Research ArticleInflammation

Depletion of H2S during obesity enhances store-operated Ca2+ entry in adipose tissue macrophages to increase cytokine production

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Science Signaling  15 Dec 2015:
Vol. 8, Issue 407, pp. ra128
DOI: 10.1126/scisignal.aac7135
  • Fig. 1 Increased proinflammatory markers in ATMs from obese mice that exhibit decreased cellular H2S concentrations correlate with increased CSE abundance and H2S production capacity compared to ATMs from lean controls.

    (A and B) ATMs isolated from lean and obese mice were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to determine the relative abundances of mRNAs encoding the classical M1 activation markers NOS2 and ITGAX (A) and the M2 activation markers MRC1, ARG1, and Chil3 (B). Data are expressed as the mean fold difference ± SEM in mRNA abundance between ATMs from three mice of each group. *P < 0.001, **P = 0.003, #P = 0.04 by unpaired t test. (C) Representative fluorescence images showing ATMs from lean (left) and obese (right) mice loaded with the H2S indicator SF7-AM. Scale bar, 50 μm. Images are representative of three mice of each group. (D) Quantification of the SF7-AM fluorescence in single ATMs taken from three lean and three obese mice. Data are means ± SEM and expressed as a percentage of the SF7-AM fluorescence measured in ATMs from lean mice. Statistical analysis was performed before data were normalized. *P < 0.001 by unpaired t test. (E) ATMs from lean and obese mice were analyzed by qRT-PCR to determine the relative abundance of CSE mRNA. Data are the mean fold differences in mRNA abundance ± SEM in ATMs between lean and obese mice and are from independent samples prepared from three lean and four obese mice. *P < 0.001 by unpaired t test. (F) ATMs isolated from three lean and four obese mice were analyzed by Western blotting to detect CSE. Actin was used as a loading control. (G) Epididymal fat pad lysates from five lean and five obese mice were analyzed by the methylene blue technique to determine the extent of H2S production. Data are means ± SEM and are expressed as a percentage of the amount of H2S produced in samples from lean mice. *P < 0.001 by unpaired t test.

  • Fig. 2 LPS decreases the cellular concentration of H2S while increasing CSE abundance and H2S production capacity in RAW264.7 cells.

    (A) RAW264.7 cells loaded with the H2S indicator SF7-AM were pretreated with 500 μM GYY4137 or vehicle before being incubated for 4 hours with either vehicle (control) or LPS (1 μg/ml). Cells were then analyzed by fluorescence microscopy. Images are representative of six independent experiments. When used, GYY4137 was present throughout the entire incubation period. Scale bar, 50 μm. (B) Analysis of SF7-AM fluorescence data. Data are means ± SEM of the SF7-AM fluorescence intensities of individual cells expressed as a percentage of control, untreated cells. Data are from single cells and were pooled from six independent samples. *P = 0.017, **P = 0.012, #P = 0.016 by one-way analysis of variance (ANOVA). (C) RAW264.7 cells were pretreated with vehicle or 500 μM GYY4137 before being incubated for 4 hours with vehicle or LPS (1 μg/ml). Cells were then analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three independent experiments. For quantification of band intensities, see fig. S1. (D) RAW264.7 cells were incubated for 4 hours with vehicle or LPS (1 μg/ml). Cell lysates were then analyzed by the methylene blue technique to detect H2S. Data are expressed as a percentage of the H2S measured in untreated cell lysates and are means ± SEM of five experiments. *P < 0.001 by unpaired t test. (E) RAW264.7 cell cultures were left untreated or were treated overnight with 50 μM GYY4137 in the absence or presence of LPS (1 μg/ml). Cell culture medium was then analyzed by the methylene blue technique to determine the concentration of H2S. Data were normalized to the amount of H2S measured in cell-free medium. Data are expressed as a percentage of the H2S found in the culture medium of untreated cells and are means ± SEM of five experiments.

  • Fig. 3 GYY4137 blocks the LPS-induced increase in SOCE.

    (A to F) Analysis of cytosolic Ca2+ flux in RAW264.7 cells and freshly isolated mouse peritoneal macrophages. To activate maximal SOCE, ER stores were depleted by the application of 10 μM CPA in the absence of extracellular Ca2+. Note that the area under the transient increase in [Ca2+] cyto in response to CPA is an index of resting ER store content, whereas the amplitude of the Ca2+ spike evoked upon adding Ca2+ back to the buffer reflects the magnitude of SOCE. (A) Typical traces recorded in RAW264.7 cells and depicting SOCE in the presence or absence of 500 μM GYY4137. (B) Measurement of SOCE in RAW264.7 cells treated for 4 hours with LPS (1 μg/ml) in the absence or presence of 500 μM GYY4137. (C) SOCE amplitudes in RAW264.7 cells treated under the indicated conditions. Data are means ± SEM of five to eight independent experiments. *P = 0.005, #P < 0.001 by one-way ANOVA. (D) Typical traces recorded in mouse peritoneal macrophages and depicting SOCE in the presence or absence of 500 μM GYY4137. (E) Traces showing SOCE recorded in peritoneal macrophages after stimulation with LPS (1 μg/ml) in the absence or presence of 500 μM GYY4137. (F) SOCE amplitude in peritoneal macrophages treated under the indicated conditions. Data are means ± SEM of five to seven independent experiments. *P = 0.029, #P < 0.001 by one-way ANOVA. (G) RAW264.7 cells were treated for 4 hours with the indicated combinations of vehicle, LPS (1 μg/ml), or 500 μM GYY4137. Cells were then analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three independent experiments. For quantification of band intensities, see fig. S2.

  • Fig. 4 Inhibition of H2S production increases SOCE in unstimulated RAW264.7 cells.

    (A) RAW264.7 cells loaded with the H2S indicator SF7-AM were incubated for 1 hour either with vehicle control (top) or with 0.4 mM PPG and 0.4 mM AOAA (bottom). Cells were then analyzed by fluorescence microcopy. Images are representative of six independent experiments. Scale bar, 50 μm. (B) Quantification of the SF7-AM fluorescence in RAW264.7 cells. Data are means ± SEM of the SF7-AM fluorescence intensities of individual cells expressed as a percentage of control cells from six independent experiments. *P = 0.005 by unpaired t test. (C) Typical SOCE traces in RAW264.7 cells in the presence or absence of both PPG and AOAA were recorded as described in Fig. 3A. (D) SOCE amplitudes in RAW264.7 cells in the presence or absence of both PPG and AOAA. Data are means ± SEM of five to seven independent experiments. *P = 0.023 by unpaired t test. (E) RAW264.7 cells stably expressing control shRNA (top) or CSE-specific shRNA (bottom) were loaded with the H2S indicator SF7-AM. Cells were then analyzed by fluorescence microcopy. Images are representative of four independent experiments. Scale bar, 50 μm. (F) Quantification of the effect of CSE knockdown on SF7-AM fluorescence in RAW264.7 cells. Data are means ± SEM of the SF7-AM fluorescence intensities of individual cells expressed as a percentage of control cells from four independent experiments. *P < 0.001 by unpaired t test. (G) Typical SOCE traces in RAW264.7 cells treated with control shRNA or CSE-specific shRNA were recorded as described in Fig. 3A. (H) Effect of CSE knockdown on SOCE amplitudes in RAW264.7 cells. Data are means ± SEM of five independent experiments. *P = 0.015 by unpaired t test.

  • Fig. 5 Increasing the cellular concentration of H2S or inhibiting SOCE attenuates proinflammatory cytokine production by LPS-treated RAW264.7 cells.

    (A) RAW264.7 cells were treated for 4 hours with vehicle or LPS (1 μg/ml) before being analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. (B) RAW264.7 cells treated for 4 hours with LPS (1 μg/ml) in the presence or absence of 500 μM GYY4137 were analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. (C) Typical SOCE traces in RAW264.7 cells in the presence or absence of 10 μM BTP2 were recorded as described in Fig. 3A. (D) SOCE amplitudes in RAW264.7 cells in the presence or absence of BTP2. (E) RAW264.7 cells treated for 4 hours with LPS (1 μg/ml) in the presence or absence of 10 μM BTP2 were analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. (F) Typical SOCE traces in RAW264.7 cells treated with control siRNA or STIM1-specfic siRNA were recorded as described in Fig. 3A. (G) SOCE amplitudes in RAW264.7 cells treated with control siRNA or STIM1-specfic siRNA. (H) RAW264.7 cells treated with control siRNA or STIM1-specific siRNA were analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. Data in all bar charts are means ± SEM of at least three independent experiments. *P < 0.001 by unpaired t test.

  • Fig. 6 Increased SOCE during obesity is dependent on the depletion of H2S and is associated with increased proinflammatory cytokine production.

    (A and B) Typical recordings of SOCE in ATMs from lean mice (A) and obese mice (B). For clarity, only the Ca2+ readdition portion of the experiment is shown, and it depicts SOCE evoked in cells treated with vehicle, 500 μM GYY4137, or both 0.4 mM PPG and 0.4 mM AOAA. (C) SOCE amplitudes in ATMs from lean and obese mice treated with vehicle, GYY4137, or PPG/AOAA. Data are means ± SEM of six to eight independent samples derived from three lean and three obese mice. *P < 0.001, #P = 0.016 by unpaired t test. (D to F) ATMs isolated from lean or obese mice were plated and cultured overnight in the presence of the indicated combinations of vehicle, 500 μM GYY4137, and 10 μM BTP2. The concentrations of IL-6 (D), IL-1β (E), and TNF-α (tumor necrosis factor–α) (F) in the cell culture medium were then measured by Luminex assay. Data are means ± SEM of independent cultures derived from seven lean and seven obese mice. P values were determined by one-way ANOVA analysis.

  • Fig. 7 GYY4137 inhibits SOCE mediated by STIM1 and Orai3 but not that mediated by STIM1 together with Orai1, or Orai2.

    (A) Typical recordings of SOCE in HEK 293 cells stably expressing STIM1 and transiently transfected with plasmids encoding Orai1, Orai2, or Orai3, as indicated. (B) SOCE amplitudes in HEK 293 cells expressing the indicated Orai proteins. Data are means ± SEM of six independent experiments. *P = 0.036 by unpaired t test. (C) Whole-cell patch clamp recordings in HEK 293 cells expressing STIM1 and Orai3 and depicting the CRAC current-voltage relationship in control cells (left) and in cells pretreated with 500 μM GYY4137 for 1 hour (right). (D) Summary of the peak CRAC current amplitudes measured during steps to −100 mV in the experiments depicted in (C). Data are means ± SEM of six cells for each treatment group. *P < 0.001 by unpaired t test. (E) HEK 293 cells were pretreated with 500 μM GYY4137 for the indicated times before SOCE amplitudes were measured as described for Fig. 3A. Data are means ± SEM of three independent experiments. *P < 0.001 by one-way ANOVA. (F) RAW264.7 cells transfected with control siRNA or Orai3-specfic siRNA were treated for 4 hours with LPS (1 μg/ml) and then were analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. Data are expressed as the fold change in mRNA abundance in cells transfected with Orai3-specific siRNA compared to that in control cells. Data are means ± SEM of six independent samples. *P = 0.039, #P = 0.033 by unpaired t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/407/ra128/DC1

    Fig. S1. LPS increases the abundance of CSE but not CBS.

    Fig. S2. LPS has no effect on the abundances of STIM and Orai isoforms.

    Fig. S3. Inhibition of endogenous H2S production increases SOCE.

    Fig. S4. Analysis of the knockdown of STIM1 in RAW264.7 cells.

    Fig. S5. SOCE is enhanced in ATMs from obese mice.

    Fig. S6. Western blotting analysis of STIM1-YFP and Orai-CFP proteins in transfected HEK 293 cells.

    Fig. S7. Analysis of the knockdown of Orai3 in RAW264.7 cells.

  • Supplementary Materials for:

    Depletion of H2S during obesity enhances store-operated Ca2+ entry in adipose tissue macrophages to increase cytokine production

    Gopal V. Velmurugan, Huiya Huang, Hongbin Sun, Joseph Candela, Mukesh K. Jaiswal, Kenneth D. Beaman, Megumi Yamashita, Murali Prakriya, Carl White*

    *Corresponding author. E-mail: carl.white{at}rosalindfranklin.edu

    This PDF file includes:

    • Fig. S1. LPS increases the abundance of CSE but not CBS.
    • Fig. S2. LPS has no effect on the abundances of STIM and Orai isoforms.
    • Fig. S3. Inhibition of endogenous H2S production increases SOCE.
    • Fig. S4. Analysis of the knockdown of STIM1 in RAW264.7 cells.
    • Fig. S5. SOCE is enhanced in ATMs from obese mice.
    • Fig. S6. Western blotting analysis of STIM1-YFP and Orai-CFP proteins in transfected HEK 293 cells.
    • Fig. S7. Analysis of the knockdown of Orai3 in RAW264.7 cells.

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    Citation: G. V. Velmurugan, H. Huang, H. Sun, J. Candela, M. K. Jaiswal, K. D. Beaman, M. Yamashita, M. Prakriya, C. White, Depletion of H2S during obesity enhances store-operated Ca2+ entry in adipose tissue macrophages to increase cytokine production. Sci. Signal. 8, ra128 (2015).

    © 2015 American Association for the Advancement of Science

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