RESUMO
Dentin is a reservoir of several potentially active molecules, and dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are the two major non-collagenous proteins. It has been established that dentin molecules are released as a consequence of osteoclast action during the resorption process. Along with osteoclasts, inflammatory cells seem to play an important role at sites of root resorption. Although the role of dentin molecules in dentinogenesis is well known, their role in pathological processes associated with dentin matrix dissolution is unclear. Recent studies have suggested that dentin components may function as chemotactic and activator signals for inflammatory cells at these sites. Herein we present evidence that demineralized dentin crude extract, DSP, and DPP induced doseand time-dependent neutrophil migration into the peritoneal cavity of mice and that this activity was inhibited by dexamethasone, but not by indomethacin or MK886. The blockade of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) receptors inhibited neutrophil accumulation. The neutrophil migration was also diminished in the absence of the chemokines cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2), but not in the absence of macrophage inflammatory protein-1alpha (MIP-1alpha). These results demonstrate that dentin induces neutrophil migration via the synthesis of IL-1beta, TNF-alpha, and chemokines and they suggest that dentin matrix proteins may have an active role in inflammatory cell recruitment during pathological processes associated with dentin and bone matrix dissolution.
Assuntos
Quimiocinas/metabolismo , Dentina/química , Infiltração de Neutrófilos/efeitos dos fármacos , Fosfoproteínas/farmacologia , Sialoglicoproteínas/farmacologia , Extratos de Tecidos/farmacologia , Animais , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CXC/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular/farmacologia , Interleucina-1/metabolismo , Proteínas Inflamatórias de Macrófagos/deficiência , Proteínas Inflamatórias de Macrófagos/genética , Proteínas Inflamatórias de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/fisiologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genéticaRESUMO
In previous work we showed that the polygonal shape of hippocampal astrocytes cultured on poly-L-lysine changes to a stellate morphology with loss of actinomyosin stress fibers on exchanging the culture medium for saline buffered with HEPES [Brain Res 946 (2002)12]. By contrast, in bicarbonate-buffered saline containing Ca(2+) astrocytes remained polygonal and continued to express stress fibers. Evidence suggests that stellation induced by saline buffered with HEPES is related to intracellular acidification due to the absence of bicarbonate. Here we studied the influence of the matrix used in preparing astrocyte cultures. Stellation in HEPES-saline occurred on a matrix of fibronectin, but not on matrices of collagen I or IV provided Ca(2+) was present. Laminin partially prevented stellation in HEPES-saline. Further, the intracellular acidification induced by HEPES-saline observed in astrocytes cultured on polylysine was abolished in cells cultured on collagens and was attentuated on a matrix of laminin. Two observations suggested the involvement of integrins and focal adhesions. (1) Treatment of cultures on collagens with a blocking antibody to the beta1 integrin subunit abolished protection against HEPES-induced stellation. (2) Compared with polylysine, astrocytes cultured on collagens expressed increased contents of phosphotyrosine proteins, focal adhesion proteins vinculin and paxillin, the beta1 integrin subunit and increased numbers of focal adhesions labelled with anti-vinculin. The observation that astrocytes cultured on collagen I or IV, in contrast to polylysine, express stress fibers and a constant intracellular pH in the absence of buffering by bicarbonate may be related to the fact that in the intact brain astrocytic processes (or end-feet) encounter and bind to collagen IV and laminin in the basement membrane of the endothelial cells which surround the cerebral capillaries. It is also possible that astrocytes retain this capacity from early development when fibrous matrix proteins are present.
Assuntos
Astrócitos/metabolismo , Bicarbonatos/metabolismo , Matriz Extracelular/fisiologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Astrócitos/citologia , Contagem de Células , Divisão Celular , Células Cultivadas , Colforsina/farmacologia , Proteínas do Citoesqueleto/farmacologia , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , HEPES/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Integrina beta1/imunologia , Integrina beta1/farmacologia , Líquido Intracelular/metabolismo , Paxilina , Fosfoproteínas/farmacologia , Ratos , Ratos Wistar , Fatores de Tempo , Vinculina/farmacologiaRESUMO
We have previously isolated and partially-sequenced a soluble phosphoprotein (p43) that acts as intermediary in the stimulation of steroid synthesis. In this report we have used synthetic peptides whose sequences match those obtained from p43 to generate antipeptide antibodies and show that these antibodies bind to purified p43 protein as determined by immunoblot analysis. The presence of p43 was detected by Western blot in both steroidogenic and non-steroidogenic tissues. One of the antibodies was also used to purify p43 on immunoaffinity chromatography columns. Proteins eluting from affinity columns produce a twelve-fold stimulation of progesterone synthesis. This effect was blocked by the use of an inhibitor of phospholipase A2. These results suggest the involvement of p43 in transducing the adrenocorticotropin signal to mitochondria in zona fasciculata cells. We also describe a partial cDNA clone with a predicted amino acid sequence that matches the sequences of the internal peptides of p43.