The responsiveness of the bovine lactoferrin promoter to cytokines and glucocorticoids : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Abstract

Lactoferrin is an iron-binding protein found in many bodily secretions and in the secondary granules of polymorphonuclear leukocytes. While there are many proposed functions for the lactoferrin protein - e.g. for iron storage, antibacterial properties, or a role in inflammation, the specific function(s) of lactoferrin have yet to be elucidated. Evidence that lactoferrin may be involved in inflammation was observed by Harmon et al. (1976) where after the induction of bovine mammary infections, a significant increase in secreted lactoferrin protein was seen during the early phase of the infection. As this increase was during the period of the acute phase response, this suggested that lactoferrin, as was the case with other proteins induced during this time, may have a role in the inflammatory response. The bovine lactoferrin (bLf) promoter contains many putative binding sites for inflammatory modulators, which suggests that the increases in lactoferrin seen during inflammation may be due to activation of lactoferrin gene transcription by these specifically-induced transcription factors. Substantiation of this suggestion would provide further evidence for a specific role for lactoferrin during inflammation. To investigate the cytokine-responsiveness of the bLf promoter, constructs corresponding to various lengths of the putative bLf promoter were linked to the luciferase reporter gene and introduced, by transient transfection, into RL95-2 human endometrial carcinoma cells. Cytokines, glucocorticoids or expression vectors for transcription factors were added to the cells, or potential 'masking' factors in the media such as phenol red or insulin were removed. The luciferase activity of the transfected cells was monitored for significant variation from the basal levels. The addition of cytokines with or without phenol red or insulin did not cause any significant changes in bLF promoter activity. In phenol red-free media, increases in luciferase reporter gene activity were observed after the co-transfection of an expression vector for NF-IL6, the addition of dexamethasone and also the addition of dexamethasone together with the co-transfection of a glucocorticoid receptor expression vector. These data provided evidence that lactoferrin transcription may be induced by inflammatory factors which support the suggestion that lactoferrin has a role in the inflammation process.