The Structure And Functional Relationship Of TR3/Nur77 In Angiogenesis

Background:Angiogenesis plays a vital role in growth, invasion and metastasis of tumors. Recently, it is recognized that anti-angiogenic therapy of tumors is a prospective modality in tumor treatment. Hepatic cell carcinoma has redundant blood supplies and anti-angiogenesis might be beneficial in development as well as prognosis of hepatic cell carcinoma. VEGF is a key cytokine in tumor angiogenesis. Anti-VEGF neutralizing antibody, VEGF kinase inhibitor and multiple kinase inhibitors are now introduced in clinic for treatment of hepatic cell carcinoma. However, the human body is a complex and VEGF has too many target organs within the human body, which result in multiple side effects and intrinsic resistance. Hence it is required to investigate more precise therapeutic target to decrease adverse effect of anti-angiogenic therapy in tumor treatment as possible.Orphan nuclear receptor TR3/Nur77 (human TR3; mouse Nur77; rat NGFI-B), also known as nerve growth factor induced gene B, is a member of nuclear receptor IV subfamily of transcription factors. It has no physiological ligand in mammalian cell lines, although previous study has identified several synthesized agonists, including cytosporone B and a series of methylene-substituted diindolymethanes. TR3/Nur77 is of vital importance in cancer cell biology, brown fat thermosis, inflammation, metabolism diseases, stress and addiction. It currently acts as a transcriptional factor and initiates or blocks transcription of downstream genes, in which manner it regulates downstream physiological processes.Our previous work demonstrated that TR3/Nur77 is the key cytokine mediating VEGF induced proliferation, migration, increased permeability of endothelial cells and angiogenesis in vivo. Application of TR3/Nur77 antisense oligonucleotide will inhibit endothelial cell proliferation, migration and tube formation induced by VEGF. And so it is the same with angiogenic activities of endothelial cells induced by histamine and serotonin in vitro. Up regulation of TR3/Nur77 in endothelial cells directly induced cell proliferation, migration and increased monolayer permeability. Compared to wild type mice, Nur77 knock-out mice has significantly inhibited tumor growth and angiogenesis. Hence we demonstrated that TR3/Nur77 is an excellent target for pro-angiogenesis and anti-angiogenesis therapies.Similar to other members of orphan nuclear receptor superfamily, TR3/Nur77 has three predominant domains:transactivation domain, DNA binding domain, and ligand binding domain. Among them, transactivity is required in mediating VEGF induced endothelial cell proliferation, migration, and increased permeability. However, requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis is still unknown. Our study is aimed to investigate the structure-function relationships and set foundation for the subsequent anti-angiogenic therapy.Aims:The present study is aimed to investigate the structural and functional relationships in TR3/Nur77 induced angiogenesis, and to identify the potential therapeutic target of anti-angiogenic therapy in tumor treatment.Methods:1、Cell culture:HEK293 cells were routinely maintained in DMEM with 10% fetal bovine serum. And HUVECs were routinely maintained in EBM/EGM SingleQuots on 30μg/ml vitrogen coated plates.2、Construction of recombinant plasmid:Recombinant PCR was employed to prepare the mutants, with corresponding oligonucleotides as primers.3、Preparation of recombinant adenovirus:pAd/CMV/V5-DESTTM Gateway(?) Vector Kit was employed to prepare adenovirus vectors expressing TR3 and its mutants. Transfect HEK293 cells with adenovirus vectors expressing TR3 and its mutants and purify the virus. Store in -80 ℃ and thaw it on ice before each use.4、Western Blot was employed to screen the protein expression of HUVECs infected with different adenovirus.5、CyQUANT(?) Cell Proliferation Assay was employed to determine proliferation of HUVECS infected with different adenovirus.6、Monolayer migration assay was employed to determine migration of HUVECS infected with different adenovirus.7、Monolayer permeability assay was employed to determine migration of HUVECS infected with different adenovirus.8、Rhodamine phalloidin staining was employed to observe cytoskeleton changes of HUVECS infected with different adenovirus.9、Computational protein structure modeling and binding site potential analysis10、SPSS17.0 software was employed for statistical analysis and results are presented as mean± SD. Student’s t-test was employed to determine statistical significance.Results:1、TR3/Nur77 promotes proliferation, migration, permeability and cytoskeleton rearrangement.2、TR3/Nur77 transcriptional activity is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.3、De-phosphorylation of Ser351 and amino acid residues GRR in the DNA-binding domain are required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton.4、Amino acid 41-60 segment of TR3/Nur77 in the transactivation domain is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.5、Phosphorylation of Thr 48,55 and 61, Ser 52 and 54, and Tyr 60 is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.6、Protein structure and binding site prediction for TR3 N-terminal transactivation domain reveals there are 3 potential protein binding sites:site 1 (T27-T34), site 2 (F56-E63) and site 3 (Y69-P77).Conclusions:The main findings of the present studies are 1)TR3/Nur77 promotes proliferation, migration, permeability and cytoskeleton rearrangement.2)TR3/Nur77 transcriptional activity is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.3)De-phosphorylation of Ser351 and amino acid residues GRR in the DNA-binding domain are required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton.4)Amino acid 41-60 segment of TR3/Nur77 in the transactivation domain is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.5) Phosphorylation of Thr 48,55 and 61, Ser 52 and 54, and Tyr 60 is required in TR3/Nur77 mediated proliferation, migration, permeability and cytoskeleton rearrangement.7)Protein structure and binding site prediction for TR3 N-terminal transactivation domain reveals there are 3 potential protein binding sites: site 1 (T27-T34), site 2 (F56-E63) and site 3 (Y69-P77).Our previous studies demonstrated that TR3 is an excellent and specific target for therapies of pro-angiogenesis and anti-angiogenesis. These studies further elucidate the structure and functional relationship of TR3 in angiogenesis by identification of novel amino acid residues that are critical for the function of TR3 in endothelial cells. Our current studies set the foundation for identification of compound(s) to inhibit or promote TR3 transcriptional activity for anti-angiogenic and pro-angiogenic therapies.