The Nuclear Factor κ-B Signaling Pathway Participates in Dysregulation of Vascular Smooth Muscle Cells in Vitroand in Human Atherosclerosis*

In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor κ-B (NFκB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFκB binding activities. SMC express two inhibitor proteins IκBβ and IκBα. Interleukin-1β stimulation caused transient loss of IκBα and a sustained decrease of IκBβ that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFκB activity, but addition of serum or platelet-derived growth factor did activate NFκB. In situ analyses showed no evidence for basal NFκB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFκB complexes in human SMC; (ii) stimulatory components in serum activate NFκB and likely account for previously reported “constitutive” NFκB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFκB activation in SMC because of sustained decreases in the inhibitory subunit IκB-β.

In the lesions of atherosclerosis, vascular smooth muscle cells (SMC) display many functions characteristic of cytokine activation that likely contribute importantly to ongoing inflammation during human atherogenesis. The transcription factor nuclear factor -B (NFB) often mediates the effects of cytokines on target cells, but the identity of Rel family members important in human SMC activation remains uncertain. In vitro, human SMC express multiple Rel family members. Of these, dimers of p65 and p50, but not a putative SMC-Rel, comprise basal and inducible NFB binding activities. SMC express two inhibitor proteins IB␤ and IB␣. Interleukin-1␤ stimulation caused transient loss of IB␣ and a sustained decrease of IB␤ that correlated with increased and persistent levels of p65/p50 protein and binding activity in the nucleus. SMC cultured under serum-free conditions displayed little NFB activity, but addition of serum or platelet-derived growth factor did activate NFB. In situ analyses showed no evidence for basal NFB activity in SMC in vivo as nonatherosclerotic arteries did not contain nuclear p65 or p50 protein. However, the nuclei of intimal SMC within human atheroma did contain both Rel proteins. We conclude that (i) dimers of p65 and p50, but not SMC-Rel, comprise NFB complexes in human SMC; (ii) stimulatory components in serum activate NFB and likely account for previously reported "constitutive" NFB activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFB activation in SMC because of sustained decreases in the inhibitory subunit IB-␤.
Vascular smooth muscle cells (SMC) 1 at sites of atherosclerotic lesions express features of an inflammatory process, such as increased expression of genes encoding growth factors, inducible surface proteins, and molecules involved in extracellular matrix remodeling (1)(2)(3). The nuclear factor -B (NFB) family of transcription factors has emerged as a regulator of many of these molecules by vascular cells. Inflammatory cytokines, oxidized lipids, and oxidative stress, factors or events present in human atheroma, can activate NFB in vitro and also elicit specific functions in SMC (1, 4 -6). Several genes up-regulated in SMC during atherogenesis, including vascular cell adhesion molecule-1 (VCAM-1), interleukin-1 (IL-1), tumor necrosis factor-␣ (TNF-␣), and c-myc also contain functional B elements in their promoter/enhancer regions (5,7). Moreover, SMC, macrophages, and endothelial cells within human atheroma exhibit nuclear localization of the NFB subunit p65 (Rel A) in situ (8). Thus, in vitro and in situ studies underscore the potential importance of the NFB pathway in dysfunction of vascular cells during atherogenesis.
NFB exists in the cytosol of many cell types as an inactive complex of Rel-related factors, bound to a member of inhibitor proteins termed IB (reviewed in Refs. 5 and 7). Rel family members include p65 (Rel A), Rel (c-Rel), Rel B, and the Drosophila homolog dorsal, each of which contain transactivation domains necessary for gene induction. Other members, p50 (NFB1) and p49 (NFB2), are synthesized, respectively, as p105 and p100 precursors, and transactivate only weakly, but can form functional dimers with members of the first group. NFB is sequestered in the cytosol in an inactive heteromeric complex by associating with one of several inhibitors denoted IB, most commonly IB␣ or IB␤, or with Rel precursor proteins. Activation of NFB follows phosphorylation of IB or p105 on serine residues, possibly by a ubiquitin-regulated Ser/ Thr kinase (9). Phosphorylated IB or p105 is enzymatically degraded or processed to p50, respectively, by the multicatalytic proteasome complex (10), and liberated NFB dimers then translocate to the nucleus and promote transactivation of target genes. One of these target genes encodes the inhibitor IB␣ that binds to and thus limits further NFB activity and gene expression (11,12). IB␤, another IB member, is not resynthesized following its degradation, and may mediate prolonged NFB activity in lymphocytes exposed to lipopolysaccharide or IL-1 (13). The diversity of dimeric complexes formed by Rel factors requires definition of the NFB system in the context of each cell type examined.
In endothelial cells, inducible expression of leukocyte adhesion molecules requires participation of a well characterized NFB system (14,15). Bovine SMC were recently found to exhibit basal, constitutive NFB activity in vitro (16). A novel, putative Rel protein termed "SMC-Rel" is thought to comprise constitutive NFB activity in SMC and may permit cell division in serum-containing medium (16,17). However, the repertoire of Rel proteins or their inhibitors expressed in SMC, the identity of NFB members involved in DNA binding, or whether constitutive NFB activity exists in human SMC in vivo has not been fully delineated. In view of the potential importance and unresolved issues regarding the role of NFB in SMC, we have addressed the identity of Rel family members and the issue of their constitutive expression in vitro and in vivo in human SMC cultures and in normal and diseased arterial specimens. As the inhibitory limb of control of NFB activity appears critical, we also tested the hypothesis that IB␣ and IB␤ may play distinct roles in the control of cytokine activation of this cell type central to the pathogenesis of atherosclerosis.
Cell Culture-Smooth muscle cells obtained from explanted sections of human saphenous veins were grown in Dulbecco's modified Eagle's Medium (Life Technologies, Inc., Grand Island, NY) supplemented with 20 mM Hepes, 10% fetal calf serum (Hyclone), 5 mM L-glutamine, plus 50 units/ml penicillin and 50 g/ml streptomycin in a humidified atmosphere of 5% CO 2 , 95% air. Cells were passaged by brief trypsinization and were used through passage 5. The cells were characterized by phase contrast microscopy and were routinely screened for mycoplasma contamination by polymerase chain reaction using a commercially available kit (ICN Biomedicals Inc., Aurora, OH). Human aortic SMC and saphenous vein endothelial cells were isolated enzymatically and cultured as described previously (18).
Western Immunoblot Analysis-Whole cell lysates were prepared as described elsewhere (19) in 2 ϫ SDS lysis buffer (1 ϫ ϭ 125 mM Tris⅐HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol). Equivalent amounts of whole cell lysates (30 -40 g of protein) or nuclear or cytosolic fractions (prepared as described below) were resolved on 10 or 12% SDS-PAGE gels, followed by electrophoretic transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated in PBS-T (phosphate-buffered saline, 0.1% Tween-20), containing 5% non-fat dry milk for 1 h at 37°C, and then incubated for 1 h with primary antibodies used at 0.4 g/ml, with the exception of IB antisera, which was used at 0.5 g/ml. Membranes were washed with PBS-T and incubated with horseradish peroxidaseconjugated donkey anti-rabbit IgG as a secondary antibody (Jackson Laboratories, West Grove, PA), diluted 1:15,000 in PBS-T, 5% dry milk. Immunocomplexes were visualized using Renaissance chemiluminescence reagents (DuPont NEN) and exposure to x-ray film.
Preparation of Nuclear and Cytoplasmic Extracts and Electrophoretic Mobility Shift Assay-Saphenous vein SMC were grown to confluence (ϳ5 ϫ 10 5 cells) in 10-cm Petri dishes in serum-containing medium. In some experiments, cells were preincubated 48 h in serum-free IT medium (Dulbecco' modified Eagle's/Ham's F-12, 1:1, insulin 5 g/ml, transferrin 5 g/ml) to reduce exposure to serum components before addition of stimuli (20). Cells were stimulated with human recombinant cytokines IL-1␤, TNF-␣, or PDGF-BB (Endogen, Cambridge, MA) for the indicated times, and were collected into chilled Microfuge tubes. Nuclear and cytosolic extracts were prepared according to Dignam et al. (21), with the additional step of washing nuclear pellets in low salt buffer prior to high salt extraction of nuclear proteins to remove any residual cytosolic contamination. Aliquots were assayed for protein concentration, dithiothreitol was added to a final 1 mM concentration, and extracts were stored at Ϫ80°C until analysis. Oligonucleotides corresponding to the B site of the murine immunoglobulin -light chain enhancer (AGTTGAGGGGACTTTCCCAGGC), mutant NFB (AGTTGAGGcGACTTTCCCAGGC; substitution in lowercase), and GAS/ISRE (AAGTACTTTCAGTTTCATATTACTCTA) (Santa Cruz Biotechnology) were radiolabeled with [␥-32 P]ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and purified by gel filtration through a 1.0-ml column of Bio-Gel P-6 polyacrylamide beads (Bio-Rad). Nuclear extracts (5-10 g) were incubated in a total volume of 20 l containing 32 P-oligonucleotide (20,000 cpm), 2 g of poly(dI⅐dC) (Boehringer Mannheim), 10 g of bovine serum albumin, 10 mM Tris⅐HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol. Reactions were incubated at room temperature for 20 min, then at 4°C for 10 min. In some experiments, increasing amounts of cold competitor oligonucleotides were added 5 min prior to addition of labeled probe. For supershift analysis, 2 g of the indicated antibodies were incubated with nuclear extracts for 15 min prior to addition of labeled probe. Binding complexes were resolved on 5% nondenaturing polyacrylamide gels via electrophoresis in 0.5 ϫ Tris/borate/EDTA buffer. Gels were dried and exposed to film for 16 -24 h.
Transient Transfection-As a measure of NFB activity, luciferase reporter constructs containing the thymidine kinase promoter (pTK) alone or downstream of three tandem NFB binding sites (pB-TK; AGCTTGGGACTTTCCATGGGACTTTCCTAGGGATTCCCC) were used. SMC were seeded at 2 ϫ 10 4 cells/cm 2 in 6-well plates and allowed to attach overnight. Cells were incubated 5 h in serum-free Opti-MEM medium (Life Technologies, Inc.) containing 1 g of reporter plasmid and 5 l of LipofectAMINE (Life Technologies, Inc.). Co-transfection with pRSV-␤-Gal (0.2 g) was used in all experiments to correct for variations in transfection efficiency. ␤-Gal activity was invariant with experimental treatments. After overnight recovery in Dulbecco's modified Eagle's medium, 10% serum, cells were incubated 48 h in serumfree IT medium or maintained in serum-containing medium. Cells were then stimulated with 10% serum, PDGF-BB, IL-1␤, or vehicle (IT medium) and incubated for an additional 24 h. Experiments were terminated by two washes with ice-cold PBS and addition of 200 l/well lysis buffer (100 mM KPO 4 , pH 7.8, 0.2% Triton X-100, 1 mM dithiothreitol). Luciferase and galactosidase activities were measured in 20-l aliquots over a period of 5 s using the Tropix detection system (Bedford, MA). Relative luciferase activity was calculated by dividing luciferase by ␤-Gal activity. For neutralization experiments, preparations of 10% FCS, 30 ng/ml PDGF-BB, or IT medium were incubated with or without 200 g of PDGF-BB neutralizing antibody for 2 h at 37°C prior to addition to cell cultures.
Indirect Immunofluorescence-Sections of human aortae and atherosclerotic carotid artery were obtained from transplantation donors and at endarterectomy, respectively. Paraffin-embedded sections were deparaffinized with xylene and rehydrated with graded steps of ethanol, then incubated with 2.5% normal goat serum in PBS. p65c and p50 polyclonal antisera or nonimmune rabbit IgG (1.5 g/ml) diluted in 2.5% goat serum in PBS were added to sections for 2 h at room temperature. After three washes with PBS, sections were layered with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 45 min. Complexes were detected with streptavidin-conjugated Texas Red or fluorescein isothiocyanate (Amersham Corp.). Some sections were counterstained for cell nuclei with 0.5 mg/ml bis-benzimide (H-33258) (Calbiochem) in PBS. Antibody specificity for immunostaining was assessed by immunocytofluorescent staining of TNF-␣-stimulated human saphenous vein endothelial cells with p65c and p50 antisera, with or without prior absorption with a 100-fold excess corresponding cognate peptides. Fluorescence was observed using an Olympus BX60F microscope (Olympus Optical Co., Ltd, Japan).

Cultured Human SMC Express Predominately NFB p65
and p50 Species-Because of the unsettled nature of the identification of Rel subunits utilized by SMC we determined the spectrum of Rel family members expressed by saphenous vein SMC by immunoblot analysis of whole cell lysates. SMC contained prominent levels of p65 (Rel-A) compared with c-Rel or Rel B, detected as weaker bands of similar size on SDS-PAGE gels (Fig. 1). c-Rel and Rel B in SMC comigrated with c-Rel and Rel B in Jurkat cell lysates and were abolished by preincubation of antisera with the corresponding cognate peptides (data not shown). Antisera to p50 (NFB1) recognized a ϳ50-kDa protein, whereas antisera to p49 (NFB2) recognized a single 100-kDa protein, suggesting that cultured SMC expressed p49 mostly as the unprocessed precursor p100. Human aortic SMC exhibited a similar profile of Rel protein expression, indicating similar expression of Rel proteins by SMC cultured from differing human vascular beds.
Human SMC Cultured in Serum-containing Medium Exhibit Two Complexes of Constitutive and Inducible NFB Binding Activity-Previous studies reported that cultured bovine SMC exhibit constitutive NFB binding activity composed of p50 complexed with a putative Rel protein termed SMC-Rel (16). To identify the Rel proteins in human SMC that participate in binding DNA under basal and cytokinestimulated conditions, nuclear extracts were prepared from saphenous vein or aortic SMC following 2 h of incubation with or without IL-1␤, a potent stimulus of NFB activation. As the NFB proteins involved in DNA binding have been identified in human endothelial cells (14), for comparison nuclear extracts were prepared from saphenous vein endothelial cells treated with or without TNF-␣. Both venous and aortic SMC expressed two bands of specific DNA-binding complexes under unstimulated conditions, referred to as complexes I and II (Fig. 2a). Stimulation with IL-1␤ for 2 h markedly increased binding of complex I and slightly increased binding of complex II. Inclusion of polymyxin B (50 g/ml) in the experiments did not inhibit binding activity, arguing against activation of NFB by lipopolysaccharide contaminants (data not shown). In comparison to SMC, unstimulated endothelial cells showed little to no complex I and low levels of complex II, and binding activity was readily induced by stimulation with TNF-␣. Of note, complexes I and II in SMC and EC comigrated in nondenaturing gels, suggesting a similar subunit composition. Unlabeled wild type NFB probe inhibited binding activity to a much greater extent than mutant NFB or GAS/ISRE probe, indicating the specificity of the DNA-binding complexes for NFB motifs (Fig. 2b). A third, faster migrating complex (NS) likely represents a low affinity protein interaction with the NFB probe used in these experiments as indicated by 1) lack of regulation by cytokine stimulation, 2) less competition or a smearing of the complex by cold competitor oligonucleotide, and 3) no interaction with recombinant IB␣ added to the binding reaction (data not shown).
The subunit composition of basal and cytokine-induced DNA-binding complexes in human SMC was ascertained by supershifting with a panel of antisera to Rel family members (Figs. 3, a and b). Complex I in both unstimulated and IL-1␤stimulated cells interacted with two different antisera to the carboxyl or amino terminus of p65 (p65c and p65a, respectively), and with antisera to p50. Complex II interacted only with antisera to p50. Moreover, addition of both p65 and p50 antisera abolished all binding activity in nuclear extracts from unstimulated SMC. Antisera to p49, c-Rel, Rel B, or nonimmune rabbit IgG did not affect either complexes I or II. Thus, in cultured human SMC, complex I likely contains heterodimers of p65 and p50, and complex II likely contains homodimers of p50. Human endothelial cells stimulated with TNF-␣ have identical subunit composition (14) (data not shown).
Loss of IB␣ and Decreased IB␤ Correlates with NFB Binding Activity and Nuclear Localization of Rel Proteins p65 and p50 -NFB signaling in cell types such as lymphocytes and macrophages is self-limited due to postinduction synthesis of the NFB inhibitor IB␣ (7); however, such an autoregulatory system has not been identified in SMC. To characterize further NFB signaling in human SMC, the fate of inhibitor proteins IB␣ and IB␤ was followed over time in nuclear and cytoplasmic fractions from SMC stimulated with IL-1␤ or TNF-␣. IB␣ occurs as a ϳ37-kDa protein in the cytosol of unstimulated SMC (Fig. 4). IB␣ rapidly (Յ0.5 h), yet transiently disappeared following stimulation with IL-1␤ or TNF-␣, and returned to detectable levels at 2 h. The nuclear fraction contained no IB␣ up to 24 h after cytokine stimulation, indicating that the disappearance of IB␣ from the cytosol resulted from proteolysis rather than translocation to the nucleus. Inhibition of IL-1␤or TNF-␣-induced depletion of IB␣ by MG132, a potent inhibitor of proteasome activity, confirmed this interpretation (data not shown) (22). IB␤ occurs as a ϳ46-kDa protein in unstimulated SMC. Two hours following treatment with IL-1␤, levels of IB␤ markedly decreased, but remained detectable. In contrast to IB␣, levels of IB␤ remained low throughout the period of treatment with IL-1␤. By comparison, TNF-␣ affected levels of IB␤ little for up to 24 h (Fig. 4). Loss of cytosolic IB␣ correlated temporally with induction of NFB binding activity in nuclear extracts from SMC cultured in parallel. Interestingly, the duration of increased NFB activity correlated inversely with levels of IB␤ protein.
Loss of cytoplasmic IB␤ in response to IL-1␤ associated with NFB-DNA binding activity that persisted for 24 h. TNF-␣ stimulation, which affected IB␤ levels little, induced transient NFB-DNA binding activity that peaked by 1 h and declined to near basal levels by 6 h (Fig. 4).
Consistent with the loss of IB inhibitor proteins and increased NFB binding activity, nuclear fractions contained increased amounts of p65 and p50 protein following IL-1␤ stimulation, determined by immunoblotting (Fig. 5). Increased levels of nuclear p65/p50 persisted for 24 h compared to unstimulated SMC, consistent with sustained NFB binding activity observed during this time (Fig. 4). Nuclear extracts from unstimulated SMC contained low amounts of both p65 and p50, consistent with basal levels of DNA binding activity (Figs. 2 and 3). Levels of cytosolic p65 and p50 did not change appreciably during the experiments, suggesting that a small fraction of the total pool of these NFB dimers translocated to the nucleus in response to cytokine stimulation. In striking contrast to the changes in p65 and p50, c-Rel remained cytosolic, and no nuclear accumulation of c-Rel was observed in response to IL-1␤, indicating selectivity in mobilization of Rel dimers in human SMC.

Serum Constituents Regulate NFB Activity in Human Smooth Muscle Cells-
The above experiments examined NFB family members in smooth muscle cells cultured in serumcontaining medium. Since serum may contain or induce production of factors that can activate NFB, experiments were performed in SMC cultured in a defined serum-free medium (IT medium). Removal of serum for 48 h strikingly decreased both basal NFB-DNA binding as well as luciferase activity from a heterologous promoter construct containing three tandem repeat NFB-binding elements (Fig. 6, a and b). Reintroduction of serum (10%) restored NFB binding and luciferase activity to "basal" levels, which persisted for at least 24 h. Plateletderived growth factor (PDGF), a serum-associated mitogen also expressed in human atheroma, alters many functions of SMC relevant in atherogenesis. PDGF-BB increased NFB-DNA binding and luciferase activity in IT-cultured SMC, but did not further increase basal levels of NFB-DNA binding in SMC maintained in serum (Figs. 6, a and b). A neutralizing PDGF antibody abolished the stimulatory effect of PDGF-BB on Bdependent luciferase activity but failed to block the stimulatory effect of 10% serum (Fig. 6c), indicating that factors other than PDGF contribute to constitutive NFB activity present in SMC cultured in serum. Human SMC responded similarly to IL-1␤ either in serum-free or serum-containing medium. Thus, "constitutive" NFB activity in cultured human SMC likely results from the presence of serum constituents or, perhaps, the proliferative status of the cell population.
Expression of p65 and p50 in Normal and Atherosclerotic Human Vessels-In the normal vessel wall, SMC do not encounter mitogens or certain other constituents of serum, an unphysiologic fluid. We therefore tested whether SMC in the normal artery wall exhibit activation of NFB. To this end, we examined sections of aortae obtained from transplantation donors for the localization of p65 and p50 by indirect immunofluorescence. Staining with rabbit polyclonal antisera to p65 or p50 (red fluorescence) and counterstaining with bis-benzimide to visualize nuclei (blue fluorescence) yielded diffuse red cytoplasmic staining around blue nuclei of medial SMC (Fig. 7a). Staining with nonimmune rabbit IgG yielded no signal. A filter that allowed single transmission of red fluorescence revealed dark, nonfluorescent nuclei surrounded by red fluorescence, indicating no nuclear staining with either p65 or p50 antisera. In contrast, sections of human atheroma obtained at carotid endarterectomy showed cells with clear nuclear red fluorescence when stained with anti-p50 and anti-p65 antibodies. Co-staining with ␣-actin antibodies (HHF-35) identified these cells as smooth muscle (green-yellow color). Specificity of p65c and p50 antisera was assessed in TNF-␣-stimulated human saphenous vein endothelial cells, as the identity of Rel subunits mobilized by TNF-␣ in this cell type is established (14). Immunofluorescent staining with p65c and p50 antisera yielded diffuse cytoplasmic but little nuclear fluorescence. Stimulation with TNF-␣ for 1 h yielded intense nuclear fluorescence for p65 and increased nuclear fluorescence for p50, indicative of nuclear translocation of these Rel subunits. Preabsorption of antisera with a 100-fold excess of corresponding cognate peptide abolished fluorescence of TNF-␣-stimulated endothelial cells (Fig. 7b). Thus, in medial SMC of the normal vessel wall, NFB proteins p65 and p50 are restricted to the cytosol, in contrast to SMC cultured in serum or in atherosclerotic lesions.

DISCUSSION
This study investigated the role of the NFB signaling system in the regulation of functions of human SMC of importance in human atherosclerosis. Recent studies suggest that SMC in culture express basal, constitutive NFB activity (16,23). Prior to this, only lymphocyte cell lines and neurons were known to exhibit constitutive NFB activity (24 -26). Curiously, the Rel proteins that comprise this basal activity differ in each cell type. In lymphocytes and neurons, the basal complexes contain Rel-B heterodimers and p65/p50 dimers, respectively. In SMC, the basal complexes are thought to contain p50 and a putative Rel protein termed SMC-Rel (16). The results reported here suggest rather that the basal NFB complexes in cultured human SMC contain p65/p50 heterodimers and p50/p50 homodimers, based on immunoblot and gel shift analyses. This discrepancy could reflect species differences between the bovine SMC used in the former study and the human SMC used herein. Indeed, bovine aortic SMC exhibit considerable levels of basal NFB activity compared with human SMC, indicating that at least regulation of the NFB system differs between these two cell types. 2 Alternatively, SMC-Rel-containing complexes may not bind to the immunoglobulin enhancer motif used in the current experiments. This is unlikely, however, since basal NFB activity in bovine SMC activated transcription from a reporter construct containing multimerized elements of the light chain enhancer (16). Moreover, experiments in human SMC using the tandem B motif from the VCAM-1 promoter also identified basal complexes as containing p65 and p50 (23). Thus, the data suggest that, unlike bovine SMC, basal NFB complexes in human SMC cultured in serum contain "classical" NFB, i.e. p65 and p50.
Since the aforementioned studies examined SMC in culture, it is possible that culture conditions provoked a low level of basal NFB activity, an activity lacking or not present in SMC of the vascular wall. Previous reports indicate that basal NFB activity in cultured bovine SMC does not depend on serum growth factors (16). In human SMC, removal of serum considerably decreased "constitutive" NFB-DNA binding as well as transcriptional activity from a reporter plasmid containing tandem B-binding elements. NFB activity was restored within hours upon reintroduction of either serum or PDGF, a growth factor considered important in atherogenesis. It is likely that serum constituents other that PDGF-BB chain contribute to the stimulatory effect of serum since a neutralizing PDGF-BB antibody failed to reduce serum-induced increases of NFB activity, despite abolishing the stimulatory effect of PDGF. This finding suggests that constitutive NFB activity in vitro results from exposure to serum, an unphysiologic medium, rather than being an intrinsic feature of SMC. Moreover, SMC exposed to growth factors such as PDGF may result in low level, persistent activation of NFB that may contribute to sustained activation of this cell type at sites of vascular lesions (33). It should be noted that some residual NFB activity remained even in SMC cultured without serum. This residual activity likely resulted from autocrine production of growth factors or IL-6, the latter of which is released from cultured SMC and can increase NFB activity (17,27). Nevertheless, these experiments cannot exclude this residual activity as basal NFB activity in SMC in serum-free culture. A more definitive answer was provided by in situ analyses of human aortae from transplantation donors for the presence of Rel proteins p65 or p50. Nuclei within medial SMC showed no immunoreactivity with Rel antibodies, arguing against the presence of basal NFB activity in SMC of the normal vessel wall. Together, the data support the view that NFB is restricted to the cytosol in quiescent SMC, as occurs in most cell types examined, and that nuclear translocation of Rel proteins and increased NFB activity occurs in response to cytokine as well as mitogenic stimulation. 2 T. Bourcier, G. Sukhova, and P. Libby, unpublished data.
FIG. 5. Subcellular location of Rel family members in human SMC stimulated with IL-1␤. Nuclear and cytosolic extracts were isolated at the indicated times from human SMC incubated with medium alone or with IL-1␤ (10 ng/ml). Extracts (40 g) were separated by 12% SDS-PAGE gels and immunoblotted with antisera to p65, p50, or c-Rel, as described under "Materials and Methods." In cell types such as macrophages and lymphocytes, activation of the NFB system is self-limited due to increased synthesis of the NFB inhibitor protein IB␣. Such an autoregulatory mechanism ensures transient activation of NFB and circumvents a potential NFB-mediated positive feedback loop of target gene expression in cells exposed to inflammatory stimuli. In SMC stimulated with IL-1␤, increased levels of p65/p50 in the nucleus persisted for at least 24 h, despite the rapid reappearance of cytosolic IB␣ to prestimulation levels. SMC thus appear to have a limited ability to curb NFB activity induced by IL-1␤. Another member of the IB family may thus regulate NFB in SMC. Indeed, the current results show that human SMC express the recently cloned NFB inhibitor IB␤ (13) and that treatment with IL-1␤ induces a sustained reduction in levels of IB␤ protein. Unlike IB␣, cytosolic pools of IB␤ are not restored following activation and thus may result in persistent activation of NFB. Indeed, sustained decreases in IB␤ appear to cause persistent NFB activity in T cells stimulated with IL-1 or lipopolysaccharide and in human endothelial cells treated with TNF-␣ (13,28). Consistent with this view, TNF-␣ fails to modulate IB␤ levels substantially in human SMC during 24 h of incubation, whereas regulation of IB␣ levels resembles that observed in response to IL-1␤. In this case, increased NFB activity is transient; indeed NFB FIG. 6. Serum mitogens increase NFB binding and heterologous promoter activity in cultured human SMC. a, EMSA analyses of nuclear extracts from SMC incubated in serum-free IT medium for 48 h, followed by stimulation with control (IT), 10% serum (ITϩFCS), 30 ng/ml PDGF-BB (ITϩPDGF), or 10 ng/ml IL-1␤ (ITϩIL1␤) for the indicated times. Unlabeled consensus (but not mutant) oligonucleotides competed with labeled NFB oligonucleotide. SMC maintained in serum throughout the period of treatment (FCS) were also stimulated with 30 ng/ml PDGF-BB (FCSϩPDGF). Similar results were obtained in three additional experiments. b, luciferase activity from human SMC transfected with a reporter construct containing the thymidine kinase promoter alone (pTK; white bars) or in combination with three tandem B binding elements (pB-TK; shaded bars) linked to the luciferase gene. Following transfection, cells were incubated in serum-free IT medium or maintained in serum for 48 h. IT-preincubated cells were stimulated with control (IT), 10% serum (FCS), or 30 ng/ml PDGF-BB and incubated for an additional 24 h. Luciferase activity was standardized to ␤-galactosidase activity and reported as relative luciferase activity. Results are the means Ϯ S.E. from two independent experiments, each performed in triplicate. The asterisk (*) indicates statistical significance relative to cells maintained in serum (FCS), at p Յ 0.05, by Student's unpaired t test. c, luciferase activity from human SMC transfected with pB-TK reporter construct. SMC in IT medium were stimulated for 24 h with control (IT), 30 ng/ml PDGF-BB, or 10% serum (FCS) in the absence or presence of 200 g of neutralizing PDGF antibody, as described under "Materials and Methods." Results are means Ϯ S.E. from a single experiment performed in triplicate. A second experiment provided similar results. activity now declines as cytosolic levels of IB␣ increase. Thus, regulation of IB␤ and possibly the nature of the NFB response to cytokine stimulation (i.e. transient versus sustained activation) likely differs between human smooth muscle cells and endothelial cells. Potential functional consequences of divergent NFB regulation in SMC and endothelial cells are of particular interest based on the close proximity of these two cell types within the vessel wall.
An alternative explanation for persistent NFB activity is that p65/p50 may have a relatively long half-life in the nuclear compartment. That IB␣ did not appear in the nucleus throughout the period of treatment precludes disruption of Rel/DNA interactions (29,30). Modification of Rel subunits, such as phosphorylation following activation, may be important in this regard (31). Alternatively, turnover of IB␣ increases following cytokine stimulation (32), which may allow a continuous low level of nuclear translocation of p65/p50, accounts for prolonged NFB activity in this cell type. Although it remains uncertain whether NFB activity in SMC exposed to IL-1 results from continued translocation of cytosolic pools of NFB or prolonged residence of nuclear NFB proteins, such sustained NFB activity may promote long term expression of gene products and maintain SMC in an activated or "primed" condition. In this regard, intimal SMC in atheroma show chronic expression of VCAM-1 (3,33), an NFB dependent process, and the current results show that NFB is present in the nucleus of these cells.
Regulation of NFB activity has emerged as a potentially important pathway in mediating specific functions of vascular endothelium pertinent to atherogenesis, such as expression of some adhesion molecules and PDGF (34). In vascular smooth muscle, activation of NFB regulates expression of the adhesion molecule VCAM-1 and occurs during cell growth induced by serum or thrombin (17,23,35). The presence of the nuclear Rel protein p50 in intimal SMC within human atheroma shown here and of p65 shown here and elsewhere (8) support a role for this transcription factor in expression of SMC products of potential importance to progression of vascular lesions, including cytokines, growth factors, and proteins involved in coagulation. This study clarified the nature of the Rel proteins expressed in human SMC and characterized activation of this pathway in response to pertinent proinflammatory stimuli, IL-1␤ and TNF-␣. An understanding of the NFB system in SMC should allow a more rational basis for elucidating potential roles of NFB in mediating specific functions of SMC in vascular diseases. FIG. 7. Immunofluorescence staining of Rel family members p65 and p50 in normal and atherosclerotic human arteries in situ. a, panel A shows staining of normal human aortic media with p50 antisera (red fluorescence) and bis-benzimide to visualize nuclei (blue fluorescence). Panel B is the identical field as in panel A, but shows only p50 red fluorescence. Panel D shows staining of human aortic media from transplant donor with p65c antisera (red fluorescence) and bisbenzimide (blue fluorescence). Panel E shows the identical field with only p65 red fluorescence. Panels C and F depict immunostaining of intima of atherosclerotic human carotid artery with antisera to p50 or p65, respectively, and ␣-actin (HHF-35), seen as yellow-green cytoplasmic staining. Red fluorescence from nuclei reflects staining with p50 or p65 antisera only. Arrows depict cell nuclei. n ϭ 6 for normal human aortic sections, n ϭ 7 for atherosclerotic human carotid sections. Original magnifications, ϫ 1000 for panels A-F. b, immunocytochemical staining with p65c or p50 antisera of cultured human saphenous vein endothelial cells incubated with control (medium) or with 10 ng/ml TNF-␣ for 1 h. TNF-␣ ϩ peptide indicates preabsorption of p65c or p50 antisera with a 100-fold excess of corresponding cognate peptide.