Characterization of a mitogen-response unit in the mouse lactoferrin gene promoter.

Abstract

Lactoferrin is present in a variety of tissues and biological fluids; however, the amount differs significantly due to differential expressions. We have previously demonstrated that the mouse lactoferrin gene is regulated by estrogen through an estrogen-response DNA element located at -349, upstream from the transcription start site (+1). In this report, we characterized by deletion and mutation analyses a cluster of mitogen-response elements located between -80 and -40 of the mouse lactoferrin promoter. We demonstrated that the chimeric chloramphenicol acetyltransferase reporter constructs (the -103 to +1 sequence of the mouse lactoferrin gene) containing the mitogen-response unit of the lactoferrin gene were stimulated by cAMP, forskolin, 12-O-tetradecanoylphorbol-13-acetate, and epidermal growth factor/recombinant transforming growth factor-alpha (EGF/TGF-alpha) in a time- and dose-dependent manner. The sequence at position -52 to -40 (mLF-CRE) of the gene conferred transcriptional activation in the presence of forskolin, cyclic AMP, and 12-O-tetradecanoylphorbol-13-acetate in transiently transfected human endometrium carcinoma RL95-2 cells, whereas the region at -80 to -60 responded to EGF/TGF-alpha stimulation. Overexpression of the catalytic unit of protein kinase C or protein kinase A in the RL95-2 cells elevated the chloramphenicol acetyl-transferase activity of the reporter construct 5-6-fold. The mobility shift assay suggested that AP1 and CREB or related proteins participated in complex formation with the mLF-CRE, whereas different proteins bound to the EGF/TGF-alpha-response element.