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 effect 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 has been associated with tumor, cancer, a weakened immune system and polygenic infections. We have previously isolated and characterized both mouse and human lactoferrin genes and their promoters. During the course of these studies, we cloned two transcription factors, estrogen receptor related receptor a1 (ERRa1) and intestine-enriched Kruppel-like factor (KLF5), which are relevant to the estrogen and EGF regulation, respectively. We cloned the IKLF5 by using CAAT/GT box of the mouse lactoferrin gene promoter as probe and screen the expression library of RL95 cells. The full-length cDNA of IKLF 5 consists of 3336 bp with a 302 bp 5'-UTR, a 1663 bp 3'-UTR, and a 1371 bp coding sequence for a 457 amino acid polypeptide. Based on its tissue distribution and sequence homology to the mouse IKLF, we renamed this protein IKLF. The human IKLF gene spans over 20 kb in length and is organized into four exons, whose intron/exon junctions follow the GT/AG rule. The three zinc fingers are encoded by three exons. Nuclear localization of IKLF was demonstrated by the green fluorescence protein (GFP) tagged IKLF in transfection experiments and Western analysis. Overexpression of IKLF in HEC-1B cells activates the activity of reporter constructs containing the CAAT/GT box of the mouse lactoferrin gene. These findings imply that IKLF is a nuclear transcription factor that binds to the CAAT/GT box, and functions as a modulator of the lactoferrin gene promoter activity. 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.
Showing the most recent 10 out of 15 publications
