RESUMO
One of the 14-3-3 protein isoforms, 14-3-3epsilon, was previously shown to be increased during skin aging. We suggest here a possible role for the 14-3-3epsilon protein in skin aging by providing evidence that 14-3-3epsilon increases the expression of the matrix-metalloproteinase (MMP)-2 gene in NIH3T3 fibroblast cells. Expression of the 14-3-3epsilon gene in NIH3T3 cells primarily up-regulated the expression of the MMP-2 gene at the transcriptional level by inducing specific DNA binding proteins bound to an upstream region of the MMP-2 promoter from -1,629 to -1,612. Inhibition of endogenous 14-3-3epsilon gene expression by RNA interference also decreased endogenous MMP-2 gene expression. Furthermore, up-regulation of the MMP-2 gene by 14-3-3epsilon was suppressed by expression of a dominant-negative mutant of p38 MAP kinase. These findings strongly suggest that increased expression of 14-3-3epsilon contributes to remodeling of extracellular matrix in skin through increasing MMP-2 gene expression via p38 MAP kinase signaling.
Assuntos
Animais , Camundongos , Proteínas 14-3-3/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloproteinase 2 da Matriz/antagonistas & inibidores , Células NIH 3T3 , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The inhibitory Smad6 and Smad7 are responsible for cross-talk between TGF-beta/bone morphogenic protein (BMP) signaling and other cellular signaling pathways, as well as negative feedback on their own signaling functions. Although inhibitory Smads are induced by various stimuli, little is known about the stimuli that increase Smad6 transcription, in contrast to Smad7. Here we demonstrate that etoposide, which induces double strand breaks during DNA replication, significantly up-regulates the transcription of the Smad6 gene in CMT-93 mouse intestinal cells by increasing specific DNA binding proteins. In addition, endogenous inhibition of the Smad6 gene by RNAi interference led to transient accumulation of G1 phase cells and reduction in incorporation of bromodeoxyuridine (BrdU). These findings strongly suggest that Smad6 plays a distinct role in the signaling of etoposide-induced DNA damage.