IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.