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The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes Background: Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods: Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFa, IFNg, and IL-1b) or by LPS plus IFNg. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results: In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion: The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies. Background neurotoxic compounds, like nitric oxide (NO). NO, one Astrocyte activation has been implicated in the patho- of the smallest known bioactive products of mammalian genesis of several neurological conditions, such as neu- cells, is biosynthesized by three distinct isoforms of NO synthase (NOS): the constitutively expressed neuronal rodegenerative diseases, infections, trauma, and ischemia. Reactive astrocytes are capable of producing a (n)NOS and endothelial (e)NOS, and the inducible (i) variety of pro-inflammatory mediators, including inter- NOS [2]. The expression of iNOS can be induced in dif- leukin-6 (IL-6), IL-1b, tumor necrosis factor-a (TNF-a), ferent cell types and tissues by exposure to immunologi- neurotrophic factors [1], as well as potentially cal and inflammatory stimuli [3]. In vitro, primary astrocyte cultures express iNOS in response to cytokines * Correspondence: [email protected] such as IL-1b [4], interferon g (IFNg), TNFa and/or the Istitute of Pharmacology, Catholic Medical School, Rome, Italy bacterial endotoxin, lipopolysaccharide (LPS) [5,6]. Once Full list of author information is available at the end of the article © 2011 Lisi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 2 of 11 http://www.jneuroinflammation.com/content/8/1/1 induced, iNOS leads to continuous NO production, and activity in microglial cultures activated by pro- which is terminated by enzyme degradation, depletion inflammatory cytokines while displaying minor effects of substrates, or cell death [7]. iNOS activity generates on astrocyte iNOS [21], thus suggesting that mTOR reg- large amounts of NO (within the μMrange)thatcan ulates glial inflammatory activation but with selective have antimicrobial, anti-atherogenic, or apoptotic effects. However, with respect to iNOS expression, both actions [8]. However, aberrant iNOS induction exerts stimulatory and inhibitory actions involving the mTOR pathway have been described. In particular, rapamycin detrimental effects and seems to be involved in the has been found to down-regulate LPS-induced iNOS pathophysiology of several human diseases [9,10]. protein expression in the mouse macrophage RAW Consistently, the expression of iNOS is tightly regu- lated by complex molecular mechanisms, involving both 264.7 cell line via mTOR dependent proteasomal activa- transcriptional and post-transcriptional processes [2]. At tion [22]. In contrast, TNF-a increases iNOS expression the post-transcriptional level an important mechanism and NO production in myoblasts via the activation of of regulation is the modulation of iNOS mRNA stability the ILK/Akt/mTOR and NFkB signaling pathway [23]; that is controlled by several RNA binding proteins and ultra-sound stimulationup-regulatesiNOSexpres- (RNA-BPs) [11]. These proteins bind to the iNOS sion by an HIF-1a-dependent mechanism involving the mRNA and allow its interaction with the exosome, the activation of ILK/Akt and mTOR pathways via integrin mRNA degrading machinery [2]. Interestingly, the mam- receptor in osteoblasts [24]. Thus, mTOR differentially malian target of rapamycin (mTOR) kinase modulates regulates iNOS activity and expression depending on theactivityofsomeoftheabovementioned RNA-BPs the cell type or activating stimulus, and this may explain [12,13] mTOR is a serine-threonine kinase that plays an the different effects that we observed on glial iNOS [21]. evolutionary conserved role in the regulation of cell In our previous studies, we observed that although growth, proliferation, survival, and metabolism, as well rapamycin reduced iNOS expression mRNA and activity as of other physiological processes such as transcription, in microglial cells, and was without effect on astrocyte mRNA turnover and protein translation [14]. Within iNOS activity [21], it caused a rapid significant increase the cells, mTOR can exist in at least two distinct com- in iNOS mRNA levels in astrocytes induced by two dif- plexes together with different partners, mTORC1 and ferent proinflammatory stimuli. Later time points were mTORC2. The mTORC1 consists of the regulatory- not examined; neither was the basis for this contrasting associated protein of mTOR, Raptor, and the adaptor result examined. In the present paper we tested the hypothesis that while at early times rapamycin increases protein mLST8/GbL(Gprotein b-subunit-like protein), iNOS mRNA, at later times it modifies iNOS mRNA and regulates several functions related to cell cycle and growth. The mTORC2 includes mLST8, the adaptor stability. Our results using primary rat astrocytes are protein Rictor, and Sin1 [15]. mTORC2 is thought to consistent with this hypothesis, and suggest that inhibi- regulate the actin cytoskeleton dynamics [16]. Indeed, tion of mTOR kinase activity in glial cells results in rapamycin is a second generation immunosuppressant anti-inflammatory actions. Together with the marked drug that blocks T-cell proliferation by inhibition of anti-inflammatory effects observed in microglial cells mTOR activity, and it is normally used to prevent trans- [21], these data further provide pre-clinical evidence for plant rejection in association with the older calcineurin a possible clinical use of mTOR inhibitors in the treat- inhibitors [17] mTORC1 activity is inhibited by rapamy- ment of inflammatory-based CNS pathologies. cin and its analogs, while mTORC2 is insensitive to the rapamycin inhibitory actions at least at immunosuppres- Methods sive concentrations [18]. Materials mTOR is also a key regulator of intracellular processes Cell culture reagents [Dulbecco’s modified Eagle’smed- in glial cells, in fact various mTOR upstream regulators ium (DMEM), DMEM-F12 and Fetal calf serum (FCS)] have been reported to play an important role in astro- were from Invitrogen Corporation (Paisley, Scotland). cyte physiology. For example, inactivation of a negative Antibiotics were from Biochrom AG (Berlin, Germany). regulator of the mTOR pathway, the tumor suppressor Bacterial endotoxin LPS (Salmonella typhimurium) was PTEN, promotes astrocyte hypertrophy and proliferation from Sigma-Aldrich (St. Louis, MO, USA). Recombinant [14]; inactivation of the tumor suppressor tuberin leads pro-inflammatory cytokines, namely human tumor to glial cell hypertrophy and formation of glial hamarto- necrosis factor a (TNFa), human interleukin-1b (IL-1b) mas [14]; up-regulation of mTOR signaling regulates and rat interferon-g (IFNg) were purchased from Endo- glutamate transporter 1 [19]; and down-regulation of gen (Pierce Biotechnology, Rockford, IL, USA). Rapamy- mTOR/S6 kinase pathway contributes to astrocyte survi- cin (RAPA) was purchased from Tocris Bioscience val during ischemia [20]. We recently showed that rapa- (Bristol, UK). b-actin (clone AC-74) mouse monoclonal mycin and its analog, RAD001, reduce iNOS expression antibody was from Sigma Aldrich; rabbit polyclonal anti- Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 3 of 11 http://www.jneuroinflammation.com/content/8/1/1 phospho [ser-2448] mTOR was purchased from Novus [26] were used to monitor effects of rapamycin on acti- Biological (Littleton, CO, USA), mouse monoclonal vation of the iNOS promoter. These cells have a low iNOS antibody was from Santa Cruz (Santa Cruz, CA). level of basal luciferase activity, which can be induced between4-and 10-folduponincubationwith LPS plus Cell cultures IFNg or with TII. C6-2.2. cells were incubated with the Primary cultures of rat cortical astrocytes were prepared indicated iNOS inducers in DMEM containing 1% FCS as previously described [25]. In brief, 1- to 2-day-old and the indicated concentrations of rapamycin. After Wistar rats were sacrificed. The brains were removed desired incubation times, the media were removed, and under aseptic conditions and placed in phosphate buffer the cells were washed once with cold phosphate-buf- ++ ++ saline with Ca and Mg (PBS-w), containing antibio- fered saline. To prepare lysates, 50 μlofCHAPS buffer tics (100 IU/ml of penicillin plus 100 μg/ml streptomy- (10 mM CHAPS, 10 mM Tris, pH 7.4) were used. Ali- cin). Under a stereomicroscope, the meninges were quots of cell lysates (40 μl) were placed into wells of an carefully removed and the cortex was dissected. The tis- opaque white 96-well microplate. A volume of luciferase sue was cut into small fragments, digested with trypsin substrate (20 μl) (Steady Glo reagent, Promega) was ++ ++ in PBS without Ca and Mg (PBS-wo), for 25 min at added to all samples, and the luminescence was mea- 37°C and for further 5 min in the presence of DNAse I. sured in a microplate luminometer (Rosys-Anthos, Aus- This step was followed by mechanically dissociation in tria). The data are presented as the percentage of Dulbecco’s MEM with Glutamax-I containing 10% fetal luciferase activity measured in the presence of NOS2 calf serum (FCS) and antibiotics as above, to obtain sin- inducers and rapamycin, relative to the activity of con- gle cells. Cell viability was roughly 45%. trol cells (incubated in media with TII). The cells thus obtained were seeded in 75-cm flasks at adensity of1×10 cells/10 ml (1 brain/flask) and incu- iNOS mRNA analysis in real time PCR bated at 37°C in a humidified atmosphere containing 5% Total cytoplasmic RNA was extracted from astrocytes CO2. The medium was changed after 24 h and then twice using TRIZOL (Invitrogen). RNA concentration was a week. Astrocytes obtained with this procedure were then measured using the Quant-iT™ RiboGreen RNA Assay subcultured twice, the first time in 75-cm flasks and the Kit (Invitrogen). In each assay, a standard curve in the second time directly in multiwell plates used for the range of 0-100 ng RNA was run using 16S and 23S experimental procedures. All the experiments were carried ribosomal RNA (rRNA) from E. coli as standard. Ali- out in 1% FCS DMEM with antibiotics. quots (1 μg) of RNA were converted to cDNA using C6 glioma cells and stable transfected C6 cells (see random hexamer primers and the ImProm-II Reverse below) were grown in DMEM containing 10% fetal calf Transcriptase (Promega, Madison, WI, USA). Quantita- serum and antibiotics, including G418. Cells were tive changes in mRNA levels were estimated by real- passed once a week and used for the experiments after time PCR (Q-PCR) using the following cycling condi- 3-4 days, when they reached 90% confluence. tions: 35 cycles of denaturation at 95°C for 20 s; anneal- ing at 59°C for 30 s; and extension at 72°C for 30 s; Nitrite assay Brilliant SYBR Green QPCR Master Mix 2× (Stratagene, NOS2 activity was assessed indirectly by measuring La Jolla, CA, USA) was used. PCR reactions were carried nitrite accumulation in the incubation media. Briefly, an out in a 20 μL reaction volume in a MX3000P real time aliquot of the cell culture media (80 μL) was mixed with PCR machine (Stratagene). The primers used for iNOS 40 μL Griess Reagent (Sigma Aldrich) and the absor- detection were: 1704F (50-CTG CAT GGA ACA GTA bance measured at 550 nm in a spectrophotometric TAA GGC AAA C-30), and 1933R (50-CAG ACA GTT microplate reader (PerkinElmer Inc., MA, USA). A stan- TCT GGT CGA TGT CAT GA-30), which yield a 230 dard curve was generated during each assay in the range base pair (bp) product. The primers used for a-tubulin of concentrations 0-100 μMusing NaNO (Sigma were: F984 (50-CCC TCG CCA TGG TAA ATA CAT- Aldrich) as standard. In this range, standard detection 30), and 1093R (50-ACT GGA TGG TAC GCT TGG resulted linear and the minimum detectable concentra- TCT-30), which yield a 110 bp product. The primers tion of NaNO was ≥ 6.25 μM. used for TTP detection were: 2F (5’- CAG CCT GAC In the absence of stimuli, basal levels of nitrites were TTC TGC GAA CCG A -3’), 102R (5’- TGG CTC ATC below the detection limit of the assay after 24 and 48 h GAC ATA AGG CTC TCG T -3’), which yield a 101 incubations. base pair (bp) product. The primers used for KSRP were: 202F (5’- CCG GGG ATA CGC AAG GAC GC iNOS promoter luciferase assay -3’), 472R (5’- CCA CCA TGC CGT CCG GAA CC -3’), C6-2.2. cells stably transfected with a 2,168-bp fragment which yield a 271 bp product. The primers used for HuR detection were: 512F (5’-AACCCCCGG GTT of the rat iNOS promoter driving luciferase expression Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 4 of 11 http://www.jneuroinflammation.com/content/8/1/1 CCT CCG AG -3’), 727R (5’- CCG AGG AAG CAT secondary antibody, diluted 1:20,000. Following three TGC CGG GG -3’), which yield a 216 base pair (bp) washes in TBST, bands were visualized by incubation in product. Relative mRNA concentrations were calculated ECL reagents (GE Healthcare) and exposure to Hyper- from the take-off point of reactions (threshold cycle, Ct) film ECL (GE Healthcare NY, USA). The same mem- using the comparative quantization method and the soft- branes were washed 3 times in TBST, blocked with 10% ware included in the unit. For this analysis, controls were (w/v) low-fat milk in TBST for 1 h at room temperature used as calibrators and the Ct values for a-tubulin and used for b-actin immunoblot. Band intensities were determined using ImageJ software (National Institutes of expression as normalizers. Thus, using the -ΔΔCt Health) from autoradiographs obtained from the mini- method, we calculated the differences (fold changes) in the expression of NOS2 target gene after a specific treat- mumexposuretimethatallowbanddetection,and ment vs. its respective control [27]. Moreover, in each background intensities (determined from an equal-sized run we calculated the PCR efficiency using serial dilution area of the film immediately above the band of interest) of one experimental sample; efficiency values were found were subtracted. between 94 and 98% for each primer set [21]. To test the hypothesis that rapamycin could be influ- Data analysis encing iNOS mRNA stability, cells were incubated with All experiments were done using 5-6 replicates per each TII or TII plus rapamycin for 6 h. Then, 4 μg/ml actino- experimental group, and repeated at least 3 times. For mycin D was added and RNA was prepared at time the RNA analysis, samples were assayed in triplicates, points from 0 to 4 h thereafter. Relative iNOS and a- and the experiments were repeated at least twice. Data tubulin mRNA amounts were determined by Q-PCR were analyzed by one- or two-way ANOVA followed by and iNOS mRNA was normalized versus a-tubulin Bonferroni’s post hoc tests or by unpaired t-test P values mRNA. The relative amount of iNOS mRNA at 0 h was < 0.05 were considered significant. taken as 100%, and the amount at later time point reported as percentage of the 0 h time point. Results Cytokine dependent mTOR activation does not Western immunoblot significantly affect astrocyte iNOS activity Astrocytes were lysed in RIPA buffer (1 mM EDTA, 150 Astrocytes were stimulated using a mixture of proin- mMNaCl, 1% igepal, 0.1% SDS, 0.5% sodium deoxycho- flammatory cytokines (TII), i.e. 10 ng/ml IL1b, 10 ng/ml late, 50 mM Tris-HCl, pH 8.0) (Sigma-Aldrich) contain- TNFa,5ng/ml IFNg, and the activation of the mTOR ing protease inhibitor cocktail diluted 1:250 (Sigma- pathway was examined by measurement of the phos- Aldrich). The protein content in each sample was deter- phorylation level of mTOR at Ser2448 [21]. Cells were mined by Bradford’s method (Biorad, Hercules, CA, incubated for 2 h, and then subjected to protein analysis USA) using bovine serum albumin as standard. A 20 μg for phospho-mTOR and b-actin. TII significantly aliquot of protein was mixed 1:2 with 2× Laemmli Buf- increased mTOR phosphorylation at Ser2448, which was fer (Biorad), boiled for 5 min, and separated through completely blocked by10 nM rapamycin (Figure 1). Sig- 10% polyacrylamide SDS gels. Apparent molecular nificant amounts of nitrites, a stable end product of NO, weights were estimated by comparison to colored mole- could be measured after 48 h incubation and were sig- cular weight markers (Sigma Aldrich). After electrophor- nificantly increased by TII [21]. However, while rapamy- esis, proteins were transferred to polyvinylidene cin significantly reduced iNOS expression and activity in difluoride membranes by semi-dry electrophoretic trans- microglial cells, it displayed only minor effects on TII fer (Biorad). The membranes were blocked with 10% stimulated astrocytes [[21], Figure 2A]. In particular, (w/v) low-fat milk in TBST (10 mM Tris, 150 mMNaCl, rapamycin within the 0-10 nM range tended to reduce 0.1% Tween-20, pH 7.6) (Biorad) for 1 h at room tem- TII dependent NO production, but this effect did not perature, and incubated in the presence of the primary reach statistical significance (Figure 2A). Similarly, rapa- antibody overnight with gentle shaking at 4°C. Primary mycin failed to inhibit NO production when astrocytes antibodies for phosphorylated mTOR, b-actin were used were exposed to another pro-inflammatory stimulus that at the final concentration of 1:1000; primary antibody is more widely used in vitro to activate glial cells, for iNOS were used at the final concentration 1:400. Pri- namely 1 μg/ml LPS and 5 ng/ml IFNg (LI) (Figure 2B). mary antibodies were removed, membranes washed 3 The modest effects of rapamycin on iNOS activity times in TBST, and further incubated for 1h at room were confirmed by western blot analysis. Under basal temperature in the presence of specific secondary anti- conditions, astrocytes do not express iNOS, but protein body, anti-rabbit IgG-HRP conjugated (Vector Labora- levels are significantly increased by 24 h stimulation in tories, Burlingame, CA, USA) diluted 1:15,000-20,000 or presence of either TII or LPS (Figure 3A). However, anti-mouse IgG-HRP conjugated (Sigma-Aldrich) protein levels of iNOS measured after 24 h treatment Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 5 of 11 http://www.jneuroinflammation.com/content/8/1/1 TII TII LPS/IFN LPS/IFNJ TII 20 *** Rapamycin, 10 nM ͲͲ + *** P 20 p pͲ mT mTOR OR ɴͲ actin 0 0 0 0 0 0 0.1 0.1 1 1 10 10 0 0 0 0 01 0.1 1 1 10 10 [Rapamycin], nM [Rapamycin], nM Figure 2 Effects of the mTOR inhibitor on NO production in TII activated astrocytes. Astrocytes were activated for 48 h with TII [Figure 2A] or 24 h with LI [Figure 2B]. The mTOR inhibitor, rapamycin was added to the cells in nM concentrations at the beginning of the experiments. NO production was assessed indirectly by measurement of nitrite accumulation in the incubation 40 40 medium (Griess method). Data are expressed as means ± SEM (n = 6). Data are representative of 3 different experiments. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Ͳ ***P < 0.001 vs. Controls. iNOS mRNA were detected after 24 h between cells treated with TII and cells co-incubated with rapamycin; 00 10 while after 48 h rapamycin caused significant reduction in iNOS mRNA levels (Figure 4A). The up-regulation of [Rapamycin], nM iNOS mRNA due to rapamycin did not directly translate Figure 1 Rapamycin reduces mTOR phosphorylation induced into increased iNOS activity, since only a slight increase by TII. (A) Whole-cell lysates were prepared from astrocytes in nitrite production was measured in astrocytes co- activated with TII for 2 h. 10 nM Rapamycin was added at the treated with TII and 10 nM rapamycin for 24 h com- beginning of the experiment and equal amounts of protein were pared to TII alone (Figure 4B). Similarly, 10 nM rapa- analyzed by western blot for phosphorylated mTOR kinase (p-mTOR) (upper gel) and consequently for b-actin (lower gel). (B) mycin significantly increased the stimulatory effect of LI Quantitation of densitometry wherein p-mTOR values are reflected relative to those for b-actin. Data are representative of two different experiments. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. *P < 0.05 vs. Controls; °P < 0.05 vs. TII. CLPS TII iNOS actin with TII alone or in presence of 10 nM rapamycin were similar (Figure 3B, C). Together these data confirm our B C previous findings that in contrast to microglia cells, TII TII rapamycin has little effect on iNOS protein expression TII or activity. 20 Rapamycin, 10 nM ͲͲ + + iNOS The mTOR pathway is involved in the regulation of iNOS ɴͲ actin expression 0 10 [Rapamycin], nM The steady state levels of iNOS mRNA were almost undetectable by Q-PCR under basal conditions (average Figure 3 Rapamycin does not modify the level of iNOS protein. (A) Whole-cell lysates were prepared from Control astrocytes or cells Ct≈31), and increased in response to TII, with statisti- activated with TII or LPS for 24 h. Equal amounts of proteins were cally significant increases observed after 2 h incubation, analyzed by western blot for iNOS (upper gel) and consequently for maximum levels detected after 4 h, and persistent eleva- b-Actin (lower gel). Two replicates per each experimental group are tion up to 48 h (the latest time point analyzed, (Figure shown in the gel. (B) Pro-inflammatory cytokines (TII), and/or 10 nM 4A). Under these conditions, 10 nM rapamycin signifi- Rapa were added at the beginning of the experiment and proteins were analyzed as described above. (C) Quantification of cantly increased iNOS mRNA levels between 4 h and 12 densitometry wherein iNOS values are reflected relative to those for h incubation (Figure 4A). However, the stimulatory b-actin. Data are representative of two different experiments. effect of rapamycin was transient, since no differences in phospho mTOR protein (a arbitrary dens. Units) Nitrites, M Nitrites s, iNOS pro otein ((arbitrary dens. Units) ɴͲ Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 6 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII AB TII//p  10 Rapamyycin *** *** *** §§§ *** *** ** 4 6 12 24 48 h h 12 24 48 72 Figure 4 Effects of the mTOR inhibitor on iNOS expression elicited by TII. (A) Total cytosolic RNA was prepared from Control, or astrocytes treated with TII and rapamycin for different times, and used for Q-PCR analysis of iNOS expression. Data are expressed as fold change vs. each respective Control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate, the experiment was repeated 2 times with similar results. Where indicated, 10 nM Rapamycin was added at the beginning of the experiment. In all time points TII significantly induced iNOS expression in comparison to Controls and rapamycin increases in 4 h the iNOS mRNA expression elicited by TII. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01, and *** P < 0.001 vs. TII; two-way ANOVA followed by Bonferroni’s post-test. (B) Nitrite production elicited by TII was assessed at different time points, 10 nM rapamycin was added together with cytokines at beginning of §§ experiment. Data are means ± SEM (n = 4). **P < 0.01, vs. Controls; and P < 0.01, vs. TII; two-way ANOVA followed by Bonferroni’s post-test. on iNOS mRNA after 4 h (Figure 5A), an effect lost help account for the stimulatory effects of rapamycin after 24 h incubations (Figure 5B). Rapamycin alone did on iNOS mRNA levels observed at earlier time points not have any significant stimulatory effect per se on in astrocytes. However, in contrast to primary astro- either iNOS activity or on its expression (data not cytes, rapamycin dose and time-dependently increased shown). nitriteproductioninC6 cells (Figure 7A, B), which Extensive characterization of glial iNOS expression suggests that additional regulatory factors are induced and regulation has been carried out with the rat C6 in astrocytes which restrict iNOS activity. glioma cell line, which shares many properties with primary astrocyte cultures, including expression and mTOR kinase regulates the rate of iNOS mRNA regulation of the iNOS gene, mRNA and protein degradation [6,28]. Using C6 cells stably transfected with a 2.2 kB The above results suggested that rapamycin could be rat iNOS promoter, we observed that TII increased the influencing iNOS mRNA stability. To test this possibi- promoter activity of iNOS, with a maximum signal lity, the levels of iNOS mRNA were quantified in pri- detected after 8 h incubation. Promoter activation mary astrocytes after different times in the presence of lasted up to 24 h, the longest time point studied (Fig- TII alone or with actinomycin D added after 6 hr incu- ure6A).Thus, the8htimepoint waschosenfor sub- bation with TII (Figure 8). The half-life of iNOS mRNA sequent studies carried out to characterize the effects after TII treatment was determined to be 4 h, and was of rapamycin on iNOS promoter activity. For this, significantly reduced to less than 1 hour by the presence rapamycin was added at the beginning of the experi- of rapamycin (Figure 8). Moreover, after 6 hr incubation ment, and after 8 h cells were washed with cold PBS in TII, rapamycin significantly increased the expression and luciferase activity measured. Rapamycin, even at of tristetraprolin (TTP), a protein involved in iNOS 10 times lower concentration, significantly increased mRNA stabilization (Figure 9A). In contrast, rapamycin iNOS promoter activity in comparison to cells treated did not modify levels of two other proteins that regulate with TII alone (Figure 6B). This increase of iNOS pro- mRNA stability, namely the KH-type splicing regulatory moter activity elicited by the mTOR inhibitor may protein (KSRP) (Figure 9B) and HuR (Figure 9C). mRNA iNOS (relative to control) Nitritess, M Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 7 of 11 http://www.jneuroinflammation.com/content/8/1/1 increase in the rate of iNOS mRNA degradation asso- LPS/IFN LPS/IFN J J ciated with the inhibition of mTOR kinase. Rapamycin treatment did not interfere with astrocyte viability or *** $ *** proliferation, as we previously reported [21]. These find- ings clearly indicate that the blocking of mTOR pathway is crucial for the production of NO, and that the overall effect observed can be regarded as anti-inflammatory 0 0 0 0 0 0 10 10 0 0 0 0 10 10 since the up-regulation of iNOS was compensated with [Rapamycin], nM [Rapamycin], nM increased iNOS degradation machinery activity. The possible involvement of mTOR during pro-inflam- Figure 5 Effects of the mTOR inhibitor RAPA on iNOS expression elicited by LI. Total cytosolic RNA was prepared from matory astrocyte activation was studied by direct evalua- Control, or astrocyte cells treated LI and rapamycin for different tion of the levels of mTOR phosphorylation at Ser2448 in times, and used for Q-PCR analysis of iNOS expression. Data are presence of proinflammatory cytokines [21]. Phosphory- expressed as fold change vs. each respective Control, taken as lation at Ser2448 is a marker of mTOR pathway activa- calibrator for comparative quantitation analysis of mRNA levels. tion, as suggested by the finding of Rosner and Astrocytes were treated for 4 h (A) or 24 h (B). Each sample was measured in triplicate, the experiment was repeated 2 times with collaborators that down-regulation of mTOR kinase similar results. Where indicated, 10 nM Rapamycin was added at the ser2448 phosphorylation correlates with decreased beginning of the experiment. LI significantly induced iNOS mTORC1 activity [29]. In our experimental conditions, expression in comparison to Controls and rapamycin increases in 4 phospho-mTOR at Ser2448 was increased by TII and h the iNOS mRNA expression elicited by LI. Data are means ± SEM §§ rapamycin completely abolished the phosphorylation, (n = 3). P < 0.01 vs. LI *P < 0.05 vs. Controls; one-way ANOVA followed by Bonferroni’s post-test. suggesting that proinflammatory activation of astrocytes involves the recruitment of the mTOR pathway in a rapa- mycin sensitive manner (Figure 1), suggesting that the Discussion drug can reduce astrocyte proinflammatory activation. In the present study we studied the involvement of the Usually under inflammatory conditions, high levels of mTOR pathway during pro-inflammatory activation of NO are generated by the up-regulation of iNOS. The rat cortical astrocytes in response to two different pro- protein can be induced by different proinflammatory sti- inflammatory stimuli, namely a mixture of cytokines muli to different extents. In general, LPS is a more ("TII”) and the bacterial endotoxin LPS augmented with robust activator, thus nitrite levels (an indirect measure IFNg ("LI”). Moreover, the pharmacological inhibition of of iNOS activity) are significantly elevated after 24 h mTOR obtained using 10 nM rapamycin up-regulated incubation, whereas in the presence of pro-inflammatory iNOS mRNA levels without any relevant effect on iNOS cytokines (TII) a significant increase in nitrite levels is activity. This opposite result is explained by the parallel generally detectable after 48 h incubation. These data may be explained by the recruitment of different signal- ing pathways by the two stimuli, and or by a different AB timing in recruitment, as happens in microglial cells TII [21]. Regardless of the stimulus used, rapamycin did not TII 115 2.27 (8) modify nitrite levels (Figure 2), while it was able to *** *** 100 r 3.4 (8) increase the amount of iNOS mRNA at the earlier time points. This up-regulation was transient, because such differences were absent at 24 h (Figure 4 and 5) and a significantreduction wasobservedat48h(Figure4A). 0 0 0 4 8 121620 24 Rapamycin alone did not display pro-inflammatory 0 1 Time effects per se, being unable to increase either iNOS [Rapamycin], nM mRNA levels or nitrite production after 4 and 24 h Figure 6 Rapamycin increases the iNOS promoter in transfected C6 cells. C6 cells were stable transfected with a 2,168- incubations (data not shown). bp fragment of the rat iNOS promoter. (A) The cells were treated The expression of iNOS is tightly regulated by both with TII or with LPS/IFNg for indicated time point and after varying transcriptional and post-transcriptional mechanisms. the incubation times the luciferase activity was measured. (B) Cells This includes regulation of iNOS promoter activity by were incubated with TII alone or with TII/1 nM Rapamycin for 8 h. binding of transcription factors (NFkB, STAT-1a)and Data are presented as the percentage of luciferase activity measured in the presence of TII and rapamycin relative to the the modulation of iNOS mRNA expression. Post-tran- activity of TII (taken as 100%). Data are means ± SEM of n = 4 scriptional regulation of gene expression is often depen- separate cell cultures; results were analyzed by unpaired t-test. dent on sequences located in the 3’-untraslated region ***p < 0.01 vs. TII. (3’-UTR) of mRNAs [30]. AU-rich (ARE) elements have iNO OS mRNA A RLU (% % control) (relativve to conttrol) iNOS p promoterr iN NOS mRNA A (relattive to control) RLU (% contro ol) iNOS promotter Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 8 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII TII/ 10 Rapamycin A B TII §§§ §§§ §§§ §§ *** *** §§§ §§§ 40 40 *** ** 12 24 48 h 0.1 110 [Rapamycin, nM] Figure 7 Effects of rapamycin on NO production in activated C6 glioma cells. (A) C6 glioma cells were activated with TII for 24 h. The mTOR inhibitor, rapamycin was added to the cells in nM concentrations at the beginning of the experiments. (B) Cells were treated with TII in presence or not of 10 nM rapamycin for different times. NO production was assessed indirectly by measurement of nitrite accumulation in the incubation medium (Griess method). Data are expressed as means ± SEM (n = 6). Data are representative of 3 different experiments. Results were analyzed by §§ §§§ one-way (A) or two-way (B) ANOVA followed by Bonferroni’s post-test. ***P<0.001 vs. Controls, and P < 0.01, P < 0.001 vs. TII. been shown to confer destabilization of mRNAs coding TII pretreatment for pro-inflammatory proteins or oncogenes [31]. In mammalian cells, ARE mediates mRNA decay mainly by TII/ 10 nM + Rapamycin recruitment of the exosome to the mRNAs, thereby pro- moting their rapid degradation. However, the mamma- *** 100 lian exosome does not seem to recognize the ARE- containing RNAs on its own but requires certain ARE *** binding protein (ARE-BPs) for this interaction. In parti- *** cular in untreated cells the RNA-Binding Protein (RNA- BP) KH-type splicing regulatory protein (KSRP) or the zinc-finger protein tristetraprolin (TTP) binds the iNOS mRNA 3’-UTR and recruits the exosome to the mRNA 0 0 [32]. This results in rapid iNOS mRNA degradation. 0 1 2 3 4 5 KSRP binds to the same ARE as HuR, another RNA-BP that oppositely stabilizes the mRNA, and both RNA-BPs Actinomycin D incubation time (h) compete for this binding sites [33]. After cytokine-treat- Figure 8 The mTOR kinase blocking increases the rate of iNOS ment, p38 MAP kinase is activated and TTP expression mRNA degradation. Astrocytes were preincubated with TII alone or with TII plus 10 nM rapamycin for 6 h. After the pretreatment, is enhanced. The protein-protein interactions between actinomycin D (4 μg/ml) was added for different periods of time, as TTP and KSRP compete for the same binding site in indicated. Cells were harvested at designated time points, total RNA the iNOS 3’-UTR sequence, causing dislodgment of was extracted and the iNOS expression was measured by real time KSRP and enhanced HuR binding to the iNOS mRNA. PCR. The mRNA level at 0 time point (before the treatment with The reduced KSRP and the enhanced HuR binding to actinomycin D) was considered as 100%. Data are representative of 3 independent experiments. Data are means ± SEM (n = 3). ***P < the iNOS mRNA 3’UTR result in marked stabilization 0.05 vs. TII/Rapa 10 nM; one-way ANOVA followed by Bonferroni’s of the iNOS mRNA and thus in enhanced iNOS expres- post-test. sion. Similarly, in our experimental model TII was Nitrites, M % iNOSS mRNA Nitrites M Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 9 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII TII/ 10 Rapamycin ** §§ 4 ** ** 612 24 BC 1.5 2.0 ** 1.5 1.0 1.0 *** *** 0.5 0.5 0.0 0.0 h h 6 12 24 6 12 24 Figure 9 Regulation of the expression of RNA-binding proteins by rapamycin in astrocytes. Total cytosolic RNA was prepared from Control, or astrocyte cells treated TII and rapamycin for different times, and used for Q-PCR analysis of iNOS expression. Data are expressed as fold change vs. each respective Control, taken as calibrator for comparative quantitation analysis of mRNA levels. Astrocytes were treated for 6- 12-24 h and TTP (A), KSRP (B) and HuR (C) were assessed. Each sample was measured in triplicate, the experiment was repeated 2 times with §§ similar results. Where indicated, 10 nM Rapamycin was added at the beginning of the experiment. Data are means ± SEM (n = 3). P < 0.01 vs. TII **P < 0.01 or ***P < 0.001 vs. Controls; two-way ANOVA followed by Bonferroni’s post-test. found to significantly increase the levels of these RNA- regulating the expression and stability of cyclin D3 BPs (Figure 9), while rapamycin transiently upregulated mRNA [34]. In fibroblasts rapamycin inhibition of G1 to the expression of TTP without affecting the other pro- S transition is mediated by an effect on cyclin D1 teins (Figure 9A). It has been shown that TTP may have mRNA and protein stability [35]. Maderosian and col- opposite effects on mRNA stability in function of the leagues showed that rapamycin regulates cyclin D1 and AKT activation state [12]. Therefore, up-regulation of c-Myc mRNA in different types of tumor cells involving TTP in our experimental model may contribute either an enhancement of TTP binding activity [12]. Recently, to the initial iNOS mRNA stabilization or be responsible Basu and collaborators demonstrated that the immuno- for the subsequent increased rate of mRNA degradation. suppressant drugs, cyclosporine and rapamycin, regulate Additional experiments are required to completely VEGF mRNA stability in opposite manners. While address the role of TTP in iNOS mRNA regulation, but cyclosporine enhances mRNA stability by induction of the present data strongly suggest a role for this protein. the RNA-BP, HuR, rapamycin increases the rate of degradation in renal cancer cells [13]. In summary, Consistent with our data, rapamycin was found to inter- fere with mRNA degradation in different cell types. For there is increasing evidence that rapamycin can regulate example in renal epithelial cells, Pallet and collaborators the activity of RNA-BPs, thus interfering with the stabi- showed that rapamycin inhibits cell proliferation by lity of different mRNAs. mRNA KSRP (re elative to contro ol) mRN NA TTP (relative tto control) mRNA HuR (re elative to contro ol) Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 10 of 11 http://www.jneuroinflammation.com/content/8/1/1 References In our experimental paradigm, rapamycin increased the 1. Dong Y, Benveniste EN: Immune function of astrocytes. Glia 2001, rate of iNOS mRNA degradation. In particular, using 36:180-90. actinomycin D to block mRNA transcription, we found 2. Pautz A, Art J, Hahn S, Nowag S, Voss C, Kleinert H: Regulation of the expression of inducible nitric oxide synthase. Nitric Oxide 2010, 23:75-93. that after induction of iNOS mRNA by TII the degrada- 3. Kleinert H, Pautz A, Linker K, Schwarz PM: Regulation of the expression of tion of iNOS mRNA occurs with a half life of about 4 h. inducible nitric oxide synthase. Eur J Pharmacol 2004, 500:255-66. Activation of astrocytes in the presence of rapamycin was 4. Chastre A, Jiang W, Desjardins P, Butterworth RF: Ammonia and proinflammatory cytokines modify expression of genes coding for characterized by an increased rate of degradation (t . ≈ 1/2 astrocytic proteins implicated in brain edema in acute liver failure. 50 min),which maybemediatedbyup-regulation of Metab Brain Dis 2010, 25:17-21. mRNA destabilizing proteins as occurs in other cell types 5. Simmons ML, Murphy S: Roles for protein kinases in the induction of nitric oxide synthase in astrocytes. Glia 1994, 11:227-34. [12,13]. The net effect of these complex actions on the 6. Feinstein DL, Galea E, Cermak J, Chugh P, Lyandvert L, Reis DJ: Nitric oxide regulation of iNOS mRNA result in similar levels of pro- synthase expression in glial cells: suppression by tyrosine kinase tein expression and in-significant differences in the inhibitors. J Neurochem 1994, 62:811-4. 7. MacMicking J, Xie QW, Nathan C: Nitric oxide and macrophage function. amount of NO generated in TII-activated astrocytes in Annu Rev Immunol 1997, 15:323-350. the presence or absence of rapamycin. 8. Bogdan C, Röllinghoff M, Diefenbach A: The role of nitric oxide in innate immunity. Immunol Rev 2000, 173:17-26. 9. Kroncke KD, Fehsel K, Kolb-Bachofen V: Inducible nitric oxide synthase in Conclusions human diseases. Clin Exp 1998, 113:147-156. Our findings suggest that rapamycin does not directly 10. Lechner M, Lirk P, Rieder J: Inducible nitric oxide synthase (iNOS) in exert pro-inflammatory actions in rat primary cultures tumor biology: the two sides of the same coin. Semin Cancer Biol 2005, 15:277-289. astrocytes, nor does it increase the proinflammatory 11. Park SK, Murphy S: Nitric oxide synthase type II mRNA stability is effects of cytokines or LPS. In fact, the iNOS mRNA translation- and transcription-dependent. J Neurochem 1996, 67(4):1766-9. up-regulation induced by rapamycin administered 12. Marderosian M, Sharma A, Funk AP, Vartanian R, Masri J, Jo OD, Gera JF: Tristetraprolin regulates Cyclin D1 and c-Myc mRNA stability in response together with a proinflammatory stimuli appears to be to rapamycin in an Akt-dependent manner via p38 MAPK signaling. transient and accompanied by an increased rate of iNOS Oncogene 2006, 25:6277-90. mRNA degradation. Such effects do not augment iNOS 13. Basu A, Datta D, Zurakowski D, Pal S: Altered vascular endothelial growth factor mRNA stability following treatments with immunosuppressive protein levels (Figure 3) nor the amount of nitrite pro- agents: Implications for cancer development. J Biol Chem 2010, duction to any significant extent. Together with the 285(33):25196-202, Epub 2010 Jun 16. marked anti-inflammatory effects observed in microglial 14. Wullschleger S, Loewith R, Hall MN: TOR signaling in growth and metabolism. Cell 2006, 124:471-84. cells [21], these data suggest possible beneficial effects 15. Weichhart T, Säemann MD: The multiple facets of mTOR in immunity. of mTOR inhibitors in the chronic treatment of inflam- Trends Immunol 2009, 30:218-26. matory-based CNS pathologies. 16. Jacinto E, Loewith R, Schmidt A, Lin S, Rüegg MA, Hall A, Hall MN: Mammalian TORcomplex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Biol 2004, 6:1122-8. 17. Säemann MD, Haidinger M, Hecking M, Hörl WH, Weichhart T: The Acknowledgements multifunctional role of mTOR in innate immunity: implications for The authors would like to thank Antony Sharp and Shao Lin for the help transplant immunity. Am J Transplant 2009, 9:2655-61. with the astrocyte cultures and transfected iNOS C6 cells. They are also 18. Oshiro N, Yoshino K, Hidayat S, Tokunaga C, Hara K, Eguchi S, Avruch J, thankful to Dr. Antonella Tramutola for help editing the figures and Prof. Yonezawa K: Dissociation of raptor from mTOR is a mechanism of Giacomo Pozzoli for his support with materials and substances. Finally, they rapamycin-induced inhibition of mTOR function. Genes Cells 2004, 9:497. would like to thank the Multiple Sclerosis International Federation (MSIF) for 19. Wu X, Kihara T, Akaike A, Niidome T, Sugimoto H: PI3K/Akt/mTOR the Du Pré Grant, awarded to LL for working in Prof Feinstein Lab. signaling regulates glutamate transporter 1 in astrocytes. Biochem Biophys Res Commun 2010, 393:514-8. Author details 1 2 20. Pastor MD, García-Yébenes I, Fradejas N, Pérez-Ortiz JM, Mora-Lee S, Istitute of Pharmacology, Catholic Medical School, Rome, Italy. Department Tranque P, Moro MA, Pende M, Calvo S: mTOR/S6 kinase pathway of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois, USA. contributes to astrocytes survival during ischemia. J Biol Chem 284:22067-78. Authors’ contributions 21. Dello Russo C, Lisi L, Tringali G, Navarra P: Involvement of mTOR kinase in LL carried out the experiments and has made substantial contributions to cytokine-dependent microglial activation and cell proliferation. Biochem conception and acquisition of data, and contributed to draft the manuscript; Pharmacol 2009, 78:1242-51. PN has been involved in the revision of the manuscript and given final 22. Jin HK, Ahn SH, Yoon JW, Park JW, Lee EK, Yoo JS, Lee JC, Choi WS, approval of the version to be published; DLF has been involved in revising Han JW: Rapamycin down-regulates inducible nitric oxide synthase by the manuscript critically for important intellectual content; CDR has made inducing proteasomal degradation. Biol Pharm Bull 2009, 32:988-92. contributions to design, analysis and interpretation of data, and drafted the 23. Tsou HK, Su CM, Chen HT, Hsieh MH, Lin CJ, Lu DY, Tang CH, Chen YH: manuscript. All authors have read and approved the final version of the Integrin-linked kinase is involved in TNF-alpha-induced inducible nitric- manuscript. oxide synthase expression in myoblasts. J Cell Biochem 2010, 109:1244-53. 24. Tang CH, Lu DY, Tan TW, Fu WM, Yang RS: Ultrasound induces hypoxia- Competing interests inducible factor-1 activation and inducible nitric-oxide synthase The authors declare that they have no competing interests. expression through the integrin/integrin-linked kinase/Akt/mammalian target of rapamycin pathway in osteoblasts. J Biol Chem 2007, Received: 3 August 2010 Accepted: 5 January 2011 282:25406-15. Published: 5 January 2011 Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 11 of 11 http://www.jneuroinflammation.com/content/8/1/1 25. Vairano M, Dello Russo C, Pozzoli G, Tringali G, Preziosi P, Navarra P: A functional link between heme oxygenase and cyclo-oxygenase activities in cortical rat astrocytes. Biochem Pharmacol 2001, 61:437-41. 26. Gavrilyuk V, Horvath P, Weinberg G, Feinstein DL: A 27-bp region of the inducible nitric oxide synthase promoter regulates expression in glial cells. J Neurochem 2001, 78:129-40. 27. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402-8. 28. Simmons ML, Murphy S: Induction of nitric oxide synthase in glial cells. J Neurochem 1992, 59:897-905. 29. Rosner M, Siegel N, Valli A, Fuchs C, Hengstschläger : mTOR phosphorylated at S2448 binds to raptor and rictor. Amino Acids 2010, 38:223-8. 30. Khabar KS: The AU-rich transcriptome: more than interferons and cytokines and its role in disease. J Interferon Cytokine Res 2005, 25:1-10. 31. Eberhardt W, Doller A, Akool el-S, Pfeilschifter J: Modulation of mRNA stability as a novel therapeutic approach. Pharmacol Ther 2007, 114:56-73. 32. Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ: Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 1999, 19:4311-23. 33. Linker K, Pautz A, Fechir M, Hubrich T, Greeve J, Kleinert H: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression- complex interplay of KSRP with TTP and HuR. Nucleic Acids Res 2005, 33:4813-4827. 34. Pallet N, Thervet E, Le Corre D, Knebelmann B, Nusbaum P, Tomkiewicz C, Meria P, Flinois JP, Beaune P, Legendre C, Anglicheau D: Rapamycin inhibits human renal epithelial cell proliferation: effect on cyclin D3 mRNA expression and stability. Kidney Int 2005, 67:2422-33. 35. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivières S, Mercep L, Ferrari S: Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem 1998, 273:14424-9. doi:10.1186/1742-2094-8-1 Cite this article as: Lisi et al.: The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes. Journal of Neuroinflammation 2011 8:1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Neuroinflammation Springer Journals

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

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Copyright © 2011 by Lisi et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Neurobiology; Immunology
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Abstract

Background: Reactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes. Methods: Primary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFa, IFNg, and IL-1b) or by LPS plus IFNg. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included. Results: In this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability. Conclusion: The present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies. Background neurotoxic compounds, like nitric oxide (NO). NO, one Astrocyte activation has been implicated in the patho- of the smallest known bioactive products of mammalian genesis of several neurological conditions, such as neu- cells, is biosynthesized by three distinct isoforms of NO synthase (NOS): the constitutively expressed neuronal rodegenerative diseases, infections, trauma, and ischemia. Reactive astrocytes are capable of producing a (n)NOS and endothelial (e)NOS, and the inducible (i) variety of pro-inflammatory mediators, including inter- NOS [2]. The expression of iNOS can be induced in dif- leukin-6 (IL-6), IL-1b, tumor necrosis factor-a (TNF-a), ferent cell types and tissues by exposure to immunologi- neurotrophic factors [1], as well as potentially cal and inflammatory stimuli [3]. In vitro, primary astrocyte cultures express iNOS in response to cytokines * Correspondence: [email protected] such as IL-1b [4], interferon g (IFNg), TNFa and/or the Istitute of Pharmacology, Catholic Medical School, Rome, Italy bacterial endotoxin, lipopolysaccharide (LPS) [5,6]. Once Full list of author information is available at the end of the article © 2011 Lisi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 2 of 11 http://www.jneuroinflammation.com/content/8/1/1 induced, iNOS leads to continuous NO production, and activity in microglial cultures activated by pro- which is terminated by enzyme degradation, depletion inflammatory cytokines while displaying minor effects of substrates, or cell death [7]. iNOS activity generates on astrocyte iNOS [21], thus suggesting that mTOR reg- large amounts of NO (within the μMrange)thatcan ulates glial inflammatory activation but with selective have antimicrobial, anti-atherogenic, or apoptotic effects. However, with respect to iNOS expression, both actions [8]. However, aberrant iNOS induction exerts stimulatory and inhibitory actions involving the mTOR pathway have been described. In particular, rapamycin detrimental effects and seems to be involved in the has been found to down-regulate LPS-induced iNOS pathophysiology of several human diseases [9,10]. protein expression in the mouse macrophage RAW Consistently, the expression of iNOS is tightly regu- lated by complex molecular mechanisms, involving both 264.7 cell line via mTOR dependent proteasomal activa- transcriptional and post-transcriptional processes [2]. At tion [22]. In contrast, TNF-a increases iNOS expression the post-transcriptional level an important mechanism and NO production in myoblasts via the activation of of regulation is the modulation of iNOS mRNA stability the ILK/Akt/mTOR and NFkB signaling pathway [23]; that is controlled by several RNA binding proteins and ultra-sound stimulationup-regulatesiNOSexpres- (RNA-BPs) [11]. These proteins bind to the iNOS sion by an HIF-1a-dependent mechanism involving the mRNA and allow its interaction with the exosome, the activation of ILK/Akt and mTOR pathways via integrin mRNA degrading machinery [2]. Interestingly, the mam- receptor in osteoblasts [24]. Thus, mTOR differentially malian target of rapamycin (mTOR) kinase modulates regulates iNOS activity and expression depending on theactivityofsomeoftheabovementioned RNA-BPs the cell type or activating stimulus, and this may explain [12,13] mTOR is a serine-threonine kinase that plays an the different effects that we observed on glial iNOS [21]. evolutionary conserved role in the regulation of cell In our previous studies, we observed that although growth, proliferation, survival, and metabolism, as well rapamycin reduced iNOS expression mRNA and activity as of other physiological processes such as transcription, in microglial cells, and was without effect on astrocyte mRNA turnover and protein translation [14]. Within iNOS activity [21], it caused a rapid significant increase the cells, mTOR can exist in at least two distinct com- in iNOS mRNA levels in astrocytes induced by two dif- plexes together with different partners, mTORC1 and ferent proinflammatory stimuli. Later time points were mTORC2. The mTORC1 consists of the regulatory- not examined; neither was the basis for this contrasting associated protein of mTOR, Raptor, and the adaptor result examined. In the present paper we tested the hypothesis that while at early times rapamycin increases protein mLST8/GbL(Gprotein b-subunit-like protein), iNOS mRNA, at later times it modifies iNOS mRNA and regulates several functions related to cell cycle and growth. The mTORC2 includes mLST8, the adaptor stability. Our results using primary rat astrocytes are protein Rictor, and Sin1 [15]. mTORC2 is thought to consistent with this hypothesis, and suggest that inhibi- regulate the actin cytoskeleton dynamics [16]. Indeed, tion of mTOR kinase activity in glial cells results in rapamycin is a second generation immunosuppressant anti-inflammatory actions. Together with the marked drug that blocks T-cell proliferation by inhibition of anti-inflammatory effects observed in microglial cells mTOR activity, and it is normally used to prevent trans- [21], these data further provide pre-clinical evidence for plant rejection in association with the older calcineurin a possible clinical use of mTOR inhibitors in the treat- inhibitors [17] mTORC1 activity is inhibited by rapamy- ment of inflammatory-based CNS pathologies. cin and its analogs, while mTORC2 is insensitive to the rapamycin inhibitory actions at least at immunosuppres- Methods sive concentrations [18]. Materials mTOR is also a key regulator of intracellular processes Cell culture reagents [Dulbecco’s modified Eagle’smed- in glial cells, in fact various mTOR upstream regulators ium (DMEM), DMEM-F12 and Fetal calf serum (FCS)] have been reported to play an important role in astro- were from Invitrogen Corporation (Paisley, Scotland). cyte physiology. For example, inactivation of a negative Antibiotics were from Biochrom AG (Berlin, Germany). regulator of the mTOR pathway, the tumor suppressor Bacterial endotoxin LPS (Salmonella typhimurium) was PTEN, promotes astrocyte hypertrophy and proliferation from Sigma-Aldrich (St. Louis, MO, USA). Recombinant [14]; inactivation of the tumor suppressor tuberin leads pro-inflammatory cytokines, namely human tumor to glial cell hypertrophy and formation of glial hamarto- necrosis factor a (TNFa), human interleukin-1b (IL-1b) mas [14]; up-regulation of mTOR signaling regulates and rat interferon-g (IFNg) were purchased from Endo- glutamate transporter 1 [19]; and down-regulation of gen (Pierce Biotechnology, Rockford, IL, USA). Rapamy- mTOR/S6 kinase pathway contributes to astrocyte survi- cin (RAPA) was purchased from Tocris Bioscience val during ischemia [20]. We recently showed that rapa- (Bristol, UK). b-actin (clone AC-74) mouse monoclonal mycin and its analog, RAD001, reduce iNOS expression antibody was from Sigma Aldrich; rabbit polyclonal anti- Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 3 of 11 http://www.jneuroinflammation.com/content/8/1/1 phospho [ser-2448] mTOR was purchased from Novus [26] were used to monitor effects of rapamycin on acti- Biological (Littleton, CO, USA), mouse monoclonal vation of the iNOS promoter. These cells have a low iNOS antibody was from Santa Cruz (Santa Cruz, CA). level of basal luciferase activity, which can be induced between4-and 10-folduponincubationwith LPS plus Cell cultures IFNg or with TII. C6-2.2. cells were incubated with the Primary cultures of rat cortical astrocytes were prepared indicated iNOS inducers in DMEM containing 1% FCS as previously described [25]. In brief, 1- to 2-day-old and the indicated concentrations of rapamycin. After Wistar rats were sacrificed. The brains were removed desired incubation times, the media were removed, and under aseptic conditions and placed in phosphate buffer the cells were washed once with cold phosphate-buf- ++ ++ saline with Ca and Mg (PBS-w), containing antibio- fered saline. To prepare lysates, 50 μlofCHAPS buffer tics (100 IU/ml of penicillin plus 100 μg/ml streptomy- (10 mM CHAPS, 10 mM Tris, pH 7.4) were used. Ali- cin). Under a stereomicroscope, the meninges were quots of cell lysates (40 μl) were placed into wells of an carefully removed and the cortex was dissected. The tis- opaque white 96-well microplate. A volume of luciferase sue was cut into small fragments, digested with trypsin substrate (20 μl) (Steady Glo reagent, Promega) was ++ ++ in PBS without Ca and Mg (PBS-wo), for 25 min at added to all samples, and the luminescence was mea- 37°C and for further 5 min in the presence of DNAse I. sured in a microplate luminometer (Rosys-Anthos, Aus- This step was followed by mechanically dissociation in tria). The data are presented as the percentage of Dulbecco’s MEM with Glutamax-I containing 10% fetal luciferase activity measured in the presence of NOS2 calf serum (FCS) and antibiotics as above, to obtain sin- inducers and rapamycin, relative to the activity of con- gle cells. Cell viability was roughly 45%. trol cells (incubated in media with TII). The cells thus obtained were seeded in 75-cm flasks at adensity of1×10 cells/10 ml (1 brain/flask) and incu- iNOS mRNA analysis in real time PCR bated at 37°C in a humidified atmosphere containing 5% Total cytoplasmic RNA was extracted from astrocytes CO2. The medium was changed after 24 h and then twice using TRIZOL (Invitrogen). RNA concentration was a week. Astrocytes obtained with this procedure were then measured using the Quant-iT™ RiboGreen RNA Assay subcultured twice, the first time in 75-cm flasks and the Kit (Invitrogen). In each assay, a standard curve in the second time directly in multiwell plates used for the range of 0-100 ng RNA was run using 16S and 23S experimental procedures. All the experiments were carried ribosomal RNA (rRNA) from E. coli as standard. Ali- out in 1% FCS DMEM with antibiotics. quots (1 μg) of RNA were converted to cDNA using C6 glioma cells and stable transfected C6 cells (see random hexamer primers and the ImProm-II Reverse below) were grown in DMEM containing 10% fetal calf Transcriptase (Promega, Madison, WI, USA). Quantita- serum and antibiotics, including G418. Cells were tive changes in mRNA levels were estimated by real- passed once a week and used for the experiments after time PCR (Q-PCR) using the following cycling condi- 3-4 days, when they reached 90% confluence. tions: 35 cycles of denaturation at 95°C for 20 s; anneal- ing at 59°C for 30 s; and extension at 72°C for 30 s; Nitrite assay Brilliant SYBR Green QPCR Master Mix 2× (Stratagene, NOS2 activity was assessed indirectly by measuring La Jolla, CA, USA) was used. PCR reactions were carried nitrite accumulation in the incubation media. Briefly, an out in a 20 μL reaction volume in a MX3000P real time aliquot of the cell culture media (80 μL) was mixed with PCR machine (Stratagene). The primers used for iNOS 40 μL Griess Reagent (Sigma Aldrich) and the absor- detection were: 1704F (50-CTG CAT GGA ACA GTA bance measured at 550 nm in a spectrophotometric TAA GGC AAA C-30), and 1933R (50-CAG ACA GTT microplate reader (PerkinElmer Inc., MA, USA). A stan- TCT GGT CGA TGT CAT GA-30), which yield a 230 dard curve was generated during each assay in the range base pair (bp) product. The primers used for a-tubulin of concentrations 0-100 μMusing NaNO (Sigma were: F984 (50-CCC TCG CCA TGG TAA ATA CAT- Aldrich) as standard. In this range, standard detection 30), and 1093R (50-ACT GGA TGG TAC GCT TGG resulted linear and the minimum detectable concentra- TCT-30), which yield a 110 bp product. The primers tion of NaNO was ≥ 6.25 μM. used for TTP detection were: 2F (5’- CAG CCT GAC In the absence of stimuli, basal levels of nitrites were TTC TGC GAA CCG A -3’), 102R (5’- TGG CTC ATC below the detection limit of the assay after 24 and 48 h GAC ATA AGG CTC TCG T -3’), which yield a 101 incubations. base pair (bp) product. The primers used for KSRP were: 202F (5’- CCG GGG ATA CGC AAG GAC GC iNOS promoter luciferase assay -3’), 472R (5’- CCA CCA TGC CGT CCG GAA CC -3’), C6-2.2. cells stably transfected with a 2,168-bp fragment which yield a 271 bp product. The primers used for HuR detection were: 512F (5’-AACCCCCGG GTT of the rat iNOS promoter driving luciferase expression Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 4 of 11 http://www.jneuroinflammation.com/content/8/1/1 CCT CCG AG -3’), 727R (5’- CCG AGG AAG CAT secondary antibody, diluted 1:20,000. Following three TGC CGG GG -3’), which yield a 216 base pair (bp) washes in TBST, bands were visualized by incubation in product. Relative mRNA concentrations were calculated ECL reagents (GE Healthcare) and exposure to Hyper- from the take-off point of reactions (threshold cycle, Ct) film ECL (GE Healthcare NY, USA). The same mem- using the comparative quantization method and the soft- branes were washed 3 times in TBST, blocked with 10% ware included in the unit. For this analysis, controls were (w/v) low-fat milk in TBST for 1 h at room temperature used as calibrators and the Ct values for a-tubulin and used for b-actin immunoblot. Band intensities were determined using ImageJ software (National Institutes of expression as normalizers. Thus, using the -ΔΔCt Health) from autoradiographs obtained from the mini- method, we calculated the differences (fold changes) in the expression of NOS2 target gene after a specific treat- mumexposuretimethatallowbanddetection,and ment vs. its respective control [27]. Moreover, in each background intensities (determined from an equal-sized run we calculated the PCR efficiency using serial dilution area of the film immediately above the band of interest) of one experimental sample; efficiency values were found were subtracted. between 94 and 98% for each primer set [21]. To test the hypothesis that rapamycin could be influ- Data analysis encing iNOS mRNA stability, cells were incubated with All experiments were done using 5-6 replicates per each TII or TII plus rapamycin for 6 h. Then, 4 μg/ml actino- experimental group, and repeated at least 3 times. For mycin D was added and RNA was prepared at time the RNA analysis, samples were assayed in triplicates, points from 0 to 4 h thereafter. Relative iNOS and a- and the experiments were repeated at least twice. Data tubulin mRNA amounts were determined by Q-PCR were analyzed by one- or two-way ANOVA followed by and iNOS mRNA was normalized versus a-tubulin Bonferroni’s post hoc tests or by unpaired t-test P values mRNA. The relative amount of iNOS mRNA at 0 h was < 0.05 were considered significant. taken as 100%, and the amount at later time point reported as percentage of the 0 h time point. Results Cytokine dependent mTOR activation does not Western immunoblot significantly affect astrocyte iNOS activity Astrocytes were lysed in RIPA buffer (1 mM EDTA, 150 Astrocytes were stimulated using a mixture of proin- mMNaCl, 1% igepal, 0.1% SDS, 0.5% sodium deoxycho- flammatory cytokines (TII), i.e. 10 ng/ml IL1b, 10 ng/ml late, 50 mM Tris-HCl, pH 8.0) (Sigma-Aldrich) contain- TNFa,5ng/ml IFNg, and the activation of the mTOR ing protease inhibitor cocktail diluted 1:250 (Sigma- pathway was examined by measurement of the phos- Aldrich). The protein content in each sample was deter- phorylation level of mTOR at Ser2448 [21]. Cells were mined by Bradford’s method (Biorad, Hercules, CA, incubated for 2 h, and then subjected to protein analysis USA) using bovine serum albumin as standard. A 20 μg for phospho-mTOR and b-actin. TII significantly aliquot of protein was mixed 1:2 with 2× Laemmli Buf- increased mTOR phosphorylation at Ser2448, which was fer (Biorad), boiled for 5 min, and separated through completely blocked by10 nM rapamycin (Figure 1). Sig- 10% polyacrylamide SDS gels. Apparent molecular nificant amounts of nitrites, a stable end product of NO, weights were estimated by comparison to colored mole- could be measured after 48 h incubation and were sig- cular weight markers (Sigma Aldrich). After electrophor- nificantly increased by TII [21]. However, while rapamy- esis, proteins were transferred to polyvinylidene cin significantly reduced iNOS expression and activity in difluoride membranes by semi-dry electrophoretic trans- microglial cells, it displayed only minor effects on TII fer (Biorad). The membranes were blocked with 10% stimulated astrocytes [[21], Figure 2A]. In particular, (w/v) low-fat milk in TBST (10 mM Tris, 150 mMNaCl, rapamycin within the 0-10 nM range tended to reduce 0.1% Tween-20, pH 7.6) (Biorad) for 1 h at room tem- TII dependent NO production, but this effect did not perature, and incubated in the presence of the primary reach statistical significance (Figure 2A). Similarly, rapa- antibody overnight with gentle shaking at 4°C. Primary mycin failed to inhibit NO production when astrocytes antibodies for phosphorylated mTOR, b-actin were used were exposed to another pro-inflammatory stimulus that at the final concentration of 1:1000; primary antibody is more widely used in vitro to activate glial cells, for iNOS were used at the final concentration 1:400. Pri- namely 1 μg/ml LPS and 5 ng/ml IFNg (LI) (Figure 2B). mary antibodies were removed, membranes washed 3 The modest effects of rapamycin on iNOS activity times in TBST, and further incubated for 1h at room were confirmed by western blot analysis. Under basal temperature in the presence of specific secondary anti- conditions, astrocytes do not express iNOS, but protein body, anti-rabbit IgG-HRP conjugated (Vector Labora- levels are significantly increased by 24 h stimulation in tories, Burlingame, CA, USA) diluted 1:15,000-20,000 or presence of either TII or LPS (Figure 3A). However, anti-mouse IgG-HRP conjugated (Sigma-Aldrich) protein levels of iNOS measured after 24 h treatment Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 5 of 11 http://www.jneuroinflammation.com/content/8/1/1 TII TII LPS/IFN LPS/IFNJ TII 20 *** Rapamycin, 10 nM ͲͲ + *** P 20 p pͲ mT mTOR OR ɴͲ actin 0 0 0 0 0 0 0.1 0.1 1 1 10 10 0 0 0 0 01 0.1 1 1 10 10 [Rapamycin], nM [Rapamycin], nM Figure 2 Effects of the mTOR inhibitor on NO production in TII activated astrocytes. Astrocytes were activated for 48 h with TII [Figure 2A] or 24 h with LI [Figure 2B]. The mTOR inhibitor, rapamycin was added to the cells in nM concentrations at the beginning of the experiments. NO production was assessed indirectly by measurement of nitrite accumulation in the incubation 40 40 medium (Griess method). Data are expressed as means ± SEM (n = 6). Data are representative of 3 different experiments. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Ͳ ***P < 0.001 vs. Controls. iNOS mRNA were detected after 24 h between cells treated with TII and cells co-incubated with rapamycin; 00 10 while after 48 h rapamycin caused significant reduction in iNOS mRNA levels (Figure 4A). The up-regulation of [Rapamycin], nM iNOS mRNA due to rapamycin did not directly translate Figure 1 Rapamycin reduces mTOR phosphorylation induced into increased iNOS activity, since only a slight increase by TII. (A) Whole-cell lysates were prepared from astrocytes in nitrite production was measured in astrocytes co- activated with TII for 2 h. 10 nM Rapamycin was added at the treated with TII and 10 nM rapamycin for 24 h com- beginning of the experiment and equal amounts of protein were pared to TII alone (Figure 4B). Similarly, 10 nM rapa- analyzed by western blot for phosphorylated mTOR kinase (p-mTOR) (upper gel) and consequently for b-actin (lower gel). (B) mycin significantly increased the stimulatory effect of LI Quantitation of densitometry wherein p-mTOR values are reflected relative to those for b-actin. Data are representative of two different experiments. Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. *P < 0.05 vs. Controls; °P < 0.05 vs. TII. CLPS TII iNOS actin with TII alone or in presence of 10 nM rapamycin were similar (Figure 3B, C). Together these data confirm our B C previous findings that in contrast to microglia cells, TII TII rapamycin has little effect on iNOS protein expression TII or activity. 20 Rapamycin, 10 nM ͲͲ + + iNOS The mTOR pathway is involved in the regulation of iNOS ɴͲ actin expression 0 10 [Rapamycin], nM The steady state levels of iNOS mRNA were almost undetectable by Q-PCR under basal conditions (average Figure 3 Rapamycin does not modify the level of iNOS protein. (A) Whole-cell lysates were prepared from Control astrocytes or cells Ct≈31), and increased in response to TII, with statisti- activated with TII or LPS for 24 h. Equal amounts of proteins were cally significant increases observed after 2 h incubation, analyzed by western blot for iNOS (upper gel) and consequently for maximum levels detected after 4 h, and persistent eleva- b-Actin (lower gel). Two replicates per each experimental group are tion up to 48 h (the latest time point analyzed, (Figure shown in the gel. (B) Pro-inflammatory cytokines (TII), and/or 10 nM 4A). Under these conditions, 10 nM rapamycin signifi- Rapa were added at the beginning of the experiment and proteins were analyzed as described above. (C) Quantification of cantly increased iNOS mRNA levels between 4 h and 12 densitometry wherein iNOS values are reflected relative to those for h incubation (Figure 4A). However, the stimulatory b-actin. Data are representative of two different experiments. effect of rapamycin was transient, since no differences in phospho mTOR protein (a arbitrary dens. Units) Nitrites, M Nitrites s, iNOS pro otein ((arbitrary dens. Units) ɴͲ Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 6 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII AB TII//p  10 Rapamyycin *** *** *** §§§ *** *** ** 4 6 12 24 48 h h 12 24 48 72 Figure 4 Effects of the mTOR inhibitor on iNOS expression elicited by TII. (A) Total cytosolic RNA was prepared from Control, or astrocytes treated with TII and rapamycin for different times, and used for Q-PCR analysis of iNOS expression. Data are expressed as fold change vs. each respective Control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate, the experiment was repeated 2 times with similar results. Where indicated, 10 nM Rapamycin was added at the beginning of the experiment. In all time points TII significantly induced iNOS expression in comparison to Controls and rapamycin increases in 4 h the iNOS mRNA expression elicited by TII. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01, and *** P < 0.001 vs. TII; two-way ANOVA followed by Bonferroni’s post-test. (B) Nitrite production elicited by TII was assessed at different time points, 10 nM rapamycin was added together with cytokines at beginning of §§ experiment. Data are means ± SEM (n = 4). **P < 0.01, vs. Controls; and P < 0.01, vs. TII; two-way ANOVA followed by Bonferroni’s post-test. on iNOS mRNA after 4 h (Figure 5A), an effect lost help account for the stimulatory effects of rapamycin after 24 h incubations (Figure 5B). Rapamycin alone did on iNOS mRNA levels observed at earlier time points not have any significant stimulatory effect per se on in astrocytes. However, in contrast to primary astro- either iNOS activity or on its expression (data not cytes, rapamycin dose and time-dependently increased shown). nitriteproductioninC6 cells (Figure 7A, B), which Extensive characterization of glial iNOS expression suggests that additional regulatory factors are induced and regulation has been carried out with the rat C6 in astrocytes which restrict iNOS activity. glioma cell line, which shares many properties with primary astrocyte cultures, including expression and mTOR kinase regulates the rate of iNOS mRNA regulation of the iNOS gene, mRNA and protein degradation [6,28]. Using C6 cells stably transfected with a 2.2 kB The above results suggested that rapamycin could be rat iNOS promoter, we observed that TII increased the influencing iNOS mRNA stability. To test this possibi- promoter activity of iNOS, with a maximum signal lity, the levels of iNOS mRNA were quantified in pri- detected after 8 h incubation. Promoter activation mary astrocytes after different times in the presence of lasted up to 24 h, the longest time point studied (Fig- TII alone or with actinomycin D added after 6 hr incu- ure6A).Thus, the8htimepoint waschosenfor sub- bation with TII (Figure 8). The half-life of iNOS mRNA sequent studies carried out to characterize the effects after TII treatment was determined to be 4 h, and was of rapamycin on iNOS promoter activity. For this, significantly reduced to less than 1 hour by the presence rapamycin was added at the beginning of the experi- of rapamycin (Figure 8). Moreover, after 6 hr incubation ment, and after 8 h cells were washed with cold PBS in TII, rapamycin significantly increased the expression and luciferase activity measured. Rapamycin, even at of tristetraprolin (TTP), a protein involved in iNOS 10 times lower concentration, significantly increased mRNA stabilization (Figure 9A). In contrast, rapamycin iNOS promoter activity in comparison to cells treated did not modify levels of two other proteins that regulate with TII alone (Figure 6B). This increase of iNOS pro- mRNA stability, namely the KH-type splicing regulatory moter activity elicited by the mTOR inhibitor may protein (KSRP) (Figure 9B) and HuR (Figure 9C). mRNA iNOS (relative to control) Nitritess, M Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 7 of 11 http://www.jneuroinflammation.com/content/8/1/1 increase in the rate of iNOS mRNA degradation asso- LPS/IFN LPS/IFN J J ciated with the inhibition of mTOR kinase. Rapamycin treatment did not interfere with astrocyte viability or *** $ *** proliferation, as we previously reported [21]. These find- ings clearly indicate that the blocking of mTOR pathway is crucial for the production of NO, and that the overall effect observed can be regarded as anti-inflammatory 0 0 0 0 0 0 10 10 0 0 0 0 10 10 since the up-regulation of iNOS was compensated with [Rapamycin], nM [Rapamycin], nM increased iNOS degradation machinery activity. The possible involvement of mTOR during pro-inflam- Figure 5 Effects of the mTOR inhibitor RAPA on iNOS expression elicited by LI. Total cytosolic RNA was prepared from matory astrocyte activation was studied by direct evalua- Control, or astrocyte cells treated LI and rapamycin for different tion of the levels of mTOR phosphorylation at Ser2448 in times, and used for Q-PCR analysis of iNOS expression. Data are presence of proinflammatory cytokines [21]. Phosphory- expressed as fold change vs. each respective Control, taken as lation at Ser2448 is a marker of mTOR pathway activa- calibrator for comparative quantitation analysis of mRNA levels. tion, as suggested by the finding of Rosner and Astrocytes were treated for 4 h (A) or 24 h (B). Each sample was measured in triplicate, the experiment was repeated 2 times with collaborators that down-regulation of mTOR kinase similar results. Where indicated, 10 nM Rapamycin was added at the ser2448 phosphorylation correlates with decreased beginning of the experiment. LI significantly induced iNOS mTORC1 activity [29]. In our experimental conditions, expression in comparison to Controls and rapamycin increases in 4 phospho-mTOR at Ser2448 was increased by TII and h the iNOS mRNA expression elicited by LI. Data are means ± SEM §§ rapamycin completely abolished the phosphorylation, (n = 3). P < 0.01 vs. LI *P < 0.05 vs. Controls; one-way ANOVA followed by Bonferroni’s post-test. suggesting that proinflammatory activation of astrocytes involves the recruitment of the mTOR pathway in a rapa- mycin sensitive manner (Figure 1), suggesting that the Discussion drug can reduce astrocyte proinflammatory activation. In the present study we studied the involvement of the Usually under inflammatory conditions, high levels of mTOR pathway during pro-inflammatory activation of NO are generated by the up-regulation of iNOS. The rat cortical astrocytes in response to two different pro- protein can be induced by different proinflammatory sti- inflammatory stimuli, namely a mixture of cytokines muli to different extents. In general, LPS is a more ("TII”) and the bacterial endotoxin LPS augmented with robust activator, thus nitrite levels (an indirect measure IFNg ("LI”). Moreover, the pharmacological inhibition of of iNOS activity) are significantly elevated after 24 h mTOR obtained using 10 nM rapamycin up-regulated incubation, whereas in the presence of pro-inflammatory iNOS mRNA levels without any relevant effect on iNOS cytokines (TII) a significant increase in nitrite levels is activity. This opposite result is explained by the parallel generally detectable after 48 h incubation. These data may be explained by the recruitment of different signal- ing pathways by the two stimuli, and or by a different AB timing in recruitment, as happens in microglial cells TII [21]. Regardless of the stimulus used, rapamycin did not TII 115 2.27 (8) modify nitrite levels (Figure 2), while it was able to *** *** 100 r 3.4 (8) increase the amount of iNOS mRNA at the earlier time points. This up-regulation was transient, because such differences were absent at 24 h (Figure 4 and 5) and a significantreduction wasobservedat48h(Figure4A). 0 0 0 4 8 121620 24 Rapamycin alone did not display pro-inflammatory 0 1 Time effects per se, being unable to increase either iNOS [Rapamycin], nM mRNA levels or nitrite production after 4 and 24 h Figure 6 Rapamycin increases the iNOS promoter in transfected C6 cells. C6 cells were stable transfected with a 2,168- incubations (data not shown). bp fragment of the rat iNOS promoter. (A) The cells were treated The expression of iNOS is tightly regulated by both with TII or with LPS/IFNg for indicated time point and after varying transcriptional and post-transcriptional mechanisms. the incubation times the luciferase activity was measured. (B) Cells This includes regulation of iNOS promoter activity by were incubated with TII alone or with TII/1 nM Rapamycin for 8 h. binding of transcription factors (NFkB, STAT-1a)and Data are presented as the percentage of luciferase activity measured in the presence of TII and rapamycin relative to the the modulation of iNOS mRNA expression. Post-tran- activity of TII (taken as 100%). Data are means ± SEM of n = 4 scriptional regulation of gene expression is often depen- separate cell cultures; results were analyzed by unpaired t-test. dent on sequences located in the 3’-untraslated region ***p < 0.01 vs. TII. (3’-UTR) of mRNAs [30]. AU-rich (ARE) elements have iNO OS mRNA A RLU (% % control) (relativve to conttrol) iNOS p promoterr iN NOS mRNA A (relattive to control) RLU (% contro ol) iNOS promotter Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 8 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII TII/ 10 Rapamycin A B TII §§§ §§§ §§§ §§ *** *** §§§ §§§ 40 40 *** ** 12 24 48 h 0.1 110 [Rapamycin, nM] Figure 7 Effects of rapamycin on NO production in activated C6 glioma cells. (A) C6 glioma cells were activated with TII for 24 h. The mTOR inhibitor, rapamycin was added to the cells in nM concentrations at the beginning of the experiments. (B) Cells were treated with TII in presence or not of 10 nM rapamycin for different times. NO production was assessed indirectly by measurement of nitrite accumulation in the incubation medium (Griess method). Data are expressed as means ± SEM (n = 6). Data are representative of 3 different experiments. Results were analyzed by §§ §§§ one-way (A) or two-way (B) ANOVA followed by Bonferroni’s post-test. ***P<0.001 vs. Controls, and P < 0.01, P < 0.001 vs. TII. been shown to confer destabilization of mRNAs coding TII pretreatment for pro-inflammatory proteins or oncogenes [31]. In mammalian cells, ARE mediates mRNA decay mainly by TII/ 10 nM + Rapamycin recruitment of the exosome to the mRNAs, thereby pro- moting their rapid degradation. However, the mamma- *** 100 lian exosome does not seem to recognize the ARE- containing RNAs on its own but requires certain ARE *** binding protein (ARE-BPs) for this interaction. In parti- *** cular in untreated cells the RNA-Binding Protein (RNA- BP) KH-type splicing regulatory protein (KSRP) or the zinc-finger protein tristetraprolin (TTP) binds the iNOS mRNA 3’-UTR and recruits the exosome to the mRNA 0 0 [32]. This results in rapid iNOS mRNA degradation. 0 1 2 3 4 5 KSRP binds to the same ARE as HuR, another RNA-BP that oppositely stabilizes the mRNA, and both RNA-BPs Actinomycin D incubation time (h) compete for this binding sites [33]. After cytokine-treat- Figure 8 The mTOR kinase blocking increases the rate of iNOS ment, p38 MAP kinase is activated and TTP expression mRNA degradation. Astrocytes were preincubated with TII alone or with TII plus 10 nM rapamycin for 6 h. After the pretreatment, is enhanced. The protein-protein interactions between actinomycin D (4 μg/ml) was added for different periods of time, as TTP and KSRP compete for the same binding site in indicated. Cells were harvested at designated time points, total RNA the iNOS 3’-UTR sequence, causing dislodgment of was extracted and the iNOS expression was measured by real time KSRP and enhanced HuR binding to the iNOS mRNA. PCR. The mRNA level at 0 time point (before the treatment with The reduced KSRP and the enhanced HuR binding to actinomycin D) was considered as 100%. Data are representative of 3 independent experiments. Data are means ± SEM (n = 3). ***P < the iNOS mRNA 3’UTR result in marked stabilization 0.05 vs. TII/Rapa 10 nM; one-way ANOVA followed by Bonferroni’s of the iNOS mRNA and thus in enhanced iNOS expres- post-test. sion. Similarly, in our experimental model TII was Nitrites, M % iNOSS mRNA Nitrites M Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 9 of 11 http://www.jneuroinflammation.com/content/8/1/1 Control TII TII/ 10 Rapamycin ** §§ 4 ** ** 612 24 BC 1.5 2.0 ** 1.5 1.0 1.0 *** *** 0.5 0.5 0.0 0.0 h h 6 12 24 6 12 24 Figure 9 Regulation of the expression of RNA-binding proteins by rapamycin in astrocytes. Total cytosolic RNA was prepared from Control, or astrocyte cells treated TII and rapamycin for different times, and used for Q-PCR analysis of iNOS expression. Data are expressed as fold change vs. each respective Control, taken as calibrator for comparative quantitation analysis of mRNA levels. Astrocytes were treated for 6- 12-24 h and TTP (A), KSRP (B) and HuR (C) were assessed. Each sample was measured in triplicate, the experiment was repeated 2 times with §§ similar results. Where indicated, 10 nM Rapamycin was added at the beginning of the experiment. Data are means ± SEM (n = 3). P < 0.01 vs. TII **P < 0.01 or ***P < 0.001 vs. Controls; two-way ANOVA followed by Bonferroni’s post-test. found to significantly increase the levels of these RNA- regulating the expression and stability of cyclin D3 BPs (Figure 9), while rapamycin transiently upregulated mRNA [34]. In fibroblasts rapamycin inhibition of G1 to the expression of TTP without affecting the other pro- S transition is mediated by an effect on cyclin D1 teins (Figure 9A). It has been shown that TTP may have mRNA and protein stability [35]. Maderosian and col- opposite effects on mRNA stability in function of the leagues showed that rapamycin regulates cyclin D1 and AKT activation state [12]. Therefore, up-regulation of c-Myc mRNA in different types of tumor cells involving TTP in our experimental model may contribute either an enhancement of TTP binding activity [12]. Recently, to the initial iNOS mRNA stabilization or be responsible Basu and collaborators demonstrated that the immuno- for the subsequent increased rate of mRNA degradation. suppressant drugs, cyclosporine and rapamycin, regulate Additional experiments are required to completely VEGF mRNA stability in opposite manners. While address the role of TTP in iNOS mRNA regulation, but cyclosporine enhances mRNA stability by induction of the present data strongly suggest a role for this protein. the RNA-BP, HuR, rapamycin increases the rate of degradation in renal cancer cells [13]. In summary, Consistent with our data, rapamycin was found to inter- fere with mRNA degradation in different cell types. For there is increasing evidence that rapamycin can regulate example in renal epithelial cells, Pallet and collaborators the activity of RNA-BPs, thus interfering with the stabi- showed that rapamycin inhibits cell proliferation by lity of different mRNAs. mRNA KSRP (re elative to contro ol) mRN NA TTP (relative tto control) mRNA HuR (re elative to contro ol) Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 10 of 11 http://www.jneuroinflammation.com/content/8/1/1 References In our experimental paradigm, rapamycin increased the 1. Dong Y, Benveniste EN: Immune function of astrocytes. Glia 2001, rate of iNOS mRNA degradation. In particular, using 36:180-90. actinomycin D to block mRNA transcription, we found 2. Pautz A, Art J, Hahn S, Nowag S, Voss C, Kleinert H: Regulation of the expression of inducible nitric oxide synthase. 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Am J Transplant 2009, 9:2655-61. with the astrocyte cultures and transfected iNOS C6 cells. They are also 18. Oshiro N, Yoshino K, Hidayat S, Tokunaga C, Hara K, Eguchi S, Avruch J, thankful to Dr. Antonella Tramutola for help editing the figures and Prof. Yonezawa K: Dissociation of raptor from mTOR is a mechanism of Giacomo Pozzoli for his support with materials and substances. Finally, they rapamycin-induced inhibition of mTOR function. Genes Cells 2004, 9:497. would like to thank the Multiple Sclerosis International Federation (MSIF) for 19. Wu X, Kihara T, Akaike A, Niidome T, Sugimoto H: PI3K/Akt/mTOR the Du Pré Grant, awarded to LL for working in Prof Feinstein Lab. signaling regulates glutamate transporter 1 in astrocytes. Biochem Biophys Res Commun 2010, 393:514-8. Author details 1 2 20. Pastor MD, García-Yébenes I, Fradejas N, Pérez-Ortiz JM, Mora-Lee S, Istitute of Pharmacology, Catholic Medical School, Rome, Italy. Department Tranque P, Moro MA, Pende M, Calvo S: mTOR/S6 kinase pathway of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois, USA. contributes to astrocytes survival during ischemia. J Biol Chem 284:22067-78. Authors’ contributions 21. Dello Russo C, Lisi L, Tringali G, Navarra P: Involvement of mTOR kinase in LL carried out the experiments and has made substantial contributions to cytokine-dependent microglial activation and cell proliferation. Biochem conception and acquisition of data, and contributed to draft the manuscript; Pharmacol 2009, 78:1242-51. PN has been involved in the revision of the manuscript and given final 22. Jin HK, Ahn SH, Yoon JW, Park JW, Lee EK, Yoo JS, Lee JC, Choi WS, approval of the version to be published; DLF has been involved in revising Han JW: Rapamycin down-regulates inducible nitric oxide synthase by the manuscript critically for important intellectual content; CDR has made inducing proteasomal degradation. Biol Pharm Bull 2009, 32:988-92. contributions to design, analysis and interpretation of data, and drafted the 23. Tsou HK, Su CM, Chen HT, Hsieh MH, Lin CJ, Lu DY, Tang CH, Chen YH: manuscript. All authors have read and approved the final version of the Integrin-linked kinase is involved in TNF-alpha-induced inducible nitric- manuscript. oxide synthase expression in myoblasts. J Cell Biochem 2010, 109:1244-53. 24. Tang CH, Lu DY, Tan TW, Fu WM, Yang RS: Ultrasound induces hypoxia- Competing interests inducible factor-1 activation and inducible nitric-oxide synthase The authors declare that they have no competing interests. expression through the integrin/integrin-linked kinase/Akt/mammalian target of rapamycin pathway in osteoblasts. J Biol Chem 2007, Received: 3 August 2010 Accepted: 5 January 2011 282:25406-15. Published: 5 January 2011 Lisi et al. Journal of Neuroinflammation 2011, 8:1 Page 11 of 11 http://www.jneuroinflammation.com/content/8/1/1 25. 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Eberhardt W, Doller A, Akool el-S, Pfeilschifter J: Modulation of mRNA stability as a novel therapeutic approach. Pharmacol Ther 2007, 114:56-73. 32. Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ: Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 1999, 19:4311-23. 33. Linker K, Pautz A, Fechir M, Hubrich T, Greeve J, Kleinert H: Involvement of KSRP in the post-transcriptional regulation of human iNOS expression- complex interplay of KSRP with TTP and HuR. Nucleic Acids Res 2005, 33:4813-4827. 34. Pallet N, Thervet E, Le Corre D, Knebelmann B, Nusbaum P, Tomkiewicz C, Meria P, Flinois JP, Beaune P, Legendre C, Anglicheau D: Rapamycin inhibits human renal epithelial cell proliferation: effect on cyclin D3 mRNA expression and stability. Kidney Int 2005, 67:2422-33. 35. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivières S, Mercep L, Ferrari S: Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem 1998, 273:14424-9. doi:10.1186/1742-2094-8-1 Cite this article as: Lisi et al.: The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes. Journal of Neuroinflammation 2011 8:1. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Journal of Neuroinflammation – Springer Journals

Published: Jan 5, 2011

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