Study On The Mechanism Through Which HPD Regulate NF-κB Pathway And NLK Regulate TGFβ Pathway

This essay includes two parts, the first is hydroxyphenylpyruvate dioxygenase (HPD) up-regulates NF-κB signaling pathway and promotes the survival and migra-tion of lung cancer cell, the second is NLK inhibits TGF-β signaling pathway by interacting with Smad4.It is said that birds of a feather flock together. The principle is also applicable in biology study. Therefore, Finding out the proteins that interact with your interested protein is an important approach to discover its function. Using yeast two-hybrid system, our lab built large scale protein-protein interaction network which included many newly found protein-protein interactions. The interaction network provides a lot of useful information to us. Two pairs of protein-protein interactions discovered by the network that we believed having important function was further studied. The two interactions are that between HPD and NEMO and that between NLK and Smad4.HPD is the key enzyme of tyrosine catabolism pathway. NEMO is the core protein that involves in NF-κB signaling pathway. Using in vitro and in vivo coimmunoprecipitation assay, we confirmed the interaction between HPD and NEMO through their N terminals, and HPD may form a protein complex with IKK complex. Ectopic expression of HPD accelerated the degradation of IκBα, enhanced the activity of NF-κB reporter and increased the expression of NF-κB target gene induced by TNFα, including BCL-xL, cIAP2, ICAM-1, VCAM-1; knock down of HPD inhibited the activity of NF-κB reporter; lacking of the enzyme activity of HPD didn't influence its function in up-regulating NF-κB pathway, hydroxyphenylpyruvic acid(HPP), the substrate of HPD, and homogentisic acid(HGA), the product of HPD, had little influence on NF-κB pathway. These results indicate HPD up-regulates NF-κB pathway independent of its enzyme activity. We found over-expression of HPD augments the ubiquitination of NEMO stimulated by TNFα. RT-PCR was used to test the expression of HPD in various organs of mice, the result showed that HPD express in liver and kidney, which is consistent with previous report. Intriguingly, besides the cell lines originated from liver, HPD was detected in many lung cancer cell lines. In order to understand the relationship between HPD and NF-κB signaling pathway in lung cancer, we compared the expression of HPD and phosphorylated p65(S276) in the lung tumor tissue and relative normal lung tissue of lung cancer patients, and found lung tumor tissue expressed higher level of HPD than normal lung tissue, the expression of HPD correlate with that of phosphorylated p65(S276). Subsequently, using lentivirus system, knock-down of HPD inhibited the colonic formation ability and migration of H1299and A549and had no influence on the cell cycle and proliferation of H1299and A549. To sum up, our data show lung cancer tissue express HPD, knock-down of HPD alleviate the malignant phenotype of lung cancer, these effects maybe relevant to the interaction between HPD and NEMO and thereby activation of NF-κB pathway. These results indicated HPD may play important role in the initiation and development of lung cancer.NLK is a conserved S/T protein kinase, recognizes S/TP motif of the target proteins, involves in many biology process through phosphorylating its target proteins. The result from CoIP and GST-pull down showed that NLK interact with Smad4. NLK down-regulate TGFβ pathway independent of its kinase activity through enhancing the ubiquitination of Smad4and decrease the stability of Smad4. The biological significance of the interaction between NLK and Smad4requires further study.