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. 2020 Jan 8;9(1):147.
doi: 10.3390/cells9010147.

IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover Through a Conserved GSK-3β-Nrf2-BNIP3 Pathway

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Free PMC article

IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover Through a Conserved GSK-3β-Nrf2-BNIP3 Pathway

Sarah Riis et al. Cells. .
Free PMC article

Abstract

The Insulin-like Growth Factor I (IGF-1) signalling pathway is essential for cell growth and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3β (GSK-3β), leading to activation of Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory Factor-1 (NRF1) and Hypoxia-inducible Factor 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic Fibroblasts (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription factor Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.

Keywords: BNIP3; HIF-1α; IGF-1; IGF-1R; NFE2L2/Nrf2; NRF1; autophagy; cancer; cell death; mitophagy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
IGF-1-induced BNIP3 expression is Nrf2-dependent. (A) Western blot showing levels of Nrf2, BNIP3, PHB1 and OXPHOS components in R− and R+ cells in the control medium (CM). (B) Gene expression levels of Nrf2 and BNIP3 in R− and R+ cells in the CM measured using RT-qPCR. (C) Mitochondrial membrane potential measured using flow cytometry in R− and R+ cells using TMRM dye. The average geometric means are shown as a relative fold change compared to R− set to a value of 1 for the first biological repeat. FCCP positive controls were included. A representative graph illustrating the fluorescence intensity of R+ cells (red) overlaid onto R− cells (black) is also shown. (D) Western blot showing Nrf2 levels in nuclear (N) and cytosolic (C) fractions of MCF-7 cells maintained in a control medium or serum starved for 4 h followed by 4 h of IGF-1 stimulation (10 ng/mL). (E) Western blot showing Nrf2 and BNIP3 levels in DU145 cells. The cells were kept in the CM or serum-starved for 4 h prior to the addition of IGF-1 (10 ng/mL) for 20 h. (F) Gene expression levels of BNIP3 in U2OS cells measured using RT-qPCR. The cells were serum-starved for 4 h prior to stimulation with IGF-1 (10 ng/mL) for 20 h. (G) MCF-7 and DU145 cells were transfected with siNeg or siNrf2. At 48 h post transfection, the cells were serum-starved for 4 h prior to the addition of IGF-1 (10 ng/mL) for 20 h. (H) Gene expression levels of Nrf2 and BNIP3 in MCF-7 cells transfected with siNeg or siNrf2 measured using RT-qPCR. At 48 h post transfection, the cells were serum-starved for 4 h prior to addition of IGF-1 at 10 ng/mL for 20 h prior to RNA extraction. In all panels, the data were derived from three independent experiments. For Western blots, protein levels were normalised to β-actin and presented as a fold change relative to the control sample set to a value of 1. For RT-qPCR, gene expression levels were normalised to the housekeeping gene UBC (human cell lines) or β-actin (MEFs) and presented as a fold change relative to control conditions set at a value of 1. Statistical analysis was performed using the Student’s t-test (AC,F), one-way ANOVA (E) or two-way ANOVA (G,H) (*: p < 0.05, **: p < 0.01, ***: p < 0.001). ATP5A: ATP Synthase F1 Subunit α, ETC V: Electron transport chain, SDHB: Succinate dehydrogenase complex iron sulfur subunit B, UQCR2: Ubiquinol-cytochrome C reductase core protein 2.
Figure 2
Figure 2
IGF-1-induces BNIP3 protein expression through inhibition of GSK-3. (A) Western blot of MCF-7 cells showing levels of AKT phosphorylated on S473 and GSK-3 phosphorylated on S9. Cells were serum-starved for 4 h prior to stimulation with IGF-1 (10 ng/mL) for 4 h in the absence or presence of the AKT inhibitor LY294002 (20 μM) that was added 30 min prior to stimulation. (B) Western blot showing levels of Nrf2, BNIP3, p62 and p-p70 S6 kinase (T371) in DU145 cells. Cells were cultured in CM in the presence or absence of 30 μM SB415286 for 2, 4 or 6 h. (C) Western blot showing levels of BNIP3, pGSK-3β (S9), p62 and p-p70 S6 kinase in MCF-7 cells. Cells were cultured in the CM in the absence or presence of 10 mM LiCl for 2, 4 or 6 h. (D) Western blot of DU145 cells transiently transfected with pcDNA3 empty vector (EV or pcDNA3-HA2-KEAP1. The cells were serum-starved for 4 h prior to stimulation with IGF-1 (10 ng/mL) for 20 h. (E) Keap1 expression levels in R− and R+ cells in control media measured using RT-qPCR. (F) mRNA expression levels of KEAP1 and Nrf2 target genes, GCLC, HO1 and NRF1 in DU145 cells transfected with pcDNA3.1 empty vector or pcDNA3.1-HA2-KEAP1 for 48 h and cultured in CM. mRNA was measured using RT-qPCR. In all panels, the data presented were derived from three independent experiments. For Western blots, the protein levels were normalised to β-actin and presented as fold-change relative to the control sample set to a value of 1. For RT-qPCR, the gene expression levels were normalised to the housekeeping gene UBC (human cell lines) or β-actin (MEFs) and presented as fold-change relative to control conditions set at a value of 1. Statistical analysis was performed using one-way ANOVA (B,C), two-way ANOVA (D) and Student’s t-test (E,F) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
Figure 3
Figure 3
Nrf2 induces BNIP3 through HIF-1α and NRF1. (A) Western blot showing levels of HIF-1α and IGF-1R in MCF-7 and DU145 cells transfected with siNeg or siNrf2. At 48 h post transfection, cells were serum-starved for 4 h prior to stimulation with IGF-1 (10 ng/mL) for 20 h. (B) Western blot showing the BNIP3 expression MCF-7 cells transfected with siNeg or siNRF1. At 48 h post transfection, cells were serum-starved for 4 h prior to stimulation with IGF-1 (10 ng/mL) for 20 h. (C) Expression levels of NRF1 in cells transfected with siNeg or siNRF1 for 72 h measured using RT-qPCR. (D) Western blot showing levels of mitochondrial markers PHB1 and TOM20 in MCF-7 cells transfected with siNeg or siNRF1 for 72 h. The cells were cultured in the CM and analysed at 72 h post transfection. In all panels, the data presented were derived from three independent experiments. For Western blots, protein levels were normalised to β-actin and presented as a fold change relative to the control sample set to a value of 1. For RT-qPCR, gene expression levels were normalised to the housekeeping gene UBC (human cell lines) or β-actin (MEFs) and presented as a fold change relative to control conditions set at a value of 1. Statistical analysis was performed using two-way ANOVA (A,C) and Student’s t-test (D,E) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).
Figure 4
Figure 4
The IGF-1-Nrf2-BNIP3 pathway regulates mitochondrial morphology and turnover. (A) Immunofluorescence showing TOM20 in MCF-7 cells transfected with siNeg or siNrf2 cultured in CM. Cells were stained with anti-TOM20 antibody at 72 h post transfection. Images were captured at 1000× magnification, and the enlarged images are 3× zooms. (B) Gene expression levels of Nrf2 in response to transfection with siNeg or siNrf2 in MCF-7 cells were measured using RT-qPCR. (C) Immunofluorescence showing TOM20 in MCF-7 cells transfected with siNeg or siBNIP3 cultured in the CM at 72 h post transfection. Images were captured at 1000× magnification, and the enlarged images are 3× zooms. (D) Gene expression levels of BNIP3 and the mitochondria dynamics regulatory genes Drp1, MFN1 and MFN2 measured using RT-qPCR in MCF-7 cells transfected with siNeg or siBNIP3 for 72 h. (E) Immunofluorescence showing TOM20 in R− and R+ cells cultured in the CM. Images were captured at 1000× magnification, and the enlarged images are 3× zooms. (F) Western blots showing BNIP3 and cleaved caspase 3 levels after 24 h serum starvation or in the CM in the absence or presence of DFP (1 mM) or CCCP (10 μM). (G) Western blots showing LC3 levels in R− and R+ cells. Cells were cultured in CM in the absence or presence of chloroquine (50 μM) added 4 h before harvesting. The autophagic flux was calculated as ΔLC3II for each condition in the presence and absence of chloroquine and presented as a fold change relative to the R− sample. (H) Western blot showing LC3 levels in R− and R+ cells cultured in CM with or without DFP (1 mM) or in response to serum starvation for 24 h. Chloroquine (50 μM) was added to the indicated cultures for the last 4 h before harvest. In all figures, the data presented was derived from three independent experiments. For Western blots, protein levels were normalised to β-actin and presented as a fold change relative to the control sample set to a value of 1. For RT-qPCR, gene expression levels were normalised to the housekeeping gene UBC and presented as a fold change relative to control conditions set at a value of 1. Statistical analysis was performed using one-way ANOVA (A,B) and the Student’s t-test (C) (*: p < 0.05, **: p < 0.01, ***: p < 0.001). CQ: chloroquine.

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References

    1. Hakuno F., Takahashi S.I. IGF1 Receptor Signaling Pathways. J. Mol. Endocrinol. 2018;61:T69–T86. doi: 10.1530/JME-17-0311. - DOI - PubMed
    1. Denduluri S.K., Idowu O., Wang Z., Liao Z., Yan Z., Mohammed M.K., Ye J., Wei Q., Wang J., Zhao L., et al. Insulin-like growth factor (IGF) signaling in tumorigenesis and the development of cancer drug resistance. Genes Dis. 2015;2:13–25. doi: 10.1016/j.gendis.2014.10.004. - DOI - PMC - PubMed
    1. Guevara-Aguirre J., Guevara A., Palacios I., Pérez M., Prócel P., Terán E. GH and GHR signaling in human disease. Growth Horm. IGF Res. 2018;38:34–38. doi: 10.1016/j.ghir.2017.12.006. - DOI - PubMed
    1. Qu X., Wu Z., Dong W., Zhang T., Wang L., Pang Z., Ma W., Du J. Update of IGF-1 receptor inhibitor (ganitumab, dalotuzumab, cixutumumab, teprotumumab and figitumumab) effects on cancer therapy. Oncotarget. 2017;8 doi: 10.18632/oncotarget.15704. - DOI - PMC - PubMed
    1. Osher E., Macaulay V.M. Therapeutic Targeting of the IGF Axis. Cells. 2019;8:895 doi: 10.3390/cells8080895. - DOI - PMC - PubMed
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