Regulation of Nuclear IGFBP-3 in Response to Intrinsic Apoptotic Stress in Bovine Mammary Epithelial Cells
Following peak lactation, the number of secretory mammary epithelial cells (MEC) in the bovine gland gradually decreases due to increased apoptosis, leading to a decrease in lactation persistency. However, the mechanisms that govern apoptosis in the bovine MEC are under-investigated. We have previously shown that anisomycin (ANS), an activator of the intrinsic apoptotic pathway, is a potent inducer of IGFBP-3 production in MAC-T cells and that knock-down of IGFBP-3 with siRNA attenuates the ability of ANS to activate apoptosis. Interestingly, IGFBP-3 is found in both the nucleus and the conditioned media in response to ANS indicating a potential for both intra- and extracellular functions. Whether nuclear IGFBP-3 arises from secreted IGFBP-3 is controversial. In the present work, MAC-T cells were transfected with a plasmid expressing GFP-tagged IGFBP-3. Analysis using fluorescent microscopy indicated that IGFBP-3 resided basally in the cytosol and translocated to the nucleus in response to ANS. Since IGFBP-3-GFP is too large to passively diffuse through nuclear pores, this supports a role for active nuclear import. Chemical inhibition of the nuclear transport protein importin-β with importazole reduced ANS-induced nuclear IGFBP-3-GFP, indicating that IGFBP-3 utilizes importin-β for nuclear import. Endoglycosidase-H digestion of nuclear fractions showed that intracellular IGFBP-3 was glycosylated, indicating it had been transported through the secretory pathway. However, inhibition of ER to Golgi transport with Brefeldin A inhibited secretion of IGFBP-3 but increased its nuclear accumulation, indicating that secretion is not required for nuclear localization. In support of these data, inhibition of clathrin-mediated endocytosis with the chemical inhibitor Pitstop2 did not impact nuclear localization of IGFBP-3. In summary, these data show that secretion is not required for ANS-induced nuclear localization of IGFBP-3 and that its nuclear import is a regulated event.
Keywords: mammary gland, lactation, IGFBP-3