Structure And Function Of ERRα1 And The Role Of ERRα1-interacting Protein, E3B1, In Breast Cancer

Nuclear receptors (NRs) constitute an important superfamily of trans-acting factors, which recruit transcriptional cofactors to DNA in a ligand-dependent fashion and mediate expression of genes associated with embryonic development, cell differentiation and homeostasis. The human estrogen receptor-related receptor α1 (hERRα1) is one of the firstly cloned orphan nuclear receptors. It binds to the ERR response element (ERRE) in the up-stream of many genes and regulates the expression of these genes, including human thyroid hormone receptor, osteopontin, lactoferrin, and aromatase. ERRα1 is expressed in normal breast tissue and also expressed at higher level in breast cancer tissue. Overexpression of ERRα1 inhibits MCF-7 breast carcinoma cell proliferation and suppresses ERα-mediated transcriptional activity in breast cancer cells, indicating the close relationship between ERRα1 and breast cancer. The direct regulation of gene transcription by nuclear receptor involves not just ligand and DNA binding but the recruitment of coregulators. Most of steroid receptors, such as estrogen receptor (ER), interact with coactivator proteins through a transcriptional activation function (AF2) structure in their ligand-binding domains. However, the information regarding the domain or amino acids residues of ERRα1 involved in the coactivator binding between ERRα1 and coactivator is still very limited. The investigation of structural determinants of ERRα1 for the coactivator interaction will provide new insight into the nature of the ERRα1 and its functional consequences. In an attempt of searching proteins that specifically interact with ERRα1 in breast tissue, E3B1(Eps8-SH3-domain binding protein-1)was identified as a novel ERRα1 binding protein in yeast-two hybrid screening of the human mammary gland cDNA library in our laboratory. Recently study demonstrated that E3B1 acts as a negative regulator in proliferation of mammalian cells and is closely associated with tumor. However, little is known about the mechanism of E3B1 on inhibition of cell proliferation. Because that E3B1 is a ERRα1-interacting protein and ERRα1 plays an important role in breast cancer development, the investigation on the role of E3B1 in the breast cancer development will provide new insight into molecular mechanism of ERRα1's role in mammary carcinoma cells. In order to further deepen our understanding of the relation on the structure and function of ERRα1 as well as functional mechanism of E3B1 on breast carcinoma, we carried out this study. The specific aims and results of my research are summarized below: 1. Structure and function of ERRα1 1) In order to illuminate the relation on structure and function of ERRα1, the conserved domain and amino acid residues of ERRα1 were analyzed, not only a series of deletions of ERRα1 but also site-directed mutagenesis of potential key amino acids were designed. The aim of the project is to reveal the relation of ERRα1 structure and its function by analyzing the functional diversity of the mutations. 2) To identify the domains of ERRα1 that interact with nuclear receptors coactivator, PNRC (Proline-rich nuclear receptor coregulatory protein). Several of yeast expression plasmids for the fusion protein of Gal4DBD and ERRα1 deleted forms including ERRα1/1-144 (AF1 and DBD), 190-423 (HBD), 1-412 and 190-412 (deletion of AF2), 145-423 (deletion of AF1 and DBD), 1-407 and 190-407 (deletion of extended AF2) were successfully constructed. The interactions between these ERRα1 fragments with coactivator PNRC were examined and compared in yeast two-hybrid assays. The results demonstrated that aa190-423 region is the coactivator-interacting domain of ERRα1. 3) To study the importance of AF2 domain in the interaction between ERRα1 and coactivator by site-directed mutagenesis and yeast two-hybrid assays. Yeast expression plasmids for the ERRα1 wild type HBD (190-423) or ERRα1/HBD with AF2 mutations, including L413A/F414A, M417A/L418A, F414A, and F399A, were successfully constructed. The results form yeast two-hybrid assays showed that all the mutated ERRα1/HBDs did not interact with coactivator PNRC. The results from AF2 deletion mutation mentioned above demonstrated the ERRα1/HBD with the deletion of AF2 domain including fragments 1-412,1-407,190-412 and 190-407 lost the interaction with PNRC. Taken together, these results demonstrated that, like most of other receptors, ERRα1 interacts with coactivator in an AF2-dependent manner. 4) To investigate the importance of the conserved residues in Helix 3 (H3) and Helix 4(H4) regions of ERRα1 LBD for the interaction between ERRα1 and coactivator. The ERRα1/HBDs containing mutations I240R, M258R, M258A, K244A, K244E, F232A, T227A, T227L, S253A, S241A, or S245A were expressed as Gal4DBD fusion proteins in yeast and tested for their interaction with coactivator PNRC. Mutant F232 totally lost its interaction with PNRC. Mutant K244A was shown very low level of interaction with PNRC while T227A,T227L,S253A,S241A,S245A,M258R affected the interaction between ERRα1/HBD and PNRC in various degrees. These experiments suggest that some amino acids residues located in Helix 3 and Helix 4 also play an important role for the ERRα1 to interact with coactivator. 5) To investigate the functional consequences of the mutations of AF2 domain in the transcriptional activity of ERRα1. The mammalian expression plasmids in pSG5 vector for the wild type ERRα1 or ERRα1 with mutations in AF2 domain, including ERRα1/1-412, ERRα1/L413A/F414A, ERRα1/M417A/L418A, ERRα1/F414A, ERRα1/F399A, and ERRα1/1-407, were generated and their transactivation function on the luciferase reporter transcription were examined by transfection. Surprisingly, all the mutations in AF2 domain of ERRα1,including aa1-412,aa1-407,F414A,L413A/F414A orM417A/L418A, were found to enhance the transactivation function of ERRα1, indicating that AF2 domain is not necessary for ERRα1's constitutive transactivation function, rather it inhibits ERRα1 transactivation activity. This finding also indicates that coactivator binding is not critical for ERRα1's transactivation function since those mutants did not interact with coactivator PNRC. 6) To define the role of other amino acid residues in H3 and H4 region in ERRα1 transcriptional function besides AF2 domain. By site-directed mutagenesis approach, the ERRα1 mutants with the mutations (same as above) in H3 or H4 region were generated and their effects on ERRα1's transactivation function were examined by transient transfection in HeLa cells. Results from this study revealed that the transactivation activities of ERRα1 mutants , F232A or K244A, were lower than that of wild type ERRα1. In addition, mutations in M258,I240,F399,T227,S241or S253 also changed transactivation activity of ERRα1, suggesting that these residues in H3 and H4 also play important role in constitutive transactivation activity of ERRα1. 2. Role and mechanism of E3B1 in mammary carcinoma.1) To investigate the expression levels of E3B1 in human breast cancer and homologous juxta-cancerous tissues. In order to know the relationship between expression of E3B1 and breast cancer, the expression levels of E3B1 in breast carcinoma and juxta-cancerous tissue were examined by RT-PCR. E3B1 was found to express in both normal and malignant breast tissues. In the most of samples examined, there were obviously higher expression levels in carcinoma tissues than in corresponding paracarcinoma tissues, revealing that E3B1 lower expression may be one of the causes responsible for breast cancer development. 2) To study the role of E3B1 in mammary carcinoma cells. In an effort to determine a function of E3B1 in mammary carcinoma cells, the overexpressing E3B1 cell line was established by stable transfection and G418 selection. It was found that the growth rate of breast cancer MCF-7 cells overexpressing E3B1 was decreased compared with that of MCF-7 cells transfected with empty vector only by MTT method. The results presented in this study demonstrated that over-expression of E3b1 can inhibit the growth of breast cancer cells. 3) To analyze the localization of E3B1 protein in cell. In order to further testify that E3B1 protein can play a role in nuclei, E3B1 was expressed as fusion protein with green fluorescence protein (GFP) and its localization in MCF-7 cells was examined by fluroscence microscope. EGFP-E3b1 fusion protein was found to localize in both cytoplasm and nuclei of MCF-7 cells while EGFP alone locates only in the cytoplasm. 4) To test the hypothesis that E3B1 inhibits breast cancer cell growth through its interaction with ERRα1 and its effect on ERRα1's transactivation function. ERRα1 has been demonstrated to antagonize the ER's stimulating effect on breast cancer cell growth and overexpression of ERRα1 has been shown to inhibit MCF-7 proliferation. E3B1 was identified through its interaction with ERRα1 in our laboratory. In the present study, using transient transfection and luciferase activity assay of reporter gene approach, we observed that E3B1 could increase transactivation activity of ERRα1 in a dose dependent manner. This result, in addition to demonstrating nuclear receptor coactivator function of E3B1, indicated that the coactivation function of E3b1 on ERRα1 transactivation function might be one of the mechanisms by which E3b1 inhibits the breast cancer cell growh. In summary, results from this study defined the detailed structural determents of ERRα1 to interact with coativator PNRC and its functional consequences. Briefly, our resultsdemonstrate that ERRα1 interacts with coactivator through its ligand-binding domain (LBD or HBD) and the integrity of AF2 structure in LBD is critical for ERRα1 for this interaction. However, unlike other steroid receptors, such as estrogen receptor, in that the integrity of AF2 domain is critical for both transactivation function and coactivator interaction, AF2 structure integrity of ERRα1 is not required for its constitutive transactivation function, although it is important for its interaction with coactivator. This new finding indicates that coactivator interaction is not critical for ERRα1's transactivation function. Overexpresion of ERRα1 was found to inhibit MCF-7 cell growth in our previously study. The results from this study demonstrated that overexpression of E3B1, an ERRα1-interacting protein, could also inhibit MCF-7 cell growth. The results further showed that E3B1 functioned as a coactivator of ERRα1 and enhanced the ERRα1's transactivation function, suggesting a role of the interaction between ERRα1 and E3B1 in regulating the proliferation of breast cancer cell.