The mitochondrial CIC (citrate carrier) catalyses the efflux of citrate from the mitochondrial matrix in exchange for cytosolic malate. In the present paper we show that CIC mRNA and protein markedly increase in lipopolysaccharide-activated immune cells. Moreover, CIC gene silencing and CIC activity inhibition significantly reduce production of NO, reactive oxygen species and prostaglandins. These results demonstrate for the first time that CIC has a critical role in inflammation.
Inflammation is an immune response to infection, tissue injury and other noxious insults . It is characterized by the production of inflammatory mediators such as cytokines, PGE2 (prostaglandin E2), NO and ROS (reactive oxygen species) by cells of the innate and adaptive immune system. The nuclear transcription factor NF-κB (nuclear factor κB) is a pivotal orchestrator of inflammation [2,3]. Exposure of cells to LPS (lipopolysaccharide) or other pro-inflammatory stimuli leads to the activation of NF-κB, which stimulates target gene expression . Many studies have shown that fatty acids modulate NF-κB activation and macrophage functions [5,6]. However, until now there has been no publications on the role of the mitochondrial CIC (citrate carrier) in inflammation. CIC is an integral inner mitochondrial membrane protein that catalyses the export of citrate from the mitochondrial matrix in exchange for cytosolic malate [7,8]. This transporter is essential for fatty acid biosynthesis because citrate in the cytosol is cleaved to acetyl-CoA and oxaloacetate by citrate lyase. Acetyl-CoA is directly used for fatty acid synthesis, and oxaloacetate produces NADPH plus H+ (also necessary for fatty acid production) via malate dehydrogenase and malic enzyme .
In the present study we show that CIC expression increases in LPS-activated immune cells and CIC silencing as well as CIC activity inhibition reduces the production of NO, ROS and PGE2 in LPS-activated U937 cells. Moreover, evidence is provided that CIC gene expression is up-regulated by NF-κB. To our knowledge, this is the first study to state the key role of CIC in the production of inflammatory mediators.
Cell culture, RNA interference and transient transfection
The PBMC (peripheral blood mononuclear cell) fraction was obtained as reported in the Supplementary Experimental section (at http://www.BiochemJ.org/bj/438/bj4380433add.htm). Human monocytic/macrophage cells from hystiocytoma, U937 cells (ICLC HTL 94002-Interlab Cell Line Collection), were cultured as described for PBMCs (see the Supplementary Experimental section). U937 cells were differentiated with 10 ng/ml PMA (Sigma) and allowed to adhere for 24 h. Where indicated, both macrophages and U937/PMA (differentiated U937) cells were treated with 2 μg/ml bacterial LPS (Sigma) for 24 h; U937/PMA cells were treated with 20 μM Tos-Phe-CH2Cl (‘TPCK’; tosylphenylalanylchloromethane; Sigma) for 1 h and then with LPS (2 μg/ml) for 24 h; and U937/PMA cells were treated with 2 mM BTA (1,2,3-benzentricarboxylic acid; Sigma) for 4 h and then with LPS (2 μg/ml) for 24 h (NO and ROS measurements) and for 48 h (PGE2). In the latter case, to aid the entry of BTA into the cells, 25 μl of a 6:1 (v/v) mixture of Fugene HD (Roche) and RPMI 1640 medium with and without BTA at a final pH of 7.3, were added to 500 μl of U937/PMA cells. After 4 h, the medium was replaced with fresh medium and the BTA-treated U937/PMA cells were incubated with LPS (2 μg/ml).
To measure CIC gene expression activity, U937/PMA cells were transiently transfected as reported previously  using 0.5 μg of pGL3 basic-LUC vector, containing the −1785/−20 bp region of the CIC gene promoter  and 10 ng of pRL-CMV (Promega) to normalize the extent of transfection . After 24 h, transfected U937/PMA cells were treated with LPS (2 μg/ml) and after a further 24 h were assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega). In RNA interference experiments, U937 cells were differentiated by adding PMA and simultaneously transfected with the specific pre-designed siRNA (small interfering RNA) targeting human SLC25A1 (solute carrier family 25 member 1; s13095, Ambion) using siPORT™ NeoFX™ Transfection Agent (Ambion). After 24 h, the medium was replaced with fresh medium and the siRNA-transfected U937/PMA cells were treated with LPS (2 μg/ml); CIC mRNA and protein, ROS and NO were measured 24 h after the addition of LPS, whereas PGE2 was measured 48 h after the addition of LPS. In these experiments, siRNA (C6A-0126; Ambion) with no significant similarity to human, mouse or rat gene sequences was used as a negative control.
Real-time PCR and Western blotting
Total RNA was extracted from 1×106 U937 cells and reverse transcribed as described previously . Real-time PCR was performed as described previously . Assay-on-demand for human CIC (Hs00761590_sH) and human β-actin (4326315E) were purchased from Applied Biosystems. All transcript levels were normalized against the β-actin expression levels. For Western blot analysis, proteins were electroblotted on to nitrocellulose membranes (Bio-Rad) and treated with anti-CIC  or anti-β-actin (Santa Cruz Biotechnology) antibodies. The immunoreaction was detected by Immobilon Western Chemiluminescent HRP (horseradish peroxidase) Substrate (Millipore).
NO, ROS and PGE2 detection
Nitrite, the oxidation product of NO, was measured using the Griess reaction . For ROS analysis, the cells were incubated with 10 μM DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate; Molecular Probes) for 30 min. The fluorescence was measured by a Victor3 plate reader (PerkinElmer) at 485 nm excitation and 530 nm emission wavelengths . PGE2 was detected by the DetectX High Sensivity PGE2 Enzyme Immunoassay Kit (K018-HX1) according to the manufacturer's instructions (Arbor Assays).
Expression of mitochondrial CIC in LPS-activated macrophages and U937 cells
If activated by an inflammatory stimulus, macrophages synthesize and release modulators of inflammation, such as the eicosanoid PGE2 . As eicosanoids originate from fatty acids, in particular from arachidonic acid, we investigated whether the upstream process of the fatty acid synthesis pathway, i.e. the exit of citrate from the mitochondrial matrix and the production of acetyl-CoA, is involved in inflammatory signalling. Acetyl-CoA is produced by citrate lyase, which cleaves the citrate transported from the mitochondria to the cytosol by CIC. Total RNA, extracted from primary blood monocytes, macrophages and LPS-stimulated macrophages, was retrotranscripted and used in real-time PCR experiments. Surprisingly, an increase of approximately 15-fold and 28-fold of CIC mRNA was found in monocyte-derived macrophages and LPS-stimulated macrophages respectively, compared with undifferentiated monocytes (Figure 1A). Consistently, a parallel increase of CIC protein content in macrophages after differentiation and after activation by LPS was found (Figure 1B).
CIC expression in immune cells
In subsequent experiments, we used differentiated U937 cells, which have a large repertoire of macrophage functions and can be stimulated with LPS to mimic the inflammatory response of activated macrophages . Both real-time PCRs and Western blots revealed that CIC mRNA and protein levels were increased in U937/PMA cells and even more in LPS-activated U937 cells as compared with untreated U937 cells, although to a lesser extent than macrophages in the presence and absence of LPS as compared with monocytes (Figures 1C and 1D). These findings clearly show that CIC expression significantly increases in immune system cells, suggesting that CIC can play a role in inflammation.
Effect of CIC silencing on the production of inflammatory molecules
To understand the role of CIC in the inflammatory pathway, we extended our investigation on a possible relationship between CIC expression and molecules involved in inflammation. U937 cells were transfected with siRNA targeting human CIC or control siRNA, differentiated by adding PMA, stimulated with LPS and assayed for CIC expression, and NO, ROS and PGE2 levels. CIC silencing efficiency was confirmed by a significant decrease in CIC mRNA and protein levels (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/438/bj4380433add.htm). The amount of NO, ROS and PGE2 was significantly decreased in silenced PMA-differentiated and LPS-stimulated U937 cells as compared with non-silenced cells (Figure 2). These results indicate a direct involvement of CIC in the inflammatory pathway.
Effect of CIC silencing on the inflammatory response
Effect of the CIC inhibitor BTA on LPS-induced inflammation
We also tested the effect of BTA, a specific inhibitor of CIC [20,21], on the production of the inflammatory mediators NO, ROS and PGE2. In this case, U937/PMA cells were treated with BTA, activated by LPS and assayed for NO, ROS and PGE2. As shown in Figure 3, a marked reduction in NO, ROS and PGE2 production was found in BTA-treated U937 cells compared with untreated cells. The BTA delivery into the cells was also verified by staining intracellular lipids (see Supplementary Figure S2 at http://www.BiochemJ.org/bj/438/bj4380433add.htm). These results show that CIC activity is essential for the production of the inflammatory mediators NO, ROS and PGE2.
Effect of CIC activity inhibition on the inflammatory response
The CIC gene promoter contains NF-κB active responsive elements
Finally, to clarify molecular mechanisms responsible for CIC gene activation during inflammation, we performed in silico analysis of the human CIC gene promoter and we found two NF-κB-responsive elements. NF-κB binding site activity was tested by transfecting LPS-activated U937/PMA cells with the pGL3 basic-LUC vector containing the −1785/−20 bp region of the CIC gene in the presence or absence of Tos-Phe-CH2Cl, a specific NF-κB inhibitor . Figure 4(A) shows that the luciferase gene reporter activity was approximately 50% lower in cells transfected with the CIC-pGL3 basic-LUC vector in the presence of Tos-Phe-CH2Cl than with the vector in the absence of Tos-Phe-CH2Cl. Moreover, when U937/PMA cells were treated with both LPS and Tos-Phe-CH2Cl, a reduction of CIC mRNA and protein levels was observed as compared with cells treated with LPS alone (Figures 4B and 4C). It is worth mentioning that NO, ROS and PGE2 contents measured in Tos-Phe-CH2Cl-treated cells were also diminished as expected (results not shown). These results demonstrate that the increased CIC gene expression in LPS-activated U937 cells is mediated by NF-κB, and the CIC gene represents a newly identified target of the inflammatory cascade.
Effect of Tos-Phe-CH2Cl on CIC gene expression
Until now, CIC function has been mainly related to liver and pancreas metabolism. For the first time, we show in the present study that CIC is involved in inflammation. Among the reported results, the following supporting evidence can be mentioned. Both CIC transcript and protein levels greatly increase when macrophages or differentiated U937 cells are activated by LPS. Moreover, CIC gene silencing and CIC activity inhibition drastically reduce the production of the inflammatory mediators NO, ROS and PGE2.
The outcome of our experiments can be explained on the basis of the important role played by CIC in intermediary metabolism. Indeed, CIC supplies acetyl-CoA necessary for PGE2 synthesis and NADPH plus H+ for NO and ROS production (Figure 5). At the same time, CIC gene expression is activated by NF-κB, the hallmark of inflammation, which activates virtually all of the genes whose expression is crucial in driving the inflammatory response . In fact, Tos-Phe-CH2Cl, a specific NF-κB inhibitor, strongly reduces CIC transcription in the inflammatory response.
Role for CIC in inflammation
Thus it seems reasonable to conclude that the PGE2, ROS and NO increase in inflammation is due to a direct effect of NF-κB not only on the COX2 (cyclo-oxygenase 2), iNOS (inducible NO synthase) and NADPH oxidase genes but also on the CIC gene, which causes an increased availability of cytosolic acetyl-CoA and NADPH plus H+, needed for the synthesis of all of the above-mentioned mediators. Furthermore, macrophage activation involves profound transcriptional and translational modifications, leading, among other effects, to changes in cellular metabolism . Thus other mitochondrial carriers, which catalyse the translocation of solutes across the inner mitochondrial membrane, may display altered expression levels and play a role in activated macrophages.
inducible NO synthase
nuclear factor κB
peripheral blood mononuclear cell
reactive oxygen species
small interfering RNA
Vittoria Infantino conceived and designed the experiments. Vittoria Infantino, Paolo Convertini, Liana Cucci, Maria Antonietta Panaro, Maria Antonietta Di Noia and Rosa Calvello performed the experiments. Vittoria Infantino and Paolo Convertini analysed the data. Ferdinando Palmieri and Vito Iacobazzi contributed reagents, materials and analysis tools. Vittoria Infantino, Paolo Convertini, Vito Iacobazzi and Ferdinando Palmieri wrote the paper.
This work was supported by Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (MIUR), the Center of Excellence in Genomics (CEGBA), the Fondazione Cassa di Risparmio di Puglia, the Apulia region, and the Italian Human ProteomeNet [grant number RBRN07BMCT_009].