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Intracellular calcium regulates nonsense-mediated mRNA decay.

Nickless A, Jackson E, Marasa J, Nugent P, Mercer RW, Piwnica-Worms D, You Z - Nat. Med. (2014)

Bottom Line: Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells.Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels.Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent a target for therapeutic intervention. Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides, including ouabain and digoxin, as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from the endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

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Cardiac glycosides block NMD through inhibition of Na+/K+-ATPase and elevation of intracellular calcium(a) Ratios of CBR/CBG bioluminescence signals in mouse dermal fibroblasts (left panel) or human U2OS cells (right panel) expressing the NMD reporter after 24 h treatment with DMSO, caffeine, or various concentrations of ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates.(b) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit or rat α3 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05 (paired t-test).(c) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, human α1 subunit, or CG-resistant mutant human α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(d) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit, or catalytically-inactive rat α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(e) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 24 h treatment with DMSO, ouabain, ouabain and Bapta-AM, or Bapta-AM. Ouabain, 0.175 µM; Bapta-AM, 25 µM. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(f) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 4 h treatment with DMSO, thapsigargin, thapsigargin and Bapta-AM, or Bapta-AM. Cells were pre-treated with either DMSO or Bapta-AM for 1 h before addition of thapsigargin. Thasipgargin, 0.2 µM; Bapta-AM, 50 µM. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(g) A model for the regulation of NMD by cardiac glycosides, Na+/K+-ATPase and intracellular calcium.
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Figure 4: Cardiac glycosides block NMD through inhibition of Na+/K+-ATPase and elevation of intracellular calcium(a) Ratios of CBR/CBG bioluminescence signals in mouse dermal fibroblasts (left panel) or human U2OS cells (right panel) expressing the NMD reporter after 24 h treatment with DMSO, caffeine, or various concentrations of ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates.(b) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit or rat α3 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05 (paired t-test).(c) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, human α1 subunit, or CG-resistant mutant human α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(d) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit, or catalytically-inactive rat α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(e) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 24 h treatment with DMSO, ouabain, ouabain and Bapta-AM, or Bapta-AM. Ouabain, 0.175 µM; Bapta-AM, 25 µM. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(f) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 4 h treatment with DMSO, thapsigargin, thapsigargin and Bapta-AM, or Bapta-AM. Cells were pre-treated with either DMSO or Bapta-AM for 1 h before addition of thapsigargin. Thasipgargin, 0.2 µM; Bapta-AM, 50 µM. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(g) A model for the regulation of NMD by cardiac glycosides, Na+/K+-ATPase and intracellular calcium.

Mentions: Since the Na+/K+-ATPase (composed of a catalytic α subunit and a structural β subunit, each with various isoforms, and a regulatory γ subunit) is the only known pharmacological target of the identified CGs, our results suggest that CGs inhibit NMD via the sodium-potassium pump23. Consistent with this idea, our reporter assay indicates that mouse skin fibroblasts, which express a naturally CG-resistant α1 subunit of Na+/K+-ATPase, exhibited more than 100 times greater resistance to ouabain, compared to human cells in which all 4 isoforms of the α subunit have a much higher affinity for CGs (Fig. 4a)24. In contrast, NMD of the reporter exhibited a similar level of sensitivity to caffeine in both human and mouse cells (Fig. 4a). Moreover, overexpression in human cells of the rat α1 subunit of Na+/K+-ATPase, which also has a low affinity for CGs, abrogated the inhibitory effects of ouabain on NMD. In contrast, expression of a similar level of rat α3 subunit of Na+/K+-ATPase, which is sensitive to CGs24,25, did not cause resistance of NMD to ouabain (Fig. 4b and Supplementary Fig. 5a). Furthermore, expression of a CG-resistant mutant version of the human α1 subunit also prevented the CG-mediated inhibition of NMD while the wild-type protein failed to do so (Fig. 4c and Supplementary Fig. 5b). To further demonstrate that CGs inhibit NMD through their binding and inhibition of Na+/K+-ATPase, we generated a catalytically inactive mutant of the rat α1 subunit (D376E) that has diminished binding and hydrolysis of ATP25. Unlike the WT rat α1 subunit, expression of this mutant in human cells could not cause resistance of NMD to CGs, indicating that blockade of the sodium/potassium pump enzymatic activity is necessary for efficient CG-induced inhibition of NMD (Fig. 4d and Supplementary Fig. 5c). Taken together, these data strongly suggest that Na+/K+-ATPase is a robust regulator of NMD in mammalian cells and that CGs inhibit NMD through binding and inhibiting this sodium-potassium pump. Given the essential role of Na+/K+-ATPase for cell viability, the observation that CGs strongly inhibited NMD at concentrations where no obvious decrease in CBG activity or cell viability was observed suggests that partial inhibition of the sodium-potassium pump is sufficient to abolish NMD (Fig. 3 and Supplementary Fig. 3b).


Intracellular calcium regulates nonsense-mediated mRNA decay.

Nickless A, Jackson E, Marasa J, Nugent P, Mercer RW, Piwnica-Worms D, You Z - Nat. Med. (2014)

Cardiac glycosides block NMD through inhibition of Na+/K+-ATPase and elevation of intracellular calcium(a) Ratios of CBR/CBG bioluminescence signals in mouse dermal fibroblasts (left panel) or human U2OS cells (right panel) expressing the NMD reporter after 24 h treatment with DMSO, caffeine, or various concentrations of ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates.(b) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit or rat α3 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05 (paired t-test).(c) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, human α1 subunit, or CG-resistant mutant human α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(d) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit, or catalytically-inactive rat α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(e) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 24 h treatment with DMSO, ouabain, ouabain and Bapta-AM, or Bapta-AM. Ouabain, 0.175 µM; Bapta-AM, 25 µM. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(f) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 4 h treatment with DMSO, thapsigargin, thapsigargin and Bapta-AM, or Bapta-AM. Cells were pre-treated with either DMSO or Bapta-AM for 1 h before addition of thapsigargin. Thasipgargin, 0.2 µM; Bapta-AM, 50 µM. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(g) A model for the regulation of NMD by cardiac glycosides, Na+/K+-ATPase and intracellular calcium.
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Figure 4: Cardiac glycosides block NMD through inhibition of Na+/K+-ATPase and elevation of intracellular calcium(a) Ratios of CBR/CBG bioluminescence signals in mouse dermal fibroblasts (left panel) or human U2OS cells (right panel) expressing the NMD reporter after 24 h treatment with DMSO, caffeine, or various concentrations of ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates.(b) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit or rat α3 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05 (paired t-test).(c) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, human α1 subunit, or CG-resistant mutant human α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(d) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit, or catalytically-inactive rat α1 subunit of Na+/K+-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(e) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 24 h treatment with DMSO, ouabain, ouabain and Bapta-AM, or Bapta-AM. Ouabain, 0.175 µM; Bapta-AM, 25 µM. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test).(f) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 4 h treatment with DMSO, thapsigargin, thapsigargin and Bapta-AM, or Bapta-AM. Cells were pre-treated with either DMSO or Bapta-AM for 1 h before addition of thapsigargin. Thasipgargin, 0.2 µM; Bapta-AM, 50 µM. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(g) A model for the regulation of NMD by cardiac glycosides, Na+/K+-ATPase and intracellular calcium.
Mentions: Since the Na+/K+-ATPase (composed of a catalytic α subunit and a structural β subunit, each with various isoforms, and a regulatory γ subunit) is the only known pharmacological target of the identified CGs, our results suggest that CGs inhibit NMD via the sodium-potassium pump23. Consistent with this idea, our reporter assay indicates that mouse skin fibroblasts, which express a naturally CG-resistant α1 subunit of Na+/K+-ATPase, exhibited more than 100 times greater resistance to ouabain, compared to human cells in which all 4 isoforms of the α subunit have a much higher affinity for CGs (Fig. 4a)24. In contrast, NMD of the reporter exhibited a similar level of sensitivity to caffeine in both human and mouse cells (Fig. 4a). Moreover, overexpression in human cells of the rat α1 subunit of Na+/K+-ATPase, which also has a low affinity for CGs, abrogated the inhibitory effects of ouabain on NMD. In contrast, expression of a similar level of rat α3 subunit of Na+/K+-ATPase, which is sensitive to CGs24,25, did not cause resistance of NMD to ouabain (Fig. 4b and Supplementary Fig. 5a). Furthermore, expression of a CG-resistant mutant version of the human α1 subunit also prevented the CG-mediated inhibition of NMD while the wild-type protein failed to do so (Fig. 4c and Supplementary Fig. 5b). To further demonstrate that CGs inhibit NMD through their binding and inhibition of Na+/K+-ATPase, we generated a catalytically inactive mutant of the rat α1 subunit (D376E) that has diminished binding and hydrolysis of ATP25. Unlike the WT rat α1 subunit, expression of this mutant in human cells could not cause resistance of NMD to CGs, indicating that blockade of the sodium/potassium pump enzymatic activity is necessary for efficient CG-induced inhibition of NMD (Fig. 4d and Supplementary Fig. 5c). Taken together, these data strongly suggest that Na+/K+-ATPase is a robust regulator of NMD in mammalian cells and that CGs inhibit NMD through binding and inhibiting this sodium-potassium pump. Given the essential role of Na+/K+-ATPase for cell viability, the observation that CGs strongly inhibited NMD at concentrations where no obvious decrease in CBG activity or cell viability was observed suggests that partial inhibition of the sodium-potassium pump is sufficient to abolish NMD (Fig. 3 and Supplementary Fig. 3b).

Bottom Line: Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells.Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels.Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.

ABSTRACT
The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent a target for therapeutic intervention. Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides, including ouabain and digoxin, as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from the endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

Show MeSH
Related in: MedlinePlus