of Work: To maintain proper growth and development of a functional organism, it is essential that genes involved in these functions are expressed in a coordinate and timely fashion. Environmental chemicals that interfere with the expression of these genes could cause various cellular abnormalities, thus endanger our health. In order to address the question on how environmental factors affect our health, we need to understand the intricate molecular event associated with gene expression. Lactoferrin gene that encodes a noneheme iron-binding glycoprotein serves as an ideal model to study the gene regulation at the molecular level. It is well documented that lactoferrin plays an important role in host defense, inflammatory response and immuno-modulation. The protein is expressed in a variety of tissues and secreted into biological fluids such as tears, saliva and seminal plasma under different regulatory mechanisms. Lactoferrin gene expression is influenced by the exposure to estrogens, retinoic acid, glucocorticoid, forsklin and EGF. Therefore, environmental chemicals, xenoestrogens and catecholestrogens that perturb the steroid hormone and growth factor signaling pathways could affect lactoferrin gene expression. Deregulation of lactoferrin gene expression and methylation status have been associated with tumor, cancer, a weakened immune system and polygenic infections. The mouse and human lactoferrin genes and their promoters have been characterized. Number of transcription factors such as COUP-TF, estrogen receptor a (ERa), estrogen receptor related receptor a1 (ERRa1) and intestine-enriched Kruppel-like factor (IKLF, KLF5) are involved in the regulation of lactoferrin gene expression by estrogen and EGF. The human estrogen receptor-related receptor (ERR a1, NR3B1a) was shown to bind a half-ERE (SFRE) 26 bp upstream from the estrogen response element (ERE) of the human lactoferrin gene promoter. We demonstrated that ERR a1 binds both SFRE and ERE elements and constitutively transactivates the lactoferrin gene promoter. ERR a1 activates reporter constructs containing various types of estrogen response elements in endometrial and non-endometrial cells in transient transfection experiments. Overexpressing the coactivator, SRC1a or GRIP1, further enhances ERR a1 induced transcriptional activity. We showed that the AF2 domain of ERR a1 is essential for the transactivation function. Protein-protein interaction between the SRC1a and ERR a1 C-terminus was confirmed with a GST """"""""pull-down"""""""" assay. We found that ER a can also bind SFRE of the lactoferrin gene and transactivate the promoter activity in a ligand-dependent manner. The present study demonstrated that ERR a1 may actively modulate the estrogen response of lactoferrin gene as well as other estrogen responsive genes. We demonstrated that IKLF transactivates the lactoferrin gene promoter in human HEC and insect SL-2 cells. The transactivation function of IKLF was enhanced by CREB binding protein (CBP) and blocked by wild type but not mutant E1A. We found that IKLF and CBP were components of complexes formed between the GC element and nuclear protein. Physical interaction between these two proteins was demonstrated by co-immunoprecipitation and GST pull-down studies. Mammalian two-hybrid experiments mapped the interaction domains to the extreme N-terminus of CBP and the N-terminus of IKLF. Phosphorylation of IKLF enhances CBP interaction thus IKLF transaction function. Inhibition of protein kinase C (PKC) activity by specific inhibitor or mutation at the potential PKC site within the interaction domain of IKLF reduces IKLF transactivation function. Results of the sutdy establish IKLF could function as a transactivator, and phosphorylation and interaction with CBP play a significant role.
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