Published online before print April 7, 2005
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Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh
1 Correspondence: Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. E-mail: sam_jones{at}ncsu.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: integrin • adhesion • migration • actin cytoskeleton • phosphatidylinositol-3 kinase
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein kinase A (PKA) has long been known to regulate migration. Many studies using pharmacological cyclic adenosine monophosphate (cAMP) analogs and cAMP-elevating agents demonstrated that PKA activity must be overcome for migration to occur [3 4 5 6 7 8 ]. It is interesting that pharmacological activation and inhibition of PKA activity reduce neutrophil migration [9 , 10 ]. The cAMP/PKA pathway regulates integrin function [8 , 11 12 13 ] as well as the actin cytoskeleton [6 , 8 , 14 15 16 17 18 ], and either function may account for the role for PKA during migration.
We recently demonstrated that a tonic PKA activity is required to maintain low-avidity ß2 integrins on neutrophils, thus preventing adhesion in the absence of an appropriate stimulus [11
]. Inhibition of PKA activity in otherwise unstimulated neutrophils increases adhesiveness via activation of ß2 integrin avidity [11
]. Moreover, Fleming et al. [12
] showed that hyperactivation of PKA by inhibition of phosphodiesterase 4 activity suppressed the formation of peripheral actin-rich adhesion structures. Finally, fibroblast migration requires PKA phosphorylation of 
4 integrins at the leading edge [8
]. These data suggest that appropriate coordination of integrin function and organization of the actin cytoskeleton in migrating neutrophils is dependent on precise spatial regulation of PKA function. However, little is known about how PKA spatially or temporally regulates integrins, adhesion, or the actin cytoskeleton during neutrophil migration.
Our hypothesis is that asymmetrical PKA activity is required for neutrophil polarization and migration. Furthermore, as inhibition of PKA activates ß2 integrins and adhesion in neutrophils [11
], we reasoned that unlike fibroblasts [8
], neutrophil migration is dependent on low PKA activity at the leading edge and high PKA activity at the trailing edge. To test this hypothesis, we determined whether global inhibition of PKA reduced formylated Met-Leu-Phe (fMLF)-induced migration and whether this was correlated with increased overall adhesiveness and increased Mß2 integrin cell-surface expression and affinity activation. We then determined whether gradients of PKA inhibitors, expected to decrease PKA activity at the leading edge of the cells relative to the rear, were sufficient to induce migration. We next determined whether global inhibition of PKA activity disorganized the actin cytoskeleton in neutrophils stimulated with fMLF. Finally, we determined whether a gradient of the PKA inhibitor was sufficient to polarize the neutrophil actin cytoskeleton.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of leukocyte suspensions
Human neutrophils were isolated from whole blood using a dextran sedimentation/Ficoll gradient centrifugation protocol as described [23
]. Neutrophil viability was greater than 98%, as indicated by the exclusion of trypan blue dye. Lymphocytes were isolated from whole blood using a dextran sedimentation/Ficoll gradient centrifugation. Monocytes were depleted by adhesion to plastic. Cells were suspended in HBSS with 20 mM HEPES, 8.9 mM sodium bicarbonate, 1.0 mM Mg2+, and 1 mM Ca2+ (HBSS++) for adhesion assays and flow cytometry.
Migration assay
Purified human neutrophils (1x107/ml) were incubated with 2 µg/ml calcein in HBSS for 30 min at room temperature (RT). The cells were washed once and resuspended in chemotaxis buffer (HBSS++ with 2% FBS) at 5 x 105/ml. Cells were treated with inhibitors at the indicated concentration or Me2SO as a control for 30 min at 37°C. Chemotaxis was then assayed using a 96-well chemotaxis chamber system (ChemoTx, Neuroprobe). The bottom wells were filled with 29 µl chemotaxis buffer containing the indicated concentration of the chemotactic agent. A framed 3-µm pore diameter polycarbonate membrane filter was then placed over the wells, and 1 x 104 cells suspended in 20 µl chemotaxis buffer were added to the top side of the filter over each well. The plates were incubated for 30 min at 37°C. The top side of the filter was then scraped with a rubber squeegee to remove residual cells and rinsed with PBS. EDTA (10 µl 5 mM) was added to the top side of the filter for 5 min and then rinsed away. The plate was centrifuged for 5 min at 1000 rpm, the filter was removed, and fluorescence (485 nm excitation, 530 nm emission wavelengths) was measured in the bottom wells using an fMax fluorescence plate reader (Molecular Devices, Sunnyvale, CA). The percentage of cells migrating into the bottom well (percent chemotaxis) was calculated by dividing the fluorescence of the well by the average total fluorescence of four lower wells containing 1 x 104 neutrophils serving as standards. In preliminary experiments, cell numbers calculated using fluorescence measurements in the lower chamber of wells containing 100 nM fMLF correlated well (r>97%) with manual cell counts (data not shown). Lymphocyte migration assays were performed as above, except a 5-µM pore-size filter was used.
Adhesion assay
Purified human neutrophils (1x107/ml) were incubated with 2 µg/ml calcein in HBSS for 30 min at RT. The cells were washed once and resuspended in HBSS++ at 2 x 106/ml. Cells were treated with inhibitors at the indicated concentration or Me2SO as a control for 20 min at 37°C. Cells (1x105) were added per well to Immulon 2 plates coated with 5% FBS in PBS. Cells were allowed to settle onto the FBS-coated surface for 6 min at RT, and then agonists were added at the indicated final concentrations. The plates were then incubated at 37°C for the indicated time. The fluorescence (485 nm excitation, 530 nm emission wavelengths) was measured using an fMax fluorescence plate reader (Molecular Devices) before and after washing twice with 150 µl PBS. Percent adhesion was calculated by dividing the fluorescence after washing by the fluorescence before washing. Fluorescence was linearly related to cell number in preliminary experiments (data not shown).
Flow cytometry
Purified neutrophils (4x106/ml in HBSS++) were pretreated with inhibitors for 30 min at 37°C and then with fMLF at the indicated concentrations for 10 min at 37°C. The cells were then placed on ice, washed once with ice-cold wash buffer (PBS, 1% FBS, 0.1% sodium azide), and resuspended in 100 µl wash buffer plus primary antibody (25 µg/ml). Cells were incubated with primary antibody F(ab')2 for 40 min on ice and then washed twice. After incubation with FITC-conjugated sheep anti-mouse IgG secondary antibody F(ab')2 at a 1:50 dilution in 200 µl wash buffer for 20 min on ice, cells were washed twice, and the relative fluorescence of 10,000 gated neutrophils was measured using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
F-actin staining
Purified neutrophils suspended in HBSS++ were pretreated with inhibitors at the indicated concentration for 30 min at 37°C. Cells (3x105) were added to wells of a 12-well cell-culture plate (Corning Inc., Corning, NY) containing 12 mm glass coverslips coated with FBS. Cells were stimulated by inserting a 200-µl micropipette tip filled with 100 µl 1% solidified low-melt agarose in PBS containing fMLF or the indicated compound into the chamber along the well edge. The cells were then incubated for 30 min at 37°C. The buffer was removed from the well, and the cells were fixed for 20 min at RT with fixation buffer (25 mM PIPES, 50 mM KCl, 10 mM MgSO4, 5 mM EGTA, and 3% paraformaldehyde, pH 7). The fixation buffer was removed, and the cells were then permeabilized with ice-cold Triton buffer (0.5% Triton X-100, 10 mM PIPES, 300 mM sucrose, 100 mM KCl, 3 mM MgCl2, and 10 mM EGTA, pH 6.8) for 10 min on ice. Coverslips were washed twice with ice-cold protein solution (0.2% gelatin, 0.2% azide, 0.1% OVA in PBS) and then incubated with rhodamine phalloidin in PBS (1:20) for 20 min at RT. Coverslips were washed twice with PBS and once with o-phenylenediamine dihydrochloride (1 mg/ml) in glycerol and mounted on glass slides. Cells were examined using an Olympus VANOX AHS-3 photomicroscope. Polarity of the actin cytoskeleton was determined from the distribution of the rhodamine phalloidin staining as described [24
]. The total number and the number of polarized cells were determined in 5 high-power fields (hpf) for each coverslip. Data were expressed as the percentage of cells with F-actin distribution typical of polarized cells in 5 hpf.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. Global inhibition of PKA activity abolishes fMLF-induced neutrophil chemotaxis. Purified neutrophils labeled with calcein were treated with vehicle control Me2SO or the indicated concentrations of KT5720 for 20 min at 37°C and placed on the upper side of a membrane separating the cells from the lower chamber of a 96-well chemotaxis system described in Materials and Methods. Chemotaxis buffer in the lower chamber contained vehicle-control Me2SO or fMLF at the indicated concentration. The plate was incubated at 37°C for 30 min, at which time neutrophil chemotaxis to the lower chamber was determined as described in Materials and Methods. Data are presented as the mean ± SE of triplicate wells reported as the percentage of neutrophils added to the top of the membrane migrating to the lower chamber. KT5720 significantly inhibited migration in response to fMLF (*, P<0.05). Data are representative of four separate experiments using neutrophils from different donors.
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(100 nM) migration, 21.0%±1.0 (P<0.05)].
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Figure 2. A gradient of PKA inhibitor is sufficient to stimulate neutrophil chemotaxis. Neutrophils labeled with calcein were placed on the upper side of a membrane separating the cells from the lower chamber of a 96-well chemotaxis system. (A) Vehicle-control Me2SO or the indicated concentration of KT5720 was added to the chemotaxis buffer in the lower well (Bottom), the cell suspension buffer (Top), or both (Top and Bottom). (B) The indicated concentration of H-89 was added to the chemotaxis buffer in the lower well. (C) The indicated concentration of each inhibitor was added to the lower well of the chemotaxis chamber. Rp-Ct-cGMP, Rp-Ct-cyclic guanosine monophosphate. (D) St-Ht31 inhibitory peptide or an inactivated control peptide was added at the indicated concentration to the chemotaxis buffer in the lower well. The plates were incubated at 37°C for 30 min, at which time, neutrophil migration to the lower chamber was determined, as described in Materials and Methods. Data are presented as the mean ± SE of triplicate wells reported as the percentage of neutrophils added to the top of the membrane migrating to the lower chamber. KT5720, H-89, and St-Ht31 in the lower chamber significantly stimulated migration (*, P<0.05). Data are representative of six separate experiments using neutrophils from different donors.
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One weakness of our pharmacological approach is that we cannot be certain of the specificity of the inhibitor compound used in our assay. To address the issue of specificity, we determined whether a gradient of a second PKA inhibitor, H-89 [27 ], was also able to stimulate migration. Like KT5720, addition of H-89 to the bottom well of a chemotaxis chamber also increased neutrophil migration (Fig. 2B) . Moreover, the PKG inhibitor Rp-Ct-cGMP, the myosin light-chain kinase inhibitor ML-7, the PKC inhibitor staurosporine, or the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin in the bottom well did not induce significant migration at any concentration tested (Fig. 2C) .
PKA function is regulated in part by intracellular localization determined by binding to A kinase anchoring proteins (AKAPs). The peptide St-Ht31, derived from the PKA-binding region of the AKAP Ht31 (N-stearate-DLIEEAASRIVDAVIEQVKAAGAY), dislocates PKA from a range of AKAPs [28 29 30 ]. As a third test of whether asymmetrical PKA activity is an important element in the mechanism that polarizes neutrophils and activates migration, we determined whether exposure to a gradient of the cell-permeant, stearated peptide St-Ht31 activated migration. Like KT5720, addition of St-Ht31 to the bottom well of a chemotaxis chamber significantly activated neutrophil migration to the bottom well in a dose-dependent manner (Fig. 2D) . In contrast, a stearated control peptide (N-stearate-DLIEEAASRPVDAVPEQVKAAGAY), with a proline substitution that abolishes the dislocating activity of Ht31, had little effect on neutrophil chemotaxis (Fig. 2D) . As with KT5720, St-Ht31, in the bottom well alone, was necessary for activation of migration (data not shown).
Global inhibition of PKA activity hyperactivates fMLF-induced neutrophil adhesion and ß2 integrin avidity
Migrating cells display adhesion asymmetry with stronger adhesion at the leading edge than at the trailing edge [31
]. Our data suggest that asymmetrical PKA activity is necessary and sufficient for neutrophil migration. PKA also regulates ß2 integrin activation and adhesion in neutrophils [11
]. We speculated that PKA might spatially regulate integrin activation to achieve adhesion polarity in migrating neutrophils. As adhesion strength is inversely related to migration speed above some optimal level [32
], our hypothesis predicts that global inhibition of PKA would increase adhesion in the rear of the cell, abolishing the polarity of adhesion and thereby increasing overall adhesion strength. To test this possibility, we determined whether global treatment with KT5720 increased fMLF-induced adhesion.
As we demonstrated previously [11 ], KT5720 treatment alone was sufficient to stimulate adhesion (Fig. 3 ). The KT5720 dose-response curve for inhibition of chemotaxis correlated closely with the dose-response curve for activating adhesion (Figs. 1 and 3) . Moreover, KT5720 treatment significantly increased fMLF-stimulated adhesion (Fig. 3) . Inhibition of fMLF-stimulated chemotaxis and hyperactivation of fMLF-induced adhesion occurred at KT5720 concentrations above 1 µM and peaked at 25 µM.
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Figure 3. Global inhibition of PKA hyperactivates fMLF-induced neutrophil adhesion. Purified neutrophils labeled with calcein were treated with vehicle-control Me2SO or the indicated concentration of KT5720 for 20 min at 37°C prior to measurement of adhesion to FBS-coated wells as described in Materials and Methods. Vehicle-control Me2SO, 10 nM fMLF, or 100 nM fMLF was added to the appropriate wells. Adhesion was measured at 3 min and presented as the mean ± SE of triplicate wells reported as adhesion index, the percentage of neutrophils remaining after washing. KT5720 significantly increased fMLF-stimulated adhesion (*, P<0.05). KT5720 also significantly increased adhesion in the absence of fMLF stimulation (*, P<0.05). Data are representative of three separate experiments using neutrophils from different donors.
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Mß2 integrin-affinity activation, as measured by binding of the mAb CBRM1/5, which specifically recognizes the high-affinity conformation of Mß2 [22
]. The ability of fMLF to increase ß2 integrin expression and Mß2 affinity activation was maximal at a concentration of 100 nM (Fig. 4
). KT5720 pretreatment significantly increased fMLF-induced, ß2 integrin cell-surface expression (Fig. 4A)
and Mß2 integrin-affinity activation (Fig. 4B)
. Neither fMLF nor KT5720 treatment affected expression of the control cell membrane protein class I major histocompatibility complex (data not shown). The effect of KT5720 on total ß2 integrin expression was maximal at a concentration of 1 µM. In contrast, the effect of KT5720 treatment on fMLF-induced adhesion and fMLF-induced migration was minimal at this concentration (Figs. 1
and 3)
. The ability of KT5720 to increase Mß2 integrin-affinity activation was observed at concentrations of KT5720 (10 and 25 µM), which effectively reduced migration and increased adhesion. The observed increase in total ß2 integrin expression and Mß2 affinity in cells treated with KT5720 and fMLF was greater than the maximum achievable with either stimulus alone (Fig. 4)
.
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Figure 4. Global inhibition of PKA increases fMLF-stimulated ß2 integrin expression and affinity. Neutrophils were incubated with vehicle-control Me2SO or KT5720 (1 or 10 µM) for 20 min at 37°C. Treated cells were stimulated with fMLF at the indicated concentration for 10 min. Binding of anti-ß2 mAb IB4 F(ab')2 (A) or the mAb CBRM1/5, which recognizes the high-affinity conformation of Mß2 (B) was measured by flow cytometry as described in Materials and Methods. Data are presented as the mean ± SE of three separate experiments. Data are reported as the relative fluorescence, the mean fluorescence of each neutrophil population normalized to the mean fluorescence of unstimulated neutrophils treated with vehicle-control. KT5720 significantly increased fMLF-stimulated binding of IB4 and CBRM1/5 (*, P<0.05). Binding of the control anti-HLA mAb W6/32 was not affected by any treatment (data not shown). KT5720 also significantly increased binding of IB4 and CBRM1/5 in otherwise unstimulated neutrophils (*, P<0.05).
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Figure 5. Global inhibition of PKA disrupts fMLF-induced actin cytoskeletal polarity. (A) Neutrophils were allowed to settle onto glass coverslips coated with FBS and then stimulated with vehicle-control Me2SO or fMLF at the indicated concentration for 10 min as described in Materials and Methods. Cells were then fixed, permeabilized with Triton X-100 buffer, and stained with rhodamine phalloidin to examine F-actin distribution. Data are presented as (A) representative images of vehicle-control- or fMLF (100 nM)-treated neutrophils and (B) mean ± SE of the percentage of cells counted in 5 hpf displaying the distribution of F-actin, typical of polarized neutrophils from three separate experiments. Stimulation with fMLF significantly increased the percentage of polarized cells (*, P<0.05). (C) Neutrophils were pretreated with vehicle-control Me2SO or KT5720 (25 µM) for 20 min and then allowed to settle on a FBS-coated coverslips. Cells were then stimulated with fMLF (100 nM) for 10 min and stained for F-actin distribution. Images are representative of three separate experiments. Global treatment with KT5720 alone induced a spread phenotype similar to fMLF-stimulated cells treated with KT5720 (data not shown). (D) Neutrophils were treated with vehicle-control Me2SO or KT5720 (25 µM) prior to or at the same time as stimulation with fMLF (100 nM) for 10 min, after which F-actin distribution was examined in permeabilized, fixed cells. Data are presented as the mean ± SE of three separate experiments, reported as the percentage of cells with a polarized actin cytoskeleton in 5 hpf. Prior and simultaneous treatment with KT5720 significantly inhibited fMLF-stimulated F-actin polarization (*, P<0.05).
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Figure 6. A gradient of PKA inhibitor is sufficient to polarize the actin cytoskeleton. Neutrophils were allowed to settle onto glass coverslips coated with FBS and were then exposed to a micropipette tip point source loaded with low-melt agarose containing vehicle-control Me2SO, KT5720 at the indicated concentration, or fMLF (100 nM) for 30 min. Cells were then fixed, permeabilized with Triton buffer, and stained with rhodamine phalloidin to examine F-actin distribution. Data are presented as (A) representative images of neutrophils exposed to tips loaded with vehicle control or KT5720 (1 µM) and (B) mean ± SE of the percentage of cells counted in 5 hpf, displaying the distribution of F-actin typical of polarized neutrophils from three separate experiments. Exposure to a point source of KT5720 or fMLF significantly increased the percentage of polarized cells (*, P<0.05).
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Figure 7. The ability of a gradient of PKA inhibitor to stimulate chemotaxis is dependent on PI-3K activity. Neutrophils labeled with calcein were incubated with vehicle-control Me2SO, wortmannin, or LY294002 at the incubated concentration for 20 min at 37°C. The cells were placed on the upper side of a membrane, separating the cells from the lower chamber of a 96-well chemotaxis system. Vehicle-control Me2SO, KT5720, control peptide, or St-Ht31 peptide was added to the chemotaxis buffer at the indicated concentrations. The plate was incubated at 37°C for 30 min, at which time neutrophil migration to the lower chamber was determined, as described in Materials and Methods. Data are presented as the mean ± SE of triplicate wells, reported as the percentage of neutrophils added to the top of the membrane migrating to the lower chamber, determined as described in Materials and Methods. Wortmannin and LY294002 significantly inhibited migration stimulated by KT5720 or St-Ht31 in the lower chamber (*, P<0.05). Data are representative of four separate experiments using neutrophils from different donors.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 integrins at the leading edge [8
]. In MDA-MB-435 carcinoma cells, phosphodiesterase and PKA activities are required for migration, highlighting the complex regulation of PKA [42
]. To add to the complexity, chemoattractants and integrin signaling increase overall cellular cAMP concentration [42
43
44
45
46
47
], and at least in neutrophils, integrin signaling modifies cAMP concentrations in cells stimulated by fMLF or other proinflammatory mediators [48
, 49
]. Taken together, the available data suggest that an intermediate PKA activity is required to enable cellular migration. However, little is known about how PKA is regulated to accomplish migration. It is unlikely that the requirement for an intermediate PKA activity is true uniformly throughout the cell. Rather, it is more likely that PKA activity is necessary in some subcellular compartments, but not others, during migration. This is akin to the findings of Zaccolo and Pozzan [50
], who demonstrated that discrete microdomains with high cAMP concentration form in stimulated cardiac myocytes and consistent with data from Goldfinger et al. [8
], demonstrating that PKA phosphorylates 4 integrins specifically at the leading edge of migrating fibroblasts. In the trailing-edge uropod, integrin inactivation is required to allow deadhesion [2 ]. At the leading edge, integrin activation must occur for new adhesions to form, and actin polymerization is induced for the assembly of appropriate actin cytoskeletal structures at sites of adhesion and in the lamellipodium [2 ]. As PKA has an important role in regulating integrin activation [11 12 13 , 47 ] and the actin cytoskeleton [6 , 14 15 16 17 18 ], we formulated a model for neutrophil migration in which asymmetrical PKA activity is established (high PKA activity in the trailing edge and low PKA activity in the leading edge), resulting in adhesive asymmetry and cytoskeletal polarity. This hypothesis is supported by our data reported here, demonstrating that global inhibition of PKA inhibits fMLF-induced migration by a mechanism that involves hyperactivation of integrin avidity and adhesion as well as remodeling of the actin cytoskeleton and a resultant loss of polarity. Moreover, gradients of PKA inhibitors, which would be expected to inhibit PKA activity asymmetrically with lower activity at the leading edge, are sufficient to polarize neutrophils, provoking actin cytoskeletal reorganization typical of chemoattractant-stimulated neutrophils, and activate migration.
Our model is consistent with the previous observation that cAMP agonists induce the formation of the trailing-edge uropod lymphocytes [51 ]. Chemokine stimulation of lymphocytes adherent to endothelial cells or ß2 or ß3 integrin substrates induced uropod formation and redistribution of ICAM-3 to the uropod. Several cAMP agonists were able to induce uropod formation and ICAM-3 redistribution, whereas H-89, a specific inhibitor of PKA, abrogated the chemokine-mediated uropod formation. Our data extend this work to demonstrate that asymmetrical PKA activity is necessary and sufficient to reorganize and polarize the actin cytoskeleton and enable migration of neutrophils and lymphocytes.
It is remarkable that our data are in direct contrast to work describing the role of spatially regulated PKA in migrating fibroblasts. In fibroblasts, PKA activity at the leading edge phosphorylates 4 integrins, and this phosphorylation are required for lamellipodia stability, assembly of 4-paxillin complexes, and migration [8
]. Thus, although PKA must be spatially regulated in neutrophils and fibroblasts, the role of PKA in generating adhesive and cytoskeletal polarity appears to be quite different in these two types of cells. This may reflect a difference in integrin use in the assay systems used to assess migration in the two types of cells (ß2 integrins in neutrophils in the work reported here and 4 integrins in fibroblasts). The and ß chains of ß2 integrins are phosphorylated [52
, 53
]. Phosphorylation of the ß chain regulates the function of ß2 integrins during activation [54
55
56
]. However, the ß chain is phosphorylated by PKC, not PKA [54
, 56
, 57
]. It is unclear which kinase phosphorylates the chain. Moreover, -chain phosphorylation appears to be constitutive [52
, 53
], not induced (or reduced), by activation. It is also possible that the disparity between our data and data obtained using fibroblasts may reflect other fundamental differences in the mechanism of migration between these two types of cells.
It is not clear from our data whether the ability of asymmetrical PKA activity to polarize neutrophils and stimulate migration is solely a result of asymmetrical adhesion established by polarized integrin activation or whether asymmetrical PKA activity concurrently regulates other aspects of the migration mechanism such as membrane protrusion or generation of the molecular motors necessary for movement. Integrin-dependent adhesion appears to be necessary for uropod formation in lymphocytes [51 ]. Moreover, formation of new adhesion sites is critical for formation of advancing lamellipodia in fibroblasts [2 , 58 ]. Thus, appropriate cellular polarization requires integrin activation and engagement. However, adhesion is not sufficient for leukocyte polarization. Indeed, stimuli such as PMA or adhesion to immobilized immune complexes are potent inducers of cell-spreading but not polarization or migration [59 , 60 ]. Our data demonstrating that inhibition of PI-3K abolishes migration in response to a gradient of KT5720 but has no effect on integrin avidity or adhesion [11 ] suggest that the ability of PKA to regulate the state of integrin activation and adhesion is not sufficient to enable migration. PKA does regulate the function of myosin motors via effects (direct and indirect) on myosin light-chain kinase and myosin light-chain phosphatase activities [61 62 63 64 65 ]. Indeed, we have demonstrated that myosin light-chain kinase inhibitors abolish KT5720-induced adhesion as well as migration in response to a gradient of KT5720 (Clayton D. Chilcoat, S. L. Jones, unpublished data). Moreover, PKA-dependent regulation of RhoA or other Rho family members [13 , 42 , 61 , 63 , 66 67 68 ] or the phosphorylation state of cytoskeletal proteins such as L-plastin and vasodilator-stimulated phosphoprotein/Ena family members, which are substrates for PKA, may be quite important for appropriate actin cytoskeletal organization as well as adhesion during neutrophil migration [69 70 71 ]. Thus, it is likely that PKA operates at multiple points in the mechanism of migration.
It is unknown how PKA activity is spatially regulated in polarized, migrating neutrophils. PKA function in polarized, migrating neutrophils may be spatially controlled by a mechanism that tunes PKA activity at the subcellular level. Fine control of subcellular PKA activity is accomplished by varying the local concentration of cAMP [50 , 72 ] or by varying the local activity of cytosolic PKA regulators, such as Rab13 [73 ]. It is worth noting that fMLF stimulation of neutrophils is associated with an increase in total intracellular cAMP concentration [43 44 45 ], raising the possibility that chemoattractant receptor signaling participates in the tuning mechanism regulating PKA activity, but the distribution of this cAMP is unclear. Signaling by engaged ß1 and ß2 (at least in lymphocytes) integrins also increases overall cellular cAMP concentration and PKA activity [42 , 46 , 47 , 74 ]. But again, the distribution of PKA activity induced by integrin engagement is not known in these cells. Moreover, there is cross-talk between ß2 integrin-induced signaling and signals generated by fMLF or other stimulators that modify intracellular cAMP concentrations in neutrophils [48 , 49 ]. Thus, the context of integrin signaling, particularly in subcellular compartments in proximity to activated integrins, may profoundly affect local PKA activity in a manner that is not reflected in overall cAMP concentration of PKA activity. Regulating binding to AKAPs may also control local PKA activity [72 , 75 ]. The ability of a gradient of the PKA anchorage inhibitor St-Ht31 to stimulate migration supports a mechanism in which the subcellular distribution of PKA is altered in polarizing neutrophils to establish asymmetrical PKA activity. It remains to be determined which of these mechanisms (or whether a combination of these mechanisms) regulating PKA operates during neutrophil migration.
PI-3K activity is necessary for neutrophil migration induced by asymmetrical PKA activity, but not for PKA inhibitor-induced adhesion. These data raise the intriguing possibility that PKA regulates PI-3K activity or function directly during polarization and/or migration. There is scant data in the literature linking PKA directly to PI-3K function. cAMP-elevating agents inhibit fMLF-induced activation of PI-3K activity in neutrophils [76 ]. However, it is not known whether this is a result of a direct effect of activated PKA on the PI-3K enzyme or whether cAMP indirectly reduces PI-3K activity by inhibiting fMLF signaling. PI-3K distribution may be regulated directly or indirectly by cAMP/PKA. Treatment of endothelial cells with 8-bromoadenosine-cAMP inhibits fibroblast growth factor-2-induced PI-3K activation and membrane localization, but this effect may not be PKA-dependent [77 ]. cAMP does not inhibit, but rather activates, PI-3K in the bile canalicular cell line, WIF-B9, suggesting that the effects of cAMP on PI-3K function are cell type-specific [78 ]. PKA has been demonstrated to phosphorylate the p85 subunit of PI-3K directly, but it is unclear how this phosphorylation affects PI-3K function [79 ].
There is a possibility that our results using PI-3K inhibitors can be explained by a regulatory role for PI-3K on PKA activity or localization. This possibility is not without support in the literature. For example, PI-3K negatively regulates PKA activity in smooth muscle cells by modulating cAMP levels [80 ]. Moreover, inhibition of PI-3K activity increases intracellular cAMP and stimulates phosphorylation of AKAP3 in sperm cells [81 ], which increases binding of PKA. The overall effect is not only to increase PKA activity but also to regulate localization as well. If PI-3K negatively regulates PKA activity, then inhibition of PI-3K would be expected to increase PKA activity, which would have the net effect of decreasing migration in response to a gradient of KT5720 if an intermediate level of PKA activity is required for migration, particularly in specific subcellular compartments. Inhibition of PI-3K would not be expected to affect PKA inhibitor-induced adhesion in our assays, as PKA is being inhibited globally. It remains to be determined whether PKA regulates PI-3K activity or vise versa in migrating neutrophils.
Received August 17, 2004; revised March 7, 2005; accepted March 14, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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}4 integrin phosphorylation regulates lamellipodial stability and 4ß1-dependent cell migration J. Cell Biol. 162,731-741
RIII cooperate in generation of a neutrophil respiratory burst: requirement for FcRRII and tyrosine phosphorylation J. Cell Biol. 125,1407-1416Mß2 avidity in polymorphonuclear neutrophils J. Biol. Chem. 273,10556-10566L/ß 2 leukocyte integrin (LFA-1) by cyclic AMP-dependent protein kinase J. Immunol. 156,2273-2279[Abstract]- and ß-chains of the CD11/CD18 leukocyte integrin family. Relationship to adhesion-dependent functions J. Immunol. 144,191-197[Abstract]RII-mediated adhesion and phagocytosis induce L-plastin phosphorylation in human neutrophils J. Biol. Chem. 271,14623-14630This article has been cited by other articles:
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