Tumor Necrosis Factor-α Induces Early-Onset Endothelial Adhesivity by Protein Kinase Cζ–Dependent Activation of Intercellular Adhesion Molecule-1
We tested the hypothesis that TNF-α induces early-onset endothelial adhesivity toward PMN by activating the constitutive endothelial cell surface ICAM-1, the β2-integrin (CD11/CD18) counter-receptor. Stimulation of human pulmonary artery endothelial cells with TNF-α resulted in phosphorylation of ICAM-1 within 1 minute, a response that was sustained up to 15 minutes after TNF-α challenge. We observed that TNF-α induced 10-fold increase in PMN adhesion to endothelial cells in an ICAM-1–dependent manner and that this response paralleled the rapid time course of ICAM-1 phosphorylation. We also observed that the early-onset TNF-α–induced endothelial adhesivity was protein synthesis–independent and associated with cell surface ICAM-1 clustering. Pretreatment of cells with the pan-PKC inhibitor, chelerythrine, prevented the activation of endothelial adhesivity. As PKCζ, an atypical PKC isoform abundantly expressed in endothelial cells, is implicated in signaling TNF-α–induced ICAM-1 gene transcription, we determined the possibility that PKCζ was involved in mediating endothelial adhesivity through ICAM-1 expression. We observed that TNF-α stimulation of endothelial cells induced PKCζ activation and its association with ICAM-1. Inhibition of PKCζ by pharmacological and genetic approaches prevented the TNF-α–induced phosphorylation and the clustering of the cell surface ICAM-1 as well as activation of endothelial adhesivity. Thus, TNF-α induces early-onset, protein synthesis–independent expression of endothelial adhesivity by PKCζ-dependent phosphorylation of cell surface ICAM-1 that precedes the de novo ICAM-1 synthesis. The rapid ICAM-1 expression represents a novel mechanism for promoting the stable adhesion of PMN to endothelial cells that is needed to facilitate the early-onset transendothelial migration of PMN.
- tumor necrosis factor-α
- intercellular adhesion molecule-1
- polymorphonuclear leukocyte adhesion
- protein kinase Cζ
Polymorphonuclear leukocytes (PMN) play an essential role as the first line of defense against the invading microorganisms. Recruitment of PMN to the site of infection is a multistep process involving sequential activation of adhesive proteins on endothelial cells and their counter-receptors on the surface of leukocytes.1–3 Intercellular adhesion molecule-1 (ICAM-1), an adhesive protein expressed on the endothelial cell surface, is a member of the immunoglobulin supergene family4 that serves as a counter-receptor for leukocyte β2-CD11/CD18 integrins.5 The interaction between ICAM-1 and β2-integrins is required for firm adhesion and the stable arrest of PMN on the endothelial cell surface5 before PMN can migrate across the endothelial barrier.3
Although ICAM-1 is normally present on the endothelial cell surface, its expression can be induced by proinflammatory mediators such as tumor necrosis factor-α (TNF-α) or thrombin.6–10 Time course studies showed that TNF-α–induced ICAM-1 expression in endothelial cells was detected within 1 hour and thereafter increased progressively over the next 24 hours.6,7 The rapid expression of ICAM-1 is required to promote the early migration of PMN, which can be maximal within 1 hour after activation by proinflammatory stimuli.10 Although the mechanisms regulating de novo expression of ICAM-1 and the role of ICAM-1 in mediating PMN-endothelial interactions have been studied in some detail,3,11,12 little is known about the role of the constitutive cell surface ICAM-1 in promoting the rapid expression of endothelial adhesivity. Studies showed significant ICAM-1 expression and ICAM-1–dependent PMN adhesion to endothelial cells within minutes after exposure to an inflammatory signal at a time when ICAM-1 would not be expected to be synthesized.10 It was postulated that rapid ICAM-1 expression involves phosphorylation-dependent activation of the cell surface protein.10 Because the cytoplasmic domain of ICAM-1 has threonine residues at positions 521, 527, and 530, we addressed the possibility that protein kinase C (PKC), a family of serine/threonine kinases,13,14 signals ICAM-1 activation. We have recently shown that TNF-α induces NADPH oxidase activation and ICAM-1 gene transcription in endothelial cells through a PKCζ-dependent pathway.8,15 Because PKCζ is critical in signaling several components of the inflammatory response,8,15,16 we addressed the possibility that PKCζ-dependent phosphorylation regulates the rapid induction of endothelial cell surface ICAM-1. We provide evidence herein that TNF-α induces early-onset endothelial adhesivity toward PMN involving a qualitative alteration in cell surface ICAM-1 that is dependent on phosphorylation by PKCζ.
Materials and Methods
125I and 32P were purchased from ICN Pharmaceuticals Inc. Cell Tracker dye and goat anti-mouse Alexa-488/568 IgG were from Molecular Probes Inc. PKCζ and PKCθ peptide inhibitors were obtained from Biosource International. ICAM-1 blocking mAb, CD-18 blocking mAb, nPKCζ rabbit polyclonal Ab, goat anti-mouse horseradish peroxidase-linked (HRP) IgG, and goat anti-rabbit HRP IgG were from Santa Cruz Biotechnology. Phospho-PKCζ antibody, which detects PKCζ when it’s phosphorylated at Thr-410, was purchased from Cell Signaling. Recombinant human TNF-α was obtained from Promega Corp. Polyvinylidene difluoride (PVDF) membrane was from Millipore Corp, a protein assay kit from Bio-Rad Laboratories, Sephadex G-25 beads and Phosphate free MEM from Sigma Aldrich Inc, and Polymorph prep was from Accurate Chemical and Scientific Corp. All other materials were purchased from Fisher Scientific Company.
Human pulmonary arterial endothelial cells (HPAECs; BioWhittaker, Walkersville, Md) were cultured as described.15 Confluent monolayers were serum starved for 4 hours in EBM-2 before TNF-α challenge.
PMN Adhesion Assay
PMN adhesion assay was performed as described.17 Briefly, HPAECs grown over 12-mm circular cover slips were labeled with 3 μmol/L fluorescent (red) Cell-Tracker dye for 30 minutes before stimulation with TNF-α, and then washed extensively to remove residual TNF-α. Freshly isolated human neutrophils (PMN) were stained with 5 μmol/L fluorescent (green) Cell-Tracker dye, coincubated with endothelial cells for 20 minutes, washed with PBS, and visualized using a fluorescent microscope. The adherent PMN were counted and expressed as PMN/0.8 mm2 of endothelial cell.
Immunoblotting and Immunofluorescence
Immunoblotting and immunofluorescence studies were performed as described.15
Preparation of Cytosolic and Membrane Fractions
Cytosolic and membrane fractions were prepared as described.15 Briefly, cells were washed with ice-cold TBS and lysed in (in mmol/L) 10 Tris-HCl (pH 7.5), 1 MgCl2, 5 EDTA, 10 EGTA, 1 NaVO4, and a cocktail of protease inhibitors (Sigma P-8340). Lysates were sonicated for 10 seconds and then centrifuged at 100 000g for 1 hour at 4°C. Supernatants were collected and designated cytosolic fraction. The remaining pellets were resuspended in the above lysis buffer containing 1% Triton X-100, sonicated, and incubated for 30 minutes at 4°C. These lysates were microfuged at 4°C, and the supernatants were designated membrane fraction.
125I-labeled Antibody Binding Assay
The binding activity of cell surface ICAM-1 was determined by the specific binding of 125I-labeled anti–ICAM-1 mAb to HPAECs as described.10 Briefly, cells were fixed with paraformaldehyde (2% PFA for 15 minutes at 4°C), and then incubated with 10 μg/mL of 125I-labeled ICAM-1 Ab for 1 hour at 4°C. The cells were washed thrice with EBM-2 containing 10% FBS and then lysed overnight with 1N NaOH, and the lysates were counted for radioactivity. The specific binding of ICAM-1 antibody was determined by preincubating the cells with unlabeled antibody.
Recombinant adenovirus containing cDNA of dominant-negative PKCζ (Ad-PKCζ K281R) was a kind gift from Dr Viswanathan Natarajan (Johns Hopkins University School of Medicine, Baltimore, Md). Confluent HPAEC monolayers were infected with Ad-DN-PKCζ at a concentration of 4×107 plaque-forming units/mL, and adenoviruses carrying LacZ gene (Ad-LacZ; Clontech) were used as controls as described.18 After incubation for 5 hours at 37°C, 5% CO2, and 95% humidity, the medium was replaced with fresh EBM-2–containing10% FBS for 48 hours before performing the experiments.
Phosphorylation of ICAM-1
Confluent HPAEC monolayers were labeled with 32P (75 μCi/mL overnight, or 150 μCi/mL for 4 hours) in phosphate-free medium at 37°C. After treatment, cells were washed 3 times with ice-cold PBS and lysed with 1 mL/plate RIPA buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.2), 1% deoxycholic acid, 1% Triton X-100, 0.25 mmol/L EDTA (pH 8.0), 5 mmol/L NaF supplemented with 100 μmol/L sodium orthovanadate, 1 mmol/L PMSF, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 1 μg/mL pepstatin]. Lysates were precleared with normal mouse IgG (0.25 μg/mL) and then immunoprecipitated with anti–ICAM-1 Ab (2 μg/mL, 4°C, overnight). Immunocomplexes were washed and resolved by SDS/PAGE. Gels were transferred to PVDF membrane, and the phosphorylated form of ICAM-1 was detected by autoradiography. The membrane was subsequently immunoblotted with an antibody against ICAM-1.19 The bands were quantified densitometrically using Scion Image, and the values were normalized to total protein in each lane.
The plasmid pGreen Lantern-1 containing green fluorescence protein (GFP) gene was purchased from GIBCO-BRL. The expression vector pcDNA3 containing tagged kinase-defective PKCζ isoform was a gift from Dr J.W. Soh (Columbia University, New York, NY).20 Endothelial transfections were performed with Superfect (Qiagen) as described.19
PKCζ Kinase Assay
PKCζ kinase activity was assayed as described.19 Briefly, cell lysates were immunoprecipitated with an antibody against PKCζ using protein A/G plus agarose (Santa Cruz). The immunocomplexes were washed twice with ice-cold PBS and once with kinase buffer (25 mmol/L Tris-HCl [pH 7.4], 5 mmol/L MgCl2, 0.5 mmol/L EGTA, 1 mmol/L DTT) and resuspended in 30 μL of kinase buffer containing 2.5 μg of histone-H1, 0.5 mmol/L cold ATP, and 20 to 30 μCi of [γ-32P]ATP. The reaction was incubated for 20 minutes at RT and terminated by addition of SDS-sample buffer. Proteins were analyzed by SDS-PAGE, and the phosphorylated form of histone-H1 was detected by autoradiography.
TNF-α Induces Rapid Endothelial Adhesivity in an ICAM-1–Dependent Manner
We determined the ability of TNF-α to induce early-onset, protein synthesis–independent endothelial adhesivity toward PMN. Time course experiments showed that endothelial adhesivity increased ≈10-fold within 1 minute, and the response sustained up to 30 minutes, and increased further (≈20-fold) at 4 hours after TNF-α challenge (Figure 1A). The rapidity of the response suggests that the modification of preexisting protein is involved in the mechanism of endothelial adhesivity. As ICAM-1 is constitutively expressed in endothelial cells, we addressed its role in mediating the rapid expression of endothelial adhesivity. Preincubation of HPAECs with anti–ICAM-1-mAb prevented TNF-α–induced endothelial adhesivity toward PMN (Figure 1B). Anti–CD18 mAb also prevented the TNF-α–induced endothelial adhesivity, but the isotype-matched control IgG had no effect. To demonstrate that the early-onset endothelial adhesivity is mediated by constitutive cell surface ICAM-1 and does not require de novo ICAM-1 expression, we determined the time course of ICAM-1 expression induced by TNF-α. Western blot analysis showed that ICAM-1 is constitutively expressed in endothelial cells and that its expression is induced only after 1 hour of TNF-α challenge (Figure 2A) consistent with the delayed secondary expression of endothelial adhesivity (Figure 1A). Moreover, the early adhesive response was insensitive to the protein synthesis inhibitor cycloheximide (CHX) (Figure 2B). These data show that TNF-α induces a rapid expression of endothelial adhesivity toward PMN and that this response is mediated by alteration in the constitutive cell surface ICAM-1.
TNF-α Induces Rapid Binding of Anti-ICAM mAb to Cell Surface ICAM-1
We addressed the possibility that the expression of rapid endothelial adhesivity is secondary to activation of the constitutive cell surface ICAM-1. Thus, we assessed the binding of anti–ICAM-1 mAb to the ICAM-1 following TNF-α exposure of endothelial cells. We used 125I-labeled anti–ICAM-1 mAb to determine the binding activity of cell surface ICAM-1. Results showed that TNF-α induced binding of anti–ICAM-1 mAb to the cell surface ICAM-1 within minutes, paralleling the expression of endothelial adhesivity (Figures 3A and 1⇑A). In a competition experiment, binding of 125I-labeled anti–ICAM-1 mAb to endothelial cells was effectively competed by the presence of excess unlabeled ICAM-1 antibody (Figure 3A), thus indicating the specificity of the response. In another experiment, we failed to detect any increase in the membrane expression of ICAM-1 after TNF-α challenge (Figure 3B). These data exclude the possibility that TNF-α–induced increase in ICAM-1 binding activity is the result of rapid translocation of a putative cytoplasmic pool of ICAM-1 to the membrane. We next determined if the increased ICAM-1 binding activity involves qualitative changes in the preexisting ICAM-1. Analysis by confocal microscopy showed that TNF-α induced a distinct punctate ICAM-1 staining suggestive of cell surface ICAM-1 clustering (Figure 3C).
TNF-α Induces Phosphorylation of the Constitutive ICAM-1
We determined the phosphorylation status of ICAM-1 after TNF-α challenge of HPAECs to ascertain if the ICAM-1 activation is attributable to its phosphorylation. We found that TNF-α induced ICAM-1 phosphorylation in a time-dependent manner; ie, the phosphorylated form of ICAM-1 was detected as early as 1 minute and was sustained up to 15 minutes after TNF-α challenge (Figure 4). This time course parallels the TNF-α–induced ICAM-1 clustering, ICAM-1 binding activity, and expression of ICAM-1–dependent endothelial adhesivity (Figure 4 versus Figures 1A, 3C, and 3⇑A).
Inhibition of PKCζ Prevents Expression of Early-Onset Endothelial Adhesivity
We addressed the possibility that PKCζ, an atypical PKC isoform abundantly expressed in endothelial cells,8 mediates the rapid induction of endothelial adhesivity through phosphorylation of ICAM-1. We used both general and isoform-specific inhibitors to evaluate the involvement of PKCζ. Pretreatment of HPAECs with chelerythrine, a pan-PKC inhibitor, prevented the early-onset endothelial adhesivity toward PMN induced by TNF-α (Figure 5A). As PKCζ signals TNF-α–induced oxidant generation and ICAM-1 gene transcription,8,15 we next addressed the possibility that PKCζ mediates the TNF-α–induced early-onset endothelial adhesivity. We used a myristoylated membrane-permeable peptide antagonist corresponding to the pseudosubstrate region of PKCζ that specifically inhibits this PKC isoform,15,21 to evaluate its role in the response. Results showed that inhibition of PKCζ prevented the expression of the rapid endothelial adhesivity in response to TNF-α challenge (Figure 5B). In control experiments, we found that pretreatment of cells with myristoylated peptide antagonist specific for PKCθ failed to inhibit this response (Figure 5B).
Inhibition of PKCζ Prevents TNF-α–Induced Binding Activity of Constitutive Cell Surface ICAM-1
We next determined the effects of inhibition of PKCζ on TNF-α–induced binding activity of the constitutive cell surface ICAM-1. We observed that inhibition of PKCζ by the specific peptide antagonist prevented the binding activity of the cell surface ICAM-1 induced by TNF-α, whereas inhibition of PKCθ had no effect (Figure 5C). These data are in accordance with the effects of inhibition of PKCζ on the TNF-α–induced expression of rapid endothelial adhesivity (Figure 5B).
Because the increased ICAM-1 binding activity is associated with the clustering of cell surface ICAM-1 (Figure 3), we determined the requirement of PKCζ in the mechanism of this response. In these experiments, we used a kinase-defective mutant of PKCζ (PKCζK281R) to inhibit the function of endogenous PKCζ. We observed that expression of PKCζK281R prevented TNF-α–induced ICAM-1 clustering (Figure 6C). We also showed that expression of constitutively active PKCζ mutant induced ICAM-1 clustering in the absence of TNF-α challenge (Figure 6D).
Inhibition of PKCζ Prevents TNF-α–Induced Phosphorylation of ICAM-1
The involvement of PKCζ in TNF-α–induced ICAM-1 clustering and the resultant endothelial adhesivity led us to investigate if PKCζ phosphorylates ICAM-1. The effect of inhibition of PKCζ on ICAM-1 phosphorylation was determined by infecting HPAECs with nonreplicating adenoviral vectors containing either kinase-inactive dominant-negative mutant of PKCζ (Ad-DN-PKCζ) or β-galactosidase (Ad-β-Gal). We found that expression of Ad-DN-PKCζ prevented TNF-α–induced phosphorylation of ICAM-1, whereas expression of Ad-β-Gal failed to inhibit the response (Figure 7).
We next determined if PKCζ directly associates with ICAM-1 or requires participation of an intermediate protein to phosphorylate ICAM-1. We first determined if TNF-α induces activation of PKCζ. Results showed that TNF-α induced PKCζ activation in a time-dependent manner and that this activation was associated with a significant translocation of PKCζ to the membrane (Figures 8A and 8B). We carried out coimmunoprecipitation studies to determine if PKCζ translocated to the membrane in order to associate with ICAM-1. Results showed increased amount of PKCζ in the ICAM-1 immunoprecipitates from TNF-α–challenged cells compared with control untreated cells (Figure 8C). The association of PKCζ with ICAM-1 was rapid (occurring within 1 minute) and was sustained up to 30 minutes of TNF-α challenge (Figure 8C), similar to the time course of ICAM-1 phosphorylation and increase in endothelial adhesivity (Figures 4 and 1⇑A). These data indicate that ICAM-1 is a direct substrate for phosphorylation by PKCζ in response to TNF-α challenge.
ICAM-1, an inducible endothelial counter-receptor for β2-integrins (CD11/CD18), mediates PMN-endothelial interactions, and thus it is essential for the mechanism of stable PMN adhesion and the consequent transendothelial PMN migration. Interaction of ICAM-1 with β2 integrins enables PMN to adhere firmly and stably to the vascular endothelium and to migrate across the endothelial barrier. As ICAM-1 is also constitutively expressed in endothelial cells and PMN are rapidly recruited as the first line of defense to site of infection,11,22 we reasoned that the constitutively expressed cell surface ICAM-1 may have an important role in mediating the rapid-onset of endothelial adhesivity toward PMN. In the present study, we provide evidence that the proinflammatory cytokine TNF-α induces early endothelial adhesivity toward PMN in an ICAM-1–dependent manner. Our data establish that this adhesive response does not involve de novo ICAM-1 protein synthesis, but it is the result of activation of the constitutive ICAM-1. This was demonstrated by the increased binding activity of the cell surface ICAM-1 to anti–ICAM-1 mAb. We further showed that PKCζ rapidly associates with ICAM-1 to phosphorylate ICAM-1, and that this event was essential for the early-onset TNF-α–induced endothelial adhesivity.
We analyzed the time course of TNF-α–induced endothelial adhesivity. Results showed that TNF-α activated endothelial adhesivity toward PMN within 1 minute after TNF-α challenge. Moreover, this response was insensitive to the protein synthesis inhibitor cycloheximide. The rapidity of the response and its insensitivity to cycloheximide excluded the involvement of a protein synthesis–dependent mechanism. The rapid time course also suggested a role of the preexisting ICAM-1 in mediating the endothelial adhesivity induced by TNF-α. To establish the involvement of constitutive cell surface ICAM-1 in the response, we determined the effect of the specific blocking antibody against ICAM-1 on PMN adhesion to endothelial cells. Preincubation of HPAECs with anti–ICAM-1 antibody prevented TNF-α–induced endothelial adhesivity toward PMN, whereas an isotype-matched control Ab failed to alter this early response. The involvement of ICAM-1 in the mechanism of early-onset endothelial adhesivity was further supported by the ability of the antibody against CD18, the counter-receptor for ICAM-1, to similarly inhibit PMN adhesion to endothelial cells. Because endothelial monolayers were extensively washed to remove residual TNF-α before the addition of freshly isolated PMN, the increased PMN adhesion cannot be ascribed to PMN activation by any residual TNF-α.
We addressed the possibility that there may be a cytoplasmic pool of ICAM-1 that rapidly translocates to the membrane in response to TNF-α, and thus can increase the cell surface ICAM-1, resulting in increased PMN adherence to the endothelial cells. However, we failed to detect any increase in membrane expression of ICAM-1 after TNF-α challenge. In related studies, we used 125I-labeled anti–ICAM-1 mAb to evaluate if TNF-α promotes endothelial adhesivity by increasing the binding activity of constitutive cell surface ICAM-1. We observed that TNF-α indeed induced anti–ICAM-1 mAb binding of cell surface ICAM-1 with a time course similar to the expression of endothelial adhesivity. These data clearly demonstrate that TNF-α–induced early-onset endothelial adhesivity is secondary to activation of the preexisting cell surface ICAM-1.
Hermanowski-Vosatka et al23 have shown that clustering of cell surface receptors allows them to bind their ligand more avidly than the same number of receptors in random array. These studies led us to assess the possibility that increased binding activity involves a qualitative change in the constitutive ICAM-1 expressed on the cell surface. Our data showed a pattern of surface clustering of ICAM-1 as evident by punctate staining of the ICAM-1 after TNF-α stimulation. These data are consistent with the findings showing the clustering of monocyte binding receptors including ICAM-1 as a mechanism for increased monocyte adhesion.24 Interestingly, both our study and that of Wojciak-Stothard et al24 showed clustering of ICAM-1, although in contrast to our study, Wojciak-Stothard et al focused on the role of de novo expression of ICAM-1. In support of ICAM-1 clustering being a critical determinant of endothelial adhesivity, Jun et al25 have recently shown that dimerized ICAM-1 has ≈1.5- to 3-fold greater affinity toward the I-domain of integrin αLβ2 (LFA-1) than monomeric ICAM-1. These studies also postulated that dimers and W-shaped tetramers form oligomers of ICAM-1, which could have important implications for regulating cell adhesion.25 These results collectively point to an important role of TNF-α in promoting ICAM-1 clustering, and the increased binding activity of cell surface ICAM-1 to PMN.
We addressed the mechanisms by which TNF-α promotes early-onset ICAM-1–dependent endothelial adhesivity. As phosphorylation is critical for activation of other adhesion proteins, eg, CD18,26,27 we investigated the possibility that cell surface activation of ICAM-1 could occur by a mechanism involving the phosphorylation of ICAM-1. Analysis of phosphorylation status showed that ICAM-1 was phosphorylated within 1 minute of TNF-α challenge, a time-course paralleling the increase in endothelial adhesivity. Clues to the kinase responsible for ICAM-1 phosphorylation came from the experiments in which we determined the effects of inhibition of PKC. These results showed that pretreatment of cells with chelerythrine, a pan-PKC inhibitor,15 prevented the induction of rapid endothelial adhesivity. As PKCζ, an abundant PKC isoform in endothelial cells, is implicated in the mechanism of TNF-α–induced oxidant signaling via NADPH oxidase activation as well as ICAM-1 gene transcription,8,15 we reasoned that PKCζ may phosphorylate the cell surface ICAM-1 to induce its activation and endothelial adhesivity. We used pharmacological and genetic approaches to address the role of PKCζ in the mechanism of TNF-α–induced endothelial adhesivity. Pretreatment of HPAECs with a myristoylated membrane-permeable peptide antagonist corresponding to the pseudosubstrate region of PKCζ, known to inhibit PKCζ activity,15,21 prevented the TNF-α–induced binding activity of the cell surface ICAM-1. Inhibition of PKCζ by this approach also prevented TNF-α–induced endothelial adhesivity. Moreover, we showed that expression of kinase-defective PKCζ mutant prevented TNF-α–induced phosphorylation and clustering of the cell surface ICAM-1. We observed that expression of the constitutively active mutant of PKCζ induced ICAM-1 clustering in the absence of TNF-α challenge. Thus, our results show an important role of PKCζ in mediating TNF-α–induced ICAM-1 clustering and the early-onset of endothelial adhesivity. These results, however, do not exclude the involvement of other PKC isoforms in the mechanism of endothelial adhesivity. Indeed, we have shown that PKCδ can signal ICAM-1 expression and thereby endothelial adhesivity in response to thrombin, which activates 7 transmembrane G protein–coupled receptors.19,28 Other studies have demonstrated the involvement of PKCα in the mechanism of ICAM-1 expression and monocyte adhesion in response to TNF-α and interferon-γ (IFN-γ) challenge of epithelial cells.29,30
This specific involvement of PKCζ in signaling ICAM-1 phosphorylation, ICAM-1 clustering, and ICAM-1–dependent rapid endothelial adhesivity led us to examine whether PKCζ can directly phosphorylate ICAM-1 or it activates an intermediate kinase that is responsible for phosphorylation. Coimmunoprecipitation studies showed that TNF-α induced the association of PKCζ with ICAM-1 and that the kinetics of this event was similar to the activation and membrane translocation of PKCζ. The present data show that ICAM-1 is a direct substrate of PKCζ; however, it is possible that PKCζ may also phosphorylate ICAM-1 through other kinases such as MAP kinases (ERK1/2 and p38), known to signal downstream of PKCζ.31,32
In summary, the present study implicates the constitutive cell surface ICAM-1 as a critical determinant of early-onset of endothelial adhesivity induced by TNF-α. Our data show that induction of binding activity of the cell surface ICAM-1 is secondary to its clustering, which in turn is the result of phosphorylation of ICAM-1 by PKCζ. Inhibition of PKCζ activity by pharmacological and genetic approaches prevented the TNF-α–induced ICAM-1 clustering and the early-onset of endothelial adhesivity. As PKCζ also regulates ICAM-1 gene expression in endothelial cells8 and ICAM-1 upregulation contributes to the amplification of PMN adhesion response,29 strategies aimed at preventing TNF-α–induced PKCζ activation may be useful in controlling inappropriate PMN sequestration and PMN-mediated vascular endothelial injury.
This work was supported by NIH grants HL46350, HL45638, and HL67424.
- Received November 19, 2002.
- Revision received April 10, 2003.
- Accepted April 10, 2003.
von Andrian UH, Chambers JD, McEvoy LM, Bargatze RF, Arfors KE, Butcher EC. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte β2-integrins in vivo. Proc Natl Acad Sci U S A. 1991; 88: 7538–7542.
Wertheimer SJ, Myers CL, Wallace RW, Parks TP. Intercellular adhesion molecule-1 gene expression in human endothelial cells: differential regulation by TNF-α and phorbol myristate acetate. J Biol Chem. 1992; 267: 12030–12035.
Ritchie AJ, Johnson DR, Ewenstein BM, Pober JS. Tumor necrosis factor induction of endothelial cell surface antigens is independent of protein kinase-C activation or inactivation: studies with phorbol myristate acetate and staurosporine. J Immunol. 1991; 146: 3056–3062.
Rahman A, Anwar KN, Malik AB. PKCζ mediates TNF-α–induced ICAM-1 gene transcription in endothelial cells. Am J Physiol Cell Physiol. 2000; 279: C906–914.
Rahman A, Bando M, Kefer J, Anwar KN, Malik AB. Protein kinase-C-activated oxidant generation in endothelial cells signals ICAM-1 gene transcription. Mol Pharmacol. 1999; 55: 575–583.
Sugama Y, Tiruppathi C, Janekidevi K, Andersen TT, Fenton JW II, Malik AB. Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion. J Cell Biol. 1992; 119: 935–944.
Frey RS, Rahman A, Kefer JC, Minshall RD, Malik AB. PKCζ regulates TNFα–induced activation of NADPH oxidase in endothelial cells. Circ Res. 2002; 90: 1012–1019.
Bhunia AK, Arai T, Bulkley G, Chatterjee S. Lactosylceramide mediates TNFα–induced ICAM-1 expression and the adhesion of neutrophil in human umbilical vein endothelial cells. J Biol Chem. 1998; 273: 34349–34357.
Suzama K, Takahara N, Suzuma I, Isshiki K, Ueki K, Leitges M, Aiello LP, King GL. Characterization of protein kinase-Cβ isoform’s action on retinoblastoma protein phosphorylation, vascular endothelial growth factor–induced endothelial proliferation, and retinal neovascularization. Proc Natl Acad Sci U S A. 2002; 99: 721–726.
Rahman A, Anwar KN, Uddin S, Xu N, Ye RD, Platanias LC, Malik AB. Protein kinase-Cδ regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol Cell Biol. 2001; 21: 5554–5565.
Soh JW, Lee EH, Prywes R, Weinstein IB. Novel roles of specific isoforms of protein kinase-C in activation of the c-fos serum response element. Mol Cell Biol. 1999; 19: 1313–1324.
House C, Kemp BE. Protein kinase-C contains a pseudosubstrate prototype in its regulatory domain. Science. 1987; 238: 1726–1728.
Hermanowski-Vosatka A, Detmers PA, Gotze O, Silverstein SC, Wright SD. Clustering of ligand on the surface of a particle enhances adhesion to receptor-bearing cells. J Biol Chem. 1988; 263: 17822–17827.
Wojciak-Stothard B, Williams L, Ridley AJ. Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol. 1999; 145: 1293–1307.
Jun CD, Carman CV, Redick SD, Shimaoka M, Erickson HP, Springer TA. Ultrastructure and function of dimeric, soluble ICAM-1. J Biol Chem. 2001; 276: 29019–29027.
Buyon JP, Philips MR, Merrill JT, Slade SG, Leszczynska-Piziak J, Abramson SB. Differential phosphorylation of the β2-integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils. J Leukoc Biol. 1997; 61: 313–321.
Rahman A, True AL, Anwar KN, Ye RD, Voyno-Yasenetskaya TA, Malik AB. Gαq and Gβγ regulate PAR-1 signaling of thrombin-induced NF-κB activation and ICAM-1 transcription in endothelial cells. Circ Res. 2002; 91: 398–405.
Chang YJ, Holtzman MJ, Chen CC. Interferon-γ–induced epithelial ICAM-1 expression and monocyte adhesion: involvement of PKC-dependent c-Src tyrosine kinase activation pathway. J Biol Chem. 2002; 277: 7118–7126.
Mizukami Y, Kobayashi S, Uberall F, Hellbert K, Kobayashi N, Yoshida K. Nuclear mitogen-activated protein kinase activation by PKCζ during reoxygenation after ischemic hypoxia. J Biol Chem. 2000; 275: 19921–19927.