RESUMEN
OBJECTIVE: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.
Asunto(s)
Desdiferenciación Celular/genética , Condrocitos/fisiología , Metaloproteinasa 13 de la Matriz/genética , Receptor Notch1/genética , Animales , Biomarcadores/metabolismo , Cartílago Articular/citología , Células Cultivadas , Condrocitos/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , ARN Mensajero/metabolismo , Receptor Notch1/biosíntesis , Transducción de SeñalRESUMEN
Data of this study showed that alphaD-alphaE helices and the conserved interdomain linker are two interfaces essential not only for the self-association but also for the functional properties of rat HSC70. Self-association which is a conserved property of HSP70 seems to be important for the activity of these proteins.
Asunto(s)
Proteínas del Choque Térmico HSC70/genética , Adenosina Trifosfatasas/metabolismo , Animales , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Proteínas del Choque Térmico HSC70/metabolismo , Estructura Cuaternaria de Proteína , Ratas , Eliminación de Secuencia , UltracentrifugaciónRESUMEN
The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His(+)/C30WT and C30DeltaL-His(+)/C30DeltaL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30DeltaL-His(+) but has no effect on the elution profile of C30WT-His(+), compared to their respective untagged forms C30DeltaL and C30WT. These observations were also confirmed by AU analysis which indicates that C30DeltaL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.
