Regulation of Nuclear IGFBP-3 in Response to Intrinsic Apoptotic Stress in Bovine Mammary Epithelial Cells

Tuesday, July 22, 2014: 2:30 PM
2103B (Kansas City Convention Center)
Allyson Agostini-Dreyer , Rutgers, the State University of NJ, New Brunswick, NJ
Amanda E. Jetzt , Rutgers, the State University of NJ, New Brunswick, NJ
Wendie S. Cohick , Rutgers, the State University of NJ, New Brunswick, NJ
Abstract Text:

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