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Originally published In Press as doi:10.1074/jbc.M410196200 on October 12, 2004

J. Biol. Chem., Vol. 279, Issue 51, 53196-53204, December 17, 2004
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G16-mediated Activation of Nuclear Factor {kappa}B by the Adenosine A1 Receptor Involves c-Src, Protein Kinase C, and ERK Signaling*

Andrew M. F. Liu and Yung H. Wong{ddagger}

From the Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

Received for publication, September 7, 2004 , and in revised form, October 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Gi-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-{kappa}B (NF{kappa}B). Here we demonstrate that an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), activated IKK{alpha}/{beta} 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 G{alpha}16 supported the CHA-induced IKK phosphorylation and NF{kappa}B-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. G{beta}{gamma} subunits also modulated IKK/NF{kappa}B, as indicated by the stimulatory actions of G{beta}1{gamma}2 and the abrogation of CHA-induced response by transducin. The participation of phospholipase C{beta}, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/NF{kappa}B 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/NF{kappa}B activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NF{kappa}B regulation. Similar results were obtained using Reh cells. These data suggest that the G16-mediated activation of IKK/NF{kappa}B by CHA required a complex signaling network composed of multiple intermediates.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear factor-{kappa}B (NF{kappa}B)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 resting state, the NF{kappa}B heterodimer is anchored and retained in the cytosol by inhibitor-{kappa}B{alpha} (I{kappa}B{alpha}). The NF{kappa}B transcription factor can be stimulated by various environmental factors including ultraviolet rays, as well as cytokines such as interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha}. These extracellular signals activate a key regulatory step in the pathway, the I{kappa}B kinase (IKK) complex, comprising the catalytic subunits (IKK{alpha} and IKK{beta}) and a linker subunit (IKK{gamma}/NEMO). This kinase complex, in turn, phosphorylates I{kappa}B{alpha} at Ser32 and Ser36 and signals for its ubiquitin-related degradation (2). The released NF{kappa}B is then translocated into the nucleus and promotes NF{kappa}B-dependent 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 NF{kappa}B. They include receptors for bradykinin (8), formyl peptide (fMLP) (9), lysophosphatidic acid (10), internalin B (11), and dopamine (12, 13). This list of NF{kappa}B-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 Gi-linked adenosine A1 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 NF{kappa}B in mediating inflammatory and immune responses, it is reasonable to predict that A1R can regulate the activity of NF{kappa}B. Signals arising from A1R can be channeled via PTX-sensitive Gi (20) and PTX-insensitive G16 (21, 22) proteins, and both pathways can potentially lead to the activation of NF{kappa}B. The fMLP receptor can employ both Gi-(23) and G16-dependent (24) mechanisms to activate NF{kappa}B, but the ability of A1R to activate NF{kappa}B has not been reported. In view of the hematopoietic-specific expression of G{alpha}16 (25), it will be particularly interesting to determine its ability to link A1R activation to changes in NF{kappa}B activity. In the present study, we examined the ability of A1R to stimulate the phosphorylation of IKK{alpha}/{beta} and the up-regulation of the transcriptional activity of NF{kappa}B-dependent luciferase reporter in human lymphoblastoma Reh cells that endogenously express G{alpha}16 (26) as well as in human embryonic kidney (HEK) 293 cells transiently expressing G{alpha}16. The possible involvement of various signaling intermediates including PKC, c-Src, and MAPKs was also determined.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 I{kappa}B{alpha} and doubly mutated I{kappa}B{alpha}-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{alpha} and IKK{beta} 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{alpha} 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 µg/ml streptomycin in a humidified atmosphere containing 5% CO2. To establish stably transfected HEK 293 cells carrying pNF{kappa}B-luc, HEK 293 cells were grown to ~40% confluence in a 100-mm dish. Cells were washed with fresh MEM medium 3 h before transfection, and 9 µg of pNF{kappa}B-luc luciferase reporter gene (Clontech) and 1 µg of pcDNA3 (as a selection marker) were introduced using the calcium phosphate method. One day after transfection, cells were subjected to selection with 500 µg/ml G418. HEK 293 cells stably expressing NF{kappa}B luciferase reporter gene (HEK 293-NF{kappa}B) were maintained in growth medium containing 250 µg/ml G418. For luciferase assay, 1 day prior to transfection, HEK 293-NF{kappa}B 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 µl of PLUS and Lipofectamine reagents (Invitrogen) in 50 µl 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 µl 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 µg/ml streptomycin in a humidified atmosphere containing 5% CO2. For immunoblotting analysis, Reh cells, serum-starved overnight, were seeded at 1 x 106 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 µM N6-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 µl 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 µl 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.

Immunoblotting Analysis—30 h after transfection, HEK 293 cells were serum-starved overnight in the presence or absence of 100 ng/ml PTX. The cells were then challenged by 10 µM CHA for 10 min followed by cell lysis in 150 µl of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 100 mM NaCl, 1% Triton X-100, 40 mM NaP2O7, 1 mM dithiothreitol, 200 µM Na3VO4, 4 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin. When kinase inhibitors were examined, the transfectants were pretreated with different kinase inhibitors for 30 min in serum-free medium. The cell lysates were shaken for 15 min, and the supernatants were collected by centrifugation at 16,000 x g for 2 min. Clarified lysates (40 µg) were resolved on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membrane (Bio-Rad). For Reh cells, treated cells were harvested by centrifugation for 2 min at 16,000 x g. The supernatant was removed and replaced with 150 µl of lysis buffer. Phospho-IKK{alpha}(Ser180)/IKK{beta}(Ser181), IKK{alpha}, phospho-ERK(Thr202/Tyr204), ERK, phospho-JNK(Thr183/Tyr185), JNK, phospho-p38 MAPK(Thr180/Tyr182), p38 MAPK, phospho-c-Src(Tyr416), and c-Src were detected by specific primary antisera and horseradish peroxidase-conjugated secondary antisera. The immunoblots were visualized by chemiluminescence with the ECL kit (Amersham Biosciences). Images detected on x-ray films were quantified by densitometric scanning using ImageJ software.

Statistical Analysis—Data were expressed as mean ± 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 < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of A1R Induces IKK{alpha}/{beta} 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 NF{kappa}B-dependent. However, no report has yet documented the functionality of A1R in regulating NF{kappa}B activity. We began our study by examining the ability of A1R to activate the NF{kappa}B signaling pathway in a human lymphoblastic leukemic Reh cell line. Agonist-induced phosphorylation of IKK{alpha}/{beta} was determined using a phospho-IKK{alpha}/{beta}-specific antiserum. As shown in Fig. 1A, the A1R-specific agonist CHA significantly stimulated the phosphorylation of IKK{alpha}/{beta} 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{alpha}/{beta} phosphorylation increased with increasing concentrations of CHA (Fig. 1B, solid squares). Phosphorylation of IKK{alpha}/{beta} in Reh cells became detectable at 100 nM CHA and reached a maximum of over 3-fold at 10 µM with an EC50 of ~100 nM.



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  		FIG. 1.
Time- and dose-dependent activation of IKK{alpha}/{beta} by CHA in Reh cells. A, Reh cells were stimulated with 10 µM CHA for different periods of time. B, Reh cells were treated with different concentrations of CHA in the absence (closed squares) or presence (opened squares) of PTX pretreatment. Cells were stimulated by 10 µM CHA for 15 min. Cell lysates were collected and analyzed by immunoblotting for activated IKK{alpha}/{beta}. Equivalent protein loading was confirmed by immunoblotting for total IKK{alpha}. Relative intensities of CHA-induced IKK{alpha}/{beta} phosphorylation are expressed as a percentage of the basal level (set as 100%). Data shown are the mean ± S.E., and immunoblots shown are representatives of three individual sets of experiments.

 
A1R has previously been demonstrated to interact with both G{alpha}i (20) and G{alpha}16 (21, 22). Having a hematopoietic lineage, the Reh cells are known to express G{alpha}16 (26). To test whether the CHA-induced IKK{alpha}/{beta} stimulation was Gi-dependent, Reh cells were pretreated with PTX (100 ng/ml, 16 h) to ADP-ribosylate the Gi proteins. The CHA-induced phosphorylation of IKK{alpha}/{beta} 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{alpha}/{beta} by A1R occurred in a time- and dose-dependent manner but did not require PTX-sensitive G proteins.

Activation of IKK/NF{kappa}B by A1R Is Mediated via PTX-insensitive G16—To identify the specific PTX-insensitive G proteins that were responsible for the A1R-mediated IKK{alpha}/{beta} phosphorylation, we employed an HEK 293-NF{kappa}B cell stably expressing the NF{kappa}B-dependent luciferase reporter gene for heterologous expression of A1R and G{alpha} subunits. As a well established recombinant system, HEK 293 cells are known to express a number of PTX-insensitive G proteins including G{alpha}s, G{alpha}12/13 and G{alpha}q/11. If any of these endogenous G proteins are capable of interacting with A1R, expression of A1R alone in HEK 293-NF{kappa}B cells will be sufficient to support CHA-induced luciferase activity. In contrast to the Reh cells, CHA was unable to induce IKK{alpha}/{beta} phosphorylation (Fig. 2A, Ctrl) or NF{kappa}B-dependent luciferase activity (Fig. 2B, Ctrl) in HEK 293-NF{kappa}B 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{alpha}z, G{alpha}14, and G{alpha}16) have previously been shown to functionally interact with A1R upon co-expression with the receptor (20, 21, 28). Hence, G{alpha}z, G{alpha}14, and G{alpha}16 were transiently co-expressed with A1R in HEK 293-NF{kappa}B cells. Co-expression of G{alpha}14 or G{alpha}z with A1R did not support CHA-induced IKK{alpha}/{beta} phosphorylation (Fig. 2A) or NF{kappa}B-dependent luciferase activity (Fig. 2B). The total expression of IKK{alpha}/{beta} was unaffected by the co-expression of G{alpha} subunits or by the application of CHA (Fig. 2A). The ineffectiveness of G{alpha}14 and G{alpha}z in supporting CHA-induced IKK{alpha}/{beta} phosphorylation and NF{kappa}B activation was not because of the lack of expression of these G{alpha} subunits (Fig. 2C). In contrast, 10 µM CHA stimulated IKK{alpha}/{beta} phosphorylation by up to 2-fold in HEK 293 cells co-expressing A1R with G{alpha}16 (Fig. 2A). The CHA-induced phosphorylation of IKK{alpha}/{beta} was accompanied by a 4-fold increase in NF{kappa}B-dependent luciferase activity (Fig. 2B) in these transfectants. Indeed, a previous study has demonstrated that the constitutively active G{alpha}16 mutant (G{alpha}16QL) can activate NF{kappa}B in HeLa cells (24). G{alpha}16QL was co-expressed with A1R to confirm the ability of G{alpha}16 to regulate NF{kappa}B in HEK 293 cells. The constitutive activity of G{alpha}16QL resulted in agonist-independent stimulation of IKK{alpha}/{beta} phosphorylation (Fig. 2D) and NF{kappa}B-driven luciferase activity (Fig. 2B). The magnitudes of the G{alpha}16QL-induced responses were similar to those obtained with CHA in G{alpha}16-expressing cells (compare Fig. 2, A and B).



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  		FIG. 2.
CHA induces NF{kappa}B activation through G{alpha}16 in a dose- and PTX-insensitive manner in HEK 293 cells. A, HEK 293 cells were transfected with A1R (200 ng) and pcDNA1 vector control (Ctrl) or G{alpha} subunits (400 ng) including G{alpha}z, G{alpha}14, and the G{alpha}16. Cells were serum-starved overnight in presence of PTX (100 ng/ml) and then incubated in the absence or presence of CHA (10 µM) for 10 min. Relative intensities of CHA-induced IKK{alpha}/{beta} phosphorylation are expressed as fold-stimulation of the basal level (set as 1). B, for luciferase assays, HEK 293-NF{kappa}B 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 µM CHA for 16 h. Luciferase activities were analyzed and expressed as a percentage of the corresponding basal NF{kappa}B activities. In the case of G{alpha}16QL, serum-free medium was used instead of CHA treatment for 16 h; % stimulation was in reference to the basal NF{kappa}B activity of G{alpha}16-transfectants. *, significantly higher than the control; paired Student's t test, p < 0.05. §, significantly higher than the G{alpha}16 basal control; paired Student's t test, p < 0.05. C, expression of different exogenous G{alpha} subunits was detected using specific anti-G{alpha} 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{alpha}/{beta} activity without CHA treatment. E and F, HEK 293-NF{kappa}B cells were transfected with G{alpha}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 NF{kappa}B-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 Fig. 1. Data shown are the mean ± S.E. from three individual experiments. Representative results from at least three sets of experiments are shown.

 
The ability of G{alpha}16 to link A1R activation to the IKK/NF{kappa}B pathway is in good agreement with the results obtained with Reh cells (Fig. 1). Since G{alpha}16 appeared to mediate the CHA-induced IKK{alpha}/{beta} phosphorylation and NF{kappa}B-dependent luciferase expression, we went on to characterize this pathway in detail. Transfectants co-expressing A1R and G{alpha}16 were serum-starved in the absence or presence of PTX prior to stimulation with increasing concentrations of CHA. The NF{kappa}B-driven luciferase activity was dose-dependently stimulated by CHA with an EC50 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{alpha}16, CHA was completely unable to stimulate the NF{kappa}B-dependent luciferase activity (Fig. 2E, pcDNA1), confirming that endogenous G proteins could not support A1R-mediated activation of NF{kappa}B even at an agonist concentration of 100 µM. The G{alpha}16 dependence and PTX insensitivity of the CHA-induced NF{kappa}B activation were further confirmed by Western blot analyses (Fig. 2F). The phosphorylation level of IKK{alpha}/{beta} increased with increasing concentrations of CHA, reaching a peak at around 10 µM. The CHA-induced phosphorylation of IKK{alpha}/{beta} was also unaffected by PTX (Fig. 2F).

CHA-induced NF{kappa}B Activation Is Inhibited by DPCPX and Mediated through IKK and I{kappa}B{alpha}—To determine the specificity of the CHA-induced response, PTX-treated HEK 293-NF{kappa}B cells co-expressing A1R and G{alpha}16 were incubated with 100 nM NF{kappa}B activation inhibitor (APQ; 6-amino-4-(4-phenoxyphenylethylamino)quinazoline) for 30 min before the CHA challenge (Fig. 3A). APQ significantly attenuated the CHA-induced NF{kappa}B-dependent 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{alpha}/{beta} phosphorylation (Fig. 3B) and NF{kappa}B activation (Fig. 3A). Next, we asked if dominant negative mutants of IKK{alpha} and IKK{beta} can attenuate the CHA-induced activation of NF{kappa}B. Introduction of IKK{alpha}, IKK{beta}, or both into HEK 293-NF{kappa}B cells did not affect the agonist-induced NF{kappa}B activation (Fig. 3C). In contrast, the dominant negative mutants of IKK{alpha} and IKK{beta} partially attenuated the CHA-mediated luciferase transcription by ~25 and 45%, respectively. When both dominant negative mutants were co-expressed in the cells, the CHA-induced NF{kappa}B activation was suppressed by 70% (Fig. 3C). Similarly, overexpression of the NF{kappa}B inhibitor protein, I{kappa}B{alpha}, significantly reduced the CHA-induced NF{kappa}B-dependent luciferase activity by ~30% as compared with the vector control (Fig. 3D). A doubly mutated I{kappa}B{alpha} protein (I{kappa}B{alpha}AA) in which the phosphorylation sites have been removed was then employed. I{kappa}B{alpha}AA is resistant to the induction of degradation by IKK{alpha}/{beta} and thus can inhibit NF{kappa}B activation. By introducing I{kappa}B{alpha}AA into HEK 293-NF{kappa}B cells in conjunction with A1R and G{alpha}16, CHA treatment was indeed significantly suppressed as compared with the vector control (Fig. 3D). The suppressive effect of this non-degradable I{kappa}B{alpha}AA protein was slightly but significantly stronger than the wild-type degradable I{kappa}B{alpha} protein.



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  		FIG. 3.
Characterization of CHA-induced NF{kappa}B activation. HEK 293-NF{kappa}B (A) and HEK 293 (B) cells were transfected with A1R and G{alpha}16. Transfectants were stimulated 10 µM CHA in the absence or presence of 1 mM of the antagonist DPCPX. Where indicated, cells were pretreated for 30 min with Me2SO (vehicle) or 100 nM of the NF{kappa}B activation inhibitor APQ. C and D, HEK 293-NF{kappa}B cells were transfected with G{alpha}16 (30 ng/well) and A1R (10 ng/well) along with pcDNA1 vector, IKK{alpha}, IKK{alpha}DN, IKK{beta}, IKK{beta}DN, I{kappa}B{alpha}, or I{kappa}B{alpha}AA (10 ng/well). Cell lysates were analyzed as described in the legend to Fig. 2 for immunodetection and luciferase activity. Data shown are the mean ± S.E. from three individual experiments. Representative results from at least three sets of experiments are shown. *, CHA-induced response was significantly greater than the basal level; paired Student's t test, p ≤ 0.05. #, CHA-induced response was significantly lower than the vehicle control; paired Student's t test, p ≤ 0.05. §, CHA-induced response was significantly lower than the pcDNA1 vector control; paired Student's t test, p ≤ 0.05. {ddagger}, CHA-induced response was significantly lower than that obtained with I{kappa}B{alpha}; paired Student's t test, p ≤ 0.05.

 
Involvement of the PLC{beta}/PKC/CaMKII Cascade in CHA-mediated IKK{alpha}/{beta} Phosphorylation and NF{kappa}B Activation—We have previously established that activation of G16-coupled A1R leads to the stimulation of PLC{beta} and Ca2+ mobilization (22, 27), and the subsequent activation of PKC and CaMKII is required for G{alpha}16-mediated stimulation of STAT3 (29). Thus, we examined if the PLC{beta}/PKC/CaMKII pathway is similarly required for G16-mediated stimulation of NF{kappa}B. First, we employed specific inhibitors against these signaling molecules to block the CHA-induced IKK{alpha}/{beta} phosphorylation and NF{kappa}B-dependent luciferase activity in PTX-treated HEK 293-NF{kappa}B cells co-expressing A1R and G{alpha}16. U73122 [GenBank] , a specific PLC{beta} inhibitor, at 10 µM inhibited the CHA-induced NF{kappa}B-dependent luciferase expression by over 30%, whereas its inactive analogue, U73343 [GenBank] , was totally ineffective (Fig. 4A). Similar observations were obtained with regard to CHA-induced IKK{alpha}/{beta} phosphorylation (Fig. 4B). Likewise, pretreating the transfectants with 100 nM calphostin C (Cal C; selective PKC inhibitor) significantly attenuated the CHA-induced NF{kappa}B-dependent transcription of luciferase by around 40% (Fig. 4A) and the phosphorylation of IKK{alpha}/{beta} were also reduced (Fig. 4C). The possible involvement of CaMKII was examined with its selective inhibitor, KN62. At 10 µM, 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.



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  		FIG. 4.
CHA stimulates IKK/NF{kappa}B through the PLC{beta}/PKC/CaMKII cascade. A, HEK 293-NF{kappa}B; B–D, HEK 293 cells were transfected with A1R and G{alpha}16. The transfected cells were treated with PTX prior to the exposure to different kinase inhibitors. The transfectants were then treated with 1% Me2SO (vehicle), 10 µM PLC{beta} inhibitor (U73122 [GenBank] ), its negative control (U73343 [GenBank] in A and B), 100 nM calphostin C (Cal C, in A and C), 10 µM KN62 (CaMKII inhibitor), its negative control (KN92 in A and D) for 30 min and followed by 16 h treatment of 10 µM CHA for luciferase expression in A or 10 min induction for Western analysis in B–D. E, overexpression of different PKC isoforms were performed by transient transfection of HEK 293-NF{kappa}B cells with 10 ng of PKC{alpha}, PKC{alpha}KR, PKC{epsilon}, or PKC{epsilon}KR in conjunction with 10 ng of A1R and 30 ng of G{alpha}16 cDNAs per well. F, HEK 293 cells were transfected with 200 ng of PKC, 200 ng of A1R, and 400 ng of G{alpha}16 cDNAs per well. After PTX pretreatment, transfectants were stimulated with 10 µM CHA and assayed for luciferase activity or IKK phosphorylation as described in the legend to Fig. 2. Data shown are the mean ± S.E. from three individual experiments. Representative results from at least three sets of experiments are shown. *, CHA-induced response was significantly enhanced as compared with the pcDNA1 vector control; paired Student's t test, p ≤ 0.05. #, CHA-induced response was significantly decreased as compared with the controls; paired Student's t test, p ≤ 0.05.

 
Next, we investigated the effects of different isoforms of PKC on A1R-mediated activation of NF{kappa}B. PKC{alpha} and PKC{epsilon} 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{alpha}16. Expressions of wild-type PKC{alpha} and PKC{epsilon} enhanced the CHA-induced NF{kappa}B-dependent luciferase activity by 25 and 30%, respectively, as compared with the vector control (Fig. 4E). Correspondingly, these wild-type PKC proteins enhanced the CHA-induced phosphorylation of IKK{alpha}/{beta} (Fig. 4F). In contrast, the KR mutants of PKC{alpha} and PKC{epsilon} 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{alpha}/{beta} (Fig. 4F). Collectively, these results demonstrate the participation of the PLC{beta}/PKC/CaMKII pathway in G16-mediated activation of IKK/NF{kappa}B.

G{beta}{gamma} Plays a Role in A1R-mediated IKK{alpha}/{beta} Phosphorylation and NF{kappa}B Activation—Because a number of studies have implicated the involvement of G{beta}{gamma} in GPCR-mediated regulation of NF{kappa}B (8, 13), we asked if G{beta}{gamma} also plays a role in G16-mediated activation of the IKK/NF{kappa}B pathway. To test this hypothesis, transducin (G{alpha}t) was co-expressed in conjunction with A1R and G{alpha}16 and potential interference from Gi-associated G{beta}{gamma} was eliminated by PTX treatment. Acting as a G{beta}{gamma} scavenger, co-expression of G{alpha}t significantly reduced CHA-stimulated luciferase activity by ~40% as compared with the vector control (Fig. 5A). As shown in Fig. 5B, the co-expression of G{alpha}t attenuated the CHA-induced IKK{alpha}/{beta} phosphorylation. To confirm the participation of G{beta}{gamma} subunits in G16-mediated NF{kappa}B activation, we overexpressed G{beta}1 and G{gamma}2 along with A1R and G{alpha}16. Co-expression of G{beta}1{gamma}2 stimulated the CHA-induced NF{kappa}B-dependent luciferase activity by over 80% (Fig. 5A). The stimulatory signal was primarily carried by G{beta}1 because its overexpression, but not that of G{gamma}2, enhanced (by ~45%) the CHA-induced NF{kappa}B activation. In agreement with the luciferase reporter data, the enhancement of NF{kappa}B activation was accompanied by increased phosphorylation of IKK{alpha}/{beta} (Fig. 5C). These results suggest that G{beta}{gamma} subunits, such as G{beta}1{gamma}2, released from activated G16 are indeed involved in CHA-induced activation of IKK{alpha}/{beta} and NF{kappa}B.



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  		FIG. 5.
NF{kappa}B activation by CHA treatment is inhibited by expression of transducin (G{alpha}t) and stimulated by the overexpression of G{beta}1{gamma}2. A, HEK 293-NF{kappa}B cells were transfected with G{alpha}t (10 ng/well) or G{beta}1 and/or G{gamma}2 (5 ng/well each) in conjunction with A1R (10 ng/well) and G{alpha}16 (30 ng/well). Transfectants were pretreated with PTX and followed by 10 µM CHA for 16 h. B and C, HEK 293 cells were transfected with G{alpha}t (200 ng/well) or G{beta}1 and G{gamma}2 (100 ng/well) together with A1R (200 ng/well) and G{alpha}16 (400 ng/well). Subsequent to PTX treatment, the transfectants were treated with 10 µM CHA for 10 min. Cell lysates were analyzed as described in the legend to Fig. 2 for luciferase activity assay and immunodetection. Data shown are the mean ± S.E. from three individual experiments. Representative results from at least three sets of experiments are shown. *, CHA-induced response was significantly enhanced over the vector control; paired Student's t test, p ≤ 0.05. #, CHA-induced response was significantly inhibited as compared with the vector control; paired Student's t test, p ≤ 0.05.

 
Ras but Not Rac1 Mediates the Activation of IKK{alpha}/{beta} Phosphorylation and NF{kappa}B—A number of studies have illustrated that small GTPases are involved in the NF{kappa}B activation (11, 30). To define the role of the small GTPases in the activation of NF{kappa}B mediated by A1R, we transiently transfected the wild-type (WT) and constitutively active (CA) mutants of Ras and Rac1 into HEK 293-NF{kappa}B and evaluated the NF{kappa}B-dependent transcription of luciferase (Fig. 6A). Overexpression of wild-type Ras and Rac1 proteins did not alter the basal NF{kappa}B-induced luciferase production. However, the constitutively active mutant of Ras (RasCA) was capable of stimulating the NF{kappa}B-dependent transcription by ~3-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{alpha}/{beta} phosphorylation whereas RacDN failed to intervene. These data suggest that the activation of IKK/NF{kappa}B cascade requires only Ras, but not Rac1.



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  		FIG. 6.
Ras participates in the CHA-induced activation of IKK/NF{kappa}B. A, HEK 293-NF{kappa}B cells were transfected with wild-type or constitutive active (CA) mutants of Rac1 or Ras small GTPases (30 ng/well) for 30 h. Cells were serum-starved for 16 h before luciferase detection. B, HEK 293 cells were transfected with Rac, dominantnegative mutant of Rac (RacDN), Ras or RasDN (200 ng/well) along with A1R (200 ng/well) and G{alpha}16 (400 ng/well). The transfectants were pretreated with PTX followed by 10 min of CHA induction. Cell lysates were analyzed as described in Fig. 2 for luciferase activity assay and immunodetection. Data shown are the mean ± S.E. from three individual experiments. Representative results from at least three sets of experiments are shown. *, CHA-induced response was significantly enhanced over the vector control; paired Student's t test, p ≤ 0.05.

 
ERK Is Important in the G16-mediated Regulation of IKK/NF{kappa}B by A1R—Activation of NF{kappa}B activity by Gi-coupled receptor through ERK has been described previously (13). Because the constitutively active G{alpha}16QL can activate all three MAPKs in HEK 293 cells (29), we asked if the coupling of A1R to G{alpha}16 can similarly stimulate the MAPKs. HEK 293 cells were transfected with A1R and G{alpha}16, treated with PTX, and then stimulated by CHA (10 µM) 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/NF{kappa}B signaling cascade, a panel of MAPK inhibitors was applied to HEK 293-NF{kappa}B cells co-expressing the A1R and G{alpha}16. Raf-1 kinase inhibitor (10 µM) and the selective MEK1/2 inhibitors (PD98059 and U0126; each at 10 µM) inhibited the CHA-induced NF{kappa}B-dependent luciferase activity by 30–50% (Fig. 7A). In contrast, the inactive analogue of U0126 (U0124; 10 µM) failed to attenuate the luciferase activity. The CHA-induced IKK{alpha}/{beta} 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/NF{kappa}B signaling was examined using SP600125, a selective JNK inhibitor. The application of SP600125 (10 µM) neither affected the NF{kappa}B-induced luciferase expression (Fig. 7A) nor the phosphorylation of IKK{alpha}/{beta} (Fig. 7C). Finally, for p38 MAPK, two p38 MAPK inhibitors, SB202190 and SB203580 at 10 µM were also unable to inhibit the CHA-induced luciferase transcription (Fig. 7A) and IKK{alpha}/{beta} phosphorylation (Fig. 7D). These findings clearly demonstrate the participation of ERK, but not JNK or p38 MAPK, in G16-mediated activation of IKK/NF{kappa}B by A1R. c-Src Participates in the G{alpha}16-mediated IKK/NF{kappa}B Activation—In the activation of NF{kappa}B by the Gi-coupled dopamine D2 receptor, the participation of the c-Src kinase is clearly evident (13). Thus, we sought to investigate whether c-Src is also involved in G16-mediated activation of IKK/NF{kappa}B. We first examined if c-Src can be activated by A1R. As shown in Fig. 8A, 10 µM CHA stimulated the phosphorylation of c-Src at Tyr416 in HEK 293 cells co-expressing A1R and G{alpha}16. A selective c-Src kinase inhibitor (PP2) was then used to confirm its involvement in CHA-induced IKK/NF{kappa}B activation. PP2 at 10 µM for 30 min significantly attenuated the CHA-induced NF{kappa}B-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 CHA-induced phosphorylation of IKK{alpha}/{beta} (Fig. 8C). Additionally, the effect of overexpressing c-Src on CHA-induced activation of IKK/NF{kappa}B was examined. Co-expression of wild-type c-Src with A1R and G{alpha}16 did not affect the ability of CHA to stimulate NF{kappa}B-dependent luciferase expression (Fig. 8D). When the dominant negative mutant of c-Src was co-expressed, the CHA-induced 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, overexpression of the dominant negative mutant of c-Src, but not wild-type c-Src, suppressed the CHA-induced phosphorylation of IKK{alpha}/{beta} (Fig. 8E). These data suggest that c-Src is involved in G16-mediated activation of IKK/NF{kappa}B by A1R.



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  		FIG. 7.
ERK is involved in the G{alpha}16-mediated activation of NF{kappa}B by CHA. HEK 293-NF{kappa}B (A) and HEK 293 (B–D) cells were transfected with A1R and G{alpha}16 as described in the legend to Fig. 2. Subsequent to PTX treatment, transfectants were treated with 1% Me2SO (vehicle), 10 µM Raf-1 kinase inhibitor, 50 µM PD98059, 10 µM U0126 (MEK1/2 inhibitors), its negative control U0124, 30 µM SP600125 (JNK inhibitor II), 10 µM SB202190, or 10 µM SB203580 (specific p38 MAPK inhibitors) for 30 min before being challenged with 10 µM CHA. Cell lysates were analyzed as described in the legend to Fig. 2 for luciferase activity assay and immunoblotting detection. The inhibitory effects of different kinase inhibitors were also examined using specific anti-phospho-MAPK and anti-MAPK antisera. Data represent the mean ± S.E. from three independent experiments. The immunoblots shown were representatives of three sets of experiments. #, CHA-induced response was significantly inhibited as compared with the vehicle control; paired Student's t test, p ≤ 0.05.

 



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  		FIG. 8.
c-Src is involved in the G{alpha}16-mediated activation of NF{kappa}B by CHA. HEK 293 (A and C) and HEK 293-NF{kappa}B(B) cells were transiently transfected with A1R and G{alpha}16. D, HEK 293-NF{kappa}B cells were transfected with c-Src, c-SrcDN, or pcDNA1 (5 ng/well) in conjunction with A1R (15 ng/well) and G{alpha}16 (30 ng/well). E, HEK 293 cells were transiently transfected with c-Src, c-SrcDN, or pcDNA1 (200 ng/well) along with A1R (200 ng/well) and G{alpha}16 (400 ng/well). After to PTX treatment, the transfectants were stimulated with 10 µM CHA for 10 min. c-Src phosphorylation was detected by anti-phospho-c-Src(Tyr416). Equivalent protein loading was monitored by anti-c-Src antibody in A. B and C, transfectants were pretreated with 25 µM PP2 (a Src inhibitor) or its negative control PP3 for 30 min before CHA treatment. Cell lysates were analyzed as described in the legend to Fig. 2 for luciferase activity assay and immunoblotting detection. Data represent the mean ± S.E. from three independent experiments. The immunoblots shown were representatives of three sets of experiments. #, CHA-induced response was significantly inhibited as compared with the controls; paired Student's t test, p ≤ 0.05.

 
CHA-induced IKK{alpha}/{beta} 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 A1R-induced IKK/NF{kappa}B activation in Reh cells, specific kinase inhibitors were employed. As shown in Fig. 9A, the involvement of PLC{beta}/PKC/CaMKII cascade was confirmed. Treatment of Reh cells with U73122 [GenBank] , Cal C, and KN62 significantly inhibited the CHA-induced IKK phosphorylation whereas the inactive analogues U73343 [GenBank] 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{alpha}/{beta} in Reh cells also required CaMKII, PKC, ERK, and c-Src.



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  		FIG. 9.
Characterization of CHA-induced phosphorylation of IKK{alpha}/{beta} in Reh cells. A–C, PTX-treated Reh cells were treated for 30 min with 1% Me2SO (vehicle), U73122 [GenBank] , U73343 [GenBank] , KN62, KN92, Raf-1 kinase inhibitor, U0126, U0124, SB202190, SB203580 (10 µM each), calphostin C (Cal C; 100 nM), SP600125 (30 µM), PP2 (25 µM), or PP3 (25 µM) as indicated. Cells were then stimulated by 10 µM CHA for 15 min, and cell lysates were analyzed as described in the legend to Fig. 2 for immunoblotting detection. Immunoblots shown are representatives of three individual sets of experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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 NF{kappa}B is an important transcription factor in regulating inflammatory and immune responses, adenosine receptors may employ G protein-dependent pathways to modulate the NF{kappa}B activity. Indeed, a number of GPCRs have been shown to activate NF{kappa}B via Gi- or Gq-dependent pathways (8–13, 23, 24). Numerous signaling components have been implicated in G protein-dependent activation of NF{kappa}B 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 G16 in A1R-mediated activation of NF{kappa}B in human lymphocytic Reh and HEK 293 cells.

Although A1R is functionally coupled to Gi proteins in HEK 293 cells (20), it cannot utilize endogenous Gi pathways to activate NF{kappa}B in both cell types (Figs. 1 and 2). In contrast, co-expression of G{alpha}16 allows A1R to efficiently stimulate the IKK/NF{kappa}B pathway (Fig. 2). The collective use of the A1R selective antagonist DPCPX, I{kappa}B{alpha} and its non-degradable mutant, IKK{alpha}/{beta} and their dominant-negative mutants, as well as the NF{kappa}B activation inhibitor APQ confirmed the specificity of the pathway. The PTX insensitivity of CHA-induced NF{kappa}B activation in Reh cells signifies the involvement of Gq or G12 family members. As G{alpha}16 is present in the Reh cells (26) and is known to interact with A1R (21, 22), while neither G{alpha}q/11 nor G{alpha}13 has been reported to associate with A1R, the activation signal for NF{kappa}B is presumably transmitted via G16. Mechanistically, both cell types appear to employ a complicated network of intermediates for signal propagation. They include an effector (PLC{beta}), 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{beta}/PKC/CaMKII, and Ras/Raf-1/MEK/ERK (Fig. 10).



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  		FIG. 10.
A mechanistic model of G16-mediated activation of IKK/NF{kappa}B by A1R. CHA-bound A1R activates G{alpha}16 and releases G{beta}{gamma}. The activated G{alpha}16 stimulates the PLC{beta} cascade. Through both G{alpha} and G{beta}{gamma}, Ras/Raf-1/MEK1/2/ERK cascade becomes activated and c-Src is activated indirectly. These activations lead to the phosphorylation of IKK{alpha}/{beta} and signal I{kappa}B{alpha} degradation. The released NF{kappa}B 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 dash-lined arrows. The experimental evidence supporting individual pathways and the interactions between their intermediates are described in the text.

 
Regulation of NF{kappa}B by Gq proteins has previously been suggested to depend on both G{alpha} and G{beta}{gamma} subunits. G{alpha}q stimulates NF{kappa}B via the PLC{beta}/PKC/CaMKII pathway (10) whereas G{beta}{gamma} propagates the signal through phosphatidylinositol-3 kinase cascade (8). Both arms of the signal appear to operate in the G16-mediated activation of NF{kappa}B. Inhibition of CHA-induced IKK/NF{kappa}B activities by U73122 [GenBank] , calphostin C, and KN62 provides evidence that PLC{beta} and its downstream effectors modulate the NF{kappa}B activation. Both Ca2+-dependent and -independent PKCs have been shown to regulate NF{kappa}B. The Ca2+-sensitive PKC{alpha} is capable of activating the IKK complex in T-lymphocytes (32) while the Ca2+-independent PKC{epsilon} is implicated in the modulation of IKK activity based on the use of PKC{epsilon}-deficient mice (33). Indeed, our data support a role of PKC{alpha} and PKC{epsilon} in G16-mediated activation of IKK/NF{kappa}B by CHA (Fig. 4). However, other PKC isoforms may also be involved since the Gi-coupled A1R is known to regulate c-fos through PKCµ (34). The deployment of PLC{beta} and PKC{alpha} in G16-mediated activation of NF{kappa}B by A1R resembles that of the formyl peptide receptor (24). Elevation of intracellular Ca2+ level can also alter the activity of NF{kappa}B 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 NF{kappa}B-dependent luciferase activity (Fig. 4) implicates the involvement of CaMKII. Thus, the classical PLC{beta}/PKC/CaMKII cascade appears to play an important role in the regulation of IKK/NF{kappa}B by the G16-coupled A1R.

Despite the fact that G{alpha}16 can propagate stimulatory signals to NF{kappa}B, the released G{beta}{gamma} also takes part in the regulation. Attenuation by the co-expression of G{alpha}t (a G{beta}{gamma} scavenger) and potentiation by the overexpression of G{beta}1{gamma}2 confirm the participation of G{beta}{gamma} in G16-mediated activation of NF{kappa}B by A1R. Signals arising from G{alpha} and G{beta}{gamma} 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{alpha}16 to Ras is provided by a novel adaptor protein named tetratricopeptide repeat 1 (39) while G{beta}{gamma} 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/NF{kappa}B by the G16-coupled A1R is attenuated in the presence of Raf-1 and MEK1/2 inhibitors (Fig. 7). Moreover, the G16-mediated IKK phosphorylation by A1R is effectively attenuated in the presence of RasDN, whereas RasCA induces NF{kappa}B-driven luciferase expression (Fig. 6). The ability of ERK to exert a stimulatory effect on IKK/NF{kappa}B is not shared by the other two MAPKs. Although both JNK (41) and p38 MAPK (42) can activate NF{kappa}B, neither is required for G16-mediated stimulation of IKK/NF{kappa}B by A1R (Fig. 7). The lack of involvement of JNK and p38 MAPK is further supported by the inability of Rac1DN to suppress the G16-mediated activation of IKK/NF{kappa}B (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 G16-mediated activation of IKK/NF{kappa}B is highly reminiscent of the regulation of STAT3 by G{alpha}16 (29).

Another site for possible signal integration is c-Src, a non-receptor 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 G16-mediated stimulation of IKK/NF{kappa}B by A1R. Although constitutively active G{alpha}16 can activate c-Src in HEK 293 cells, the interaction is probably indirect because G{alpha}16 does not directly associate with c-Src (29). It is interesting to note that in human epithelial cells, TNF-{alpha}-induced cyclooxygenase-2 expression is mediated via c-Src/NF{kappa}B 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 G16-mediated activation of NF{kappa}B (Fig. 4), it is conceivable that they can bridge the gap between G{alpha}16 and c-Src. Additionally, c-Src can be activated by G{beta}{gamma} (45) even though no direct binding between them can be established (46). Indeed, activation of NF{kappa}B by the dopamine D2 receptor in HeLa cells is mediated via c-Src in a G{beta}{gamma}-dependent manner (13). Direct interaction between c-Src and the IKK complex (47) leads to the phosphorylation of IKK (43) and, subsequently, I{kappa}B{alpha} (48). These findings provide a pathway connecting the agonist-stimulated GPCR and G proteins to IKK/NF{kappa}B.

In considering the signal routing from G16 to NF{kappa}B, 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) signaling, and each of these regulatory intermediates has been shown to modulate the NF{kappa}B activation through the upstream IKK complex. The present study has revealed an intricate signaling network for G16-coupled receptors to regulate IKK/NF{kappa}B pathway. Some of the signaling intermediates such as PLC{beta} and PKC{alpha} have previously been shown to mediate the activation of NF{kappa}B by constitutively active G{alpha}16 (24). Other molecular players like G{beta}{gamma} and c-Src are known to be required for Gi-mediated stimulation of NF{kappa}B (13). Yet we are far from fully appreciating all the intricacies of the complex signaling network. Because dysregulation of NF{kappa}B activity has been implicated in the pathogenesis of a variety of human diseases, G16-coupled receptors may represent attractive targets for therapeutic intervention. This is especially applicable to inflammatory and immune diseases as G{alpha}16 is primarily expressed in hematopoietic cells.


   FOOTNOTES
 
* This work was supported in part by Grants from the Research Grants Council of Hong Kong (HKUST 6095/01M, 2/99C, and 3/03C), the University Grants Committee (AoE/B-15/01), and the Hong Kong Jockey Club. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of the Croucher Senior Research Fellowship. To whom correspondence should be addressed: Dept. of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. Tel.: 852-2358-7328; Fax: 852-2358-1552; E-mail: boyung{at}ust.hk.

1 The abbreviations used are: NF{kappa}B, nuclear factor-{kappa}B; A1R, adenosine A1 receptor; CaMKII, calmodulin-dependent protein kinase II; CHA, N6-cyclohexyladenosine; ERK, extracellular signal-regulated kinase; GPCRs, G protein-coupled receptors; HEK 293, human embryonic kidney 293; IKK, I{kappa}B kinase; JNK, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; MEK, MAPK/ERK kinase; PKC, protein kinase C; PLC{beta}, phospholipase C{beta}; PTX, pertussis toxin; c-Src, cellular Src; APQ, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine. Back


   ACKNOWLEDGMENTS
 
We thank the following individuals for kindly providing the various cDNAs: Drs. Alain Israel, Richard Ye, Zhenguo Wu, and Shengcai Lin. We thank Dr. David New for helpful discussions and valuable comments.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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