RESUMO
We have previously observed that preirradiation with naturally occurring doses of near-infrared (IR) protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. This effect was observed in temperature-controlled conditions, without heat shock protein (Hsp72-70) induction. Moreover, IR inhibited UVB-induced apoptosis by modulating the Bcl2/Bax balance, pointing to a role of p53. Here, we show for the first time that p53-deficient SaOs cells are not protected from UVB cytotoxicity by IR preirradiation, suggesting that the response to IR is p53-dependent. Thus, we investigated the effect of IR on the p53 signaling pathway. Normal human dermal fibroblasts exposed in vitro to IR accumulated p53 protein, involving p53 stabilization and phosphorylation of serine 15 (Ser15) and Ser20. IR-induced p53 accumulation correlated with increased expression of p21 and GADD45, showing that IR also stimulates p53 transcriptional activity. By modulating UVB-induced targets of the p53 signaling pathway, IR irradiation appears to anticipate the UVB response and to prepare cells to better resist subsequent UV-induced stress. This is reinforced by the fact that IR preirradiation reduces the formation of UVB-induced thymine dimers.
Assuntos
Raios Infravermelhos , Pele/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Adolescente , Adulto , Apoptose/efeitos da radiação , Proteínas de Ciclo Celular/biossíntese , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Técnicas In Vitro , Pessoa de Meia-Idade , Proteínas Nucleares/biossíntese , Fosforilação , Dímeros de Pirimidina/biossíntese , Dímeros de Pirimidina/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Pele/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have previously observed that near-infrared (IR) pre-irradiation protects normal human dermal fibroblasts from ultraviolet (UV) cytotoxicity in vitro. Here, we show that IR pre-irradiation of human fibroblasts inhibited UVB activation of caspase-9 and -3, leading us to study early events in the mitochondrial apoptotic pathway after IR irradiation. IR irradiation led to a partial release of cytochrome c and Smac/Diablo but not apoptosis-inducing factor (AIF). This was accompanied by a slight but transient decrease in the mitochondrial membrane potential (Deltapsim) and by the insertion of Bax into mitochondrial membrane. Early apoptotic events in the mitochondrial pathway thus occurred after IR irradiation despite a lack of caspase-9 and -3 activation. This could be explained by the induction by IR of the expression of heat shock protein Hsp27, which is known to prevent apoptosome assembly. Furthermore, the balance between pro-apoptotic (i.e., Bax) and anti-apoptotic (i.e., Bcl-2 or Bcl-xL) proteins, which was rather pro-apoptotic after IR exposure, became anti-apoptotic 24 h later, suggesting a protective effect. Together, these actions could also contribute to prepare the cell to resist UVB-triggered apoptosis. Finally, isolated rat liver mitochondria-released cytochrome c in response to IR, demonstrating that mitochondria were a primary target of IR radiation.
Assuntos
Apoptose/efeitos da radiação , Derme/citologia , Fibroblastos/efeitos da radiação , Raios Infravermelhos , Mitocôndrias/efeitos da radiação , Adolescente , Adulto , Caspase 3 , Caspase 9 , Caspases/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/metabolismo , Humanos , Potenciais da Membrana/efeitos da radiação , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2RESUMO
Physical forces play an important role in regulating cell functions. We applied mechanical strain to human fibroblasts by magnetic attraction of superparamagnetic arginine-glycine-aspartic acid (RGD)-coated beads. We confirmed that the MAP kinases Erk and p38 are activated by mechanical strain, and went further by demonstrating the activation of Elk-1 by mechanical strain, mainly through a MEK-Erk pathway. Transfection of a dominant negative form of the G protein rac-1 (rac T17N), and inhibition of PI3K, an effector of rac-1, efficiently prevented Elk-1 activation by mechanical forces. Transfection with C3 transferase, known to inhibit rhoA, and inhibition of rock (a downstream effector of rhoA), gave similar results. However, contrary to the active form of rhoA (rho G14V), transfection of the active form of rac-1 (rac G12V) induced Elk activation and mimicked the effects of mechanical strain. These results point out that the two small G proteins rhoA and rac-1 participate in cell sensitivity to mechanical strain and lead to the modulation of the Erk pathway.