Activation of Nuclear Factor-κB in Cultured Endothelial... : Journal of Cardiovascular Pharmacology (original) (raw)
Nuclear factor κB (NFκB) is a transcription factor common to many cell types (1), including endothelial cells (2,3). Activation of NFκB results from the dissociation of p50/p65 subunits from the inhibitory IκB protein and translocation of dimer subunits to the nucleus. The composition of the NFκB binding in nuclear fractions may consist of various combinations of p50 and p65 homo- or heterodimers, depending on the cell type, species, and experimental conditions. In general, it is thought that the p50 moiety is necessary for nuclear translocation, whereas the p65 moiety contributes to transcription, although p50 homodimers can also produce transcription in vitro (4). NFκB activation plays a prominent role in the transient expression of several genes responding to physiological and pathologic stimuli (5). For example, there are NFκB binding sites in the promoter region for genes coding several immune and inflammatory molecules including interleukin-1, interleukin-6, interleukin-8, intercellular adhesion molecule 1, vascular cell adhesion molecule, E-selectin, monocyte chemoattractant protein 1, tissue factor, and inducible nitric oxide synthase.
The activation of NFκB within endothelial cells is a rather new area of interest, as it could play a key role in the pathologic characteristics associated with various forms of inflammatory and immune diseases. There may be other diseases in which NFκB activation may be important as well. For example, it is known that diabetes (6,7) and hyperglycemia (8) alone cause increased expression of various adhesive proteins. In contrast, to our knowledge, no one has yet demonstrated that hyperglycemic states increase the expression of the important transcription factor NFκB.
One of the most potent stimuli for NFκB activation is oxidative stress (1,9). It has been widely assumed that hyperglycemia present in diabetes produces oxidative stress and that oxidative stress leads to vascular dysfunction (10). Accordingly, in our study, we hypothesized that increases in extracellular glucose concentration would increase the expression of NFκB in endothelial cells and that this process was mediated via a protein kinase C (PKC)-dependent pathway.
MATERIALS AND METHODS
Bovine aortic endothelial cells (BAECs) were obtained from the N.I.A. Cell Culture Repository (Camden, NJ, U.S.A.). The cells were grown in T75 flasks containing minimal essential medium (MEM) supplemented with 1% glutamine, 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 μg/ml gentamicin, 8 μg/ml tylosin, and 0.25 μg/ml amphotericin in a humidified atmosphere containing 5% CO2 and 95% air. The medium was sterilized with a 0.2-μm filter and stored at 4°C until use.
BAECs for glucose incubations were used from passages 4 through 9. At confluence, the medium was removed and replaced with glutamine- and serum-free media containing glucose (5.5-35 m_M_) for periods of 1, 2, 4, or 24 h. In some BAECs, the glucose-induced NFκB activation was blocked by inhibition of nuclear translocation by using 30 μg/ml of the peptide SN-50 (Biomol, Plymouth Meeting, PA, U.S.A.) containing the nuclear-localization sequence of NFκB p50 linked to a membrane-permeable motif of the sequence for Kaposi fibroblast growth factor (11). In other studies, cells were incubated with 100 n_M_ staurosporine (a nonselective PKC inhibitor) or 50-200 n_M_ calphostin C (a potent and highly selective PKC inhibitor) for 30 min before incubation with 35 m_M_ glucose for 2 h to evaluate whether NFκB activation by elevated glucose is regulated by PKC.
BAECs were harvested by scraping and transferred along with medium to polypropylene tubes and pelleted for 10 min at 150 g. All subsequent steps were performed at 4°C. Pellets from cells were washed twice with ice-cold phosphate-buffered saline (PBS) followed by washing with buffer containing 10 m_M_ HEPES (pH 7.9), 1.5 m_M_ MgCl2, 10 m_M_ KCl, 0.5 m_M_ dithiothreitol, 0.5 m_M_ phenylmethylsulfonyl fluoride (PMSF), and 10 ng/ml leupeptin. Cells were suspended in the same buffer containing 0.1% NP-40, lysed by vortexing, and incubated on ice for 10 min. Nuclear fractions were pelleted for 10 min at 12,000 g and nuclear proteins extracted in a buffer containing 20 m_M_ HEPES (pH 7.9), 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 m_M_ MgCl2, 0.2 m_M_ ethylenediaminetetraacetic acid (EDTA), 0.5 m_M_ dithiothreitol, 0.5 m_M_ PMSF, and 10 ng/ml leupeptin, set on ice 10 min, and then centrifuged for 10 min at 12,000 g. Supernatants were mixed with 1.5 vol of buffer containing 20 m_M_ HEPES (pH 7.9), 20% glycerol, 0.2 m_M_ EDTA, 50 m_M_ KCl, 0.5 m_M_ dithiothreitol, 0.5 m_M_ PMSF, and 10 ng/ml leupeptin, incubated on ice for 10 min, and then centrifuged for 5 min at 12,000 g. Nuclear extracts were stored at −80°C for later analysis. Protein content of the nuclear fractions was determined by using the bicinchoninic acid assay (12).
Electrophoretic mobility shift assay (EMSA)
Double-stranded NFκB oligonucleotide (5′-AGTTGAGGG GACTTTCCCAGGC-3′) was end-labeled by using T4 polynucleotide kinase according to the manufacturer's specifications (Promega, Madison, WI, U.S.A.). DNA-binding reactions were performed at room temperature for 20 min in 50 m_M_ TRIS (pH 7.5), 50 m_M_ NaCl, 1 m_M_ EDTA, 1 m_M_ dithiothreitol, 2 μg poly d[I-C], 10,000-20,000 cpm 32P end-labeled oligonucleotides, 0.05% NP-40, and 10 μg nuclear protein. Samples were subjected to EMSA on native 4% polyacrylamide gels at 180 V. Gels were transferred to Whatman paper, dried, and placed in film holders with XAR-5 film at −80°C. Densitometry was performed by using DeskScan 1.5.2 and NIH Image 1.52 software.
To verify specific NFκB activity, competitive EMSA was used in which 50- or 100-fold excess of unlabeled oligonucleotide was used in the binding reaction and allowed to react for 45 min before the addition of the 32P end-labeled oligonucleotide. In addition, immunosupershift EMSA was performed by incubating 2-4 μg of NFκB p50 or NFκB p65 (C-20 type) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) in the binding reaction buffer for 1 h before the addition of the 32P end-labeled oligonucleotide.
RESULTS AND DISCUSSION
Preliminary studies showed two regions containing NFκB-binding activity in nuclear fractions of BAECs (Fig. 1). By using cold oligonucleotide-competitive EMSA, we observed a complete elimination of the lower band and a 50% reduction in the upper band. The presence of two NFκB-binding activities with the lower specific for p50 and the upper specific for p65 by using human umbilical vein endothelial cells was previously reported (13); however, use of these antibodies for bovine cells was not previously published. Preincubation with p50 antibody caused a shift and partial reduction of the lower band. The very weak immunoshift for the p65 (C-20) antibody probably reflects the weak affinity for this antibody in bovine cells. Indeed, similar weak binding was demonstrated by using this antibody in rabbit coronary microvascular endothelial cells (14). Because the competitive EMSA with cold oligonucleotide revealed a mixture of nonspecific and specific NFκB activity for BAECs (i.e., the upper band) but complete elimination of the lower band, qualitative and quantitative analysis of specific NFκB activity was limited to the lower band.
Competitive electrophoretic mobility shift assay (EMSA) using cold oligonucleotide or immunosupershift of nuclear factor κB (NFκB) activity in nuclear fractions of bovine aortic endothelial cells (BAECs) by using antibodies to p50 and p65 subunits. Similar results were shown in three independent experiments.
Incubation with 25 or 35 m_M_ glucose (but not 15 m_M_ glucose) for 2 h increased NFκB activity compared with pair-matched cells incubated with 5.5 m_M_ glucose (Fig. 2). Furthermore, the increase in NFκB activity was a relatively early event, occurring within 1 h after addition of 35 m_M_ glucose (Fig. 3). Maximal increase in NFκB activity appeared at 2 and 4 h, and the increased activity persisted at 24 h relative to corresponding time-matched control cells incubated with 5.5 m_M_ glucose. Quantitatively, the increase in NFκB activity after exposure to 35 m_M_ glucose for 2 h was significantly increased (mean ± SEM, 336 ± 69%; n = 10 paired studies; p < 0.01) compared with that seen in pair-matched control cells containing 5.5 m_M_ glucose). Thus these studies suggest that relatively short periods of exposure of endothelial cells to increased glucose concentration may potentially stimulate a cascade of important intracellular events initiated by activation of NFκB.
Glucose concentration-dependent increase in nuclear factor κB (NFκB) activity in nuclear fractions of bovine aortic endothelial cells (BAECs) after 2-h exposure to various glucose concentrations. Similar results were shown in three independent studies.
Time-dependent changes in the expression of nuclear factor κB (NFκB) activity in nuclear fractions of bovine aortic endothelial cells (BAECs) exposed to 5.5 or 35 m_M_ glucose concentration. Example shown is typical of two experiments.
Our studies also verified that nuclear translocation of NFκB p50 subunits are necessary for increased nuclear binding of NFκB elicited by increased glucose levels, because the commercially available SN-50 peptide effectively blocked the increase in NFκB activity in the lower band (Fig. 4). This is consistent with the immunosupershift data using the p50 antibody. Exposure for 2 h with 35 m_M_ glucose (lane 2) produced a marked increase in NFκB activity compared with that of 5.5 m_M_ glucose (lane 1). The SN-50 peptide (lane 3) significantly blocked (mean ± SEM, 83 ± 17% of control cells; n = 3; p < 0.01) the glucose-induced activation of NFκB activity. This effect is consistent with previous reports in which the increase in nuclear binding of NFκB activity of a murine endothelial cell line induced by lipopolysaccharide treatment was prevented by SN-50 peptide (11).
Effect of 2-h exposure with 35 versus 5.5 m_M_ glucose on nuclear factor κB (NFκB) expression in nuclear fractions of bovine aortic endothelial cells (BAECs) and its prevention by preincubation with 30 μg/ml SN-50 peptide containing the nuclear-localization sequence of NFκB p50 linked to the membrane-permeable motif of the sequence for Kaposi fibroblast growth factor. This effect was confirmed in three independent experiments.
NFκB activation in various cell types may potentially occur via PKC-dependent (1) and PKC-independent pathways (15,16). Furthermore, the highly specific PKC inhibitor, calphostin C, has been shown to inhibit H2O2-induced increase in NFκB in porcine aortic endothelial cells (17). Calphostin C also has been shown to prevent glucose-induced endothelial dysfunction in rat cerebral arterioles (18).
To evaluate whether the increase in NFκB elicited by increased glucose levels occurs via a PKC-dependent pathway, we performed additional experiments by using staurosporine and calphostin C. Our studies indicate that the activation of NFκB by increased glucose concentration is likely mediated by PKC because it was blocked by the nonselective PKC inhibitor staurosporine (not shown) and blocked in a concentration-dependent manner by the highly selective PKC inhibitor calphostin C (Fig. 5). This observation is consistent with studies showing that increased glucose concentration and diabetes increase PKC in vascular smooth-muscle and endothelial cells (19).
Increase in nuclear factor κB (NFκB) activity in nuclear fractions of bovine aortic endothelial cells (BAECs) exposed to increased glucose (35 M) concentration for 2 h and its concentration-dependent reversal in endothelial cells incubated with increased glucose levels in the presence of calphostin C. Vehicle is the dimethylsulfoxide (DMSO) equivalent used only at the highest calphostin C concentration. Similar findings were observed in three independent experiments.
Recently cytokines (2,20,21), shear stress (22), and atherosclerosis (23) have been implicated as factors increasing NFκB expression in endothelial cells under in vitro or in vivo conditions. In our study, we showed that increases in glucose concentration caused rapid activation of NFκB in BAECs. To our knowledge, this is the first report in any cell type wherein short-term elevation in glucose concentration is yet another stimulus inducing early cellular changes in this important gene-transcription factor.
It was also recently demonstrated that culturing of endothelial cells with diabetic erythrocytes promotes NFκB activation through increased expression of advanced glycosylation end products on the surface of diabetic erythrocytes (24). Our study suggests that increases in glucose concentration per se also can activate NFκB. Taken together with our new studies, these observations suggest the possibility that diabetes may trigger the expression of NFκB.
Our studies indicate that rather acute increases in glucose concentration can have a marked effect on the expression of this important transcription factor, which regulates a number of important genes contributing to vascular complications associated with diabetes mellitus. In addition, our studies may have important implications regarding the observations in the Diabetes Control and Complications Trial (25), showing increased vascular complications in diabetic patients in which tight glycemic control is not effective.
Acknowledgment: This work was supported, in part, by grant HL47072 from the National Institutes of Health, Heart and Lung Institutes.
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Keywords:
Hyperglycemia; Transcription factor; Nuclear factor κB; Endothelium; Diabetes mellitus; Protein kinase C
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