Dataset Information

IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells.

ABSTRACT: IGF-binding protein (IGFBP)-3 is a multifunctional protein that can exert IGF-independent effects on apoptosis. Anisomycin (ANS) is a potent inducer of IGFBP-3 production in bovine mammary epithelial cells (MECs), and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. IGFBP-3 is present in the nucleus and the conditioned media in response to ANS. The goal of this study was to determine whether ribotoxic stress induced by ANS or a second ribotoxin, deoxynivalenol (DON), specifically regulates transport of IGFBP-3 to the nucleus and to determine the pathway by which it traffics. In ribotoxin-treated cells, both endogenous IGFBP-3 and transfected IGFBP-3 translocated to the nucleus. Inhibition of the nuclear transport protein importin-? with importazole reduced ribotoxin-induced nuclear IGFBP-3. Immunoprecipitation studies showed that ANS induced the association of IGFBP-3 and importin-?, indicating that ribotoxins specifically induce nuclear translocation via an importin-??dependent mechanism. To determine whether secretion of IGFBP-3 is required for nuclear localization, cells were treated with Pitstop 2 or brefeldin A to inhibit clathrin-mediated endocytosis or overall protein secretion, respectively. Neither inhibitor affected nuclear localization of IGFBP-3. Although the IGFBP-3 present in both the nucleus and conditioned media was glycosylated, secreted IGFBP-3 exhibited a higher molecular weight. Deglycosylation experiments with endoglycosidase Hf and PNGase indicated that secreted IGFBP-3 completed transit through the Golgi apparatus, whereas intracellular IGFBP-3 exited from the endoplasmic reticulum before transit through the Golgi. In summary, ANS and DON specifically induced nuclear localization of nonsecreted IGFBP-3 via an importin-??mediated event, which may play a role in their ability to induce apoptosis in MECs.

SUBMITTER: Agostini-Dreyer A

PROVIDER: S-EPMC6371081 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells.

Agostini-Dreyer Allyson A Jetzt Amanda E AE Skorupa Jennifer J Hanke Jennifer J Cohick Wendie S WS

Journal of the Endocrine Society 20181224 3

IGF-binding protein (IGFBP)-3 is a multifunctional protein that can exert IGF-independent effects on apoptosis. Anisomycin (ANS) is a potent inducer of IGFBP-3 production in bovine mammary epithelial cells (MECs), and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. IGFBP-3 is present in the nucleus and the conditioned media in response to ANS. The goal of this study was to determine whether ribotoxic stress induced by ANS or a second ribotoxin, deoxynivalenol (DON), specifically regulates ...[more]

PMID: 30788454

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Project description:Porphyromonas gingivalis, an opportunistic pathogen usurps gingival epithelial cells (GECs) as primary intracellular niche for its colonization in the oral mucosa. However, the precise characterization of the intracellular trafficking and fate of P. gingivalis in GECs remains incomplete. Therefore, we employed high-resolution three-dimensional-transmission-electron-microscopy to determine the subcellular location of P. gingivalis in human primary GECs upon invasion. Serial sections of infected-GECs and their tomographic reconstruction depicted ER-rich-double-membrane autophagosomal-vacuoles harboring P. gingivalis. Western-blotting and fluorescence confocal microscopy showed that P. gingivalis significantly induces LC3-lipidation in a time-dependent-manner and co-localizes with LC3, ER-lumen-protein Bip, or ER-tracker, which are major components of the phagophore membrane. Furthermore, GECs that were infected with FMN-green-fluorescent transformant-strain (PgFbFP) and selectively permeabilized by digitonin showed rapidly increasing large numbers of double-membrane-vacuolar-P. gingivalis over 24 hours of infection with a low-ratio of cytosolically free-bacteria. Moreover, inhibition of autophagy using 3-methyladenine or ATG5 siRNA significantly reduced the viability of intracellular P. gingivalis in GECs as determined by an antibiotic-protection-assay. Lysosomal marker, LAMP-1, showed a low-degree colocalization with P. gingivalis (∼20%). PgFbFP was used to investigate the fate of vacuolar- versus cytosolic-P. gingivalis by their association with ubiquitin-binding-adaptor-proteins, NDP52 and p62. Only cytosolic-P. gingivalis had a significant association with both markers, which suggests cytosolically-free bacteria are likely destined to the lysosomal-degradation pathway whereas the vacuolar-P. gingivalis survives. Therefore, the results reveal a novel mechanism for P. gingivalis survival in GECs by harnessing host autophagy machinery to establish a successful replicative niche and persistence in the oral mucosa.

Dataset Information

IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells.

Publications

IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells.

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