-13 has wide substrate specificity compared with other MMPs and appears to be involved in periodontitis. Previously, we reported that roxithromycin (RXM) inhibits vascular endothelial growth factor expression induced by tumour necrosis factor-alpha in human periodontal ligament cells, but little is known about the effect of RXM on MMP-13 expression in human gingival epithelial cells. We therefore examined the effect of RXM on MMP-13 mRNA expression and production in cultured human gingival epithelial cells. MATERIAL AND METHODS: Human epithelial cell lines (Ca9-22, TU4, SCCTF and HSC-3) were plated in tissue culture dishes. Then, the culture supernatants and sediments were collected and the production of MMP-13 was analysed using enzyme-linked immunosorbent assay; the expression of MMP-13 mRNA and runt-related gene 2 mRNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We also studied the effect of Runx2 short interfering RNA (siRNA) on the induction of MMP-13. RESULTS: Roxithromycin downregulated the induction of MMP-13 in Ca9-22 cells. Roxithromycin suppressed the expression of MMP-13 mRNA not only in Ca9-22 cells, but also in other human epithelial cell lines. Roxithromycin strongly inhibited the expression of Runx2 mRNA. Furthermore, Runx2 siRNA inhibited the induction of MMP-13 in Ca9-22 cells. CONCLUSION: These results indicate that RXM suppresses MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures.
Assuntos
Antibacterianos/farmacologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Indução Enzimática/efeitos dos fármacos , Gengiva/enzimologia , Inibidores de Metaloproteinases de Matriz , Roxitromicina/farmacologia , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Regulação para Baixo , Células Epiteliais/enzimologia , Gengiva/citologia , Humanos , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/fisiologiaRESUMO
The expression of the vanilloid receptor subtype 1 (VR1, TRPV1) was detected in human dental pulp fibroblasts (PF-10) using RT-PCR, Western blotting, and immunocytochemical analysis. As revealed by ELISA, capsaicin induced IL-6 expression in PF-10 cells, and the VR1 antagonist capsazepine dose-dependently inhibited capsaicin-induced IL-6 production, indicating that capsaicin-induced IL-6 expression is related to VR1 activation. The interaction between capsaicin and mitogen-activated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) were detected after capsaicin stimulation. p38 MAPK is involved in capsaicin-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. The result of EMSA showed that capsaicin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappa B (NF-kappaB) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.
Assuntos
Polpa Dentária/fisiopatologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Droga/metabolismo , Adulto , Capsaicina/metabolismo , Células Cultivadas , Polpa Dentária/citologia , Feminino , Humanos , NF-kappa B/metabolismo , Canais de Cátion TRPVRESUMO
The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined. Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E. coli-type lipid A (LA-15-PP) enhanced VEGF mRNA expression. LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level. Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.
Assuntos
Fatores de Crescimento Endotelial/biossíntese , Lipopolissacarídeos/farmacologia , Linfocinas/biossíntese , Monócitos/metabolismo , Fator de Transcrição Sp1/fisiologia , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Humanos , Lipídeo A/farmacologia , Linfocinas/genética , RNA Mensageiro/análise , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The effects of interleukin (IL)-10 on the expression of IL-6 and IL-8 mRNA in human dental pulp cell cultures were investigated by using the Northern blot analysis. On stimulation with Prevotella intermedia lipopolysaccharide (PiLPS), IL-10 was produced in peripheral blood but was not detected in human dental pulp cell culture supernatants. IL-10 inhibited IL-8 mRNA expression, which is normally stimulated by PiLPS, IL-1alpha, and tumor necrosis factor-alpha and inhibited IL-6 mRNA expression, which is normally stimulated by IL-1alpha. In addition, IL-10 inhibited the activation of nuclear factor-kappaB, which is normally induced by PiLPS. We conclude that IL-10 inhibits expression of IL-6 and IL-8 mRNA in dental pulp cell cultures by inhibiting the activation of nuclear factor-kappaB.
Assuntos
Polpa Dentária/metabolismo , Interleucina-10/farmacologia , Interleucina-6/antagonistas & inibidores , Interleucina-8/antagonistas & inibidores , Adolescente , Northern Blotting , Células Cultivadas , Polpa Dentária/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/sangue , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Lipopolissacarídeos/farmacologia , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Prevotella intermedia/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/análise , Proteínas Recombinantes/farmacologiaRESUMO
Interleukin-10 receptor (IL-10R) expression in human, dental pulp, fibroblast cultures was investigated by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. After exposure to lipopolysaccharide (LPS) from Prevotella intermedia, the IL-10R mRNA levels increased after 4 h, peaked at 7 h, and dropped back to the unstimulated level at 24 h. Maximal production of the IL-10R protein in dental pulp fibroblast cultures was detected by Western blot analysis after 12 h of LPS stimulation. In contrast, the human skin fibroblast (SF-MA) and human monocyte (U937) cell lines expressed IL-10R mRNA. Anti-CD14 antibodies inhibited P. intermedia LPS-induced IL-10R mRNA expression. These results indicate that P. intermedia LPS induces IL-10R gene expression in human, dental pulp fibroblasts in vitro.
Assuntos
Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Prevotella intermedia/química , Receptores de Interleucina/biossíntese , Adolescente , Células Cultivadas , Polpa Dentária/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , RNA Mensageiro/análise , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/metabolismo , Células U937/metabolismoRESUMO
To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.
Assuntos
Polpa Dentária/imunologia , Polpa Dentária/metabolismo , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Substância P/fisiologia , Comunicação Autócrina , Western Blotting , Células Cultivadas , Ciclo-Oxigenase 2 , Polpa Dentária/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Membrana , NF-kappa B/metabolismo , Prevotella intermedia/química , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/análise , Receptores de Interferon/antagonistas & inibidores , Receptores de Interleucina/biossíntese , Receptores de Interleucina-10 , Receptores da Neurocinina-1/biossíntese , Receptores da Neurocinina-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substância P/biossíntese , Substância P/genética , Regulação para Cima , Receptor de Interferon gamaRESUMO
Substance P (SP) induces the expression of proinflammatory cytokines, such as interleukin (IL)-6, which are implicated in pulp inflammation. To determine the signal pathway of SP-induced IL-6, we examined the activities of the mitogen-activated protein kinases (MAPKs) in human dental pulp cell (PF-10) cultures. SP induced the phosphorylation of p38 MAPK within 5 min; this activation persisted for up to 40 min and was independent of the activation of extracellular signal-related kinases (ERK-1 and ERK-2) that were induced after SP stimulation of PF-10 cells. As shown by electrophoretic mobility shift assay p38 MAPK was not involved in SP-induced activation of nuclear factor-kappa B (NF-kappaB). However, p38 MAPK mediated SP-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. Our results suggest that the activation of p38 MAPK is important for NF-kappaB-independent regulator of neurogenic inflammation in dental pulp tissues.
Assuntos
Polpa Dentária/efeitos dos fármacos , Polpa Dentária/metabolismo , Interleucina-6/biossíntese , Substância P/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos , Humanos , Interleucina-6/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Rats of the DA strain are highly susceptible to 4NQO-induced TCs, whereas WF rats are barely susceptible. In (DA x WF)F2 rats, 5 QTL, Tscc1-5, are responsible for most of the phenotypic variations, though they do not account for all of the phenotypic differences between WF and DA rats. Analysis of 40 tongue tumors >5 mm in diameter from (DA x WF)F1 rats for LOH at the Tscc loci revealed a high frequency of LOH in chromosomal regions where the Tscc2, -3 and -4 loci map. In most cases of LOH, the allele of the barely susceptible WF strain was lost, suggesting that these loci in the WF strain encode tumor-suppressor genes. Analysis of the same tumors for somatic mutations in oncogenes indicated frequent alteration of Ha-ras, which maps in the Tscc3 region, but rare mutation of the p15(INK4B) and p16(INK4A) genes or the p53 and Msh2 genes. Frequent LOH was also found on rat chromosomes 5 (RNO5) and 6 (RNO6). Tumors of large size accumulated LOH at multiple loci, suggesting the involvement of Tscc loci in tumor progression.