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Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 10 (2004) 112–118 www.elsevier.com/locate/yniox
Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W Neil Beeharry,a,* Julie A. Chambers,b Richard G.A. Faragher,a Kay E. Garnett,b and Irene C. Greena a
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK b AstraZeneca, CVGI, Mereside, Alderley Park, Macclesfield SK10 4TG, UK Received 22 January 2004 Available online
Abstract RNA interference has been used to silence gene expression and evaluate the contribution of a gene product to cell function. Here, we investigated conditions under which expression of an inducible protein, nitric oxide synthase 2 (NOS2), is decreased by RNA interference. Cytokine treatment of insulin-producing RINm5F cells results in NOS2 induction and cell death. Conditions used here favoured cytokine-induced apoptosis, for the first time—rather than necrosis, previously recorded. In RINm5F cells, transfected with NOS2-specific small interfering RNA followed by a 12 h exposure to cytokines, there was a significant reduction in NOS2 protein, nitrite, and apoptosis. There were no changes in these three parameters when experiments were carried out with unrelated vimentin siRNA. To interpret the NOS2-siRNA result further, we compared it with complete pharmacological inhibition of nitric oxide (NO) production by the NOS2 competitive inhibitor, 1400W, which lowered apoptosis by only 50% in the RINm5F cells. We conclude that the use of NOS2-specific siRNA has resulted in the subsequent lowering of expression of a cytokine-inducible protein whose function can be quantified. siRNA results have compared favourably with use of a pharmacological inhibitor of NOS2, in revealing the subtle, partial contribution of cytokine-induced NO to apoptosis induction in these insulin-producing cells. Ó 2004 Elsevier Inc. All rights reserved. Keywords: siRNA; Insulin; RINm5F cells; Nitric oxide synthase 2; 1400W; Cytokines
RNA interference (RNAi)1 [1] has been demonstrated to be an effective mechanism to silence gene expression of target mRNAs in a number of mammalian cell types [2,3]. This study investigates the reduction in expression of an inducible rather than a constitutive protein in an insulin-producing rat cell line. We and others [4,5] have previously studied the conditions for induction of nitric * Corresponding author. Fax: +44-1273-679333. E-mail addresses: [email protected] (N. Beeharry), [email protected] (J.A. Chambers), r.g.a.faragher@ bton. ac.uk (R.G.A. Faragher), [email protected] (K.E. Garnett), [email protected] (I.C. Green). 1 Abbreviations used: RNAi, RNA interference; siRNA, small interfering RNA; IL-1b, interleukin 1b; TNF-a, tumour necrosis factora; IFN-c, interferon-c; NOS2, nitric oxide synthase 2; 1400W, (N-(3-(aminomethyl)benzyl)acetamidine); HPI, Hoechst-propidium iodide; NO, nitric oxide.
1089-8603/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.02.003
oxide synthase 2 (NOS2) by cytokines in primary cells and cell lines. The nitric oxide (NO) dependency of cytokine effects on gene and protein expression, cell signalling, hormone secretion, and especially cell death is of interest in connection with autoimmune cell killing, with special reference to insulin-secreting b-cells in type 1 diabetes [6–9]. The present study investigates the use of RNAi as a new tool for studying the role of NO in cell protection [10,11] or cell death [12–15]. Expression of NOS2 in b-cells in the presence of arginine results in NO generation which we studied here in parallel with its contribution to cell death by apoptosis. It was shown that NO donors [16] or cytokines [17] decreased the viability of RINm5F cells, however, the type of cell death induced by cytokines was shown to be necrosis in >95% of the cells [17] and this necrosis was recorded as NO dependent, results not shown [17]. There are no previous
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reports in RINm5F cells of the contribution of cytokinederived NO to cell death by apoptosis, however in INS-1 cells using 1400W, this was shown to be approximately 50% [18]. Preliminary experiments were carried out to establish RNAi technology in insulin-secreting cell lines RINm5F and INS-1. Vimentin was chosen as a constitutive protein targeted for siRNA-mediated mRNA ablation; vimentin siRNA was successfully transfected into human fibroblasts, RINm5F cells, and INS-1 cells using conditions optimised by fluorescent siRNAs. Concomitant decreases in vimentin protein were confirmed by Western blotting and immunocytochemistry (unpublished observations N. Beeharry and K. Garnett). In the main body of the work, we investigated whether NOS2-specific siRNA could decrease expression of an inducible protein, NOS2, which is normally absent in RINm5F cells. Induction of NOS2 is achieved by treatment of cells with cytokines [19,20]. A combination of three cytokines was used here to give the highest level of induction of the protein. NOS2 is typically not seen until 4 h, so production of NO from the induced protein is for a significantly shorter time period than the whole cell cytokine treatment in culture (12 h used here). Measurable activities of NOS2 protein in generating NO and in causing apoptosis were assayed with and without siRNA transfection. Functional effects of NOS2-specific siRNA were compared with effects of a pharmacological inhibitor of NO production, 1400W (N-(3-(aminomethyl)benzyl)acetamidine).
Materials and methods RINm5F cell culture Cell culture media were from Gibco (Paisley, Scotland) and all other chemicals were from Sigma–Aldrich (Poole, Dorset, UK). RINm5F insulin-secreting cells [21] were cultured in RPMI-1640 medium with additions of 2 mmol/L L -glutamine, 50 U/ml penicillin, 50 lg/ml streptomycin, and 5% heat-inactivated foetal calf serum (FCS). In some experiments, RINm5F cells were exposed to combined cytokines: IL-1b 100 pmol/L and IFN-c 50 U/ ml (R&D Systems, Abingdon, UK) and TNF-a 100 U/ ml (AMS Biotechnology Europe, Abingdon, UK) in the presence or absence of 1400W (Alexis, Nottingham, UK). siRNA design Two 21 nucleotide siRNA duplexes were designed homologous to positions 90–109 (AACCCCAGGUGC UAUUCCC) (duplex 1) and 360–379 (GAGUUUGAC CAGAGGACCC) (duplex 2) of the rat NOS2 consen-
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sus coding sequence (GenBank Accession No. NM_012611) using EurogentecÕs on-line siRNA design software (http://www.eurogentec.com/upload/si_RNA/ design_request_form.doc). Sequence specificity was verified by submitting siRNA sequences to a BLAST similarity search for short near exact matches against the nucleotide sequence database [22]. Deprotected and desalted oligonucleotides were obtained from Oswel (Southampton, Hants, UK). The sense and antisense complementary RNA oligonucleotides were annealed according to the manufacturerÕs recommendations to form double stranded RNA, the siRNA duplex. Commercially available vimentin siRNA based on Elbashir et al. [2] was purchased from Dharmacon (Lafayette, CO, USA) and was used to optimise transfection conditions and, since it is unrelated to NOS2, served as a negative control. RINm5F cell transient transfection RINm5F cells were seeded into 12-well plates at a density of 3 105 cells/well for Western blotting, or into 48-well plates at a density of 8 104 cells/well for transfection, nitrite determination and apoptosis detection. Cells were transiently transfected using TransMessenger transfection reagent (Qiagen, Crawley, UK) for 4 h in the presence and absence of NOS2 siRNA duplex 1 or 2 (100 nmol/L), or vimentin siRNA (100 nmol/L). For transient transfection of RINm5F cells, 5 ll of Enhancer R was diluted in 90 ll Buffer EC-R. Five microlitres of siRNA duplex was added to the Enhancer R/Buffer EC-R mixture (95 ll) and incubated at room temperature for 5 min. After this time, 5 ll of TransMessenger transfection reagent was added to the siRNA/Enhancer R mixture (100 ll) and incubated at room temperature for a further 10 min, allowing transfection-complex formation. Following transfection-complex formation, serum-free 300 ll RPMI was added to the mixture (105 ll). RINm5F cells were rinsed once with sterile PBS and incubated with transfection complexes for 4 h. Following transfection, the complexes were removed from the cells and fresh RPMI (containing FCS and antibiotics) was added, allowing cells to recover before cytokine treatments. NOS2 siRNA duplex 2 was more effective than duplex 1 and was used to generate the data shown in Figs. 1–3. Determining transfection efficiency Insulin-containing cells [23] were seeded at a density of 3 105 cells/well in a 6-well plate containing coverslips. Cells were transfected with fluorescein-labelled siRNA duplex (100 nmol/L) (Dharmacon, Colorado, USA) using either Transmessenger transfection reagent (Qiagen) or Oligofectamine (Invitrogen, Paisley, UK) using conditions shown under Results. Transfected cells
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Fig. 1. Cytokine-induced NOS2 protein expression is decreased by NOS2-specific siRNAs. NOS2 protein was detected by Western blotting in extracts of RINm5F cells following exposure to combined cytokines and (A) NOS2 siRNA (100 nmol/L), (B) vimentin siRNA (100 nmol/L) or (C) a NOS2 inhibitor 1400W (200 lmol/L). In (A) and (B), NOS2 levels in untransfected cells or cells transfected with TransMessenger transfection reagent alone are also shown. The images shown are representative of three individual experiments.
were visualised using fluorescence microscopy and the percentage of transfected cells was determined. The highest rate of transfection (60–70%) was found using a ratio of 1:4 Enhancer R:TransMessenger transfection reagent. Detection of apoptotic and membrane-damaged cells by Hoechst-propidium iodide (HPI) staining Briefly, non-transfected or transfected RINm5F cells, following 12 h combined cytokine exposure, were trypsinised, washed, and stained with Hoechst 33342–Bisbenzimide H 33342 fluorochrome trihydrochloride (Calbiochem, Nottingham, UK) and propidium iodide (Sigma–Aldrich, Poole, UK). Detection was as described previously [24,25]. Apoptosis percent was determined in an average 2500 nuclei viewed for each treatment, typically eight treatments per experiment; five experiments were carried out.
Fig. 2. Cytokine-induced nitrite production is prevented by (A) NOS2 siRNA and (B) 1400W. Nitrite production was measured using the Griess assay in medium from (A) RINm5F cells transfected with TransMessenger transfection reagent alone or NOS2 siRNA (100 nmol/L) followed by 12 h exposure to cytokines. Data are expressed as nitrite accumulated (lmol/L) means SEM. N ¼ 3 independent experiments, each in duplicate. *p < 0:001, **p < 0:0001 vs. control RINm5F cells; y p < 0:005 vs. TransMessenger transfection reagent cells. (B) Nitrite production was measured as above from RINm5F cells exposed to combined cytokines 1400W for 12 h. Data are expressed as nitrite accumulated (lmol/L) means SEM. N ¼ 4 independent experiments, each in duplicate. *p < 0:0001 vs. control RINm5F cells; y p < 0:0001 vs. cytokines treated RINm5F cells.
ECL Western blotting analysis system (Amersham Biosciences, Amersham, UK). Nitrite assay Determination of nitrite accumulation in culture medium as evidence of NO production was by the Griess assay [19,27].
Western blotting
Statistical analysis
Western blotting for NOS2 was as described [25,26], using the polyclonal NOS2 antibody (Beckton–Dickinson, Oxford, UK); and detection was carried out using
Comparisons between treatments were made using StudentsÕ t test and confirmed by analysis of variance as indicated in legends.
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Ratios containing the highest amount of TransMessenger transfection reagent gave an acceptably high rate of transfection. The optimal condition used gave a clear biological effect of siRNA without transfection being toxic; higher concentrations of TransMessenger transfection reagent (>10 ll) or longer transfection times resulted in cell death (unpublished observations N. Beeharry). Cytokine-induced NOS2 protein expression is decreased by NOS2-specific siRNA
Fig. 3. Effect of decreased NOS2 expression on cytokine-induced apoptosis in RINm5F cells. (A) Apoptotic nuclei were scored following RINm5F cell culture with cytokines TransMessenger transfection reagent or NOS2 siRNA (100 nmol/L). Data are expressed as % of apoptotic nuclei (means SEM), N ¼ 5. Statistical significance was determined by ANOVA. *p < 0:05, **p < 0:01, ***p < 0:005 vs. control RINm5F cells; y p < 0:05 vs. TransMessenger transfection reagent + cytokine treated RINm5F cells. (B) Apoptotic nuclei were determined as above, after RINm5F cells were exposed to cytokines the specific NOS2 inhibitor 1400W for 12 h. Data are expressed as % apoptotic nuclei in the suspension (means SEM). Statistical significance was determined by ANOVA. *p ¼ 0:05, **p ¼ 0:005 vs. control RINm5F cells; y p < 0:05 vs. cytokine treated RINm5F cells.
To determine whether we could inhibit nitric oxide synthase protein induction through the use of NOS2specific siRNAs, we transiently transfected RINm5F cells with NOS2 siRNA (100 nmol/L) for 4 h prior to combined cytokine treatment. Following 12 h exposure to cytokines, NOS2 protein levels were determined by Western blot analysis. NOS2 protein, absent in untreated cells, was induced to a comparable level in RINm5F cells exposed to combined cytokines in the absence and presence of the TransMessenger transfection reagent (Fig. 1A). However, in RINm5F cells previously transfected with NOS2 siRNA (100 nmol/L), on exposure to combined cytokines, induced NOS2 protein expression was significantly lower (Fig. 1A). To demonstrate the specificity of cytokine-induced NOS2 expression reduction by NOS2 siRNA, RINm5F cells were transiently transfected with vimentin siRNA (100 nmol/L), an unrelated cytoskeletal protein. Fig. 1B shows that upon stimulation with cytokines, NOS2 protein expression was not decreased by the unrelated RNA. NOS2 induction was also unaffected by the selective pharmacological inhibitor 1400W (200 lmol/L), which affects protein activity rather than expression (Fig. 1C). Cytokine-induced nitrite production is prevented by NOS2 siRNA
Results Transfection efficiency Table 1 shows that the TransMessenger transfection reagent was superior to Oligofectamine in facilitating the introduction of fluorescein-labelled siRNA into cells.
To investigate a functional effect of NOS2 siRNAmediated inhibition, nitrite accumulation was measured after transfection and cytokine treatment. As expected, cytokine exposure significantly increased nitrite accumulation in RINm5F medium (p < 0:0001), and this
Table 1 Transfection efficiencies of labelled siRNA duplexes in insulin-containing cells Transfection reagent
Ratio of enhancer R:TransMessenger transfection reagent
TransMessenger transfection reagent Transfection efficiency
1:1 5%
1:2 20–30%
1:4 60–70%
4 ll <5%
6 ll <5%
Volume of Oligofectamine Oligofectamine Transfection efficiency
2 ll <5%
Insulin-containing cells were seeded at a density of 3 105 cells/well in a 6-well plate containing coverslips. Cells were transfected with fluoresceinlabelled siRNA duplex (100 nmol/L) using either Transmessenger transfection reagent or Oligofectamine for 4 h. Transfected cells were visualised using fluorescence microscopy and the percentage of transfected cells was determined.
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was comparable to that in medium from RINm5F cells exposed to TransMessenger transfection reagent alone (Fig. 2A). RINm5F cells transiently transfected with NOS2 siRNA (100 nmol/L), prior to cytokine treatment, generate significantly less nitrite (TransMessenger transfection reagent treated cells + cytokines 4.5 0.4 vs. NOS2 siRNA transfected + cytokines 2.5 0.2 lmol/ L nitrite produced, n ¼ 3; p < 0:005) (Fig. 2A). Fig. 2B shows that the NOS2-specific inhibitor, 1400W at 200 lmol/L, is effective at inhibiting cytokine-induced nitrite production (cytokines 5.5 0.5 vs. cytokines + 1400W 0.3 0.2 lmol/L nitrite produced, n ¼ 4; p < 0:001). Effect of NOS2 inhibition on cytokine-induced apoptosis Having established that by transiently transfecting RINm5F cells with NOS2 siRNA (100 nmol/L) we could reduce NOS2 protein expression and nitrite production by 50%, we sought to determine whether this inhibition was sufficient to prevent cytokine-induced apoptosis. RINm5F cells were transiently transfected with TransMessenger transfection reagent or NOS2 siRNA (100 nmol/L) prior to 12 h cytokine exposure. Apoptotic nuclei were quantified using the HPI staining methodology, in each experiment 20,000 nuclei were scored. Cytokine treatment significantly increased the percentage of apoptotic nuclei in all experiments in untransfected and TransMessenger transfection reagent treated cells. The fold effect of cytokine treatment over control was 5.4-fold and minimally, in one experiment, 2.5-fold. In RINm5F cells transfected with NOS2 siRNA compared to transfection reagent only and then exposed to cytokines, the percentage of apoptotic nuclei was reduced in each of five experiments, by an average of 35% (minimum 24%, maximum 50.2%) (Fig. 3A). The plating densities used ensured that necrosis was kept to a minimum (0.60 0.1% in control cells). There was no increase in necrosis in cytokine treated cells (p ¼ 0:09, n ¼ 9). To evaluate the NO-dependent component of cytokine-induced apoptosis, RINm5F cells were exposed to cytokines in the presence and absence of 1400W (Fig. 3B). Although the percentage of apoptotic nuclei was significantly decreased by 1400W (0.43 0.1% vs. 0.9 0.1; p < 0:05), apoptosis was still higher than in control cells (control 0.16 0.06 vs. combined cytokines + 1400W 0.43 0.1; p < 0:05), indicating that some apoptosis in RINm5F cells, even at this early time point, is independent of NO.
Discussion Following the demonstration that mammalian cells can undergo post-transcriptional gene silencing by RNAi through the introduction of sequence-specific
siRNAs [2], this technique is increasingly being used to study the function of specific genes. The gene target in many reports has been constitutively active proteins [2,3] and following RNAi experimentation, loss of product is sometimes correlated with an altered biochemical/morphological phenotype [3]. RNAi has been used successfully to knock down expression of a constitutively active neuronal isoform NOS1, which produces relatively very low amounts of NO, and acts as a signalling molecule in snail Lymnaea stagnalis [28]. To our knowledge, this is the first demonstration of the use of RNAi to ablate cytokine-induced NOS2 induction, which subsequently attenuates substantial NO production. We demonstrate that RINm5F cells treated with cytokines for 12 h show high expression of NOS2 protein and, when previously transfected with NOS2 siRNA, exhibited a significant lowering as evidenced by Western blotting. There were no changes in NOS2 expression as a result of the transfection reagent or an unrelated siRNA, vimentin, following exposure to combined cytokines. NOS2 catalyses the conversion of arginine to citrulline and NO, and under in vitro conditions NO is rapidly converted into the more stable compound nitrite. By decreasing NOS2 protein levels, we demonstrate that combined cytokine-induced nitrite produced by RINm5F cells is decreased by 50%, consistent with reduced NOS2 protein expression. Modulating levels of NO produced by NOS2 following its induction by cytokine exposure of cells can influence cell function and survival. NO is regarded as a bifunctional mediator of cell viability (reviewed in [29]); at low concentrations NO serves as a signalling molecule and is cytoprotective, e.g., in cultured hepatocytes [30], whereas at higher concentrations NO causes dysfunction, necrosis [8,17], and apoptosis [13,18], though cytokine-induced apoptosis was found to be NO independent, even after short treatment times in INS-1 cells [31]. We measured necrosis in each experiment and found it unchanged by cytokine treatment with and without TransMessenger transfection reagent, NOS2 siRNA or 1400W. This result was not unexpected given the short treatment time we used, however b-cells are subject to cytokine-induced NO-dependent necrosis after treatments lasting 3 days [17] and 9 days [8]. Pertinent to our findings of lower NOS2, protein and nitrite production in NOS2 siRNA—but not vimentin siRNA— transfected cells was a functional change in cytokineinduced apoptosis. Although the level of apoptosis detected is low, this is consistent with time needed for apoptosis signalling, the assay which detected end-stage apoptosis, and the very short exposure time to cytokines of 12 h. A brief exposure time to cytokines was chosen to see direct effects of NO and minimise secondary effects of cytokines on gene and protein expression. For
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example, manganese superoxide dismutase is induced following combined cytokine exposure [11]; it, in turn, can influence NO-induced toxicity [32]. To ensure that the effect studied was the most NO dependent, we drew on previous experience that as treatment time increases, cytokine effects may become more NO independent [33], as demonstrated in other systems [8,34]. Parallel experiments used 1400W, a specific inhibitor of NOS2 [35], to provide a comparison with use of interference RNA. Our data using 1400W verify its inhibitory effectiveness in that, although NOS2 protein is expressed in the presence of cytokines and 1400W, NO production is, unlike with NOS2 siRNAs, completely inhibited. However, the finding that 1400W is only partially protective against combined cytokineinduced apoptosis suggests a significant degree of NO-independent cell death (50%). NOS2 siRNA transfection reduced cytokine-induced apoptosis by 35% (maximally 50%), which confirms its efficacy. Our findings are consistent with data from INS-1 cells [18], where NO was shown to be only partially—approximately 50%—responsible for cytokine-induced apoptosis, but both are different to newer findings in INS-1 cells [31]. In primary b-cells from rat islets, we found that inhibition of NOS2 activity using NMMA almost completely prevented low dose cytokine-induced apoptosis after 2 days [6,13]. However in iNOS ()/)) mouse islets [8] and in human islet cells [34], cytokineinduced apoptosis measured after 9 days was shown to be independent of NO. It has been proposed that NO and ROS may synergise to induce b-cell death following treatment with cytokines [16]. ROS could be generated by cytokine treatment in two ways, one independent of NOS2 activity [36], and the other as a consequence of NOS2 generation of superoxide [37]. Both pharmacological inhibition of NOS2, using 1400W, and NOS2 siRNA would remove the NO-dependent action and the NO synergistic action of cytokines. However, 1400W alone may not, but NOS2 siRNA treatment would, prevent NOS2 generated superoxide production. In other conditions mimicking type 1 diabetes, the ability to remove NOS2 generated superoxide is valuable. Using oligonucleotide gene arrays, it was shown that in b-cells, following combined cytokine treatment, cytoprotective genes as well as genes which cause cellular dysfunction are induced [38]. The genes altered were involved in regulation of transcription factor expression, insulin synthesis and secretion, antioxidant status, glucose metabolism, and in cellular protection. The novel approach presented here in attenuating the effects of pro-inflammatory cytokines and maintaining cell viability by using NOS2 siRNA demonstrates that this methodology offers the possibility of identifying key regulators which, as in the case of NOS2, may be induced and mediate some cytokine effects.
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Acknowledgments This work was supported in part by the Biotechnology and Biological Sciences Research Council (BBSRC) to R.G.A.F. and the BBSRC and AstraZeneca plc through industrial CASE studentships to N.B. and K.E.G. We are very grateful to Katrin Jennert-Burston for advice on siRNA design and critical reading of the manuscript.
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[26] V.D. Belin, J.G. Mabley, R.F.L. James, S.M. Swift, H.A. Clayton, M.A. Titheradge, I.C. Green, Glucagon decreases cytokine induction of nitric oxide synthase and action on insulin secretion in RIN5F cells and rat and human islets of Langerhans, Cytokine 11 (1999) 585–592. [27] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite and [15 N]nitrate in biological fluids, Anal. Biochem. 126 (1982) 131– 138. [28] S.A. Korneev, I. Kemenes, V. Straub, K. Staras, E.I. Korneeva, G. Kemenes, P.R. Benjamin, O.S.M., Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene, J. Neurosci. 22 (2002) RC227 (1–5). [29] Y.-M. Kim, C.A. Bombeck, T.R. Billiar, Nitric oxide as a bifunctional regulator of apoptosis, Circ. Res. 84 (1999) 253–256. [30] J.R. Li, C.A. Bombeck, S.F. Yang, Y.M. Kim, T.R. Billiar, Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes, J. Biol. Chem. 274 (1999) 17325–17333. [31] B. Kutlu, A.K. Cardozo, M.I. Darville, M. Kruhoffer, N. Magnusson, T. Orntoft, D.L. Eizirik, Discovery of gene networks regulating cytokine-induced dysfunction and apoptosis in insulinproducing INS-1 cells, Diabetes 52 (2003) 2701–2719. [32] A.K. Azevedo-Martins, S. Lortz, S. Lenzen, R. Curi, D.L. Eizirik, M. Tiedge, Improvement of the mitochondrial antioxidant defense status prevents cytokine-induced nuclear factor-kappa B activation in insulin-producing cells, Diabetes 52 (2003) 93–101. [33] A.M. Dunger, J. Cunningham, C.A. Delaney, J.E. Lowe, M.H.L. Green, A.J. Bone, I.C. Green, Tumour necrosis factor-a and interferon-c inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement, Diabetes 45 (1996) 183–189. [34] C.A. Delaney, D. Pavlovic, A. Hoorens, D.G. Pipeleers, D.L. Eizirik, Cytokines induce deoxyribonucleic acid strand breaks and apoptosis in human pancreatic islet cells, Endocrinology 138 (1997) 2610–2614. [35] E.P. Garvey, J.A. Oplinger, E.S. Furfine, R.J. Kiff, F. Laszlo, B.J.R. Whittle, R.G. Knowles, 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo, J. Biol. Chem. 272 (1997) 4959–4963. [36] W.L. Suarez-Pinzon, K. Strynadka, A. Rabinovitch, Destruction of rat pancreatic islet B-cells by cytokines involves the production of cytoxic aldehydes, Endocrinology 137 (1996) 5290–5296. [37] Y. Xia, J.L. Zweier, Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages, Proc. Natl. Acad. Sci. USA 94 (1997) 6954–6958. [38] A.K. Cardozo, M. Kruhøffer, R. Leeman, T. Ørntoft, D.L. Eizirik, Identification of novel cytokine-induced genes in pancreatic b-cells by high-density oligonucleotide arrays, Diabetes 50 (2001) 909–920.
Biology and Chemistry
Nitric Oxide 10 (2004) 112–118 www.elsevier.com/locate/yniox
Analysis of cytokine-induced NO-dependent apoptosis using RNA interference or inhibition by 1400W Neil Beeharry,a,* Julie A. Chambers,b Richard G.A. Faragher,a Kay E. Garnett,b and Irene C. Greena a
School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Lewes Road, Brighton BN2 4GJ, UK b AstraZeneca, CVGI, Mereside, Alderley Park, Macclesfield SK10 4TG, UK Received 22 January 2004 Available online
Abstract RNA interference has been used to silence gene expression and evaluate the contribution of a gene product to cell function. Here, we investigated conditions under which expression of an inducible protein, nitric oxide synthase 2 (NOS2), is decreased by RNA interference. Cytokine treatment of insulin-producing RINm5F cells results in NOS2 induction and cell death. Conditions used here favoured cytokine-induced apoptosis, for the first time—rather than necrosis, previously recorded. In RINm5F cells, transfected with NOS2-specific small interfering RNA followed by a 12 h exposure to cytokines, there was a significant reduction in NOS2 protein, nitrite, and apoptosis. There were no changes in these three parameters when experiments were carried out with unrelated vimentin siRNA. To interpret the NOS2-siRNA result further, we compared it with complete pharmacological inhibition of nitric oxide (NO) production by the NOS2 competitive inhibitor, 1400W, which lowered apoptosis by only 50% in the RINm5F cells. We conclude that the use of NOS2-specific siRNA has resulted in the subsequent lowering of expression of a cytokine-inducible protein whose function can be quantified. siRNA results have compared favourably with use of a pharmacological inhibitor of NOS2, in revealing the subtle, partial contribution of cytokine-induced NO to apoptosis induction in these insulin-producing cells. Ó 2004 Elsevier Inc. All rights reserved. Keywords: siRNA; Insulin; RINm5F cells; Nitric oxide synthase 2; 1400W; Cytokines
RNA interference (RNAi)1 [1] has been demonstrated to be an effective mechanism to silence gene expression of target mRNAs in a number of mammalian cell types [2,3]. This study investigates the reduction in expression of an inducible rather than a constitutive protein in an insulin-producing rat cell line. We and others [4,5] have previously studied the conditions for induction of nitric * Corresponding author. Fax: +44-1273-679333. E-mail addresses: [email protected] (N. Beeharry), [email protected] (J.A. Chambers), r.g.a.faragher@ bton. ac.uk (R.G.A. Faragher), [email protected] (K.E. Garnett), [email protected] (I.C. Green). 1 Abbreviations used: RNAi, RNA interference; siRNA, small interfering RNA; IL-1b, interleukin 1b; TNF-a, tumour necrosis factora; IFN-c, interferon-c; NOS2, nitric oxide synthase 2; 1400W, (N-(3-(aminomethyl)benzyl)acetamidine); HPI, Hoechst-propidium iodide; NO, nitric oxide.
1089-8603/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.02.003
oxide synthase 2 (NOS2) by cytokines in primary cells and cell lines. The nitric oxide (NO) dependency of cytokine effects on gene and protein expression, cell signalling, hormone secretion, and especially cell death is of interest in connection with autoimmune cell killing, with special reference to insulin-secreting b-cells in type 1 diabetes [6–9]. The present study investigates the use of RNAi as a new tool for studying the role of NO in cell protection [10,11] or cell death [12–15]. Expression of NOS2 in b-cells in the presence of arginine results in NO generation which we studied here in parallel with its contribution to cell death by apoptosis. It was shown that NO donors [16] or cytokines [17] decreased the viability of RINm5F cells, however, the type of cell death induced by cytokines was shown to be necrosis in >95% of the cells [17] and this necrosis was recorded as NO dependent, results not shown [17]. There are no previous
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reports in RINm5F cells of the contribution of cytokinederived NO to cell death by apoptosis, however in INS-1 cells using 1400W, this was shown to be approximately 50% [18]. Preliminary experiments were carried out to establish RNAi technology in insulin-secreting cell lines RINm5F and INS-1. Vimentin was chosen as a constitutive protein targeted for siRNA-mediated mRNA ablation; vimentin siRNA was successfully transfected into human fibroblasts, RINm5F cells, and INS-1 cells using conditions optimised by fluorescent siRNAs. Concomitant decreases in vimentin protein were confirmed by Western blotting and immunocytochemistry (unpublished observations N. Beeharry and K. Garnett). In the main body of the work, we investigated whether NOS2-specific siRNA could decrease expression of an inducible protein, NOS2, which is normally absent in RINm5F cells. Induction of NOS2 is achieved by treatment of cells with cytokines [19,20]. A combination of three cytokines was used here to give the highest level of induction of the protein. NOS2 is typically not seen until 4 h, so production of NO from the induced protein is for a significantly shorter time period than the whole cell cytokine treatment in culture (12 h used here). Measurable activities of NOS2 protein in generating NO and in causing apoptosis were assayed with and without siRNA transfection. Functional effects of NOS2-specific siRNA were compared with effects of a pharmacological inhibitor of NO production, 1400W (N-(3-(aminomethyl)benzyl)acetamidine).
Materials and methods RINm5F cell culture Cell culture media were from Gibco (Paisley, Scotland) and all other chemicals were from Sigma–Aldrich (Poole, Dorset, UK). RINm5F insulin-secreting cells [21] were cultured in RPMI-1640 medium with additions of 2 mmol/L L -glutamine, 50 U/ml penicillin, 50 lg/ml streptomycin, and 5% heat-inactivated foetal calf serum (FCS). In some experiments, RINm5F cells were exposed to combined cytokines: IL-1b 100 pmol/L and IFN-c 50 U/ ml (R&D Systems, Abingdon, UK) and TNF-a 100 U/ ml (AMS Biotechnology Europe, Abingdon, UK) in the presence or absence of 1400W (Alexis, Nottingham, UK). siRNA design Two 21 nucleotide siRNA duplexes were designed homologous to positions 90–109 (AACCCCAGGUGC UAUUCCC) (duplex 1) and 360–379 (GAGUUUGAC CAGAGGACCC) (duplex 2) of the rat NOS2 consen-
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sus coding sequence (GenBank Accession No. NM_012611) using EurogentecÕs on-line siRNA design software (http://www.eurogentec.com/upload/si_RNA/ design_request_form.doc). Sequence specificity was verified by submitting siRNA sequences to a BLAST similarity search for short near exact matches against the nucleotide sequence database [22]. Deprotected and desalted oligonucleotides were obtained from Oswel (Southampton, Hants, UK). The sense and antisense complementary RNA oligonucleotides were annealed according to the manufacturerÕs recommendations to form double stranded RNA, the siRNA duplex. Commercially available vimentin siRNA based on Elbashir et al. [2] was purchased from Dharmacon (Lafayette, CO, USA) and was used to optimise transfection conditions and, since it is unrelated to NOS2, served as a negative control. RINm5F cell transient transfection RINm5F cells were seeded into 12-well plates at a density of 3 105 cells/well for Western blotting, or into 48-well plates at a density of 8 104 cells/well for transfection, nitrite determination and apoptosis detection. Cells were transiently transfected using TransMessenger transfection reagent (Qiagen, Crawley, UK) for 4 h in the presence and absence of NOS2 siRNA duplex 1 or 2 (100 nmol/L), or vimentin siRNA (100 nmol/L). For transient transfection of RINm5F cells, 5 ll of Enhancer R was diluted in 90 ll Buffer EC-R. Five microlitres of siRNA duplex was added to the Enhancer R/Buffer EC-R mixture (95 ll) and incubated at room temperature for 5 min. After this time, 5 ll of TransMessenger transfection reagent was added to the siRNA/Enhancer R mixture (100 ll) and incubated at room temperature for a further 10 min, allowing transfection-complex formation. Following transfection-complex formation, serum-free 300 ll RPMI was added to the mixture (105 ll). RINm5F cells were rinsed once with sterile PBS and incubated with transfection complexes for 4 h. Following transfection, the complexes were removed from the cells and fresh RPMI (containing FCS and antibiotics) was added, allowing cells to recover before cytokine treatments. NOS2 siRNA duplex 2 was more effective than duplex 1 and was used to generate the data shown in Figs. 1–3. Determining transfection efficiency Insulin-containing cells [23] were seeded at a density of 3 105 cells/well in a 6-well plate containing coverslips. Cells were transfected with fluorescein-labelled siRNA duplex (100 nmol/L) (Dharmacon, Colorado, USA) using either Transmessenger transfection reagent (Qiagen) or Oligofectamine (Invitrogen, Paisley, UK) using conditions shown under Results. Transfected cells
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Fig. 1. Cytokine-induced NOS2 protein expression is decreased by NOS2-specific siRNAs. NOS2 protein was detected by Western blotting in extracts of RINm5F cells following exposure to combined cytokines and (A) NOS2 siRNA (100 nmol/L), (B) vimentin siRNA (100 nmol/L) or (C) a NOS2 inhibitor 1400W (200 lmol/L). In (A) and (B), NOS2 levels in untransfected cells or cells transfected with TransMessenger transfection reagent alone are also shown. The images shown are representative of three individual experiments.
were visualised using fluorescence microscopy and the percentage of transfected cells was determined. The highest rate of transfection (60–70%) was found using a ratio of 1:4 Enhancer R:TransMessenger transfection reagent. Detection of apoptotic and membrane-damaged cells by Hoechst-propidium iodide (HPI) staining Briefly, non-transfected or transfected RINm5F cells, following 12 h combined cytokine exposure, were trypsinised, washed, and stained with Hoechst 33342–Bisbenzimide H 33342 fluorochrome trihydrochloride (Calbiochem, Nottingham, UK) and propidium iodide (Sigma–Aldrich, Poole, UK). Detection was as described previously [24,25]. Apoptosis percent was determined in an average 2500 nuclei viewed for each treatment, typically eight treatments per experiment; five experiments were carried out.
Fig. 2. Cytokine-induced nitrite production is prevented by (A) NOS2 siRNA and (B) 1400W. Nitrite production was measured using the Griess assay in medium from (A) RINm5F cells transfected with TransMessenger transfection reagent alone or NOS2 siRNA (100 nmol/L) followed by 12 h exposure to cytokines. Data are expressed as nitrite accumulated (lmol/L) means SEM. N ¼ 3 independent experiments, each in duplicate. *p < 0:001, **p < 0:0001 vs. control RINm5F cells; y p < 0:005 vs. TransMessenger transfection reagent cells. (B) Nitrite production was measured as above from RINm5F cells exposed to combined cytokines 1400W for 12 h. Data are expressed as nitrite accumulated (lmol/L) means SEM. N ¼ 4 independent experiments, each in duplicate. *p < 0:0001 vs. control RINm5F cells; y p < 0:0001 vs. cytokines treated RINm5F cells.
ECL Western blotting analysis system (Amersham Biosciences, Amersham, UK). Nitrite assay Determination of nitrite accumulation in culture medium as evidence of NO production was by the Griess assay [19,27].
Western blotting
Statistical analysis
Western blotting for NOS2 was as described [25,26], using the polyclonal NOS2 antibody (Beckton–Dickinson, Oxford, UK); and detection was carried out using
Comparisons between treatments were made using StudentsÕ t test and confirmed by analysis of variance as indicated in legends.
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Ratios containing the highest amount of TransMessenger transfection reagent gave an acceptably high rate of transfection. The optimal condition used gave a clear biological effect of siRNA without transfection being toxic; higher concentrations of TransMessenger transfection reagent (>10 ll) or longer transfection times resulted in cell death (unpublished observations N. Beeharry). Cytokine-induced NOS2 protein expression is decreased by NOS2-specific siRNA
Fig. 3. Effect of decreased NOS2 expression on cytokine-induced apoptosis in RINm5F cells. (A) Apoptotic nuclei were scored following RINm5F cell culture with cytokines TransMessenger transfection reagent or NOS2 siRNA (100 nmol/L). Data are expressed as % of apoptotic nuclei (means SEM), N ¼ 5. Statistical significance was determined by ANOVA. *p < 0:05, **p < 0:01, ***p < 0:005 vs. control RINm5F cells; y p < 0:05 vs. TransMessenger transfection reagent + cytokine treated RINm5F cells. (B) Apoptotic nuclei were determined as above, after RINm5F cells were exposed to cytokines the specific NOS2 inhibitor 1400W for 12 h. Data are expressed as % apoptotic nuclei in the suspension (means SEM). Statistical significance was determined by ANOVA. *p ¼ 0:05, **p ¼ 0:005 vs. control RINm5F cells; y p < 0:05 vs. cytokine treated RINm5F cells.
To determine whether we could inhibit nitric oxide synthase protein induction through the use of NOS2specific siRNAs, we transiently transfected RINm5F cells with NOS2 siRNA (100 nmol/L) for 4 h prior to combined cytokine treatment. Following 12 h exposure to cytokines, NOS2 protein levels were determined by Western blot analysis. NOS2 protein, absent in untreated cells, was induced to a comparable level in RINm5F cells exposed to combined cytokines in the absence and presence of the TransMessenger transfection reagent (Fig. 1A). However, in RINm5F cells previously transfected with NOS2 siRNA (100 nmol/L), on exposure to combined cytokines, induced NOS2 protein expression was significantly lower (Fig. 1A). To demonstrate the specificity of cytokine-induced NOS2 expression reduction by NOS2 siRNA, RINm5F cells were transiently transfected with vimentin siRNA (100 nmol/L), an unrelated cytoskeletal protein. Fig. 1B shows that upon stimulation with cytokines, NOS2 protein expression was not decreased by the unrelated RNA. NOS2 induction was also unaffected by the selective pharmacological inhibitor 1400W (200 lmol/L), which affects protein activity rather than expression (Fig. 1C). Cytokine-induced nitrite production is prevented by NOS2 siRNA
Results Transfection efficiency Table 1 shows that the TransMessenger transfection reagent was superior to Oligofectamine in facilitating the introduction of fluorescein-labelled siRNA into cells.
To investigate a functional effect of NOS2 siRNAmediated inhibition, nitrite accumulation was measured after transfection and cytokine treatment. As expected, cytokine exposure significantly increased nitrite accumulation in RINm5F medium (p < 0:0001), and this
Table 1 Transfection efficiencies of labelled siRNA duplexes in insulin-containing cells Transfection reagent
Ratio of enhancer R:TransMessenger transfection reagent
TransMessenger transfection reagent Transfection efficiency
1:1 5%
1:2 20–30%
1:4 60–70%
4 ll <5%
6 ll <5%
Volume of Oligofectamine Oligofectamine Transfection efficiency
2 ll <5%
Insulin-containing cells were seeded at a density of 3 105 cells/well in a 6-well plate containing coverslips. Cells were transfected with fluoresceinlabelled siRNA duplex (100 nmol/L) using either Transmessenger transfection reagent or Oligofectamine for 4 h. Transfected cells were visualised using fluorescence microscopy and the percentage of transfected cells was determined.
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was comparable to that in medium from RINm5F cells exposed to TransMessenger transfection reagent alone (Fig. 2A). RINm5F cells transiently transfected with NOS2 siRNA (100 nmol/L), prior to cytokine treatment, generate significantly less nitrite (TransMessenger transfection reagent treated cells + cytokines 4.5 0.4 vs. NOS2 siRNA transfected + cytokines 2.5 0.2 lmol/ L nitrite produced, n ¼ 3; p < 0:005) (Fig. 2A). Fig. 2B shows that the NOS2-specific inhibitor, 1400W at 200 lmol/L, is effective at inhibiting cytokine-induced nitrite production (cytokines 5.5 0.5 vs. cytokines + 1400W 0.3 0.2 lmol/L nitrite produced, n ¼ 4; p < 0:001). Effect of NOS2 inhibition on cytokine-induced apoptosis Having established that by transiently transfecting RINm5F cells with NOS2 siRNA (100 nmol/L) we could reduce NOS2 protein expression and nitrite production by 50%, we sought to determine whether this inhibition was sufficient to prevent cytokine-induced apoptosis. RINm5F cells were transiently transfected with TransMessenger transfection reagent or NOS2 siRNA (100 nmol/L) prior to 12 h cytokine exposure. Apoptotic nuclei were quantified using the HPI staining methodology, in each experiment 20,000 nuclei were scored. Cytokine treatment significantly increased the percentage of apoptotic nuclei in all experiments in untransfected and TransMessenger transfection reagent treated cells. The fold effect of cytokine treatment over control was 5.4-fold and minimally, in one experiment, 2.5-fold. In RINm5F cells transfected with NOS2 siRNA compared to transfection reagent only and then exposed to cytokines, the percentage of apoptotic nuclei was reduced in each of five experiments, by an average of 35% (minimum 24%, maximum 50.2%) (Fig. 3A). The plating densities used ensured that necrosis was kept to a minimum (0.60 0.1% in control cells). There was no increase in necrosis in cytokine treated cells (p ¼ 0:09, n ¼ 9). To evaluate the NO-dependent component of cytokine-induced apoptosis, RINm5F cells were exposed to cytokines in the presence and absence of 1400W (Fig. 3B). Although the percentage of apoptotic nuclei was significantly decreased by 1400W (0.43 0.1% vs. 0.9 0.1; p < 0:05), apoptosis was still higher than in control cells (control 0.16 0.06 vs. combined cytokines + 1400W 0.43 0.1; p < 0:05), indicating that some apoptosis in RINm5F cells, even at this early time point, is independent of NO.
Discussion Following the demonstration that mammalian cells can undergo post-transcriptional gene silencing by RNAi through the introduction of sequence-specific
siRNAs [2], this technique is increasingly being used to study the function of specific genes. The gene target in many reports has been constitutively active proteins [2,3] and following RNAi experimentation, loss of product is sometimes correlated with an altered biochemical/morphological phenotype [3]. RNAi has been used successfully to knock down expression of a constitutively active neuronal isoform NOS1, which produces relatively very low amounts of NO, and acts as a signalling molecule in snail Lymnaea stagnalis [28]. To our knowledge, this is the first demonstration of the use of RNAi to ablate cytokine-induced NOS2 induction, which subsequently attenuates substantial NO production. We demonstrate that RINm5F cells treated with cytokines for 12 h show high expression of NOS2 protein and, when previously transfected with NOS2 siRNA, exhibited a significant lowering as evidenced by Western blotting. There were no changes in NOS2 expression as a result of the transfection reagent or an unrelated siRNA, vimentin, following exposure to combined cytokines. NOS2 catalyses the conversion of arginine to citrulline and NO, and under in vitro conditions NO is rapidly converted into the more stable compound nitrite. By decreasing NOS2 protein levels, we demonstrate that combined cytokine-induced nitrite produced by RINm5F cells is decreased by 50%, consistent with reduced NOS2 protein expression. Modulating levels of NO produced by NOS2 following its induction by cytokine exposure of cells can influence cell function and survival. NO is regarded as a bifunctional mediator of cell viability (reviewed in [29]); at low concentrations NO serves as a signalling molecule and is cytoprotective, e.g., in cultured hepatocytes [30], whereas at higher concentrations NO causes dysfunction, necrosis [8,17], and apoptosis [13,18], though cytokine-induced apoptosis was found to be NO independent, even after short treatment times in INS-1 cells [31]. We measured necrosis in each experiment and found it unchanged by cytokine treatment with and without TransMessenger transfection reagent, NOS2 siRNA or 1400W. This result was not unexpected given the short treatment time we used, however b-cells are subject to cytokine-induced NO-dependent necrosis after treatments lasting 3 days [17] and 9 days [8]. Pertinent to our findings of lower NOS2, protein and nitrite production in NOS2 siRNA—but not vimentin siRNA— transfected cells was a functional change in cytokineinduced apoptosis. Although the level of apoptosis detected is low, this is consistent with time needed for apoptosis signalling, the assay which detected end-stage apoptosis, and the very short exposure time to cytokines of 12 h. A brief exposure time to cytokines was chosen to see direct effects of NO and minimise secondary effects of cytokines on gene and protein expression. For
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example, manganese superoxide dismutase is induced following combined cytokine exposure [11]; it, in turn, can influence NO-induced toxicity [32]. To ensure that the effect studied was the most NO dependent, we drew on previous experience that as treatment time increases, cytokine effects may become more NO independent [33], as demonstrated in other systems [8,34]. Parallel experiments used 1400W, a specific inhibitor of NOS2 [35], to provide a comparison with use of interference RNA. Our data using 1400W verify its inhibitory effectiveness in that, although NOS2 protein is expressed in the presence of cytokines and 1400W, NO production is, unlike with NOS2 siRNAs, completely inhibited. However, the finding that 1400W is only partially protective against combined cytokineinduced apoptosis suggests a significant degree of NO-independent cell death (50%). NOS2 siRNA transfection reduced cytokine-induced apoptosis by 35% (maximally 50%), which confirms its efficacy. Our findings are consistent with data from INS-1 cells [18], where NO was shown to be only partially—approximately 50%—responsible for cytokine-induced apoptosis, but both are different to newer findings in INS-1 cells [31]. In primary b-cells from rat islets, we found that inhibition of NOS2 activity using NMMA almost completely prevented low dose cytokine-induced apoptosis after 2 days [6,13]. However in iNOS ()/)) mouse islets [8] and in human islet cells [34], cytokineinduced apoptosis measured after 9 days was shown to be independent of NO. It has been proposed that NO and ROS may synergise to induce b-cell death following treatment with cytokines [16]. ROS could be generated by cytokine treatment in two ways, one independent of NOS2 activity [36], and the other as a consequence of NOS2 generation of superoxide [37]. Both pharmacological inhibition of NOS2, using 1400W, and NOS2 siRNA would remove the NO-dependent action and the NO synergistic action of cytokines. However, 1400W alone may not, but NOS2 siRNA treatment would, prevent NOS2 generated superoxide production. In other conditions mimicking type 1 diabetes, the ability to remove NOS2 generated superoxide is valuable. Using oligonucleotide gene arrays, it was shown that in b-cells, following combined cytokine treatment, cytoprotective genes as well as genes which cause cellular dysfunction are induced [38]. The genes altered were involved in regulation of transcription factor expression, insulin synthesis and secretion, antioxidant status, glucose metabolism, and in cellular protection. The novel approach presented here in attenuating the effects of pro-inflammatory cytokines and maintaining cell viability by using NOS2 siRNA demonstrates that this methodology offers the possibility of identifying key regulators which, as in the case of NOS2, may be induced and mediate some cytokine effects.
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Acknowledgments This work was supported in part by the Biotechnology and Biological Sciences Research Council (BBSRC) to R.G.A.F. and the BBSRC and AstraZeneca plc through industrial CASE studentships to N.B. and K.E.G. We are very grateful to Katrin Jennert-Burston for advice on siRNA design and critical reading of the manuscript.
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