Study Of The Functions And Mechanisms Of NLK On The IL-1? Induced C/EBP Activation And The SRF Mediated Myogenesis

NLK (Nemo-like kinase) is an evolutionarily conserved MAPK (mitogen-activated protein kinase) and CDK (cyclin-dependent kinase) related kinase. Just like MAPK and CDK, NLK is involved in regulating several important signal pathways. NLK is evolutionarily conserved from worms to humans. Studies from invertebrates, mice and human showed NLK is associated with phenotypes and diseases with poor understood mechanisms. Therefore, to explore novel funtions of NLK on regulating signal pathways may not only contribute to understanding NLK related phenotypes, but also sever as theoretical basis for the prevention and treatment of diseases associated with NLK. The study in this thesis elucidated the functions and mechanisms of NLK on the Interleukin-1? (IL-1?) induced C/EBP activation and the SRF mediated myogenesis.IL-1? is a key proinflammatory cytokine that initiates several signaling cascades, including those involving CCAAT/enhancer binding proteins (C/EBPs). The mechanism by which IL-1? propagates a signal that activates C/EBP has remained elusive. In the first part of this thesis, we found that IL-1?-induced C/EBP activation was positively regulated by NLK through a luciferase reporter screen. Overexpression of NLK activated C/EBP and potentiated IL-1?-triggered C/EBP activation, whereas knockdown or knockout of NLK had the opposite effect. Mechanistically, we found that NLK interacted with activating transcription factor 5 (ATF5) by the combination of 3 ×Flag knock-in technology, affinity purification and mass spectrometry. Moreover, we found that NLK inhibited the ubquitination and proteasome-mediated degradation of ATF5 in a kinase activity independent manner. The results also demonstrated that NLK cooperated with ATF5 to activate C/EBP, whereas NLK could not activate C/EBP upon knockdown of ATF5. In addition, several reports suggest that TAK1 is a downstream effector of IL-1(3 that acts upstream of NLK. Our results showed that TAK1 mimicked the ability of NLK to stabilize ATF5 and activate C/EBP. Thus, the study in this part reveal the TAK1-NLK pathway as a novel regulator of basal or IL-1?-triggered C/EBP activation though stabilization of ATF5.Muscle is a highly complex and heterogeneous tissue serving a multitude of functions in the organism. The normal myogenesis is very important to the body. Normal muscle cell differentiation was precisely control by a variety of complex signaling pathways that triggered by extracellular and intracellular signal. SRF signaling pathway plays a key role in muscle cell differentiation for its regulation of contractile genes and cytoskeleton genes. However, the regulation of SRF signaling pathway remains elusive.In the second part of this thesis, we found that cells displayed a flattened and enlarged morphology and better attachment ability upon knockout of NLK, which hinted the regulation of cytoskeleton genes by NLK. Then we found that the expression of most of the SRF targeting genes,including contractile genes and cytoskeleton genes,were changed significantly upon knockout of NLK by RNA sequencing anaylsis. Furthermore, additional experiments demonstrated that NLK positively regrulated SRF/ELK1 signaling pathway but nagtively regulated SRF/MKL pathway. Mechanistically, we found that NLK participated SRF signaling pathway through its regulation of MICAL2 transcription and Rho GTPase/actin pathway. Moreover, the mRNA and protein levels of NLK were gradually decreased during the differentiation of myoblast, which was prometed upon knock-down of NLK. Therefore, the work in this part demonstrated the function and michanism of NLK druing the myoblast differentiation by the transcriptional regulation of MICAL2 which regulates SRF signaling.