Activities Via Forward-and Reverse-signaling Through C-28F TM-TNF-a Mutein

Tumor necrosis factor-a(tumor necrosis factor-α, TNF-α) is a pleiotropic cytokine and exerts its biological activities in two forms, namely secreted TNF-α(S-TNF-α) and transmembrane TNF-α(TM-TNF-α). TM-TNF-αis the precursor of S-TNF-α. TM-TNF-αcontains a signal peptide consisting of 76 amino acid residues and S-TNF-αdose not. It has been shown in our previous studies that TM-TNF-αhad a broader tumoricidal spectrum than sTNF-αand was able to kill the tumor cells which are resistant to S-TNF-α. TM-TNF-αinduced mainly apoptosis while sTNF-αcaused chiefly necrosis.TM-TNF-αis not only as a ligand that deliveries forward signal via TNF receptor to target cell but also as a receptor that transmits reverse signal to effect cells. TM-TNF-αbearing cells often express TNFR, so it's difficult to exclude the interference of the forward signaling in the study of reverse signaling via TM-TNF-α. In our previous work on the relationship between the structure and the functions of TM-TNF-α, we found that the replacement of cysteine with phenylanaline at -28 position of this membrane molecule ( C-28F TM-TNF-α) resulted in the loss of TM-TNF-αcytotoxicity,but the mutein remained the binding ability to the receptor. Therefore, we presume that C-28F TM-TNF-αonly deliveries reverse signaling but dose not transduce forward signaling. In the present work whether the C-28F TM-TNF-αonly deliveries reverse signaling was studied which may provide a useful tool to further research the reverse signaling via TM-TNF-α. The main results as follows:1. The failure of forward signal transduction via C-28F TM-TNF-αmutein(1) Disability of C-28F TM-TNF-αmutein to induce secretion of NO by U937 cells. It was found that non-stimulated U937 cells could secrete basal level of NO, while wild type TM-TNF-αtransfected COS-7 cells caused more secretion of NO by U937 cells. In contrast, C-28F TM-TNF-αmutein transfected COS-7 cells did not display this function.(2) Disability of C-28F TM-TNF-αmutein to induce iNOS mRNA transcription by U937 cells. The results from RT-PCR and Realtime PCR were shown that non-stimulated U937 cells transcribed basal level of iNOS mRNA, while wild type TM-TNF-αtransfected COS-7 cells increased the transcription of iNOS mRNA by 90%. However, C-28F TM-TNF-αmutein transfected COS-7 cells did not have this effect.(3) Disability of C-28F TM-TNF-αmutein to kill HL-60 cells. By double staining of target cells with annexin V-FITC and PI the wild type TM-TNF-αtransfected COS-7 cells have shown to able to induce cell death by 79%, the C-28F TM-TNF-αmutein transfected COS-7 cells, however, were unable to kill the tumor cells as the other controls, suggesting that C-28F TM-TNF-αmutein loses the cytotoxicity of TM-TNF-αto S-TNF-αresistant tumor cells.(4) Disability of C-28F TM-TNF-αmutein to inhibit I-κB degradation in Raji cells. The results of Western blotting showed that the wild type TM-TNF-αtransfected COS-7 cells could efficiently inhibit the degradation of cytoplasmic I-κB in Raji cells, conversely, no obvious effect on inhibition of I-κB degradation was observed by incubation of Raji cells with C-28F TM-TNF-αtransfected COS-7 cells, since the level of cytoplasmic I-κB was similar to that in the controls.2. The reservation of reverse signal transduction via C-28F TM-TNF-αmutein(1) The enhancement of S-TNF-αinduced transcription of IL-8 mRNA via reverse signaling mediated by wild type as well as C-28F TM-TNF-α. The results of RT-Realtime PCR revealed that S-TNF-αcould stimulate U937 cells to transcribe IL-8 mRNA. Activation of reverse signaling via endogenous TM-TNF-αwith sTNFR1 obviously promoted S-TNF-?α? induced transcription of IL-8 mRNA. This effect could be enhanced by transfection of U937 cells with wild type as well as C-28F TM-TNF-α, suggesting that C-28F mutein remained reverse signaling of TM-TNF-α.(2) Facilitation of I-κB degradation induced by reverse signaling via wild type as well as C-28F TM-TNF-α. The results of Western blotting revealed that transfection with wild type and C-28F TM-TNF-αc?ould delivery reverse signaling and led to the degradation of I-κB by 20%, while transfection with TM-TNF-αlacking cytoplasmic segment had no effect.Conclusion: C-28F TM-TNF-αis able to delivery reverse signaling, but unable to transduce forward signaling, so that it can be used as a molecule specific for reverse signaling in the further study of TM-TNF-α.