G16-mediated Activation of Nuclear Factor κB by the Adenosine A1 Receptor Involves c-Src, Protein Kinase C, and ERK Signaling*

The Gi-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-κB (NFκB). Here we demonstrate that an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), activated IKKα/β phosphorylation through PTX-insensitive G proteins in human lymphoblastoma Reh cells. To delineate the mechanism of action, different PTX-insensitive G proteins were expressed in human embryonic kidney 293 cells. Only Gα16 supported the CHA-induced IKK phosphorylation and NFκB-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. Gβγ subunits also modulated IKK/NFκB, as indicated by the stimulatory actions of Gβ1γ2 and the abrogation of CHA-induced response by transducin. The participation of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/NFκB activation were demonstrated by employing specific inhibitors and dominant-negative mutants. Inhibition of c-Src and numerous intermediates along the extracellular signal-regulated (ERK) kinase cascade including Ras, Raf-1 kinase, and MEK1/2 abolished the CHA-induced IKK/NFκB activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NFκB regulation. Similar results were obtained using Reh cells. These data suggest that the G16-mediated activation of IKK/NFκB by CHA required a complex signaling network composed of multiple intermediates.

Nuclear factor-B (NFB) 1 is a ubiquitous heterodimeric transcription factor, which plays important roles in the regulation of numerous inducible genes involved in modulating inflammation, cell survival, and differentiation (1). In the rest-ing state, the NFB heterodimer is anchored and retained in the cytosol by inhibitor-B␣ (IB␣). The NFB transcription factor can be stimulated by various environmental factors including ultraviolet rays, as well as cytokines such as interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣. These extracellular signals activate a key regulatory step in the pathway, the IB kinase (IKK) complex, comprising the catalytic subunits (IKK␣ and IKK␤) and a linker subunit (IKK␥/NEMO). This kinase complex, in turn, phosphorylates IB␣ at Ser 32 and Ser 36 and signals for its ubiquitin-related degradation (2). The released NFB is then translocated into the nucleus and promotes NFBdependent transcription (3).
As one of the largest superfamilies of cell surface detectors, the heptahelical G protein-coupled receptors (GPCRs) are known to regulate numerous cellular processes, ranging from photon detection to cell differentiation. The GPCRs transduce signals through the coupling of one or more heterotrimeric G proteins to generate diverse cellular responses. Increasing evidence indicates that GPCRs can actively control gene transcription and expression in different cell types (4). Practically all GPCRs are capable of activating the mitogen-activated protein kinase (MAPK) pathways (5), thereby regulating numerous cellular responses including apoptosis (6) and inflammation (7). A variety of GPCRs have now been shown to regulate inflammation and cell survival processes by controlling the activation of NFB. They include receptors for bradykinin (8), formyl peptide (fMLP) (9), lysophosphatidic acid (10), internalin B (11), and dopamine (12,13). This list of NFB-regulating GPCRs is far from complete and is rapidly expanding.
The nucleoside adenosine controls a variety of physiological responses and the adenosine receptors can be considered as novel therapeutic targets of various diseases (14). In particular, the G i -linked adenosine A 1 receptor (A1R) has been shown to play critical roles in regulating apoptotic and inflammatory activities. The apoptotic effects induced by ethanol (15) and hydrogen peroxide (16) are attenuated by the activation of A1R, and the severity of multiple sclerosis is reduced by the administration of A1R-specific agonist in female 129/Sv mice (17). Immune responses, for instance the adherence of neutrophil to endothelium (18) and chemotaxis in human dendritic cells (19), are also regulated by A1R. However, the precise mechanisms by which A1R modulates these cellular events remain elusive. Given the central role of NFB in mediating inflammatory and immune responses, it is reasonable to predict that A1R can regulate the activity of NFB. Signals arising from A1R can be channeled via PTX-sensitive G i (20) and PTX-insensitive G 16 (21,22) proteins, and both pathways can potentially lead to the activation of NFB. The fMLP receptor can employ both G i - (23) and G 16 -dependent (24) mechanisms to activate NFB, but the ability of A1R to activate NFB has not been reported. In view of the hematopoietic-specific expression of G␣ 16 (25), it will be particularly interesting to determine its ability to link A1R activation to changes in NFB activity. In the present study, we examined the ability of A1R to stimulate the phosphorylation of IKK␣/␤ and the up-regulation of the transcriptional activity of NFB-dependent luciferase reporter in human lymphoblastoma Reh cells that endogenously express G␣ 16 (26) as well as in human embryonic kidney (HEK) 293 cells transiently expressing G␣ 16 . The possible involvement of various signaling intermediates including PKC, c-Src, and MAPKs was also determined.

EXPERIMENTAL PROCEDURES
Materials-All chemicals except for PTX were purchased from Sigma-Aldrich or CalBiochem (San Diego, CA). PTX was from List Laboratories (Campbell, CA). Cell culture reagents, including Lipofectamine Plus and Lipofectamine 2000, were from Invitrogen. The origin of cDNAs for receptors and G proteins were as described previously (22,27). The cDNAs of wild-type IB␣ and doubly mutated IB␣-AA were gifts from Dr. Alain Israel (Institut Pasteur, France) whereas Akt and its dominant-negative mutant cDNAs were obtained from Dr. Zhenguo Wu (Hong Kong University of Science and Technology, Hong Kong). The cDNAs for wild type and dominant-negative mutants of IKK␣ and IKK␤ were from Dr. Richard D. Ye (University of Illinois). The luciferase reporter gene was obtained from Clontech Laboratories (Palo Alto, CA), and the luciferin substrate kit was a product of Roche Diagnostics (Mannheim, Germany). Various antisera were products of Cell Signaling Technology (Beverly, MA) and Amersham Biosciences (Piscataway, NJ). Specific anti-G␣ antibodies were purchased from CalBiochem (San Diego, CA), PerkinElmer Life Sciences (Boston, MA), and Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Culture and Transfection-Human embryonic kidney HEK 293 cells (CRL-1573, American Type Culture Collection) were maintained at 37°C in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere containing 5% CO 2 . To establish stably transfected HEK 293 cells carrying pNFB-luc, HEK 293 cells were grown to ϳ40% confluence in a 100-mm dish. Cells were washed with fresh MEM medium 3 h before transfection, and 9 g of pNFB-luc luciferase reporter gene (Clontech) and 1 g of pcDNA3 (as a selection marker) were introduced using the calcium phosphate method. One day after transfection, cells were subjected to selection with 500 g/ml G418. HEK 293 cells stably expressing NFB luciferase reporter gene (HEK 293-NFB) were maintained in growth medium containing 250 g/ml G418. For luciferase assay, 1 day prior to transfection, HEK 293-NFB cells were seeded at a density of 15,000 cells/well into white 96-well microplates designed for luminescent work (Nunc). Cells were transfected with cDNAs encoding various receptors (12.5 ng) and G proteins (37.5 ng) using 0.2 l of PLUS and Lipofectamine reagents (Invitrogen) in 50 l of Opti-MEM per well. In the case where other signaling molecules were investigated, 10 ng of receptor, 30 ng of G protein, and 10 ng of the signaling molecule cDNAs were transfected per well instead. After 3 h, 25 l of Opti-MEM containing 30% fetal bovine serum were added to the well. For immunoblotting analysis, HEK 293 cells were seeded at 500,000 cells/well into 6-well microplates 1 day prior to transfection, and the cells were transfected with 200 ng of receptor and 400 ng of G protein cDNAs per well using Lipofectamine 2000 reagent (Invitrogen). To investigate other signaling molecules, an extra 200 ng of cDNA encoding the gene of interest was also transfected.
Human lymphoblastoma cell line Reh (CRL-8286, American Type Culture Collection) was cultured at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere containing 5% CO 2 . For immunoblotting analysis, Reh cells, serum-starved overnight, were seeded at 1 ϫ 10 6 cells/ml into a 1.5-ml Eppendorf tube for drug treatment.
Luciferase Reporter Assay-Transfectants were grown in culture medium for 30 h and then maintained in serum-free medium in the presence or absence of 10 M N 6 -cyclohexyladenosine (CHA) for 16 h. Where indicated, cells were pretreated with PTX (100 ng/ml, 4 h) and/or various kinase inhibitors (30 min) before the CHA challenge. Subsequently, the growth medium was removed and replaced by 25 l of lysis buffer provided in the Luciferase Reporter Gene Assay kit (Roche Applied Science). The 96-well microplate was shaken on ice for 30 min. Luciferase activity was determined using a microplate luminometer LB96V (EG&G Berthold, Germany). Injector M connected to lysis buffer and injector P connected to the luciferin substrate were set to inject 25 l of each component into each well. A 1.6-s delay time followed by a 2-s measuring time period was assigned to injector M whereas injector P was measured for 10 s after the luciferin was introduced into the well. Results were collected by WinGlow version 1.24 and expressed as relative luminescent units. Statistical calculation was performed using KyPlot version 2.0.
Statistical Analysis-Data were expressed as mean Ϯ S.E. of at least three independent sets of experiments. The probability of the observed difference being a coincidence was evaluated by analysis of variance and paired Student's t test using KyPlot software. Differences at values of p Ͻ 0.05 were considered significant.

Activation of A1R Induces IKK␣/␤ Phosphorylation in Human Lymphoblastic Reh Cell through PTX-insensitive G Proteins-A1R
has been shown to induce adherence in neutrophils (18) and chemotaxis (19) in plasmacytoid dendritic cells. These inflammatory events are likely to be NFB-dependent. However, no report has yet documented the functionality of A1R in regulating NFB activity. We began our study by examining the ability of A1R to activate the NFB signaling pathway in a human lymphoblastic leukemic Reh cell line. Agonist-induced phosphorylation of IKK␣/␤ was determined using a phospho-IKK␣/␤-specific antiserum. As shown in Fig. 1A, the A1Rspecific agonist CHA significantly stimulated the phosphorylation of IKK␣/␤ in a time-dependent manner in Reh cells. Maximum stimulation was observed with a 15-min CHA treatment, and the response was sustained up to 60 min. The levels of IKK␣/␤ phosphorylation increased with increasing concentrations of CHA (Fig. 1B, solid squares). Phosphorylation of IKK␣/␤ in Reh cells became detectable at 100 nM CHA and reached a maximum of over 3-fold at 10 M with an EC 50 of ϳ100 nM.
A1R has previously been demonstrated to interact with both G␣ i (20) and G␣ 16 (21,22). Having a hematopoietic lineage, the Reh cells are known to express G␣ 16 (26). To test whether the CHA-induced IKK␣/␤ stimulation was G i -dependent, Reh cells were pretreated with PTX (100 ng/ml, 16 h) to ADP-ribosylate the G i proteins. The CHA-induced phosphorylation of IKK␣/␤ in Reh cells was resistant to PTX treatment (Fig. 1B, open squares), indicating the lack of involvement of PTX-sensitive G proteins in this pathway. Collectively, these data suggested that activation of IKK␣/␤ by A1R occurred in a time-and dose-dependent manner but did not require PTX-sensitive G proteins.
Activation of IKK/NFB by A1R Is Mediated via PTX-insensitive G 16 -To identify the specific PTX-insensitive G proteins that were responsible for the A1R-mediated IKK␣/␤ phosphorylation, we employed an HEK 293-NFB cell stably expressing the NFB-dependent luciferase reporter gene for heterolo-gous expression of A1R and G␣ subunits. As a well established recombinant system, HEK 293 cells are known to express a number of PTX-insensitive G proteins including G␣ s , G␣ 12/13 and G␣ q/11 . If any of these endogenous G proteins are capable of interacting with A1R, expression of A1R alone in HEK 293-NFB cells will be sufficient to support CHA-induced luciferase activity. In contrast to the Reh cells, CHA was unable to induce IKK␣/␤ phosphorylation ( Fig. 2A, Ctrl) or NFB-dependent luciferase activity (Fig. 2B, Ctrl) in HEK 293-NFB cells expressing the A1R; CHA was, however, fully capable of inhibiting adenylyl cyclase in the transfectants (data not shown). These data implied that PTX-insensitive G proteins, which are absent in HEK 293 cells might be responsible for the CHA effects observed in Reh cells. Indeed, three such PTX-insensitive G proteins (G␣ z , G␣ 14 , and G␣ 16 ) have previously been shown to functionally interact with A1R upon co-expression with the receptor (20,21,28). Hence, G␣ z , G␣ 14 , and G␣ 16 were transiently co-expressed with A1R in HEK 293-NFB cells. Co-expression of G␣ 14 or G␣ z with A1R did not support CHAinduced IKK␣/␤ phosphorylation ( Fig. 2A) or NFB-dependent luciferase activity (Fig. 2B). The total expression of IKK␣/␤ was unaffected by the co-expression of G␣ subunits or by the application of CHA ( Fig. 2A). The ineffectiveness of G␣ 14 and G␣ z in supporting CHA-induced IKK␣/␤ phosphorylation and NFB activation was not because of the lack of expression of these G␣ subunits (Fig. 2C). In contrast, 10 M CHA stimulated IKK␣/␤ phosphorylation by up to 2-fold in HEK 293 cells co-expressing A1R with G␣ 16 (Fig. 2A). The CHA-induced phosphorylation of IKK␣/␤ was accompanied by a 4-fold increase in NFB-dependent luciferase activity (Fig. 2B) in these transfectants. Indeed, a previous study has demonstrated that the constitutively active G␣ 16 mutant (G␣ 16 QL) can activate NFB in HeLa cells (24). G␣ 16 QL was co-expressed with A1R to confirm the ability of G␣ 16 to regulate NFB in HEK 293 cells. The constitutive activity of G␣ 16 QL resulted in agonist-independent stimulation of IKK␣/␤ phosphorylation (Fig. 2D) and NFB-driven luciferase activity (Fig. 2B). The magnitudes of the G␣ 16 QL-induced responses were similar to those obtained with CHA in G␣ 16expressing cells (compare Fig. 2, A and B).
The ability of G␣ 16 to link A1R activation to the IKK/NFB pathway is in good agreement with the results obtained with Reh cells (Fig. 1). Since G␣ 16 appeared to mediate the CHAinduced IKK␣/␤ phosphorylation and NFB-dependent luciferase expression, we went on to characterize this pathway in detail. Transfectants co-expressing A1R and G␣ 16 were serumstarved in the absence or presence of PTX prior to stimulation with increasing concentrations of CHA. The NFB-driven luciferase activity was dose-dependently stimulated by CHA with an EC 50 of around 100 nM (Fig. 2E), similar to that obtained with Reh cells. The CHA-induced response was essentially unaffected by PTX throughout the agonist dose range tested. In the absence of G␣ 16 , CHA was completely unable to stimulate the NFB-dependent luciferase activity (Fig. 2E, pcDNA1), confirming that endogenous G proteins could not support A1Rmediated activation of NFB even at an agonist concentration of 100 M. The G␣ 16 dependence and PTX insensitivity of the CHA-induced NFB activation were further confirmed by Western blot analyses (Fig. 2F). The phosphorylation level of IKK␣/␤ increased with increasing concentrations of CHA, reaching a peak at around 10 M. The CHA-induced phosphorylation of IKK␣/␤ was also unaffected by PTX (Fig. 2F).
CHA-induced NFB Activation Is Inhibited by DPCPX and Mediated through IKK and IB␣-To determine the specificity of the CHA-induced response, PTX-treated HEK 293-NFB cells co-expressing A1R and G␣ 16 were incubated with 100 nM NFB activation inhibitor (APQ; 6-amino-4-(4-phenoxyphenylethylamino)quinazoline) for 30 min before the CHA challenge (Fig. 3A). APQ significantly attenuated the CHA-induced NFBdependent luciferase activity as compared with the vehicle control. Furthermore, blockade by a selective A1R antagonist DPCPX (1 mM) confirmed the specific involvement of A1R in mediating the IKK␣/␤ phosphorylation (Fig. 3B) and NFB activation (Fig. 3A). Next, we asked if dominant negative mutants of IKK␣ and IKK␤ can attenuate the CHA-induced activation of NFB. Introduction of IKK␣, IKK␤, or both into HEK 293-NFB cells did not affect the agonist-induced NFB activation (Fig. 3C). In contrast, the dominant negative mutants of IKK␣ and IKK␤ partially attenuated the CHA-mediated luciferase transcription by ϳ25 and 45%, respectively. When both dominant negative mutants were co-expressed in the cells, the CHA-induced NFB activation was suppressed by 70% (Fig.  3C). Similarly, overexpression of the NFB inhibitor protein, IB␣, significantly reduced the CHA-induced NFB-dependent luciferase activity by ϳ30% as compared with the vector control (Fig. 3D). A doubly mutated IB␣ protein (IB␣AA) in which the phosphorylation sites have been removed was then employed. IB␣AA is resistant to the induction of degradation by IKK␣/␤ and thus can inhibit NFB activation. By introducing IB␣AA into HEK 293-NFB cells in conjunction with A1R and G␣ 16 , CHA treatment was indeed significantly suppressed as compared with the vector control (Fig. 3D). The suppressive effect of this non-degradable IB␣AA protein was slightly but significantly stronger than the wild-type degradable IB␣ protein.
Involvement of the PLC␤/PKC/CaMKII Cascade in CHA-mediated IKK␣/␤ Phosphorylation and NFB Activation-We have previously established that activation of G 16 -coupled A1R leads to the stimulation of PLC␤ and Ca 2ϩ mobilization (22,27), and the subsequent activation of PKC and CaMKII is required for G␣ 16 -mediated stimulation of STAT3 (29). Thus, we examined if the PLC␤/PKC/CaMKII pathway is similarly required for G 16 -mediated stimulation of NFB. First, we employed specific inhibitors against these signaling molecules to block the CHA-induced IKK␣/␤ phosphorylation and NFB-dependent luciferase activity in PTX-treated HEK 293-NFB cells co-expressing A1R and G␣ 16 . U73122, a specific PLC␤ inhibitor, at 10 M inhibited the CHA-induced NFB-dependent luciferase expression by over 30%, whereas its inactive analogue, U73343, was totally ineffective (Fig. 4A). Similar observations were obtained with regard to CHA-induced IKK␣/␤ phosphorylation (Fig. 4B). Likewise, pretreating the transfectants with 100 nM calphostin C (Cal C; selective PKC inhibitor) significantly attenuated the CHA-induced NFB-dependent transcription of luciferase by around 40% (Fig. 4A) and the phosphorylation of IKK␣/␤ were also reduced (Fig. 4C). The possible involvement of CaMKII was examined with its selective inhibitor, KN62. At 10 M, KN62 significantly attenuated the CHA-induced responses (Fig. 4, A and D). The inactive analogue of this CaMKII inhibitor, KN92, was incapable of inhibiting the CHA-induced responses.
Next, we investigated the effects of different isoforms of PKC on A1R-mediated activation of NFB. PKC␣ and PKC⑀ were selected for the study as representatives of calcium-sensitive and -insensitive PKC isoforms, respectively. The wild-type and kinase-deficient mutant (KR) of these PKC isoforms were transfected into the cells along with A1R and G␣ 16 . Expressions of wild-type PKC␣ and PKC⑀ enhanced the CHA-induced NFB-dependent luciferase activity by 25 and 30%, respectively, as compared with the vector control (Fig. 4E). Correspondingly, these wild-type PKC proteins enhanced the CHAinduced phosphorylation of IKK␣/␤ (Fig. 4F). In contrast, the KR mutants of PKC␣ and PKC⑀ significantly inhibited the A1R-mediated luciferase expressions by over 50% compared with the vector control (Fig. 4E) and comparable results were obtained by monitoring the phosphorylation of IKK␣/␤ (Fig.  4F). Collectively, these results demonstrate the participation of  1). B, for luciferase assays, HEK 293-NFB cells were transfected with A1R (12.5 ng/well) and different G proteins (37.5 ng/well) using Lipofectamine PLUS reagent for 30 h. Transfectants were treated with PTX for 4 h and stimulated by 10 M CHA for 16 h. Luciferase activities were analyzed and expressed as a percentage of the corresponding basal NFB activities. In the case of G␣ 16 QL, serum-free medium was used instead of CHA treatment for 16 h; % stimulation was in reference to the basal NFB activity of G␣ 16 -transfectants. *, significantly higher than the control; paired Student's t test, p Ͻ 0.05. §, significantly higher than the G␣ 16 basal control; paired Student's t test, p Ͻ 0.05. C, expression of different exogenous G␣ subunits was detected using specific anti-G␣ antibodies. Cell lysates from parental (C) and transfected (T; as in A) HEK 293 cells were immunoblotted. D, HEK 293 cells were transfected as described in A. Transfectants were serum-starved, lysed, and assayed for basal IKK␣/␤ activity without CHA treatment. E and F, HEK 293-NFB cells were transfected with G␣ 16 and A1R. Where indicated, transfectants were treated with 100 ng/ml PTX for 4 h prior to the CHA treatment. Different concentrations of CHA were used to stimulate the NFB-dependent luciferase expression for 16 h in E and IKK phosphorylation for 10 min in F. Cell lysates were analyzed by immunodetection as described in the legend to G␤␥ Plays a Role in A1R-mediated IKK␣/␤ Phosphorylation and NFB Activation-Because a number of studies have implicated the involvement of G␤␥ in GPCR-mediated regulation of NFB (8,13), we asked if G␤␥ also plays a role in G 16mediated activation of the IKK/NFB pathway. To test this hypothesis, transducin (G␣ t ) was co-expressed in conjunction with A1R and G␣ 16 and potential interference from G i -associated G␤␥ was eliminated by PTX treatment. Acting as a G␤␥ scavenger, co-expression of G␣ t significantly reduced CHAstimulated luciferase activity by ϳ40% as compared with the vector control (Fig. 5A). As shown in Fig. 5B, the co-expression of G␣ t attenuated the CHA-induced IKK␣/␤ phosphorylation. To confirm the participation of G␤␥ subunits in G 16 -mediated NFB activation, we overexpressed G␤ 1 and G␥ 2 along with A1R and G␣ 16 . Co-expression of G␤ 1 ␥ 2 stimulated the CHAinduced NFB-dependent luciferase activity by over 80% (Fig.  5A). The stimulatory signal was primarily carried by G␤ 1 because its overexpression, but not that of G␥ 2 , enhanced (by ϳ45%) the CHA-induced NFB activation. In agreement with the luciferase reporter data, the enhancement of NFB activation was accompanied by increased phosphorylation of IKK␣/␤ (Fig. 5C). These results suggest that G␤␥ subunits, such as G␤ 1 ␥ 2 , released from activated G 16 are indeed involved in CHA-induced activation of IKK␣/␤ and NFB.
Ras but Not Rac1 Mediates the Activation of IKK␣/␤ Phosphorylation and NFB-A number of studies have illustrated that small GTPases are involved in the NFB activation (11,30). To define the role of the small GTPases in the activation of NFB mediated by A1R, we transiently transfected the wildtype (WT) and constitutively active (CA) mutants of Ras and Rac1 into HEK 293-NFB and evaluated the NFB-dependent transcription of luciferase (Fig. 6A). Overexpression of wildtype Ras and Rac1 proteins did not alter the basal NFBinduced luciferase production. However, the constitutively active mutant of Ras (RasCA) was capable of stimulating the NFB-dependent transcription by ϳ3-fold whereas the constitutively active mutant of Rac1 (RacCA) was ineffective (Fig.  6A). To further investigate the involvement of these small GTPases in the A1R-stimulated IKK phosphorylation, we then introduced the dominant negative mutants of Rac1 (RacDN) and Ras (RasDN) into the cells (Fig. 6B). Indeed, overexpression of RasDN abrogated the CHA-stimulated IKK␣/␤ phosphorylation whereas RacDN failed to intervene. These data suggest that the activation of IKK/NFB cascade requires only Ras, but not Rac1.
ERK Is Important in the G 16 -mediated Regulation of IKK/ NFB by A1R-Activation of NFB activity by G i -coupled receptor through ERK has been described previously (13). Because the constitutively active G␣ 16 QL can activate all three MAPKs in HEK 293 cells (29), we asked if the coupling of A1R to G␣ 16 can similarly stimulate the MAPKs. HEK 293 cells were transfected with A1R and G␣ 16 , treated with PTX, and then stimulated by CHA (10 M) for 10 min. Immunodetection with phosphospecific antisera revealed that all three MAPKs were activated upon CHA treatment (data not shown). Next, to investigate the involvement of MAPKs in the activation of IKK/NFB signaling cascade, a panel of MAPK inhibitors was applied to HEK 293-NFB cells co-expressing the A1R and G␣ 16 . Raf-1 kinase inhibitor (10 M) and the selective MEK1/2 inhibitors (PD98059 and U0126; each at 10 M) inhibited the CHA-induced NFB-dependent luciferase activity by 30 -50% (Fig. 7A). In contrast, the inactive analogue of U0126 (U0124; 10 M) failed to attenuate the luciferase activity. The CHA-induced IKK␣/␤ phosphorylation were similarly affected by these inhibitors (Fig. 7B). U0126, raf-1 kinase inhibitor, and PD98059, but not U0124, were able to inhibit CHA-induced ERK phosphorylation (Fig. 7B). The effect of JNK on IKK/ NFB signaling was examined using SP600125, a selective JNK inhibitor. The application of SP600125 (10 M) neither affected the NFB-induced luciferase expression (Fig. 7A) nor the phosphorylation of IKK␣/␤ (Fig. 7C). Finally, for p38 MAPK, two p38 MAPK inhibitors, SB202190 and SB203580 at 10 M were also unable to inhibit the CHA-induced luciferase transcription (Fig. 7A) and IKK␣/␤ phosphorylation (Fig. 7D). These findings clearly demonstrate the participation of ERK, but not JNK or p38 MAPK, in G 16 -mediated activation of IKK/ NFB by A1R.
c-Src Participates in the G␣ 16 -mediated IKK/NFB Activation-In the activation of NFB by the G i -coupled dopamine D 2 receptor, the participation of the c-Src kinase is clearly evident (13). Thus, we sought to investigate whether c-Src is also involved in G 16 -mediated activation of IKK/NFB. We first examined if c-Src can be activated by A1R. As shown in Fig. 8A, 10 M CHA stimulated the phosphorylation of c-Src at Tyr 416 in HEK 293 cells co-expressing A1R and G␣ 16 . A selective c-Src kinase inhibitor (PP2) was then used to confirm its involvement in CHA-induced IKK/NFB activation. PP2 at 10 M for 30 min significantly attenuated the CHA-induced NFB-dependent luciferase activity, whereas the inactive PP3 had no inhibitory effect under identical conditions (Fig. 8B). Similar results were obtained on the ability of PP2 to inhibit CHAinduced phosphorylation of IKK␣/␤ (Fig. 8C). Additionally, the effect of overexpressing c-Src on CHA-induced activation of IKK/NFB was examined. Co-expression of wild-type c-Src with A1R and G␣ 16 did not affect the ability of CHA to stimulate NFB-dependent luciferase expression (Fig. 8D). When the dominant negative mutant of c-Src was co-expressed, the CHAinduced luciferase activity was attenuated by over 40% as compared with the level generated by the wild-type c-Src (Fig.  8D). In agreement with the reporter gene assays, overexpres-FIG. 5. NFB activation by CHA treatment is inhibited by expression of transducin (G␣ t ) and stimulated by the overexpression of G␤ 1 ␥ 2 . A, HEK 293-NFB cells were transfected with G␣ t (10 ng/well) or G␤ 1 and/or G␥ 2 (5 ng/well each) in conjunction with A1R (10 ng/well) and G␣ 16 (30 ng/well). Transfectants were pretreated with PTX and followed by 10 M CHA for 16 h. B and C, HEK 293 cells were transfected with G␣ t (200 ng/well) or G␤ 1 and G␥ 2 (100 ng/well) together with A1R (200 ng/well) and G␣ 16 (400 ng/well). Subsequent to PTX treatment, the transfectants were treated with 10 M CHA for 10 min. Cell lysates were analyzed as described in the legend to Fig. 2  sion of the dominant negative mutant of c-Src, but not wildtype c-Src, suppressed the CHA-induced phosphorylation of IKK␣/␤ (Fig. 8E). These data suggest that c-Src is involved in G 16 -mediated activation of IKK/NFB by A1R.
CHA-induced IKK␣/␤ Phosphorylation in Human Reh Cells Also Requires PLC/PKC/CaMKII, ERK, and c-Src-To test whether the different signaling intermediates identified from the recombinant HEK 293 system were also involved in A1Rinduced IKK/NFB activation in Reh cells, specific kinase inhibitors were employed. As shown in Fig. 9A, the involvement of PLC␤/PKC/CaMKII cascade was confirmed. Treatment of Reh cells with U73122, Cal C, and KN62 significantly inhibited the CHA-induced IKK phosphorylation whereas the inactive analogues U73343 and KN92 were ineffective. Among the three MAPKs, again only inhibition of ERK was capable of attenuating the activation of IKK, as demonstrated by the application of Raf-1 inhibitor and U0126 (Fig. 9B). Neither the inactive analogue of U0126 (U0124) nor JNK and p38 MAPK inhibitors generated an inhibitory effect on the CHA-induced IKK activation. Lastly, the c-Src inhibitor, PP2, notably reduced the effect of CHA in stimulating IKK and the failure of its inactive analogue PP3 verified the specific involvement of c-Src (Fig.  9C). These data demonstrated that the A1R-induced activation of IKK␣/␤ in Reh cells also required CaMKII, PKC, ERK, and c-Src. DISCUSSION Adenosine has recently been shown to possess anti-inflammatory actions in rodents (34) and it inhibits cytokine production in mature plasmacytoid dendritic cells (19). Given that NFB is an important transcription factor in regulating inflammatory and immune responses, adenosine receptors may employ G protein-dependent pathways to modulate the NFB activity. Indeed, a number of GPCRs have been shown to activate NFB via G i -or G q -dependent pathways (8 -13, 23, 24).
Numerous signaling components have been implicated in G protein-dependent activation of NFB but there is no clear indication as to which pathway is predominant. The signaling specificity becomes even more complicated for those GPCRs that employ multiple G proteins for signal transduction. The present study provides evidence to support a role of G 16 in A1R-mediated activation of NFB in human lymphocytic Reh and HEK 293 cells.
Although A1R is functionally coupled to G i proteins in HEK 293 cells (20), it cannot utilize endogenous G i pathways to activate NFB in both cell types ( Figs. 1 and 2). In contrast, co-expression of G␣ 16 allows A1R to efficiently stimulate the IKK/NFB pathway (Fig. 2). The collective use of the A1R selective antagonist DPCPX, IB␣ and its non-degradable mutant, IKK␣/␤ and their dominant-negative mutants, as well as the NFB activation inhibitor APQ confirmed the specificity of the pathway. The PTX insensitivity of CHA-induced NFB activation in Reh cells signifies the involvement of G q or G 12 family members. As G␣ 16 is present in the Reh cells (26) and is known to interact with A1R (21,22), while neither G␣ q/11 nor G␣ 13 has been reported to associate with A1R, the activation signal for NFB is presumably transmitted via G 16 . Mechanistically, both cell types appear to employ a complicated network of intermediates for signal propagation. They include an effector (PLC␤), a small GTPase (Ras), a non-receptor tyrosine kinase (c-Src), serine kinases (Raf-1, PKC, and CaMKII), and also a MAPK (ERK). These signaling molecules can be divided into two major cascades: PLC␤/PKC/CaMKII, and Ras/Raf-1/ MEK/ERK (Fig. 10).
Regulation of NFB by G q proteins has previously been suggested to depend on both G␣ and G␤␥ subunits. G␣ q stimulates NFB via the PLC␤/PKC/CaMKII pathway (10) whereas G␤␥ propagates the signal through phosphatidylinositol-3 kinase cascade (8). Both arms of the signal appear to operate in the G 16 -mediated activation of NFB. Inhibition of CHA-induced IKK/NFB activities by U73122, calphostin C, and KN62 provides evidence that PLC␤ and its downstream effectors modulate the NFB activation. Both Ca 2ϩ -dependent and -independent PKCs have been shown to regulate NFB. The Ca 2ϩsensitive PKC␣ is capable of activating the IKK complex in T-lymphocytes (32) while the Ca 2ϩ -independent PKC⑀ is implicated in the modulation of IKK activity based on the use of PKC⑀-deficient mice (33). Indeed, our data support a role of PKC␣ and PKC⑀ in G 16 -mediated activation of IKK/NFB by CHA (Fig. 4). However, other PKC isoforms may also be involved since the G i -coupled A1R is known to regulate c-fos through PKC (34). The deployment of PLC␤ and PKC␣ in G 16 -mediated activation of NFB by A1R resembles that of the formyl peptide receptor (24). Elevation of intracellular Ca 2ϩ level can also alter the activity of NFB through the actions of CaMKs. In particular, CaMKII is known to mediate phorbol ester-induced activation of IKK (35). The fact that KN62, but not KN92, suppresses the CHA-induced IKK phosphorylation and NFB-dependent luciferase activity (Fig. 4) implicates the involvement of CaMKII. Thus, the classical PLC␤/PKC/ CaMKII cascade appears to play an important role in the regulation of IKK/NFB by the G 16 -coupled A1R.
Despite the fact that G␣ 16 can propagate stimulatory signals to NFB, the released G␤␥ also takes part in the regulation. Attenuation by the co-expression of G␣ t (a G␤␥ scavenger) and potentiation by the overexpression of G␤ 1 ␥ 2 confirm the participation of G␤␥ in G 16 -mediated activation of NFB by A1R. Signals arising from G␣ and G␤␥ subunits are often integrated at downstream loci (36,37). One locus for signal integration is the small GTPase Ras (38). Recently, the linkage of G␣ 16 to Ras is provided by a novel adaptor protein named tetratricopeptide repeat 1 (39) while G␤␥ has long been shown to activate Ras in HEK 293 cells (38). Ras is known to initiate the Raf-1/MEK/ ERK signaling cascade and ERK has previously been demonstrated to activate the IKK complex through direct interaction (40). Thus, it is not surprisingly that activation of IKK/NFB by the G 16 -coupled A1R is attenuated in the presence of Raf-1 and MEK1/2 inhibitors (Fig. 7). Moreover, the G 16 -mediated IKK phosphorylation by A1R is effectively attenuated in the presence of RasDN, whereas RasCA induces NFB-driven lu-ciferase expression (Fig. 6). The ability of ERK to exert a stimulatory effect on IKK/NFB is not shared by the other two MAPKs. Although both JNK (41) and p38 MAPK (42) can activate NFB, neither is required for G 16 -mediated stimulation of IKK/NFB by A1R (Fig. 7). The lack of involvement of JNK and p38 MAPK is further supported by the inability of Rac1DN to suppress the G 16 -mediated activation of IKK/NFB (Fig. 6). Stimulation of A1R by CHA can, nevertheless, lead to the activation and phosphorylation of JNK and p38 MAPK, and such activities can be effectively abolished by specific inhibitors of the two kinases. The requirement of Ras/Raf-1/MEK/ERK pathway, but not JNK or p38 MAPK, for G 16 -mediated activation of IKK/NFB is highly reminiscent of the regulation of STAT3 by G␣ 16 (29).
Another site for possible signal integration is c-Src, a nonreceptor tyrosine kinase, which becomes phosphorylated upon activation of A1R (Fig. 8A). The combined use of selective inhibitors and dominant negative mutants of c-Src (Fig. 8, B-E) clearly demonstrates the involvement of c-Src in G 16 -mediated stimulation of IKK/NFB by A1R. Although constitutively active G␣ 16 can activate c-Src in HEK 293 cells, the interaction is probably indirect because G␣ 16 does not directly associate with c-Src (29). It is interesting to note that in human epithelial cells, TNF-␣-induced cyclooxygenase-2 expression is mediated via c-Src/NFB in a PKC-dependent manner (43). Similarly, suppression of the c-fos gene promoter by c-SrcDN indicates that c-Src is required for CaMKII-induced activity in cultured rat mesangial cells (44). As both PKC and CaMKII participate in G 16 -mediated activation of NFB (Fig. 4), it is conceivable that they can bridge the gap between G␣ 16 and c-Src. Additionally, c-Src can be activated by G␤␥ (45) even though no direct binding between them can be established (46). Indeed, activation of NFB by the dopamine D 2 receptor in HeLa cells is mediated via c-Src in a G␤␥-dependent manner (13). Direct interaction between c-Src and the IKK complex (47) leads to the phosphorylation of IKK (43) and, subsequently, IB␣ (48). These findings provide a pathway connecting the agonist-stimulated GPCR and G proteins to IKK/NFB.
In considering the signal routing from G 16 to NFB, it is important to note that signal diversification as well as convergence may occur at multiple loci (Fig. 10). For instance, c-Src is known to play regulatory roles in Ras (45) and ERK (49) sig- FIG. 10. A mechanistic model of G 16mediated activation of IKK/NFB by A1R. CHA-bound A1R activates G␣ 16 and releases G␤␥. The activated G␣ 16 stimulates the PLC␤ cascade. Through both G␣ and G␤␥, Ras/Raf-1/MEK1/2/ERK cascade becomes activated and c-Src is activated indirectly. These activations lead to the phosphorylation of IKK␣/␤ and signal IB␣ degradation. The released NFB is translocated to the nucleus and promotes transcription of the luciferase reporter. Solid-lined arrows illustrate findings based on previous studies and the putative interactions are indicated with dashlined arrows. The experimental evidence supporting individual pathways and the interactions between their intermediates are described in the text.
naling, and each of these regulatory intermediates has been shown to modulate the NFB activation through the upstream IKK complex. The present study has revealed an intricate signaling network for G 16 -coupled receptors to regulate IKK/ NFB pathway. Some of the signaling intermediates such as PLC␤ and PKC␣ have previously been shown to mediate the activation of NFB by constitutively active G␣ 16 (24). Other molecular players like G␤␥ and c-Src are known to be required for G i -mediated stimulation of NFB (13). Yet we are far from fully appreciating all the intricacies of the complex signaling network. Because dysregulation of NFB activity has been implicated in the pathogenesis of a variety of human diseases, G 16 -coupled receptors may represent attractive targets for therapeutic intervention. This is especially applicable to inflammatory and immune diseases as G␣ 16 is primarily expressed in hematopoietic cells.