LACTOFERRIN EXPRESSION IN MAMMARY EPITHELIAL-CELLS IS MEDIATED BY CHANGES IN CELL-SHAPE AND ACTIN CYTOSKELETON

Lactoferrin is a secreted iron binding protein which is expressed duri ng normal functional development of mammary epithelium. Murine mammary epithelial cell lines competent for milk protein expression were used to identify microenvironmental factors that regulate lactoferrin expr ession. While lactoferrin was not expressed in adherent monolayer cult ures under standard subconfluent conditions on plastic, lactoferrin mR NA and protein steadily accumulated when the cells aggregated to form spheroids on a reconstituted basement membrane gel. However, unlike ot her milk proteins such as beta-casein, lactoferrin expression was also induced at high cell density in the absence of exogenously added base ment membrane or prolactin. These results led us to examine whether ch anges in cell growth, cell-cell interactions and/or cell shape were re sponsible for regulation of lactoferrin gene expression. Rounded, non- proliferating cells in suspension in serum-free medium expressed lacto ferrin even as single cells. Conversely, lactoferrin expression could be inhibited in non-proliferative cells in serum-free medium by mainta ining them in contact with an air-dried extracellular matrix which cau sed the cells to retain flat, spread morphologies. These findings indi cated that cessation of cell growth was not sufficient, that cell-cell interactions were not required, and that cell culture conditions whic h minimize cell spreading may be important in maintaining lactoferrin expression. Additional data supporting this latter concept were genera ted by treating spread cells with cytochalasin D. The resulting disrup tion of microfilament assembly induced both cell rounding and lactofer rin expression. Shape-dependent regulation of lactoferrin mRNA was bot h transcriptional and post-transcriptional. Surprisingly, treatment of rounded cells with a transcription inhibitor, actinomycin D, produced a stabilization of lactoferrin mRNA, suggesting that transcription of an unstable factor is required for degradation of lactoferrin mRNA. I mportantly, lactoferrin mRNA expression was regulated similarly in ear ly passage normal human mammary epithelial cells. In vivo, the changin g extracellular matrix components of the mammary gland during differen t stages of normal and abnormal growth and differentiation may provide different physical constraints on the configurations of cell surface molecules. These physical constraints may be communicated to the cell interior through mechanical changes in the cytoskeleton. Unlike beta-c asein whose expression is upregulated by specific integrin-mediated si gnals, lactoferrin may be representative of a class of proteins synthe sized in the mammary gland using basal transcriptional and translation al machinery. The suppression of lactoferrin expression that is observ ed in monolayer culture and in malignant tissues may reflect inappropr iate cell shapes and cytoskeletal structures that are manifested under these conditions.