The Nuclear Factor kappa -B Signaling Pathway Participates in Dysregulation of Vascular Smooth Muscle Cells in Vitro and in Human Atherosclerosis*

(Received for publication, November 21, 1996, and in revised form, March 7, 1997)

Todd Bourcier Dagger , Galina Sukhova and Peter Libby §

From the Vascular Medicine and Atherosclerosis Unit, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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 kappa -B (NFkappa B) 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 NFkappa B binding activities. SMC express two inhibitor proteins Ikappa Bbeta and Ikappa Balpha . Interleukin-1beta stimulation caused transient loss of Ikappa Balpha and a sustained decrease of Ikappa Bbeta 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 NFkappa B activity, but addition of serum or platelet-derived growth factor did activate NFkappa B. In situ analyses showed no evidence for basal NFkappa B 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 NFkappa B complexes in human SMC; (ii) stimulatory components in serum activate NFkappa B and likely account for previously reported "constitutive" NFkappa B activity in cultured SMC; and (iii) exposure to inflammatory cytokines may produce prolonged NFkappa B activation in SMC because of sustained decreases in the inhibitory subunit Ikappa B-beta .


INTRODUCTION

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 (1-3). The nuclear factor kappa -B (NFkappa B) 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 NFkappa B 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-alpha (TNF-alpha ), and c-myc also contain functional kappa B elements in their promoter/enhancer regions (5, 7). Moreover, SMC, macrophages, and endothelial cells within human atheroma exhibit nuclear localization of the NFkappa B subunit p65 (Rel A) in situ (8). Thus, in vitro and in situ studies underscore the potential importance of the NFkappa B pathway in dysfunction of vascular cells during atherogenesis.

NFkappa B exists in the cytosol of many cell types as an inactive complex of Rel-related factors, bound to a member of inhibitor proteins termed Ikappa B (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 (NFkappa B1) and p49 (NFkappa B2), are synthesized, respectively, as p105 and p100 precursors, and transactivate only weakly, but can form functional dimers with members of the first group. NFkappa B is sequestered in the cytosol in an inactive heteromeric complex by associating with one of several inhibitors denoted Ikappa B, most commonly Ikappa Balpha or Ikappa Bbeta , or with Rel precursor proteins. Activation of NFkappa B follows phosphorylation of Ikappa B or p105 on serine residues, possibly by a ubiquitin-regulated Ser/Thr kinase (9). Phosphorylated Ikappa B or p105 is enzymatically degraded or processed to p50, respectively, by the multicatalytic proteasome complex (10), and liberated NFkappa B dimers then translocate to the nucleus and promote transactivation of target genes. One of these target genes encodes the inhibitor Ikappa Balpha that binds to and thus limits further NFkappa B activity and gene expression (11, 12). Ikappa Bbeta , another Ikappa B member, is not resynthesized following its degradation, and may mediate prolonged NFkappa B activity in lymphocytes exposed to lipopolysaccharide or IL-1 (13). The diversity of dimeric complexes formed by Rel factors requires definition of the NFkappa B system in the context of each cell type examined.

In endothelial cells, inducible expression of leukocyte adhesion molecules requires participation of a well characterized NFkappa B system (14, 15). Bovine SMC were recently found to exhibit basal, constitutive NFkappa B activity in vitro (16). A novel, putative Rel protein termed "SMC-Rel" is thought to comprise constitutive NFkappa B 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 NFkappa B members involved in DNA binding, or whether constitutive NFkappa B 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 NFkappa B 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 NFkappa B activity appears critical, we also tested the hypothesis that Ikappa Balpha and Ikappa Bbeta may play distinct roles in the control of cytokine activation of this cell type central to the pathogenesis of atherosclerosis.


MATERIALS AND METHODS

Antibodies

Rabbit affinity-purified polyclonal antisera to NFkappa B proteins p65, p50 (NFkappa B1), p49/52 (NFkappa B2), cRel, Rel B, Ikappa Balpha /MAD-3, and Ikappa Bbeta were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibody HHF-35 (mouse IgG1) recognizing muscle-specific actin was purchased from Enzo Diagnostics (Sysosset, NY). Polyclonal rabbit neutralizing antibody to human PDGF-BB was purchased from Genzyme (Cambridge, MA). A nonimmune rabbit IgG fraction was from Dako (Carpenteria, CA).

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 µg/ml streptomycin in a humidified atmosphere of 5% CO2, 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 × SDS lysis buffer (1 × = 125 mM Tris·HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol). Equivalent amounts of whole cell lysates (30-40 µg 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 µg/ml, with the exception of Ikappa B antisera, which was used at 0.5 µg/ml. Membranes were washed with PBS-T and incubated with horseradish peroxidase-conjugated 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 (~5 × 105 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 µg/ml, transferrin 5 µg/ml) to reduce exposure to serum components before addition of stimuli (20). Cells were stimulated with human recombinant cytokines IL-1beta , TNF-alpha , 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 -80 °C until analysis. Oligonucleotides corresponding to the kappa B site of the murine immunoglobulin kappa -light chain enhancer (AGTTGAGGGGACTTTCCCAGGC), mutant NFkappa B (AGTTGAGGcGACTTTCCCAGGC; substitution in lowercase), and GAS/ISRE (AAGTACTTTCAGTTTCATATTACTCTA) (Santa Cruz Biotechnology) were radiolabeled with [gamma -32P]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 µg) were incubated in a total volume of 20 µl containing 32P-oligonucleotide (20,000 cpm), 2 µg of poly(dI·dC) (Boehringer Mannheim), 10 µg of bovine serum albumin, 10 mM Tris·HCl, 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 µg 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 × Tris/borate/EDTA buffer. Gels were dried and exposed to film for 16-24 h.

Transient Transfection

As a measure of NFkappa B activity, luciferase reporter constructs containing the thymidine kinase promoter (pTK) alone or downstream of three tandem NFkappa B binding sites (pkappa B-TK; AGCTTGGGACTTTCCATGGGACTTTCCTAGGGATTCCCC) were used. SMC were seeded at 2 × 104 cells/cm2 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 µg of reporter plasmid and 5 µl of LipofectAMINE (Life Technologies, Inc.). Co-transfection with pRSV-beta -Gal (0.2 µg) was used in all experiments to correct for variations in transfection efficiency. beta -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 serum-free IT medium or maintained in serum-containing medium. Cells were then stimulated with 10% serum, PDGF-BB, IL-1beta , 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 µl/well lysis buffer (100 mM KPO4, pH 7.8, 0.2% Triton X-100, 1 mM dithiothreitol). Luciferase and galactosidase activities were measured in 20-µl aliquots over a period of 5 s using the Tropix detection system (Bedford, MA). Relative luciferase activity was calculated by dividing luciferase by beta -Gal activity. For neutralization experiments, preparations of 10% FCS, 30 ng/ml PDGF-BB, or IT medium were incubated with or without 200 µg 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 µg/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-alpha -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).


RESULTS

Cultured Human SMC Express Predominately NFkappa B 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 (NFkappa B1) recognized a ~50-kDa protein, whereas antisera to p49 (NFkappa B2) 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.


Fig. 1. Repetoire of Rel family members expressed by unstimulated human smooth muscle cells cultured in serum-containing medium. Whole cell lysates (30 µg) of saphenous vein SMC were immunoblotted with antisera to p65, c-Rel, Rel B, p50/105, or p49/100, as described under "Materials and Methods." Molecular mass markers are indicated on the right. Cultured SMC from human aorta displayed a similar profile of Rel protein expression (not shown). Similar results were obtained in at least two additional experiments, using separate SMC isolates.
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Human SMC Cultured in Serum-containing Medium Exhibit Two Complexes of Constitutive and Inducible NFkappa B Binding Activity

Previous studies reported that cultured bovine SMC exhibit constitutive NFkappa B 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 cytokine-stimulated conditions, nuclear extracts were prepared from saphenous vein or aortic SMC following 2 h of incubation with or without IL-1beta , a potent stimulus of NFkappa B activation. As the NFkappa B 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-alpha . 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-1beta for 2 h markedly increased binding of complex I and slightly increased binding of complex II. Inclusion of polymyxin B (50 µg/ml) in the experiments did not inhibit binding activity, arguing against activation of NFkappa B 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-alpha . Of note, complexes I and II in SMC and EC comigrated in nondenaturing gels, suggesting a similar subunit composition. Unlabeled wild type NFkappa B probe inhibited binding activity to a much greater extent than mutant NFkappa B or GAS/ISRE probe, indicating the specificity of the DNA-binding complexes for NFkappa B motifs (Fig. 2b). A third, faster migrating complex (NS) likely represents a low affinity protein interaction with the NFkappa B 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 Ikappa Balpha added to the binding reaction (data not shown).


Fig. 2. Human SMC exhibit constitutive and IL-1beta -inducible NFkappa B-DNA binding activity. a, human saphenous vein (HSVSMC) or aortic SMC (HAoSMC) were stimulated for 2 h with IL-1beta (10 ng/ml), or human endothelial cells (HSVEC) with TNF-alpha (10 ng/ml). Nuclear extracts were prepared and subjected to EMSA in the presence or absence of excess unlabeled competitor oligonucleotide. Complexes I and II are indicated on the left; NS represents a nonspecific complex. b, competition of IL-1beta -induced NFkappa B binding activity with a 2-, 20-, or 200-fold excess unlabeled consensus or mutant NFkappa B oligonucleotide, or with consensus GAS/ISRE oligonucleotide, demonstrating specificity of IL-1beta -induced DNA binding complexes. Similar results were obtained in at least three independent experiments, using separate SMC isolates.
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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-1beta -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-alpha have identical subunit composition (14) (data not shown).


Fig. 3. Identification of NFkappa B family members comprising constitutive or IL-1beta -stimulated DNA binding complexes in SMC cultured in serum. Human SMC were incubated in a, medium alone, or b, medium + IL-1beta (10 ng/ml) for 2 h. Rel proteins were identified by performing EMSA reactions on nuclear extracts (10 µg) in the presence of 2 µg of the indicated Rel antisera or with nonimmune rabbit IgG. Complexes I, II, and supershifted complexes are indicated on the left. Probe indicates EMSA reaction without nuclear extract. Similar results were obtained in four independent experiments, each using separate SMC isolates.
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Loss of Ikappa Balpha and Decreased Ikappa Bbeta Correlates with NFkappa B Binding Activity and Nuclear Localization of Rel Proteins p65 and p50

NFkappa B signaling in cell types such as lymphocytes and macrophages is self-limited due to postinduction synthesis of the NFkappa B inhibitor Ikappa Balpha (7); however, such an autoregulatory system has not been identified in SMC. To characterize further NFkappa B signaling in human SMC, the fate of inhibitor proteins Ikappa Balpha and Ikappa Bbeta was followed over time in nuclear and cytoplasmic fractions from SMC stimulated with IL-1beta or TNF-alpha . Ikappa Balpha occurs as a ~37-kDa protein in the cytosol of unstimulated SMC (Fig. 4). Ikappa Balpha rapidly (<= 0.5 h), yet transiently disappeared following stimulation with IL-1beta or TNF-alpha , and returned to detectable levels at 2 h. The nuclear fraction contained no Ikappa Balpha up to 24 h after cytokine stimulation, indicating that the disappearance of Ikappa Balpha from the cytosol resulted from proteolysis rather than translocation to the nucleus. Inhibition of IL-1beta - or TNF-alpha -induced depletion of Ikappa Balpha by MG132, a potent inhibitor of proteasome activity, confirmed this interpretation (data not shown) (22). Ikappa Bbeta occurs as a ~46-kDa protein in unstimulated SMC. Two hours following treatment with IL-1beta , levels of Ikappa Bbeta markedly decreased, but remained detectable. In contrast to Ikappa Balpha , levels of Ikappa Bbeta remained low throughout the period of treatment with IL-1beta . By comparison, TNF-alpha affected levels of Ikappa Bbeta little for up to 24 h (Fig. 4). Loss of cytosolic Ikappa Balpha correlated temporally with induction of NFkappa B binding activity in nuclear extracts from SMC cultured in parallel. Interestingly, the duration of increased NFkappa B activity correlated inversely with levels of Ikappa Bbeta protein. Loss of cytoplasmic Ikappa Bbeta in response to IL-1beta associated with NFkappa B-DNA binding activity that persisted for 24 h. TNF-alpha stimulation, which affected Ikappa Bbeta levels little, induced transient NFkappa B-DNA binding activity that peaked by 1 h and declined to near basal levels by 6 h (Fig. 4).


Fig. 4. Time course of cytokine-induced loss of cytosolic Ikappa Balpha and Ikappa Bbeta correlates with increased nuclear NFkappa B binding activity. Cytosolic and nuclear extracts were isolated at the indicated times from human SMC treated with medium alone, IL-1beta (10 ng/ml), or TNF-alpha (10 ng/ml). Upper panels show levels of Ikappa Balpha or Ikappa Bbeta protein in cytosolic extracts, determined by immunoblot analysis. Lower panels show time course of NFkappa B binding activity by EMSA in nuclear extracts from IL-1beta - or TNF-alpha -stimulated SMC cultured in parallel.
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Consistent with the loss of Ikappa B inhibitor proteins and increased NFkappa B binding activity, nuclear fractions contained increased amounts of p65 and p50 protein following IL-1beta stimulation, determined by immunoblotting (Fig. 5). Increased levels of nuclear p65/p50 persisted for 24 h compared to unstimulated SMC, consistent with sustained NFkappa B 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 NFkappa B 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-1beta , indicating selectivity in mobilization of Rel dimers in human SMC.


Fig. 5. Subcellular location of Rel family members in human SMC stimulated with IL-1beta . Nuclear and cytosolic extracts were isolated at the indicated times from human SMC incubated with medium alone or with IL-1beta (10 ng/ml). Extracts (40 µg) were separated by 12% SDS-PAGE gels and immunoblotted with antisera to p65, p50, or c-Rel, as described under "Materials and Methods."
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Serum Constituents Regulate NFkappa B Activity in Human Smooth Muscle Cells

The above experiments examined NFkappa B family members in smooth muscle cells cultured in serum-containing medium. Since serum may contain or induce production of factors that can activate NFkappa B, experiments were performed in SMC cultured in a defined serum-free medium (IT medium). Removal of serum for 48 h strikingly decreased both basal NFkappa B-DNA binding as well as luciferase activity from a heterologous promoter construct containing three tandem repeat NFkappa B-binding elements (Fig. 6, a and b). Reintroduction of serum (10%) restored NFkappa B binding and luciferase activity to "basal" levels, which persisted for at least 24 h. Platelet-derived growth factor (PDGF), a serum-associated mitogen also expressed in human atheroma, alters many functions of SMC relevant in atherogenesis. PDGF-BB increased NFkappa B-DNA binding and luciferase activity in IT-cultured SMC, but did not further increase basal levels of NFkappa B-DNA binding in SMC maintained in serum (Figs. 6, a and b). A neutralizing PDGF antibody abolished the stimulatory effect of PDGF-BB on kappa B-dependent luciferase activity but failed to block the stimulatory effect of 10% serum (Fig. 6c), indicating that factors other than PDGF contribute to constitutive NFkappa B activity present in SMC cultured in serum. Human SMC responded similarly to IL-1beta either in serum-free or serum-containing medium. Thus, "constitutive" NFkappa B activity in cultured human SMC likely results from the presence of serum constituents or, perhaps, the proliferative status of the cell population.


Fig. 6. Serum mitogens increase NFkappa B 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 (IT+FCS), 30 ng/ml PDGF-BB (IT+PDGF), or 10 ng/ml IL-1beta (IT+IL1beta ) for the indicated times. Unlabeled consensus (but not mutant) oligonucleotides competed with labeled NFkappa B oligonucleotide. SMC maintained in serum throughout the period of treatment (FCS) were also stimulated with 30 ng/ml PDGF-BB (FCS+PDGF). 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 kappa B binding elements (pkappa B-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 beta -galactosidase activity and reported as relative luciferase activity. Results are the means ± S.E. from two independent experiments, each performed in triplicate. The asterisk (*) indicates statistical significance relative to cells maintained in serum (FCS), at p <=  0.05, by Student's unpaired t test. c, luciferase activity from human SMC transfected with pkappa B-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 µg of neutralizing PDGF antibody, as described under "Materials and Methods." Results are means ± S.E. from a single experiment performed in triplicate. A second experiment provided similar results.
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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 NFkappa B. 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 alpha -actin antibodies (HHF-35) identified these cells as smooth muscle (green-yellow color). Specificity of p65c and p50 antisera was assessed in TNF-alpha -stimulated human saphenous vein endothelial cells, as the identity of Rel subunits mobilized by TNF-alpha in this cell type is established (14). Immunofluorescent staining with p65c and p50 antisera yielded diffuse cytoplasmic but little nuclear fluorescence. Stimulation with TNF-alpha 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-alpha -stimulated endothelial cells (Fig. 7b). Thus, in medial SMC of the normal vessel wall, NFkappa B proteins p65 and p50 are restricted to the cytosol, in contrast to SMC cultured in serum or in atherosclerotic lesions.


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 bis-benzimide (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 alpha -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 = 6 for normal human aortic sections, n = 7 for atherosclerotic human carotid sections. Original magnifications, × 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-alpha for 1 h. TNF-alpha + peptide indicates preabsorption of p65c or p50 antisera with a 100- fold excess of corresponding cognate peptide.
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DISCUSSION

This study investigated the role of the NFkappa B 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 NFkappa B activity (16, 23). Prior to this, only lymphocyte cell lines and neurons were known to exhibit constitutive NFkappa B 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 NFkappa B 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 NFkappa B activity compared with human SMC, indicating that at least regulation of the NFkappa B system differs between these two cell types.2 Alternatively, SMC-Rel-containing complexes may not bind to the kappa  immunoglobulin enhancer motif used in the current experiments. This is unlikely, however, since basal NFkappa B activity in bovine SMC activated transcription from a reporter construct containing multimerized elements of the kappa  light chain enhancer (16). Moreover, experiments in human SMC using the tandem kappa 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 NFkappa B complexes in human SMC cultured in serum contain "classical" NFkappa B, 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 NFkappa B activity, an activity lacking or not present in SMC of the vascular wall. Previous reports indicate that basal NFkappa B activity in cultured bovine SMC does not depend on serum growth factors (16). In human SMC, removal of serum considerably decreased "constitutive" NFkappa B-DNA binding as well as transcriptional activity from a reporter plasmid containing tandem kappa B-binding elements. NFkappa B 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 NFkappa B activity, despite abolishing the stimulatory effect of PDGF. This finding suggests that constitutive NFkappa B 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 NFkappa B that may contribute to sustained activation of this cell type at sites of vascular lesions (33). It should be noted that some residual NFkappa B 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 NFkappa B activity (17, 27). Nevertheless, these experiments cannot exclude this residual activity as basal NFkappa B 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 NFkappa B activity in SMC of the normal vessel wall. Together, the data support the view that NFkappa B is restricted to the cytosol in quiescent SMC, as occurs in most cell types examined, and that nuclear translocation of Rel proteins and increased NFkappa B activity occurs in response to cytokine as well as mitogenic stimulation.

In cell types such as macrophages and lymphocytes, activation of the NFkappa B system is self-limited due to increased synthesis of the NFkappa B inhibitor protein Ikappa Balpha . Such an autoregulatory mechanism ensures transient activation of NFkappa B and circumvents a potential NFkappa B-mediated positive feedback loop of target gene expression in cells exposed to inflammatory stimuli. In SMC stimulated with IL-1beta , increased levels of p65/p50 in the nucleus persisted for at least 24 h, despite the rapid reappearance of cytosolic Ikappa Balpha to prestimulation levels. SMC thus appear to have a limited ability to curb NFkappa B activity induced by IL-1beta . Another member of the Ikappa B family may thus regulate NFkappa B in SMC. Indeed, the current results show that human SMC express the recently cloned NFkappa B inhibitor Ikappa Bbeta (13) and that treatment with IL-1beta induces a sustained reduction in levels of Ikappa Bbeta protein. Unlike Ikappa Balpha , cytosolic pools of Ikappa Bbeta are not restored following activation and thus may result in persistent activation of NFkappa B. Indeed, sustained decreases in Ikappa Bbeta appear to cause persistent NFkappa B activity in T cells stimulated with IL-1 or lipopolysaccharide and in human endothelial cells treated with TNF-alpha (13, 28). Consistent with this view, TNF-alpha fails to modulate Ikappa Bbeta levels substantially in human SMC during 24 h of incubation, whereas regulation of Ikappa Balpha levels resembles that observed in response to IL-1beta . In this case, increased NFkappa B activity is transient; indeed NFkappa B activity now declines as cytosolic levels of Ikappa Balpha increase. Thus, regulation of Ikappa Bbeta and possibly the nature of the NFkappa B 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 NFkappa B 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 NFkappa B activity is that p65/p50 may have a relatively long half-life in the nuclear compartment. That Ikappa Balpha 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 Ikappa Balpha increases following cytokine stimulation (32), which may allow a continuous low level of nuclear translocation of p65/p50, accounts for prolonged NFkappa B activity in this cell type. Although it remains uncertain whether NFkappa B activity in SMC exposed to IL-1 results from continued translocation of cytosolic pools of NFkappa B or prolonged residence of nuclear NFkappa B proteins, such sustained NFkappa B 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 NFkappa B dependent process, and the current results show that NFkappa B is present in the nucleus of these cells.

Regulation of NFkappa B 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 NFkappa B 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-1beta and TNF-alpha . An understanding of the NFkappa B system in SMC should allow a more rational basis for elucidating potential roles of NFkappa B in mediating specific functions of SMC in vascular diseases.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grant R37 HL34636-13 (to P. L).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. Section 1734 solely to indicate this fact.
Dagger    Recipient of the National Research Service Award HL09483-01.
§   To whom correspondence should be addressed: Vascular Medicine and Atherosclerosis Unit, Dept. of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-6628; Fax: 617-732-6961.
1   The abbreviations used are: SMC, smooth muscle cells; IL, interleukin; TNF-alpha , tumor necrosis factor-alpha ; PDGF, platelet-derived growth factor; EMSA, electromobility shift assay; beta -Gal, beta -galactosidase; FCS, fetal calf serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; VCAM-1, vascular cell adhesion molecule-1.
2   T. Bourcier, G. Sukhova, and P. Libby, unpublished data.

ACKNOWLEDGEMENTS

We thank Marysia Muszynski and Elissa Simon-Morrissey (Brigham and Women's Hospital) for their skillful assistance, Dr. Edward Murray (Roche, Welwyn Garden City, Herts, UK) for the generous gift of luciferase reporter constructs, and Dr. Rosalind Fabumni for helpful discussions.


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