TWEAK affects keratinocyte G2/M growth arrest and induces apoptosis through the translocation of the AIF protein to the nucleus.
Bottom Line: Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins.Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes.Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase.
Affiliation: Inserm, U976, Paris, France.
The soluble TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) binds to the fibroblast growth factor-inducible 14 receptor (FN14, TNFRSF12A) on the cell membrane and induces multiple biological responses, such as proliferation, migration, differentiation, angiogenesis and apoptosis. Previous reports show that TWEAK, which does not contain a death domain in its cytoplasmic tail, induces the apoptosis of tumor cell lines through the induction of TNFα secretion. TWEAK induces apoptosis in human keratinocytes. Our experiments clearly demonstrate that TWEAK does not induce the secretion of TNFα or TRAIL proteins. The use of specific inhibitors and the absence of procaspase-3 cleavage suggest that the apoptosis of keratinocytes follows a caspase- and cathepsin B-independent pathway. Further investigation showed that TWEAK induces a decrease in the mitochondrial membrane potential of keratinocytes. Confocal microscopy showed that TWEAK induces the cleavage and the translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, thus initiating caspase-independent apoptosis. Moreover, TWEAK induces FOXO3 and GADD45 expression, cdc2 phosphorylation and cdc2 and cyclinB1 degradation, resulting in the arrest of cell growth at the G2/M phase. Finally, we report that TWEAK and FN14 are normally expressed in the basal layer of the physiological epidermis and are greatly enhanced in benign (psoriasis) and malignant (squamous cell carcinoma) skin pathologies that are characterized by an inflammatory component. TWEAK might play an essential role in skin homeostasis and pathology.
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Mentions: TWEAK induces cell death in a variety of tumor cell lines by increasing cell apoptosis , , , . A previous report showed that TWEAK decreases the viability of HaCaT cells and NHK, and that this decrease is enhanced by the simultaneous addition of the inflammatory cytokine IFNγ . Therefore, we investigated whether the decreased growth that is observed in HaCaT cells and primary keratinocytes relies on increased apoptosis. The cells were incubated with variable concentrations of TWEAK and/or IFNγ for 48 h. We observed a slight increase in the number of HaCaT and NHK annexin V-single positive cells, and the number of annexinV/PI double positive cells increased in a dose-dependent manner. The incubation of cells with IFNγ doubled the population of annexinV single positive cells, and the number of annexinV/PI double positive late apoptotic cells was markedly increased (8-fold in HaCaT and 2-fold in NHK cells, respectively) compared to controls. An additive maximal apoptotic effect was observed following the simultaneous addition of low doses (10 ng/ml) of TWEAK and IFNγ. The results are shown in Figure 2A for HaCaT cell line and in Figure 2B for normal human keratinocytes (NHK).