Mouse lactoferrin gene. Promoter-specific regulation by EGF and cDNA cloning of the EGF-response-element binding...

Expression of the lactoferrin gene in a variety of tissues is regulated differentially. We have previously demonstrated that the lactoferrin gene is regulated by estrogen and mitogen in mouse uterus. The mouse lactoferrin gene responded to forskolin, cAMP, TPA and EGF stimulation via two adjacent enhancer elements, the CRE and EGFRE and collectively referred to as the Mitogen Response Unit (MRU). We found that CRE is responsible for forskolin, cAMP and TPA whereas EGFRE is for EGF stimulation. We examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the CAT reporter construct, whereas, the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters (SV 40 and TK). Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. Mutation made at either elements or insertion of extra nucleotides between the two elements, severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells protected the EGFRE, CRE and noncanonical TATA from DNAase I digestion in a footprinting analysis. Nuclear protein which interacted with the CRE were previously identified as API and CREB. In this study, we isolated a cDNA clone from an RL95-2 expression library that encodes the EGFRE binding protein. Partial sequence of the cDNA clone revealed 100% nucleotide identity with a GC-box binding protein, BTEB2. Protein-protein interaction among the transcription factors could fine-tune the mouse lactoferrin expression in various tissues.