Integrins are type I membrane and heterodimeric (αβ) cell adhesion receptors. Intracellular signals triggered by ligand-bound integrins are important for cell growth, differentiation, and migration. Integrin αMβ2 plays key roles in myeloid cell adhesion, phagocytosis, and degranulation. In this study, we show that protein kinase C (PKC) δ is involved in αMβ2 signaling. In human monocytic U937 cells and peripheral blood monocytes, αMβ2 clustering induced PKCδ translocation to the plasma membrane, followed by Tyr311 phosphorylation and activation of PKCδ by the src family kinases Hck and Lyn. Interestingly, αMβ2-induced PKCδ Tyr311 phosphorylation was not mediated by the tyrosine kinase Syk, which is a well reported kinase in β2 integrin signaling. Analysis of the β2 cytoplasmic tail showed that the sequence Asn727-Ser734 is important in αMβ2-induced PKCδ Tyr311 phosphorylation. It has been shown that αMβ2 clustering regulates the expression the transcription factor Foxp1 that has a role in monocyte differentiation. We show that Foxp1 expression was reduced in monocytes that were allowed to adhere to human microvascular endothelial cells. However, the expression of Foxp1 was not affected in monocytes that were treated with PKCδ-targeting small interfering RNA, suggesting that PKCδ regulates Foxp1 expression. These results demonstrate a role of PKCδ in αMβ2-mediated Foxp1 regulation in monocytes.

Integrins are heterodimeric transmembrane receptors that mediate cell–cell and cell–extracellular matrix interactions (1). The α and β subunits of an integrin associate by noncovalent interaction. Each subunit contains a large extracellular region, a single transmembrane, and a cytoplasmic tail. Cytoplasmic signals derived from the integrins are important for cell survival and differentiation (13). The leukocyte-restricted β2 integrins have four members: αLβ2 (LFA-1 and CD11aCD18), αMβ2 (Mac-1 and CD11bCD18), αXβ2 (p150,95 and CD11cCD18), and αDβ2 (CD11dCD18). The importance of the β2 integrins is exemplified by the poor capacity of leukocytes to migrate into sites of inflammation in leukocyte adhesion deficiency I patients (4, 5). In these patients, the expression of β2 integrin on the leukocytes is defective as a result of mutations in the β2 subunit. Mutations in the cytosolic protein kindlin 3 in leukocyte adhesion deficiency III patients also lead to defective β2 integrin function, resulting in poor leukocyte adhesion (6).

Integrin αMβ2 is expressed primarily on myeloid, NK, and γδ T cells (7, 8). It has a wide range of ligands, including ICAM-1, junctional adhesion molecule-3, complement protein iC3b, fibrinogen, microbial saccharides, and denatured proteins (912). In myeloid cells, αMβ2 mediates cell adhesion, phagocytosis, and degranulation (13). In dendritic cells, a role of αMβ2 in maintaining tolerance has been reported (14). Fibrinogen-induced clustering of αMβ2 has been shown to regulate monocyte differentiation into macrophages by downregulating the expression of forkhead transcription factor Foxp1, a transcriptional repressor of the c-fms gene that encodes the M-CSF receptor (15, 16). Shi et al. (15) also reported a reduction in Foxp1 expression in monocytes isolated from wild-type mice, but not αMβ2-deficient mice, treated with thioglycollate to induce peritonitis. Clustering of αMβ2 on monocytic THP-1 cells has been shown to trigger Toll/IL-1 pathway that affects NF-κB activity (17). However, little evidence is available that demonstrates this signaling pathway regulates Foxp1 expression.

The protein kinase Cs (PKCs) are a large multigene family of serine/threonine kinases involved in cell growth and apoptosis. They are grouped into three classes: conventional (α, βI, βII, and γ), novel (δ, ε, η, and θ), and atypical (ζ and ι/λ) (18). PMA, an analog of the second messenger 1,2-diacylglycerol, is a well reported inducer of monocyte differentiation by activating the conventional and novel family members of the PKC (18). Overexpression of PKCδ, a member of the novel PKCs, induces myeloid cell differentiation (19), and the catalytic domain of PKCδ is implicated in this process (20). In this study, we investigated the early signaling events triggered by clustering αMβ2 on monocytes. We show that clustering of αMβ2, but not αLβ2, induces PKCδ translocation to the plasma membrane and src kinase-dependent phosphorylation of PKCδ. Our results also led us to conclude that PKCδ is involved in Foxp1 regulation by αMβ2.

Src-family kinase inhibitor (PP2), inactive analog of PP2 (PP3), LY2940002 (PI3K inhibitor), and piceatannol (Syk inhibitor) were from Calbiochem (San Diego, CA). PMA, polyvinylpyrrolidone 10000, recombinant human PKCδ, and mouse IgG were from Sigma-Aldrich (St. Louis, MO). All other general chemicals were from Sigma-Aldrich unless stated otherwise. Monoclonal Ab MHM24 (anti-αL, function-blocking) (21) hybridoma was provided by Dr. A. J. McMicheal (Institute of Molecular Medicine, Oxford, U.K.). Monoclonal Ab LPM19c (anti-αM, function-blocking) (22) hyrbidoma was provided by Dr. K. Pulford (LRF Diagnostic Unit, Oxford, U.K.). Monoclonal Ab KIM185 (anti-β2, activating Ab) hybridoma was from American Type Culture Collection (23). Monoclonal Abs were purified from culture supernatants using HiTrap protein A/G columns (GE Healthcare, Amersham, U.K.). Goat anti-human IgG (Fc specific), FITC-conjugated sheep anti-mouse IgG, and goat anti-mouse IgG were from Sigma-Aldrich. HRP-conjugated donkey anti-rabbit IgG and HRP-conjugated sheep anti-mouse IgG were from GE Healthcare. AlexaFluor 488-conjugated goat anti-rabbit IgG was from Invitrogen (Carlsbad, CA). Rabbit anti-PKCδ (C-20), rabbit anti-Hck, and mouse anti–c-Yes Abs were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-PKCδ pTyr311 was from Cell Signaling Technology (Beverly, MA). Rabbit anti-Foxp1, rabbit anti-αM (EP1345Y), and mouse anti-Lyn Abs were from Abcam (Cambridge, U.K.). Mouse anti-β actin, mouse anti-Hck, mouse anti-PKCα, and mouse anti-PKCε Abs were from BD Biosciences (San Jose, CA). Monoclonal Ab 4B4 (integrin β1 specific) was from Beckman Coulter (Fullerton, CA), and mAb P1F6 (integrin αVβ5 specific) was from Abcam. Phospho-Erk1/2 mAb was from Cell Signaling Technology. Mouse anti-Syk mAb was from BD Pharmingen (San Diego, CA). The human recombinant ICAM-1-Fc was prepared as described previously (24).

U937, K562, and Jurkat (E6-1) cells were from American Type Culture Collection. The stable K562 cells expressing αMβ2 were provided by Dr. L. Zhang (University of Maryland, Baltimore, MD). These cells were cultured in complete RPMI 1640 medium that contained 10% (v/v) heat-inactivated FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin (HyClone, Scoresby, Australia) at 37°C in a humidified 5% CO2 incubator. K562 cells were transfected with αM and either wild-type β2 or β2 mutant plasmids (10 μg each) by electroporation using a pipette-type microporator MP-100 (NanoEn Tek, Seoul, South Korea), according to the manufacturer’s instructions. Human lung microvascular endothelial (HMVE) cells that were immortalized with human telomerase protein were provided by Dr. R. Shao. (University of Massachusetts at Amherst, Springfield, MA) (25). HMVE cells were cultured in endothelial cell medium (PAA Laboratories, Somerset, U.K.) in culture dish precoated with 0.1% (w/v) gelatin (Life Technologies, Grand Island, NY). Pan human monocytes were isolated from buffy coat (from Bloodbank, Health Sciences Authority, Singapore, following regulatory guidelines) by Ficoll-Paque (GE Healthcare) sedimentation, followed by magnetic isolation using CD14 microbeads and an LS column (Miltenyi Biotec, Auburn, CA). The purity of the isolated monocytes was assessed by flow cytometry using anti-CD14 mAb (Miltenyi Biotec). Isolated monocytes were maintained in RPMI 1640 medium containing 10% (v/v) human AB serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. The same medium was used for monocyte Ab cross-linking experiments.

Full-length integrin αM and β2 cDNAs in the expression plasmid pcDNA3.0 (Invitrogen) were reported previously (24). Integrin amino acids were numbered based on the mature protein (26). The β2 mutants used in this study β2D709R, β2M740, β2S734, β2N727, β2R711, and β2K702 were described previously (26, 27). Full-length human p59 Hck cDNA was obtained by RT-PCR of mRNA isolated from U937 cells using relevant forward and reverse primers and cloned into expression vector pcDNA3.1 (Invitrogen) using the KpnI and XhoI restriction sites. mRNA extraction kit was from Roche Diagnostic Systems (Somerville, NJ). RT-PCR kit was from Qiagen (Valencia, CA). All site-directed mutations were performed using the Quikchange site-directed mutagensis kit (Stratagene) with relevant primer pairs. All constructs generated were verified by DNA sequencing (First Base, Singapore).

Hck cDNA encoding aa 78–526 as previously reported (28) was cloned into pFastBac Dual (Invitrogen) using the EcoRI and XbaI restriction sites to generate expression bacmid pFastBac-Dual-78-Hck with an N terminus His6-tag. Bacmid was first amplified in Escherichia coli DH-5α, followed by amplification in Max Efficiency DH10Bac. Bacmid was transfected into SF9 insect cells using Cellfectin transfection reagent. Hck expression in the transfectants was assessed by Western blotting using rabbit anti-His Ab. P2 virus was collected, and the optimal P2 inoculum and density of SF9 cells for Hck production determined. Briefly, 2 × 106 adherent SF9 cells in SF9 medium containing 0.5% (v/v) of heat-inactivated FBS were infected with P2 virus. Cells were collected after 3 d, lysed, and rHck purified using Ni-NTA purification system (Qiagen).

Flow cytometry analyses of cells expressing integrins were performed as described previously (24). Cells were stained with relevant primary Ab (10 μg/ml) in PBS for 30 min at 4°C. Cells were washed in PBS and incubated in PBS containing FITC-conjugated sheep anti-mouse IgG (1/400 dilution) for 30 min at 4°C. Stained cells were fixed in PBS containing 1% (v/v) formaldehyde and analyzed on a BD FACSCalibur with CellQuest software (BD Biosciences, San Jose, CA) installed.

Cell adhesion on immobilized ICAM-1 was performed essentially as described previously (24). Fluorescence signal of adherent cells was measured in a fluorescent plate reader (FL600) (Bio-Tek Instruments, Winooski, VT). The percentage of bound cells was calculated: fluorescence signal after wash/fluorescence signal before wash × 100. Under activating condition, 10 μg/ml mAb KIM185 was included. Adhesion specificity mediated by αMβ2 was assessed by including mAb LPM19c (10 μg/ml) in the assay.

For cell adhesion to immobilized Abs, a similar assay was performed, except that the well of the microtitre plate was coated with 5 μg/ml goat anti-mouse IgG in bicarbonate buffer (pH 9.0) overnight at 4°C. Subsequently, the well was coated with mAb MHM24, LPM19c, or control mouse IgG (10 μg/ml each) for 1 h at 37°C. For cross-linking of integrins on U937 cells or monocytes, cells were plated into Ab-coated microtiter wells aforementioned. After incubation for different time points at 37°C in a humidified 5% CO2 incubator, all cells (bound and unbound) in the wells were collected and lysed for subsequent analyses.

For cross-linking experiments using F(ab′)2, the F(ab′)2 of LPM19c and control IgG were prepared using the F(ab′)2 micropreparation kit (Pierce, Rockford, IL), following the manufacturer’s instructions. Digestion of mAbs by immobilized pepsin was monitored by SDS-PAGE under reducing and nonreducing conditions. F(ab′)2 was immobilized onto tissue culture dish as described previously (29). U937 cells were plated into the F(ab′)2-coated dish and incubated for 30 min at 37°C in a humidified 5% CO2 incubator. All cells were collected for subsequent analyses.

U937 cells were seeded into Ab-coated microtiter wells. Cells were collected by washing the wells in PBS containing 0.5 mM EDTA. Recovered cells were spun down at 1000 × g for 10 min onto poly-l-lysine–coated slides using a cytospin centrifuge (Thermo Electron, San Jose, CA). For monocytes, cells were allowed to adhere to poly-l-lysine slides coated with mAbs for 10 min at room temperature. Cells were washed twice in PBS, followed by fixation in PBS containing 3.7% (w/v) paraformaldehyde for 5 min at room temperature. Cells were washed twice in PBS and permeabilized in modified cytoskeletal buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl2, and 1 mM EGTA [pH 6.8]) containing 0.3% (v/v) Triton X-100 and protease inhibitors mixture (Roche Diagnostic Systems) for 1 min at room temperature. Cells were washed and incubated in PBS containing rabbit anti-PKCδ Ab (1 μg/ml) for 1 h at room temperature. Thereafter, cells were washed and incubated in PBS containing Alexa Fluor 488 goat anti-rabbit IgG (1/1000 dilution) for 30 min at room temperature in the dark. Images were acquired using a ×40 objective lens on a fluorescence microscope (Olympus, Melville, NY) equipped with MetaMorph software.

For live imaging of human peripheral blood monocytes adhering and migrating on HMVE cells, monocytes were labeled with 1 μM fluorescence label CellTracker Green (Molecular Probes, Eugene, OR) in complete RPMI 1640 medium for 20 min at 37°C. Cells were washed twice in medium and seeded onto HMVE cells that were grown to confluence in coverslip glass bottom tissue culture dish (MatTek, Ashland, MA). The cells were viewed with a Zeiss Axiovert 200M inverted fluorescence microscope (with ×20 objective) housed in a closed system with 5% CO2 and temperature maintained at 37°C. Images of migrating monocytes were captured with time by a CoolSnap HQ charge-coupled device camera (Roper Scientific, Tucson, AZ) for 15 min. Labeled monocytes were detected with 485/525 ex/em filters. Images were generated using the software Metamorph (Molecular Devices, Sunnyvale, CA).

Cells were lysed in protein solublization buffer, and proteins were resolved on 10% SDS-PAGE gel under reducing conditions and electrotransfered onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The same procedure was used for analyzing immunoprecipitates. Protein bands were probed with relevant primary Ab, followed by HRP-conjugated secondary Ab and detected using the ECL-plus kit (Amersham Biosciences, Little Chalfont, U.K.).

To analyze PKCδ localization in the subcellular fractions, U937 cells were resuspended in buffer A (20 mM Tris-HCl [pH 7.5], 0.25 M sucrose, 2 mM EGTA, 2 mM EDTA, and protease inhibitor mixture) and subjected to sonication on ice for 5 s. Cell lysate was centrifuged at 600 × g to remove the nuclei and cell debris. Cell lysate was centrifuged at 100,000 × g for 1 h, and the resulting supernatant was collected as the soluble fraction. The pellet was solubilized in buffer B (20 mM Tris-HCl [pH 7.5], 1% (w/v) SDS, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, and protease inhibitors mixture) as particulate fraction. Proteins were resolved on SDS-PAGE gel under reducing conditions, electroblotted, and probed with rabbit anti-PKCδ (C-20) or anti-pTyr311 PKCδ Ab.

To examine Hck interaction with PKCδ, U937 cells were lysed and subjected to subcellular fractionation. The particulate fraction was collected and solubilized in buffer B as described above. Hck was immunoprecipitated with rabbit anti-Hck Ab and protein A-Sepharose beads (Amersham Biosciences). The beads were washed in buffer C (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% (v/v) Nonidet P-40, and protease inhibitors mixture). Proteins precipitated were resolved on SDS-PAGE gel, followed by immunoblotting with these Abs: mouse anti-Hck, rabbit anti-PKCδ (C-20), or rabbit anti-pTyr311 PKCδ. Similar procedure was performed to detect Lyn interaction with PKCδ in U937 cells.

To detect a highly extended αMβ2, K562 transfectants expressing wild-type αMβ2 or αMβ2D709R were incubated with mAb LPM19c or the conformation reporter mAb KIM127 (10 μg/ml each) in culture medium with or without MnCl2 (0.5 mM) as activating agent for 30 min at 37°C (30). Cells were washed extensively in medium and lysed in buffer C. Immune complexes in the whole-cell lysates were precipitated with protein A-Sepharose. Bound proteins were resolved on SDS-PAGE under reducing conditions. The αM protein band was detected by immunoblotting using rabbit anti-αM Ab.

U937 cells were lysed in buffer C containing protease inhibitor mixture for 30 min at 4°C. Cell lysate was precleared with irrelevant rabbit Ab and protein A-Sepharose beads. Hck or Lyn was precipitated with relevant Ab and protein A-Sepharose beads and washed three times in lysis buffer and two times in kinase buffer (2.5 mM HEPES [pH 7.4], 2.5 mM MgCl2, 5 mM NaF, and 20 μM Na3VO4). Beads containing immunoprecipitated Hck or Lyn were incubated with 100 ng rPKCδ in kinase buffer containing 3 mM ATP for 30 min at 37°C. Reaction was terminated by adding protein solubilization buffer and boiling. For PKCδ phosphorylation by rHck, 100 ng each of rPKCδ and purified rHck were used.

For γ[32P]ATP PKCδ kinase assay, PKCδ in the particulate fraction of U937 cells was immunoprecipitated with rabbit anti-PKCδ Ab and protein A-Sepharose. The immunoprecipitate was washed twice in PKC kinase buffer (20 mM Tris-HCl [pH 7.5], 5 mM MgCl2, and 0.2 mM CaCl2). Each reaction was performed in PKC kinase buffer containing 20 μg histone H1 (Calbiochem) and 5 μM ATP (unlabeled ATP and 2 μCi γ[32P]ATP, sp. act. 3000 Ci/mmol) (PerkinElmer, Wellesley, MA) for 10 min at 30°C (31). Reaction was terminated by adding protein solubilization buffer and boiling. Proteins were resolved on SDS-PAGE gel, followed by autoradiography.

Accell SMART pool small interfering RNA (siRNA) targeting human Syk, Hck, or PKCδ (0.4 nmol each) was used for 3 × 104 U937 cells in 100 μl Accell delivery medium to reduce expression of Syk, Hck, or PKCδ as recommended by the manufacturer (Thermo Scientific Dharmacon). The control Accell green nontargeting siRNA was used at the same concentration. Cells were collected 3 or 4 d later after adding the relevant siRNAs for subsequent analyses. Cell viability was at least 80% as determined by trypan blue exclusion.

siRNA targeting PKCδ in human peripheral blood monocytes was performed by incubating 2 × 106 cells in Opti-MEMI reduced serum medium containing 33 nM PKCδ-targeting siRNA or control siRNA and Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The cell suspension in polypropylene tubes was incubated for 16 h at 37°C in a humidified 5% CO2 incubator. Cell viability was ∼70% as determined by trypan blue exclusion. Cells were subsequently collected for Ab cross-linking or HMVE adhesion studies. For the latter, cells were seeded onto HMVE cells grown to confluence on culture dish or culture dish without HMVE and incubated for 8 h at 37°C in a humidified 5% CO2 incubator. Cells were then collected by incubating in PBS containing 0.5 mM EDTA. Monocytes were isolated using CD14 microbeads aforementioned. Cells were lysed, and Foxp1 expression detected by immunoblotting.

We initially examined the early signaling events derived from clustering of integrin subunits in U937 cells. U937 cells expressed a high level of αLβ2 and a moderate level of αMβ2 as determined by flow cytometry analyses using relevant Abs (Fig. 1A). Minimal αXβ2 was detected (data not shown). In line with the expression profiles, a higher percentage of U937 cells adhered to anti–αLβ2-coated microtiter wells as compared with anti-αMβ2 or control IgG (Fig. 1B). The same procedure was used to induce clustering of integrin, but cells (bound and unbound) were harvested and lysed. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311. Phosphorylation of Tyr311 has been reported to initiate a series of phosphorylation reactions on other tyrosines that have important roles in regulating PKCδ activity (32). Cross-linking of αMβ2, but not αLβ2 (or control IgG), induced marked PKCδ Tyr311 phosphorylation (Fig. 1C). Cross-linking of αXβ2, which has minimal expression on U937 cells, did not induce PKCδ Tyr311 phosphorylation (data not shown). Cross-linking of β1 or β5 integrin on U937 cells also did not induce PKCδ Tyr311 phosphorylation (Supplemental Fig. 1A). It has been shown previously that the responses in neutrophils to anti-integrin mAbs are dependent on the engagement of the FcRs on the neutrophils by the Fc portions of the mAbs used (29). In this study, αMβ2-specific F(ab′)2, but not control IgG F(ab′)2, induced PKCδ Tyr311 phosphorylation in U937 cells (Fig. 1D). These data show that αMβ2 clustering alone triggers PKCδ Tyr311 phosphorylation.

FIGURE 1.

Clustering of αMβ2-induced PKCδ Tyr311 phosphorylation in U937 cells. A, Expression levels of αLβ2 and αMβ2 on U937 cells were determined by flow cytometry using mAb MHM24 and mAb LPM19c, respectively (gray histogram). An irrelevant mAb was used for background staining (black histogram). B, Adhesion of cells to mAb MHM24 (αL), mAb LPM19c (αM), or control IgG (Ig)-coated microtitre wells. C, The effect of integrin clustering on PKCδ Tyr311 phosphorylation was assessed by plating cells on immobilized mAbs. Cells were incubated for 30 min at 37°C in a humidified incubator with 5% CO2. All cells (bound and unbound) were collected and lysed. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311. The membrane was stripped of Abs and reblotted with anti-PKCδ to detect total PKCδ. D, As in C, but LPM19c F(ab′)2 immobilized on tissue culture dish was used to induce αMβ2 clustering. Ig, control IgG F(ab′)2. Data are representative of two independent experiments. E, Time course analysis of PKCδ Tyr311 phosphorylation. F, Cells were treated with inhibitors (20 μM each) for 1 h. Cells were then plated on mAb LPM19c (αM) or control IgG (Ig) for 30 min. G, Cells were treated with inhibitors (20 μM each) for 30 min. Con*, without inhibitor. Cells were then treated with PMA (100 ng/ml) for 1 h. Con, without PMA. In C–G, β-actin was used as a loading control. C, E, F, and G, PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/ total PKCδ intensity). Data are the average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). αL, MHM24; αM, mAb LPM19c; con, control, without any additive; Ig, control IgG; PP2, src family kinase inhibitor; PP3, inactive analog of PP2; LY, PI3K inhibitor LY294002; PIC, Syk kinase inhibitor picetannol.

FIGURE 1.

Clustering of αMβ2-induced PKCδ Tyr311 phosphorylation in U937 cells. A, Expression levels of αLβ2 and αMβ2 on U937 cells were determined by flow cytometry using mAb MHM24 and mAb LPM19c, respectively (gray histogram). An irrelevant mAb was used for background staining (black histogram). B, Adhesion of cells to mAb MHM24 (αL), mAb LPM19c (αM), or control IgG (Ig)-coated microtitre wells. C, The effect of integrin clustering on PKCδ Tyr311 phosphorylation was assessed by plating cells on immobilized mAbs. Cells were incubated for 30 min at 37°C in a humidified incubator with 5% CO2. All cells (bound and unbound) were collected and lysed. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311. The membrane was stripped of Abs and reblotted with anti-PKCδ to detect total PKCδ. D, As in C, but LPM19c F(ab′)2 immobilized on tissue culture dish was used to induce αMβ2 clustering. Ig, control IgG F(ab′)2. Data are representative of two independent experiments. E, Time course analysis of PKCδ Tyr311 phosphorylation. F, Cells were treated with inhibitors (20 μM each) for 1 h. Cells were then plated on mAb LPM19c (αM) or control IgG (Ig) for 30 min. G, Cells were treated with inhibitors (20 μM each) for 30 min. Con*, without inhibitor. Cells were then treated with PMA (100 ng/ml) for 1 h. Con, without PMA. In C–G, β-actin was used as a loading control. C, E, F, and G, PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/ total PKCδ intensity). Data are the average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). αL, MHM24; αM, mAb LPM19c; con, control, without any additive; Ig, control IgG; PP2, src family kinase inhibitor; PP3, inactive analog of PP2; LY, PI3K inhibitor LY294002; PIC, Syk kinase inhibitor picetannol.

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Cross-linking of αMβ2 induced transient phosphorylation of PKCδ Tyr311, which peaked at 30–45 min, then diminished at 60 min following plating of U937 cells on anti-integrin Abs (Fig. 1E). The src family kinase (SFK) is required for this event, because clustering of αMβ2 failed to induce PKCδ Tyr311 phosphorylation in cells that were pretreated with the SFK inhibitor (PP2) (Fig. 1F). By contrast, the inactive analog of PP2 (PP3), PI3K inhibitor (LY294002), and Syk inhibitor (piceatannol) had no significant effect on PKCδ Tyr311 phosphorylation. Similarly, PMA-induced PKCδ Tyr311 phosphorylation was diminished in cells that were pretreated with PP2, but not with other inhibitors (Fig. 1G).

Signal transduction by β2 integrins in macrophages has been shown to involve the ITAM of DAP12 and the Src homology 2 domains of Syk in an elegant study that used mouse knockout models (33). Syk serves as an important tyrosine kinase in signal transduction following integrin engagement in hematopoietic cells (34). It is therefore surprising that piceatannol treatment (20 μM) had no significant effect on αMβ2-induced PKCδ Tyr311 phosphorylation (Fig. 1F). To verify the efficacy of the piceatannol treatment, we examined the phosphorylation of Erk1/2 that has been reported to be induced by β2 integrin cross-linking (35) and is Syk dependent (33). Increasing the concentration of piceatannol from 20 to 100 μM had minimal effect on PKCδ Tyr311 phosphorylation induced by αMβ2 cross-linking in U937 cells (Fig. 2A). By contrast, concentration-dependent inhibition of Erk1/2 phosphorylation by piceatannol was observed. We also made use of siRNA to reduce the expression of endogenous Syk in U937 cells, and we examined PKCδ Tyr311 phosphorylation level in these cells (Fig. 2B, 2C). No significant difference in αMβ2-induced PKCδ Tyr311 phosphorylation between control siRNA and Syk-targeting siRNA-treated cells was observed, although significant reduction of Syk expression was detected in cells treated with Syk-targeting siRNA. These data suggest that αMβ2-induced PKCδ Tyr311 phosphorylation does not involve Syk. However, these data do not exclude the possibility that Syk phosphorylates other Tyr(s) on PKCδ. It is also interesting to note that in endothelial cells, Syk activation by thrombin is dependent on PKCδ (36). Further studies are needed to address possible cross-talk between PKCδ and Syk in hematopoietic cells.

FIGURE 2.

Effect of piceatannol treatment and reduced Syk expression by siRNA on PKCδ Tyr311 phosphorylation in U937 cells. A, Cells were treated with different concentrations of piceatannol (PIC) for 1 h. Cells were then plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Immunoblot analyses of Erk1/2 and PKCδ Tyr311 phosphorylations were performed. Data are representative of two independent experiments. B, Reduction of endogenous Syk expression in cells treated with Syk-targeting siRNA as determined by immunoblotting whole-cell lysates with anti-Syk. C, Cells treated with control siRNA or Syk-targeting siRNA were plated onto mAbs. The effect of reduced Syk expression on PKCδ Tyr311 phosphorylation was assessed by immunoblotting. In all figures, β-actin was used as a loading control. Densitometry analyses of protein bands were performed for B and C. Data are the average of three experiments and expressed as means ± SD. *p < 0.05 (Student t test). αM, mAb LPM19c; Ig, control IgG; PIC, piceatannol.

FIGURE 2.

Effect of piceatannol treatment and reduced Syk expression by siRNA on PKCδ Tyr311 phosphorylation in U937 cells. A, Cells were treated with different concentrations of piceatannol (PIC) for 1 h. Cells were then plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Immunoblot analyses of Erk1/2 and PKCδ Tyr311 phosphorylations were performed. Data are representative of two independent experiments. B, Reduction of endogenous Syk expression in cells treated with Syk-targeting siRNA as determined by immunoblotting whole-cell lysates with anti-Syk. C, Cells treated with control siRNA or Syk-targeting siRNA were plated onto mAbs. The effect of reduced Syk expression on PKCδ Tyr311 phosphorylation was assessed by immunoblotting. In all figures, β-actin was used as a loading control. Densitometry analyses of protein bands were performed for B and C. Data are the average of three experiments and expressed as means ± SD. *p < 0.05 (Student t test). αM, mAb LPM19c; Ig, control IgG; PIC, piceatannol.

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To determine whether the subcellular localization of PKCδ changed following integrin cross-linking, we performed cellular fractionation experiments. In resting cells, PKCδ was localized primarily in the cytosolic fraction (Fig. 3A); following cross-linking the amount of cytosolic PKCδ decreased and PKCδ pTyr311 appeared in the particulate fraction. This was verified by immunofluorescence analyses of αMβ2–cross-linked U937 cells that showed PKCδ translocated to the plasma membrane, which was also observed in PMA-treated cells (Fig. 3B). PKCδ translocation was not observed in anti–αLβ2-treated or control IgG-treated cells. The number of cells with translocated PKCδ was also counted in each field and plotted (Fig. 3C). We then examined the activity of the translocated PKCδ. The particulate fractions of cells treated with integrin–cross-linking Abs or cells treated with PMA were immunoprecipitated with anti-PKCδ, followed by in vitro kinase assay using γ[32P]ATP and the substrate histone H1 (Fig. 3D) (31). PKCδ activity was detected in αMβ2–cross-linked or PMA-treated cells but was not detected in other samples. Taken together, these data suggest that αMβ2 clustering in U937 cells triggers specific PKCδ Tyr311 phosphorylation that leads to activation and membrane localization of the kinase.

FIGURE 3.

αMβ2 clustering induced PKCδ translocation to the plasma membrane in U937 cells. A, Cells were plated onto mAb LPM19c (αM) or control IgG as in Fig. 1C. No cross-link, cells seeded into empty microtiter wells. Cells were harvested and lysed. Cell lysates were then fractionated by high-speed centrifugation. The soluble fraction containing the cytosol and the particulate fraction containing the membranes were immunoblotted with anti-PKCδ pTyr311. The membrane was then stripped and reblotted with anti-PKCδ to detect total PKCδ. B, Cells treated with anti-integrin mAb, control IgG, or PMA (100 ng/ml) were collected and seeded onto poly-l-lysine slides. Immunofluorescence staining of PKCδ was performed. Bar, 10 μm. Magnification ×40. C, The number of cells with membrane translocated PKCδ was counted by visual inspection. To determine the percentage cells with membrane translocated PKCδ, the number of cells with translocated PKCδ in a field/total number of cells in the same field was calculated. Data are means ± SD of cells in three different fields. D, Cells treated with anti-integrin mAb, control IgG, or PMA (100 ng/ml) were collected and lysed. The particulate fractions were immunoprecipitated with anti-PKCδ. The activity of precipitated PKCδ was assessed by in vitro kinase assay using γ[32P]ATP and histone H1. Whole-cell lysates were also immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. αM, mAb LPM19c.

FIGURE 3.

αMβ2 clustering induced PKCδ translocation to the plasma membrane in U937 cells. A, Cells were plated onto mAb LPM19c (αM) or control IgG as in Fig. 1C. No cross-link, cells seeded into empty microtiter wells. Cells were harvested and lysed. Cell lysates were then fractionated by high-speed centrifugation. The soluble fraction containing the cytosol and the particulate fraction containing the membranes were immunoblotted with anti-PKCδ pTyr311. The membrane was then stripped and reblotted with anti-PKCδ to detect total PKCδ. B, Cells treated with anti-integrin mAb, control IgG, or PMA (100 ng/ml) were collected and seeded onto poly-l-lysine slides. Immunofluorescence staining of PKCδ was performed. Bar, 10 μm. Magnification ×40. C, The number of cells with membrane translocated PKCδ was counted by visual inspection. To determine the percentage cells with membrane translocated PKCδ, the number of cells with translocated PKCδ in a field/total number of cells in the same field was calculated. Data are means ± SD of cells in three different fields. D, Cells treated with anti-integrin mAb, control IgG, or PMA (100 ng/ml) were collected and lysed. The particulate fractions were immunoprecipitated with anti-PKCδ. The activity of precipitated PKCδ was assessed by in vitro kinase assay using γ[32P]ATP and histone H1. Whole-cell lysates were also immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. αM, mAb LPM19c.

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To further examine the role of SFKs in αMβ2-induced PKCδ Tyr311 phosphorylation, we performed coimmunoprecipitation assays. Hck is abundantly expressed in myeloid cells, and it localizes around αMβ2-containing phagosomes (37). PKCδ coimmunoprecipitated with Hck from αMβ2–cross-linked U937 cells, and PKCδ pTyr311 was detected (Fig. 4A). PKCδ was not detected in the control IgG sample. Immunoprecipitated Hck from these cells was also used in kinase assay with rPKCδ (Fig. 4B). A significant increase in rPKCδ pTyr311 was detected in sample containing Hck immunoprecipitated from αMβ2–cross-linked cells as compared with cells treated with control IgG. To test direct phosphorylation of PKCδ by Hck, we performed kinase assay with rPKCδ and rHck (Fig. 4C). rHck phosphorylated Tyr311 in rPKCδ, and this was effectively abrogated by inhibitor PP2 but not by the inactive analog PP3. Next, we examined the effect of reducing Hck expression on αMβ2-induced PKCδ phosphorylation. Hck-targeting siRNA reduced the expression of Hck by ∼50% in U937 cells as compared with the control siRNA-treated cells (Fig. 4D). Cells treated with Hck-targeting siRNA, but not with control siRNA, showed a significant reduction in PKCδ Tyr311 phosphorylation when plated onto anti-αMβ2 (Fig. 4E). The expression levels of total PKCδ in both groups of cells were comparable. These data suggest that αMβ2-induced PKCδ Tyr311 phosphorylation involves Hck.

FIGURE 4.

The interaction of Hck and PKCδ was assessed. A, U937 cells were plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Whole-cell lysates were fractionated by high-speed centrifugation. The particulate fractions were solubilized and immunoprecipitated with anti-Hck or Ig. Immunoprecipitates were resolved by SDS-PAGE. Association of PKCδ with Hck and PKCδ Tyr311 phosphorylation were assessed by immunoblotting. The H chain of anti-Hck IgG used in the immunoprecipitation is indicated. B, As in A, but immunoprecipitated Hck was incubated with rPKCδ (100 ng) in kinase buffer. The extent of rPKCδ Tyr311 phosphorylation in the reaction samples was assessed by immunoblotting. Total rPKCδ and Hck were used as loading controls. C, In vitro kinase assay using rPKCδ and rHck (100 ng each) was performed without or with additive (PP2 or PP3, 20 μM each). The effects of inhibitors on rPKCδ Tyr311 phosphorylation were assessed by immunoblotting. Total rPKCδ and rHck were used as loading controls. D, Immunoblot analysis of Hck expression in U937 cells treated with control siRNA or Hck-targeting siRNA. β-Actin was used as a loading control. E, Immunoblot analyses of PKCδ pTyr311 and total PKCδ of siRNA-treated U937 cells plated onto mAbs. β-Actin was used as a loading control. In B–E, densitometry analyses of protein bands were performed. Data are the average of three experiments and expressed as means ± SD. *p < 0.05 (Student t test). In E, the signals of PKCδ pTyr311 in the Ig groups are close to 0. αM, mAb LPM19c; Ig, control IgG.

FIGURE 4.

The interaction of Hck and PKCδ was assessed. A, U937 cells were plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Whole-cell lysates were fractionated by high-speed centrifugation. The particulate fractions were solubilized and immunoprecipitated with anti-Hck or Ig. Immunoprecipitates were resolved by SDS-PAGE. Association of PKCδ with Hck and PKCδ Tyr311 phosphorylation were assessed by immunoblotting. The H chain of anti-Hck IgG used in the immunoprecipitation is indicated. B, As in A, but immunoprecipitated Hck was incubated with rPKCδ (100 ng) in kinase buffer. The extent of rPKCδ Tyr311 phosphorylation in the reaction samples was assessed by immunoblotting. Total rPKCδ and Hck were used as loading controls. C, In vitro kinase assay using rPKCδ and rHck (100 ng each) was performed without or with additive (PP2 or PP3, 20 μM each). The effects of inhibitors on rPKCδ Tyr311 phosphorylation were assessed by immunoblotting. Total rPKCδ and rHck were used as loading controls. D, Immunoblot analysis of Hck expression in U937 cells treated with control siRNA or Hck-targeting siRNA. β-Actin was used as a loading control. E, Immunoblot analyses of PKCδ pTyr311 and total PKCδ of siRNA-treated U937 cells plated onto mAbs. β-Actin was used as a loading control. In B–E, densitometry analyses of protein bands were performed. Data are the average of three experiments and expressed as means ± SD. *p < 0.05 (Student t test). In E, the signals of PKCδ pTyr311 in the Ig groups are close to 0. αM, mAb LPM19c; Ig, control IgG.

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Besides Hck, the SFKs Lyn and c-Yes have been reported to interact with the β2 integrin cytoplasmic tail in pull-down assays that used recombinant integrin β cytoplasmic tails (38). In U937 cells, the expression of Lyn was modest as compared with Hck, and the expression of c-Yes was minimal (Fig. 5A). The lack of c-Yes expression was not due to poor Ab reactivity, because moderate expression of c-Yes was detected in the T cell line Jurkat (39). The lack of c-Yes mRNA in the promyelocyte HL60 cells has also been reported (40). Thus, we only examined Lyn in the following studies. Cells treated with anti-αMβ2 were lysed, and Lyn was immunoprecipitated (Fig. 5B). PKCδ coprecipitated with Lyn. PKCδ pY311 was also detected in the Lyn immunoprecipitate, albeit at a low level, which could be attributed to the moderate expression level of Lyn in these cells. By contrast, PKCδ was not detected in Lyn immunoprecipitate of cells treated with the control IgG. Higher level of rPKCδ Tyr311 phosphorylation was detected in Lyn immunoprecipitate of cells treated with anti-αMβ2 but not with control IgG (Fig. 5C). These data suggest that Lyn is also involved in αMβ2-induced PKCδ Tyr311 phosphorylation, although Hck, which is highly expressed as compared with Lyn, may have a predominant role in these cells.

FIGURE 5.

The interaction of Lyn and PKCδ was assessed. A, Immunoblot analyses of Hck, Lyn, and c-Yes expression in U937 cells. c-Yes expression in Jurkat was also analyzed. β-Actin was used as a loading control. B, U937 cells were plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Whole-cell lysates were immunoprecipitated with anti-Lyn or Ig. Immunoprecipitates were resolved on SDS-PAGE. Association of PKCδ with Lyn and PKCδ Tyr311 phosphorylation were assessed by immunoblotting. C, Lyn immunoprecipitates as in B were incubated with rPKCδ (100 ng) in kinase buffer. The extent of rPKCδ Tyr311 phosphorylation in the reaction samples was assessed by immunoblotting. Total rPKCδ was used as a loading control. Densitometry analyses of protein bands were performed. Data are average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test).

FIGURE 5.

The interaction of Lyn and PKCδ was assessed. A, Immunoblot analyses of Hck, Lyn, and c-Yes expression in U937 cells. c-Yes expression in Jurkat was also analyzed. β-Actin was used as a loading control. B, U937 cells were plated onto mAb LPM19c (αM) or control IgG (Ig) as in Fig. 1C. Whole-cell lysates were immunoprecipitated with anti-Lyn or Ig. Immunoprecipitates were resolved on SDS-PAGE. Association of PKCδ with Lyn and PKCδ Tyr311 phosphorylation were assessed by immunoblotting. C, Lyn immunoprecipitates as in B were incubated with rPKCδ (100 ng) in kinase buffer. The extent of rPKCδ Tyr311 phosphorylation in the reaction samples was assessed by immunoblotting. Total rPKCδ was used as a loading control. Densitometry analyses of protein bands were performed. Data are average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test).

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The integrin cytoplasmic tails are essential for integrin outside-in signaling upon ligand binding (41). The SFKs Hck, Lyn, and c-Yes have been reported to interact with the β2 integrin cytoplasmic tail (38). We therefore examined the β2 cytoplasmic tail to determine the region that is important for αMβ2-induced PKCδ Tyr311 phosphorylation. The myeloid K562 cells express PKCδ but not the β2 integrins. We used stable-transfected K562 cells expressing αMβ2 (Fig. 6A) to show that cross-linking of αMβ2 on these cells triggers PKCδ Tyr311 phosphorylation (Fig. 6B). Phosphorylation was transient as determined by time course analysis (Fig. 6C), and it was SFK dependent (Fig. 6D). These data are consistent with the data of U937 cells in previous sections. A panel of β2 mutants with cytoplasmic tail deletions was then cotransfected with wild-type αM into empty K562 cells (Fig. 6E, 6F). Transfectants were treated with anti-αMβ2 (or control IgG), and the level of PKCδ pTyr311 in these cells was assessed (Fig. 6G). We showed that the region Asn727-Ser734 in the β2 tail is required for αMβ2-induced PKCδ Tyr311 phosphorylation. Interestingly, this region has previously been reported to be required for colocalization of Hck with αMβ2 (26)

FIGURE 6.

Defining the region in the integrin β2 cytoplasmic tail required for αMβ2-induced PKCδ Tyr311 phosphorylation. A, Flow cytometry analyses of stable-transfected K562 cells expressing αMβ2 and untransfected K562 cells. Gray histograms represent staining with mAb LPM19c (anti-αM). Black histograms represent staining with an irrelevant mAb. B, αMβ2-expressing K562 or untransfected K562 cells were plated onto anti-integrin mAbs (or control IgG) as in Fig. 1C. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311. The membrane was stripped of Abs and reblotted with anti-PKCδ to detect total PKCδ. C, Time course analysis of PKCδ Tyr311 phosphorylation in αMβ2-expressing K562 cells that were plated onto mAb LPM19c (αM) or control IgG (Ig). D, αMβ2-expressing K562 cells were treated with inhibitor (20 μM each) for 1 h. Cells were then plated onto αM or Ig for 30 min. E, β2 cytoplasmic tail sequences of truncated mutants used in this study. The sequence of the αM cytoplasmic tail is also included. The unshaded and shaded boxes indicate the residues in β2 and αM cytoplasmic tails, respectively, that forms a salt bridge. Dotted line represents the boundary between the juxtamembrane residues of the transmembrane domain and the intracellular residues based on the transmembrane boundary assignment in integrin αIIbβ3 (42). F, Flow cytometry analyses of αMβ2 and mutants expressed on K562 transfectants. Gray histograms represent staining with mAb LPM19c. Black histograms represent staining with an irrelevant mAb. G, Immunoblot analysis of PKCδ pTyr311 in K562 cells that express αMβ2 or mutants and were treated with immobilized mAb LPM19c (+) or control IgG (−) for 30 min at 37°C. In C and D, PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/ total PKCδ intensity). Data are the average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). αM, mAb LPM19c; Con, no inhibitor; Ig, control IgG; WT, wild-type β2.

FIGURE 6.

Defining the region in the integrin β2 cytoplasmic tail required for αMβ2-induced PKCδ Tyr311 phosphorylation. A, Flow cytometry analyses of stable-transfected K562 cells expressing αMβ2 and untransfected K562 cells. Gray histograms represent staining with mAb LPM19c (anti-αM). Black histograms represent staining with an irrelevant mAb. B, αMβ2-expressing K562 or untransfected K562 cells were plated onto anti-integrin mAbs (or control IgG) as in Fig. 1C. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311. The membrane was stripped of Abs and reblotted with anti-PKCδ to detect total PKCδ. C, Time course analysis of PKCδ Tyr311 phosphorylation in αMβ2-expressing K562 cells that were plated onto mAb LPM19c (αM) or control IgG (Ig). D, αMβ2-expressing K562 cells were treated with inhibitor (20 μM each) for 1 h. Cells were then plated onto αM or Ig for 30 min. E, β2 cytoplasmic tail sequences of truncated mutants used in this study. The sequence of the αM cytoplasmic tail is also included. The unshaded and shaded boxes indicate the residues in β2 and αM cytoplasmic tails, respectively, that forms a salt bridge. Dotted line represents the boundary between the juxtamembrane residues of the transmembrane domain and the intracellular residues based on the transmembrane boundary assignment in integrin αIIbβ3 (42). F, Flow cytometry analyses of αMβ2 and mutants expressed on K562 transfectants. Gray histograms represent staining with mAb LPM19c. Black histograms represent staining with an irrelevant mAb. G, Immunoblot analysis of PKCδ pTyr311 in K562 cells that express αMβ2 or mutants and were treated with immobilized mAb LPM19c (+) or control IgG (−) for 30 min at 37°C. In C and D, PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/ total PKCδ intensity). Data are the average of three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). αM, mAb LPM19c; Con, no inhibitor; Ig, control IgG; WT, wild-type β2.

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Integrin-mediated cell adhesion includes two major events pre- and postligand occupancy. Inside-out activation of integrin by cytoplasmic proteins is required for integrin ligand binding, whereas engagement of multivalent or multimeric ligand by the activated integrin promotes receptor clustering that induces outside-in signaling (43). We therefore extended our investigation to address the importance of αMβ2 clustering in the induction of PKCδ Tyr311 phosphorylation. We first examined PKCδ Tyr311 phosphorylation in U937 cells that were allowed to adhere to immobilized αMβ2 ligand ICAM-1 (9). Cells adhered to ICAM-1 only when treated with the β2 integrin-activating mAb KIM185 (data not shown) (23). PKCδ pTyr311 was detected in cells that adhered to ICAM-1 (Fig. 7A). Similar observations were made using immobilized fibrinogen (data not shown). Other than its activating property, mAb KIM185 can potentially cross-link αMβ2 that triggers downstream signaling. We therefore generated an intrinsically activated αMβ2 that has a salt-bridge mutation β2D709R (Fig. 7B) (27). K562 cells were then transfected with wild-type αMβ2 or αMβ2D709R. Cell surface expression of wild-type αMβ2 or αMβ2D709R was determined by flow cytometry (Fig. 7C). The activated conformation of αMβ2D709R was verified by immunoprecipitation with the reporter mAb KIM127 that recognizes an epitope in the integrin epidermal growth factor 2-fold of an extended β2 (44). Immunoprecipitates were immunoblotted with anti-αM to detect the αM subunit that associates with the β2 subunit (Fig. 7D). Immunoprecipitation with αM-specific mAb LPM19c was included as a control. MnCl2 instead of mAb KIM185 was used as the activating agent so as not to interfere with the immunoprecipitation procedure. The αM protein band was detected in the mAb KIM127 immunoprecipitate of wild-type αMβ2-expressing cells only in the presence of MnCl2. The αM protein band was detected in the mAb KIM127 immunoprecipitates of αMβ2D709R-expressing cells with or without MnCl2 treatment. These data verify the extended conformation of αMβ2D709R. Consistent with the activated conformation of αMβ2D709R detected, transfectants expressing αMβ2D709R adhered constitutively to immobilized ICAM-1, whereas cells expressing wild-type αMβ2 required the presence of mAb KIM185 (Fig. 7E). Adhesion specificity was verified by including mAb LPM19c that is function blocking (45).

FIGURE 7.

Ligand-induced clustering of αMβ2 triggers PKCδ Tyr311 phosphorylation. A, U937 cells were plated onto immobilized ICAM-1 for 30 min at 37°C in a humidified incubator with 5% CO2. Cells were harvested and lysed. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. Activating mAb KIM185 (10 μg/ml) was required for cell adhesion. β-Actin was used as a loading control. B, An illustration showing a resting αMβ2 with the cytoplasmic residues αM Arg1120 and β2 Asp709 forming a salt bridge (left). The charge reversion mutation Asp709 to Arg (D709R) in the β2 cytoplasmic tail disrupts the salt bridge, and it promotes activation of αMβ2 to bind ligand (right). C, Expression analyses of αMβ2 and αMβ2D709R on K562 transfectants by flow cytometry using mAb LPM19c (anti-αM). D, The conformation of αMβ2D709R was verified by immunoprecipitation using the mAb KIM127 that recognizes an epitope in the extended β2 integrin. Monoclonal Ab LPM19c was used as a control Ab. MnCl2 (0.5 mM) was included as an activating agent instead of mAb KIM185, because it does not interfere with the immunoprecipitation procedure. E, Adhesion of K562 transfectants expressing αMβ2 or αMβ2D709R to immobilized ICAM-1. For the adhesion of αMβ2-expressing K562, the addition of activating mAb KIM185 was required. Adhesion specificity mediated by αMβ2 and mutant was demonstrated using mAb LPM19c (10 μg/ml). F, K562 transfectants were seeded into microtitre wells with or without ICAM-1 coating in the presence or absence of mAb KIM185. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. β-Actin was used as a loading control. PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/total PKCδ intensity). Data are average of three independent experiments expressed as means ± SD. *p < 0.05 (Student t test).

FIGURE 7.

Ligand-induced clustering of αMβ2 triggers PKCδ Tyr311 phosphorylation. A, U937 cells were plated onto immobilized ICAM-1 for 30 min at 37°C in a humidified incubator with 5% CO2. Cells were harvested and lysed. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. Activating mAb KIM185 (10 μg/ml) was required for cell adhesion. β-Actin was used as a loading control. B, An illustration showing a resting αMβ2 with the cytoplasmic residues αM Arg1120 and β2 Asp709 forming a salt bridge (left). The charge reversion mutation Asp709 to Arg (D709R) in the β2 cytoplasmic tail disrupts the salt bridge, and it promotes activation of αMβ2 to bind ligand (right). C, Expression analyses of αMβ2 and αMβ2D709R on K562 transfectants by flow cytometry using mAb LPM19c (anti-αM). D, The conformation of αMβ2D709R was verified by immunoprecipitation using the mAb KIM127 that recognizes an epitope in the extended β2 integrin. Monoclonal Ab LPM19c was used as a control Ab. MnCl2 (0.5 mM) was included as an activating agent instead of mAb KIM185, because it does not interfere with the immunoprecipitation procedure. E, Adhesion of K562 transfectants expressing αMβ2 or αMβ2D709R to immobilized ICAM-1. For the adhesion of αMβ2-expressing K562, the addition of activating mAb KIM185 was required. Adhesion specificity mediated by αMβ2 and mutant was demonstrated using mAb LPM19c (10 μg/ml). F, K562 transfectants were seeded into microtitre wells with or without ICAM-1 coating in the presence or absence of mAb KIM185. Whole-cell lysates were immunoblotted with anti-PKCδ pTyr311 and anti-PKCδ. β-Actin was used as a loading control. PKCδ pTyr311 and total PKCδ protein bands were quantified by densitometry and plotted as relative units (PKCδ pTyr311 intensity/total PKCδ intensity). Data are average of three independent experiments expressed as means ± SD. *p < 0.05 (Student t test).

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We then examined PKCδ Tyr311 phosphorylation in wild-type αMβ2 (resting) and αMβ2D709R (constitutively activated) transfectants plated onto immobilized ICAM-1 (Fig. 7F). In the absence of ICAM-1, PKCδ pTyr311 was not detected in both transfectants. Activation of wild-type αMβ2 by mAb KIM185 did not trigger PKCδ Tyr311 phosphorylation in the absence of ICAM-1 (Fig. 7F, left panel). In the presence of immobilized ICAM-1, pTyr311 PKCδ was detected in αMβ2D709R cells and in KIM185-activated wild-type αMβ2 cells (Fig. 7F, right panel). These data suggest that the process of clustering is required for αMβ2-induced signaling that regulates the function of PKCδ.

Treatment of the promyelocyte HL60 cells with PMA has been reported to downregulate Foxp1 expression (15). A reduction in Foxp1 expression level has also been observed in αMβ2-cross-linked monocytes, although the detailed signaling pathway remains to be clarified (46). In line with these observations, U937 cells treated with PMA, which induces cell differentiation, showed a significant reduction in Foxp1 expression after 4 h (Fig. 8A). Our present study shows that PKCδ phosphorylation and its activation are early events triggered by αMβ2 clustering. We hypothesize that PKCδ may be involved in the regulation of Foxp1 expression that is induced by αMβ2 clustering. To this end, PKCδ expression was reduced in U937 cells by the method of siRNA. Significant reduction in PKCδ expression was observed in cells treated with PKCδ-targeting siRNA but not in cells treated with control siRNA (Fig. 8B). The reaction was specific because the expression levels of PKCα and PKCε were unaffected. Cross-linking of αMβ2 on cells treated with PKCδ-targeting siRNA, but not control siRNA, failed to reduce the expression of Foxp1 (Fig. 8C). Foxp1 expressions were also unaffected in cells treated with control IgG. These data suggest that PKCδ is involved in the regulation of Foxp1 by αMβ2.

FIGURE 8.

PKCδ is involved in the regulation of Foxp1 by αMβ2. A, Time course analysis of Foxp1 expression by immunoblotting whole-cell lysates of U937 cells treated with PMA (100 ng/ml) for various time points (0–8 h). B, Cells were treated with control siRNA or PKCδ-targeting siRNA. Whole-cell lysates were immunoblotted with anti-PKCδ, anti-PKCα, or anti-PKCε. C, Cells treated with control siRNA or PKCδ-targeting siRNA were plated onto mAb LPM19c (αM) or control Ig (Ig) and incubated for 8 h at 37°C in a humidified incubator with 5% CO2. Cells were then harvested and lysed. Whole-cell lysates were immunoblotted with anti-Foxp1. In all figures, β-actin was used as a loading control. All densitometry measurements and plots are average from three independent experiments and expressed as means ± SD. Relative units, PKCδ intensity/β-actin or Foxp1 intensity/β-actin intensity. *p < 0.05 (Student t test). αM, mAb LPM19c; Ig, control Ig.

FIGURE 8.

PKCδ is involved in the regulation of Foxp1 by αMβ2. A, Time course analysis of Foxp1 expression by immunoblotting whole-cell lysates of U937 cells treated with PMA (100 ng/ml) for various time points (0–8 h). B, Cells were treated with control siRNA or PKCδ-targeting siRNA. Whole-cell lysates were immunoblotted with anti-PKCδ, anti-PKCα, or anti-PKCε. C, Cells treated with control siRNA or PKCδ-targeting siRNA were plated onto mAb LPM19c (αM) or control Ig (Ig) and incubated for 8 h at 37°C in a humidified incubator with 5% CO2. Cells were then harvested and lysed. Whole-cell lysates were immunoblotted with anti-Foxp1. In all figures, β-actin was used as a loading control. All densitometry measurements and plots are average from three independent experiments and expressed as means ± SD. Relative units, PKCδ intensity/β-actin or Foxp1 intensity/β-actin intensity. *p < 0.05 (Student t test). αM, mAb LPM19c; Ig, control Ig.

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We also examined the contribution of PKCδ to αMβ2-regulated Foxp1 expression in human peripheral blood monocytes. Cross-linking of αMβ2, but not αLβ2 (or control IgG), induced PKCδ Tyr311 phosphorylation in monocytes (Fig. 9A). Cross-linking of β1 or β5 integrin also did not induce PKCδ Tyr311 phosphorylation (Supplemental Fig. 1B). Immunofluorescence analysis showed that PKCδ translocated to the plasma membrane in cells treated with anti-αMβ2 but not with anti-αLβ2 (or control IgG) (Fig. 9B). Next, we reduced the expression of PKCδ in monocytes using PKCδ-targeting siRNA (Fig. 9C). The targeting was specific because the expression levels of PKCα and PKCε were unaffected. Cross-linking of αMβ2 significantly reduced Foxp1 expression in control siRNA-treated cells but not in cells treated with PKCδ-targeting siRNA (Fig. 9D). No difference in Foxp1 expression was observed in these cells treated with control IgG.

FIGURE 9.

Clustering of αMβ2 induced PKCδ Tyr311 phosphorylation and Foxp1 downregulation in human peripheral blood monocytes. A, Monocytes were plated onto anti-integrin mAb or control IgG (Ig) and processed as in Fig. 1C. B, The localization of PKCδ in monocytes after plating on anti-integrin mAb or control IgG was assessed by immunofluorescence staining. Bar, 10 μm. Magnification ×40. C, Immunoblot analyses of PKCδ, PKCα, and PKCε in monocytes treated with control siRNA or PKCδ-targeting siRNA. D, Monocytes treated with control siRNA or PKCδ-targeting siRNA were plated onto Abs as in A for 8 h. Whole-cell lysates were immunoblotted with anti-Foxp1. E, Still images (phase contrast and fluorescence) from live-cell imaging of monocytes on HMVE cells. Bar, 50 μm. Magnification ×20. F, Monocytes treated with control siRNA or PKCδ-targeting siRNA were allowed to adhere and migrate on HMVE cells for 8 h. Monocytes without siRNA treatment were also plated onto HMVE or onto culture dish without HMVE. Monocytes were then isolated by anti-CD14 magnetic bead separation and Foxp1 expression assessed by immunoblotting. All densitometry measurements and plots are average from three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). Ig, control IgG.

FIGURE 9.

Clustering of αMβ2 induced PKCδ Tyr311 phosphorylation and Foxp1 downregulation in human peripheral blood monocytes. A, Monocytes were plated onto anti-integrin mAb or control IgG (Ig) and processed as in Fig. 1C. B, The localization of PKCδ in monocytes after plating on anti-integrin mAb or control IgG was assessed by immunofluorescence staining. Bar, 10 μm. Magnification ×40. C, Immunoblot analyses of PKCδ, PKCα, and PKCε in monocytes treated with control siRNA or PKCδ-targeting siRNA. D, Monocytes treated with control siRNA or PKCδ-targeting siRNA were plated onto Abs as in A for 8 h. Whole-cell lysates were immunoblotted with anti-Foxp1. E, Still images (phase contrast and fluorescence) from live-cell imaging of monocytes on HMVE cells. Bar, 50 μm. Magnification ×20. F, Monocytes treated with control siRNA or PKCδ-targeting siRNA were allowed to adhere and migrate on HMVE cells for 8 h. Monocytes without siRNA treatment were also plated onto HMVE or onto culture dish without HMVE. Monocytes were then isolated by anti-CD14 magnetic bead separation and Foxp1 expression assessed by immunoblotting. All densitometry measurements and plots are average from three independent experiments and expressed as means ± SD. *p < 0.05 (Student t test). Ig, control IgG.

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It has been reported that clustering of αMβ2 modulates monocyte differentiation via Foxp1 regulation (15, 16). Adhesion of monocytes to the endothelium prior to their egress into tissues could have an important role in their differentiation. To address this, monocytes without and with siRNA treatment were plated onto HMVE cells that express ICAM-1 (data not shown) or culture dish without HMVE (Fig. 9E, representative still images of monocytes labeled with fluorescent dye CellTracker Green on HMVE). We did not observe significant difference in the migration profiles of monocytes treated with control siRNA and PKCδ-targeting siRNA (data not shown). Monocytes were then isolated from the cell mixture, and the expression of Foxp1 in these cells assessed by immunoblotting (Fig. 9F). Monocytes without siRNA treatment showed reduced expression of Foxp1 when plated onto HMVE but not onto culture dish. Monocytes treated with control siRNA, but not with PKCδ-targeting siRNA, showed a reduction in Foxp1 expression when plated onto HMVE. These data provide additional support to a role of PKCδ in αMβ2-regulated Foxp1 expression.

In this study, we made use of K562 transfectants, U937 cells, and human peripheral blood monocytes to examine the membrane proximal signaling event triggered by integrin αMβ2 clustering. We also examined its effect on the expression of transcription factor Foxp1 that regulates monocyte differentiation (15, 16). We show that clustering of αMβ2 induces PKCδ translocation to the plasma membrane. This could allow PKCδ to localize in SFK-enriched microdomains and thus promote SFK-dependent PKCδ phosphorylation. Indeed, c-Src–dependent PKCδ Tyr311 and Tyr565 phosphorylations in human platelets treated with thrombin or PMA have been reported previously (47). In another study on cardiomyocytes, PMA induced PKCδ Tyr311 phosphorylation by localizing PKCδ to SFK-enriched caveolae (48).

Our data suggest that SFK is required for αMβ2-induced PKCδ Tyr311 phosphorylation. The mechanism of SFK activation that is triggered by integrin clustering has been reported with reference to c-Src and platelet integrin αIIbβ3 (49). c-Src interacts directly with the β3 cytoplasmic tail of αIIbβ3 (38). Direct binding of c-Src to αIIbβ3 may promote c-Src priming, and thus, the clustering of ligand-bound αIIbβ3 will increase the local concentration of c-Src, leading to its transautophosphorylation and full activation (49). In pull-down experiments that used recombinant β2 cytoplasmic tail, a high level of Hck and Lyn, minimal c-Yes, but not c-Src or Fyn was detected (38). Thus, the mechanism of SFK activation induced by αMβ2 clustering may be similar to that of c-Src activation by αIIbβ3 clustering.

PKCδ is phosphorylated by c-Src, Fyn, and c-Abl in different cell types (5052). However, c-Src or Fyn failed to interact with the β2 cytoplasmic tail as compared with Hck and Lyn in the aforementioned study (38). Hck is highly expressed in myeloid cells, and it colocalizes with αMβ2 in macrophages (37). In this study, we report that Hck is involved in αMβ2-induced PKCδ Tyr311 phosphorylation. Our previous data showed that the region Asn727-Ser734 in the β2 cytoplasmic tail is required for αMβ2 and Hck colocalization in Chinese hamster ovary transfectant (26). Consistent with this observation, we showed in this study that the β2 Asn727-Ser734 sequence has an important role in αMβ2-induced PKCδ Tyr311 phosphorylation. We also show that in addition to Hck, Lyn is involved in PKCδ phosphorylation. Lyn interacts with and phosphorylates PKCδ Tyr311 in platelets (53). It is therefore possible that Hck and Lyn could perform similar function in this context, which is not unexpected because both kinases have been shown to interact with the integrin β2 cytoplasmic tail (38), and overlapping and complementary functions of SFKs are known (54).

Peritoneal macrophages derived from Hck or Fgr (another SFK involved in β2 integrin signaling) knockout mice showed normal integrin signaling when plated on fibronectin, whereas Hck and Fgr double knockout attenuated this process (55). These data are in line with the observations made in earlier studies suggesting functional overlap between Hck and Fgr (56, 57). Interestingly, Fgr negatively regulates β2 integrin signaling in monocytes by its direct association with Syk (58). Fgr also negatively regulates αMβ2-mediated phagocytosis (59). U937 cells expressed a low level of Fgr as compared with Hck (data not shown). In this study, we have not examined the involvement of Fgr in PKCδ phosphorylation and activation. We reasoned that ultimately the use of mouse knockout models in future work will better define the specific contributions of each of these SFKs in PKCδ regulation.

Our study shows that the clustering of αMβ2, but not αLβ2, triggers significant PKCδ Tyr311 phosphorylation in U937 cells and peripheral blood monocytes. This observation recapitulates specific signaling derived from integrins with different α subunits that pair with a common β subunit in the same cell. It also points to the importance of the highly divergent α cytoplasmic tails as compared with the β cytoplasmic tails in generating specificity in signaling. Interestingly, CC chemokine stimulation of monocytes triggers different αLβ2 and αMβ2 activation kinetics (60). Ligand-bound wild-type αMβ2 has been shown to delay apoptosis of transfected K562 cells, and the effect was abrogated when the αM cytoplasmic tail was substituted with the αL tail (61). Other examples include the differential effects on adhesion and migration of cells that involves the α cytoplasmic tails of integrins α2β1, α4β1, and α5β1 (62, 63). The mechanism by which signaling specificity is achieved remains to be defined.

Our data also show that αMβ2 activation in the absence of receptor clustering is not an effective trigger of PKCδ Tyr311 phosphorylation. Constitutively activated αMβ2, in the form of αMβ2D709R, failed to trigger PKCδ Tyr311 phosphorylation in K562 transfectants in the absence ICAM-1 binding. This is in concordance with the rest of the data in this study showing that Ab cross-linking of resting αMβ2 triggers cytoplasmic signaling. How does clustering of resting αMβ2 induce cytoplasmic signaling? It is possible that a population of primed SFK is constitutively tethered to the β2 cytoplasmic tail of resting αMβ2 similar to the proposed model of c-Src and αIIbβ3 interaction (38), and the clustering of these resting αMβ2 is sufficient to activate the associated SFK. Reminiscent to this model of membrane receptor signaling is the capacity to induce cytoplasmic signaling in T cells by using Abs to cross-link cell surface TCR/CD3 (64). In a physiological setting, clustering of integrin by the engagement of multimeric or multivalent ligand induces integrin outside-in signaling that regulates cell growth and differentiation (2, 43).

It has been reported that the clustering of αMβ2 on monocytes triggers differentiation signal that downregulates the expression of transcription factor Foxp1 (16, 46). The signaling pathway linking αMβ2 to the regulation of Foxp1 is unclear. Clustering of αMβ2 on THP-1 cells triggers Toll/IL-1 signaling, and the IL-1 receptor-associated kinase 1 is implicated in this pathway (17). However, evidence demonstrating the involvement of IL-1 receptor-associated kinase 1 in Foxp1 regulation is lacking. Our data based on siRNA studies show that PKCδ is involved in Foxp1 regulation. The observation that Foxp1 was downregulated in monocytes that adhered to HMVE also lends support for the importance of adhesion-derived signal in monocyte differentiation when monocytes transmigrate across endothelium. Finally, PKCδ has been implicated in MEK and ERK1/2-dependent monocyte differentiation (65). Future studies will examine the contribution of MEK-ERK pathway in the regulation of Foxp1.

We thank K.G. Lim, M. Cooray, and S.C. Ng for technical assistance. We thank S.K.A. Law (Nanyang Technological University, Singapore) for information on growing the hybridomas and purification of mAbs. We also thank Andrew N.S. Tan (Nanyang Technological University) for providing helpful information on the growth conditions of the HMVE cells.

Disclosures The authors have no financial conflicts of interest.

This work was supported by the Agency for Science, Technology and Research Biomedical Research Council Grants 04/1/22/19/358 and 06/1/22/19/445 (to S.-M.T.).

The online version of this article contains supplemental material.

Abbreviations used in this paper:

αL

MHM24

αM

mAb LPM19c

con

control, without any additive

HMVE

human lung microvascular endothelial

Ig

control IgG

LY

PI3K inhibitor LY294002

PIC

Syk kinase inhibitor picetannol

PKC

protein kinase C

PP2

src family kinase inhibitor

PP3

inactive analog of PP2

SFK

src family kinase

siRNA

small interfering RNA.

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