Constitutive expression of IGF-binding protein-3 by mammary epithelial cells alters signaling through Akt and p70S6 kinase.

IGF-binding protein-3 (IGFBP-3) potentiates IGF-I action in the non-transformed mammary epithelial cell line, MAC-T, via a mechanism that is independent of its ability to bind IGF-I. The goal of the present study was to determine if IGFBP-3 might enhance IGF action by influencing intracellular signaling events downstream of the IGF receptor. IGF-I stimulated a time-dependent activation of Akt in which phosphorylation of Ser(473) was detectable by 1 min and maximal at 15 min. In contrast, no activation of extracellular signal-regulated kinase (ERK)1/2 by IGF-I was observed although basal phosphorylation was readily detectable. In MAC-T cells constitutively expressing IGFBP-3 (+BP3), phosphorylation of Akt following stimulation with IGF-I was enhanced relative to mock-transfected cells (Mock). The enhancement was detectable within 1 min of IGF-I treatment and persisted for up to 10 h. The increased phosphorylation observed by Western blotting corresponded to a 1.7-fold increase in Akt kinase activity. The enhanced Akt response was elicited by factors that activate the IGF receptor but exhibit reduced affinity for IGFBP-3, such as Long R(3)IGF-I, B chain IGF-I and insulin. In contrast, [Leu(60)]IGF-I, which binds IGFBP-3 but has reduced affinity for the IGF receptor, failed to induce comparable activation, suggesting that an association between IGF-I and IGFBP-3 is not required for the effect. The enhanced Akt activation could not be mimicked by addition of exogenous IGFBP-3. Akt phosphorylation was also enhanced by transforming growth factor-alpha in +BP3 cells, indicating that the effect was not specific to IGF-I. Similar to Akt, phosphorylation of p70S6 kinase (p70(S6K)) by IGF-I was also enhanced in +BP3 cells relative to Mock cells at both 15 min and 10 h. However, this was largely an effect of lower basal activation of p70(S6K) in +BP3 cells. These data indicate that endogenous IGFBP-3 potentiates IGF action in MAC-T cells by enhancing signaling via the phosphatidylinositol 3-kinase pathway at a point that is downstream of IGF receptor activation. Further studies will delineate specific mechanisms by which IGFBP-3 may influence intracellular events that regulate growth in mammary epithelial cells.

Postpubertal development of the rat mammary gland is preserved during iron deficiency.

We previously showed that moderate iron deficiency is associated with increased susceptibility to chemically induced breast carcinogenesis. Epidemiological and experimental data suggest that breast cancer risk may be modulated by the developmental and proliferative state of the mammary epithelium. The adverse effects of iron deficiency on organ growth are well documented. However, the role of iron in mammary gland development has not been examined. Therefore, we studied the effect of iron deficiency on mammary gland development and epithelial cell kinetics in female Sprague-Dawley rats. Weanling rats were fed experimental diets that provide 6 (severe), 12 (moderate) or 35 (control) mg Fe/kg diet. After 6 wk of treatment, hematocrit and blood hemoglobin were lower in iron-restricted rats than in controls, with significant differences from controls observed in rats receiving 6 mg Fe/kg diet (P < 0.05). Liver iron was reduced 90 and 80% in severe and moderate groups, respectively, compared with controls. Puberty onset and 17-beta-estradiol levels were unaltered by iron status, but plasma progesterone was significantly lower in iron-restricted groups (P < 0.05). Microscopic examination of mammary gland whole mounts revealed an increased density of terminal end buds in thoracic glands from iron-restricted rats, indicative of decreased differentiation, although the differences were not statistically significant compared with controls (P = 0.21). Mammary epithelial cell proliferation, determined in contralateral glands by measuring 5-bromo-2'-deoxyuridine incorporation, did not differ between rats receiving 12 and 35 mg Fe/kg diet. In conclusion, these results suggest that alveolar development of the mammary gland and the proliferative capacity of the mammary epithelium are refractory to iron deficiency during early postpubertal growth of the rat.

Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction.

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. In addition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell lines were developed that constitutively express IGFBP-3. MAC-T cells transfected with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cells compared to Mock cells. However, DNA synthesis was increased by IGF-I (1-50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. IGF-I (20 ng/ml) increased DNA synthesis 11- and threefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells were more sensitive to the lower concentrations of IGF-I (1-5 ng/ml). In contrast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance responsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the differences observed between +BP3 and Mock cells were not attributable to sequestration of endogenous IGF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis was also increased in +BP3 cells, compared to controls, in response to 5 microg/ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated response did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does require an IGF-I-IGFBP-3 interaction.