The Activation Of Jnk Regulates Mule Expression At Transcriptional Level By C-Jun

In this thesis, we report that it's not only Mule regulates TNFα-induced JNK activation and apoptosis, the activation of JNK also gives Mule a positive feedback, and regulates Mule expression at transcriptional level by c-Jun.BACK GROUND: JNK signaling pathway has a critical role in regulating a wide range of cellular activities from cell proliferation and differentiation, to programmed cell death, and also in the development of a number of diseases, such as diabetes and cancers. The activation of JNK is through sequential protein phosphorylation, mediated by a MAP kinase module, in response to a variety of extracellular stimuli, including physical stresses, cytokines, T-cell costimulation, and growth factors. JNK has two ubiquitously expressed isoforms, JNK 1 and JNK 2. It has reported that JNK1, but not JNK2 is required for TNFαinduced JNK activation, c-Jun expression, and apoptosis.As a transcription factor, Miz1 plays an important role in regulation of DNA damage response, cell cycle arrest, proliferation, differentiation, and apoptosis by activating or repressing transcription. In addition to being a transcription factor, Miz1 acts as a signal- and pathway-specific modulator or regulator (SMOR) that specifically regulates TNFαinduced JNK1 activation and cell death. Upon TNFαstimulation, the HECT domain containing E3 ubiquitin ligase Mule polyubiquitinated Miz1, relieving its suppression on JNK1 activation, which leads to the activation of JNK signaling pathway and apoptosis.AIMS: Although it is already a general consensus that Mule has ability to control the levels of key molecules, and regulate the related signaling transduction, Mule is still a not well-understood"fresh"protein. Its gigantic size suggests that, Mule may hide numerous additional possibilities. And how Mule is regulated in the highly dynamic network of signals remains unclear. Here, this thesis tries to confirm that it's not only Mule regulates TNFα-induced JNK activation and apoptosis, the activation of JNK also gives Mule a feedback, and regulates its expression.METHODS: First of all, expression of Mule, Miz1, JNK, p38, and c-Jun in WT, Jnk1-/- and Jnk2-/- MEFs were analyzed by immunoblotting. After we introduced JNK1 to Jnk1-/- MEFs, over expressed JNK1 and knock down JNK1 expression by siRNA in HEK293 cells, expression levels of Mule were analyzed by immunoblotting respectively. Then semiquantitative RT-PCR was used to analyze Mule mRNA expression levels in WT, Jnk1-/- and Jnk2-/- MEFs. We used USCS to get the sequence of predicted Mule promoter and fused the Mule promoter to a promoterless luciferase reporter gene, pGL2-Basic Vector, named as MP-LUC. The Mule promoter activity in HEK293 cells transfected RSV-c-Jun or empty vector, and with TNFαstimulation in WT, Jnk1-/- and Jnk2-/- MEFs were measured by luciferase reporter assay. Lastly, we identified the AP-1 site of Mule promoter by using TFSEARCH program (Version 1.3). We performed Chromatin immunoprecipitation (ChIP) assays to examine the binding of c-Jun to the AP-1 site of the Mule promoter without stimulation, and then following TNFαstimulation in vivo.RESULTS: Immunoblotting analysis revealed that Mule protein levels were severely reduced in Jnk1-/- cells where the expression of c- Jun was also lower than Jnk2-/- and WT MEFs. Reintroduction of HA-JNK1 into Jnk1-/- cells partially restored Mule expression. Over expression of JNK1 in HEK293 cells by transfection with HA-JNK1 further increased Mule expression. The knockdown of JNK1 expression by siRNA strongly reduced Mule expression levels. The Mule mRNA expression profile in WT, Jnk1-/- and Jnk2-/- MEFs measured by semiquantitative RT-PCR was the same as the protein expression profile in WT, Jnk1-/- and Jnk2-/- MEFs. The Mule promoter activity was stimulated by cotransfected RSV-c-Jun, and was gradually increased along with the increased c-Jun protein expression. MP-LUC activity was more dramatically simulated by TNFαin Jnk2-/- MEFs than in WT MEFs. And the levels of MP-LUC reporter expression following TNFαstimulation did not have obvious change. ChIP assay show that in nonstimulated wt fibroblasts, c-Jun had already been recruited to the AP-1 site of Mule promoter, and the recruitment was further stimulated by TNFαinduced JNK activation.CONCLUSION: Transcriptional factor c-Jun, one major member of AP-1 family, is an important downstream effector of JNK activation. After specifically phosphorylated and activated by JNK, c-Jun binds to AP-1 site of Mule promoter, and further stimulates Mule transcription. The JNK activation gives Mule a positive feedback, and regulates Mule expression at transcriptional level by c-Jun.