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. 2009 Oct;86(4):981-8.
doi: 10.1189/jlb.0708454. Epub 2009 Jul 14.

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2

Yanlin Wang  1 , Luping Huang, Maen Abdelrahim, Qingsong Cai, Anh Truong, Roger Bick, Brian Poindexter, David Sheikh-Hamad
Affiliations

Stanniocalcin-1 suppresses superoxide generation in macrophages through induction of mitochondrial UCP2

Yanlin Wang et al. J Leukoc Biol. 2009 Oct.

Abstract

Mammalian STC1 decreases the mobility of macrophages and diminishes their response to chemokines. In the current experiments, we sought to determine the impact of STC1 on energy metabolism and superoxide generation in mouse macrophages. STC1 decreases ATP level in macrophages but does not affect the activity of respiratory chain complexes I-IV. STC1 induces the expression of mitochondrial UCP2, diminishing mitochondrial membrane potential and superoxide generation; studies in UCP2 null and gp91phox null macrophages suggest that suppression of superoxide by STC1 is UCP2-dependent yet is gp91phox-independent. Furthermore, STC1 blunts the effects of LPS on superoxide generation in macrophages. Exogenous STC1 is internalized by macrophages within 10 min and localizes to the mitochondria, suggesting a role for circulating and/or tissue-derived STC1 in regulating macrophage function. STC1 induces arrest of the cell cycle at the G1 phase and reduces cell necrosis and apoptosis in serum-starved macrophages. Our data identify STC1 as a key regulator of superoxide generation in macrophages and suggest that STC1 may profoundly affect the immune/inflammatory response.

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Figures

Figure 1.
Figure 1.
(A) STC1 decreases intracellular ATP levels. Raw264.7 cells grown (37°C, 5% CO2) in DMEM medium containing 10% FBS or freshly isolated peritoneal macrophages suspended in DMEM plus 10% FBS were treated with varying concentrations of rSTC1 for 1 h. Values represent the mean + sem of three independent experiments. (B) Cells were treated with rSTC1 (100 ng/mL) for increasing times. (C) Cells were treated with rSTC1 (100 ng/mL) for 3 h, washed in PBS (×3), and resuspended in fresh medium for 24 additional hours before ATP analysis (washout experiment). (D) Cells were treated with denatured rSTC1 (100 ng/mL) or vehicle for 3 h. Data represent the mean + sem of three independent experiments. Compared with vehicle, *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) Labeling for STC1 is detected intracellularly in quiescent, freshly isolated peritoneal macrophages. Ten minutes after the addition of rSTC1 to the medium (100 ng/mL), intracellular labeling for STC1 increases tenfold and colocalizes with the mitochondrial marker, Mitotracker. DAPI = 4′,6-Diamidino-2-phenylindole.
Figure 2.
Figure 2.
STC1 has no effect on the activity of complexes I–IV of the respiratory chain. Raw264.7 cells grown (37°C, 5% CO2) in DMEM medium containing 10% FBS were treated with rSTC1 (100 ng/mL) for 3 h, lysed, and analyzed for respiratory chain complex activity as described in Materials and Methods. Data represent the mean + sem of three independent experiments. Differences were not statistically significant.
Figure 3.
Figure 3.
STC1 induces UCP2 expression in macrophages. Raw264.7 cells grown (37°C, 5% CO2) in DMEM medium containing 10% FBS were treated with rSTC1 (100 ng/mL) for varying periods of time. Cells were lysed in modified RIPA buffer, and UCP2 protein abundance was determined using Western blotting. (A) Representative blot is shown. (B) Graph represents the mean ± sem of three independent experiments. Densitometry values of UCP2 were normalized to GAPDH. *, P < 0.05, compared with 0 h; **, P < 0.01, compared with 0 h.
Figure 4.
Figure 4.
STC1 decreases mitochondrial membrane potential. Raw264.7 cells grown (37°C, 5% CO2) in DMEM medium containing 10% FBS were treated with rSTC1 (150 ng/mL) for 5 h and 24 h, followed by mitochondrial membrane potential measurement using JC-1 florescence-based assay (red:green fluorescence ratio). Data represent the mean + sem of eight independent experiments. *, P < 0.001.
Figure 5.
Figure 5.
STC1-induced reduction in superoxide generation in macrophages is UCP2-dependent. Freshly isolated peritoneal macrophages from wild-type (Wt and WT) and UCP2−/− mice were suspended in DMEM medium containing 10% FBS (37°C, 5% CO2). Sixteen hours prior to the experiment, cells were shifted to 1% FBS-containing medium and then were treated with rSTC1 (100 ng/mL) for an 24 additional hours. Fifteen minutes prior to completion of the experiment, cells were treated with DHE, and red fluorescence (ethidium bromide) was quantitated using flow cytometry. Bar graph represents the mean + sem of three independent experiments.
Figure 6.
Figure 6.
STC1-induced reduction in superoxide generation in macrophages is gp91phox-independent; STC1 blocks LPS-induced superoxide generation in macrophages. An equal number of freshly isolated peritoneal macrophages from wild-type (A) and gp91phox−/− (B) mice were treated with PBS or rSTC1 (100 ng/mL) for 2 h, followed by 60 min incubation with 100 μL Y-NBT solution containing LPS (1 μ/mL) or vehicle [STC1 treatment was continued (at 100 ng/mL) in rSTC1-pretreated cells]. NBT deposited inside the cells (represents superoxide generation in 1 h) was dissolved, and A620 nm of the lysate was measured. Data are normalized to superoxide generation in control cells (no STC1 and no LPS). Bar graphs represent the means + sem of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 7.
Figure 7.
STC1 enhances macrophage viability. (A) Raw264.7 cells grown (37°C, 5% CO2) in DMEM medium containing 10% FBS were shifted to DMEM containing 1% FBS for 16 h, followed by treatment with STC1 (100 ng/mL) in DMEM containing 1% FBS for 24 additional hours. Medium was then collected for LDH analysis. Data are expressed as percent of controls and represent the means + sem of three independent experiments. *, P < 0.01. (B) Raw264.7 cells were grown (37°C, 5% CO2) in DMEM medium containing 10% FBS. Cells were then shifted to DMEM containing 1% FBS in the presence of STC1 (100 ng/mL) or vehicle for 48 h. Cells were harvested using cell lifter, fixed in 70% ethanol, stained with propidium iodide, and treated with RNase for 30 min. Cell-cycle analysis was performed using flow cytometry as described previously [333435]; numbers indicate fraction of total cells (%) in each phase of the cell cycle. Peak F represents apoptotic cells; peak C represents cells in the G1 phase; peak D represents cells in the S phase; Peak E represents cells in the G2/M phase. The differences between peaks F and C were statistically significant when comparing STC1-treated cells versus controls. FL3 = Fluorescence 3.
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