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Vol. 18, Issue 2, 512-522, February 2007

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A Chemoattractant-mediated G_i-coupled Pathway Activates Adenylyl Cyclase in Human Neutrophils

Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4256

Submitted May 16, 2006; Revised November 15, 2006; Accepted November 21, 2006
Monitoring Editor: John York

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to G_s-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical G_s-meditated pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Many types of cells have the ability to migrate directionally when exposed to gradients of chemoattractants. This chemotactic response is essential for a variety of physiological processes and is initiated when chemoattractants bind surface receptors and activate a wide range of signal transduction cascades, which ultimately lead to cellular polarization and migration (Parent, 2004). The acquisition of polarity is accompanied by a dramatic redistribution of cytoskeletal components, where F-actin and numerous actin-binding proteins are enriched at the front or leading edge and myosin II is assembled on the sides and at the back or trailing edge (Van Haastert and Devreotes, 2004; Bagorda et al., 2006).

A striking chemotactic behavior is exhibited by neutrophils as they move across vascular barriers and navigate to sites of inflammation (Niggli, 2003b; Parent, 2004). The agents that induce directed migration of neutrophils include a large and diverse group of chemoattractants originating from different sources (Uhing and Snyderman, 1999). These agents include formylated peptides secreted by bacteria (such as N-formylmethionyl-leucyl-phenylalanine [fMLP]), products of the complement cascade (such as complement factor 5a [C5a]), and phospholipid metabolites (such as leukotriene B4 [LTB₄]) as well as a large family of chemokines that are derived from endothelial, epithelial, and stromal cells (Baggiolini, 1998). Remarkably, neutrophils can also relay the signal to surrounding cells by stimulating the production and release of more attractants, such as LTB₄ and interleukin (IL)-8, which act in a paracrine manner to spread the chemotactic response to surrounding cells (Bazzoni et al., 1991; Baggiolini et al., 1994; Kannan, 2002). Chemoattractants bind to serpentine transmembrane receptors that couple to heterotrimeric G proteins (Murphy, 1996). Receptor activation leads to the dissociation of the G protein into - and -subunits, which go on to stimulate a variety of effectors that ultimately lead to cellular polarization and migration.

Several studies have established that the addition of chemoattractants to neutrophils causes an increase in the production of cAMP (Simchowitz et al., 1980; Smolen et al., 1980; Spisani et al., 1996; Suzuki et al., 1996; Ali et al., 1998). Although a clear correlation between total cAMP levels and chemotaxis has not been established, many studies have shown that, in neutrophils, increases in cAMP levels inhibit chemotaxis (Harvath et al., 1991; Elferink and VanUffelen, 1996; VanUffelen et al., 1998; Ariga et al., 2004). Furthermore, cAMP has been shown to regulate chemotactic migration in other cell types. O'Conner et al. 1998, 2000 have determined that the chemotactic migration of invasive carcinoma cells depends on cAMP in a RhoA-dependent manner, and studies using embryonic Xenopus spinal neurons have clearly established that the directionality of growth cones induced by many guidance factors can be switched by competitive analogues of cAMP or inhibitors of protein kinase A (PKA) (Ming et al., 1997). Although the mechanism by which cAMP controls chemotaxis remains to be established, it has been demonstrated that a variety of cytoskeletal and signaling components, including Rho and VASP, are regulated by PKA—the main intracellular target of cAMP (Howe et al., 2002). It therefore seems that modulation of intracellular cAMP levels is commonly used to regulate cell migration.

In neutrophils, the mechanism by which chemoattractants increase cAMP levels, which is synthesized from ATP by adenylyl cyclases, remains to be determined. Mammalian genomes harbor nine distinct G protein-coupled adenylyl cyclases (ACI-ACIX), composed of two sets of six transmembrane domains each followed by a conserved catalytic domain within a cytoplasmic loop (referred to as C1 and C2) (Tang and Hurley, 1998; Sunahara and Taussig, 2002). The nine ACs show distinct expression profiles and modes of regulation by G proteins, calcium, posttranslational modifications, and a variety of small molecules. Whereas all isoforms are stimulated by G_s, only ACV and ACVI are sensitive to inhibition by G_i. In contrast, G-subunits can be either stimulatory, as for ACII, ACIV, and ACVII, or inhibitory, as for ACI and ACVIII. ACIX is the most divergent isoform of ACs and constitutes a separate subfamily of ACs (Tang and Hurley, 1998; Sunahara and Taussig, 2002). Like all transmembrane ACs, ACIX is stimulated by G_s, but unlike ACI-ACVIII, ACIX is insensitive to forskolin stimulation, a diterpene from the plant Coleus forskohlii (Premont et al., 1996; Hacker et al., 1998). In mammalian cells, the canonical pathway leading to AC activation involves the activation of the heterotrimeric G proteins G_s. The cascade is initiated by the release of G_s and G subunits from the heterotrimer, which then go on to activate ACs, either alone (for G_s) or synergistically (Tang and Gilman, 1991; Lustig et al., 1993). It has been established that chemoattractants mediate most of their effects, including increases in cAMP levels, through the pertussis toxin (PTX)-sensitive G protein G_i (Suzuki et al., 1996; Niggli, 2003b). Interestingly, a non-G_s pathway also regulates the chemoattractant-mediated activation of the adenylyl cyclase ACA in the social amoebae Dictyostelium (Saran et al., 2002; Kriebel and Parent, 2004). Genetic and biochemical analyses have established that ACA requires G-subunits, the cytosolic regulator cytosolic regulator of adenylyl cyclase (CRAC), and the target of rapamycin complex 2 (TORC2) (Insall et al., 1994; Lilly and Devreotes, 1995; Chen et al., 1997; Lee et al., 1999; Comer et al., 2005; Lee et al., 2005). Because many chemoattractant-mediated signal transduction cascades are conserved between neutrophils and Dictyostelium, we hypothesize that the events leading to the activation of AC in neutrophils are analogous to the events leading to the activation of ACA in Dictyostelium. We therefore set out to investigate the signal transduction events controlling the chemoattractant-mediated increase in cAMP levels in human neutrophils.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents
The following reagents were obtained commercially from Sigma-Aldrich (St. Louis, MO): fMLP, LTB₄, IL-8, C5a, isoproterenol (Iso), forskolin, 1,9 dideoxyforskolin, 3-isobutyl1-methylxanthine (IBMX), latrunculin B (LatB), diisopropyl fluorophosphate (DFP), nocodazole (Noco), blebbistatin, Clostridium difficile toxin B, Y-27632, and histopaque 1077. LY294002 was obtained from AG Scientific. Texas Red X-phalloidin was obtained from Invitrogen (Carlsbad, CA). iScript and iQ SYBER Green Supermix were obtained from Bio-Rad (Hercules, CA). Radiolabeled [³²P]-ATP was obtained from MP Biomedicals (Irvine, CA). Phospho-AKT (Ser473) and AKT antibodies were obtained from Cell Signaling Technology (Beverly, MA). TRIzol and Superscript II were obtained from Invitrogen.

Isolation of Peripheral Blood Neutrophils
Heparinized whole blood was obtained by venipuncture from healthy donors. Neutrophils were isolated by dextran sedimentation coupled to differential centrifugation over Histopaque 1077, similar to the method described previously (Boyum, 1974). Three rounds of hypotonic lysis with 0.2 and 1.6% saline removed residual red blood cells. Purified neutrophils were resuspended in modified Hanks' balanced salt solution (mHBSS; 10 mg/ml glucose, 150 mM NaCl, 4 mM KCl, 1.2 mM MgCl₂, and 20 mM HEPES) and kept on ice until used. For the preparation of samples used for immunoblotting, neutrophils were treated with 2 mM diisofluorophosphate (DFP) for 20 min and resuspended in modified Hanks' balanced salt solution with protease inhibitors.

Human Embryonic Kidney (HEK)293T Cells
HEK293T cells were maintained in DMEM media + 10% serum on 100-mm plates. Cells were grown to 80% confluence for experimental samples. Cells were dislodged with Versene, washed twice with mHBSS, and placed on ice. Cells were warmed to room temperature (RT) before adenylyl cyclase assay.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis
Neutrophil RNA was purified using the TRIzol reagent according to manufacturer instructions. One microgram of total RNA was used in RT reactions with human AC isoform-specific primers (see Supplemental Table 1). We used 100 ng of human brain mRNA as a positive control. RT reactions using Superscript II were performed at 42°C for 2 h. Subsequent PCR analysis used 40 cycles to identify low abundance RNA message. Aliquots were run on Tris borate-EDTA gels and stained with ethidium bromide.

Real-Time Quantitative RT-PCR
PCR amplification using a Bio-Rad iCycler system was carried out in a 20-µl reaction mixture in 96-well PCR plate. The reaction components were 0.2 µl of cDNA, 1X iQ Syber Green Supermix, and 100 nM each primer (see Supplemental Table 1). The reactions were incubated at 95°C for 30 s followed by 50 cycles of amplification at 95°C (30 s) for melting and at 55°C (30 s) for annealing and extension. After cycling, melt curves of the resultant PCR products were acquired. Serial dilutions of cloned PCR products were included for controls. The results are represented as the ratio of 1/(AC isoform threshold cycle/GAPDH threshold cycle) to establish relative expression. The data represents the analysis of five independent experiments.

AKT Phosphorylation Assay
Neutrophils were treated with DMSO or 100 µM LY294002 for 30 min at 37°C. Cells were then stimulated with 50 nM fMLP for 5 min. Aliquots were taken for Western analysis before and 5 min after fMLP addition. Total cell lysates were run on a 10% polyacrylamide gel and transferred to polyvinylidene difluoride membrane. Detection of Phospho-AKT (Ser473) and total AKT proteins was performed using commercially available antibodies, and detection was achieved using chemiluminescence with a goat anti-rabbit horseradish peroxidase-coupled antibody.

Chemotaxis Assay
Neutrophils were resuspended at 1 x 10⁷/ml in mHBSS. One hundred microliters of cells were placed in the upper chamber of a 5-µm pore size transwell chamber (3421; Corning Life Sciences, Acton, MA). Chemoattractant resuspended in mHBSS was placed in the lower compartment. The apparatus was incubated at 37°C for 1.5 h. Cells that had migrated through the well into the lower chamber were counted using an automated Coulter counter (Beckman Coulter, Fullerton, CA).

Adenylyl Cyclase Assay
Neutrophils were resuspended at 2 x 10⁷/ml in mHBSS and stimulated with various compounds. Aliquots of cells were taken at specific time points and rapidly lysed through a 5-µm membrane. Samples were transferred to an AC assay mix containing 10 mM Tris-HCl, 1 mM MgCl₂, 100 µM ATP, 100 µM cAMP, 100 µM dithiothreitol (DTT), 200 µM IBMX, and 0.5 µCi of [³²P]-ATP (final concentrations) and incubated for 5 min. IBMX and DTT are added to the reaction mix to inhibit phosphodiesterase activity. Furthermore, a high concentration of cold cAMP is present to ensure that any residual phosphodiesterase activity mainly acts on the cold cAMP and spare the [³²P]cAMP generated from the [³²P]ATP. The reactions were stopped with the addition of 0.1% SDS and 1 mM ATP. Radiolabeled cAMP produced in the reaction was purified by sequential chromatography over Dowex 50W x4 and Alumina columns (Salomon, 1979). AC measurements by this assay exhibit considerable variation from experiment to experiment. Although the relative extent of activation is highly reproducible, the absolute activity can vary significantly up to twofold from day to day. Therefore, we have chosen to present AC activation data as an average of -fold stimulation from at least three independent experiments, each performed in duplicate on any given day. The basal and peak AC stimulation after the addition of 10 µM fMLP ranged from 2.4 to 4.3 and 6.8 to 12.9 pmol/min/mg protein, respectively.

Microscopy
For microscopic observation of actin polymerization, neutrophils were allowed to adhere to gelatin-coated coverslips for 10 min. Neutrophils were stimulated with 10 nM fMLP and then fixed (1% formaldehyde, 0.125% glutaraldehyde, and 0.01% Triton X-100 in mHBSS) after 10 min. Cells were blocked in 3% bovine serum albumin in phosphate-buffered saline and stained with Texas Red X-phalloidin (Invitrogen).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Chemoattractants Rapidly and Transiently Stimulate Transmembrane Adenylyl Cyclases in a G_i-dependent Manner
To determine the mechanism that regulates the chemoattractant-mediated accumulation of cAMP, we measured the effect of chemoattractants on AC activity using an activation trap assay. In this assay, cells are stimulated with various concentrations of chemoattractants, and, at specific times, cells are rapidly filter lysed into a [³²P]ATP-containing mixture, and AC activity is assayed by measuring the [³²P]cAMP produced (see Materials and Methods). As depicted in Figure 1A, addition of the formylated peptide fMLP to neutrophils induces a rapid, dose-dependent, and transient stimulation of AC activity, peaking at 1 min and returning to basal levels after 7–8 min. To determine whether this response is dependent on G protein-coupled transmembrane ACs, we pretreated cells with the potent P-site inhibitor dideoxyadenosine, which specifically acts on transmembrane ACs (Han et al., 2005). We found that it completely inhibits the response to 1 µM fMLP (Figure 1B). We next determined whether the ability of fMLP to stimulate AC activity is a common feature of chemoattractants. We find that a wide range of chemoattractants exhibit this property. Figure 1C shows the response induced by the end target chemoattractant C5a as well as endogenous agents such as LTB₄ and IL-8. Together, these findings establish that the chemoattractant-mediated elevation in cAMP production observed in neutrophils is mediated by an increase in AC activity.

View larger version (39K): [in this window] [in a new window]   Figure 1. Chemoattractants rapidly and transiently stimulate transmembrane AC activity. (A) Neutrophils were stimulated with various concentrations of fMLP. Aliquots were taken at 0, 1, 4, and 8 min and filter lysed into a reaction mix containing [32P]ATP to measure de novo synthesis of [32P]cAMP (see Materials and Methods). (B) Cells were pretreated with the P-site inhibitor dideoxyadenosine at 300 nM for 10 min at RT, and AC activity in response to 1 µM fMLP was measured as described in A. (C) Assays were performed as described in A in response to fMLP, C5a, LTB4, or IL-8. Results are presented as -fold stimulation of AC activity. (D) Neutrophils were incubated with 450 µg/ml PTX or with buffer for 2 h at 37°C and AC activity was measured just before (–fMLP) and 1 min after the addition of 10 µM fMLP (+fMLP). For all experiments, data are presented as the average -fold stimulation over basal for at least three independent experiments done in duplicate for each time point (see Materials and Methods).

The Chemoattractant-mediated Adenylyl Cyclase Activation in Neutrophils Is Insensitive to Forskolin
RT-PCR analysis performed on RNA extracts showed that, of the nine mammalian AC enzymes, isoforms III, IV, VII, and IX are expressed in human neutrophils (Figure 2A, inset). Further analysis using real-time PCR established that ACIII is the least abundant isoform and that isoforms IV, VII, and IX are expressed at similar levels (Figure 2A).

View larger version (35K): [in this window] [in a new window]   Figure 2. ACIX may be the predominant isoform present in neutrophils. (A) Purified RNA from whole blood neutrophils was subjected to RT-PCR by using AC isoform-specific primers (see Supplemental Table 1). The agarose gel electrophoresis is presented as an inset in A. First-strand cDNA was subjected to real-time PCR analysis for the presence of ACIII, IV, VII, and IX mRNAs as a function of GAPDH mRNA present in each sample. Relative expression is represented as the threshold cycle of each isoform/the threshold cycle for GAPDH for each sample. (B) HEK293T or neutrophils were filter lysed and buffer-, 100 µM GTPS-, or 100 µM forskolin-mediated AC activity was assayed as described in Materials and Methods. (C) Neutrophils were stimulated with 1 µM fMLP and, at specific times, filter lysed into a reaction mix containing DMSO (control) or 100 µM forskolin. AC activity was measured as described in Materials and Methods. (D) Neutrophils were stimulated with 100 µM isoproterenol, and at specific times, filter lysed into a reaction mix containing DMSO (control) or 100 µM forskolin. AC activity was measured as described in Materials and Methods. For all experiments, data are presented as the average -fold stimulation over basal for at least three independent experiments done in duplicate for each time point (see Materials and Methods).

Chemoattractant-mediated Activation of Adenylyl Cyclases Adapts to a Constant Level of Stimulus
In sharp contrast to the sustained activation profile observed after stimulation with isoproterenol, the chemoattractant-mediated activation of AC is transient, showing an excitation peak followed by a return of the response to basal levels (Figure 3A). This activation profile is reminiscent of the AC response observed following chemoattractant addition to Dictyostelium cells (Dinauer et al., 1980). To determine whether the transient chemoattractant-mediated AC activation in neutrophils is due to the loss of active fMLP during the 8-min assay period, we stimulated cells with a saturating dose of fMLP every 2 min during the assay period and measured enzyme activity. Under these conditions, we again found that the activation profile retains its transient nature, indicating that the AC turns off in the presence of persistent fMLP signal (Figure 3B). These findings show that the chemoattractant-mediated activation of AC is similar to what we observe in Dictyostelium, displaying a rapid peak of activation that occurs 1 min after stimulation, followed by a turn off phase where the activity goes back down to basal levels after 8 min.

View larger version (38K): [in this window] [in a new window]   Figure 3. Chemoattractant-mediated activation of AC adapts to constant levels of stimulus. (A) Neutrophils were stimulated with either 10 µM fMLP or 100 µM isoproterenol, and AC activity was assayed as described in the legend of Figure 1. (B) Neutrophils were stimulated with a single dose (10 µM) or multiple doses (10 µM) of fMLP at 2-min intervals. AC activity was measured as described in the legend of Figure 1. (C) Neutrophils were stimulated with 10 µM fMLP or 100 µM isoproterenol, or costimulated with fMLP and isoproterenol and AC activity was measured as described in Materials and Methods. (D) Neutrophils were stimulated with 10 µM fMLP and after lysis, transferred to a reaction mix supplemented with buffer or 100 µM isoproterenol. As a control, cells were stimulated with buffer, lysed, and transferred to a reaction mix containing 100 µM isoproterenol. For results presented in C and D, representative data of at least three independent experiments done in duplicate for each time point are shown (see Materials and Methods).

The Cytoskeleton Does Not Regulate Chemoattractant-mediated Activation of Adenylyl Cyclases
On chemoattractant stimulation, neutrophils rapidly assemble a dense network of cortical F-actin that becomes polarized to the leading edge of chemotaxing cells (Niggli, 2003b; Parent, 2004). Because F-actin has been reported to amplify chemoattractant signals via a positive feedback loop to the effector PI3K, we set out to determine whether the actin cytoskeleton could also regulate the chemoattractant-mediated activation of AC (Niggli, 2000; Weiner et al., 2002). For these experiments, we pretreated neutrophils with 40 µg/ml latrunculin B for 30 min, which completely inhibited the fMLP-mediated burst in F-actin polymerization as well as chemotaxis to fMLP (Figure 4A; data not shown). In sharp contrast, we found that latrunculin B treatment had no effect on fMLP-mediated AC activity (Figure 4B). In agreement with these findings, we also found that RhoGTPases are not required, as neutrophils treated with C. difficile toxin B show normal fMLP-mediated AC activation (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 4. The cytoskeleton does not regulate chemoattractant-mediated activation of adenylyl cyclase. (A) Neutrophils were treated with or without 40 µg/ml latrunculin B for 30 min to disrupt actin filaments and allowed to adhere to a gelatin-coated coverslip for 10 min. Cells were stimulated with 10 nM fMLP for 10 min followed by fixation and staining with Texas Red X-phalloidin to visualize F-actin localization. (B) Control and latrunculin B-treated cells were stimulated with 10 µM fMLP, and AC activity was assessed as described in legend of Figure 1. (C) Neutrophils were treated with or 30 µM nocodazole for 30 min to disrupt microtubules. Actin assembly was assessed for control and nocodazole-treated cells as described in A. (D) Control and nocodazole-treated cells were stimulated with 10 µM fMLP, and AC activity was assessed as described in legend of Figure 1. (E and F) Neutrophils were treated with 100 µM blebbistatin (E) or 10 µM Y-27632 (F) for 30 min and processed for fMLP-mediated AC activity as described in legend of Figure 1.

We next evaluated the involvement of myosin II and Rho-associated protein kinases (ROCK) on AC activation. For these experiments we treated neutrophils with the myosin II inhibitor blebbistatin (100 µM) or the ROCK inhibitor Y-27632 (10 µM) and assessed fMLP-mediated AC activation. As published previously, we found that both treatments gave rise to cells with multiple pseudopods and a general lost of uropod (data not shown) (Niggli, 2003a; Xu et al., 2003; Xu et al., 2005). However, neither drugs affected the extent by which fMLP stimulates AC activity (Figure 4, E and F). These findings suggest that the integrity of the back of neutrophils is not required for the chemoattractant-mediated activation of AC.

Chemoattractant Stimulation of Adenylyl Cyclase Is PI3K Independent but Requires Cellular Integrity
To further identify the signals involved in mediating the activation of AC by chemoattractants in neutrophils, we studied the role of PI3K in this response. Because the activation of the Dictyostelium ACA is dependent on PI3K activity, we reasoned that a similar pathway could also be operative in neutrophils (Comer et al., 2005). We treated neutrophils with the PI3K inhibitor LY294002 and assessed its efficiency by measuring the extent by which fMLP-mediated Akt/PKB phosphorylation and chemotaxis were inhibited. We determined that both responses were almost completely inhibited by pretreating neutrophils with 100 µM LY294002 for 30 min (Figure 5A; data not shown). In contrast, we found that similar treatment had no effect on fMLP-mediated AC activation (Figure 5B). Similar results were obtained with LTB₄-mediated stimulation (data not shown). These findings establish that the signaling cascade that leads to the chemotactic activation of AC in neutrophils is independent of PI3K activity.

View larger version (33K): [in this window] [in a new window]   Figure 5. Chemoattractant stimulation of AC is PI3K independent and requires cellular integrity. (A) Neutrophils were treated with or without 100 µM LY294002 for 30 min to inhibit PI3K activity, placed in a transwell chamber, and chemotaxis to 10 nM fMLP was assessed as described in the legend of Figure 1. (B) LY294002-treated and control cells were stimulated with 10 µM fMLP, and AC activity was assessed as described in the legend of Figure 1. Similar results were obtained with 50 nM fMLP (data not shown). (C) Neutrophils were filter lysed into a cup and stimulated with buffer, 100 µM GTP S, 100 µM isoproterenol, or 10 µM fMLP, and AC was measured as described in Materials and Methods. (D) Neutrophils were filter lysed into a cup and stimulated with buffer, 100 µM isoproterenol, 10 µM fMLP, or costimulated with 100 µM isoproterenol and 10 µM fMLP for 1 min and AC was measured as described in Materials and Methods.

To rule out the possibility that chemoattractant receptor-G protein coupling is altered in neutrophil lysates, we performed costimulation experiments with isoproterenol and fMLP. We found that costimulation of lysates gives rise to a level of activation that is higher compared with the activation profile of isoproterenol alone (Figure 5D). We envision that in cell lysates, the G-subunits released after fMLP stimulation provides further positive input on the G_s-mediated isoproterenol response leading to higher AC activity. Collectively, these results show that the chemoattractant-mediated activation of AC exhibits properties that are distinct from the canonical -adrenergic, G_s-mediated activation, which solely relies on plasma membrane-bound components.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We show that chemoattractant addition to human neutrophils gives rise to a rapid and transient activation of AC activity. Whereas chemoattractants, particularly fMLP, have previously been shown to increase total cAMP levels in neutrophils, the mechanism by which this occurs has remained unclear (Simchowitz et al., 1980; Smolen et al., 1980; Spisani et al., 1996; Suzuki et al., 1996; Ali et al., 1998). Cyclic AMP production is regulated by its synthesis through the activation of ACs and its degradation by phosphodiesterases. Neutrophil cAMP levels peak 15–30 s after exposure to fMLP and rapidly return to basal levels after 5 min (Simchowitz et al., 1980; Verghese et al., 1985). Pretreatment of neutrophils with phosphodiesterase inhibitors renders the cAMP elevation sustained and shifts the peak of accumulation to a plateau achieved at 60 s after fMLP stimulation (Simchowitz et al., 1980; Iannone et al., 1989). In addition, other chemokines such as C5a and LTB₄ have been shown to elicit an increase in cAMP levels within neutrophils with similar kinetics (Simchowitz et al., 1980; Gorman et al., 1984). Using a pharmacological approach, it has been suggested that fMLP-stimulated cAMP accumulation is partly mediated by a transient inhibition of phosphodiesterase activity (Verghese et al., 1985). Whereas our studies do not address the impact of fMLP on phosphodiesterase activity, they clearly establish that chemoattractants activate AC. We were able to directly assess the effect of chemoattractants on AC activity by using an activation trap assay commonly used in Dictyostelium (Devreotes et al., 1987). Using filter lysis, this assay allows the rapid and accurate determination of AC activity after whole cell stimulation. We show that the activation is mediated via G_i, in a PI3K-independent manner, and it is lost when chemoattractants are added to cell lysate.

Our findings provide evidence that ACIX is the predominant AC isoform present in human neutrophils. We found that AC activity in neutrophil lysates is mostly forskolin insensitive, a property that is exclusively characteristic of ACIX (Premont et al., 1996; Hacker et al., 1998). The lack of reliable and specific antibodies against the AC isoforms precluded us from directly measuring protein levels. In a recent study, Chang et al. 2003 studied the AC expression profile in rat neutrophils. They showed that whereas all nine AC isoforms are expressed at the mRNA level, ACI, II, VI, and IX predominate (Chang et al., 2003). In addition, and in accordance with our findings using isoproterenol and forskolin, they observed that forskolin inhibits GTPS stimulated cAMP accumulation. Similarly, it has been reported that forskolin attenuates GTPS-mediated AC activation in membranes derived from guinea pig neutrophils as well as in HEK293 cells exogenously expressing ACIX (Suzuki et al., 1996; Hacker et al., 1998). These findings could be explained by the ability of forskolin to bind a novel, unknown, partner that specifically interferes with G_s-mediated activation. However, because ACIX is the only AC known to exhibit this particular behavior in response to G_s/forskolin costimulation when expressed in HEK293 cells, this explanation is unlikely. As previously proposed by Hacker et al. 1998, we favor a mechanism in which the binding of forskolin to ACIX renders the enzyme resistant to G_s stimulation (Hacker et al., 1998). Because we also found that fMLP stimulation of AC is not altered by forskolin, we propose that signals downstream of chemotactic and -adrenergic receptors act on distinct sites of ACIX.

Our findings establish that G_i-coupled signaling is required for chemoattractant-induced AC activity. At first glance, this result seems counterintuitive, because G_i inhibits a subset of ACs (Sunahara and Taussig, 2002). However, because the four ACs expressed in neutrophils are insensitive to G_i (Hacker et al., 1998; Sunahara and Taussig, 2002) and G-subunits do not activate ACs on their own (Tang and Gilman, 1991), we propose that chemoattractant-mediated activation of AC in neutrophils requires the released G subunits from G_i, along with an additional factor having G_s-like activity. Although basal G_s activity could synergize with the G subunits released from G_i to stimulate AC activity after neutrophil exposure to chemoattractants (Iannone et al., 1989), our studies suggest that another mechanism may be involved as well. Chemoattractant-mediated activation is specifically lost in cell lysates. Yet, under the same conditions, we are able to measure a synergistic effect on AC activity in the presence of fMLP and isoproterenol. Consequently, if such a G_s/G synergy were at play during chemoattractant-mediated activation, it would have been detected in cell lysates. Instead, we envision that other components are involved. Interestingly, others have shown that chemoattractants use noncanonical pathways. Xenopus oocyte complementation studies revealed that coupling between fMLP or C5a receptors and calcium influx occurs only in oocytes preinjected with RNAs-derived from phagocytic cells (Murphy and McDermott, 1991; Schultz et al., 1992a,b). In addition, in sharp contrast to what is observed in neutrophils, fMLP stimulation of HEK293 cells engineered to express fMLP receptors leads to an inhibition of isoproterenol-mediated cAMP accumulation (Uhing et al., 1992). We have not thoroughly tested the requirement for small GTPases in the chemoattractant-mediated activation of AC; however, our findings with C. difficile toxin B establish that RhoGTPases are not required. Together, these observations suggest that neutrophils require unique factor(s) to transduce chemotactic signals to ACs.

Chemoattractant-mediated activation of the Dictyostelium adenylyl cyclase ACA, which shares homology with mammalian transmembrane ACs, requires cytosolic components (Kriebel and Parent, 2004). In these cells, chemoattractant receptors are coupled to the heterotrimeric G protein G2. Genetic analyses revealed that cells lacking either G2- or G-subunits do not exhibit chemoattractant-induced ACA activity. However, although the GTPS-mediated activation of ACA is also lacking in g^– cells, g2^– cells show a robust response to GTPS, presumably mediated by the release of G subunits from G proteins other than G₂ (Kesbeke et al., 1988; Wu et al., 1995; Parent and Devreotes, 1996; Kriebel and Parent, 2004). Such findings establish that, in Dictyostelium, G-subunits are not required for the chemoattractant-mediated activation of ACs. Instead, G-subunits work in concert with the pleckstrin homology (PH)-containing cytosolic regulator CRAC (Insall et al., 1994). On chemoattractant addition, CRAC is transiently recruited to the plasma membrane, in a PI3K-dependent manner (Parent et al., 1998; Comer et al., 2005). Although we have yet to determine the mechanism by which CRAC regulates ACA, we do know that the redistribution of CRAC to the plasma membrane, and PI3K activity, are essential (Comer et al., 2005; Comer and Parent, 2006). Extensive searches of the human, Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae genome data banks failed to identify CRAC homologues. Furthermore, the findings reported here show that AC activation does not seem to depend on PI3K activity. Together, these results exclude the requirement for a CRAC-like component for the chemoattractant-mediated activation of AC in neutrophils. Nevertheless, the striking homology of the chemoattractant-mediated AC activation profile between neutrophils and Dictyostelium, at the biochemical level, underscores the evolutionary conversation of the signaling cascades. Moreover, as the chemoattractant-mediated ACA activation also requires components of the TORC2 pathway (Chen et al., 1997; Lee et al., 1999, 2005), other novel factors may also be required in neutrophils.

In summary, our findings establish that, in neturophils, the pathway that links chemoattractant receptors/G_i to AC activation is fundamentally different from the canonical G_s to AC pathway. Moreover, it is similar to the well-characterized cascade leading to ACA activation in Dictyostelium. Additional studies will be required to identify the components required to transduce G_i-coupled signals to AC.

	ACKNOWLEDGMENTS

 
We thank Dr. Phil Murphy and Joan Sechler for help with the neutrophil isolation procedure as well as the Department of Transfusion Medicine at the National Institutes of Health and the many donors for providing blood products. We also thank Drs. Pierre Coulombe, Alan Kimmel, Joe Brzostowski, and Lakshmi Balagopalan for reading the manuscript and making many helpful suggestions and the members of the Parent laboratory for reviewing the manuscript and for insightful discussions. This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. D.C.M. is a recipient of a National Science and Engineering Research Council of Canada postdoctoral fellowship. R.L.S. is a recipient of a Howard Hughes Medical Institute-National Institutes of Health Research Scholars Award.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0418) on November 29, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Carole A. Parent (parentc{at}helix.nih.gov)

Abbreviations used: AC, adenylyl cyclase; C5a, complement factor 5a; fMLP, N-formylmethionyl-leucyl-phenylalanine; GTPS, guanosine 5'-O-(3-thio)triphosphate; LTB₄, leukotriene B4; PTX, pertussis toxin.

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