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(The FASEB Journal. 2000;14:368-378.)
© 2000 FASEB

Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1ß in vascular smooth muscle cells

LAUREN C. FOSTER*, PHILIPPE WIESEL*,{ddagger}, GORDON S. HUGGINS*,{dagger},¶, REA PAÑARES*, MICHAEL T. CHIN*,{dagger},{ddagger}, ANDREA PELLACANI*,{dagger},{ddagger} and MARK A. PERRELLA*,{dagger},§1

* Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, Massachusetts 02115, USA;
{dagger} Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA;
{ddagger} Cardiovascular and
§ Pulmonary and Critical Care Divisions, Brigham and Women’s Hospital, Boston, Massachusetts 02115, USA; and
Cardiac Unit, Massachusetts General Hospital, Boston, Massachusetts 02115, USA

1Correspondence: Program of Developmental Cardiovascular Biology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA. E-mail: perrella{at}cvlab.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD44 is a multifunctional cell adhesion molecule that participates in pathological states such as inflammation and tumorigenesis. CD44 is induced on vascular smooth muscle cells after arterial wall injury and may mediate their proliferation and migration into the neointima during arteriosclerosis. We have demonstrated elsewhere that the proinflammatory cytokine interleukin (IL)-1ß up-regulates CD44 mRNA and protein expression in cultured rat aortic smooth muscle cells (RASMC) by increasing gene transcription. By transient transfection of 5'-deletion constructs into RASMC, we show in the present study that a conserved AP-1 site 110 base pairs from the transcription start site of the mouse CD44 promoter is important for basal activity. Mutation of the AP-1 site significantly reduced induction of promoter activity by IL-1ß, and electrophoretic mobility shift assays demonstrated that Fos and c-Jun were present in the CD44 AP-1 binding complex after IL-1ß stimulation. In addition, cotransfection of the architectural transcription factor high mobility group (HMG)-I(Y) protein with c-Fos and c-Jun markedly increased trans-activation of the CD44 promoter. Taken together, our studies demonstrate that AP-1 proteins are a central regulatory component used by IL-1ß to modulate expression of CD44 during an inflammatory response in vascular smooth muscle cells and that transcription of CD44 by AP-1 proteins is enhanced by HMG-I(Y).—Foster, L. C., Wiesel, P., Huggins, G. S, Pañares, R., Chin, M. T., Pellacani, A., Perrella, M. A. Role of activating protein-1 and high mobility group-I(Y) protein in the induction of CD44 gene expression by interleukin-1ß in vascular smooth muscle cells.


Key Words: inflammation • gene transcription • adhesion molecule • arteriosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ADHESION MOLECULES ON vascular smooth muscle cells mediate a variety of cell–cell and cell–matrix interactions that regulate pathophysiological processes. We have demonstrated elsewhere that the adhesion molecule CD44 is induced on neointimal smooth muscle cells after arterial wall injury in vivo, both in a model of mechanical injury to the rat carotid artery and in a mouse model of transplant arteriosclerosis (1 , 2) . The coordinate expression within arteriosclerotic lesions of CD44 and its extracellular matrix ligands, including hyaluronan (HA) and osteopontin, may have important functional consequences in disease progression (1 , 3 4 5 6) . Specific binding of HA to the CD44 receptor increases DNA synthesis in vascular smooth muscle cells (1) . CD44 mediates cell migration on HA substrates (7 , 8) , and CD44 isoforms containing variant exon 6 (v6) confer metastatic and invasive potential to carcinoma cells (9 , 10) . Furthermore, osteopontin is chemotactic for smooth muscle cells (11) . Therefore, CD44 may contribute to smooth muscle cell accumulation in the neointima through its effects on proliferation and motility. CD44 may also play a role in airway inflammation and smooth muscle cell hyperplasia associated with asthma: activated T lymphocytes adhere to airway smooth muscle cells via integrins and CD44, an interaction that induces smooth muscle cell DNA synthesis (12) . By analogy, the expression of CD44 on smooth muscle cells may promote the adherence of leukocytes within the vessel wall and thereby act to maintain the chronic inflammatory response characteristic of arteriosclerosis.

The expression of CD44 is modulated by a variety of stimuli on different cell types. In vivo, the level of CD44 isoforms increases on lymphocytes after their activation during the immune response (13) and on macrophages at sites of inflammation (14) . In vitro, CD44 expression is regulated by cytokines and the state of cellular activation and/or differentiation (15 16 17 18) . Little is known, however, about the molecular regulation of the CD44 gene. In the context of lymphocyte activation through B cell antigen receptor stimulation, the EGR1 transcription factor transactivates the human CD44 promoter (19) . In mouse fibroblasts, epidermal growth factor acts through a novel cis-acting element to induce CD44 expression, which is accompanied by enhanced cell attachment to HA (20 , 21) .

Cytokines and growth factors regulate the phenotype and activation state of cells involved in the pathogenesis of arteriosclerosis (22 , 23) . Macrophages, endothelial cells, and vascular smooth muscle cells in atherosclerotic lesions express interleukin (IL)-1ß, a pleiotropic cytokine that mediates cellular responses to infection and tissue injury (24 25 26) . Proinflammatory cytokines such as IL-1ß modulate vascular smooth muscle cell growth, migration, and metabolic activity during atherogenesis (27 28 29) The target genes of IL-1ß include the HA binding proteins CD44, intercellular adhesion molecule (ICAM)-1, and TSG-6 (2 , 30 31 32 33) . We are interested in the regulation of CD44 gene expression on arterial smooth muscle cells. Recently, we demonstrated that IL-1ß increases CD44 gene transcription in cultured rat aortic smooth muscle cells (RASMC) (2) . The response to IL-1ß stimulation is mediated by a 1.4-kb fragment of 5'-flanking sequence in the mouse CD44 gene.

The present study was designed to further investigate the molecular mechanisms responsible for the increase in CD44 transcription by IL-1ß in vascular smooth muscle cells. Beyond identifying the interaction of specific DNA sequences and their cognate nuclear transcription factors, we also wanted to determine whether architectural transcription factors contribute to trans-activation of CD44. Architectural transcription factors do not drive transcription themselves; rather, they alter chromatin structure and assemble transcription factors into nucleoprotein complexes that drive transcription efficiently (34 35 36) . One such architectural factor, high mobility group (HMG)-I(Y) protein, regulates cytokine-responsive genes that are critical mediators of inflammation (37 , 38) . HMG-I(Y) itself is also induced by inflammatory cytokines (39) . Moreover, HMG-I(Y) expression increases after cellular transformation (40 41 42) , and HMG-I(Y) may represent a marker of metastatic aggressiveness in tumors (43 44 45) . Because these characteristics of HMG-I(Y) are similar to those of CD44, we determined whether HMG-I(Y) contributes to the trans-activation of CD44.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Recombinant human IL-1ß (Collaborative Biomedical, Bedford, Mass.) was stored at -80°C until use. Antibodies against the protein encoding c-Jun (rabbit anti-avian) and Fos (rabbit anti-human, recognizing both c-Fos and v-Fos) were obtained from Upstate Biotechnology (Lake Placid, N.Y.). Anti-Nrf1 antibody (rabbit anti-human) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Distamycin A was obtained from Sigma (St. Louis, Mo.).

Cell culture
RASMC were harvested from adult male Sprague-Dawley rats (200–250 g) by enzymatic dissociation according to the method of Gunther et al. (46) . The cells were cultured in Dulbecco’s modified Eagle’s medium (DME; Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (FCS), penicillin (100 units/ml), streptomycin (100 µg/ml), and 25 mM Hepes (pH 7.4). RASMC were passaged every 4–7 days, and experiments were performed on cells 4–7 passages from primary culture. Mouse F9 cells were maintained in DME/F12 medium (Life Technologies) supplemented with 10% FCS, penicillin (100 units/ml), streptomycin (100 µg/ml), and 5 x 10–5 M ß-mercaptoethanol (47) . Drosophila SL2 cells (American Type Culture Collection, Rockville, Md.) (48) were maintained at 23°C in Schneider’s insect medium (Sigma) supplemented with 12% FCS and gentamicin (50 µg/µl) (49) . Rat fetal aortic smooth muscle cells (A7r5) were cultured in DME supplemented with 10% FCS, penicillin (100 units/ml), and streptomycin (100 µg/ml).

Plasmids
pGL2-Basic and pGL2-Control contained the firefly luciferase gene (Promega, Madison, Wisc.). pGL2-Basic had no promoter, whereas pGL2-Control was driven by the SV40 promoter and enhancer. Phagemid pOPRSVI-CAT (Stratagene, La Jolla, Calif.) contained the prokaryotic chloramphenicol acetyltransferase (CAT) gene driven by the Rous sarcoma virus–long terminal repeats promoter. Reporter constructs containing fragments of the mouse CD44 5'-flanking sequence were named according to the location of the fragment from the transcription start site in the 5' and 3' directions. A gene fragment amplified from mouse genomic DNA containing 1,262 base pairs (bp) of the CD44 5'-flanking sequence upstream and 109 bp downstream of the transcription initiation site was cloned into pGL2-Basic and named CD44(-1,262/+109) as described (2) . Mouse CD44 genomic DNA was used as a template to generate a series of 5'-deletion constructs, CD44(-632/+109), CD44(-425/+109), CD44(-249/+109), CD44(-181/+109), and CD44(-97/+109). The AP-1 site at -110 was mutated (-110 to -104, TTAGTCA to CTAGGCA) in the -1,262/+109 fragment by using a polymerase chain reaction (PCR)-based site-directed mutagenesis technique (50) , and this construct was named CD44(-1,262/+109 AP-1m). All constructs were generated by PCR with Pfu polymerase (Stratagene) and subcloned into pGL2-Basic. Plasmids were sequenced by the dideoxy chain termination method with ThermoSequenaseTM DNA polymerase (Amersham, Arlington Heights, Ill.) to confirm the identity and the orientation of the insert.

Mouse c-fos and rat c-jun cDNA clones were obtained from Michael E. Greenberg (Children’s Hospital, Boston, Mass.) and cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif.). The Drosophila expression plasmids pPAC, phsp82LacZ, and pPACHMGI have been described elsewhere (49) . Constructs for expression in Drosophila cells were made by inserting the cDNAs coding for c-Jun and c-Fos into the BamHI site of pPAC.

Transfection and reporter assays
RASMC were transfected by a diethylaminoethyl (DEAE)-dextran method (51) . In brief, cells were plated onto six-well tissue culture dishes and allowed to grow for 48–72 h (until 80–90% confluent). Luciferase plasmid DNA (2 µg) and pOPRSVI-CAT (1 µg) (to correct for differences in transfection efficiency) were added to RASMC in a solution containing 500 µg/ml of DEAE-dextran. RASMC were then shocked with a 5% dimethyl sulfoxide solution for 1 min and allowed to recover in medium containing 10% FCS. Twelve hours after transfection, RASMC were placed in 2% FCS and stimulated with IL-1ß (10 ng/ml) for 48 h. Distamycin A (5 µM) was added simultaneously with IL-1ß.

SL2 cells were transfected by the calcium phosphate method according to Di Nocera and Dawid (52) In brief, SL2 cells were plated into six-well tissue culture dishes 24 h before transfection. Plasmid CD44(-1,262/+109) or CD44(-1,262/+109 AP-1m) was added at 1 µg per well and phsp82LacZ (to correct for differences in transfection efficiency) was added at 100 ng per well. Plasmids pPAC-fos and pPAC-jun were added at 0.1 or 0.5 µg per well. Plasmid pPACHMGI was added at 0.5 µg per well alone or in combination with pPAC-fos and pPAC-jun. Empty pPAC vector was added to equalize the total DNA concentration within each transfection group.

F9 cells were transfected by the calcium phosphate DNA coprecipitation method as described (47) . Cells (0.1 x 106) were plated 16–18 h before transfection, incubated for 6 h with a precipitate containing 5 µg of CD44(-1,262/+109), 1.5 µg of pOPRSVI-CAT (to correct for differences in transfection efficiency), and 0.1 or 0.5 µg of pcDNA3-jun or empty pcDNA3 expression vector, and harvested after 24 h. A7r5 cells were transfected with the FuGENETM reagent according to the instructions of the manufacturer (Boehringer Mannheim, Indianapolis, Ind.).

Cell extracts were prepared by a detergent lysis method (Promega), and luciferase activity was measured in duplicate for all samples by using the Promega luciferase assay system and an EG&G (Gaithersburg, Md.) AutoLumat LB953 luminometer. The CAT assay was performed by a modified two-phase fluor-diffusion method as described (51) . ß-Galactosidase activity was assayed as described (49) . The ratio of luciferase activity to CAT or ß-galactosidase activity in each sample served as a measure of normalized luciferase activity. Each construct was transfected at least six times, and data for each construct are presented as the mean ± SE

Electrophoretic mobility shift assay
RASMC were starved in 0.4% calf serum for 72 h, stimulated with IL-1ß (10 ng/ml) for 3 h, and washed in cold phosphate buffered saline (PBS). Nuclear extracts were prepared as described (51) . Protein concentration was determined with the DC protein assay kit (Bio-Rad, Hercules, Calif.). Oligonucleotides were synthesized according to the mouse CD44 promoter sequence, annealed, and labeled with [{gamma}-32P]-ATP by using T4 polynucleotide kinase.

CD44 AP-1 (bp -120 to -95): 5'-CGTTGGCTGCTTAGTCACAGCCCCCT-3'

CD44 AP-1m (bp -120 to -95): 5'-CGTTGGCTGCCTAGGCACAGCCCCCT-3'

CD44 AP-1s (bp -112 to -102): 5'-GCTTAGTCACAACACTGATTCG-3'

Consensus AP-1: 5'-CGGTTGATGAGTCAGCCGGAA-3'

CD44 AP-1ext (bp -132 to -101):

5'-TCTTTAAACTTCCGTTGGCTGCTTAGTCACAG-3'

CD44 AP-1ext AP-1mut (bp -132 to -101):

5'-TCTTTAAACTTCCGTTGGCTGCCTAGGCACAG-3'

CD44 AP-1ext AT-mut (bp -132 to -101):

5'-TCGGACCTCTTCCGTTGGCTGCTTAGTCACAG-3'

Binding reactions were performed in a 25 µl volume containing 20,000 cpm labeled probe, 10 µg nuclear extract, 1 µg poly (dI-dC)•poly (dI-dC) (Sigma), 25 mM Hepes (pH 7.9), 50 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol, with or without oligonucleotide competitors, as indicated. Reactions were incubated for 20 min at room temperature, and DNA-protein complexes were analyzed by electrophoresis on a 5% native polyacrylamide gel in 0.25 x Tris borate-EDTA (TBE) buffer at 4°C. To characterize specific DNA-binding proteins, we incubated nuclear extracts with various antibodies for 12 h at 4°C before the addition of probe. In studies involving distamycin A, reactions were performed in 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 1 mM dithiothreitol, 5% glycerol, and 50 ng poly (dG-dC)•poly (dG-dC). Labeled probe was treated with distamycin A (5 µM) before the addition of nuclear extract. In experiments assessing HMG-I(Y) binding, recombinant protein (100 ng) was substituted for nuclear extract in the binding reaction.

Statistics
Data from the transfections experiments were compared by analysis of variance, and statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion analysis of mouse CD44 promoter
To identify the cis-acting element(s) within the 5'-flanking sequence of the mouse CD44 gene that mediates the IL-1ß response, we generated a series of 5'-deletion mutants of the CD44 promoter by PCR and subcloned them into the pGL2-Basic luciferase reporter vector (Fig. 1A ). Plasmids were transiently transfected into RASMC along with pOPRSVI-CAT (to correct for differences in transfection efficiency) and stimulated with IL-1ß for 48 h. The corrected luciferase activity was normalized to that of CD44(-1,262/+109) not stimulated with IL-1ß. Serial 5'-deletions from bp 1262 to -181 did not have a significant effect on basal promoter activity or induction by IL-1ß (Fig. 1B ). However, the deletion of sequences between bp -181 and -97 caused a dramatic 72% reduction in the basal level of CD44 promoter activity and resulted in the loss of promoter induction in response to IL-1ß. This reduction implies that cis-acting elements in this region are essential for cytokine stimulation of CD44 gene expression. A candidate positive regulatory element in this region is an AP-1 site at position -110 to -104 that is conserved between the mouse and human CD44 promoters.



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  		Figure 1. 5'-deletion analysis of CD44 promoter activity in response to IL-1ß stimulation. A) Schematic representation of deletion sites in relation to consensus DNA sequences for known transcription factors. B) Functional analysis of mouse CD44 promoter by transient transfection into RASMC of luciferase constructs containing serial 5'-deletions. All constructs were cotransfected with pOPRSVI-CAT to correct for differences in transfection efficiency. After transfection, RASMC were incubated in the absence (-, white bars) or presence (+, black bars) of IL-1ß (10 ng/ml) for 48 h. Normalized luciferase activity is presented as a percentage of the activity of CD44(-1,262/+109) in the absence of IL-1ß (mean ± SE, n>=6 in each group). Striped bar indicates luciferase activity of pGL2-Control. * indicate significant differences (P<0.0006) in comparison with unstimulated (-IL-1ß) controls. Crosses indicate significant differences (P<0.0001) in comparison with all other constructs.

To determine the functional importance of this AP-1 site, we used site-directed mutagenesis to generate a 1.4 kb CD44 5'-promoter construct containing an AP-1 site mutation, CD44(-1,262/+109AP-1m). A comparable mutation of the AP-1 motif in the human CD44 promoter (TTAGTCA to CTAGGCA) disrupts the function of this site (53) . In comparison with the native CD44(-1,262/+109) construct, the CD44(-1,262/+109AP-1m) construct displayed a 51% reduction in promoter activity in RASMC after stimulation by IL-1ß (Fig. 2 ). Taken together these data indicate that the AP-1 site at bp -110 to -104 appears to be important for cytokine induction of the CD44 promoter.



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  		Figure 2. The AP-1 binding motif at position -110 to -104 is important for induction of CD44 gene transcription by IL-1ß. The AP-1 site was specifically mutated within the CD44(-1,262/+109) construct as described under Methods. Both the CD44(-1,269/+109) wild-type construct and the CD44(-1,262/+109 AP-1m) mutated construct were transfected transiently into RASMC. The constructs were cotransfected with pOPRSVI-CAT to correct for differences in transfection efficiency. After transfection, RASMC were incubated in the absence (-, white bars) or presence (+, black bars) of IL-1ß (10 ng/ml) for 48 h. Normalized luciferase activity is presented as a percentage of the activity of CD44(-1,262/+109) in the absence of IL-1ß (mean ± SE, n=12 in each group). * indicates significant difference (P<0.0001) in comparison with the other groups.

IL-1ß induces nuclear protein binding to the CD44 AP-1 site
To further characterize the IL-1ß regulatory element(s) in the CD44 gene, we performed electrophoretic mobility shift assays (EMSA) with a 32P-labeled oligonucleotide probe encoding region -120 to -95 of the CD44 5'-flanking sequence, which contains the conserved AP-1 site. This probe generated multiple DNA-protein complexes (Fig. 3A , bands 1–4) when incubated with nuclear extracts from RASMC. The specificity of these binding complexes was assessed by including unlabeled oligonucleotide competitors in the binding reactions. Only band 2 appeared to be specific for binding at the AP-1 site, because it was competed away by a 100-fold molar excess of an unlabeled identical probe (I) but not by an unrelated probe (NI) or a probe containing a specific mutation at the AP-1 site (AP-1m), as described above. The band 2 binding complex was also specifically competed away by an AP-1 consensus binding site oligonucleotide (data not shown). The remaining specific bands (1 and 3) were a result of proteins binding to the DNA sequences flanking the AP-1 site, as they were present when the 32P-labeled AP-1m probe was incubated with RASMC nuclear extract. Band 4 was nonspecific, because it was competed away by the unrelated probe (NI). The subsequent time course experiment was conducted in the presence of an excess of unlabeled AP-1m competitor in order to focus specifically on DNA-protein complex formation at the AP-1 site. CD44 AP-1 binding activity was inducible by IL-1ß in a time-dependent fashion; maximal binding occurred after 2–3 h of cytokine stimulation and returned toward baseline after 5 h (Fig. 3B ).



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  		Figure 3. IL-1ß induces nuclear proteins to interact with the CD44 AP-1 site. A) Nuclear proteins bind to the CD44 5'-flanking sequence. 32P-labeled CD44 AP-1 oligonucleotide probe (bp -120 to -95) was incubated with 10 µg of RASMC nuclear extract. Unlabeled competitors were added at a 100-fold molar excess as indicated. I, identical competitor; NI, unrelated competitor; AP-1m, AP-1 site mutant competitor. The CD44 probe containing a mutated AP-1 site (AP-1m, bp -110 to -104) was also labeled with 32P and incubated with RASMC nuclear extract to assess the contribution of the AP-1 site flanking sequences to binding activity. Arrow corresponds to band 2 and indicates the specific AP-1 binding complex. B) Induction of AP-1 DNA-protein complex formation by IL-1ß. Nuclear extracts were harvested from RASMC stimulated with

Identity of nuclear proteins that bind to the CD44 AP-1 site
EMSA were also used to identify nuclear protein(s) that binds at the CD44 AP-1 site after IL-ß stimulation. A minimal CD44 AP-1 site probe (AP-1s, bp -112 to -102) was used for these studies. The DNA-protein complex formed with the AP-1s probe displayed a specificity and migration pattern similar to that of the specific CD44 AP-1 complex (band 2) identified in Fig. 3A . Nuclear extracts from RASMC stimulated with IL-1ß for 3 h were incubated with 32P-labeled AP-1s in the presence or absence of antibodies specific for the AP-1 family members Fos and c-Jun (Fig. 4 ; arrow indicates specific DNA-protein complex formed in the absence of antibody). The anti-Fos polyclonal antibody disrupted the CD44 AP-1 binding complex, whereas the anti–c-Jun antibody produced a supershifted complex (indicated by an asterisk). No shifted bands were observed in control samples treated with an antibody specific for Nrf1 (data not shown), an NF-E2 family member whose recognition element resembles an AP-1 site. This experiment indicates that members of the AP-1 family of proteins (Fos and c-Jun) were present in the CD44 AP-1 binding complex after IL-1ß stimulation.



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  		Figure 4. Identification of nuclear factors in the CD44 AP-1 binding complex. Nuclear extract (5 µg) from RASMC stimulated with IL-1ß for 3 h was incubated with antibody against Fos or c-Jun for 12 h at 4°C. 32P-labeled CD44 AP-1s probe was added subsequently and the mixture was allowed to incubate for 20 min at room temperature. Electrophoretic mobility shift assays were performed as described under Methods. Arrow indicates CD44 AP-1 DNA-protein complex. * indicates supershifted complex.

Trans-activation of the CD44 promoter by AP-1
To determine whether the CD44 promoter could be activated by AP-1 proteins in culture, we cotransfected the CD44(-1,262/+109) luciferase reporter construct with a c-Jun expression vector. Transient transfection was performed in F9 embryonic carcinoma cells that, in the undifferentiated state, do not contain functional AP-1 activity because they lack AP-1 subunits that include c-Jun (54) . c-Jun increased the activity of the CD44(-1,262/+109) promoter construct significantly and in a dose-dependent fashion (Fig. 5 , black bars) in comparison with control cells treated with empty vector (Fig. 5 , white bars). Transfection of 0.5 µg of the c-Jun expression plasmid produced a 6.8-fold stimulation of the CD44 promoter after 24 h. AP-1 proteins also trans-activated the CD44 promoter when coexpressed in A7r5 fetal smooth muscle cells (data not shown).



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  		Figure 5. Activation of CD44 gene transcription by c-Jun. The CD44(-1,262/+109) reporter construct (5 µg) was transiently transfected into F9 cells with c-Jun expression plasmid (black bars) or empty pcDNA3 vector (control, white bars) at the concentrations indicated. All constructs were cotransfected with pOPRSVI-CAT to correct for differences in transfection efficiency. Normalized luciferase activity is presented as a percentage of the activity of the respective control. Results represent the mean ± SE of at least three independent experiments (n=15). * indicate significant differences (P<0.0007) in comparison with control values. Crosses indicate significant differences (P<0.0001) in comparison with all other groups.

HMG-I(Y) potentiates CD44 promoter transactivation by AP-1
IL-1ß induces expression of the architectural transcription factor HMG-I(Y) in RASMC (39) and is an important component in the regulation of cytokine-inducible promoters/enhancers such as endothelial-leukocyte adhesion molecule-1 (E-selectin), ICAM-1, vascular cell adhesion molecule (VCAM)-1 (37) , and inducible nitric oxide synthase (iNOS or NOS2) (39) . To determine whether HMG-I(Y) plays a role in trans-activation of the CD44 promoter, we performed cotransfection experiments in Drosophila SL2 cells. These cells were chosen because they express low levels of endogenous HMG-I(Y) in comparison with mammalian cells (55) . We transiently transfected the CD44(-1,262/+109) promoter construct into SL2 cells along with expression plasmids for the AP-1 subunits c-Fos and c-Jun. c-Jun alone or in combination with c-Fos produced a significant and dose-dependent increase in CD44 promoter activity (Fig. 6A ).



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  		Figure 6. HMG-I(Y) enhances AP-1–dependent trans-activation of the CD44 promoter. A) AP-1 stimulates the CD44 promoter in SL2 cells. CD44 promoter construct CD44(-1,262/+109) (1 µg) was transiently transfected into Drosophila SL2 cells with expression plasmids encoding c-Fos, c-Jun, or both c-Fos and c-Jun at the indicated concentrations. Normalized luciferase activity is plotted as the fold induction over activity in control cells (white bars) expressing no (-) c-Fos or c-Jun. Values represent the mean ± SE (n=6 in each group). * indicate significant differences (P<0.0001) in comparison with control values. Crosses indicate a significant difference (P<0.0001) in comparison with groups receiving a lower dose of expression plasmids. B) HMG-I(Y) enhances CD44 promoter trans-activation by AP-1 in SL2 cells. Drosophila SL2 cells were transiently transfected with the reporter construct CD44(-1,262/+109) alone (1 µg, white bar) or with CD44(-1,262/+109) and expression plasmids encoding HMG-I(Y) (0.5 µg, gray bar), c-Fos and c-Jun (0.1 µg each,

To assess the effect of HMG-I(Y) on AP-1–mediated trans-activation of the CD44 promoter, we transfected the HMG-I(Y) expression plasmid pPACHMGI alone or in combination with the AP-1 subunits c-Fos and c-Jun. HMG-I(Y) did not have a significant independent effect on CD44(-1,262/+109) promoter activity. When HMG-I(Y) was expressed together with c-Fos and c-Jun, however, a potentiated increase in promoter activity of 27.9 ± 1.1-fold was observed, as compared with an increase in promoter activity of 6.9 ± 0.2-fold after expression of c-Fos and c-Jun in the absence of HMG-I(Y) (Fig. 6B ). When AP-1 site position -110 to -104 was mutated, the ability of HMG-I(Y) to potentiate CD44 trans-activation by c-Fos and c-Jun was disrupted (Fig. 6C ). These data suggest that enhanced trans-activation of the CD44 promoter by HMG-I(Y) requires an intact AP-1 site at position -110 to -104 of the CD44 5'-flanking sequence. To ensure that this HMG-I(Y) response was not related to a nonspecific effect, we also expressed HMG-I(Y) with the promoter for heat shock protein (HSP) 82. HMG-I(Y) had no effect on HSP 82 promoter activity, either in the presence or absence of c-Fos and c-Jun (data not shown).

To assess the contribution of HMG-I(Y) in the formation of nucleoprotein complexes on the CD44 promoter, we first performed EMSA with an oligonucleotide probe (CD44 AP-1ext) spanning bp -132 to -101 of the CD44 promoter, which contains the AP-1 site (-110 to -104) and an adjacent AT-rich region (-130 to -125). HMG-I(Y) is known to bind AT-rich sequences in the minor groove of DNA (56) . Recombinant HMG-I(Y) protein bound to this probe (Fig. 7A , arrow). To localize the site of HMG-I(Y) binding, we also generated probes containing a mutation in the AP-1 site or the AT-rich region. Mutation of the AP-1 site did not disrupt HMG-I(Y) binding; however, mutation of the AT-rich region led to a marked (56%) decrease in HMG-I(Y) binding (Fig. 7A ).



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  		Figure 7. HMG-I(Y) binds to the CD44 promoter and contributes to induction of promoter activity by IL-1ß. A) EMSA were performed with the radiolabeled oligonucleotide probe CD44 AP-1ext (bp -132 to -101). Within region -132 to -101, the oligonucleotides contained the wild-type sequence, a mutated AP-1 site (AP-1m), or a mutated AT-rich sequence (AT-rich m). Radiolabeled probes were incubated in the presence (+) or absence (-) of recombinant HMG-I(Y) (100 ng). Arrow indicates the HMG-I(Y) protein-DNA complex. B) Nuclear proteins binding to the CD44 5'-flanking sequence. 32P-labeled, CD44 AP-1ext oligonucleotide probe (bp -132 to -101) was incubated with 10 µg of RASMC nuclear extract. Oligonucleotide probes were pretreated with distamycin A (5 µM) or vehicle (EtOH) for 10 min at room temperature as indicated. Arrow indicates the specific DNA-protein complex. C) RASMC were transiently transfected with the CD44(-1,262/+109) construct and cotransfected with

A specific DNA-protein complex was formed (Fig. 7B , arrow) when a probe containing the wild-type sequence (bp -132 to -101) was incubated with nuclear extract from RASMC stimulated with IL-1ß. This complex was effectively competed away by a 100-fold molar excess of unlabeled identical probe but not by an unrelated probe (data not shown). We then used distamycin A, an antibiotic that binds to AT-rich DNA sequences [clusters of at least 4 AT bp in the minor groove of DNA (57) ] and disrupts the binding of HMG-I(Y) to DNA (58) . Distamycin A disrupted specific protein binding to the oligonucleotide probe, but the vehicle for distamycin A did not (Fig. 7B ). Moreover, when the AT-rich sequence in the oligonucleotide probe was mutated, formation of the specific DNA-protein complex decreased dramatically (65%) (data not shown). This decrease in oligonucleotide probe binding is similar to the decrease in recombinant HMG-I(Y) protein binding observed for the AT-rich mutant probe (Fig. 7A ). These data suggest that the AT-rich sequence (-130 to -125) adjacent to the CD44 AP-1 site (-110 to -104) mediates protein binding to the CD44 promoter and that this region is part of a transcriptionally active enhancer region after cytokine stimulation.

To evaluate the role of HMG-I(Y) in cytokine regulation of CD44 gene transcription, we performed transient transfection experiments in the presence of distamycin A. In RASMC that had been transfected with the CD44(-1,262/+109) reporter construct and treated with 5 µM distamycin A, IL-1ß–induced CD44 promoter activity decreased by 31% (Fig. 7C ). Inhibition of CD44 promoter activity by distamycin A appears to be specific because it did not affect the promoter activity of the CRP2/SmLim gene (data not shown), which is also expressed in vascular smooth muscle cells (59) , or that of the pOPRSVI-CAT plasmid (data not shown). Taken together, these data suggest that the conserved AP-1 site (-110 to -104) and the neighboring AT-rich sequence (-130 to -125) are important for the assembly of a higher-order nucleoprotein complex on the CD44 promoter after IL-1ß stimulation. Also, inhibition of HMG-I(Y) binding suppressed full induction of the CD44 promoter by IL-1ß.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously described the transcriptional regulation of CD44 gene expression in vascular smooth muscle cells by the proinflammatory cytokine IL-1ß (2) . A 1.4 kb fragment of CD44 5'-flanking sequence is sufficient to drive high-level promoter activity in RASMC and to mediate induction by IL-1ß. The objective of the present study was to characterize the IL-1ß–responsive element(s) in the CD44 promoter. Sequential deletion analysis of the CD44 5'-flanking sequence revealed that a conserved AP-1 site at position -110 to -104 was important for both basal promoter activity in RASMC and induction by IL-1ß. EMSA showed that IL-1ß induced nuclear protein binding to the CD44 AP-1 site and identified Fos and c-Jun proteins in the binding complex. Moreover, overexpression of c-Jun in transient transfection experiments trans-activated the CD44 promoter to levels comparable with those obtained with IL-1ß, confirming the contribution of AP-1 proteins in the regulation of CD44 gene transcription. Specific mutation of the CD44 AP-1 site inhibited stimulation of CD44 promoter activity in response to IL-1ß by 51%. This suggests that AP-1 proteins act, in conjunction with other factors, to mediate cytokine stimulation of CD44 gene expression.

Proteins of the HMG-I family contribute to the regulation of gene transcription by binding to AT-rich regions in the minor groove of DNA and facilitating the assembly of functional nucleoprotein complexes (enhanceosomes), which they foster by modifying DNA conformation and recruiting nuclear proteins to an enhancer (57) . The three members of the HMG-I family are HMG-I, HMG-Y, and HMG-C. HMG-I(Y) refers to the first two proteins, which are alternatively spliced products of a single gene (60) . HMGI-(Y) plays an important role in viral induction of the interferon-ß gene (55 , 61) . In addition, cytokine-inducible enhancers of some endothelial-leukocyte cell adhesion molecules use HMGI-(Y) in concert with transcription factors (such as NF-{kappa}B and c-Jun/ATF-2 heterodimers) to direct transcriptional activity (37) . We demonstrate here for the first time that HMG-I(Y) potentiates trans-activation of the CD44 promoter by AP-1 proteins.

Our laboratory demonstrated recently that HMG-I(Y) potentiates iNOS trans-activation by NF-{kappa}B (62) . Studies by others have suggested that binding sites for HMG-I(Y) partially or fully overlap binding sites for transcription factors that are incorporated into enhanceosome complexes (63) . However, in the iNOS promoter/enhancer, HMG-I(Y) forms a ternary complex with NF-{kappa}B even though it binds to the promoter/enhancer 15 bp away from the NF-{kappa}B site. Similarly, HMG-I(Y) binds to an AT-rich site (-130 to -125) that is located 15 bp from the critical AP-1 binding site (-110 to -104) in the CD44 promoter.

The functional importance of AP-1 proteins in the regulation of CD44 expression has been demonstrated in the context of cellular transformation (53) . The conserved AP-1 binding site at position -110 to -104 mediates an increase in CD44 promoter activity after transformation of rat embryo fibroblasts by ras, which is consistent with the role of CD44 in tumor progression and metastasis (53) . This AP-1 site has also been suggested to mediate CD44 promoter induction by phorbol esters (64) . The invasive potential of fibroblasts transformed by epidermal growth factor and/or oncogenic fos requires AP-1–mediated induction of CD44 (65) . In addition, tumor progression from small cell to non-small cell lung carcinoma is accompanied by an increase in AP-1 binding activity and an increase in the expression of CD44 (66) . Expression of HMG-I(Y) protein, like that of CD44, increases after cellular transformation (40 41 42) and correlates with a malignant phenotype in humans (43 44 45) . Thus, the ability of HMG-I(Y) to interact with CD44 and to regulate its gene transcription may have pathophysiologic relevance to cellular transformation and tumor aggressiveness.

This study establishes a role for AP-1 proteins (c-Fos and c-Jun), in conjunction with HMG-I(Y), to assemble higher-order transcriptional complexes during induction of CD44 gene expression by the proinflammatory cytokine IL-1ß in vascular smooth muscle cells. Messenger RNA and protein levels of c-Fos and c-Jun are induced rapidly in aortic smooth muscle cells after arterial wall injury (67) . HMG-I proteins are also induced in neointimal smooth muscle cells after balloon catheter injury to the rat carotid artery (Chin, M. T., and Lee, M.-E., unpublished observations), and the expression of these HMG-I proteins correlates well with the temporal pattern of CD44 expression on proliferating smooth muscle cells in the developing neointima after artery denudation (1) . Therefore, the coordinate action of AP-1 and HMG-I(Y) proteins may function to promote CD44 expression during neointima formation in arteriosclerosis.


   ACKNOWLEDGMENTS
 
We dedicate this manuscript to the memory of Dr. Edgar Haber, who was a constant source of inspiration and support of our work. We also extend our gratitude to Dr. Mu-En Lee for his helpful suggestions and his enthusiasm and support of our work, and to Dr. Mu-En Lee and Dr. Raymond Reeves for their critical reviews of the manuscript. Mouse c-fos and rat c-jun cDNA clones were kindly provided by Dr. Michael E. Greenberg. We thank Mr. Thomas McVarish for his editorial assistance and Mr. Bonna Ith for his technical assistance. This study was supported in part by National Institutes of Health grants HL03194 and HL60788 (M. A. P.), HL03745 (M. T. C.), and HL03667 (G. S. H.), an American Heart Grant-in-Aid (M. A. P.), a grant from Novartis and SICPA Foundation (P. W.), and a grant from the Bristol-Myers Squibb Pharmaceutical Research Institute.


   FOOTNOTES
 
Received for publication June 17, 1999. Revised for publication September 30, 1999.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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