March 2008
Volume 49, Issue 3
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Retinal Cell Biology  |   March 2008
Implication of S-Adenosylhomocysteine Hydrolase in Inhibition of TNF-α- and IL-1β-Induced Expression of Inflammatory Mediators by AICAR in RPE Cells
Author Affiliations
  • Suofu Qin
    From Retinal Disease Research, Department of Biological Sciences, Allergan, Inc., Irvine, California.
  • Ming Ni
    From Retinal Disease Research, Department of Biological Sciences, Allergan, Inc., Irvine, California.
  • Gerald W. De Vries
    From Retinal Disease Research, Department of Biological Sciences, Allergan, Inc., Irvine, California.
Investigative Ophthalmology & Visual Science March 2008, Vol.49, 1274-1281. doi:10.1167/iovs.07-1109
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      Suofu Qin, Ming Ni, Gerald W. De Vries; Implication of S-Adenosylhomocysteine Hydrolase in Inhibition of TNF-α- and IL-1β-Induced Expression of Inflammatory Mediators by AICAR in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(3):1274-1281. doi: 10.1167/iovs.07-1109.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. AMP-activated protein kinase (AMPK) has been suggested to be a novel signaling pathway in regulating inflammation. The role of AMPK in retinal pigment epithelial cell inflammatory response is addressed using AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR).

methods. Protein expression and activation of signaling molecules were detected by immunoblotting. Cytokines were determined by ELISA kits. AMPKα expression was knockdown by siRNAs.

results. AICAR inhibited tumor necrosis factor (TNF)-α- or interleukin (IL)-1β-induced production of IL-6, IL-8, and monocyte chemotactic protein (MCP)-1 and of intercellular adhesion molecule (ICAM)-1 expression in human RPE cells. The inhibitory effect on cytokine production and ICAM-1 expression persisted in the RPE cells in which AMPK was knocked down by AMPK siRNA. Moreover, an adenosine kinase inhibitor 5′-iodotubercidin, which effectively abolished AMPK activation caused by AICAR, did not reverse the anti-inflammatory effect of AICAR. In comparison, anti-inflammatory effects of AICAR were mimicked by adenosine but not inosine, the metabolites of AICAR. Finally, with the exception of TNF-α-induced IL-6 production, adenosine dialdehyde, an inhibitor of S-adenosylhomocysteine hydrolase, was found to block cytokine production and ICAM-1 expression.

conclusions. Regardless of the ability of AICAR to activate AMPK, the inhibitory effects of AICAR on cytokine production and ICAM-1 expression were not associated with AMPK. The mechanism of AICAR inhibition may be attributed to the interference of adenosylmethionine-dependent methylation.

The retinal pigment epithelium (RPE) is a monolayer of pigmented cells forming a part of the blood-retina barrier. 1 The basal membrane of the RPE is in contact with Bruch membrane, whereas the apical surface faces the photoreceptor outer segments. The close structural interactions of RPE cells with the outer retina indicate that the major functions of the RPE layer are to maintain the survival and normal functioning of photoreceptors by controlling the exchange of nutrients and waste products, 2 controlling the volume and chemical composition of the subretinal space, 3 phagocytizing shed outer segments, 3 shuttling retinoids to synthesize visual pigments, 4 and producing immunologic factors to establish immune privilege of the eye and to mediate inflammatory responses. 5 6 Failure of any one of these functions can result in degeneration of the retina and loss of visual function. 
Age-related macular degeneration (AMD), consisting of atrophic and exudative AMD, is an idiopathic retinal degenerative disease that predominates in the elderly in the Western world as a cause of irreversible, profound vision loss. 7 Atrophic AMD, characterized by drusen formation and geographic atrophy, accounts for approximately 75% of cases. Exudative AMD is characterized by choroidal neovascularization (CNV) under the RPE and retina, with subsequent hemorrhage and retinal detachment. 8 The molecular mechanisms whereby pathogenic factors contribute to the development of AMD remain elusive. However, growing evidence indicates that inflammation contributes to disease formation. The inflammatory response evolves in the early asymptomatic and dry drusenoid stage and in the early stage of CNV. 9 Initial evidence for the role of inflammation in CNV formation is derived from anatomic studies. 10 Further support for a role of inflammation in CNV comes from macrophage depletion and knockout mouse studies. Macrophage depletion inhibits experimental CNV. 11 12 CNV induction is also markedly decreased in intercellular adhesion molecule (ICAM)-1 or CD18 (ICAM-1 receptor)-deficient mice, 13 suggesting that leukocyte infiltration plays an important role in the angiogenic reaction. Molecular evidence for the role of inflammation in drusen biogenesis, the biomarker of atrophic AMD, has recently been described by Hageman, 6 Johnson, 14 and Anderson, 15 and an inflammation hypothesis for drusen biogenesis has been proposed. 6 In this model, entrapped RPE debris between the RPE basal lamina and Bruch membrane is construed as the critical seeding event in drusen formation, triggering local upregulation of proinflammatory mediators and activation of the complement cascade. RPE cells are considered a major source of proinflammatory mediators. In vivo disruption of RPE inhibits the development of experimental autoimmune uveitis. 16 Proinflammatory cytokines, phagocytosis of oxidized photoreceptor outer segments, or complement fragments lead to the release of chemotactic cytokines such as interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, and expression of ICAM-1. 17 18 19 20 ICAM-1 expression in concert with locally released chemokines promotes extravasation of inflammatory cells into the retina. 21  
AMP-activated protein kinase (AMPK) is a metabolic-sensing Ser/Thr kinase expressed in all cell types and exists as a heterotrimer consisting of a catalytic α subunit and regulatory β and γ subunits. 22 The catalytic subunit of AMPKα has two major isoforms, α1 and α2. The α1 isoform is primarily cytoplasmic, whereas α2 is predominantly nuclear and plays a role in transcriptional regulation. 23 24 25 Through Thr172 phosphorylation by an upstream kinase LKB1, 26 AMPK is activated by energy deficiency to coordinate a switch from ATP-consuming pathways to catabolic pathways, such as carbohydrate and fatty acid metabolism, to produce a positive energy balance. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) is a commonly used indirect activator of AMPK. AICAR enters cells through the adenosine transporter and is quickly phosphorylated to 5-amino-imidazole-4-carboxamide-1-β-d-ribotide (ZMP). A rise in intracellular ZMP results in the activation of AMPK by mimicking AMP. 27 AICAR has been shown to inhibit experimental autoimmune encephalomyelitis by blocking adhesion molecule expression on endothelial cells 28 and proinflammatory cytokine production from glial cells 29 in the central nervous system, implicating a possible role of AMPK in inflammatory processes. We therefore hypothesize that AMPK might regulate RPE cell inflammatory response to proinflammatory cytokines. The goal of this study was to provide direct evidence that AMPK regulates the synthesis of inflammatory mediators and to elucidate the mechanism(s) by which AMPK inhibits RPE cell biosynthesis of inflammatory mediators. In contrast to our hypothesis, we found that although AICAR does indeed inhibit IL-1β- or TNFα-induced increases in IL-6, IL-8, MCP-1, and ICAM-1, it does so independently of AMPK activation. 
Materials and Methods
Materials
DMEM/F12 (1:1) medium and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA). AICAR, adenosine, adenosine dialdehyde, dipyridamole, iodotubercidin, and anti–β-actin antibody were from Sigma (St. Louis, MO). Polyclonal anti-ICAM-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Control siRNA and validated siRNAs targeting AMPKα1 (sense 5-GGUUGGCAAACAUGAAUUGtt-3) and AMPKα2 (sense 5-GGUUUCUUAAAAACAGCUGtt-3) were purchased from Applied Biosystems (Foster City, CA). Enhanced chemiluminescence reagents were from GE Healthcare (Piscataway, NJ). Human retinal pigment epithelium cell line ARPE19 was from ATCC (Rockville, MD). Anti-pThr172 AMPKα, anti-pSer79 ACC, and anti-AMPKα antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against AMPKα1 and AMPKα2 were obtained from Bethyl Laboratories (Montgomery, TX). Recombinant tumor necrosis factor (TNF-α), IL-1β, and ELISA kits for IL-6, IL-8, and MCP-1 were from R&D Systems (Minneapolis, MN). 
RPE Cell Culture and siRNA Transfection
Human RPE cell line ARPE19 was obtained from ATCC at passage 20, and RPE cells between passages 22 and 28 were used for experiments. ARPE19 cells have structural and functional properties characteristic of RPE cells in vivo and are a valuable cell line for in vitro studies of RPE function. 30 31 The cells were cultured 1:1 in Dulbecco modified Eagle medium/F12 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO2. Cells were passed approximately every 3 to 4 days by digestion with 0.05% trypsin/0.02% EDTA. In addition, 10 × 105 cells per 10-cm dish were seeded for 24 hours and transfected with control siRNA and validated siRNAs targeting human AMPKα1 and AMPKα2 (Ambion, Austin, TX) diluted in medium (Opti-MEM 1; Invitrogen) at a concentration of 25 nM (Lipofectamine 2000; Invitrogen). Eight hours after transfection, the medium was changed with fresh complete medium, and cells were cultivated for 24 hours before they were reseeded for experiments. 
Cell Extraction and Immunoblotting Assays
ARPE19 cells (2.5 × 105/well, 6-well plate) were seeded for 3 days. Before stimulation with agonists, cells were serum starved for 24 hours in media with 0.1% FBS. After treatment, cells were washed twice with cold PBS containing 2 mM NaF and 2 mM vanadate and then were lysed in RIPA lysis buffer. Total cell lysates were resolved by SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA), and detected with appropriate primary antibodies. The blots were subsequently incubated with secondary antibodies conjugated to horseradish peroxidase, and images were developed using the enhanced chemiluminescence system (Amersham, Piscataway, NJ). The secondary antibody was horseradish peroxidase-conjugated anti-mouse or rabbit IgG antibody from Santa Cruz Biotechnology. 
Cytokine Determination
Confluent RPE cells were preincubated with the reagents for 60 minutes, followed by 24-hour stimulation by 10 ng/mL TNF-α or IL-1β in 2 mL of 0.5% FBS-containing media. The protein concentrations of IL-6, IL-8, and MCP-1 in the culture media were assayed by ELISA using protocols provided by the manufacturer (R&D Systems). 
Statistical Analysis
Statistical significance was determined by paired two-tailed Student’s t-test. P < 0.05 was considered significant for all experiments. Values were presented as mean ± SEM. 
Results
AICAR Inhibits TNF-α- or IL-1β-Induced Production of IL-6, IL-8, and MCP-1 In Vitro
RPE cell activation has been linked to the development of AMD as a result of the expression of inflammatory mediators. Major cytokines produced by RPE cells include IL-6, IL-8, and MCP-1. TNF-α and IL-1β have been routinely used to induce inflammatory cytokine responses in vitro. Thus, to mimic the inflammatory responses, RPE cells were treated with 10 ng/mL TNF-α or IL-1β, and protein levels of IL-6, IL-8, and MCP-1 in culture medium were determined 24 hours later. Basal levels of IL-6, IL-8, and MCP-1 were 31 ± 4.3, 82 ± 20, and 2206 ± 212 pg/mL, respectively. TNF-α treatment raised the levels of IL-6, IL-8, and MCP-1 to 54-, 128-, and 29-fold of control (Fig. 1A) , whereas IL-1β stimulation led to an increase in IL-6, IL-8, and MCP-1 by 143-, 104-, and 15-fold over control, respectively (Fig. 1B) . To investigate the effect of AICAR on the TNF-α- or IL-1β-triggered inflammatory response, confluent RPE cells were preincubated with different concentrations of AICAR for 1 hour before stimulation with TNF-α or IL-1β for 24 hours. AICAR dose dependently inhibited the production of IL-6, IL-8, and MCP-1 induced by TNF-α and IL-1β. Pretreatment with 2 mM and 1 mM AICAR reduced approximately 90% of TNF-α- and IL-1β-induced production of IL-6, IL-8, and MCP-1 (Fig. 1) , suggesting that AICAR can abolish TNF-α- or IL-1β-induced inflammatory responses in RPE cells. 
AICAR Activates AMPK, and AMPK Downregulates Basal Levels of IL-6 and IL-8
AICAR is an AMPK activator that is taken up into cells by a nucleoside transporter and is phosphorylated by adenosine kinase to the monophosphorylated form (ZMP) and that activates AMPK by mimicking AMP. 27 Thr172 and Ser79 phosphorylation of AMPKα and acetyl-CoA carboxylase (ACC), one of the AMPK downstream targets, respectively, were taken as markers of AMPK activation. Immunoblotting revealed that treatment with AICAR led to a transient phosphorylation of Thr172 of AMPKα and reached a peak within 30 to 60 minutes (Fig. 2A , top). Thr172 phosphorylation of AMPKα correlated with its increased capability of phosphorylating Ser79 of ACC (Fig. 2A , middle), demonstrating that AICAR activates AMPK signaling in RPE cells. 
To test whether AMPK is of functional relevance in TNF-α- and IL-1β-induced production of cytokines and to evaluate possible AMPKα isoform-specific functions, we then used siRNA to knock down AMPKα. Validated siRNA constructs complementary to AMPKα1 or AMPKα2 (Applied Biosystems) were transfected into ARPE19 cells. AMPKα expression was measured 72 hours after transfection by Western blot analysis using isoform-specific antibodies. AMPK siRNAs (α1 and α2) selectively suppressed AMPKα1 and AMPKα2 protein, respectively (Fig. 2B) . Optical density analysis of the data from three independent experiments revealed that AMPKα1 and AMPKα2 proteins were knocked down to 6% and 1% of respective controls. AMPKα1 repression caused a 37% compensational increase in AMPKα2 protein. Combined treatment with both α1 and α2 AMPK siRNAs led to 66% and 92% reductions of AMPKα1 and AMPKα2 protein, respectively. Single-dose siRNA treatment suppressed the expression of AMPKα for at least 5 days after transfection (data not shown). Without any effect on the production of MCP-1 (Fig. 2C , grey bars), the suppression of AMPKα1 selectively enhanced IL-6 production by 3.7-fold, with IL-6 levels inversely correlated with AMPKα1 protein (Fig. 2C , open bars), whereas AMPKα2 knockdown stimulated IL-8 production by fourfold (Fig. 2C , black bars). These effects appeared to be isoforms specific because combined knockdown of α1 and α2 did not further elevate the levels of IL-6 and IL-8. 
Inhibition of TNF-α-Induced Cytokine Production by AICAR Is Independent of AMPK Activation
We then addressed the requirement of AMPK for the anti-inflammatory effects of AICAR in human RPE cells. RPE cells transfected with siRNAs against α1, α2, or α1+ α2 AMPK were treated with 10 ng/mL TNF-α. The TNF-α-induced production of IL-6 (Fig. 3A) , IL-8 (Fig. 3B) , and MCP-1 (Fig. 3C)in AMPKα-knockdown cells was comparable to that in wild-type and negative siRNA-transfected RPE cells. In contrast to our hypothesis, knockdown of AMPKα did not affect the anti-inflammatory effect of AICAR because the TNF-α-induced production of IL-6, IL-8, and MCP-1 was inhibited by AICAR to the same extent in control and AMPKα-knockdown RPE cells (Fig. 3) . Similar data were observed when RPE cells were stimulated with IL-1β (data not shown). Thus, the production of IL-6, IL-8, and MCP-1 induced by TNF-α and IL-1β and the inhibition of their production by AICAR are independent of AMPK. These observations indicate that AMPK is not critical for the anti-inflammatory effect of AICAR in RPE cells, though a contribution of the AMPKα isoform or the remaining AMPKα protein in the combined α1 + α2 knockdown experiments could not be ruled out completely. 
Dipyridamole but Not Iodotubercidin Abolishes the Inhibition of AICAR on Cytokine Production Induced by TNF-α and IL-1β
Extracellular AICAR can exert its effect on cells either by cell surface receptors such as adenosine receptors or after entering cells, which is mediated by adenosine transporters. Dipyridamole, an inhibitor of nucleoside transporters, was used to examine whether AICAR executes its inhibition through extrinsic or intrinsic pathways. Dipyridamole treatment did not affect RPE cell viability and did not alter the basal levels of IL-6, IL-8, and MCP-1, but it completely abolished the inhibition of AICAR on the production of IL-6, IL-8, and MCP-1 induced by TNF-α (Fig. 4A)and IL-1β (Fig. 4B) , suggesting that the inhibition of TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1 by AICAR depends on AICAR uptake by nucleoside transporters. In comparison, AICAR inhibition of TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1 was not affected by treatment with iodotubercidin, an adenosine kinase inhibitor (Fig. 4) . Iodotubercidin at the concentration used was not cell toxic, further confirming the observation that AMPK activation by AICAR plays no role in mediating AICAR inhibition on TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1. 
Inhibition of TNF-α- and IL-1β-Induced Cytokine Production by Adenosine and Adenosine-2′, 3′-Dialdehyde
The data obtained thus far indicate that AICAR acts intracellularly independent of its conversion to ZMP, which activates AMPK. Adenosine and its analogues are reported to inhibit the inflammatory response in a variety of inflammatory diseases. 32 33 AICAR was reported to be converted to inosine in cultured Chinese hamster ovary fibroblast 34 and to raise the adenosine concentration in the rat heart. 35 With this in mind, we investigated whether the purine intermediates adenosine and inosine inhibit TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1. Confluent RPE cells were incubated with 1 mM adenosine or inosine, followed by 24-hour exposure to 10 ng/mL TNF-α or IL-1β. Treatment with adenosine but not inosine inhibited TNF-α- or IL-1β-induced the production of IL-6, IL-8, and MCP-1 (Figs. 5A 5B) . The anti-inflammatory effect of adenosine, like that of AICAR, required its intracellular translocation but was independent of adenosine conversion to AMP because the nucleoside transporter inhibitor, but not the adenosine kinase inhibitor, abrogated the inhibitory effect of adenosine on the TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1 (Figs. 5A 5B)
Adenosine can be enzymatically converted to S-adenosylhomocysteine, an inhibitor of S-adenosylmethionine-dependent methyltransferases, by S-adenosylhomocysteine hydrolase (SAHH), thereby interfering with a number of cellular methylation reactions. 36 The importance of this reaction has been inferred from experiments in which the methylation of R17 on histone 3 is required to initiate the transcription of a subset of inflammatory genes. 37 38 39 Inhibition of the TNF-α- or IL-1β-induced production of IL-6, IL-8, and MCP-1 by adenosine in RPE cells required the intracellular translocation of adenosine but was independent of AMPK activation and adenosine conversion to inosine (Figs. 5A 5B) , suggesting that the observed anti-inflammatory activity might be attributed to the formation of S-adenosylhomocysteine. We therefore explored the possible involvement of the SAHH pathway in the inflammatory response observed in RPE cells using an SAHH inhibitor, adenosine-2′, 3′-dialdehyde (adox). Treatment with 20 μM adox significantly inhibited the IL-1β-induced production of IL-6, IL-8, and MCP-1 (Fig. 5C) . In response to TNFα, adox could block only TNF-α-induced production of IL-8 and MCP-1 (Fig. 5C , black and grey bars) without any effect on IL-6 production (Fig. 5C , open bars), suggesting that the anti-inflammatory effect of adenosine (AICAR) observed in RPE cells was mediated, at least in part, by the inhibition of adenosylmethionine-dependent methyltransferases. 
Inhibition of IL-1β- or TNF-α-Induced ICAM-1 Expression by AICAR, Adenosine, and Adox
In addition to proinflammatory cytokines, cell adhesion molecules also serve as major inflammatory mediators. The ARPE19 cells expressed very low levels of ICAM-1 constitutively, as determined by Western blot analysis. Exposure of ARPE19 cells to IL-1β or TNF-α for 24 hours strongly induced the expression of ICAM-1, and preincubation with AICAR significantly inhibited ICAM-1 induction by IL-1β and TNF-α (Fig. 6A) . Knockdown of AMPKα by AMPKα siRNA did not affect IL-1β- and TNF-α-induced ICAM-1 expression or AICAR inhibition on ICAM-1 induction (Fig. 6A) . Similar to AICAR, adenosine treatment abrogated ICAM-1 induction by IL-1β and TNFα (Fig. 6B) . Inhibition of IL-1β- and TNF-α-dependent ICAM-1 induction by adenosine was abolished by dipyridamole but not by iodotubercidin, again demonstrating that intracellular translocation but not AMP conversion was critical for the anti-inflammatory effect of adenosine. Treatment with adox significantly inhibited IL-1β-dependent ICAM-1 induction but less efficiently inhibited the TNF-α-induced production of ICAM-1 (Fig. 6C) . These data imply that, similar to inhibition of cytokine production, AICAR blocks TNF-α- and IL-1β-induced ICAM-1 expression by interfering adenosylmethionine-dependent methylation reactions. 
Discussion
As a pharmacologic activator of AMPK, AICAR has recently been shown to have anti-inflammatory properties in vivo 28 and in vitro. 29 40 41 The anti-inflammatory effects of AICAR were reported to be mediated by AMPK activation, 29 40 41 though its AMPK-independent effects have also been reported. 42 43 44 In our study, AICAR mediates its anti-inflammatory effects independently of the activation of AMPK in RPE cells in response to TNF-α or IL-1β stimulation, even though AICAR does activate AMPK. The evidence obtained by molecular and pharmacologic approaches supports that AMPK is not involved in mediating the inhibition of cytokine production and ICAM-1 expression. Knockdown of AMPKα by siRNA against AMPKα does not result in the reversal of AICAR-mediated inhibition of TNF-α- or IL-1β-induced cytokine production and ICAM-1 expression (Figs. 3 6A , and data not shown). Knockdown does not mean complete knockout. AMPKα1 is the major contributor of total phosphorylated AMPK. Though it is lower in the knockdown cells, phosphorylated AMPK is still detectable (data not shown). Thus, that the remaining AMPK might be functional cannot be ruled out, even though it is unlikely. Moreover, the inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor iodotubercidin also fails to abrogate its inhibition on TNF-α- or IL-1β-induced cytokine production and ICAM-1 expression (Figs. 4 6A)
Anti-inflammatory effects of AICAR in RPE cells require its cytosolic translocation as the nucleoside transporter inhibitor completely abolishes its inhibition of cytokine production (Fig. 4) . AICAR can be converted intracellularly to adenosine, inosine, and other adenosine intermediates. 34 35 Adenosine and inosine have been reported to have anti-inflammatory activities, depending on cell type and agonist used. 32 33 In RPE cells, adenosine, but not inosine, was found to mimic the anti-inflammatory effects of AICAR in the same way—that is, the requirement of the intracellular relocation of adenosine for its anti-inflammatory activity and AMPK independence (Figs. 5A 5B 6B) . Intracellular adenosine is enzymatically metabolized to S-adenosylhomocysteine, a potent inhibitor of S-adenosylmethionine-dependent methyltransferases. 45 Elevated levels of S-adenosylhomocysteine consequently inhibit S-adenosylmethionine-dependent transmethylation reactions. 36 Methylation of R17 on histone 3 has been demonstrated to be required for initiating the transcription of a subset of inflammatory genes, including IL-8 and MCP-1. 37 38 39 S-adenosylhomocysteine is hydrolyzed to adenosine and homocysteine by SAHH. Pharmacologic inhibition of SAHH will indirectly block S-adenosylmethionine-dependent methyltransferases. Therefore, adox, a potent SAHH inhibitor, was applied to test whether the SAHH pathway is involved in mediating RPE cell inflammatory responses after stimulation with IL-1β or TNF-α. Adox indeed inhibited cytokine production and ICAM-1 expression, with the exception that it failed to affect TNF-α-induced IL-6 production (Figs. 5C 6C) , suggesting that the anti-inflammatory effects of AICAR likely result from the intracellular action of S-adenosylhomocysteine and are independent of AMPK. 
RPE cells are documented to play a role in the creation and maintenance of the immune privilege of the subretinal space by providing the outer blood-retinal barrier and by expressing cell surface molecules and secreting soluble mediators that influence the immune system. For example, thrombospondin and pigment epithelial-derived factor secreted by RPE cells inhibit the activation of lymphocytes and macrophages, respectively, 46 47 thereby limiting the severity and duration of retinal inflammation. On the other hand, RPE cells might maintain immune privilege by keeping the expression of proinflammatory mediators under control. In this regard, RPE cells constitutively express very low levels of glucocorticoid-induced TNF-related receptor ligand (GITRL). Ectopic expression of GITRL abrogated RPE-mediated immunosuppression of CD3+ T cells by inhibiting the secretion of tumor growth factor-β by RPE cells. 48 We observed that knockout of AMPKα1 selectively upregulates IL-6 expression by approximately fourfold, whereas the knockout of AMPKα2 results in a fourfold increase in IL-8 secretion without any effect on MCP-1 level (Fig. 2E) . AMPKα isoform-dependent suppression of IL-6 and IL-8 suggests that AMPK could contribute to the maintenance of RPE cell immune privilege under physiologic conditions, even though there may be no role for AMPK in regulating RPE cell pathologic inflammatory responses. In addition, the unique suppression of IL-6 and IL-8, but not MCP-1, by AMPK indicates that signal transducers other than AMPK are involved in controlling RPE cell immune privilege. As suggested by Bian et al., 17 the inflammatory induction of MCP-1 is mediated through the phosphoinositide 3-kinase pathway, but that of IL-8 is not. 
In conclusion, AICAR inhibited TNF-α- or IL-1β-induced cytokine production and ICAM-1 expression in RPE cells. This inhibition required the intracellular translocation of AICAR. Moreover, pharmacologic and genetic evidence demonstrated that anti-inflammatory effects of AICAR were independent of AMPK activation but were caused by intracellular action on indirectly interfering S-adenosylmethionine-dependent methylation reactions. Intriguingly, isoform-specific upregulation of IL-6 and IL-8 levels in AMPKα knockout RPE cells suggested that AMPK signaling might contribute to the maintenance of subretinal space immune privilege. Further experimental data are required to support this notion. 
 
Figure 1.
 
AICAR inhibits TNF-α- and IL-1β-induced cytokine production. Confluent RPE cells were preincubated with different concentrations of AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) as indicated. After 24 hours of incubation, protein concentrations of IL-6, IL-8, and MCP-1 released into the culture medium were determined using ELISA kits. Data were shown as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus the TNF-α- or IL-1β-treated group.
Figure 1.
 
AICAR inhibits TNF-α- and IL-1β-induced cytokine production. Confluent RPE cells were preincubated with different concentrations of AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) as indicated. After 24 hours of incubation, protein concentrations of IL-6, IL-8, and MCP-1 released into the culture medium were determined using ELISA kits. Data were shown as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus the TNF-α- or IL-1β-treated group.
Figure 2.
 
AMPKα1 and AMPKα2 downregulate basal levels of IL-6 and IL-8, respectively. (A) AMPKα activation by AICAR. Confluent RPE cells were treated with 2 mM AICAR for the indicated time intervals. AMPKα activation was assessed by immunoblotting with anti-pThr172 AMPKα and anti-pSer79 ACC (acetyl-CoA carboxylase, AMPK substrate). (B) Knockdown of AMPKα1 and AMPKα2 by siRNA. Human RPE cells were transfected with either control siRNA or siRNAs directed against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM), respectively. Total cell lysates were subjected to Western blotting using antibodies against AMPKα1 and AMPKα2 to detect protein levels. (C) Upregulation of IL-6 and IL-8 by knockdown of AMPKα1 and AMPKα2. Confluent RPE cells were cultured in 0.5% FBS-containing medium for 24 hours, and protein concentrations of IL-6, IL-8, and MCP-1 in culture medium were assayed by ELISA kits. Shown are mean ± SEM of three independent experiments. *P < 0.05 versus wild-type RPE cells.
Figure 2.
 
AMPKα1 and AMPKα2 downregulate basal levels of IL-6 and IL-8, respectively. (A) AMPKα activation by AICAR. Confluent RPE cells were treated with 2 mM AICAR for the indicated time intervals. AMPKα activation was assessed by immunoblotting with anti-pThr172 AMPKα and anti-pSer79 ACC (acetyl-CoA carboxylase, AMPK substrate). (B) Knockdown of AMPKα1 and AMPKα2 by siRNA. Human RPE cells were transfected with either control siRNA or siRNAs directed against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM), respectively. Total cell lysates were subjected to Western blotting using antibodies against AMPKα1 and AMPKα2 to detect protein levels. (C) Upregulation of IL-6 and IL-8 by knockdown of AMPKα1 and AMPKα2. Confluent RPE cells were cultured in 0.5% FBS-containing medium for 24 hours, and protein concentrations of IL-6, IL-8, and MCP-1 in culture medium were assayed by ELISA kits. Shown are mean ± SEM of three independent experiments. *P < 0.05 versus wild-type RPE cells.
Figure 3.
 
Knockdown of AMPKα does not prevent AICAR-induced inhibition of cytokine production. Human RPE cells were transfected with either control siRNA or siRNAs against AMPKα1, AMPKα2, or α1+α2 (final siRNA concentration, 25 nM), respectively. Confluent RPE cells were pretreated with 2 mM AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α for 24 hours in 0.5% FBS-containing medium. Cytokine levels of IL-6 (A), IL-8 (B), and MCP-1 (C) in culture media were determined by ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 3.
 
Knockdown of AMPKα does not prevent AICAR-induced inhibition of cytokine production. Human RPE cells were transfected with either control siRNA or siRNAs against AMPKα1, AMPKα2, or α1+α2 (final siRNA concentration, 25 nM), respectively. Confluent RPE cells were pretreated with 2 mM AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α for 24 hours in 0.5% FBS-containing medium. Cytokine levels of IL-6 (A), IL-8 (B), and MCP-1 (C) in culture media were determined by ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 4.
 
Dipyridamole but not iodotubercidin abolishes the inhibitory effect of AICAR. Confluent RPE cells were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 4.
 
Dipyridamole but not iodotubercidin abolishes the inhibitory effect of AICAR. Confluent RPE cells were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 5.
 
Inhibition of TNF-α- and IL-1β-induced cytokine production by adenosine and adenosine-2′,3′-dialdehyde. (A, B) Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by 1 mM adenosine or inosine for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments. (C) Confluent RPE cells in six-well plate were preincubated with 20 μM adox for 1 hour before exposure to 10 ng/mL TNF-α or IL-1β for 24 hours in 0.5% FBS-containing medium. *P < 0.05, **P < 0.01 compared with groups treated with IL-1β or TNF-α only.
Figure 5.
 
Inhibition of TNF-α- and IL-1β-induced cytokine production by adenosine and adenosine-2′,3′-dialdehyde. (A, B) Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by 1 mM adenosine or inosine for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments. (C) Confluent RPE cells in six-well plate were preincubated with 20 μM adox for 1 hour before exposure to 10 ng/mL TNF-α or IL-1β for 24 hours in 0.5% FBS-containing medium. *P < 0.05, **P < 0.01 compared with groups treated with IL-1β or TNF-α only.
Figure 6.
 
Inhibition of IL-1β- or TNF-α-induced ICAM-1 expression by AICAR, adenosine, and adox. (A) No requirement of AMPK for AICAR inhibition of IL-1β- or TNF-α-dependent ICAM-1 expression. Human RPE cells were transfected with control siRNA or siRNAs against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM) and reseeded in six-well plate. Confluent RPE cells were treated with AICAR (AI) for 1 hour, followed by 10 ng/mL IL-1β or TNF-α for 24 hours in 0.5% FBS-containing medium. Cell lysates were separated on SDS-PAGE, and protein levels were determined with anti-ICAM-1 antibody. (B) Effect of dipyridamole and iodotubercidin on adenosine inhibition of ICAM-1 expression. Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes, followed by adenosine (Ade) for 1 hour, and were then stimulated with 10 ng/mL TNF-α or IL-1β for 24 hours. (C) Differential effect of adox on ICAM-1 expression induced by IL-1β and TNF-α. Confluent RPE cells were preincubated with 10 or 20 μM adox for 1 hour, followed by 24-hour stimulation by 10 ng/mL IL-1β or TNF-α. Equal loading was verified by immunoblotting with anti–β-actin.
Figure 6.
 
Inhibition of IL-1β- or TNF-α-induced ICAM-1 expression by AICAR, adenosine, and adox. (A) No requirement of AMPK for AICAR inhibition of IL-1β- or TNF-α-dependent ICAM-1 expression. Human RPE cells were transfected with control siRNA or siRNAs against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM) and reseeded in six-well plate. Confluent RPE cells were treated with AICAR (AI) for 1 hour, followed by 10 ng/mL IL-1β or TNF-α for 24 hours in 0.5% FBS-containing medium. Cell lysates were separated on SDS-PAGE, and protein levels were determined with anti-ICAM-1 antibody. (B) Effect of dipyridamole and iodotubercidin on adenosine inhibition of ICAM-1 expression. Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes, followed by adenosine (Ade) for 1 hour, and were then stimulated with 10 ng/mL TNF-α or IL-1β for 24 hours. (C) Differential effect of adox on ICAM-1 expression induced by IL-1β and TNF-α. Confluent RPE cells were preincubated with 10 or 20 μM adox for 1 hour, followed by 24-hour stimulation by 10 ng/mL IL-1β or TNF-α. Equal loading was verified by immunoblotting with anti–β-actin.
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Figure 1.
 
AICAR inhibits TNF-α- and IL-1β-induced cytokine production. Confluent RPE cells were preincubated with different concentrations of AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) as indicated. After 24 hours of incubation, protein concentrations of IL-6, IL-8, and MCP-1 released into the culture medium were determined using ELISA kits. Data were shown as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus the TNF-α- or IL-1β-treated group.
Figure 1.
 
AICAR inhibits TNF-α- and IL-1β-induced cytokine production. Confluent RPE cells were preincubated with different concentrations of AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) as indicated. After 24 hours of incubation, protein concentrations of IL-6, IL-8, and MCP-1 released into the culture medium were determined using ELISA kits. Data were shown as the mean ± SEM of three independent experiments. *P < 0.05 and **P < 0.01 versus the TNF-α- or IL-1β-treated group.
Figure 2.
 
AMPKα1 and AMPKα2 downregulate basal levels of IL-6 and IL-8, respectively. (A) AMPKα activation by AICAR. Confluent RPE cells were treated with 2 mM AICAR for the indicated time intervals. AMPKα activation was assessed by immunoblotting with anti-pThr172 AMPKα and anti-pSer79 ACC (acetyl-CoA carboxylase, AMPK substrate). (B) Knockdown of AMPKα1 and AMPKα2 by siRNA. Human RPE cells were transfected with either control siRNA or siRNAs directed against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM), respectively. Total cell lysates were subjected to Western blotting using antibodies against AMPKα1 and AMPKα2 to detect protein levels. (C) Upregulation of IL-6 and IL-8 by knockdown of AMPKα1 and AMPKα2. Confluent RPE cells were cultured in 0.5% FBS-containing medium for 24 hours, and protein concentrations of IL-6, IL-8, and MCP-1 in culture medium were assayed by ELISA kits. Shown are mean ± SEM of three independent experiments. *P < 0.05 versus wild-type RPE cells.
Figure 2.
 
AMPKα1 and AMPKα2 downregulate basal levels of IL-6 and IL-8, respectively. (A) AMPKα activation by AICAR. Confluent RPE cells were treated with 2 mM AICAR for the indicated time intervals. AMPKα activation was assessed by immunoblotting with anti-pThr172 AMPKα and anti-pSer79 ACC (acetyl-CoA carboxylase, AMPK substrate). (B) Knockdown of AMPKα1 and AMPKα2 by siRNA. Human RPE cells were transfected with either control siRNA or siRNAs directed against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM), respectively. Total cell lysates were subjected to Western blotting using antibodies against AMPKα1 and AMPKα2 to detect protein levels. (C) Upregulation of IL-6 and IL-8 by knockdown of AMPKα1 and AMPKα2. Confluent RPE cells were cultured in 0.5% FBS-containing medium for 24 hours, and protein concentrations of IL-6, IL-8, and MCP-1 in culture medium were assayed by ELISA kits. Shown are mean ± SEM of three independent experiments. *P < 0.05 versus wild-type RPE cells.
Figure 3.
 
Knockdown of AMPKα does not prevent AICAR-induced inhibition of cytokine production. Human RPE cells were transfected with either control siRNA or siRNAs against AMPKα1, AMPKα2, or α1+α2 (final siRNA concentration, 25 nM), respectively. Confluent RPE cells were pretreated with 2 mM AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α for 24 hours in 0.5% FBS-containing medium. Cytokine levels of IL-6 (A), IL-8 (B), and MCP-1 (C) in culture media were determined by ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 3.
 
Knockdown of AMPKα does not prevent AICAR-induced inhibition of cytokine production. Human RPE cells were transfected with either control siRNA or siRNAs against AMPKα1, AMPKα2, or α1+α2 (final siRNA concentration, 25 nM), respectively. Confluent RPE cells were pretreated with 2 mM AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α for 24 hours in 0.5% FBS-containing medium. Cytokine levels of IL-6 (A), IL-8 (B), and MCP-1 (C) in culture media were determined by ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 4.
 
Dipyridamole but not iodotubercidin abolishes the inhibitory effect of AICAR. Confluent RPE cells were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 4.
 
Dipyridamole but not iodotubercidin abolishes the inhibitory effect of AICAR. Confluent RPE cells were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by AICAR for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments.
Figure 5.
 
Inhibition of TNF-α- and IL-1β-induced cytokine production by adenosine and adenosine-2′,3′-dialdehyde. (A, B) Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by 1 mM adenosine or inosine for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments. (C) Confluent RPE cells in six-well plate were preincubated with 20 μM adox for 1 hour before exposure to 10 ng/mL TNF-α or IL-1β for 24 hours in 0.5% FBS-containing medium. *P < 0.05, **P < 0.01 compared with groups treated with IL-1β or TNF-α only.
Figure 5.
 
Inhibition of TNF-α- and IL-1β-induced cytokine production by adenosine and adenosine-2′,3′-dialdehyde. (A, B) Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes followed by 1 mM adenosine or inosine for 1 hour and were then stimulated with 10 ng/mL TNF-α (A) or IL-1β (B) for 24 hours. Protein levels of IL-6, IL-8, and MCP-1 released in culture medium were determined using ELISA kits. Shown are mean ± SEM of three independent experiments. (C) Confluent RPE cells in six-well plate were preincubated with 20 μM adox for 1 hour before exposure to 10 ng/mL TNF-α or IL-1β for 24 hours in 0.5% FBS-containing medium. *P < 0.05, **P < 0.01 compared with groups treated with IL-1β or TNF-α only.
Figure 6.
 
Inhibition of IL-1β- or TNF-α-induced ICAM-1 expression by AICAR, adenosine, and adox. (A) No requirement of AMPK for AICAR inhibition of IL-1β- or TNF-α-dependent ICAM-1 expression. Human RPE cells were transfected with control siRNA or siRNAs against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM) and reseeded in six-well plate. Confluent RPE cells were treated with AICAR (AI) for 1 hour, followed by 10 ng/mL IL-1β or TNF-α for 24 hours in 0.5% FBS-containing medium. Cell lysates were separated on SDS-PAGE, and protein levels were determined with anti-ICAM-1 antibody. (B) Effect of dipyridamole and iodotubercidin on adenosine inhibition of ICAM-1 expression. Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes, followed by adenosine (Ade) for 1 hour, and were then stimulated with 10 ng/mL TNF-α or IL-1β for 24 hours. (C) Differential effect of adox on ICAM-1 expression induced by IL-1β and TNF-α. Confluent RPE cells were preincubated with 10 or 20 μM adox for 1 hour, followed by 24-hour stimulation by 10 ng/mL IL-1β or TNF-α. Equal loading was verified by immunoblotting with anti–β-actin.
Figure 6.
 
Inhibition of IL-1β- or TNF-α-induced ICAM-1 expression by AICAR, adenosine, and adox. (A) No requirement of AMPK for AICAR inhibition of IL-1β- or TNF-α-dependent ICAM-1 expression. Human RPE cells were transfected with control siRNA or siRNAs against AMPKα1 and AMPKα2 (final siRNA concentration, 25 nM) and reseeded in six-well plate. Confluent RPE cells were treated with AICAR (AI) for 1 hour, followed by 10 ng/mL IL-1β or TNF-α for 24 hours in 0.5% FBS-containing medium. Cell lysates were separated on SDS-PAGE, and protein levels were determined with anti-ICAM-1 antibody. (B) Effect of dipyridamole and iodotubercidin on adenosine inhibition of ICAM-1 expression. Confluent RPE cells in six-well plate were treated with 1 μM dipyridamole (DPY) or 0.1 μM iodotubercidin (IO) for 30 minutes, followed by adenosine (Ade) for 1 hour, and were then stimulated with 10 ng/mL TNF-α or IL-1β for 24 hours. (C) Differential effect of adox on ICAM-1 expression induced by IL-1β and TNF-α. Confluent RPE cells were preincubated with 10 or 20 μM adox for 1 hour, followed by 24-hour stimulation by 10 ng/mL IL-1β or TNF-α. Equal loading was verified by immunoblotting with anti–β-actin.
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