The function and regulation of insulin-like growth factor binding protein-5 in HC11 cells

Our group has previously shown that IGFBP-5 expression increases dramatically during involution of the mammary gland and subsequently we developed a transgenic mouse model expressing IGFBP-5 specifically in the mammary gland, which proved that the binding protein was a causative factor in mammary epithelial cell death. In this study, the aim was to develop an in vitro system to investigate the regulation of IGFBP-5 expression and the function of the binding protein on cell death/survival and tissue remodelling. The mouse mammary epithelial cell line, HC11, was chosen for this and initially we characterised the IGFBP expression profiles in these cells. We demonstrated that although IGFBP-5 protein levels are up-regulated by up to 10-fold during differentiation of HC11 cells induced by treatment with a lactogenic hormone mix (DIP) and that IGFBP-2 secretion was down regulated, there was a clear dissociation between the process of cell differentiation and the regulation of these IGFBPs. Furthermore, the mRNA expression profile of the IGFBPs was also established using quantitative RT-PCR to examine similarities and differences in IGFBP mRNA expression profiles between the mammary gland and the HC11 cell line. In addition to the significant up-regulation of IGFBP-5 message in differentiated HC11 cells, we report that IGFBP-5 mRNA levels were increased by a dramatic 54-fold in the involuting mouse mammary gland. We decided to study the DIP-induced increase in IGFBP-5 levels in HC11 cells as an in vitro model in which to study the potential molecular signals responsible for the induction of IGFBP-5 expression in the involuting mammary gland. Using transient gene transfer methods, we demonstrated that there is an enhancer element(s) between positions-1004 to -156 in the IGFBP-5 promoter that results in significant induction of gene expression in HC11 cells and that there is a potential site for a novel transcriptional regulator(s) of IGFBP-5 expression at position -556, which merits further investigation. As HC11 cells also secrete a plasminogen activator in vitro, which results in cleavage of focal adhesions and cell death, and because IGFBP-5 has been previously shown to bind to PAI-1, we decided to use this system to determine the biological consequences of IGFBP-5/PAI-1 interaction. Our data demonstrates that IGFBP-5 can induce cell death both by sequestering IGF-I and by activation of plasmin which, in turn, induces degradation of the extracellular matrix. We have also shown that IGFBP-5 can enhance the activation of tPA, in a PAI-1- and IGF-independent fashion and that this can result in cell death. These studies thus identified the possibility that IGFBP-5 may act as a central coordinator of the apoptosis and ECM degradation that occurs during tissue remodelling