RESUMO
Transforming growth factor-ß1 (TGF-ß1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-ß1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-ß1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-ß1-induced EMT, and the cells were sensitized to the TGF-ß1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-ß1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-ß1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-ß1 in transformed cells.
Assuntos
Movimento Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Fatores de Transcrição , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Transforming growth factor-beta1 (TGF-beta1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-beta1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-beta1, while N17TRac1 was inhibitory. TGF-beta1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-beta1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-beta1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.
Assuntos
Células Epiteliais/metabolismo , Queratinócitos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Caderinas/metabolismo , Desdiferenciação Celular , Movimento Celular , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
TGF-beta1 is a potent inductor of malignance in cancer cells. TGF-beta1 stimulates the expression of extracellular matrix degrading proteases, cell migration and it is also involved in the epithelial-mesenchymal transition (EMT). In the present work, we analyzed the role of Spred2 in the urokinase-type plasminogen activator (uPA) stimulation, EMT and cell migration by TGF-beta1. We found that both the expression of mRNA and the protein level of Spred2 were lower in transformed keratinocytes PDV compared with immortalized keratinocytes MCA-3D. The transient ectopic expression of Spred2 in PDV cells inhibited the TGF-beta1-transactivated SRE-Luc reporter which is related with the ERK1,2 signal. The stable ectopic expression of Spred2 in PDV cells (SP cells) led to the loss of ERK 1,2 activation by TGF-beta1, although Smad2 activation was not affected, and the knockdown of Spred2 enhanced the activation of ERK1,2 signal by TGF-beta1. The increment of uPA expression induced by TGF-beta1 was suppressed in SP cells. In contrast, the stimulus on PAI-1 expression was not affected and comparable to parental PDV cells. SP cells under TGF-beta1 treatment were unable to display the EMT, since the overexpression of Spred2 abolished the TGF-beta1-induced disruption of the E-cadherin cell to cell interactions, reorganization of the actin cytoskeleton and upregulation of the mesenchymal marker vimentin. Finally, SP cells could not respond to the TGF-beta1 stimulus on cell migration. Taken together, the data in the present study suggests that Spred2 is a regulator of TGF-beta1-induced malignance in transformed keratinocytes.