Oppositional Regulation of Noxa by JNK1 and JNK2 during Apoptosis Induced by Proteasomal Inhibitors

<div><p>Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2−/− cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2−/− cells, but not the delayed death occurring in JNK1−/− and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1α, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2−/− cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner.</p></div

JNK1-dependent upregulation of Noxa mRNA and protein by MG-132.

<p>(<b>A</b>) Measurement of the mitochondrial membrane potential (ΔΨm) of JNK1−/− and JNK2−/− cells exposed to MG-132 in the absence (control) or presence of Chx. Values represent the mean of two independent experiments +/− SD. (<b>B</b>) Western blots showing the status of the indicated Bcl-2 family members and β-actin in extracts of the three MEF lines treated as indicated. One representative experiment out of three is shown. (<b>C</b>) Semi-quantitative PCR for determination of MG-132-induced expression of Noxa and Bim mRNAs in the indicated MEF lines normalized to GAPDH mRNA. Data are the mean of three independent experiments +/− SD. *** p<0.005 according to ANOVA Tukeýs test as indicated. The inlet shows one representative agarose gel.</p

Influence of MG-132 on expression of transcription factors known to be involved in Noxa regulation.

<p>Western blot analyses for the indicated transcription factors and β-actin in the three MEF lines treated as indicated. One representative experiment out of three is shown.</p

Knockdown of Noxa protects JNK2−/− cells, but not JNK1−/− cells from MG-132-induced apoptosis.

<p>(<b>A</b>, <b>D</b>) Western blots showing the status of BimEL, Noxa and β-actin in JNK2−/− and JNK1−/− cells that were either left untreated or exposed for the indicated times to MG-132 72 hours post transfection with control, Noxa or Bim siRNAs. One representative experiment out of five and three is shown, respectively. Please note that the autoradiograph in <b>D</b> was overexposed in order to detect trace amounts of Noxa in JNK1−/− cells. (<b>B</b>, <b>E</b>) Fluorometric determination of caspase-3 (DEVDase) activities in JNK2−/− and JNK1−/− cells treated as described in <b>A</b> and <b>D</b>, respectively. Please note that DEVDase activities of JNK2−/− and JNK1−/− cells were analyzed at different time points (8 h and 16 h) according to their sensitivities. (<b>C</b>, <b>F</b>) Cytometric determination of cell death (propidium iodide uptake) in JNK2−/− and JNK1−/− cells treated as described in <b>A</b> and <b>D</b>, respectively. Values are the mean of four and three independent experiments, respectively, +/− SD. * p<0.05; *** p<0.001 according to ANOVA Tukeýs test (<b>B</b>) or ANOVA on ranks Student-Newman-Keuls-Method (<b>C</b>) in an all pairwise multiple comparison as indicated.</p

Knockdown of c-Myc protects JNK2−/−, but not JNK1−/− cells from MG-132-induced apoptosis without affecting expression of Noxa and Bim.

<p>(A, D) Western blots showing the status of the indicated proteins in JNK2−/− and JNK1−/− cells that were either left untreated or exposed for the indicated times to MG-132 72 hours post transfection with the indicated siRNAs. One representative experiment out of four and three is shown, respectively. (B, E) Fluorometric determination of caspase-3 (DEVDase) activities in JNK2−/− and JNK1−/− cells treated as described in A and D, respectively. Please note that DEVDase activities of JNK2−/− and JNK1−/− cells were analyzed at different time points (8 h and 16 h) according to their sensitivities. Values are the mean of three independent experiments +/− SD, respectively. (C, F) Cytometric determination of cell death (propidium iodide uptake) in JNK2−/− and JNK1−/− cells treated as described in A and D. Values are the mean of three independent experiments +/− SD. * p<0.05 according to ANOVA on ranks Student-Newman-Keuls-Method (B) or *** p<0.003 according to ANOVA Tukey′s test (C) in an all pairwise multiple comparison as indicated.</p

Influence of MG-132 on apoptosis and Noxa expression in JNK1+/+ cells.

<p>(<b>A</b>) Cytometric determination of cell death (propidium iodide uptake) of JNK1+/+, JNK1−/− and JNK2−/− cells treated with MG-132 as indicated. Values represent the mean of three independent experiments +/− SD. (<b>B</b>) Western blots for the status of Noxa and β-actin in JNK1+/+ and JNK2−/− cells treated as indicated. (<b>C</b>) Semi-quantitative PCR for the determination of the MG-132-induced expression levels of Noxa mRNA in the indicated MEF lines normalized to GAPDH mRNA. Data are the mean of three independent experiments +/− SD. The inlet shows one representative agarose gel. (<b>D-F</b>) Knockdown of Noxa does not protect JNK1+/+ cells from MG-132-induced apoptosis. (<b>D</b>) Western blots showing the status of Noxa and β-actin in JNK1+/+ cells that were either left untreated or exposed for the indicated times to MG-132 72 hours post transfection with control or Noxa siRNAs. One representative experiment out of three is shown. (<b>E</b>, <b>F</b>) Fluorometric and cytometric determination of caspase-3 (DEVDase) activities and cell death (propidium iodide uptake), respectively, in JNK1+/+ cells treated as described in <b>D</b>. Values are the mean of three independent experiments +/− SD. For panels A (ANOVA) and C (ANOVA on ranks) * p<0.05 according to the Student-Newman-Keuls-Method in a time-matched comparison as indicated.</p

Rapid induction of apoptosis by MG-132 only in JNK2−/− cells.

<p>The three MEF lines were exposed to MG-132 for the indicated times in the absence or presence of Chx, ActD or Q-VD-OPh. Cell death was determined either cytometrically by quantification of propidium iodide-stained cells (<b>A</b>) and hypodiploid nuclei (<b>C</b>), or by fluorometric assessment of caspase-3-like DEVDase activities (<b>B</b>, <b>D</b>). Values represent the mean of three (<b>A–C</b>) and two (<b>D</b>) independent experiments +/− SD. (<b>E</b>) Extracts of the indicated MEF lines were analyzed by Western blotting for their JNK status using the detection of β-Actin as loading control. (<b>F</b>) Western blots showing the status of active caspase-3 and β-actin in extracts of the three MEF lines treated as indicated. One representative experiment out of three is shown. For all panels, * p<0.05; *** p<0.005 according to ANOVA Tukeýs test in a time-matched comparison of cells that were treated with MG-132 alone vs. cells treated with MG-132 in the presence of Chx or Q-VD-OPh, respectively.</p

p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation

<div><p>The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γ^KOδ^D910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ^-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ^-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ^-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ^-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ^-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.</p></div

Bronchoalveolar infiltration of eosinophils, neutrophils, T and B cells is reduced in OVA-treated p110γ^-/-, p110δ^-/-, and p110γ/δ^-/- mice.

<p>To determine the number of eosinophils, neutrophils, T and B cells in the BALF from OVA-treated and PBS-treated KO and corresponding WT mice, cells were collected, and analysed by flow cytometry. Cell counts were normalised as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159310#pone.0159310.g002" target="_blank">Fig 2</a>. (<b>A</b>) Eosinophils (eos) in BALF from p110γ^-/- and WT mice (left), from p110δ^-/- and WT mice (middle), and from p110γ/δ^-/- and WT mice (right). (<b>B</b>) Neutrophils (neutros) in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>C</b>) T cells in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>D</b>) B cells in BALF from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). Data (n = 3–6) are presented as means + SD. Data were analysed by One-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. ⁺⁺⁺ P < 0.001, ⁺⁺ P < 0.01, ⁺ P < 0.05. ⁺ indicate differences between WT PBS and WT OVA groups. ***P < 0.001, **P < 0.01, *P < 0.05. Asterisks indicate differences between OVA-treated groups.</p

Lung tissue infiltration by eosinophils, neutrophils, T and B cells is only reduced in OVA-treated p110δ^-/- and p110γ/δ^-/- mice.

<p>To determine OVA-induced infiltration of immune cell populations into the lung tissue, leukocytes were prepared from lungs after BAL and exsanguination of PBS-treated and OVA-treated KO and corresponding WT mice. Cell populations were analysed by flow cytometry. Cell counts were normalised as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159310#pone.0159310.g002" target="_blank">Fig 2</a>. (<b>A</b>) Eosinophils (eos) in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>B</b>) Neutrophils (neutros) in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>C</b>) T cells in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). (<b>D</b>) B cells in lung tissue from p110γ^-/- and WT mice (left), p110δ^-/- and WT mice (middle), and p110γ/δ^-/- and WT mice (right). Data (n = 3–6) are presented as means + SD. Data were analysed by One-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. ⁺⁺⁺ P < 0.001, ⁺⁺ P < 0.01, ⁺ P < 0.05. ⁺ indicate differences between WT PBS and WT OVA groups. ***P < 0.001, **P < 0.01, *P < 0.05. Asterisks indicate differences between OVA-treated groups.</p