Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels - PubMed (original) (raw)

Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels

Paul T Mungai et al. Mol Cell Biol. 2011 Sep.

Abstract

AMP-activated protein kinase (AMPK) is an energy sensor activated by increases in [AMP] or by oxidant stress (reactive oxygen species [ROS]). Hypoxia increases cellular ROS signaling, but the pathways underlying subsequent AMPK activation are not known. We tested the hypothesis that hypoxia activates AMPK by ROS-mediated opening of calcium release-activated calcium (CRAC) channels. Hypoxia (1.5% O(2)) augments cellular ROS as detected by the redox-sensitive green fluorescent protein (roGFP) but does not increase the [AMP]/[ATP] ratio. Increases in intracellular calcium during hypoxia were detected with Fura2 and the calcium-calmodulin fluorescence resonance energy transfer (FRET) sensor YC2.3. Antioxidant treatment or removal of extracellular calcium abrogates hypoxia-induced calcium signaling and subsequent AMPK phosphorylation during hypoxia. Oxidant stress triggers relocation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, to the plasma membrane. Knockdown of STIM1 by short interfering RNA (siRNA) attenuates the calcium responses to hypoxia and subsequent AMPK phosphorylation, while inhibition of L-type calcium channels has no effect. Knockdown of the AMPK upstream kinase LKB1 by siRNA does not prevent AMPK activation during hypoxia, but knockdown of CaMKKβ abolishes the AMPK response. These findings reveal that hypoxia can trigger AMPK activation in the apparent absence of increased [AMP] through ROS-dependent CRAC channel activation, leading to increases in cytosolic calcium that activate the AMPK upstream kinase CaMKKβ.

PubMed Disclaimer

Figures

Fig. 1.

Exogenous oxidants activate AMPK and its activity. 143B cells were untreated (white bars) or treated with _tert_-butyl hydroperoxide (tBH) (black bars) for 1 or 2 h. Cells were also treated with the AMPK inhibitor compound C (Comp.C). Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) and of ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were determined by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown.

Fig. 2.

Activation of AMPK in hypoxia by oxidant signaling. (A) 143B cells expressing the ratiometric redox sensor roGFP were exposed to hypoxia (1.5% O2) (n = 8) or normoxia (21% O2) (n = 5). The graph depicts the fold change in oxidation during hypoxia compared to normoxia over time. Values are expressed as means ± standard errors of the means (SEM). *, P < 0.05. (B) 143B cells were exposed to hypoxia (1.5% O2) (n = 7), normoxia (21% O2) (n = 5), or tBH (25 μM) (n = 3) in the presence of 2′,7′-dichlorofluorescin (DCFH). The graph depicts the fold change in oxidation of DCFH in hypoxia or normoxia with tBH compared to normoxia with respect to time. Values are expressed as means ± SEM. (C) 143B cells were exposed to hypoxia (1.5% O2) (n = 3) or normoxia (21% O2) (n = 4) alone (white bars) or in the presence of the chemical antioxidant _N_-acetyl-

-cysteine (NAC) (black bars). Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) and of ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were measured by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM. *, P < 0.05 compared to normoxia; †, P < 0.05 compared to hypoxia control. (D) Rat pulmonary arterial smooth muscle cells (PASMC) were exposed to hypoxia (1.5% O2) (n = 7) or normoxia (21% O2) (n = 8) alone (white bars) or in the presence of NAC (black bars). Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) of and ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were measured by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM. *, P < 0.05 compared to normoxia; †, P < 0.05 compared to hypoxia control. WT, wild type.

Fig. 3.

ROS-dependent activation of AMPK activity in intact lung. (A) Mice were housed for 28 days in normoxia (21% O2), hypoxia (10% O2), or hypoxia with NAC in their drinking water. Images of representative lung sections stained for ACC phosphorylated at Ser-79 (p-ACC) (panels A to C), total ACC (panels D to F), and secondary antibody alone (panels G to I) are shown. (B) Total integrated pixel intensities for lung section areas stained for ACC phosphorylated at Ser-79 (p-ACC) and total ACC compared to hematoxylin were quantified. The graph depicts the p-ACC/ACC ratios. Values are expressed as means ± SEM (n = 3). *, P < 0.05 compared to normoxia; †, P < 0.05 compared to hypoxia. a.u., arbitrary units.

Fig. 4.

AMPK activation in hypoxia: role of AMP. (A) 143B cells and PASMC were exposed to hypoxia (1.5% O2) (n = 5), normoxia (21% O2) (n = 6), or normoxia with 2-deoxy-

-glucose (2DG) (n = 3) for 2 h. Cell lysates were analyzed by HPLC to measure adenine nucleotide concentrations. Graphs depict AMP, ADP, and ATP concentrations under each condition. Values are expressed as means ± SEM. (B) Graphs depict AMP/ATP ratios in 143B cells and PASMC under each experimental condition. Values are expressed as means ± SEM. *, P < 0.05 compared to normoxia or hypoxia.

Fig. 5.

AMPK activation in hypoxia: role of LKB1. Embryonic fibroblasts from mice genetically deficient for LKB1−/− (LKB1−/− MEF) were exposed to hypoxia (1.5% O2) (black bars) or normoxia (21% O2) (white bars). Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) and of ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were measured by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM (n = 9). *, P < 0.05 compared to normoxia. (B) Wild-type mouse embryonic fibroblasts (MEF WT) were transfected with negative-control siRNA (white bars) or siRNA for LKB1 (black bars) and exposed to hypoxia (1.5% O2) or normoxia (21% O2) for 2 h. Levels of ACC phosphorylated at Ser-79 (p-ACC) as well as total ACC were measured by Western blotting. Graphs depict the fold change in p-ACC/ACC ratios. Representative Western blots are shown. Values are expressed as means ± SEM (n = 3). *, P < 0.05 compared to normoxia control.

Fig. 6.

AMPK activation in hypoxia: role of CaMKKβ. (A) LKB1−/− MEFs were transfected with negative-control siRNA (white bars) or siRNA for CaMKKβ (black bars) and exposed to hypoxia (1.5% O2) or normoxia (21% O2) for 2 h. Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) and ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were measured by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM (n = 5). *, P < 0.05 compared to normoxia control; †, P < 0.05 compared to hypoxia control. (B) The same protocol as for panel A was used to study the role of CaMKKβ in AMPK activation during hypoxia. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM (n = 5). *, P < 0.05 compared to normoxia control; †, P < 0.05 compared to hypoxia control.

Fig. 7.

Role of calcium in hypoxia-induced AMPK activation. (A) 143B cells expressing the ratiometric FRET calcium sensor YC2.3 were exposed to hypoxia (1.5% O2) (n = 5) or hypoxia in the presence of NAC (n = 3). The graph depicts the fold change in FRET ratio during hypoxia compared to normoxia with respect to time. Values are expressed as means ± SEM. *, P < 0.05. (B) 143B cells in control (white bars) or calcium-free (black bars) BSS were exposed to hypoxia (1.5% O2) or normoxia (21% O2) for 2 h. Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) as well as total AMPKα were measured by Western blotting. Graphs depict the fold change in p-AMPK/AMPK ratios. Representative Western blots are shown. Values are expressed as means ± SEM (n = 5). *, P < 0.05 compared to normoxia control; †, P < 0.05 compared to hypoxia control. (C) 143B cells expressing roGFP or loaded with Fura2-AM were exposed to hypoxia (1.5% O2) or hypoxia and the calcium chelator BAPTA. The first graph depicts the change in oxidation of roGFP over time. The second graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 4).

Fig. 8.

SOCE and oxidant-induced Ca2+ responses in 143B cells. (A) 143B cells were loaded with Fura2-AM and treated with thapsigargin (TG) in calcium-free Ringer solution to deplete ER stores, and then 2 mM Ca2+ Ringer solution was added to trigger store-operated Ca2+ influx. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 34). (B) The same protocol as for panel A was used to study intracellular Ca2+ responses to tBH. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 37). (C) 143B cells were loaded with Fura2-AM and treated with thapsigargin (TG) in calcium-free Ringer solution to deplete ER stores, and then 2 mM Ca2+ Ringer solution was added to trigger store-operated Ca2+ influx, after which tBH was added to study oxidant-mediated intracellular Ca2+ responses after SOCE. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 36).

Fig. 9.

Pharmacological properties of SOCE in 143B cells. (A) 143B cells were loaded with Fura2-AM and treated with thapsigargin (TG) in calcium-free Ringer solution to deplete ER stores, and then 2 mM Ca2+ Ringer solution was added to trigger store-operated Ca2+ influx, after which 2-APB was added to characterize _I_CRAC in these cells. The graph represents the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 31). (B) The same protocol as for panel A was used to characterize _I_CRAC with lanthanum (La3+) in 143B cells. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 16). (C) 143B cells (n = 42) and 143B cells treated with the CRAC channel inhibitor BTP-2 (n = 41) were loaded with Fura2-AM and treated with thapsigargin (TG) in calcium-free Ringer solution to deplete ER store, and then, 2 mM Ca2+ Ringer solution was added to trigger store-operated Ca2+ influx. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM.

Fig. 10.

Pharmacological properties of oxidant-induced Ca2+ responses in 143B cells. (A) 143B cells were loaded with Fura2-AM and transferred to calcium-free Ringer solution, and then 2 mM Ca2+ Ringer solution was added in the presence of tBH to trigger oxidant-mediated intracellular Ca2+ entry, after which 2APB was added to characterize _I_CRAC in these cells. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 49). (B) The same protocol as in for panel A was used to inhibit _I_CRAC with lanthanum (La3+) in 143B cells. The graph represents the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 16). (C) 143B cells (n = 42) and 143B cells treated with the CRAC channel inhibitor BTP-2 (n = 41) were loaded with Fura2-AM and treated with tBH in calcium-free Ringer solution, and then, 2 mM Ca2+ Ringer solution was added to examine oxidant-mediated intracellular Ca2+ responses. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM.

Fig. 11.

Role of STIM1 in oxidant-induced Ca2+ responses. (A) 143B cells transfected with negative-control siRNA (n = 42) or with siRNA for STIM1 (n = 34) were loaded with Fura2-AM and treated with thapsigargin (TG) in calcium-free Ringer solution to deplete ER stores, and then 2 mM Ca2+ Ringer solution was added to enable store-operated Ca2+ influx. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM. (B) 143B cells transfected with negative-control siRNA (n = 45) or with siRNA for STIM1 (n = 28) were loaded with Fura2-AM and treated with tBH in calcium-free Ringer solution, and then, 2 mM Ca2+ Ringer solution was added to examine oxidant-mediated intracellular Ca2+ responses. The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM. (C) The same protocol as for panel B was used to study the role of STIM1 in intracellular Ca2+ responses to hypoxia (1.5% O2). The graph depicts the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 5).

Fig. 12.

Role of STIM1 in hypoxia-induced AMPK activation. (A) 143B cells were transfected with negative-control siRNA (white bars) or siRNA for STIM1 (black bars) and exposed to hypoxia (1.5% O2) or normoxia (21% O2) for 2 h. Levels of AMPK phosphorylated at Thr-172 (p-AMPKα) and of ACC phosphorylated at Ser-79 (p-ACC) as well as total AMPKα and ACC were measured by Western blotting. Graphs depict the fold change in the p-AMPK/AMPK or p-ACC/ACC ratio. Representative Western blots are shown. Values are expressed as means ± SEM (n = 6). *, P < 0.05 compared to normoxia control; †, P < 0.05 compared to hypoxia control. (B) Control 143B cells (white bars) and 143B treated with nifedipine (black bars) were analyzed for AMPK activation during hypoxia as for panel A. Graphs depict the fold change in p-AMPK/AMPK ratios. Representative Western blots are shown. Values are expressed as means ± SEM (n = 4). *, P < 0.05 compared to normoxia control.

Fig. 13.

Oxidant stress involvement in STIM1 redistribution and store-activated calcium signaling. (A) 143B cells were fixed and immunostained for STIM1 after normoxia (21% O2) (panel A), hypoxia (1.5% O2) (panels B and C), treatment with thapsigargin (panel D), and treatment with tBH (panels E and F). (B) 143B cells were loaded with Fura2-AM and imaged during hypoxia (1.5 O2) or normoxia (21% O2) in normal-Ca2+ BSS or low-calcium BSS. Graphs depict the change in the 340/380 intensity ratio over time. Values are expressed as means ± SEM (n = 3).

Fig. 14.

A model for ROS- and STIM1-dependent activation of AMPK in hypoxia. Oxidant signaling in hypoxia causes ER calcium release and translocation of STIM1 to ER domains near the plasma membrane. Orai proteins tethered by STIM1 form CRAC channels facilitating Ca2+ influx. Increased cytosolic Ca2+ activates CaMKKβ, which phosphorylates AMPK.

References

1. Bardeesy N., et al. 2002. Loss of the Lkb1 tumour suppressor provokes intestinal polyposis but resistance to transformation. Nature 419:162–167 - PubMed
1. Birnbaum M. J. 2005. Activating AMP-activated protein kinase without AMP. Mol. Cell 19:289–290 - PubMed
1. Budinger G. R., et al. 1996. Cellular energy utilization and supply during hypoxia in embryonic cardiac myocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 270:L44–L53 - PubMed
1. Carling D., Sanders M. J., Woods A. 2008. The regulation of AMP-activated protein kinase by upstream kinases. Int. J. Obes. 32(Suppl. 4):S55–S59 - PubMed
1. Chandel N. S., Budinger G. R. S., Choe S. H., Schumacker P. T. 1997. Cellular respiration during hypoxia. J. Biol. Chem. 272:18808–18816 - PubMed

Hypoxia triggers AMPK activation through reactive oxygen species-mediated activation of calcium release-activated calcium channels - PubMed (original) (raw)