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Von Hippel-Lindau Regulates interleukin-32β Stability in Ovarian Cancer Cells

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Von Hippel-Lindau Regulates interleukin-32β Stability in Ovarian Cancer Cells

Hyo Jeong Yong et al. Oncotarget.

Abstract

Hypoxia-induced interleukin-32β (IL-32β) shifts the metabolic program to the enhanced glycolytic pathway. In the present study, the underlying mechanism by which hypoxia-induced IL-32β stability is regulated was investigated in ovarian cancer cells. IL-32β expression increased under hypoxic conditions in ovarian cancer cells as it did in breast cancer cells. The amount of IL-32β was regulated by post-translational control rather than by transcriptional activation. Under normoxic conditions, IL-32β was continuously eliminated through ubiquitin-dependent degradation by the von-Hippel Lindau (VHL) E3 ligase complex. Oxygen deficiency or reactive oxygen species (ROS) disrupted the interaction between IL-32β and VHL, leading to the accumulation of the cytokine. The fact that IL-32β is regulated by the energy-consuming ubiquitination system implies that it plays an important role in oxidative stress. We found that IL-32β reduced protein kinase Cδ (PKCδ)-induced apoptosis under oxidative stress. This implies that the hypoxia- and ROS-stabilized IL-32β contributes to sustain survival against PKCδ-induced apoptosis.

Keywords: apoptosis; hypoxia; interleukin-32; protein kinase C; von Hippel-Lindau.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. IL-32β levels increase under hypoxic conditions in ovarian cancer cells
(A) The IL-32 levels in the human ovarian cancer cell lines IGROV1, SKOV3, and OVCAR8 were determined by RT-PCR, qRT-PCR, and immunoblot assays. The qRT-PCR results were normalized with the level of IL-32β and IL-32γ in IGROV1 cells, respectively. The data shown represent one of three independent experiments carried out in triplicate. (B, C) SKOV3 and OVCAR8 cells were treated with 150 μM CoCl2 and the IL-32β levels were determined by RT-PCR and immunoblot assays at the indicated times. The bands were quantified using ImageJ software and the numbers indicate the comparison of each lane. (D) SKOV3 and OVCAR8 cells were treated with 150 μM CoCl2 and 5 μg/mL CHX. The IL-32β levels were determined by immunoblot assay at the indicated times.
Figure 2
Figure 2. ROS inhibit ubiquitin-dependent IL-32β degradation
(A) SKOV3 and OVCAR8 cells were treated with 5 μM MG-132 and the IL-32β levels were determined by immunoblot assay at the indicated times. (B) SKOV3 and OVCAR8 cells were pretreated with 1 μg/mL pepstatin A for 1 h, and then treated with 150 μM CoCl2. (C) SKOV3 and OVCAR8 cells were pretreated with NAC for 1 h, and then treated with 150 μM CoCl2. (D) SKOV3 and OVCAR8 cells were pretreated with NAC for 1h, and then treated with 150 μM CoCl2 and MG-132. (E) HEK 293T cells were transfected with Myc-IL-32β and HA-Ub, and then treated with 150 μM CoCl2 or 5 μM MG-132. The cell lysates were immunoprecipitated with anti-Myc antibody and the interaction was examined by immunoblot assay.
Figure 3
Figure 3. IL-32β is regulated by E3 ligase VHL
(A) SKOV3 and OVCAR8 cells were transfected with VHL siRNA, CHIP siRNA, and Keap1 siRNA. The VHL, CHIP and Keap1 levels were determined by RT-PCR. The IL-32β levels were determined by immunoblot assay. (B) SKOV3 cells were co-transfected with IL-32β and HA-VHL-expressing plasmids. The IL-32β and VHL levels were determined by immunoblot assay. (C) OVCAR8 cells were transfected with HA-VHL. The IL-32β and VHL levels were determined by immunoblot assay.
Figure 4
Figure 4. The interaction between IL-32β and VHL is disrupted by ROS
(A) HEK 293T cells were transfected with Myc-IL-32β and HA-VHL, and then treated with 100 μM H2O2 for the indicated times. The cell lysates were immunoprecipitated with anti-Myc antibody and the interaction was examined by immunoblot assay. (B) HEK 293T cells were transfected with the Myc-IL-32 isoforms and HA-VHL. The cell lysates were immunoprecipitated with anti-HA antibody and the interaction was examined by immunoblot assay. (C) HEK 293T cells were transfected with Myc-IL-32β, Myc-IL-32 exon 7, and HA-VHL. The cell lysates were immunoprecipitated with anti-HA antibody and the interaction was examined by immunoblot assay. (D) HEK 293T cells were transfected with Myc-IL-32β and HA-VHL19, and then treated with 100 μM H2O2 for the indicated times. The cell lysates were immunoprecipitated with anti-Myc antibody and the interaction was examined by immunoblot assay. (E) SKOV3 and OVCAR8 cells were treated with DMOG for 24 h. The IL-32β levels were determined by immunoblot assay. (F) SKOV3 and OVCAR8 cells were treated with DFO for 24 h. The IL-32β levels were determined by immunoblot assay. (G) SKOV3 and OVCAR8 cells were transfected with PHD2 siRNA. The PHD2 levels were determined by RT-PCR. The IL-32β levels were determined by immunoblot assay. (H) HEK 293T cells were transfected with Myc-IL-32β, HA-VHL wild type (WT), or HA-VHL mutant. The cell lysates were immunoprecipitated with anti-HA antibody and the interaction was examined by immunoblot assay.
Figure 5
Figure 5. PKCδ forms a complex with IL-32β and VHL
(A) HEK 293T cells were transfected with Myc-IL-32β, HA-VHL, and Flag-PKC. The cell lysates were immunoprecipitated with anti-Flag antibody and the interaction was examined by immunoblot assay. (B) HEK 293T cells were transfected with Myc-IL-32β, HA-VHL, and Flag-PKCδ, and then placed in a hypoxia chamber for 24 h. The cell lysates were immunoprecipitated with anti-HA antibody and the interaction was examined by immunoblot assay. (C) HEK 293T cells were transfected with Myc-IL-32β and Flag-PKCδ, and then placed in a hypoxia chamber for 24 h. The cell lysates were immunoprecipitated with anti-Flag antibody and the interaction was examined by immunoblot assay. (D) OVCAR8 cells were transfected with Myc-IL-32β, HA-VHL and Flag-PKCδ, and then treated with 150 μM CoCl2 for 24 h. The cell lysates were immunoprecipitated with anti-HA antibody and the interaction was examined by immunoblot assay.
Figure 6
Figure 6. IL-32β prevents PKCδ-induced apoptosis under oxidative stress
(A, B) SKOV3 cells were transfected with GFP-IL-32β and Flag-PKCδ for 24 h, whereas OVCAR8 cells were transfected with IL-32 siRNA and PKCδ siRNA for 48 h. The transfected cells were treated with 100 μM H2O2 for 24 h. The caspase-3/7 activity was assessed using a luminescent assay. The cleaved-PARP levels were determined by immunoblot assay. (C) SKOV3 cells were transfected with GFP-IL-32β and Flag-PKCδ. OVCAR8 cells were transfected with IL-32 siRNA and PKCδ siRNA, and then treated with 150 μM CoCl2 for 24 h. The proliferation was assessed using CellTiter-Blue. (D) SKOV3 cells were transfected with GFP-IL-32β and Flag-PKCδ, whereas OVCAR8 cells were transfected with IL-32 siRNA and PKCδ siRNA. The transfected cells were seeded onto the upper part of a Transwell chamber in serum-free medium with or without 150 μM CoCl2. The migrated SKOV3 and OVCAR8 cells were counted after 24 or 48 h incubation, respectively. Scale bars = 10 μm.
Figure 7
Figure 7. A schematic diagram summarizes IL-32β function in human ovarian cancer cells
Hypoxia-induced ROS disrupted the interaction between IL-32β and VHL, leading to the IL-32β accumulation. The increased IL-32β bound to PKCδ in the oxidative stress condition and PKCδ-mediated apoptosis was inhibited.

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