Research ArticleCell Biology

Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduce mitochondrial ROS production

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Science Signaling  28 Mar 2017:
Vol. 10, Issue 472, eaag2588
DOI: 10.1126/scisignal.aag2588
  • Fig. 1 Oxidative stress triggers a loss of mitoSTAT3.

    Wild-type (WT) MEFs or 4T1 cells were treated with 1 mM H2O2 for the indicated times, and mitochondrial (mito) (A) or cytosolic (cyto) (B) extracts were probed for STAT3. Quantification of mitoSTAT3 as compared to a loading control is depicted in (A). *P < 0.05 (compared to control); #P < 0.05 (compared to control). Blots are representative of three independent experiments. (C) WT MEFs treated with the indicated concentrations of H2O2 and extracts probed for mitoSTAT3 (top) and phosphorylated (p) Tyr705 and total STAT3 in the cytosol (bottom) (tubulin, cytosolic loading control; CypD, mitochondrial loading control). Blots are representative of two independent experiments. (D) Isolated mitochondria from WT MEFs treated for the indicated times with H2O2 were immunoblotted for mitoSTAT3 or mitoSTAT1 and quantified. *P < 0.01; **P < 0.04; #P < 0.005. Blots are representative of three independent experiments. a.u., arbitrary units. (E) MDA-231 and SK-BR3 human breast cancer cells were treated for the indicated times with H2O2 and immunoblotted for STAT3 and GRIM19 (mitochondrial loading control). Blots are representative of two independent experiments. (F) WI-38 human lung fibroblasts were treated with H2O2, and purified mitochondria were immediately lysed in sample buffer. Blots were probed for mitoSTAT3, pERK1/2, and NDUFA9 (mitochondrial loading control). Blots are representative of two independent experiments.

  • Fig. 2 Cytokine treatment of cells induces mitoSTAT3 loss and recovery.

    (A and B) WT MEFs were treated with either OSM (A) and IL-6 (B) for the indicated times, and mitochondrial extracts were immunoblotted for STAT3, pERK, total ERK (ERK1/2), and a loading control (NDUFA9 and/or CypD) (top). Cytosolic extracts probed for pTyr705 STAT3 are shown to confirm cell activation (bottom). Blots are representative of two independent experiments. (C) Mitochondria from 4T1 cells treated with OSM (top) or MDA-231 cells treated with IL-6 (bottom) were probed for STAT3 and loading control (GRIM19 or CypD). Blots are representative of two independent experiments. (D) Rat hepatoma cell lines expressing the chimeric G-CSF–gp130 receptor were stimulated with recombinant human G-CSF and immunoblotted for STAT3 in the mitochondrial fraction or pTyr705 STAT3 in the cytosolic fraction. Blots are representative of two independent experiments. (E) Purified mitochondria (P-mito) from control or OSM-stimulated WT MEFs were fractionated into cytosolic (cyto), ER (endoplasmic reticulum), crude mitochondria (C-mito), pure mitochondria (P-mito), and a mitochondria-associated membrane (MAM) fraction and blotted against a total homogenate fraction (Homog.). The abundance of calreticulin (ER and MAM marker), tubulin (cytosolic marker), NDUFA9 (mitochondrial marker), histone H3 (nuclear marker), and STAT3 is shown. Blots are representative of two independent experiments.

  • Fig. 3 Inhibition of JAKs prevents mitoSTAT3 loss upon cytokine treatment.

    (A and B) MEFs were incubated with or without the JAK inhibitor ruxolitinib for 1 hour before the addition of OSM (A) or H2O2 (B), and mitochondrial and cytosolic extracts were immunoblotted for STAT3 or pTyr705 STAT3, respectively. Blots are representative of three independent experiments. Densitometric quantification of mitochondrial results from (A) is depicted. *P < 0.01; #P < 0.001; ns, not significant. (C) Mitochondria were isolated from MDA-231 breast cancer cells treated with ruxolitinib for 1 hour before the addition of OSM or H2O2 for the indicated times and immunoblotted for STAT3 and NDUFA9 (loading control). Blots are representative of two independent experiments. (D) Mitochondrial superoxide production was assayed by flow cytometry analysis using the MitoSOX dye in WT MEFs treated for 30 min with either OSM or H2O2. *P = 0.0002. n = 3 independent experiments. (E) WT MEFs were pretreated for 1 hour with the mitochondrial restricted antioxidant mitoTEMPO and then stimulated for 30 min with H2O2 with mitochondrial extracts blotted for STAT3, pERK1/2, or NDUFA9 (loading control). Blots are representative of two independent experiments. (F) WT MEFs were pretreated with the MEK inhibitor PD0325901 for 1 hour, followed by H2O2 treatment for the indicated times, and isolated mitochondria were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted for STAT3, pERK1/2, tERK1/2, and NDUFA9 (loading control). Blots are representative of two independent experiments.

  • Fig. 4 Recovery of mitoSTAT3 is dependent on new protein synthesis and Ser727 phosphorylation of STAT3.

    (A) STAT3−/− MEFs were engineered to stably express mitochondrially targeted STAT3 (MLS-STAT3) and subjected to H2O2 treatment. Whole-cell extract (WCE) and mitochondria were Western blotted for STAT3 or the respective loading control. Blots are representative of three independent experiments and densitometric quantification is shown. (B) Mitochondria from MDA-231 cells were pretreated for 4 hours with vehicle or MG132 and treated with OSM or H2O2 and extracts probed for STAT3. Cytosolic extracts from vehicle- or MG132-treated cells were probed for ubiquitin (Ub) (right). Blots are representative of two independent experiments. (C and D) Mitochondrial extracts from MDA-231 cells treated with or without cycloheximide and stimulated with OSM or H2O2 were subjected to Western blotting (C). Results from OSM experiments were quantified in (D) by densitometry analysis. *P = 0.009; #P = 0.023. n = 3 independent experiments. (E and F) STAT3−/− MEFs expressing WT STAT3 (STAT3α) or the nonphosphorylatable mutant (STAT3 S727A) were treated for the indicated times with OSM, and mitochondrial lysates were immunoblotted for STAT3 (E). Quantification of these results is presented in (F). *P < 0.005; **P < 0.002; ***P < 0.02; #P ≤ 0.0002. n = 3 independent experiments.

  • Fig. 5 mitoSTAT3 interacts with CypD.

    (A) Lysates from WT MEF whole-cell extracts treated with H2O2 were subjected to pull-down with bead-bound glutathione S-transferase (GST)–CypD (Coomassie stain) and probed for STAT3 (input; bottom). Blots are representative of two independent experiments. (B and C) WT MEFs (B) and 4T1 cells (C) were incubated with H2O2 for various times before isolation of the mitochondria. Extracts were incubated with GST-CypD in the presence (lane 6) or absence of CsA, and STAT3 was assayed by immunoblotting (top). Bottom panels show the input (10% of that used for the pull downs). Blots are representative of two independent experiments. (D) Mitochondria from 4T1 cells either untreated or treated with H2O2 were subjected to GST-CypD or GST pull-down with immunoblotting for STAT3 or GST. Input is shown in the bottom panels. Blots are representative of two independent experiments. (E) mitoSTAT3 immunoprecipitations (IPs) from MEFs and 4T1 cells treated 30 min with H2O2. Immunoblots were probed for STAT3 (bottom) or CypD (top). Input (10% of that used for immunoprecipitation) is shown. Blots are representative of two independent experiments. (F) GST-CypD pull-down of mitochondrial extracts from MDA-231 cells treated with IL-6 and the soluble IL-6Rα with input is shown below. Blots were probed for STAT3. ATP5O is shown as a loading control for the pull-down and input samples. Blots are representative of two biological replicates.

  • Fig. 6 Binding of mitoSTAT3 to CypD is mediated through STAT3’s N terminus and can be recapitulated in a cell-free system.

    (A) 293T cells expressing various chimeric versions of Flag-tagged STAT3/STAT1 were treated with H2O2, and whole-cell extracts were incubated with GST-CypD and probed for Flag (top). Bottom panels show the input (10% of that used for the pull-downs) with Coomassie stain of the GST-CypD input. Blots are representative of three independent experiments. (B) Diagram of the chimeric constructs used in (A), indicating the critical region (green bar) in STAT3 that mediates its binding to CypD (13). NTD, N-terminal domain; CC, coiled-coil domain; DBD, DNA binding domain; TAD, transcriptional activation domain. (C) 293T cells expressing STAT3/1S or STAT1/3S were treated with H2O2, subjected to GST-CypD pull-down and probed for Flag [pull-down (top) and input (bottom)]. Blots are representative of two independent experiments. (D) 293T cell lysates expressing STAT3/1H were warmed to 30°C and incubated with either GST-CypD or GST alone and immunoblotted for Flag. CsA was incubated with warmed lysates to also show the specificity of the interaction (lane 5). Bottom panel depicts Coomassie stain for GST-CypD and GST. Blots are representative of two independent experiments. (E) Mitochondrial lysates from MDA-231 cells tested in the cell-free system were incubated at 30°C, and a GST-CypD pull-down was performed in the presence or absence of CsA with immunoblotting for STAT3 and ATP5O. Blots are representative of two independent experiments. (F) Extracts from 293T cells expressing the chimeric protein STAT3/1H were warmed to 30°C in the established cell-free system in the presence of vanadate (Na3VO4), calyculin A, or the pan-phosphatase inhibitor cocktail PhosSTOP (Roche). Lysates (left; input) were subjected to GST-CypD pull-down (right) and probed for Flag (STAT3). Coomassie stain of GST-CypD is shown (right, bottom) to confirm equal loading of recombinant CypD. Blots are representative of two independent experiments.

  • Fig. 7 A tyrosine phosphatase–mediated event drives mitoSTAT3-CypD interactions.

    (A) 293T cells expressing STAT3/1S were pretreated with either vanadate (Na3VO4) or staurosporine, followed by treatment with H2O2 for 1 hour. Untreated lysates were also subjected to λ phosphatase (λ phos) treatment or mock treatment. GST-CypD pull-down samples (top) and the corresponding input (bottom) were probed for Flag (STAT3), with the input also immunoblotted for tubulin (loading control) and pERK1/2. Coomassie stain of GST-CypD is shown (bottom) to confirm equal loading of recombinant CypD. Blots are representative of two independent experiments. (B) Extracts from 293T cells expressing STAT3/1H were dialyzed before mock treatment (30°C) with or without the addition of ATP (20 mM). Staurosporine was included during the mock treatment period for lysates in lanes 4 and 5. Binding of STAT3 and CypD was assessed by GST-CypD pull-down. Blots are representative of three independent experiments. (C) Whole-cell extracts from STAT3−/− MEFs were incubated with recombinant STAT3 on ice or either during (+pre) or after (+post) warming of the extract at 30°C for 30 min. Input (bottom) and GST-CypD pull-downs were probed for STAT3. Blots are representative of three independent experiments. (D) MDA-231 human cancer cells were pretreated with CsA and then treated with OSM, and mitochondrial lysates were probed for STAT3. Blots are representative of two independent experiments. (E) CypD+/+ and CypD−/− MEFs were incubated with cycloheximide, and mitoSTAT3 abundance was assessed from purified mitochondrial extracts. Blots are representative of two independent experiments. (F) WT, STAT3−/− MEFs, or STAT3−/− MEFs reconstituted with STAT3 WT or STAT3/1S were treated with H2O2, stained with MitoSOX and then analyzed by flow cytometry. Results are normalized to each respective untreated sample. *P < 0.01; **P < 0.001; #P = 0.0005. n = 5 independent experiments.

  • Fig. 8 Model of mitoSTAT3 regulation.

    Upon initial stimulation (left), mitoSTAT3 and/or mitochondrial proteases (mitoproteases) are posttranslationally modified, which induces their association and leads to the proteolytic cleavage of mitoSTAT3. Presumably, proteolytic fragments of mitoSTAT3 may be further degraded or may contribute to extramitochondrial signaling. With continued stimulation (right), new protein synthesis (either of STAT3 or a chaperone protein required for STAT3 mitochondrial targeting) couples with Ser727 phosphorylation of STAT3 to mediate targeting of STAT3 to the mitochondria. Likely, mitoSTAT3 is also tyrosine-phosphorylated (pY; non-Tyr705 site). After its import into the mitochondria, mitoSTAT3 is dephosphorylated by a protein tyrosine phosphatase (PTP). This facilitates its interaction with CypD, which is likely important for mitoSTAT3’s proper folding and mitochondrial stability. This association determines ROS abundance and is also likely important for other downstream effects of mitoSTAT3, including regulation of the ETC and maintenance of the permeability transition pore (mitoSTAT3’s actions). IMS, intermembrane space.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/472/eaag2588/DC1

    Fig. S1. Ser727 and Tyr705 of STAT3 are not required for H2O2-induced mitoSTAT3 loss.

    Fig. S2. Selectivity of the mitoSTAT3 signaling pathway and relevance in vivo.

    Fig. S3. Inhibition of relevant kinase pathways does not affect stimulation-induced decreases in mitoSTAT3.

    Fig. S4. Inhibition of mitoproteases does not affect proteolysis of mitoSTAT3.

    Fig. S5. mitoSTAT3 inducibly binds to CypD after H2O2 or cytokine stimulation.

    Fig. S6. Ser727 is dispensable for the mitoSTAT3-CypD interaction.

  • Supplementary Materials for:

    Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduce mitochondrial ROS production

    Jeremy A. Meier, Moonjung Hyun, Marc Cantwell, Ali Raza, Claudia Mertens, Vidisha Raje, Jennifer Sisler, Erin Tracy, Sylvia Torres-Odio, Suzana Gispert, Peter E. Shaw, Heinz Baumann, Dipankar Bandyopadhyay, Kazuaki Takabe, Andrew C. Larner*

    *Corresponding author. Email: andrew.larner{at}vcuhealth.org

    This PDF file includes:

    • Fig. S1. Ser727 and Tyr705 of STAT3 are not required for H2O2-induced mitoSTAT3 loss.
    • Fig. S2. Selectivity of the mitoSTAT3 signaling pathway and relevance in vivo.
    • Fig. S3. Inhibition of relevant kinase pathways does not affect stimulation-induced decreases in mitoSTAT3.
    • Fig. S4. Inhibition of mitoproteases does not affect proteolysis of mitoSTAT3.
    • Fig. S5. mitoSTAT3 inducibly binds to CypD after H2O2 or cytokine stimulation.
    • Fig. S6. Ser727 is dispensable for the mitoSTAT3-CypD interaction.

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    Citation: J. A. Meier, M. Hyun, M. Cantwell, A. Raza, C. Mertens, V. Raje, J. Sisler, E. Tracy, S. Torres-Odio, S. Gispert, P. E. Shaw, H. Baumann, D. Bandyopadhyay, K. Takabe, A. C. Larner, Stress-induced dynamic regulation of mitochondrial STAT3 and its association with cyclophilin D reduce mitochondrial ROS production. Sci. Signal. 10, eaag2588 (2017).

    © 2017 American Association for the Advancement of Science

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