Regulation of insulin-like growth factor binding protein (IGFBP)-3 expression by mitogenic and stress-activated signaling pathways in mammary epithelial cells

Both growth stimulatory and inhibitory agents increase the expression of insulin-like growth factor binding protein (IGFBP)-3, which, in turn, modulates their effects on mammary epithelial cell (MEC) growth and survival. In the bovine MEC line, MAC-T, IGF-I, TGF-α, anisomycin (ANS) and forskolin (FSK) were each found to increase IGFBP-3 expression. Furthermore, FSK in combination with IGF-I or ANS had a synergistic effect. The primary goal of this work was to delineate the intracellular signaling pathways used by these factors to regulate IGFBP-3 expression. IGF-I activated the PI3K pathway, with no discernable effect on ERK 1/2 phosphorylation. In contrast, TGF-α activated both the P13K and MAPK pathways.{09}The onset of ANS-stimulated JNK and p38 MAPK activation was rapid, though sustained for hours, while the activation of ERK 1/2 and p70S6K occurred later and was relatively transient. The PI3K inhibitor LY 249002 completely inhibited the ability of IGF-I and TGF-α to stimulate IGFBP-3 expression (P < 0.001). Interestingly, LY 294002 also inhibited the IGFBP-3 mRNA response to FSK, though FSK did not activate PDK-1 or Akt. Inhibition of ERK 1/2 activation with the MAPK inhibitor PD 98059 abolished increases in IGFBP-3 mRNA levels induced by both TGF-α and ANS, and decreased IGF-I-stimulated IGFBP-3 mRNA levels by 65% (P < 0.001). Both PD 98059 and LY 249002 were able to inhibit phosphorylation of p70S6K by IGF-I or TGF-α. In contrast to TGF-α, IGF-I retained the ability to partially increase IGFBP-3 mRNA levels in the presence of PD 98059, indicating that IGF-I activates additional substrates of the PI3K pathway that are involved in IGFBP-3 regulation. Inhibitors of p38 MAPK or ERK 1/2 completely abrogated ANS-stimulated IGFBP-3 expression while an inhibitor of JNK was much less effective. Inhibition of p38 MAPK decreased phosphorylation of ATF-2, ATF-1 and CREB. In contrast, inhibition of JNK decreased ANS-stimulated phosphorylation of ATF-2 only, suggesting that ATF-1 and CREB are critical for regulation of IGFBP-3 by ANS. Inhibiting ERK 1/2 had no effect on the activation of any of these molecules by ANS; however, phosphorylation of p70S6K was completely inhibited. In conclusion, IGFBP-3 mRNA levels are regulated via activation of ERK 1/2, PI3K and p38 MAPK in MAC-T cells.