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.
Grill
C J
CJ
Department of Animal Sciences, Rutgers, The State University of New Jersey, 108 Foran Hall, 59 Dudley Road, New Brunswick, New Jersey 08901-8520, USA.
Sivaprasad
U
U
Cohick
W S
WS
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D015398:Signal Transduction
D018972:Insulin-Like Growth Factor Binding Protein 3; D011518:Proto-Oncogene Proteins; D007334:Insulin-Like Growth Factor I; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases