Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells

Theodore J. Kalogeris1,2, F. Stephen Laroux2, Adam Cockrell2, Hiroshi Ichikawa2, Naotsuka Okayama2, Travis J. Phifer1, J. Steven Alexander2, and Matthew B. Grisham2

Departments of 1 Surgery and 2 Molecular and Cellular Physiology, Louisiana State University Medical Center, Shreveport, Louisiana 71130


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappa B (NF-kappa B) activation in tumor necrosis factor (TNF)-alpha -stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappa B activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappa B, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

inflammatory mediators; nuclear factor-kappa B; adhesion molecules; leukocyte adhesion; U-937 cell line; neutrophils; human umbilical vein endothelial cells; ECV cell line


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A FUNDAMENTAL AND EARLY event in inflammation is adhesion of leukocytes to the endothelium. This is mediated by binding of leukocytes to endothelial cell adhesion molecules (ECAMs). Inflammatory mediators such as tumor necrosis factor (TNF)-alpha stimulate transcription and subsequent cell surface expression of ECAMs such as vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin (20, 30). It has been demonstrated that expression of ECAMs in response to TNF is controlled by the activity of the transcription factor nuclear factor-kappa B (NF-kappa B) (21).

Under basal conditions, NF-kappa B is sequestered in an inactive form in the cytoplasm by an inhibitory binding protein, Ikappa Balpha . When stimulated by inflammatory stress, Ikappa Balpha undergoes postranslational modification (e.g., phosphorylation, polyubiquitination) that leads to its degradation and dissociation from NF-kappa B (4, 19). The released NF-kappa B is then translocated to the nucleus, where it activates transcription of genes having NF-kappa B binding sites in their promoters.

Proteolytic degradation of Ikappa Balpha is thought to be mediated by the 26S proteasome complex and is required for activation of NF-kappa B (19). Recently, it was shown that, in cells exposed to TNF-alpha , pretreatment with peptide aldehydes, which inhibit chymotryptic activities of the proteasome and possibly other proteases, prevents both activation of NF-kappa B (19, 21) and increased ECAM expression and neutrophil adhesion in endothelial cells (21). Thus it has been proposed that the latter processes are mediated by proteasome activity. However, direct evidence supporting this hypothesis has not yet been presented.

Although the peptide aldehydes are potent and relatively specific proteasome inhibitors (19, 22), their Michaelis-Menten constants for proteasome inhibition (5-12 nM) are similar to those for other proteases, such as cathepsin B and calpain (10 nM) (19). Other, nonproteasome inhibitors of cathepsins and calpain have no effect on either NF-kappa B precursor processing or NF-kappa B activation (19). However, it is possible that these other proteases (especially calpain) might be involved in events required for ECAM surface mobilization and leukocyte adhesion that are downstream from, or parallel to, NF-kappa B-dependent ECAM gene expression. For example, known calpain substrates include cytoskeletal proteins (actin-binding and microtubule-associated proteins), adhesion molecules, and the Fos and Jun components of transcription factor activating protein-1 (AP-1) (25). Thus, in interpreting the results of studies using the peptide aldehyde proteasome inhibitors, it is important to distinguish between the effects of these compounds on proteasome activity and their possible effects on other aspects of cell function that are likely also important in ECAM surface expression and leukocyte adhesion. It remains necessary to examine the specific role of the proteasome in mediating the effect of TNF on ECAM expression and leukocyte adhesion.

Another, recently described, class of proteasome inhibitors is boronic acid peptides (1). These inhibitors are thought to form a more stable bond at the active site of the proteasome than the peptide aldehydes and are more potent and selective for proteasome inhibition (1). Moreover, whereas peptide aldehydes cannot be used in vivo due to their acute toxicity, peptide boronates have been used in animals (6). Use of peptide boronates in studies of ECAM expression and leukocyte adhesion have not been reported.

The most specific proteasome inhibitor described to date is lactacystin, a Streptomyces metabolite that is structurally distinct (10) from the peptide aldehyde and peptide boronate proteasome inhibitors. Lactacystin is converted to a beta -lactone intermediate (the active inhibitor), which binds irreversibly to the subunit X of the proteasome and acylates the active site NH2-terminal threonine (1, 8). It has been demonstrated using radiolabeled lactacystin that this inhibitor binds only to the proteasome complex in cell extracts (8, 9). Finally, it was shown that lactacystin selectively inhibits proteasome activity, with no detectable effect on the activity of any other protease, including cathepsin B and calpain (9). To date, lactacystin remains the most specific proteasome inhibitor known (1, 9). Thus lactacystin should be a useful tool in determining, unambiguously, the role of the proteasome in mediating the effects of proinflammatory stimuli such as TNF on ECAM expression and leukocyte-endothelial cell adhesion.

The objective of this study was to compare the effects of two structurally unrelated yet selective proteasome inhibitors, lactacystin and the tripeptide boronate PS-341, on TNF-alpha -stimulated NF-kappa B activation, surface expression of ECAMs (VCAM-1, ICAM-1, and E-selectin), and endothelial adhesion of monocytes and neutrophils. We examined these parameters in endothelial cells derived from primary culture and in a transformed cell line (ECV) derived from human umbilical vein endothelial cells (HUVEC). Our results demonstrate the importance of the proteasome in mediating TNF-stimulated NF-kappa B activation, ECAM expression, and leukocyte adhesion.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents

Antibodies to VCAM-1 (1.G11B1), ICAM-1 (15.2), and E-selectin (1.2B6) were purchased from Southern Biotechnology Associates (Birmingham, AL). Antibody to Ikappa Balpha was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA), and alkaline phosphatase-coupled goat anti-rabbit IgG was purchased from Sigma Chemical (St. Louis, MO). The proteasome inhibitor PS-341 was a generous gift of Dr. Peter Elliot (Proscript, Cambridge, MA). Lactacystin was purchased from Calbiochem/Behring (Torrey Pines, CA).

Cell Culture

HUVEC were isolated from human umbilical cords using a modification of the procedures described by Jaffe et al. (13). These primary cultures were seeded in T-25 flasks in endothelial growth medium (EGM) plus bovine pituitary brain extract (Clonetics, San Diego, CA). For experiments, they were trypsinized and passaged to either 24-well plates (monocyte adhesion and ECAM surface expression measurements) or 100-mm dishes (analysis of NF-kappa B activation). All HUVEC were used at the first passage. Preliminary experiments indicated that cell responses (U-937 adhesion) were similar from 1 to 8 days after attaining confluence; data reported herein are from cells at 4 days postconfluence. Spontaneously transformed HUVEC (ECV cell line) (29) were obtained from the American Type Culture Collection (ATCC; Manassas, VA) at unknown passage and grown in EGM as described above for HUVEC. Promonocytic U-937 cells (28) were obtained at unknown passage from ATCC and maintained in suspension culture in RPMI 1640 (Sigma) with 10% fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA) and 2 mM L-glutamine (GIBCO-BRL, Gaithersburg, MD). They were passaged every 3 days after achieving a density of 4 × 105 cells/ml.

Isolation of Neutrophils

Human neutrophilic polymorphonuclear leukocytes were isolated from venous blood of healthy adults using standard dextran sedimentation and gradient separation on Histopaque 1077 (Sigma) (12, 31). This procedure yields a polymorphonuclear leukocyte population that is 95-98% viable (by trypan blue exclusion) and 98% pure (by acetic acid-crystal violet staining).

Monocyte and Neutrophil Adhesion Assay

U-937 cells and freshly isolated neutrophils were washed twice with labeling medium (RPMI 1640 plus 1% FBS) and then incubated for 1 h (37°C; 5% CO2) with 51CrO4 (sodium salt; DuPont NEN, Boston, MA; 3-5 µCi/5 × 107 cells; 2-ml incubation volume). Labeled leukocytes were washed four times with labeling medium and then resuspended in fresh labeling medium at 2 × 107 cells/ml. After control or experimental treatments, endothelial cell monolayers in 24-well plates were washed twice with labeling medium, and then 450 µl of labeling medium were added to each well. For static adhesion assays, 50 µl of labeled monocyte or neutrophil suspension (1 × 106 cells) were added to each well of endothelial cells (2:1 ratio of leukocytes to endothelial cells), and the plates were gently agitated and placed in a cell culture incubator for 30 min. At the end of the incubation period, the medium from each well was aspirated and saved for radioactive counting. The monolayer was gently washed three times with cold PBS; collected washes were combined with medium and counted, yielding a measure of nonadherent leukocytes. Preliminary experiments indicated that three washes of the monolayer decreased control or basal adhesion by 50-100% (compared with a single wash), while effecting a minimal decrease (<2%) in TNF-stimulated adhesion. After the final wash, monolayers were lysed for 1 h with 1 M NaOH; counting of the lysate [in counts/min (cpm)] yielded a measure of adherent leukocytes. Adhesion was expressed as the percent of total collected radioactivity present in the lysate: %adhesion = [cpm in lysate / (cpm in lysate + cpm in supernatant and washes)] × 100.

Effect of TNF on Leukocyte Adhesion to Endothelial Monolayers

Stock solutions of PS-341 and lactacystin were prepared in DMSO immediately before use. TNF (human recombinant; Calbiochem, La Jolla, CA) was dissolved in Hanks' buffered saline solution (HBSS) with 0.5% BSA and stored in small aliquots at -70°C. Freshly thawed aliquots were used for each experiment. Endothelial cells were seeded at ~1 × 105 cells/well in 24-well plates. At 4 days postconfluence, medium was aspirated and replaced with fresh EGM. Lactacystin (10 µM) and PS-341 (10 µM) or DMSO were added, and the cells were incubated for 1 h before addition of TNF (10 ng/ml) or HBSS plus BSA. Cells were exposed to TNF for 4 h, after which adhesion assays were carried out as described above. Before adhesion assays were conducted, endothelial monolayers were gently but thoroughly washed free of all treatment substances with labeling medium.

Effect of TNF on Endothelial Expression of ECAMs

Surface expression of VCAM-1, E-selectin, and ICAM-1 was assayed using the method of Khan et al. (15). Endothelial cells were grown in 24-well culture plates. After exposure to TNF with or without pretreatment with PS-341 or lactacystin, wells were gently washed once with PBS and then incubated with antibodies to VCAM-1, E-selectin, or ICAM-1 (1.G11B1, 1.2B6, or 15.2, respectively; Southern Biotechnology Associates) diluted 1:400 in PBS plus 5% FCS at 37°C for 30 min. Wells were washed twice with PBS and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) in PBS plus 5% FCS at 37°C for 30 min. Wells were washed four times with PBS and then incubated with 0.003% hydrogen peroxide plus 0.1 mg/ml 3,3',5,5'-tetramethylbenzidine (Sigma) for 30 min in the dark. The color reaction was stopped by addition of 75 µl of 8 N H2SO4, and the samples were transferred to 96-well plates. Plates were read on a microplate reader at 450 nm, blanking on wells stained with only secondary antibody. For a given experiment, each treatment was performed in triplicate.

Activation of NF-kappa B

Isolation of nuclear extracts. Methods used were a modification of Schreiber et al. (26). HUVEC and ECV were incubated for 4 h with either vehicle or TNF (10 ng/ml), after pretreatment for 1 h with or without PS-341 (0.01, 0.1, 1.0, or 10 µM) or lactacystin (0.01, 0.1, 1.0, 10, 20, or 50 µM). Incubation medium was aspirated, and the cells were scraped from the plate in PBS. Cells were pelleted (1,500 rpm, 5 min) at 4°C, the PBS was decanted, and the pellets were resuspended in 0.4 ml of buffer A [in mM: 10 HEPES (pH 7.9), 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol (DTT), and 0.5 phenylmethylsulfonyl fluoride (PMSF), with 1 µg/ml leupeptin and 1 µg/ml aprotinin] by gentle pipetting through a large-bore pipette tip. The cell suspension was allowed to swell on ice for 15 min, after which 25 µl of 10% NP-40 were added and the suspension was vortexed for 10 s. Homogenates were centrifuged at 10,000 g for 30 s at 25°C; the nuclear pellet was washed once with and resuspended in 50 µl of buffer C [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The resuspended nuclear fraction was sonicated on ice and then centrifuged at 12,000 g for 1 min at 4°C. The supernatant from this spin (nuclear extract) and the cytosolic extract were assayed for protein concentration using the Bio-Rad DC protein assay (Hercules, CA) and then immediately used for gel shift assay.

Electrophoretic mobility shift assay. NF-kappa B consensus oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC (Promega, Madison, WI) was end labeled with [32P]ATP using T4 polynucleotide kinase (Promega gel shift assay system) according to manufacturer's instructions. Nuclear extracts (20 µg protein) were preincubated for 10 min at 25°C in a solution containing (in mM) 1 MgCl2, 2.5 EDTA, 2.5 DTT, 250 NaCl, and 50 Tris · HCl (pH 7.5), with 0.25 µg/µl poly(dI-dC) in 20% glycerol. Labeled oligonucleotide (0.07 pmol) was then added (total assay volume 20 µl), and binding reactions were incubated for 30 min at 25°C. Reactions were stopped by addition of 4 µl of 10× gel loading buffer [250 mM Tris · HCl (pH 7.4), 0.2% bromphenol blue, 0.2% xylene cyanol, and 40% glycerol]. Specificity of binding was verified by including a 100-fold molar excess of unlabeled oligonucleotide in some reactions. Reaction mixtures were applied to a nondenaturing, 4% polyacrylamide gel and electrophoresed at 125 V (constant voltage) for 3 h. Gels were fixed in 10% acetic acid-40% methanol for 15 min, washed in distilled water, dried, and then exposed to X-ray film (Kodak X-OMAT) for 4-16 h at -70°C. Activation of NF-kappa B (relative to non-TNF-treated control) was determined by performing densitometric analysis (ImageQuant software, Molecular Dynamics, Sunnyvale, CA) on shifted bands from scanned autoradiographs.

Cytosolic Ikappa Balpha in TNF-Stimulated HUVEC

Western blotting was used to detect relative levels of the NF-kappa B cytosolic sequestrant Ikappa Balpha at various times of exposure of HUVEC to TNF, in the presence or absence of either PS-341 or lactacystin. HUVEC in P-100 dishes were pretreated with either lactacystin, PS-341, or vehicle for 1 h and then incubated with either TNF or TNF vehicle (0.5% BSA in HBSS) for 10 min or 4 h. Monolayers were then washed with ice-cold PBS, scraped from the dish into lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% SDS, 1% NP-40, 1 µg/ml aprotinin, and 100 µg/ml PMSF], and homogenized by sonication. Homogenates were centrifuged at 2,000 g for 15 min to remove cellular debris, and protein content was measured using a modified Lowry method (Bio-Rad DC protein assay); similar amounts of supernatant protein were then subjected to SDS-PAGE followed by Western blotting. Blots were probed with rabbit anti-human Ikappa Balpha antiserum followed by alkaline phosphatase-coupled goat anti-rabbit IgG; color development was obtained using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt chromogenic system.

Statistical Analysis

Data were analyzed by one-way ANOVA with multiple comparisons, using Bonferroni's method (18). Means were considered significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leukocyte Adhesion to Endothelial Cells

We examined the effects of preincubation of HUVEC monolayers with PS-341 or lactacystin on both basal and TNF-stimulated adhesion of U-937 cells and neutrophils (Fig. 1). Neither PS-341 nor lactacystin (incubated with HUVEC for 5 h) affected basal adhesion of monocytes and neutrophils to HUVEC. Exposure to TNF (10 ng/ml) for 4 h stimulated U-937 and neutrophil adhesion to HUVEC by ~10-fold and 3- to 10-fold, respectively. Preincubation of HUVEC with either PS-341 or lactacystin for 1 h before exposure to TNF significantly attenuated leukocyte adhesion. Lactacystin completely blocked TNF-stimulated neutrophil adhesion and decreased TNF-stimulated U-937 adhesion by 62% (Fig. 1). PS-341 (10 µM) decreased TNF-stimulated neutrophil and U-937 adhesion to HUVEC by 67 and 49%, respectively. A higher dose of PS-341 (50 µM) was also tested; this dose had no further effect on either monocyte or neutrophil adhesion to HUVEC compared with 10 µM (data not shown).


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Fig. 1.   Effect of proteasome inhibitors lactacystin and PS-341 on tumor necrosis factor (TNF)-stimulated leukocyte adhesion to human umbilical vein endothelial cells (HUVEC). Endothelial cells were pretreated for 1 h with vehicle alone (0.1% DMSO) or vehicle containing lactacystin or PS-341 and then exposed to TNF for 4 h. Endothelial cells were then washed free of TNF, 51Cr-labeled U-937 cells or polymorphonuclear neutrophils (PMN) were added, and leukocyte adhesion was assayed as described in MATERIALS AND METHODS. Values are means ± SD for 3 replicates in each of 4 separate experiments. * Significant effect of TNF compared with respective controls lacking TNF (P < 0.001); for a given leukocyte type, differing letters indicate significant attenuation of TNF effect by proteasome inhibitor (P < 0.001). cpm, Counts/min.

We were unable to demonstrate increased adhesion of either U-937 cells or neutrophils to ECV monolayers in response to TNF at doses up to 20 ng/ml (data not shown).

Effect of TNF on Endothelial Expression of ECAMs

We next examined the effects of the two proteasome inhibitors on basal and TNF-stimulated ECAM surface expression in HUVEC. TNF enhanced surface expression of VCAM-1, ICAM-1, and E-selectin (Fig. 2). Lactacystin had no effect on basal ECAM expression but completely blocked the increases in ECAM surface expression produced by TNF. Although PS-341 significantly attenuated surface expression of all ECAMs examined (VCAM-1, 69% decrease; ICAM-1, 48% decrease; E-selectin, 41% decrease), all three ECAMs remained significantly elevated compared with basal expression. Higher doses of PS-341 (50 or 100 µM) had no further inhibitory effect on TNF-stimulated ECAM expression (data not shown).


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Fig. 2.   Effect of proteasome inhibitors lactacystin and PS-341 on TNF-stimulated surface expression of endothelial cell adhesion molecules (ECAMs) in HUVEC. Endothelial cells were treated as described for Fig. 1, and then cell surface ELISA for vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and E-selectin was performed as described in MATERIALS AND METHODS. Values are means ± SD for 3 replicates in each of 3 separate experiments. * Significant effect of TNF compared with respective controls lacking TNF (P < 0.001); for a given adhesion molecule, differing letters indicate significant attenuation of TNF effect by proteasome inhibitor (P < 0.001). OD450, optical density at 450 nm.

We then examined the effects of TNF with or without lactacystin on ECAM expression in the ECV cells. Basal expression of VCAM-1 and E-selectin could not be demonstrated, and TNF did not significantly stimulate surface expression of these ECAMs after either 4 or 16 h of exposure to TNF (data not shown). Basal expression of ICAM-1 was comparable to or slightly higher than that in HUVEC (Fig. 3). Incubation with TNF for 4 h produced a significant, 45% increase in surface ICAM-1 expression (considerably lower than the 300-700% increase produced in HUVEC; Fig. 2). Preincubation with lactacystin (50 µM) attenuated the TNF-elicited increase in ICAM-1 surface expression by 60%. Similar increases in ICAM-1 expression were observed after 16-h incubation of ECV with TNF, but lactacystin did not inhibit this increase.


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Fig. 3.   Effect of lactacystin on surface ICAM-1 expression in ECV cells. Values are means ± SD for 3 replicates in each of 3 separate experiments. * Significant effect of TNF compared with respective controls lacking TNF (P < 0.001); differing letters indicate significant attenuation of TNF effect by lactacystin (P < 0.05).

Activation of NF-kappa B

Activation of NF-kappa B was examined by electrophoretic mobility shift assay, using labeled kappa B consensus oligonucleotides incubated with nuclear extracts from TNF-treated HUVEC and ECV. Both cell types showed activation in response to TNF (Figs. 4 and 5): a 13- to 19-fold stimulation in ECV and a 1.6- to 2.5-fold stimulation in HUVEC. Antibody supershift analysis showed that the major TNF-inducible shifted complexes in both cells contained both p50 and p65. Further evidence of specificity came from incubating nuclear extracts with a 100-fold excess of unlabeled kappa B oligonucleotide: this treatment completely prevented mobility shift of the labeled oligonucleotide (data not shown). Pretreatment of both cell types with PS-341 produced a dose-dependent attenuation in activation (data not shown), with the highest dose, 10 µM, producing 63 and 48% lower levels of activation in ECV and HUVEC, respectively (Fig. 4). Lactacystin at 10 µM produced 71 and 39% decreases in TNF-induced NF-kappa B activation in ECV and HUVEC, respectively. Thus both proteasome inhibitors were similarly effective at preventing activation of NF-kappa B in both HUVEC and ECV.


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Fig. 4.   Effect of proteasome inhibitors on nuclear factor-kappa B (NF-kappa B) activation induced by TNF in HUVEC. Endothelial cells were pretreated with either vehicle (same as Fig. 1) or vehicle containing lactacystin or PS-341 (10 µM) for 1 h and then incubated with TNF (10 ng/ml) for 1 h. Nuclear extracts were then obtained, and electrophoretic mobility shift assay was performed as described in MATERIALS AND METHODS. Left: representative autoradiographs. Right: densitometric analysis of shifted complexes. Units are multiples of control value, which was set to 1.


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Fig. 5.   Effect of proteasome inhibitors on NF-kappa B activation induced by TNF in ECV cells. Endothelial cells were treated and assayed for activation of NF-kappa B as in Fig. 4. Left: representative autoradiographs. Right: densitometric analysis of shifted complexes. Units are multiples of control value, which was set to 1.

Degradation of Cytosolic Ikappa Balpha in HUVEC

Using immunoblot methods, we examined the effect of pretreatment of HUVEC with PS-341 or lactacystin on TNF-induced changes in cytosolic levels of Ikappa Balpha (Fig. 6). Compared with control cells, treatment of HUVEC with TNF for 10 min resulted in a marked decrease in Ikappa Balpha levels. Ikappa Balpha levels after 4 h exposure to TNF remained well below those in control cells; however, these 4-h levels were elevated compared with the 10-min levels, consistent with previous reports showing de novo synthesis of Ikappa Balpha in response to cytokine stimulation of endothelial cells (21). The other major finding was the appearance of minor, higher molecular weight bands similar to those reported previously as phosphorylated forms of Ikappa Balpha . Pretreatment of TNF-treated cells with either PS-341 or lactacystin attenuated the TNF-induced decrease in Ikappa Balpha levels (especially in cells treated with TNF for 10 min), produced a relative accumulation of phosphorylated forms of Ikappa Balpha , and, finally, resulted in multiple lower molecular weight immunoreactive forms. The major one of these lower molecular weight forms migrated at ~22 kDa, and its relative levels were dependent on the time of exposure to TNF plus proteasome inhibitor and were not detectable in extracts from HUVEC treated with TNF alone. Thus appearance of these bands was strictly dependent on pretreatment of HUVEC with proteasome inhibitors.


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Fig. 6.   Effect of lactacystin and PS-341 on cytosolic levels of NFkappa B inhibitor (Ikappa Balpha ) in HUVEC, analyzed by Western blot. Cells were pretreated with proteasome inhibitor and then incubated with TNF (10 ng/ml) for 10 min or 4 h. Cell extracts (prepared as described in MATERIALS AND METHODS) were subjected to SDS-PAGE on 10% gels, followed by immunoblotting. Similar amounts of cell protein (60 µg) were loaded in each well. Ikappa Balpha -P, phosphorylated Ikappa Balpha .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The recent availability of specific proteasome inhibitors now allows unequivocal examination of the role of proteasome activity in mediating diverse cellular functions. In the present work, we examined the effect of two such inhibitors, the peptide boronate PS-341 and the structurally distinct bacterial metabolite lactacystin, on several measures of inflammatory activation of endothelial cells. Both compounds significantly attenuated TNF-stimulated leukocyte adhesion and ECAM surface expression in HUVEC. These effects were correlated with inhibition of NF-kappa B activation as measured by electrophoretic mobility shift assay. Our results unequivocally support the hypothesis that proteasome-dependent processes, including activation of NF-kappa B, are necessary for TNF-elicited increases in both ECAM expression and leukocyte adhesion in endothelial cells.

In the experiments examining leukocyte adhesion to HUVEC, our results clearly demonstrate that lactacystin is more effective at blocking TNF-elicited adhesion of both monocytes and neutrophils. The relatively incomplete blockade of both TNF-stimulated monocyte and neutrophil adhesion in HUVEC by PS-341 (49 and 67% inhibition, respectively) compared with lactacystin (62 and 100% inhibition, respectively) was directly correlated to similar differences in effect on TNF-stimulated ECAM surface expression between these two inhibitors: whereas lactacystin virtually completely prevented TNF-elicited upregulation of VCAM-1 (92% inhibition), ICAM-1 (84% inhibition), and E-selectin (97% inhibition), the inhibitory effect of PS-341 on TNF-elicited stimulation of these ECAMs was less marked (VCAM-1, 69% inhibition; ICAM-1, 48% inhibition; E-selectin, 41% inhibition). Because VCAM-1, ICAM-1, and E-selectin could play a role in mediating monocyte adhesion to endothelial cells and because ICAM-1 and E-selectin are probably involved in adhesion of neutrophils, it is reasonable to conclude that differences between lactacystin and PS-341 in their effects on ECAM surface expression probably account for the relative effects of the two inhibitors on leukocyte adhesion.

Although lactacystin completely prevented the upregulation of surface expression of ICAM-1, VCAM-1, and E-selectin and polymorphonuclear neutrophil adhesion in HUVEC, it did not completely block the increase in monocyte adhesion (62% inhibition). A higher dose of lactacystin (50 µM) had no further effect; these results suggest the existence of TNF-stimulated adhesive determinants, other than those assayed in the present study, that mediate a component of TNF-stimulated U-937 adhesion to endothelial cells and are not influenced by proteasome inhibition. Other molecular determinants for monocyte-endothelial adhesion have recently been described (3, 16); however, it is unknown whether cytokine-elicited stimulation of their surface expression depends on NF-kappa B activation or proteasome activity.

Immunoblot analysis of cytosolic Ikappa Balpha supports the role of the proteasome in activating NF-kappa B in HUVEC. TNF elicited a decrease in cytosolic levels of Ikappa Balpha ; this was blocked by preincubation with both lactacystin and PS-341; importantly, inhibition of degradation of Ikappa Balpha was associated with a relative accumulation of phosphorylated forms of this protein similar to those reported previously (4, 19, 21), indicating that neither lactacystin nor PS-341 affects these earlier steps in Ikappa Balpha processing. The other major finding was the appearance of smaller molecular weight immunoreactive bands in extracts from HUVEC that had been pretreated with proteasome inhibitor for 1 h and then treated with TNF. These bands were not detectable in control or TNF-treated HUVEC without proteasome inhibitor pretreatment. These observations suggest that prolonged inhibition of the proteasome has no effect on eventual degradation of excess accumulated Ikappa Balpha by nonproteasomal cellular proteases (11), which under conditions of normal proteasome function would not encounter a sufficient level of accumulated Ikappa Balpha substrate to produce detectable smaller molecular weight proteolytic products. Examples of proteasome-independent NF-kappa B activation have been reported previously (2, 24); our findings in HUVEC are consistent with such activity, and this latter finding could explain the apparent discrepancy between the effects of proteasome inhibitors on ECAM expression and apparent NF-kappa B activation (see below).

Our findings confirm those of previous workers (20) that only partial inhibition of NF-kappa B activation (as assessed by gel shift assay) produces a greater inhibition of ECAM expression. The reasons for this finding are not clear but have been previously considered (20). One possibility may involve other aspects of cytokine-stimulated activation of NF-kappa B besides nuclear translocation and DNA binding, such as phosphorylation of p65 (17). Alternatively, the apparent discrepancy between the level of NF-kappa B activation and the extent of ECAM expression may reflect amplification in the effects of relatively small changes in the levels of active, nuclear NF-kappa B on the transcription of NF-kappa B-dependent genes (i.e., such genes may require only a threshold, submaximal level of NF-kappa B activation to be transcriptionally active) (1, 20), especially since NF-kappa B works with other transcription factors to induce ECAM expression. Our Western blot data on cytosolic levels of Ikappa Balpha suggest another explanation, namely, that the ability of NF-kappa B to activate genes with kappa B promoters may depend on whether its presence in the nucleus is dependent on proteasome activity.

The differences in effects of PS-341 and lactacystin on ECAM surface expression in HUVEC did not precisely correlate with differences in the extent of attenuation of NF-kappa B activation by these two inhibitors; indeed, the 40% decrease in TNF-elicited NF-kappa B activation by lactacystin was associated with almost complete inhibition of ECAM expression, whereas PS-341 produced a slightly greater suppression of NF-kappa B activation yet attenuated TNF-stimulated increases in ECAM surface expression by only 40-70%. The reasons for these differences are not clear. All available evidence indicates that both PS-341 and lactacystin are specific in their proteasome-inhibitory activity (1). Thus one explanation for the above differences may be that TNF-stimulated increases in ECAM surface expression depend on additional, as yet uncharacterized, cellular functions of the proteasome besides activation of NF-kappa B, functions that may vary in their susceptibility to inhibition by lactacystin and PS-341. Our Western blot analysis of cytosolic levels of Ikappa Balpha in the presence or absence of proteasome inhibition is consistent with this hypothesis.

In the transformed, HUVEC-derived cell line ECV, basal surface expression of VCAM-1 and E-selectin was undetectable by our assay system, and TNF did not elicit an increase in surface expression of these proteins. Similarly, we were unable to detect a significant increase in either polymorphonuclear neutrophils or U-937 adhesion to ECV treated with TNF. In contrast to VCAM-1 and E-selectin, basal expression of ICAM-1 in ECV was similar to that found in HUVEC, and TNF produced a significant increase in ICAM-1 surface expression, which was partially blocked by lactacystin. It should be noted that the TNF-elicited upregulation of ICAM-1 in ECV was markedly blunted (58% increase above basal) compared with this response in HUVEC (300-700% increase above basal). In HUVEC, lactacystin completely blocked TNF-stimulated increases in ICAM-1 expression, but in ECV, in contrast, lactacystin only partially (60%) blocked the TNF-stimulated increase in ICAM-1 expression. The reason for this is not clear, but one possibility is that other pathways for activation of ICAM-1 expression [e.g., AP-1-dependent mechanisms (23)] may play a more important role in the control of ICAM expression during proteasome blockade in ECV than they do in HUVEC. The reason for lactacystin not blocking increases in ICAM-1 expression in response to 16 h of exposure to TNF is not certain. This observation might suggest proteasome-independent processes playing a role in long-term ICAM-1 expression, but a simpler and more likely explanation is the relatively short half life of the biologically active intermediate in vivo metabolite of lactacystin (the beta -lactone) (8).

Our findings in ECV are essentially in agreement with previous conclusions (27) that ECV are a poor model for examining the effects of proinflammatory stimuli on ECAM expression. Our results extend these conclusions by suggesting where the relative defect in ECV may lie. Recently, Cobb et al. (5) demonstrated marked induction of VCAM-1 and ICAM-1 promoter-luciferase reporter genes by cytokines in ECV cells transfected with such constructs. The results of Cobb et al. (5) combined with our findings that TNF stimulates NF-kappa B activation in ECV that is similar to that in HUVEC yet does not induce NF-kappa B-dependent ECAM expression make it seem likely that the defect in ECAM expression in ECV lies between NF-kappa B binding to promoter sites and activation of transcription of certain NF-kappa B-dependent ECAMs. Experiments addressing this issue are needed, because the precise molecular defect in ECV remains unknown.

The effects of lactacystin and PS-341 on ECAM surface expression in HUVEC indicate that proteasome activity is both necessary and sufficient to elicit increases in surface expression of ICAM-1, VCAM-1, and E-selectin in response to stimulation by TNF. These events were correlated with lactacystin-induced inhibition of NF-kappa B activation, but, in view of the difference in the extent of inhibition of ECAMs by lactacystin and PS-341, it seems unlikely that the mechanism for inhibition of TNF-stimulated ECAM expression by lactacystin is solely the prevention of NF-kappa B activation. Rather, our data suggest that the proteasome may play additional roles in mediating the effects of TNF on adhesion molecule expression. Thus whether the effects of lactacystin are entirely mediated by direct effects of proteasome inhibition on availability of NF-kappa B for binding to ECAM NF-kappa B promoter sites or whether inhibition of other proteasome-dependent processes such as nitric oxide production (6, 7, 14) might play a contributory role will require further investigation.


    ACKNOWLEDGEMENTS

We thank Laura L. Coe for isolating and culturing the HUVEC used in these studies.


    FOOTNOTES

This work was supported by funds from the Louisiana State University Medical Center Dept. of Surgery and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52148 (T. J. Kalogeris) and DK-43785 (to M. B. Grisham).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. B. Grisham, Dept. of Molecular and Cellular Physiology, 1501 Kings Hwy., Shreveport, LA 71130 (E-mail: mgrish{at}lsumc.edu).

Received 6 July 1998; accepted in final form 6 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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