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The Journal of Immunology, 2001, 166: 6885-6892.
Copyright © 2001 by The American Association of Immunologists

G{alpha}16 Couples Chemoattractant Receptors to NF-{kappa}B Activation1

Ming Yang*, Hairong Sang*, Arshad Rahman*, Dianqing Wu{dagger}, Asrar B. Malik* and Richard D. Ye2,*

* Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612; and {dagger} Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The guanine nucleotide-binding regulatory protein {alpha}-subunit, G{alpha}16, is primarily expressed in hemopoietic cells, and interacts with a large number of seven-membrane span receptors including chemoattractant receptors. We investigated the biological functions resulting from G{alpha}16 coupling of chemoattractant receptors in a transfected cell model system. HeLa cells expressing a {kappa}B-driven luciferase reporter, G{alpha}16, and the formyl peptide receptor responded to fMLP with a ~7- to 10-fold increase in luciferase activity. This response was accompanied by phosphorylation of I{kappa}B{alpha} and elevation of nuclear {kappa}B-DNA binding activity, indicating activation of NF-{kappa}B. In contrast to G{alpha}16, expression of G{alpha}q, G{alpha}13, and G{alpha}i2 resulted in a marginal increase in {kappa}B luciferase activity. A GTPase-deficient, constitutively active G{alpha}16 mutant (Q212L) could replace agonist stimulation for activation of NF-{kappa}B. Furthermore, expression of G{alpha}16 (Q212L) markedly enhanced TNF-{alpha}-induced {kappa}B reporter activity. The G{alpha}16-mediated NF-{kappa}B activation was paralleled by an increase in phospholipase C-{beta} activity, and was blocked by pharmacological inhibitors of protein kinase C (PKC) and by buffering of intracellular Ca2+. The involvement of a conventional PKC isoform was confirmed by the finding that expression of PKC{alpha} enhanced the effect of G{alpha}16, and a dominant negative PKC{alpha} partially blocked G{alpha}16-mediated NF-{kappa}B activation. In addition to formyl peptide receptor, G{alpha}16 also enhanced NF-{kappa}B activation by the C5a and C3a receptors, and by CXC chemokine receptor 2 and CCR8. These results suggest a potential role of G{alpha}16 in transcriptional regulation downstream of chemoattractant receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Guanine nucleotide-binding regulatory proteins (G proteins)3 are signal transducers that couple cell surface receptors to numerous intracellular signaling pathways (1). In the immune system, G proteins play important functions in lymphocyte homing, migration of leukocytes to site of inflammation, integrin activation, and generation of superoxide anions (2). Heterotrimeric G proteins consist of receptor-interacting {alpha}-subunits, which possess intrinsic GTPase activity, and tightly associated {beta}{gamma}-subunits that are targeted to plasma membranes. Agonist binding triggers the activation of heterotrimeric G proteins by virtue of guanine nucleotide exchange, converting the proteins to a GTP-bound state that activates downstream effectors either directly or through the released G{beta}{gamma} subunits (3). Four classes of heterotrimeric G proteins have been identified based on their primary structure (4). In leukocytes, receptors for chemoattractants and chemokines typically couple to the G{alpha}i proteins for various cell responses associated with host defense. Pertussis toxin (PTX), which catalyzes ADP ribosylation of the G{alpha}i and G{alpha}o proteins and prevents interaction of these G proteins with chemoattractant receptors, effectively blocks these cellular functions (2).

Recent studies demonstrate that chemoattractant and chemokine receptors play important roles in cell growth and differentiation. Macrophage-inflammatory protein 1{alpha} has been found to regulate the development of hemopoietic cells (5). Targeted deletion of the murine IL-8 receptor homolog results in expansion of granulocytes (6), whereas mice lacking CXC chemokine receptor 4 (CXCR4) or its ligand SDF-1 are developmentally defective (7). A number of CXC chemokines that possess N-terminal Glu-Leu-Arg motif have been found to stimulate angiogenesis (8). In addition, CXC chemokines such as melanoma growth-stimulating activity/growth-related oncogene-{alpha} stimulate the growth of melanocytes (9). These functions of chemoattractants and chemokines result in part from their abilities to regulate gene transcription. Indeed, several published studies have demonstrated that chemoattractants and chemokines stimulate the activation of NF-{kappa}B (10, 11, 12), a transcription factor that regulates the expression of a large number of cytokine and chemokine genes including that for melanoma growth-stimulating activity/growth-related oncogene-{alpha} and IL-8 (13). It is important to note that although G{alpha}i is known for coupling chemoattractant and chemokine receptors, its role in transcription regulation has been unclear (14). As an example, an earlier study suggests that the C5a receptor can use a PTX-insensitive G protein for NF-{kappa}B activation (11). Given the established functions of G{alpha}q and G{alpha}12 classes of G proteins in regulating cell growth, it is possible that these G proteins couple chemoattractant and chemokine receptors for transcription regulation.

G{alpha}16 and its mouse equivalent G{alpha}15 are members of the G{alpha}q family with restricted expression in hemopoietic cells (15). A unique feature of G{alpha}16 is that it interacts with a large number of receptors with seven-transmembrane domain structure. In exogenous expression systems (16, 17), receptors for IL-8, C5a, and fMLP have been shown to use G{alpha}16 for phospholipase C-{beta} (PLC{beta}) activation (16, 18). A recent study demonstrated that leukocytes from mice with targeted deletion of the G{alpha}15 gene exhibited reduced phosphoinositide hydrolysis in response to C5a stimulation (19). Using a G{alpha}16 antisense approach, it has been shown that reduction of G{alpha}16 protein level in an erythroleukemia cell line could impair the expression of the {beta}-globin gene (20). However, whether G{alpha}16-mediated activation of PLC{beta} results in gene transcription has not been established because of technical difficulties in transfecting hemopoietic cells and the variation of G{alpha}16 expression levels in these cells at different developmental stages.

In this study, we sought to determine the potential involvement of G{alpha}16 in transcriptional regulation. We examined the ability of G{alpha}16 to stimulate NF-{kappa}B activation in a well-characterized cell transfection system by coexpression of G{alpha}16 with selected chemoattractant receptors and a {kappa}B-driven luciferase reporter. Our results indicate that G{alpha}16 efficiently couples chemoattractant receptors to NF-{kappa}B activation, suggesting a potential function of G{alpha}16 beyond activation of PLC{beta}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

fMLP was purchased from Sigma (St. Louis, MO). GF 109203X, Gö 6976, and 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) were obtained from Calbiochem (La Jolla, CA). The anti-G{alpha}q Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-G{alpha}i2 Ab was prepared against a synthetic peptide with the sequences of CAKNNLKDCGLF. The anti-G{alpha}13 Ab was a gift from Tohru Kozasa (University of Illinois at Chicago). The anti-G{alpha}16 Ab was obtained from Torrey Pines Biolabs (San Diego, CA). Abs against the PLC{beta} isoforms were purchased from Santa Cruz Biotechnology. The expression vector pCMV-formyl peptide receptor (pCMV-FPR) was constructed by subcloning of the human FPR cDNA into the pCMV4 expression vector (21). The G{alpha}16 expression vectors (22) were provided by Cindy Knall and Gary Johnson (University of Colorado, Denver, CO). Construction and characterization of the PLC{beta} expression vectors were described elsewhere (23). The protein kinase C (PKC) expression constructs were provided by I. B. Weinstein (Columbia University, New York, NY) and were described elsewhere (24). Other plasmids used in this study were described in a recent publication (25).

Cell culture, transfection, and luciferase reporter assay

HeLa cells were maintained in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 50 µg/ml streptomycin. Cells (~40% confluence) in six-well plates were transfected with plasmid expression vectors coding for a 3x {kappa}B-directed luciferase reporter (25), G{alpha}16, FPR, and/or other expression constructs as indicated. Where needed, pCMV{beta} vector DNA (Promega, Madison, WI) was added to make total DNA of 1 µg/sample. Transient transfection was performed as described (25), using LipofectAmine Plus reagent (Life Technologies, Rockville, MD) according to manufacturer instructions. Twenty-four hours after transfection, cells were serum-starved for 16–18 h, washed twice with PBS, and assayed with or without agonist stimulation. Reporter lysis buffer (Promega) was then added to the cells. The expressed luciferase activity was measured in a Femtomaster FB12 luminometer (Zylux, Maryville, TN). Relative expression level of the transfected constructs was standardized by the use of the pRLCMV plasmid (Promega) to overcome variations between samples. Unless otherwise indicated, all luciferase assays were performed with duplicate samples, and 2–4 independent experiments were usually conducted. Normalized data were plotted using the Prism (version 3.0) software (GraphPad, San Diego, CA).

Inositol phosphate (IP) production

IP production was measured as previously described (26). Briefly, 24 h after transfection, HeLa cells were labeled with myo-[3H]inositol (3 µCi/ml) (Amersham Pharmacia) in inositol-free DMEM. Twenty-four hours later, the cells were washed twice with the same medium supplemented with 20 mM HEPES (pH 7.4) and 50 mM LiCl. Thereafter, cells were stimulated with fMLP (100 nM) at 37°C for 45 min. Reactions were stopped by removing the medium followed by addition of 1 ml ice-cold methanol. The cells were then transferred into a vial and 1.5 ml of chloroform, 0.5 ml of water were added. After vigorous vortex, the phases were separated by centrifugation. Aliquots of the upper phase (450 µl) were loaded onto packed columns containing Dowex AG1-8 (250 mg/column; Bio-Rad, Hercules, CA). Free inositol was eluted with 10 ml of water and 10 ml of 60 mM ammonium formate. Total IPs were eluted with 1 M ammonium formate (2 ml) dissolved in 100 mM formic acid, and the associated radioactivity was measured by scintillation counting in a Beckman LS 3801 liquid scintillation counter. All measurements were performed in duplicate for a total of 2–3 independent experiments. Data were analyzed using the Prism software (GraphPad).

EMSA

Nuclear protein extracts were prepared as described (27). Double-stranded NF-{kappa}B oligonucleotide, containing the sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Promega), was end-labeled using [{gamma}-32P]ATP and T4 polynucleotide kinase. EMSA was performed according to a previously described procedure (27), using 6% acrylamide gels and 0.5x Tris/boric acid/EDTA (TBE) buffer. The gels were dried and autoradiograph was taken using a PhosphoImager cassette (Molecular Dynamics, Sunnyvale, CA). Analysis of the result was performed using software from Molecular Dynamics.

Immunoblotting analysis

Proteins from whole cell extracts were separated on 6–10% acrylamide SDS-PAGE gels by electrophoresis at 30 mA. Proteins were electrotransferred to nitrocellulose membrane at 100 V for 1 h at 4°C. The membrane was pretreated with 5% nonfat milk in Tween 20 + TBS (TTBS; 20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.05% Tween 20) for 1–2 h at room temperature. Incubation with primary Ab was performed at 4°C in TTBS with 5% BSA for 16 h. The membrane was then washed with TTBS three times, for 10 min each, and incubated with horseradish peroxidase-conjugated secondary Ab for 1 h at room temperature (23°C). After three washes with TTBS, the bound Ab was detected by enhanced chemiluminescence (Pierce, Rockford, IL). The phosphorylated I{kappa}B{alpha} was detected using a polyclonal Ab from Cell Signaling Technology (Beverly, MA). The Ab recognizes phosphorylated serine-32 of I{kappa}B{alpha} as a species with slightly higher mobility in SDS gel.

Calcium mobilization assay

Mobilization of calcium was measured in Indo-1/AM-labeled cells. Cells were dissociated with trypsin-free buffer (Life Technologies) and washed once with HBSS. Cells were adjusted to 5 x 106/ml in HBSS and incubated with 5 µM Indo-1/AM at 37°C for 25 min. After a brief wash with HBSS, the cells were resuspended to 1 x 106/ml in HBSS. Continuous fluorescent measurements of calcium-bound and free Indo-1/AM were made using a PTI (Photon Technology International, Monmouth Junction, NJ) spectrofluorometer, detecting at 405 and 485 nm, with an excitation wavelength of 340 nm. Changes of the intracellular Ca2+ concentration was calculated as 250 · (F - Fmin)/(Fmax - F), where 250 is the Kd of Indo-1 for calcium (in nM), F is the fluorescence emission at 405 nm over that at 485 nm (A/B), Fmax is the ratio A/B when Triton X-100 is added (0.1%), and Fmin is the ratio A/B when EGTA (0.5 mM) is added to chelate the released Ca2+.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine whether chemoattractant receptors use G{alpha}16 for transcriptional activation, we adopted a cell transfection system in which NF-{kappa}B activation has been extensively characterized. HeLa cells were transiently transfected with expression vectors for a {kappa}B-directed luciferase reporter, G{alpha}16 and the human FPR. No significant changes in luciferase activity were observed in unstimulated cells or in cells that expressed only the FPR but not G{alpha}16 (Fig. 1Go, A and B). However, when the transfected cells were challenged with fMLP, dose-dependent increases in the luciferase activity were observed, indicating NF-{kappa}B activation. fMLP was found to activate NF-{kappa}B at nanomolar concentrations. The induced luciferase activity peaked at ~100 nM of fMLP, giving a ~7- to 10-fold induction over basal level in a typical experiment (Fig. 1GoA). Cotransfection of expression vectors coding for other G proteins, including G{alpha}q and G{alpha}13, did not produce marked increases in NF-{kappa}B activation as seen with G{alpha}16 (Fig. 2GoB). An unexpected finding was that coexpression of G{alpha}i2, believed to couple FPR to downstream signaling pathways in neutrophils, brought only marginal increase in NF-{kappa}B activation. Except for G{alpha}16, all G proteins tested are present in HeLa cells, and cotransfection with the respective G{alpha} constructs under the experimental conditions only slightly increased their cellular protein levels (Fig. 1GoC). Given the transfection efficiency of ~20–30%, there could be a greater increase in the G protein levels in the transfected cells. Increasing the amounts of the G{alpha}16 construct resulted in dose-dependent increases of cellular G{alpha}16 protein levels that could be detected in Western blot with a threshold of ~80 ng of input DNA (Fig. 1GoC). Accordingly, there were increases in the fMLP-induced luciferase activity when more G{alpha}16 was present (Fig. 1GoD).



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  		FIGURE 1. G{alpha}16 mediates fMLP-induced NF-{kappa}B activation in transfected HeLa cells. A, Dose-dependent induction of {kappa}B-directed luciferase activity. HeLa cells were transfected with expression constructs for G{alpha}16, FPR, and a {kappa}B reporter (200 ng each). The cells were stimulated with culture medium (vehicle) or with increasing concentrations of fMLP. Luciferase activities were measured 5 h after agonist stimulation and expressed as relative luciferase activity (RLA) with basal level (vehicle) set at 1. B, FPR preferentially couples to G{alpha}16 for NF-{kappa}B activation. The cells were cotransfected with expression vectors encoding G{alpha}q, G{alpha}13, G{alpha}16, or G{alpha}i2 (200 ng each), respectively. FPR was cotransfected in all samples except the control (mock). The cells were stimulated with fMLP (100 nM, solid bars) or with culture medium (vehicle, open bars), and luciferase activities were measured 5 h later. C, Immunoblotting analysis of G{alpha} protein expression using specific Abs. The cells were either mock-transfected (0) or transfected with indicated amounts of DNA for each G{alpha} tested. Cells were lysed 2 days after transfection for immunoblotting. D, G{alpha}16 dose-dependently up-regulates fMLP-induced {kappa}B reporter expression. Increasing amounts of G{alpha}16 expression plasmid were cotransfected with the FPR construct into HeLa cells. After 2 days the cells were stimulated with fMLP, and the expressed luciferase activities were measured 5 h later. All experiments were performed three times with similar results. Duplicate samples were included in each experiment in A, B, and D, and the mean ± SD values are shown. A representative immunoblotting figure is shown in C.

 


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  		FIGURE 2. A constitutively active G{alpha}16 stimulates NF-{kappa}B activation. A, HeLa cells were transfected with increasing amounts of G{alpha}16QL and fixed amount (200 ng) of a {kappa}B luciferase reporter. Forty-eight hours later, the cells were assayed for the luciferase activities. Bottom panel, Representative immunoblot indicating dose-dependent expression of the G{alpha}16QL protein. B, Synergistic effect of G{alpha}16QL on TNF-{alpha}-induced NF-{kappa}B activation. Cells were cotransfected with, or without, G{alpha}16QL (5 ng). Forty-eight hours later, two of the four samples were stimulated with TNF-{alpha} (40 ng/ml) for 5 h. Luciferase activities were measured and expressed as fold induction over basal. Duplicated samples were included in each experiment, and results are shown as mean ± SD from one of the three experiments, all with similar results.

 
Replacement of a glutamine at position 212 with a leucine (Q->L) impairs the intrinsic GTPase activity of G{alpha}16, resulting in a constitutively active G protein (22). To confirm the role of G{alpha}16 in mediating NF-{kappa}B activation, we cotransfected a G{alpha}16QL expression construct together with the {kappa}B luciferase reporter into HeLa cells. As shown in Fig. 2Go, G{alpha}16QL expression led to a potent activation of NF-{kappa}B in the absence of FPR and fMLP stimulation. Maximal luciferase reporter activity was detected in cells transfected with 5–10 ng of G{alpha}16QL (Fig. 2GoA). G{alpha}16QL-induced luciferase reporter activity progressively decreased when more DNA was used for transfection, reaching to ~35% of the peak value with 200 ng of input DNA (data not shown). Using immunoblotting, we determined that the G{alpha}16QL was expressed equally well in the transfected cells compared with the wild-type G{alpha}16, indicating that the high potency in activating NF-{kappa}B at lower concentrations is an intrinsic property of this G{alpha}16 mutant. It was not clear why higher levels of G{alpha}16QL produced less activation of NF-{kappa}B. Expression of G{alpha}16QL at optimal concentrations dramatically enhanced TNF-{alpha}-induced {kappa}B luciferase activity (Fig. 2GoB), indicating a possible synergistic effect between these two inducers of NF-{kappa}B activation.

Activation of NF-{kappa}B results from nuclear translocation of the NF-{kappa}B proteins and their subsequent binding to {kappa}B sequences (28). Recent studies have revealed an additional mechanism for NF-{kappa}B activation that involves modification of nuclear proteins, and may not be accompanied by increased nuclear translocation of the p65 and p50 proteins (29, 30). To determine whether G{alpha}16 stimulates nuclear translocation of the NF-{kappa}B proteins, a process that involves phosphorylation and degradation of I{kappa}B{alpha} by the I{kappa}B kinases (IKKs), EMSAs were performed using a [32P]-labeled probe containing consensus {kappa}B binding sequence. As shown in Fig. 3GoA, expression of FPR alone (lane 3) did not stimulate {kappa}B binding activity. fMLP induced nuclear translocation and binding of the NF-{kappa}B proteins only in the presence of both FPR and G{alpha}16 (lane 6). The fMLP-induced response is less potent than the TNF-{alpha}-induced response (lane 2). The difference could be attributed in part to the small population of cells (~20–30%) that were transfected and, therefore, responsive to fMLP stimulation, whereas all cells could respond to TNF-{alpha}. The fMLP-induced NF-{kappa}B activation involves mostly IKK2 (IKK{beta}) because coexpression of a dominant negative IKK2 (IKK2DN) construct, but not the IKK1DN (IKK{alpha}DN) construct, markedly blocked the {kappa}B binding activity in EMSA (compare lane 8 to lane 7). Expression of a "super repressor" of I{kappa}B{alpha} (I{kappa}B{alpha}M) devoid of constitutive and inducible serine phosphorylation (31), abolished fMLP-stimulated {kappa}B binding activity in EMSA (lane 9). That fMLP-stimulated NF-{kappa}B activation follows I{kappa}B{alpha} phosphorylation was further demonstrated by the detection of a phosphorylated I{kappa}B{alpha} 10 min after ligand addition, using a specific polyclonal Ab against phosphorylated serine-32 of I{kappa}B{alpha} (Fig. 3GoB).



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  		FIGURE 3. G{alpha}16-mediated NF-{kappa}B activation involves IKK and I{kappa}B{alpha}. A, A representative EMSA autoradiograph from three similar experiments showing induction of {kappa}B-specific DNA binding activity in cells stimulated with fMLP (100 nM) in the presence of G{alpha}16. The FPR, G{alpha}16, IKK1 and IKK2 DN, and I{kappa}B{alpha}M expression plasmids were used at 200 ng each. TNF-{alpha} (40 ng/ml) was used as a positive control. The cells were stimulated for 40 min before nuclear extract preparation. The specific NF-{kappa}B/DNA complex ({kappa}B) and a nonspecific species (NS) are marked by arrows. A semiquantification indicated a reduction of 14 and 74% in the intensity of the DNA-protein complexes in cells transfected with IKK1DN (lane 7) and IKK2DN (lane 8), respectively, as compared with cells without the IKK DN constructs (lane 6). B, Detection of phosphorylated I{kappa}B{alpha}. FPR and G{alpha}16-transfected cells were stimulated with fMLP (100 nM) for the time indicated. Cytosolic I{kappa}B{alpha} was detected by immunoblotting using a polyclonal Ab specific for I{kappa}B{alpha} phosphorylated at Serine-32. Protein size markers were shown on the left. This experiment was conducted twice, and nearly identical results were obtained. C and D, Results showing inhibition of {kappa}B reporter expression by the IKK DN and I{kappa}B{alpha}M constructs. All plasmids were used at 200 ng for transfection, and fMLP (B) was used at 100 nM. Luciferase expression was measured 48 h after transfection or 5 h after fMLP stimulation (B). The mean ± SD values of duplicate samples are shown in C and D and are representative of one of the three experiments.

 
Recent studies have shown that IKK2 is primarily responsible for NF-{kappa}B activation induced by inflammatory stimuli, whereas IKK1 plays an important role in development (32, 33, 34). Consistent with the EMSA data, results from reporter assays indicate that coexpression of IKK2DN almost completely blocked the luciferase activities induced by fMLP (Fig. 3GoC) and G{alpha}16QL (Fig. 3GoD). A much smaller inhibitory effect was seen in cells coexpressing IKK1DN. As expected, the I{kappa}B{alpha} "super repressor" completely abolished NF-{kappa}B activation by fMLP and G{alpha}16QL in luciferase reporter assays (Fig. 3Go, C and D). These data suggest that NF-{kappa}B activation by G{alpha}16 involves phosphorylation of I{kappa}B{alpha} by IKK, primarily IKK2.

To further explore the signaling mechanism by which G{alpha}16 mediates NF-{kappa}B activation, we examined involvement of PLC{beta}, a downstream effector of G{alpha}16 (35, 36). As shown in Fig. 4GoA, expression of G{alpha}16QL in HeLa cells increased IP production. The elevated IP level was further enhanced (~40%) by cotransfection of a PLC{beta}2 construct (Fig. 4GoA). A similar increase in IP production was observed in cells transfected with the FPR and G{alpha}16 and stimulated with fMLP (data not shown). The potentiation effect of the coexpressed PLC{beta}2 on IP production paralleled a marked increase (~100%) in {kappa}B reporter activity, but expression of PLC{beta}2 alone had no effect (Fig. 4GoB). Subsequently, we investigated the function of other PLC{beta} isoforms that are known effectors of G{alpha}16 (36). As determined by immunoblotting using specific PLC{beta} Abs, HeLa cells contain both PLC{beta}1 and PLC{beta}3, but not PLC{beta}2 (Fig. 4GoC). Cotransfection with plasmids for these PLC{beta} isoforms slightly increased their intracellular protein levels, although the actual increase in the transfected cells may be higher. Accordingly, there was a ~40 and a ~80% increase of {kappa}B luciferase activity over the level induced by G{alpha}16QL in cells cotransfected with the PLC{beta}1 and PLC{beta}3 constructs, respectively (Fig. 4GoD).



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  		FIGURE 4. G{alpha}16 activation of NF-{kappa}B involves PLC{beta}. A, G{alpha}16QL induces production of IP. IP production was measured 48 h after transfection in cells expressing G{alpha}16QL (5 ng) or together with PLC{beta}2 (200 ng), which potentiated the effect of G{alpha}16QL. B, Coexpression of PLC{beta}2 enhanced G{alpha}16QL-induced {kappa}B reporter expression. Experimental conditions were similar to those in A, except that luciferase reporter activities were measured. C, Expression of the three PLC{beta} isoforms in untransfected (Unt) and transfected (Txf) cells. The cells were transfected with 200 ng each of the expression vectors for PLC{beta}1, {beta}2, or {beta}3. Forty-eight hours later, cells were lysed for immunoblotting with specific anti-PLC{beta} polyclonal Abs. A representative blot is shown. D, Relative potency of PLC{beta} isoforms in promoting G{alpha}16QL-induced {kappa}B luciferase expression. For each PLC{beta} isozyme, 200 ng of DNA was cotransfected into the HeLa cells. Assays were conducted 48 h after transfection. For all experimental data shown in A, B, and D, duplicate samples were measured, and mean ± SD values are presented. The IP production assay was performed twice, and the luciferase assays shown in B and D were performed three times, all with similar results.

 
Activation of PLC{beta} leads to production of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (37, 38). The former triggers release of free calcium from intracellular stores, whereas the latter activates PKC. Although NF-{kappa}B can be activated by some PKC isoforms, the mechanism that involves G protein and PLC{beta} has not been directly explored. We found that in the transfected HeLa cells, fMLP-induced NF-{kappa}B activation could be readily blocked by GF 109203X, an inhibitor of conventional and novel PKC isoforms, and by Gö 6976, which inhibits the conventional PKC isoforms (Fig. 5GoA). Buffering of intracellular free calcium by treatment with BAPTA also blocked NF-{kappa}B activation. These results together suggest a role of conventional PKC in G{alpha}16-mediated NF-{kappa}B activation. To further test this possibility, expression constructs coding for representative PKC isozymes (PKC{alpha}, PKC{delta}, PKC{epsilon}, and PKC{zeta}) were cotransfected into HeLa cells, and {kappa}B luciferase assays were performed. Among the four PKC isoforms, only PKC{alpha} potentiated fMLP-induced luciferase activity (Fig. 5GoB and data not shown). Consistent with this finding, a DN PKC{alpha} (PKC{alpha}DN) with a Lys->Arg switch at position 368, partially inhibited NF-{kappa}B activation by fMLP (Fig. 5GoB) and by G{alpha}16QL (Fig. 5GoC). Expression of a constitutively active PKC{alpha} stimulated {kappa}B luciferase activity to an extent similar to that induced by G{alpha}16QL (Fig. 5GoC).



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  		FIGURE 5. A role of PKC{alpha} in G{alpha}16-mediated NF-{kappa}B activation. A, Inhibition of fMLP-induced and G{alpha}16-mediated {kappa}B reporter activity. HeLa cells were transfected with FPR, the {kappa}B luciferase reporter, and G{alpha}16 (200 ng each). Forty-three hours later, the cells were stimulated with fMLP (100 nM), and the induced luciferase activities were measured 5 h after stimulation. In some samples, the cells were treated for 30 min with GF 109203X (GF; 2 µM), Gö 6976 (GO; 10 µM), or BAPTA (BA; 20 µM) before fMLP stimulation. The fMLP-induced luciferase activity (average of 10- and 12-fold increase over basal level in two experiments) was set as 100%, against which the inhibitor-treated samples were compared. B, Effects of the wild-type (WT) and DN PKC{alpha} on fMLP-induced {kappa}B reporter expression. Cells were cotransfected to express FPR, G{alpha}16, PKC{alpha}WT (200 ng each), and PKC{alpha}DN (200 and 800 ng as indicated). Forty-three hours after transfection, the cells were stimulated with fMLP (100 nM), and the induced luciferase activities were measured after 5 h. C, Effects of a constitutively active (CA) and a DN PKC{alpha} on G{alpha}16QL-induced luciferase activity. The assay was performed 48 h after cells were transfected with G{alpha}16QL (5 ng), in the presence or absence of PKC{alpha}DN (200 ng). In another sample, PKC{alpha}CA (200 ng) was cotransfected with the {kappa}B luciferase reporter in the absence of G{alpha}16QL. All luciferase reporter assays shown in B and C were conducted three times, with similar results. Presented here are mean ± SD values from one of the representative experiments from each assay.

 
A large number of chemoattractant and chemokine receptors have been shown to couple to G{alpha}16 for PLC{beta} activation (16, 17, 18). Therefore, we examined whether G{alpha}16 coupling to NF-{kappa}B activation is restricted to the FPR, or it also applies to other receptors. Selected chemoattractant and chemokine receptors were transiently expressed in HeLa cells, which were then stimulated with the respective agonists for {kappa}B luciferase assays. In the absence of cotransfected G{alpha}16, cells expressing the receptors responded to agonist stimulation (100 nM each) with minimally enhanced luciferase activities (Fig. 6GoA, hatched bars), which could be blocked by PTX (100 ng/ml, 16 h; data not shown). Cotransfection of a G{alpha}16 expression construct dramatically enhanced the agonist-stimulated luciferase activity by 18- and 14-fold for C5aR and C3aR, and by 4.5- and 4.1-fold for CXCR2 and CCR8, respectively (Fig. 6GoA, filled bars). An explanation for these observations is that these receptors preferentially couple to G{alpha}16 for NF-{kappa}B activation. Therefore, we examined whether G{alpha}16-mediated NF-{kappa}B activation could accurately reflect signaling through the chemoattractant receptors. Using the FPR as an example, we conducted calcium mobilization and {kappa}B luciferase assays to test cell responsiveness to a group of synthetic peptides derived from recently identified FPR and FPRL1 agonists (39, 40, 41, 42). As shown in Fig. 6Go, B and C, the abilities of these peptides to stimulate {kappa}B luciferase activation are consistent with their potency in the induction of calcium mobilization. The P2 peptide (WKYMVm-NH2), known to bind and activate FPR and FPRL1 (39), elicited responses similar to those induced by fMLP in both the luciferase and calcium mobilization assays. Carboxyl- or amino-terminal modifications of P2 resulted in peptides (P1, P3, and P4) with markedly reduced potency in both calcium mobilization and luciferase assays. The P5 and P6 peptides, which preferentially activate FPRL1 over FPR (41), did not induce either calcium mobilization or {kappa}B luciferase expression in FPR-transfected cells.



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  		FIGURE 6. The {kappa}B luciferase reporter expression induced by selected chemoattractants and chemokines, and comparison to calcium mobilization. A, HeLa cells cotransfected to express the {kappa}B luciferase reporter and one of the four chemoattractant and chemokine receptors was stimulated with C5a, C3a, IL-8, or I-309 (100 nM each) for 5 h. The induced luciferase activities were measured and expressed as mean ± SD values from duplicate measurements. In some samples, G{alpha}16 was cotransfected into the HeLa cells ({blacksquare}). Basal luciferase activity (without agonist stimulation) was set as 1. B, Induction of the {kappa}B luciferase reporter by various synthetic peptides. HeLa cells cotransfected with FPR, G{alpha}16, and the {kappa}B-directed luciferase reporter were stimulated with 100 nM each of fMLP and peptides 1–6 (P1 to P6). The induced luciferase reporter activities were measured 5 h after agonist stimulation. The mean ± SD values of one representative experiment (from a total of three) are shown. C, Stable FPR transfectants of RBL-2H3 cells were stimulated with the same group of peptides (100 nM), and the induced calcium mobilization was measured. The peptides used are: P1, WKYMVM-NH2; P2, WKYMVm-NH2; P3, WKYMVM-COOH; P4, N-formyl-WKYMVM-COOH; P5, LESIFRSLLFRVM-COOH; P6, SLLWLTCRPWEAM-COOH. Veh, vehicle (culture medium). These peptides were derivatives of previously characterized agonists for FPR (39 42 ) and FPRL1 (41 ). Arrow in C marks the starting point of peptide stimulation. Changes in the intracellular calcium levels are indicated in the left, with each ticker representing a 150 nM increment. Representative tracings from one of the two experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
G{alpha}16 has been known to couple a large number of G protein-coupled receptors (GPCRs) for PLC{beta} activation. However, the biological consequence of G{alpha}16 activation by these GPCRs has not been determined. The lack of this information is partly due to technical difficulties in biochemical characterization of the G{alpha}16 protein (36) and in the transfection of hemopoietic cells in which G{alpha}16 is expressed (18). A number of recent studies suggest that G{alpha}16 coupling of chemoattractant and chemokine receptors may be important for leukocyte functions. It was shown that transmembrane signaling of the leukotriene B4 receptor is mediated by both G{alpha}i and G{alpha}16 (43). In human T cells, alteration of the G{alpha}16 expression level by cell-activating agents affects TCR-mediated functions (44). In other hemopoietic cells, G{alpha}16 is known to couple the P2U purinergic receptor to calcium mobilization (20), and has been suggested to mediate some of the functions of thromboxane A2 receptor (45). G{alpha}16 is expressed at higher levels in early stages of hemopoietic cells, and in poorly differentiated leukemia cells (46). These findings suggest an association of G{alpha}16 with hemopoietic cell growth and differentiation.

In this study, we used transiently transfected cells to assess the potential function of G{alpha}16 in NF-{kappa}B activation. Our results demonstrate that, among four different G proteins examined, FPR preferentially couples to G{alpha}16 for this function. Overexpression of G{alpha}i2, which is known to couple the FPR for leukocyte activation, only marginally increased fMLP-induced NF-{kappa}B activation. We have also tested the C5a and C3a receptors, as well as two representative chemokine receptors (CXCR2 and CCR8), for their abilities to activate NF-{kappa}B. In the absence of G{alpha}16, C5a (100 nM) induced a ~2-fold increase in the expression of the {kappa}B luciferase reporter. A G{alpha}i protein(s) likely mediates this activity because it could be blocked by PTX (data not shown). Activation of G{alpha}i results in the release of G{beta}{gamma} proteins that mediate leukocyte chemotaxis and other functions. Thus, G{beta}{gamma} but not G{alpha}i presumably is responsible for the induced NF-{kappa}B activation. In comparison, expression of G{alpha}16 resulted in a potent induction of NF-{kappa}B activation with all the five receptors tested. These findings suggest that the chemoattractant and chemokine receptors preferentially couple to G{alpha}16 over G{alpha}i for NF-{kappa}B activation.

Our experimental data indicate that the activated G{alpha}16 (Q212L) is a highly potent inducer for NF-{kappa}B activation. In comparison, maximal induction of the same {kappa}B luciferase reporter by an activated G{alpha}q (Q209L) requires DNA input in the range of 100–200 ng per sample (data not shown). The high potency of G{alpha}16 is likely an intrinsic property of this G{alpha} protein, and may contribute to its ability to couple the chemoattractant receptors for NF-{kappa}B activation. We noted that overexpression of the activated G{alpha}16 could reduce the expression of the {kappa}B-driven luciferase reporter (Fig. 2GoA and data not shown). This may result from down-regulation of PKC{alpha} after protracted stimulation, as observed in cells treated with phorbol ester overnight. It is also possible that G{alpha}16 (Q212L) inhibits cell growth (22) when expressed at high concentrations, thereby reducing the ability to activate NF-{kappa}B.

We observed that activated G{alpha}16 markedly enhanced the TNF-{alpha}-induced NF-{kappa}B activation (Fig. 2GoB), suggesting that chemoattractants and chemokines work together with cytokines and growth factors for transcription activation. TNF-{alpha} is known to act on TNFR1, which couples to TNFR-associated factor for activation of the downstream kinases responsible for IKK activation. GPCR that couples to G{alpha}16 activates a different signaling pathway involving PLC{beta} and PKC{alpha}. Because both TNFR1- and G{alpha}16-mediated NF-{kappa}B activation involve IKK, these two signaling pathways likely converge on or before IKK activation and thereby create an additive or synergistic effect on NF-{kappa}B activation. This possibility can be more readily tested when stably transfected cells expressing both FPR and G{alpha}16 become available. The synergism between TNF-{alpha} and G{alpha}16 may also result from modification of NF-{kappa}B coactivators, but additional experiments will be necessary to examine this possibility. In leukocytes and other hemopoietic cells, activation of G{alpha}16 may synergize the effect of G{alpha}i-coupled receptors. This possibility has been suggested by a recent study demonstrating that preactivation of PLC{beta} by G{alpha}16 permits subsequent stimulation by G{alpha}i-coupled receptors in transiently transfected COS-7 cells (47). Indeed, in macrophages derived from G{alpha}15-/- mice, C5a-stimulated phosphoinositide accumulation and Ca2+ release were significantly reduced (19). These findings, combined with the regulation of G{alpha}16 expression level at various stages of cell differentiation (15, 48), suggest the potential of G{alpha}16 in regulating transcription and other cellular functions in conjunction with other cytokine receptors and GPCRs. Future studies will focus on the identification of GPCRs that functionally couple to G{alpha}16 at early stages of hemopoiesis, when this G protein most likely plays an important role in cell growth and differentiation.

G{alpha}16 was first characterized for its ability to stimulate PLC{beta} activation (23, 36). Our results extend this function to the activation of a transcription factor, suggesting that other G proteins may also activate NF-{kappa}B through PLC{beta}. Indeed, several GPCRs that primarily couple to G{alpha}q have been shown to stimulate NF-{kappa}B activation (27, 49, 50). We have shown that all three PLC{beta} isoforms found in non-neuronal cells can function in this capacity. Although PLC{beta}2 appears to be highly efficient in mediating G{alpha}16-induced NF-{kappa}B activation, cells lacking PLC{beta}2 (such as HeLa) may use PLC{beta}1 and PLC{beta}3 for NF-{kappa}B activation by this G protein. Because PLC{beta} expression levels vary under our experimental conditions, we cannot conclude whether PLC{beta}2 is more efficient than other PLC{beta} isoforms in mediating NF-{kappa}B activation. Our data indicate that the conventional PKC isoform, PKC{alpha}, is largely responsible for G{alpha}16-induced and PLC{beta}-mediated NF-{kappa}B activation (Fig. 5Go). PKC{alpha} is a downstream effector of PLC{beta} and can be activated by diacylglycerol, one of the PLC{beta}-generated second messengers. PLC{beta} activation also produces inositol 1,4,5-trisphosphate, which induces release of Ca2+ from intracellular stores (37). It is known that activation of PKC{alpha} requires Ca2+. Consistent with this, buffering of intracellular Ca2+ by BAPTA blocked G{alpha}16-induced NF-{kappa}B activation. PKC{alpha} has been known to play a role in NF-{kappa}B activation, such as in cells stimulated by phorbol esters (51, 52). Our results provide an example of NF-{kappa}B activation by PKC{alpha} through a pathway that involves G proteins and PLC{beta}. However, we noted that G{alpha}16-induced NF-{kappa}B activation was not completely blocked by the PKC inhibitors or by the DN PKC{alpha}. Therefore, it is possible that G{alpha}16 also triggers other signaling pathways that contribute to NF-{kappa}B activation.

We have tested five selected chemoattractant and chemokine receptors and found that these G{alpha}i-coupling receptors were able to stimulate G{alpha}16 and activate NF-{kappa}B to various degrees. Whether all G{alpha}i-coupling receptors possess this property remains to be investigated, but our results from the FPR study suggest that the ability of selected peptide ligands to induce NF-{kappa}B activation corresponds to their calcium mobilization potency. The P2 peptide WKYMVm-NH2 (42) has been shown to activate both FPR and a related receptor, FPRL1 (39). We demonstrate here that modifications of the carboxyl terminus or amino terminus (P1, P3, and P4; see Fig. 6Go) severely impaired the potency of this peptide in both calcium mobilization and NF-{kappa}B activation assays. Our results also confirmed that peptides P5 and P6, which are known agonists for FPRL1 (41), do not activate FPR at the concentration used (100 nM). These findings suggest a potential usage of G{alpha}16-mediated transcription activation in agonist and antagonist selection (53). Reporter-based assays provide a rapid and sensitive readout for GPCR signaling and have been widely used in both basic research and new drug discovery (54). With the development of cell lines that stably express G{alpha}16 and a {kappa}B reporter, the G{alpha}16-based assay may find its use in high-throughput screening of drug candidates for a large number of chemoattractant and chemokine receptors.


   Acknowledgments
 
We thank Darren Browning for helpful discussions, Tohru Kozasa for the anti-G{alpha}13 Ab, and Cindy Knall and Gary Johnson for the G{alpha}16 constructs used in this study.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (AI40176, AI41670, and AI33503), the Arthritis Foundation, and the American Heart Association. R.D.Y. is an Established Investigator of the American Heart Association. Back

2 Address correspondence and reprint requests to Dr. Richard D. Ye, Department of Pharmacology, MC868, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail address: yer{at}uic.edu Back

3 Abbreviations used in this paper: G protein, guanine nucleotide-binding regulatory protein; FPR, formyl peptide receptor; GPCR, G protein-coupled receptors; PKC, protein kinase C; PLC{beta}, phospholipase C-{beta}; PTX, pertussis toxin; IKK, I{kappa}B kinase; BAPTA, 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid; CXCR, CXC chemokine receptor; TTBS, Tween 20 + TBS; IP, inositol phosphate; DN, dominant negative. Back

Received for publication December 5, 2000. Accepted for publication March 23, 2001.


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R. A. Bakker, P. Casarosa, H. Timmerman, M. J. Smit, and R. Leurs
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The Proinflammatory Mediators C3a and C5a Are Essential for Liver Regeneration
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Requirement of G{beta}{gamma} and c-Src in D2 Dopamine Receptor-Mediated Nuclear Factor-{kappa}B Activation
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C. Marty, D. D. Browning, and R. D. Ye
Identification of Tetratricopeptide Repeat 1 as an Adaptor Protein That Interacts with Heterotrimeric G Proteins and the Small GTPase Ras
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B. Chandrasekar, P. C. Melby, H. M. Sarau, M. Raveendran, R. P. Perla, F. M. Marelli-Berg, N. O. Dulin, and I. S. Singh
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T Cell Chemokine Receptor Expression in Aging
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IL-12-dependent nuclear factor-{kappa}B activation leads to de novo synthesis and release of IL-8 and TNF-{alpha} in human neutrophils
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Regulation of nuclear factor {kappa}B activation by G-protein-coupled receptors
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