Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response

Administration of 10 ng ml^-1 tetracycline to Tex293 clone-22 cells for 16 h generated 150 to 200 nM NO, as measured using an NO electrode. This was accompanied by a decline in oxygen consumption from 25.5 ( 1.5) to 13.7 ( 1.5) M min^-1 (per 10⁷ cells; n = 3). Treatment of EcR293 clone-11 subclone-1 cells with 10 M muristerone A for 16 h resulted in a similar decline in oxygen consumption from 7.3 ( 1.5) to 4.4 ( 0.6) M min^-1 (per 10⁷ cells; n = 3). Mitochondria were obtained from these cell lines after 16 h treatment and NO-mediated calcium efflux from the mitochondria was evaluated using the calcium-sensitive dye arsenazo III¹³. The rate of calcium release from mitochondria was 6.0 ( 1.5) and 39 ( 0.3) nmol mg^-1 of mitochondrial protein min^-1 in untreated and NO-generating Tex293 clone-22 cells respectively, and 18 ( 3.2) and 32.0 ( 5.2) nmol mg^-1 of mitochondrial protein min^-1 in untreated and NO-generating EcR293 clone-11 subclone-1 cells respectively (n = 3; P < 0.05). In both cell lines, the mitochondrial calcium release could be inhibited completely by cyclosporin A (500 nM). This finding agrees with reports that exogenous NO¹⁴ and peroxynitrite^{13, 15} cause release of calcium from mitochondria, which is prevented by cyclosporin A.

To study the downstream consequences of the NO-dependent inhibition of respiration, differential hybridization experiments were performed using normalized cDNA arrays. Fifty nanograms of polyA⁺ RNA from untreated EcR293 clone-11 subclone-1 cells were labelled with the fluorescent molecule Cy3, whereas 50 ng polyA⁺ RNA from muristerone A-treated (24 h) cells were labelled with Cy5. RNA was hybridised to a Gem array containing 8,372 unique annotated genes/EST clusters. Scanning the array at two wavelengths showed that glucose-regulated protein 78 (Grp78) was highly upregulated (threefold) in NO-generating cells (see Supplementary Information, Fig. S1). A similar threefold upregulation of Grp78 was observed after hybridisation to a second Gem array using poly A⁺ mRNA extracted from Tex293 clone-22 cells expressing iNOS (data not shown). The array results were supported by northern (Fig. 1a) and western (Fig. 1b) blotting experiments. The NOS inhibitor N-iminoethyl-L-ornithine (L−NIO; 20 M) attenuated the increase in Grp78 in NO-generating cells, whereas the soluble guanylate cyclase (sGC) inhibitor ODQ (10 M) had no marked effect (Fig. 1b). Thus, increases in NO are linked to the expression of Grp78 independently of activation of sGC. Administration of exogenous NO, using DETA-NONOate (500 M), to the T-REx-293 parental cell line also resulted in an upregulation of Grp78 protein within 2 h, which was detectable for up to 20 h (Fig. 1c).

Figure 1. NO increases expression of Grp78 and cleavage of p90 ATF6. (a) Northern blot analysis of Grp78 and iNOS mRNA in the non-NO-generating parental cell lines T-REx-293 and EcR-293, and in their respective NO-generating derived cell lines Tex293 clone-22 and EcR293 clone-11 subclone-1 after treatment with tetracycline (10 ng ml-1) or with muristerone A (5 and 10 M). -actin was used as an internal control (n = 3). (b) Western blot analysis of Grp78 protein from untreated (None), muristerone A (10 M, 16h; MuA)-treated, muristerone A plus ODQ (10 M)-treated and muristerone A plus L-NIO (20 M)-treated EcR293 clone-11 subclone-1 cells. -tubulin was used as a control (n = 3). Scanning densitometry of six western blots is shown below. Asterisk indicates a significant difference (P < 0.05) from non-NO-generating cells. (c) Western blot analysis of Grp78 protein in T-REx-293 cells at different times after treatment with the NO donor DETA-NONOate (500 M). Scanning densitometry of three western blots is shown below. Asterisk indicates a significant difference (P < 0.05) from untreated cells. (d) Cleavage of p90 ATF6 in Tex293 clone-22 cells. Treatment with tetracycline significantly increased the amount of soluble transcription factor p50 ATF6 and reduced the amount of uncleaved protein p90 ATF6 (whole-cell extracts). Pre-treatment of tetracycline-treated cells with the NOS inhibitor L-NIO (20 M) attenuated the increase in p50 ATF6. Scanning densitometry of three western blots is shown below. Data are presented as the percentage conversion to p50, calculated as the p50 value divided by the sum of p50 + p90, divided by the tubulin value, multiplied by 100 (that is, p50/(p50 + p90)/tubulin, 100). Asterisk indicates a significant difference (P < 0.05) from untreated cells; double asterisk indicates a significant difference (P < 0.05) from NO-generating cells in the absence of L-NIO. Full Figure and legend (57K)

Previous studies demonstrated that the soluble transcription factor p50 ATF6 is involved in Grp78 ERSE-binding^{4, 16, 17}, consequently p50 ATF6 was investigated in NO-generating cells. Treatment of Tex293 clone-22 cells with tetracycline resulted in an increase in p50 ATF6 that was attenuated by pre-treatment of cells with L-NIO (20 M; Fig. 1d).

ATF6 is a membrane-associated transcription factor controlled by a process called 'regulated intramembrane proteolysis' (RIP)⁵, involving cleavage of a membrane-bound form of the protein by the sequential action of two proteases: the site-1 protease, S1P, and the site-2 protease, S2P⁴. Because cleavage of several proteins by S1P is calcium-dependent^{18, 19, 20}, we examined the calcium-dependency of NO-mediated ATF6 cleavage and Grp78 upregulation. For this, EcR293 clone-11 subclone-1 cells, whose generation of NO after treatment with muristerone A (10 M) was controlled using the iNOS inhibitor S-methylisothiourea (100 M), were used. After overnight incubation, cells were washed to remove the inhibitor and maintained for 1 h in L-arginine-free medium. High concentrations of NO were then generated by the addition of l-arginine (1 mM) in either normal or calcium-free DMEM medium²¹. Some of the cells in calcium-free medium were also treated with the calcium ionophore A23187, which stimulates the loss of intracellular calcium. After 4 h, the cells were lysed and subjected to western blot analysis. Whereas ATF6 cleavage was observed in NO-generating cells suspended in both normal and calcium-free medium, a dose-dependent decrease in P50 ATF6 was seen in cells whose intracellular calcium was depleted by A23187 (Fig. 2a), showing that ATF6 cleavage is dependent on intracellular but not extracellular calcium. The NO-mediated increase in Grp78 protein concentration was also diminished in a dose-dependent manner after treatment with A23187 (Fig. 2a). To further investigate the calcium-dependency of ATF6 cleavage, EcR293 clone-11 subclone-1 cells were transfected with a 3Flag−ATF6 plasmid²² (see Methods section). Equal amounts (50 g protein) of the whole-cell extract were aliquoted and treated with CaCl₂ (1 mM), with or without EGTA (20 mM). After treatment with CaCl₂ the p90 ATF6 band in the insoluble membrane pellet fraction was markedly reduced, with a concomitant increase in the soluble p50 Flag-tagged ATF6 component (Fig. 2b). The p50 ATF6 cleavage was abolished by EGTA, consistent with the role of calcium in the processing pathway.

Figure 2. Calcium dependence of NO-mediated ATF6 cleavage and upregulation of Grp78. (a) Western blot analysis showing ATF6 cleavage and Grp78 upregulation in NO-generating EcR293 clone-11 subclone-1 cells suspended in normal or calcium-free medium and treated with A23187. Cells shown in lane 1 (-NO) were not treated with muristerone A (n = 3). (b) Immunoblot analysis of calcium-dependent cleavage of Flag-tagged ATF6 in cell-free extracts in vitro. Top, insoluble membrane pellet fraction; bottom, soluble fraction after immunoprecipitation (IP) with Flag antibody. (c) The role of S1P and S2P in NO-mediated cytoprotection. EcR293 clone-11 subclone-1 cells were transfected with siRNA duplex (200 nM per well, 12 wells) on day one and day two. Cells were then treated with muristerone A and thapsigargin and the viability determined. All values are the mean values + s.d. of three replicates. Double asterisk indicates a significant difference (P < 0.01) between no siRNA treatment and either S1P siRNA-treated or S2P siRNA-treated samples. Bottom, real-time RT−PCR quantification using the total RNA prepared from cells treated with target-specific siRNA, sense siRNA, and non-silencing (NS) siRNA for 48h. GAPDH was used as an internal control. The values represent the amount of mRNA relative to that in the untreated cells (arbitrary value = 1). Asterisk indicates a significant difference (P < 0.05) between siRNA-treated and non-silencing siRNA-treated samples. Full Figure and legend (74K)

Cytoprotection by NO was investigated further in cells treated with the calcium chelator 1,2-bis (2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), and cyclosporin A, which disrupts mitochondrial calcium flux. NO-dependent cytoprotection against thapsigargin (37 nM; Fig. 3a) was abolished after treatment with either BAPTA-AM or cyclosporin A (Fig. 3a). Neither BAPTA-AM (10 M) nor cyclosporin A (2.5 M) showed any effect on thapsigargin-induced cell toxicity in non-NO-generating cells (Fig. 3a). Western blot analysis showed that treatment of NO-generating cells with thapsigargin resulted in an increase in Grp78 protein (5.1-fold; Fig. 3a), which was significantly greater than that seen for cells treated with thapsigargin (2.3-fold; Fig. 3a) or muristerone A alone (3.0-fold; Fig. 3a). Our data suggest that relatively high increases in Grp78 expression (> fivefold) are necessary to afford cytoprotection, as smaller increases were ineffective under our experimental conditions. A similar fivefold increase in Grp78 expression has been reported in CHO cells transfected with a hamster Grp78 cDNA, where it was shown to mediate resistance to toxicity from topoisomerase inhibitors⁶.

Figure 3. NO-mediated cytoprotection is calcium dependent. (a) Cells incubated in the absence (-MuA) or presence (+MuA) of muristerone A for 16 h were washed with PBS, treated with thapsigargin (37 nM) and cell viability determined by the LDH assay after a further 48 h. In some samples, BAPTA-AM (10 M) or cyclosporin A (CyA; 2.5 M) was administered at the same time as MuA. Values are the mean values + s.d. of three replicates. Double asterisk indicates a significant difference (P < 0.01) between NO-generating and non-NO-generating cells. Middle, western blot of Grp78 protein from the above treatment groups; bottom, scanning densitometry of three western blots (asterisk indicates a significant difference (P < 0.05) from untreated cells and double asterisk indicates a significant difference (P < 0.05) from thapsigargin-treated non-NO-generating cells). (b) Western blot analysis of whole-cell extracts (50 g protein) showing the effect of BAPTA-AM and cyclosporin A on NO-induced cleavage of ATF6 in EcR293 clone-11 subclone-1 cells. Percentage conversion to p50 was calculated as described in the legend to Fig. 1d. -tubulin was used as a control (n = 3). Full Figure and legend (57K)

Figure 4. NO-mediated Grp78 expression in mitochondrial DNA-deficient cell lines. Western blot analysis of Grp78 and iNOS protein in EcR293 clone-11 subclone-1 cells (rho+) cells and the derived rho0 cell line, either untreated (None) or treated with muristerone A (16 h; MuA). -tubulin was used as a control (n = 3). Asterisk indicates a significant difference (P < 0.05) from non-NO-generating cells. Full Figure and legend (18K)

EcR293 clone-11 subclone-1 cells were treated with muristerone A (10 M) in the presence or absence of BAPTA-AM (10 M) or cyclosporin A (2.5 M) for 16 h. They were then treated with thapsigargin (37 nM), and viability was determined after 48 h. Cyclosporin A, BAPTA-AM and thapsigargin were from Sigma (St Louis, MO). Cell viability was assayed using an LDH cytotoxicity detection kit (Roche, Lewes, UK). Cells were treated with 1% Triton X-100 overnight to generate 100% killing, whereas untreated cells were used as negative controls.

Mitochondria were isolated using a mitochondria isolation kit (MITO-ISO1; Sigma) following the manufacturer's recommended conditions. Calcium efflux was measured using the described Method¹³. Briefly, mitochondria (2 mg protein ml^-1) and arsenazo III (30 M) were added to medium containing mannitol (195 mM), sucrose (25 mM), Hepes (40 mM at pH 7.2), CaCl₂ (60 nmol mg protein^-1) and rotenone (13 M). After 2−3 min, potassium succinate (3.33 mM) was added. The Spectra Max plus spectrophotometer (Molecular Devices, Sunnyvale, CA) was used to measure the difference in absorbance between 675 nm and 685 nm of the calcium-sensitive dye Arsenazo III.

EcR293 clone-11 subclone-1 cells were treated with muristerone A (10 M) in the presence of the iNOS inhibitor S-methylisothiourea (100 M). After overnight incubation, cells were washed for 1 h in calcium-free DMEM to remove the inhibitor and were maintained for 1 h in L-arginine-free medium. High concentrations of NO were then generated immediately by resuspending cells in normal or calcium-free DMEM medium containing the NOS substrate L-arginine (1 mM) and, in some treatment groups, the calcium-specific ionophore A23187 (0.1 or 0.3 M, Calbiochem). After 4 h, the cells were lysed and subjected to western blot analysis.

EcR293 clone-11 subclone-1 cells were treated for three months with ethidium bromide (50 ng ml^-1) plus uridine (50 g ml^-1) in 10% fetal calf serum DMEM (containing pyruvate; Gibco, Carlsbad, CA) to deplete mitochondrial DNA¹⁰. EcR293 clone-11 subclone-1 rho⁰ cells thus produced were found to be auxotrophic for pyrimidines and pyruvate. No mitochondrial DNA was detected by real-time PCR analysis (n = 3). For quantitative PCR analysis of mtDNA, a pair of specific primers for the mitochondria gene ND2 (NADH dehydrogenase sub-unit 2) was used with the forward primer 5'-CACCCTTAATTCCATCCACC-3' and reverse primer 5'-TGGGCAAAAAGCCGGTA-3'. For quantitative PCR analysis of nuclear DNA, a pair of specific primers for 28S rDNA was used with the forward primer 5'- GAATCCGCTAAGGAGTGTGTA-3' and reverse primer 5'-CTCCAGCGCCATCCATTT-3'. The following cycling conditions were used for the subsequent PCR: 96 °C for 35 s, 56 °C for 2 min and 72 °C for 2 min for 40 cycles. Real-time PCR analysis was performed using Applied Biosystems Prism 7700 sequence detection and an SYBR Green quantiTech probe PCR kit (Qiagen, Crawley, UK). All the reactions were performed in triplicate.

Twenty-one-base RNA oligonucleotides (including a two-nucleotide 3' overhang of two deoxythymidine residues) directed against human S1P were supplied by Eurogentec (Seraing, Belgium). The siRNA sequences targeting human S1P (GenBank accession number, NM 003791) correspond to the coding regions 252−271 and 371−390 (relative to the first nucleotide of the start codon) with the following sequences: S1 P-1, 5'-AAUUGGAGAAUUAUACCUCTT-3' and 3'-TTUUAACCUCUUAAUAUGGAG-5'; S1 P-2, 5'-AACGGGUCACGCCCCAACGTT-3' and 3'-TTUUGCCCAGUGCGGGGUUGC-5'. The siRNA sequences targeting human S2P (GenBank accession number, NM 015884) correspond to the coding region sequences 306−325 and 550−569. The sequences of the two S2P siRNA duplexes used in this study were as follows: S2 P-1, 5'-AAGGAUGCUUUACCAAUGGTT-3' and 3'-TTUUCCUACGAAAUGGUUACC-5'; S2 P-2, 5'-AAUUUACCCGUCAAUCAACTT-3' and 3'-TTUUAAAUGGGCAGUUAGUUG-5'. Single-stranded siRNAs (50 M) were incubated in annealing buffer (100 mM potassium acetate, 30 mM Hepes-KOH at pH 7.4 and 2.0 mM magnesium acetate) for 1 min at 90 °C followed by 1 h at 37 °C. EcR293 clone-11 subclone-1 cells were transfected with 200 nM S1P-1 and S1P-2 siRNA duplexes per well after one day and then again after two days using oligofectamine (Life Technologies, Rockville, MD) in Opti-MEM-1 medium (Life Technologies). After two days, the cells were treated with muristerone A (10 M). After three days, thapsigargin (37 nM) was added, and cell viability assayed 48 h later using the LDH assay (Roche). S1P sense oligonucleotides and non-silencing siRNAs (Qiagen) were used as controls. A pair of PCR primers (forward primer, 5'-AACACTTGAAGATCATCCAAACAT-3'; reverse primer, 5'-TGCCAGAAGCCAGAGCCCAGGGAG-3') were derived from the human S1P sequence corresponding to 180 bp of the coding region (nucleotides 343−523; spanning exons 3 and 4). A pair of PCR primers (forward primer, 5'-AGGATGCTTTACCAATGGTTCA-3'; reverse primer, 5'-CCAAGGAGAAAAATGAGCTAAACA-3') were derived from the human S2P sequence (GenBank accession number, AF019612) corresponding to 76 bp of the coding region (nucleotides 307−383). Total RNA was isolated from cells treated with SIP siRNAs, SIP sense siRNAs, and non-silencing siRNA. First-strand cDNA was generated using random primers and reverse transcriptase. The subsequent PCR was performed using the following cycling conditions (for 30 cycles): 96 °C for 35 s, 56 °C for 2 min and 72 °C for 2 min. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; Applied Biosystems, Foster City, CA) was used as a PCR control. For detection of Grp78 mRNA, a pair of PCR primers (forward primer, 5'-CGCCAAGCGGCTCATC-3'; reverse primer, 5'-AACCACCTTGAACGGCAAGA-3') were derived from the human Grp78 sequence corresponding to 123 bp of the coding region (nucleotides 441−564; NM 005347). For detection of iNOS mRNA, a pair of PCR primers (forward primer, 5'-TCAAATCTCGGCAGAATCTACAAA-3'; reverse primer, 5'-CAGGAGAGTTCCACCAGGATG-3') were derived from the human iNOS sequence corresponding to 66 bp of the coding region (nucleotides 2357−2423; NM 153292). Real-time PCR analysis was performed as above and all reactions were performed in triplicate. The relative amounts of all mRNAs were calculated using the relative cycle threshold method (Perkin-Elmer, Norwalk, CT).

EcR293 clone-11 subclone-1 cells were transfected with a 3Flag−ATF6 plasmid containing a human ATF6 cDNA, with three copies of the Flag epitope tag at the N-terminus cloned into the p3Flag−CMV 7.1 vector²². Seven hours after transfection, whole-cell extracts were prepared by treatment with a mild lysis solution (CytoSignal, Irvine, CA) followed by three freeze-thaw cycles (-70 °C freeze; 30 °C thaw). Cell-free extracts were aliquoted and exposed to a range of calcium concentrations at 20−22 °C for 30 min. After exposure to calcium, cell-free extracts were subjected to centrifugation (16,000g; 15 min) to separate the insoluble membrane fraction from the soluble components. The soluble fraction was then subjected to immunoprecipitation with an anti-Flag-M2-specific monoclonal antibody (Sigma) using an IMMUNOcatcher kit (CytoSignal), according to the manufacturer's instructions.

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This work was supported by the Medical Research Council, UK. We thank E. A. Higgs for help in the preparation of this manuscript. We also thank R. Prywes for providing the 3Flag−ATF6 plasmid.

Competing interests statement:  The authors declare that they have no competing financial interests.