| Nogo-A is a myelin derived central nervous system neurites outgrowth inhibitor, and the mechanism of its role in neurites out-growth inhibition has been well studied since it was cloned in 2000. It is well known that the oligodendrocyte expressed Nogo-A transduces its neurite-outgrowth inhibitory signal through the NgR/ p75NTR(or TROY)/LINGO-1 receptor complex on the membrane of neurons. A plenty of in vivo and in vitro strategies had been applied to block the Nogo-associated inhibitory singaling pathway, most of them had got significant improvements in neurites outgrowth and CNS regeneration. Unexpectedly, in 2003 three independent labs had produced Nogo knockout mice with three different phenotypes by different knockout methods, but actually these experiments did not support the dominant inhibitory role of Nogo-A in CNS regeneration.Nogo-A is not only highly expressed in mature oligodendrocytes, but also in various kinds of neurons in the central nervous system. Previous work of our lab showed that most of Nogo-A localized in endoplasmic reticulum, polyribosomes and associated with the chromatins of the nucleus in neurons, suggesting that Nogo-A may play important roles in neurons. Moreover, according to the bioformatical data of Nogo-A protein sequence, the N-terminal of Nogo-A contained several potential NLS sites, acidic amino acids and proline-rich domain that can interact with proteins containing WW and SH3 domains, which indicated the possible role of Nogo-A as a regulator of the neural transcription.As a part of the researches to reveal the functions of neural Nogo-A, human embryonic kidney cell line 293FT was used as a model to investigate the involvement of Nogo-A in transcriptional regulation and its downstream signaling pathway. In addition, the possible role of Nogo-A in neuron differentiation and neurites growth was also checked using the PC12 differentiation model.The main results were obtained as follows:1. In HEK293FT cells, the pathway profiling reporter system was used to screen for the downstream targets of Nogo-A, it was found that overexpression of Nogo-A could activate NF-κB signaling specifically, and the NF-κB activating ability of Nogo-A is dose dependent.2. Then, followed by using different Nogo-A alternative splicing isoforms and a serial of truncations, it was confirmed that the activation of NF-κB by Nogo-A relied on its N-terminal proline rich domain.3. Furthermore, by co-expressing the dominant negative mutants of some NF-κB pathway associated proteins, it revealed that the IκBα,TRAF6,Rac/Cdc42 were involved in Nogo-A inducing activation of NF-κB.4. In addition, the HEK293FT cells were treated with the purified GST-NogoA truncated proteins in vitro, it was found that they did not had any significant effect on the NF-κB activity, suggesting that the activation of NF-κB by Nogo-A was not dependent on its putative receptors.5. Nogo-A specific RNAi was used to knockdown the Nogo-A expression in PC12 cells, consequently, the NF-κB activation by NGF inducing was effectively blocked.6. In NGF-treated PC12 cells , overexpression of Nogo-A by transcient transfection had no effect on the PC12 differentiation and neurites growth compared with the control ; Besides,the PC12 cell lines which stably expressed PMIR-Nogo-A and PMIR-Control was constructed independently, it was found that stably knockdown of Nogo-A expression in PC12 cells did not influence their differentiation induced by NGF.Above all, our results indicated that Nogo-A could significantly activate NF-κB, and this activation depended primarily on its N-terminal proline rich domain, and the IκBα,TRAF6,Rac/Cdc42 were also involved in Nogo-A induced activation of NF-κB. Furthermore, it was found that Nogo-A was not involved in NGF induced PC12 differentiation, but the discovery of Nogo-A-NF-κB pathway will contribute to unveil the crucial role of Nogo-A in neuron.
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