RESUMEN
To investigate the role of glycolysis and the M2 isoform of pyruvate kinase (PKM2) in odontogenic keratocysts (OKCs), the glycolytic flux of primary odontogenic keratocyst fibroblasts (OKC-Fs) and normal oral mucosa fibroblasts (OM-Fs) was determined by glucose uptake, lactate production, and cell proliferation assays. Wound healing assay and Matrigel-coated chamber system were used to investigate the effects of PKM2 on migration and invasion capacities of OKC-Fs. Co-culture of OKC-Fs with osteoclast precursors (RAW264.7 cells) was used to clarify the role of glycolysis in the osteoclastogenic effects of OKC-Fs. In addition, hypoxia-inducible factor 1α and some key enzymes related to glycolysis, including PKM2, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A, were detected to assess the activation of glycolysis in OKC stroma by immunohistochemistry. Results showed that the glucose uptake and lactate production were significantly higher in OKC-Fs than OM-Fs. PKM2 was elevated in OKC-Fs compared with that in OM-Fs. PKM2 significantly regulated glycolysis, proliferation, migration, invasion, and osteoclastogenic effects of OKC-Fs. Additionally hypoxia-inducible factor 1α, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A were markedly overexpressed in OKC stroma, and correlated with PKM2. Moreover, the expression of PKM2 was regulated by oxygen concentration in vitro. In sum, PKM2-mediated glycolysis regulated the growth, aggressiveness, and osteoclastogenesis of OKC.
Asunto(s)
Glucólisis , Quistes Odontogénicos/enzimología , Osteogénesis , Piruvato Quinasa/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Ratones , Invasividad Neoplásica , Quistes Odontogénicos/patología , Oxígeno/metabolismo , Isoformas de Proteínas , Piruvato Quinasa/genética , Células RAW 264.7RESUMEN
INTRODUCTION: The aim of this study was to analyze the presence of myofibroblasts and matrix metalloproteinase 2 (MMP-2) in radicular cysts (RCs), dentigerous cysts (DCs), and keratocystic odontogenic tumors (KOTs). METHODS: For the study, 29 RCs, 19 DCs, and 15 KOTs were selected. Immunohistochemical reactions were performed by using anti-MMP-2 and anti-α-smooth muscle actin (SMA) antibodies. For the analysis, 10 high-power fields were observed in each case to determine the percentage of positive cells, which was classified as negative, weak, or strong. RESULTS: The presence of myofibroblasts (α-SMA-positive cells) was most common in KOTs (46.67%), followed by DCs (36.84%) and RCs (31.04%); however, it was not statistically significant (P = .8). The stromal MMP-2 expression was positive in all lesions but 1 case of KOT. Most cases of RC and DC presented strong MMP-2 expression in the stroma, whereas half of the KOTs showed similar classification. The MMP-2 expression was commonly found in the epithelial lining of the lesions; it was strong in almost all KOTs. No correlation between epithelial and stromal MMP-2 and α-SMA expressions was observed. CONCLUSIONS: Myofibroblasts and MMP-2 are frequent in RCs, DCs, and KOTs and eventually can contribute to bone resorption, favoring the progression and growth of these lesions.
Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Miofibroblastos/metabolismo , Quistes Odontogénicos/enzimología , Quistes Odontogénicos/patología , Tumores Odontogénicos/enzimología , Tumores Odontogénicos/patología , Actinas/metabolismo , Células Epiteliales/metabolismo , Humanos , Inmunofenotipificación , Metaloproteinasa 2 de la Matriz/análisis , Miofibroblastos/patología , Estudios Retrospectivos , Estadísticas no ParamétricasRESUMEN
OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expression of collagen IV, matrix metalloproteinase (MMP) 9 and tissue inhibitor of MMP (TIMP) 2 in dentigerous cysts (DCs), radicular cysts (RCs), keratocystic odontogenic tumors (KOTs), and ameloblastomas. STUDY DESIGN: Twenty cases of DCs, 20 RCs, 20 KOTs, and 20 ameloblastomas were selected and analyzed by immunohistochemistry. RESULTS: Most DCs and RCs showed continuous and >50% staining for collagen IV in the basement membrane of the epithelium, whereas predominantly discontinuous thin and ≤ 50% staining was observed in KOTs and ameloblastomas, with a significant difference in staining percentage (P < .001). MMP-9 was diffusely distributed and localized in both epithelial and mesenchymal cells of all of the lesions analyzed. The staining percentage was higher in the epithelium (P = .058) and mesenchyme (P = .005) of KOTs and ameloblastomas. Moreover, the distribution pattern, location, and percentage of expression of TIMP-2 were similar in the lesions studied, except for ameloblastoma, with a significant difference in staining percentage (P < .001). CONCLUSION: These results demonstrate that the interaction between collagen IV, MMP-9, and TIMP-2 is an important factor for the establishment of differences in the biologic behavior of the odontogenic cysts and tumors studied.
Asunto(s)
Ameloblastoma/patología , Colágeno Tipo IV/análisis , Metaloproteinasa 9 de la Matriz/análisis , Quistes Odontogénicos/patología , Inhibidores de Proteasas/análisis , Inhibidor Tisular de Metaloproteinasa-2/análisis , Ameloblastoma/enzimología , Membrana Basal/enzimología , Membrana Basal/patología , Colorantes , Tejido Conectivo/enzimología , Tejido Conectivo/patología , Quiste Dentígero/enzimología , Quiste Dentígero/patología , Células Endoteliales/enzimología , Células Endoteliales/patología , Células Epiteliales/enzimología , Células Epiteliales/patología , Epitelio/enzimología , Epitelio/patología , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Inmunohistoquímica , Mesodermo/enzimología , Mesodermo/patología , Quistes Odontogénicos/enzimología , Quiste Radicular/enzimología , Quiste Radicular/patologíaRESUMEN
OBJECTIVE: Proteases are considered critical in peri-cystic tissue degradation required in jaw cyst expansion. We studied the expression of the plasminogen activation system (plasminogen activators; their inhibitor type-1, PAI-1; the receptor for the urokinase-type plasminogen activator, uPAR) in follicular and inflammatory cysts of the jaw, to identify a possible role of this system in jaw cyst enlargement. MATERIALS AND METHODS: Jaw cysts were collected by therapeutic enucleation. ELISA and casein zymography were used to measure and characterize plasminogen activators in cyst fluid. By immunohistochemistry we examined the presence of uPAR in cyst walls and inflammatory cells, and by Western blotting the molecular forms of uPAR within the cyst fluid. RESULTS: Inflammatory cysts fluid contained higher amounts of plasminogen activators of the urinary-type (uPA), and lower amounts of PAI-1, when compared to follicular cysts fluid. Epithelial layers of both types of cysts and inflammatory cells expressed uPAR. Native 3-domain uPAR was scarcely detectable within cysts, where its cleavage was accounted for by uPA. CONCLUSION: These data suggest a plasminogen activation-dependent mechanism of cyst enlargement, where only the outward uPAR expressed on epithelial cells and on inflammatory cells direct the peri-cystic protease cascade, in a way similar to tumor enlargement within tissues.
Asunto(s)
Quistes Maxilomandibulares/patología , Quistes Odontogénicos/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/análisis , Activador de Plasminógeno de Tipo Uroquinasa/análisis , Adulto , Anciano , Anciano de 80 o más Años , Western Blotting , Líquido Quístico/química , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Inmunohistoquímica , Inflamación , Quistes Maxilomandibulares/enzimología , Antígenos Comunes de Leucocito/análisis , Masculino , Persona de Mediana Edad , Quistes Odontogénicos/enzimología , Inhibidor 1 de Activador Plasminogénico/análisis , Activadores Plasminogénicos/análisisRESUMEN
OBJECTIVE: To investigate the matrix metalloproteinase (MMP)-13 expression in associated and non-nevoid basal cell carcinoma syndrome (NBCCS) Odontogenic Keratocysts (OCKs) in order to contribute to a better understanding of the differences in the growth pattern between them. MATERIALS AND METHODS: Thirty-nine paraffin-embedded blocks of OCKs, 26 sporadic OCKs and 11 NBCCS-associated KCOTs were studied by immunohistochemistry to evaluate MMP-13 expression both in epithelial and stromal layers. A semi-quantitative scale was used to evaluate immunostaining. Obtained data were compared between the two groups, using Fischer's exact test and the chi-square test. RESULTS: Only 13 of 26 sporadic OCKs showed a positive immunostaining, whilst 11 KCOTs resulted in positive labelling for MMP-13 expression. Moreover, syndromic cysts displayed a more intense and diffuse MMP-13 labelling of the stromal tissue. Instead, in non-syndromic forms, the staining pattern of MMP-13 in stromal tissue was completely absent. Fisher's exact test showed a statistically significant greater prevalence of KCOTs-immunolabelled cysts with respect to sporadic OCKs. CONCLUSIONS: Results from this study point out that the biological behaviour of these cysts could be related not only to the epithelial layer but also to stromal tissue in that... MMP-13 overexpression in stromal tissue of NBCCS-associated KCOTs could clarify the higher aggressiveness of these cysts.
Asunto(s)
Carcinoma Basocelular/enzimología , Metaloproteinasa 13 de la Matriz/análisis , Quistes Odontogénicos/enzimología , Tumores Odontogénicos/enzimología , Carcinoma Basocelular/patología , Células Epiteliales/enzimología , Células Epiteliales/patología , Epitelio/enzimología , Humanos , Inmunohistoquímica , Quistes Odontogénicos/patología , Tumores Odontogénicos/patología , Células del Estroma/enzimología , Células del Estroma/patologíaRESUMEN
Aldehyde dehydrogenases (ALDHs) represent a superfamily of NAD(P)(+)-dependent enzymes which catalyze the oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some ALDHs have been identified as corneal crystallins and thereby contribute to the protective and refractive properties of the cornea. ALDH3A1 is highly expressed in the cornea of most mammals with the exception of rabbit that abundantly expresses ALDH1A1 in the cornea instead of ALDH3A1. In this study, we examined the gene expression of other ALDHs and found high messenger levels of ALDH1B1, ALDH2 and ALDH7A1 in mouse cornea and lens. Substantial evidence supports a protective role for ALDH3A1 and ALDH1A1 against ultraviolet radiation (UVR)-induced oxidative damage to ocular tissues. The mechanism by which this protection occurs includes UVR filtering, detoxification of reactive aldehydes generated by UVR exposure and antioxidant activity. We recently have identified ALDH3A1 as a nuclear protein in corneal epithelium. Herein, we show that ALDH3A1 is also found in the nucleus of rabbit keratocytes. The nuclear presence of ALDH3A1 may be involved in cell cycle regulation.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Núcleo Celular/enzimología , Córnea/citología , Córnea/enzimología , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Línea Celular Transformada , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Quistes Odontogénicos/enzimología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Conejos , Retinal-Deshidrogenasa , Transfección/métodosRESUMEN
OBJECTIVE: The objective of this study was to analyze the expression of matrix metalloproteinases (MMPs) 1, 7, and 26 in odontogenic keratocysts (OKCs) associated with Gorlin syndrome (SOKCs) and nonsyndrome OKCs (NSOKCs). STUDY DESIGN: Twenty-one SOKCs and 20 NSOKCs were evaluated for epithelial expression of MMP-1, MMP-7, and MMP-26 and for mesenchymal expression of MMP-1 by immunohistochemistry. RESULTS: Strong epithelial positivity to MMP-1 was observed in 76% of SOKCs and in 15% of NSOKCs (P < .05). Strong mesenchymal immunoreactivity to MMP-1 was observed in 38% of SOKCs and in 20% of NSOKCs (P > .05). Epithelial immunoreactivity to MMP-7 was strongly positive in 67% of SOKCs and in 40% of NSOKCs (P > .05). For MMP-26, strong positivity was found in 62% of SOKCs, in contrast to 35% of NSOKCs (P > .05). CONCLUSION: MMPs-1, -7 and -26 may play important roles in the biology of OKCs. Furthermore, the presence of these proteases at higher levels in SOKCs may help to explain increased OKC aggressiveness associated with nevoid basal cell carcinoma syndrome.
Asunto(s)
Síndrome del Nevo Basocelular/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 7 de la Matriz/biosíntesis , Metaloproteinasas de la Matriz Secretadas/biosíntesis , Quistes Odontogénicos/enzimología , Femenino , Humanos , Inmunohistoquímica , Queratinas , Masculino , SíndromeRESUMEN
BACKGROUND: Keratocystic odontogenic tumor (KCOT), also known as odontogenic keratocyst, is a benign cystic neoplasm, which may be associated with nevoid basal cell carcinoma syndrome (NBCCS) and if it does, will occur as multiple cystic lesions. KCOT is locally destructive despite its bland histological features. However, the neoplastic nature of KCOT is not well established. Heparanase is an endo-d-glucuronidase enzyme that specifically cleaves heparan sulfate (HS) and the increase of its level in tumors promotes invasion, angiogenesis, and metastasis. METHODS: To investigate the neoplastic character of KCOT, we studied the localization patterns of heparanase in KCOT, focusing on the differences between sporadic and NBCCS-associated KCOTs, by immunohistochemistry and in situ hybridization. To compare the expression pattern of these cysts with non-tumorous odontogenic developmental cyst, dentigerous cyst was included. RESULTS: All the odontogenic cysts showed positive immunoreaction for heparanase protein in various intensities. The expression pattern of heparanase gene corresponded to that of protein expression. Interestingly, intense gene and protein expressions were observed in KCOT associated with NBCCS compared with sporadic ones and dentigerous cyst. CONCLUSIONS: The results implied that heparanase expression may be correlated with the neoplastic properties of KCOT, particularly in NBCCS-associated cases.
Asunto(s)
Síndrome del Nevo Basocelular/enzimología , Glucuronidasa/biosíntesis , Quistes Odontogénicos/enzimología , Tumores Odontogénicos/enzimología , Síndrome del Nevo Basocelular/complicaciones , Quiste Dentígero/enzimología , Regulación Neoplásica de la Expresión Génica , Glucuronidasa/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Inmunohistoquímica , Hibridación in Situ , Invasividad Neoplásica , Quistes Odontogénicos/complicaciones , Tumores Odontogénicos/complicacionesRESUMEN
Visando contribuir para um melhor entendimento do crescimento das lesões odontogênicas e do papel das metaloproteinases da matriz (MMPs) nesse processo, realizou-se uma análise da expressão imuno-histoquímica das MMPs -1, -2 e -9, em 15 cistos radiculares (CR), 10 cistos radiculares residuais (CRR), 10 cistos dentígeros (CD) e 10 ceratocistos odontogênicos (CO). Analisou-se, no epitélio e no mesênquima, a imunopositividade das lesões, atribuindo-se os escores: (-) ausência de marcação, (+) marcação focal e (++) marcação difusa. De uma maneira geral verificou-se, no limitante epitelial das lesões, expressão predominantemente difusa da MMP-1 (CRR: 100 por cento, CD: 70 por cento) e focal para 53 por cento dos CRs e 60 por cento dos COs. Ela variou de focal (CR: 60 por cento e CO: 100 por cento) a difusa (CRR: 60 por cento e CD: 50 por cento) para a MMP-2 e marcadamente focal para a MMP-9 (100 por cento dos CR, CRR e CO e 60 por cento dos CD). No mesênquima, detectou-se expressão destacadamente maior nos COs: 100 por cento difusa para a MMP-1, enquanto a grande maioria de todos os cistos foi focal; a MMP-2 expressou-se com escore focal em 100 por cento dos casos, contrastando-se a forte ausência de marcação nos outros cistos; para a MMP-9, 50 por cento foram difusas e 50 por cento focais, enquanto a maioria dos outros cistos não exibiu marcação. Os resultados deste estudo sugerem que o crescimento dos cistos odontogênicos pode ser influenciado pela secreção das MMPs. A expressão mais exuberante das MMPs no mesênquima dos COs confirma sua participação ativa no crescimento da lesão, o que pode justificar em parte sua maior agressividade em relação às outras lesões císticas.
In an attempt to contribute to a better understanding about the growth of odontogenic lesions and the role of matrix metalloproteinases (MMPs) in this event, the immunoexpression of MMP-1, -2 and -9 was evaluated in 15 radicular cysts (RCs), 10 residual radicular cysts (RRCs), 10 dentigerous cysts (DCs) and 10 odontogenic keratocysts (OKs). To the analysis of epithelium and supporting mesenchyme, the following scores were utilized: negative (-), focal (+) and diffuse (++). Our results showed that the epithelial lining of the RRCs (100 percent) and DCs (70 percent) was diffuse to MMP-1, while to the RCs (53 percent) and OKs (60 percent) was predominantly focal. MMP-2 immunoscores ranged from focal in the lining of OKs (100 percent) and RCs (60 percent) to diffuse in RRCs (60 percent) and DCs (60 percent). With relation to MMP-9, the lining of all lesions was immunoexpressed focally (100 percent of RCs, RRCs and OKs and 60 percent of DCs). In relation to mesenchyme, the immunoexpression was higher in all OKs: the MMP-1 scores were diffuse (100 percent), while most of the other studied lesions were focal; all OKs focally immunoexpressed the MMP-2, what differed from the other cysts which did not express this MMP. To MMP-9, the mesenchyme of the OKs were immunoscored as focal (50 percent) and diffuse (50 percent), while most of other cysts had not shown staining. These results suggest that the growth of the odontogenic cysts may be influenced by the secreted MMPs. The most exuberant expression of the MMPs in mesenchyme of the OKs confirms its active participation in the growth, what may in part justify its bigger aggressiveness in comparison to other cystic lesions.
Asunto(s)
Humanos , Quistes Odontogénicos/enzimología , Quistes Odontogénicos/patología , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 9 de la Matriz , InmunohistoquímicaRESUMEN
OBJECTIVE: The activation of intracellular signaling cascades involving serine/threonine kinases ERK1/2 has been variably reported either to stimulate or inhibit epithelial cell differentiation in response to extracellular signals. The purpose of our study was to determine the distribution of the signaling molecule ERK1 and its activated form pERK1/2 in the epithelial components of developmental and inflammatory odontogenic cysts in relation to parameters of differentiation and proliferation. STUDY DESIGN: Thirty samples of dental follicles, dentigerous cysts, and radicular cysts were immunostained with antibodies to ERK1, pERK1/2, and proliferating cell nuclear antigen (a marker for proliferation). The tissues were subclassified according to the pattern of histomorphological differentiation (ie, squamous differentiation) and the proliferation rate of their epithelial components. The significance of differences in the proportion of ERK1- and pERK1/2-expressing cells among the tissue groups was determined by chi-square analysis or Fisher's exact test. RESULTS: ERK1 and pERK1/2 were found to be expressed in a significantly higher proportion of cells with differentiated and highly proliferating epithelial components, as compared with those of nondifferentiated, quiescent epithelial rests. The epithelium of radicular cysts exhibited the highest proportion of pERK1/2-positive cells. In both dentigerous and radicular cyst samples, pERK1/2 expression was significantly higher in the inflamed tissues. CONCLUSIONS: These data demonstrate that ERK1 and its active form pERK1/2 are associated with differentiating and actively proliferating epithelia of odontogenic cysts, and are consistent with pERK1/2 involvement in the activation of odontogenic epithelia in response to inflammation.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/fisiología , Quistes Odontogénicos/enzimología , Diferenciación Celular , División Celular , Distribución de Chi-Cuadrado , Técnicas de Cultivo , Saco Dental/enzimología , Células Epiteliales/enzimología , Humanos , Técnicas para Inmunoenzimas , Inflamación/enzimología , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
Interleukin-1alpha (IL-1alpha) is strongly expressed in odontogenic keratocysts. In this study, we investigated the effects of IL-1alpha on the activation of matrix metalloproteinase-2 (MMP-2) in the fibroblasts isolated from odontogenic keratocysts. Odontogenic keratocyst fibroblasts secreted a latent form of MMP-2 (proMMP-2) spontaneously. Type I collagen induced the activation of the proMMP-2, and recombinant human IL-1alpha (rhIL-1alpha) further enhanced the type I collagen-induced activation of proMMP-2 in a dose-dependent manner. The rhIL-1alpha-induced activation of proMMP-2 was inhibited by anti-human IL-1alpha antibody. A reverse-transcription/polymerase chain-reaction (RT-PCR) and Western immunoblotting demonstrated that the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) mRNA and protein was increased in the fibroblasts when the cells were cultured on type I collagen, and the expression was further enhanced by rhIL-1alpha. Thus, IL-1alpha may up-regulate proMMP-2 activation by increasing the expression of MT1-MMP in the fibroblasts isolated from odontogenic keratocysts synergistically with type I collagen.
Asunto(s)
Colágeno Tipo I/fisiología , Fibroblastos/enzimología , Interleucina-1/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloendopeptidasas/biosíntesis , Quistes Odontogénicos/enzimología , Western Blotting , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos/biosíntesis , Fibroblastos/efectos de los fármacos , Humanos , Interleucina-1/fisiología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estadísticas no ParamétricasRESUMEN
Matrix metalloproteinases (MMPs) collectively degrade extracellular matrix and basement membrane proteins in chronic inflammation and bone-destructive lesions. This study examined the ability of immunoglobulin-producing plasma cells, typically present in sites of chronic inflammation, to express collagenases (MMP-8 and -13) in vivo and in vitro. Phorbol-12-myristate-13-acetate, interleukin-6, and tumour necrosis factor-alpha and heparin with the tumour promoter or cytokines potently enhanced (up to nine-fold) MMP-8 and -13 expression by the RPMI 8226 myeloma cell line, as evidenced by western blotting and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemical analysis and in situ hybridization revealed that plasma cells expressed MMP-8 and -13 focally in periapical granulomas, odontogenic cysts, and malignant plasmacytomas. MMP-8 and MMP-13 from plasma cells can participate in bone organic matrix destruction at sites of chronic inflammation and neoplastic growth. Since MMP-13 was more frequently expressed than MMP-8 in plasma cells of strongly recurring keratocysts and malignant plasmacytomas, it is concluded that plasma cell MMP-13 has a particularly important role in benign and malignant bone-destructive lesions.
Asunto(s)
Enfermedades Óseas/enzimología , Metaloproteinasas de la Matriz/análisis , Células Plasmáticas/enzimología , Neoplasias Óseas/enzimología , Distribución de Chi-Cuadrado , Colagenasas/análisis , Colagenasas/genética , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 8 de la Matriz/análisis , Metaloproteinasa 8 de la Matriz/genética , Mieloma Múltiple/enzimología , Quistes Odontogénicos/enzimología , Granuloma Periapical/enzimología , Plasmacitoma/enzimología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/enzimologíaRESUMEN
Interleukin-1alpha (IL-1alpha) and matrix metalloproteinase-9 (MMP-9) are thought to be involved in odontogenic cyst expansion. In this study, we investigated the effects of IL-1alpha on the secretion and activation of MMP-9 in odontogenic jaw cysts. An active form of MMP-9 was present in odontogenic keratocyst (6 of 8 cases) fluids more frequently than dentigerous cyst (3 of 10 cases) and radicular cyst (3 of 10 cases) fluids, although proMMP-9 was present in all cyst fluids. Odontogenic keratocyst fragments in explant culture secreted a larger amount of IL-1alpha than dentigerous cyst and radicular cyst fragments in explant culture, and spontaneously secreted both proMMP-9 and an active form of MMP-9. The fragments of dentigerous cysts and radicular cysts secreted a small amount of proMMP-9, but no active form of MMP-9. Exogenously added recombinant human IL-1alpha (rhlL-1alpha) increased the secretion and activation of proMMP-9 in the fragments of dentigerous cysts and radicular cysts. The epithelial cells isolated from odontogenic keratocysts secreted IL-1alpha and proMMP-9 without stimulation. Under the cultivation on a fibronectin-coated dish, rhIL-1alpha increased the secretion of proMMP-9 from the epithelial cells in a dose-dependent manner. Moreover, rhIL-1alpha induced the secretion of proMMP-3 and plasminogen activator urokinase (u-PA) from the epithelial cells, and converted the secreted proMMP-3 to the active form in the presence of plasminogen. The secreted proMMP-9 was also activated in the presence of rhIL-1alpha and plasminogen. Hence, our results suggest that IL-1alpha may up-regulate not only proMMP-9 secretion but also proMMP-9 activation by inducing proMMP-3 and u-PA production in the cyst epithelial cells by autocrine/paracrine regulatory mechanisms.
Asunto(s)
Interleucina-1/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Quistes Odontogénicos/patología , Comunicación Autocrina/efectos de los fármacos , Células Cultivadas , Medios de Cultivo , Líquido Quístico/enzimología , Quiste Dentígero/enzimología , Quiste Dentígero/patología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/metabolismo , Células Epiteliales/enzimología , Fibronectinas , Humanos , Interleucina-1/administración & dosificación , Interleucina-1/farmacología , Metaloproteinasa 3 de la Matriz/efectos de los fármacos , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Quistes Odontogénicos/enzimología , Comunicación Paracrina/efectos de los fármacos , Activadores Plasminogénicos/efectos de los fármacos , Activadores Plasminogénicos/metabolismo , Quiste Radicular/enzimología , Quiste Radicular/patología , Proteínas Recombinantes , Regulación hacia Arriba/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/efectos de los fármacos , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Inflammatory and developmental cysts of the jaws are relatively common bone destructive lesions in the human maxillofacial skeleton but their pathogenesis is still poorly understood. In this study the role of mast cells (MC), and mast cell tryptase in particular, was evaluated in the pathophysiology of bone resorption and jaw cyst formation in different types of cysts. The distribution of MC and the amount of tryptase in histological tissue sections were determined by immunohistochemistry using monoclonal antihuman tryptase antibodies and the results were quantitated by using an image analyzing system. The amount of tryptase was further studied by Western-blotting and measurement of trypsin-like activity from the neutral salt extracts obtained from different types of jaw cysts. In contrast to control tissue, high trypsin-like activities and abundant immunoreactive tryptase were observed in the extracts of all types of cysts studied (radicular, dentigerous and keratocyst). In tissue sections the highest amount of tryptase-positive staining was observed in radicular cysts (mean 6.2% of reference area) and the lowest amount in keratocysts (mean 2.1% of reference area, P < 0.01). MC were found to be located in inflammatory cell-rich tissue areas and just beneath the cyst epithelium. Importantly, MC located at the border of bone were observed to be degranulated, indicating high activity of MC and release of tryptase at the regions of early bone destruction. Based on previous findings addressing the role of mast cell tryptase in proteolytic cascades, and the known association of MC with osteoporosis, we suggest that mast cells and mast cell tryptase may contribute significantly to jaw cyst tissue remodelling during growth of a cyst, and to the destruction of the surrounding bone, resulting in jaw cyst expansion.
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Mediadores de Inflamación/análisis , Mastocitos/enzimología , Quistes Odontogénicos/patología , Serina Endopeptidasas/análisis , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales , Western Blotting , Remodelación Ósea , Resorción Ósea/enzimología , Resorción Ósea/patología , Degranulación de la Célula , Niño , Quimasas , Colorantes , Quiste Dentígero/enzimología , Quiste Dentígero/patología , Epitelio/enzimología , Epitelio/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Maxilares/enzimología , Maxilares/patología , Persona de Mediana Edad , Quistes Odontogénicos/enzimología , Quiste Radicular/enzimología , Quiste Radicular/patología , TriptasasRESUMEN
An imbalance in human leucocyte elastase (HLE) activity is widely recognized to play an important pathological role in a number of human diseases. An earlier report has described greater transcription of elafin, an endogenous inhibitor of HLE, in epithelia of odontogenic keratocysts of the jaw than in normal oral mucosa. The elafin gene was now localized to chromosome 20q11.2-13.1 using a combination of somatic cell-hybrid panel screening and fluorescence in situ hybridization using a biotinylated DNA probe prepared from isolated yeast artificial chromosomes. No other positive fluorescent signals were observed. This eliminates the elafin gene as a candidate gene for naevoid basal-cell carcinoma syndrome, as the gene for this syndrome localizes to chromosome 9q23.1-31. The elafin yeast artificial chromosome DNA is to be subcloned to identify polymorphic microsatellite markers that will establish whether this gene is frequently amplified in oral neoplastic tissue.
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Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Proteínas de la Membrana/genética , Quistes Odontogénicos/enzimología , Proteínas/genética , Inhibidores de Serina Proteinasa/genética , Síndrome del Nevo Basocelular/enzimología , Síndrome del Nevo Basocelular/genética , Cromosomas Humanos Par 20/genética , Cromosomas Humanos Par 9/genética , Sondas de ADN , Epitelio/enzimología , Fluorescencia , Amplificación de Genes , Humanos , Células Híbridas , Hibridación in Situ , Elastasa de Leucocito/antagonistas & inhibidores , Elastasa de Leucocito/metabolismo , Repeticiones de Microsatélite/genética , Mucosa Bucal/enzimología , Neoplasias de la Boca/enzimología , Neoplasias de la Boca/genética , Quistes Odontogénicos/genética , Polimorfismo Genético/genética , Proteínas Inhibidoras de Proteinasas Secretoras , Transcripción Genética/genéticaRESUMEN
The levels of succinate, lactate, glutamate, glycerophosphate and glucose-6-phosphate dehydrogenases within the linings of keratinizing and non-keratinizing odontogenic cysts were investigated using static end-point and continuously monitored Nitroblue Tetrazolium-based histochemical methods. The use of TV image analysis for quantification of formazan final reaction products was validated by demonstrating significant relationships between the integrated absorbance at 585 nm and the amount of formazan in, and thickness of, gelatin films containing reduced tetrazolium salt (r = 1.0, p < 0.001). Absorbance readings of stained sections gave mean coefficients of variation of 1.8 +/- 0.9% between day of measurement, and of 5.65 +/- 1.32% between serial sections. End-point assays indicated that the linings of odontogenic keratocysts contained higher levels of glucose-6-phosphate dehydrogenases (p < 0.0002) and lower levels of lactate dehydrogenase (p < 0.002) than those of radicular cysts. Succinate, glutamate and glycerophosphate dehydrogenase activities were similar in both cyst types. Results from continuously monitored assays, performed for glucose-6-phosphate and succinate dehydrogenases, demonstrated linear reaction rates over the first 2.75 min of reaction. The calculated enzyme activities from continuous assays were between 1.49 and 3.49 times higher than those determined from end-point assays and confirmed that levels of glucose-6-phosphate dehydrogenase were significantly higher in the linings of odontogenic keratocysts than those of radicular cysts (p < 0.004). By contrast, succinate dehydrogenase activity was significantly higher in radicular cyst linings (p < 0.03). These results highlight the benefits of an approach to in situ determination of enzyme activity using image analysis and continuous monitoring methodologies. Overall, the high level of glucose-6-phosphate dehydrogenase found in keratocyst linings is consistent with their clinical behaviour and higher level of proliferation and synthetic activity whereas the level of lactate dehydrogenase in radicular cysts probably reflects the presence of local tissue damage within these inflammatory lesions.
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Quistes Odontogénicos/enzimología , Oxidorreductasas/metabolismo , Quiste Radicular/enzimología , Histocitoquímica , Humanos , Procesamiento de Imagen Asistido por Computador , Queratinas/metabolismo , Quistes Odontogénicos/patología , Quiste Radicular/patología , Reproducibilidad de los ResultadosRESUMEN
Neutral salt extracts of 14 specimens of jaw cysts were prepared. Histopathological analysis showed that the specimens consisted of 6 radicular cysts, 6 dentigerous cysts, 1 residual cyst, and 1 odontogenic keratocyst. One periapical granuloma, 1 dental follicle and a sample of clinically healthy oral mucosa were similarly processed and used as controls. Measurement of collagenase activity by monitoring the formation of specific degradation products of type I and II collagen in solution by SDS-PAGE demonstrated that all the cyst extracts contained collagenase, some of which was endogenously activated. Cyst wall collagenase preferably degraded type I over type II collagen, which suggests that the degradation was due to MMP-1 (matrix metalloproteinase-1) rather than the MMP-8 type. This was further supported by the doxycycline-inhibition profile of cyst collagenase, which was similar to that of MMP-1. Part of the cyst wall collagenase was in latent proenzyme form and probably derived, at least in part, from the newly synthesized intracellular collagenase pool. Latent cyst collagenase was efficiently activated with phenylmercuric chloride and to a lesser extent by gold (I) thioglucose and NaOCl. Western-blotting, using specific antibodies against collagenase from human polymorphonuclear neutrophilic leukocytes (MMP-8) and from fibroblasts (MMP-1), revealed a typical 55/45 kDa doublet; also MMP-8 in the latent 80 kDa form and fragmented to 65 kDa active species were found. These results suggest the presence of MMP-1 and, to a lesser extent, MMP-8 type collagenase in the cyst wall.(ABSTRACT TRUNCATED AT 250 WORDS)
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Colagenasas/química , Quistes Odontogénicos/enzimología , Quiste Radicular/enzimología , Adolescente , Adulto , Anciano , Western Blotting , Niño , Colágeno/metabolismo , Colagenasas/metabolismo , Quiste Dentígero/enzimología , Doxiciclina/farmacología , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Femenino , Humanos , Masculino , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 8 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Persona de Mediana EdadRESUMEN
Fragments of keratocysts removed at operation were maintained in explant culture and the media were assayed for the biological activity of the potent osteolytic cytokines--interleukin (IL)-1, interleukin (IL)-6 and tumour necrosis factor (TNF). Media were also assayed for their ability to stimulate bone resorption. All six cysts examined released IL-1 and IL-6 bioactivity but TNF bioactivity was unmeasurable. Dialysed cyst media stimulated bone resorption and this could be completely inhibited by a monospecific antibody which neutralized IL-1 alpha and IL-1 beta. Immunohistochemical staining of cryostat sections of keratocysts revealed the presence of IL-1 alpha and IL-6 in cyst epithelial cells but not in other cell types. Sections did not react with antibodies to IL-1 beta or TNF. It is therefore proposed that IL-1 alpha is the major osteolytic cytokine produced by keratocysts and that IL-6 and IL-1 may contribute to keratocyst growth by promoting epithelial cell proliferation and bone resorption, respectively.
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Interleucina-1/análisis , Quistes Odontogénicos/química , Adulto , Animales , Resorción Ósea , Cartílago/enzimología , Colagenasas/análisis , Encía/química , Enfermedades de las Encías/enzimología , Enfermedades de las Encías/metabolismo , Enfermedades de las Encías/patología , Humanos , Inmunohistoquímica , Interleucina-1/farmacología , Interleucina-6/análisis , Activación de Linfocitos , Ratones , Quistes Odontogénicos/enzimología , Quistes Odontogénicos/patología , Conejos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
This article briefly reviews the origin, classification and pathogenesis of the various odontogenic cysts. Keratocysts and follicular cysts are said to be developmental lesions arising from the remnants of the dental lamina and the cell rests of the dental follicle respectively. The radicular cysts are the most commonly occurring lesions associated with the apices of non-vital teeth. They are said to arise from proliferation of the cell rests of Malassez in chronically inflamed granulomata. It is noted that bone resorption is the major requirement for any bony lesion to expand; hence the interest in the role of diverse cellular and chemical mediators of bone resorption in disease. The current concepts of the role, in cyst initiation and growth, of enzymes including cellular metabolites and cytokines are presented. Evidence on the activities of collagenase, arachidonic acid metabolites, leukotrienes, hydroxyeicosatetraenoic acids, interleukin--1 and prostaglandins is cited. It is observed that the understanding of these cellular and molecular biological behaviour patterns may yield more appropriate information necessary for the development of more effective management modalities for such tissue degrading lesions as odontogenic cysts.