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Histone Deacetylase-1 Is Enriched at the Platelet-derived Growth Factor-D Promoter in Response to Interleukin-1β and Forms a Cytokine-inducible Gene-silencing Complex with NF-κB p65 and Interferon Regulatory Factor-1*

Understanding the mechanisms governing cytokine control of growth factor expression in smooth muscle cells would provide invaluable insight into the molecular regulation of vascular phenotypes and create future opportunities for therapeutic intervention. Here, we report that the proinflammatory cytokine interleukin (IL)-1β suppresses platelet-derived growth factor (PDGF)-D promoter activity and mRNA and protein expression in smooth muscle cells. NF-κB p65, induced by IL-1β, interacts with a novel element in the PDGF-D promoter and inhibits PDGF-D transcription. Interferon regulatory factor-1 (IRF-1) is also induced by IL-1β and binds to a different element upstream in the promoter. Immunoprecipitation and chromatin immunoprecipitation experiments showed that IL-1β stimulates p65 interaction with IRF-1 and the accumulation of both factors at the PDGF-D promoter. Mutation of the IRF-1 and p65 DNA-binding elements relieved the promoter from IL-1β-mediated repression. PDGF-D repression by IL-1β involves histone deacetylation and interaction of HDAC-1 with IRF-1 and p65. HDAC-1 small interfering RNA ablates complex formation with IRF-1 and p65 and abrogates IRF-1 and p65 occupancy of the PDGF-D promoter. Thus, HDAC-1 is enriched at the PDGF-D promoter in cells exposed to IL-1β and forms a cytokine-inducible gene-silencing complex with p65 and IRF-1.

exocytic secretion, whereas PDGF-C and PDGF-D are secreted as latent factors that require activation by extracellular proteases. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulfide-linked homodimer and is a specific agonistic ligand for PDGF receptor ␤ (5). PDGF-D mRNA is widely expressed in organs such as the heart, pancreas, kidney, and ovary and has been linked to lung, prostate, and ovarian cancers. PDGF-D promotes tumor growth by accelerating tumor cell proliferation and stimulating tumor neovascularization (6,7). PDGF-D has also been shown to induce Notch-1-dependent angiogenesis (8) and maturation of blood vessels during angiogenesis (9). PDGF-D is expressed upon by vascular injury and stimulates smooth muscle cell (SMC) proliferation, and induces cardiac fibrosis in transgenic mice (10,11). The mechanisms regulating the PDGF-D promoter are poorly understood. We recently demonstrated that angiotensin II induces PDGF-D transcription in SMCs through Ets-1 and Sp1 and endogenous hydrogen peroxide generation (12,13). More recently, we demonstrated that Sp1 regulation of PDGF-D promoter activity involves phosphorylation of multiple residues in the zinc finger of Sp1 (14). Whether histone modification in chromatin by enzymes such as histone deacetylases (HDACs) regulate PDGF-D transcription is also unexplored. HDACs play a central role in the epigenetic regulation of gene expression. Recent findings from our group indicate that IL-1␤ facilitates HDAC dissociation from the PDGF receptor ␣ promoter and that HDAC inhibition potentiates induction of PDGF receptor ␣ transcription (15).
Proinflammatory cytokines activate a complex network of intracellular signaling pathways that alter gene expression and cellular phenotype, which include nuclear factor-B (NF-B) and interferon regulatory factor-1 (IRF-1). NF-B is a ubiquitous transcription factor that can be activated by diverse array of proatherogenic stimuli such as inflammatory cytokines, oxidant stress, and hemodynamic forces (16,17). Interferon regulatory factor-1 (IRF-1) was isolated by virtue of its affinity to specific DNA sequences in the interferon-␤ promoter that mediate virus responsiveness. The IRFs are a family of factors that regulate cytokine signaling, cellular growth regulation, hematopoietic development, and pathogen response (18). Our understanding of NF-B regulation of PDGF ligand expression is limited. KLF5 transactivation of PDGF-A involves cooperative interactions with NF-B p50 but not Egr-1 (19,20) in endothelial cells exposed to phorbol ester (21). We showed that NF-B binds to the PDGF-B promoter in vascular endothelial cells exposed to fluid shear stress and activates the PDGF-B promoter (22,23). Tumor necrosis factor-␣ activation of PDGF-B gene expression involves NF-B (24). It is not known whether NF-B controls PDGF-C or PDGF-D transcription in any cell type or whether IRF-1 is involved in transcription of any ligand chain. Here, we were surprised to find that IL-1␤ suppresses PDGF-D promoter activity, mRNA, and protein in SMCs, and this involves transcriptional repression by NF-B p65 and IRF-1, both of which form a complex in response to the cytokine and interact with the PDGF-D promoter. This study provides the first direct link between NF-B and the PDGF-D promoter and IRF-1 with any member of the PDGF family.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-Primary rat and human aortic SMCs were obtained from Cell Applications (San Diego, CA) and cultured in Waymouth's media containing 10% fetal bovine serum, 10 units/ml penicillin, and 10 g/ml streptomycin at 37°C in a humidified atmosphere of 5% CO 2 /air. Cells were used between passages 3 and 8. Cells were incubated for 24 h in serum-free media to achieve quiescence before being stimulated with indicated concentrations of IL-1␤. Human recombinant IL-1␤ and oridonin were purchased from Calbiochem. Trichostatin was bought from Sigma. Rabbit polyclonal antibodies to NF-B p65, NF-B p50, c-Rel, RelB, IRF-1, and HDAC-1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human p65 protein was obtained from Active Motif. Recombinant HDAC-1 and IRF-1 were obtained from Abnova Corp. Protein A/G agarose was bought from Amersham Biosciences. Mutant PDGF-D promoter constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene).
RNA Extraction and Reverse Transcription-PCR-Serumstarved SMCs were treated with 10 ng/ml of IL-1␤ at the indicated time or with different concentrations of cytokine for 4 h. For overexpression studies, cells were transfected with 20 or 40 g of p65 expression plasmid or empty vector. Total RNA was prepared using TriZOL reagent, and 2 g was used for synthesis of cDNA in a 25 l volume in the presence of 0.5 g of oligo dT primer (Sigma), 1 M of dNTP mix (Roche), 40 units of RNase inhibitor (Promega), 200 units of SuperScript TM II reverse transcriptase (Invitrogen), and 4 l of 5ϫ first strand buffer (Invitrogen) in diethyl pyrocarbonate-treated water. Reactions were allowed to proceed at 42°C for 50 min and at 70°C for 15 min. 1 l of cDNA was amplified for PDGF-D fragment in a final volume of 20 l. The primers for amplifying PDGF-D were: forward, 5Ј-GTG CAG AGT CCT ACT ATT CCC-3Ј and reverse, 5Ј-GAG GTG GTC TTG AGC TGC AG-3Ј. PCR was performed by denaturing at 94°C for 5 min; 94°C for 1 min; 59°C for 1 min; 72°C for 1 min; and extending at 72°C for 7 min. The primers for rat VCAM-1 were: forward, 5Ј-GGT CGC GAT CTT CGG AGC-3Ј and reverse, 5Ј-CTT GTA GTT CTC TGA CAG TCT CCC TTT CTT-3Ј. The cycling conditions were: 94°C 2 min; 96°C for 30 s; 60°C for 30 s; 72°C for 1 min; and 72°C for 2 min. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The primers for GAPDH were: forward, 5Ј-ACC ACA GTC CAT GCC ATCAC-3Ј and reverse, 5Ј-TCC ACC ACC CTG TTG CTG TA-3Ј. The amplification conditions for GAPDH were: 94°C, 1 min; 94°C, 30 s; 58°C, 10 s; 72°C, 1 min for 20 cycles; and 72°C, 4 min.
Real-time Quantitative PCR-Quantitative PCR was carried out using Rotor-Gene 3000 (Corbett Life Science). The reaction was set in a final volume of 10 l containing 1 l of cDNA, 5 l of 2ϫ SYBR Green Master Mix (Applied Biosystems), 0.2 l of 10 M of forward and reverse primer (Sigma) and 3.6 l of DNase-free water. Primers used were: rat PDGF-D: (forward) 5Ј-ATC GGG ACA CTT TTG CGA CT-3Ј, (reverse) 5Ј-GTG CCT GTC ACC CGA ATG TT-3Ј; and rat GAPDH: (forward) 5Ј-ACA AGA TGG TGA AGG TCG GTG-3Ј, (reverse) 5Ј-AGA AGG CAG CCC TGG TAA CC-3Ј. The PCR conditions were: 95°C for 10 min, followed by 40 cycles at 95°C for 20 s, 60°C for 45 s, and 72°C for 20 s.
Transient Transfection Assays-SMCs in 100-mm petri dishes at 60 -70% confluence were transfected with 10 g of the indicated luciferase reporter construct. 1 g of pRL-null was also transfected to normalize transfection efficiencies. Transfection was performed using FuGENE6 reagent (Roche). After a 24-h incubation, cells were harvested, and cell lysates were prepared for luciferase activity assessment using Dual Luciferase assay reporter system (DLR) (Promega) on a luminometer (model TD-20/20; Turner Designs, Quantum Science). Firefly luciferase activity was normalized to Renilla. In some experiments, cells were arrested in serum-free medium for 6 h before overnight transfection of plasmid and then treated with IL-1␤.
Western Blotting-Whole cell lysates and nuclear protein were prepared as described (12). The concentration of protein in both whole cell and nuclear extracts were determined with a BCA protein assay kit (Pierce). 20 g total cell lysates or 50 g nuclear extracts were resolved in 12% SDS-polyacrylamide gel and were transferred onto Immobilon polyvinylidene difluoride transfer membranes (Millipore). Membranes were blocked for 1 h at 22°C with 5% skim milk in 0.05% Tween 20 phosphate-buffered saline and then incubated overnight with primary antibody at 4°C. Primary antibodies were used at a final dilution of 1:300 -500. Membranes were washed three times with 0.05% Tween 20 phosphate-buffered saline followed by incubation with swine anti-rabbit or rabbit anti-goat IgG conjugated with horseradish peroxidase (diluted at 1:2000) for 1 h at room temperature. Membranes were then incubated with chemiluminescence (PerkinElmer Life Sciences) and then exposed to film for 1-15 min.
Immunoprecipitation-500 g of whole cell lysates or 200 g of nuclear extracts from growth quiescent or IL-1␤-stimulated SMCs were used in immunoprecipitation analysis. Protein G-Sepharose beads were precleaned with radioimmune precipitation assay buffer containing protease inhibitor mixture. Precleaned whole cell lysates were incubated with 5 g of primary antibody overnight at 4°C with gentle rotating followed by incubation with 30 l of bead suspension overnight at 4°C. Sepharose bound immunoprecipitates were washed, boiled in sample SDS buffer, and separated on 12% SDS-PAGE and then immunoblotted as described above.
Chromatin Immunoprecipitation-Human SMCs in a 75 cm 2 flask (80ϳ90% confluent) were arrested in serum-free media for 24 h and then stimulated with or without IL-1␤ for 1 h. After cross-linking with 37% formaldehyde and shearing with sonicator, the cells were washed twice with phosphatebuffered saline, pH 7.4 before chromatin immunoprecipitation (ChIP) using the appropriate antibody.

RESULTS
Proinflammatory Cytokine IL-1␤ Reduces Levels of PDGF-D mRNA, Promoter Activity, and Protein Expression-Because PDGF-D and IL-1␤ are both generated in response to vascular injury and regulate SMC proliferation (10,11,(27)(28)(29)(30) and because other members of the PDGF family are induced by IL-1␤, we began this study with the hypothesis that PDGF-D expression would be controlled by IL-1␤. Growth-quiescent rat aortic SMCs were incubated with IL-1␤ (10 ng/ml) for various times (0 -24 h) and PDGF-D mRNA levels were measured by reverse-transcription PCR. Surprisingly, the cytokine caused a progressive decrease in levels of PDGF-D mRNA despite unbiased loading (Fig. 1A). In contrast, using the same samples, we found that IL-1␤ stimulates VCAM-1 mRNA expression (Fig.  1A). VCAM-1 is a well recognized IL-1␤-dependent gene, positively regulated by IL-1␤ in an NF-B-dependent manner in multiple cell types (31,32). Quantitative real-time PCR and transient transfection analysis (with Ϫ1168-PDGF-D-luciferase, a PDGF-D promoter-luciferase construct bearing Ϫ1168/ ϩ72 of the PDGF-D promoter in pGL3-basic) confirmed IL-1␤ suppression of PDGF-D mRNA and promoter activity, respectively (Fig. 1B). Further expression analysis with increasing concentrations of IL-1␤ (0.1-50 ng/ml) revealed that cytokine inhibition of PDGF-D mRNA levels was dose-dependent ( Fig. 1C) with no biphasic effect. The decline in PDGF-D levels at this concentration of IL-1␤ is not due to toxic effects of the cytokine; the y axis of Fig. 1C indicates the ratio of PDGF-D relative to GAPDH mRNA expression. Western blot analysis demonstrated that PDGF-D protein levels (50 and 21 kDa) (12) were down-regulated by IL-1␤ (Fig. 1D).

NF-B p65, Induced by IL-1␤ in SMCs, Interacts with a Novel Element in the PDGF-D Promoter and Inhibits PDGF-D Transcription-
The transcription factor NF-B plays a major role in coordinating innate and adaptive immunity, proliferation, and apoptosis. Inspection of the PDGF-D promoter revealed the existence of a putative NF-B binding element at position Ϫ471 GGGATTCTAC Ϫ462 (Table 1). EMSA, using a double-stranded oligonucleotide spanning this element ( 32 Plabeled Oligo D Ϫ481/Ϫ457 ) in NF-B binding conditions (32), revealed that IL-1␤ induced the formation of two distinct nucleoprotein complexes within 1 h, A and B ( Fig. 2A, left). Antibody elimination analysis revealed that Complex A contains NF-B p65 and HDAC-1, whereas Complex B contains p65 but no HDAC-1 ( Fig. 2A). No supershifts were obtained with antibodies to p50, c-Rel, RelB, or IRF-1 ( Fig. 2A, left). Mutation of the Ϫ470 GGAT Ϫ467 sequence to Ϫ470 TTCG Ϫ467 in 32 P-labeled mOligo D Ϫ481/Ϫ457 disrupted complex formation in EMSA with control or IL-1␤-treated nuclear extracts ( Fig.  2A, upper right). Moreover, wild-type oligonucleotide bound recombinant p65-p65 homodimers in a dose-dependent manner, whereas the mutant probe failed to interact ( Fig. 2A, lower  right). Western blotting of cytoplasmic and nuclear extracts of untreated and IL-1␤-treated cells confirmed the existence of immunoreactive p65, p50, c-Rel, RelB, IRF-1, and HDAC-1 in the extracts (Fig. 2B, left). Of these, levels of p65 and IRF-1 were increased in the nucleus. EMSA performed with 32 P-labeled Oligo PD-B, which bears a high affinity consensus palindromic binding element for NF-B (22), revealed that the nucleoprotein complex that formed with these IL-1␤-treated extracts contained p65 but not p50, c-Rel, or RelB (Fig. 2B, right).
To provide functional evidence for the influence of p65 on PDGF-D promoter activity, we overexpressed p65 in a transient transfection system with the PDGF-D promoter-luciferase construct. p65 suppressed PDGF-D promoter activity (Fig. 2C) and inhibited PDGF-D mRNA levels (Fig. 2C). p50 has no effect on PDGF-D transcription, either alone or in conjunction with p65 (Fig. 2D). Additionally, the diterpenoid oridonin, which inhibits p65 DNA binding (33), rescued the PDGF-D promoter from IL-1␤ suppression (Fig. 2E). p65 siRNA inhibits IL-1␤suppression of PDGF-D transcription, whereas nonsense siRNA has no effect (Fig. 2F, top). Western blotting confirmed that the siRNA, but not the nonsense siRNA, inhibits cellular p65 levels (Fig. 2F, bottom).

IRF-1 Is Induced by IL-1␤ in SMCs and Interacts with a Second Novel Binding Element in the PDGF-D Promoter-Further
inspection of the PDGF-D promoter revealed a putative IRF-1 recognition element in the PDGF-D promoter at Ϫ840 GAAAAGTGAAA Ϫ830 . Western blotting demonstrated that IRF-1 accumulated in SMC nuclei within 1 h of exposure to IL-1␤ (Fig.  3A, right). EMSA studies with 32 Plabeled Oligo D Ϫ847/Ϫ823 spanning this element revealed IL-1␤-inducible nucleoprotein complex formation within 1 h (Fig. 3A, left). Mutation of the Ϫ840 GAAAAGTG-AAA Ϫ830 sequence to Ϫ840 GAC-AAGTGACT Ϫ830 in 32 P-labeled mOligo D Ϫ847/Ϫ823 abrogated an IL-1␤-inducible complex formation (Fig. 3A). Antibody elimination experiments demonstrated the presence of IRF-1 in this IL-1␤-inducible complex (Fig. 3B). This is the first association of IRF-1 with any PDGF promoter. The EMSA also showed that this complex, formed with 32 P-labeled Oligo D Ϫ847/Ϫ823 contains HDAC-1 (Fig.  3B). HDACs play a central role in the epigenetic regulation of gene expression. HDAC-1 is a predominantly nuclear 482-amino acid HDAC isoform (34). Germline deletion of HDAC-1 results in embryonic lethality, which cannot be sufficiently compensated by HDAC2 (35), and like several other HDACs, is a drug target in cancer (34). HDAC-1 is also a component of the p65/ 32 P-labeled Oligo D Ϫ481/Ϫ457 complex in Fig. 2A (left).
IL-1␤ Induces Interaction of p65 and IRF-1 Together and with the PDGF-D Promoter-Previous studies in certain other genes, such as VCAM-1 (36), have demonstrated that p65 and IRF-1 cooperatively activate gene expression. To the best of our knowledge, this has not been shown in SMCs nor is there any report of negative cooperation between these factors. We hypothesized that these factors bind not only one another but also the PDGF-D promoter in response to SMC exposure to IL-1␤. Immunoprecipitation analysis and Western blotting was performed with lysates of cells that had been exposed to IL-1␤ for various times (0 -2 h). We pulled down with IRF-1 antibodies and then immunoblotted for p65. Alternatively, we immunoprecipitated with p65 antibodies and then probed for IRF-1. p65 and IRF-1 formed a complex within 1 h of IL-1␤ exposure using either of these conditions, and complex formation was transient (Fig. 4A, left). ChIP analysis was performed to demonstrate the inducible interaction of endogenous proteins with A, time course of IL-1␤ suppression of PDGF-D mRNA expression. SMCs were treated with 10 ng/ml of IL-1␤ for the times indicated, mRNA was extracted, and semi-quantitative reverse transcription-PCR was performed for PDGF-D and VCAM-1. GAPDH was also amplified as a loading control. B, quantitative real-time PCR (left) and transient transfection analysis (right) confirms IL-1␤ suppression of PDGF-D mRNA levels and PDGF-D promoter activity, respectively. In the latter, SMCs were transfected with 10 g of p1168D-luciferase (luc) construct and 1 g of pRL-null and then exposed to IL-1␤ for 8 h. firefly luciferase activity was normalized to Renilla. C, dose-response of IL-1␤ suppression of PDGF-D mRNA levels. SMCs were treated with different amounts of IL-1␤ for 4 h prior to extraction of total RNA and assessment of PDGF-D mRNA levels by real-time quantitative PCR. D, IL-1␤ suppression of PDGF-D protein expression. SMCs were treated with IL-1␤ for various times, and 20 g total protein was used in Western blot analysis with PDGF-D antibodies. No Addition refers to samples that did not receive IL-1␤. Data are representative of at least three experiments. Error bars represent S.E. performed in triplicate. An asterisk denotes p Ͻ 0.05. the authentic PDGF-D promoter. IRF-1, p65, and HDAC-1 were not detected at the PDGF-D promoter under basal conditions but were both bound to the promoter within 1 h of SMC exposure to IL-1␤ (Fig. 4B). EMSA with recombinant proteins revealed that 32 P-labeled Oligo D Ϫ481/Ϫ457 directly bound p65, whereas HDAC-1 and IRF-1 did not bind (Fig. 4C). Conversely, 32 P-labeled mOligo D Ϫ847/Ϫ823 was bound by recombinant IRF-1, but not p65 or HDAC-1 (Fig. 4C). These binding data with recombinant proteins, taken together with the ChIP and EMSA studies, indicate that HDAC-1 occupancy of the PDGF-D promoter is dynamically regulated by IL-1␤ and suggest that it partners both IRF-1 and p65 without directly contacting the IRF-1 and p65 binding elements.
NF-B and IRF-1 DNA-binding Elements in the PDGF-D Promoter Are Required for NF-B, IRF-1, and IL-1␤ Repression of PDGF-D Promoter Activity-Transient transfection and mutational analysis revealed that IRF-1 suppression of the PDGF-D promoter was dependent on the Ϫ840 GAAAAGTGAAA Ϫ830 element and that p65 suppression of PDGF-D promoter activity was dependent on the Ϫ471 GGGATTCTAC Ϫ462 element (Fig.  5A, left). IL-1␤ repression of the PDGF-D promoter was reversed by mutation in either site (Fig. 5A, right).
To demonstrate the requirement of endogenous IRF-1 in IL-1␤ repression of the PDGF-D promoter, we performed an additional transient transfection experiment. IL-1␤-mediated suppression of PDGF-D transcription was rescued by 100 nM of IRF-1 siRNA, but not by an identical concentration of irrelevant siRNA (Fig. 5B, upper). Western blotting confirmed that the siRNA, but not the nonsense siRNA, inhibited IL-1␤-inducible IRF-1 expression (Fig. 5B, lower).

TABLE 1 Proximal region of the human PDGF-D promoter
The NF-B and IRF-1 binding sites are indicated in blue. ChIP amplicon (Ϫ863 to Ϫ445) is indicated in red. Four putative Ets sites described previously (12) (Ets-D1, -D2, -D3, and -D4) are in bold and underlined. The transcriptional start site is italicized and bolded (ccAGCGC). The primers originally used for primer extension analysis (12) are bolded/underlined and labeled as primer A and B. Capital letters represent 5Ј-untranslated region (UTR). atg denotes the predicted translational start site. Putative binding sites for other factors are indicated.

Repression of PDGF-D Transcription by IL-1␤ Involves Histone
Deacetylation and Interaction of HDAC-1 with IRF-1 and p65-The role of the chromatin environment in regulation of IL-1␤ PDGF-D expression was next investigated. Rat aortic SMCs were pretreated for 18 h with trichostatin A, a HDAC inhibitor, and then treated with IL-1␤ for another 4 h. Repression of PDGF-D mRNA expression by IL-1␤ was completely blocked by trichostatin A (Fig. 6A, left). In contrast, trichostatin A had no effect on PDGF-D levels in the absence of IL-1␤ (Fig. 6A, left). This data implicates HDACs in the negative control of PDGF-D in SMCs exposed to the cytokine. Co-immunoprecipitation experiments revealed that IL-1␤ indeed triggers the inducible interaction between IRF-1, p65, and HDAC-1 (Fig. 6A, right).
Negative regulation of PDGF-D expression by IL-1␤ is clearly an unusual phenomenon for this cytokine, which is well known for its stimulatory roles. The present study demonstrates the requirement of HDAC-1 in this process. IL-1␤ induces HDAC-1 complex formation with IRF-1 and HDAC-p65 (Fig. 6A, right) and the enrichment of HDAC-1, p65, and IRF-1 at the PDGF-D promoter (Fig. 4B).
To provide further insight on the role of HDAC-1 in IL-1␤-dependent transcriptional repression, we performed additional ChIP experiments, this time using two separate PDGF-D promoter amplicons. One amplicon ( Ϫ689 PDGF-D Ϫ445 ) spans the NF-B recognition motif

. NF-B p65 induced by IL-1␤ interacts with a novel element in the PDGF-D promoter and inhibits PDGF-D transcription.
A, left, EMSA was performed with 32 P-labeled Oligo D Ϫ481/Ϫ457 and nuclear extracts from SMCs treated with IL-1␤ for 1 h. Two inducible complexes were formed, A and B. The upper inset, from a longer exposure of the autoradiogram, more clearly indicates Complex A. These complexes were eliminated with 4 g of (supershift grade) antibodies to p65, p50, c-Rel, RelB, IRF-1, or HDAC-1 for 20 min prior to the addition of the probe. Middle, EMSA with 32 P-labeled mOligo D Ϫ481/Ϫ457 bearing a mutation in the putative NF-B element demonstrates no inducible complex with nuclear extracts. Right, EMSA with recombinant human (rh) p65 protein and 32 P-labeled Oligo D Ϫ481/Ϫ457 or 32 P-labeled mOligo D Ϫ481/Ϫ457 . ns denotes nonspecific band. B, 50 g of cytoplasmic or nuclear extract from SMCs treated with or without IL-1␤ for 1 h were used for Western blotting. C, p65 inhibits PDGF-D transcription (left) and mRNA expression (right). SMCs were co-transfected with 10 g of p1168D-luciferase (luc) and 3 g of the expression plasmid (left). Alternatively, SMCs were transfected with 20 or 40 g of p65-pcDNA3 (made up to total 40 g with pcDNA3). After 24 h, luciferase activity was measured in the lysates, or total RNA was extracted, and real-time quantitative PCR for PDGF-D was performed. D, left, p50 has no effect on PDGF-D transcription, either alone or in conjunction with p65. Transient transfection was performed with various combinations of p65-pcDNA3 (3 g), p50-pUNO (3 g), and 10 g of p1168D-luciferase. Right, p65 activates the control reporter construct, pNF-B-luc (22). E, SMCs were pre-treated with 10 g/ml of the p65 inhibitor Oridonin for 1 h prior to incubation with 10 ng/ml of IL-1␤ for 4 h. Total RNA was extracted, and real-time quantitative PCR was performed to assess PDGF-D mRNA levels. F, SMCs were transfected with p1168D-luciferase and 100 nM siRNA, and after 24 h, incubated with 10 ng/ml IL-1␤ for 8 h prior to harvest and assessment of luciferase activity (left) or total p65 levels by Western blotting (right). Data are representative of at least two independent experiments. Band density was quantified by scanning densitometry. siRNAns, nonspecific siRNA. No Addition refers to samples that did not receive IL-1␤. An asterisk denotes p Ͻ 0.05.

FIGURE 3. IRF-1 is induced by IL-1␤ and interacts with an element in the PDGF-D promoter.
A, left, EMSA was performed by incubating 32 P-labeled Oligo D Ϫ847/Ϫ823 and nuclear extracts of SMCs treated with IL-1␤ for various times, or 32 P-labeled Oligo D Ϫ847/Ϫ823 bearing a mutation in the putative IRF-1 binding motif. Alternatively, Western blot analysis (right) demonstrates the presence of IRF-1 in the extracts. B, EMSA was performed using nuclear extracts of SMCs exposed to IL-1␤ for 1 h incubated with antibodies (Ab; 4 g) to IRF-1, HDAC-1, and the indicated factors for 20 min prior to the addition of 32 P-labeled Oligo D Ϫ847/Ϫ823 . ns, non-specific. No Addition refers to samples that did not receive IL-1␤. Data are representative of at least two independent experiments. p65 and IRF-1, at both regions of the promoter (Fig, 6B). HDAC-1 siRNA rescues the PDGF-D promoter from IL-1␤ repression (Fig.  6D, upper). Importantly, HDAC-1 siRNA also blocks the IL-1␤-inducible interaction of IRF-1 with p65, as demonstrated by either pulling down with IRF-1 antibodies and immunoblotting with p65 antibodies or pulling down with p65 antibodies and immunoblotting for IRF-1 (Fig. 4A, right). These results indicate that HDAC-1 is enriched at the PDGF-D promoter in cells exposed to IL-1␤ and forms a cytokine-inducible gene silencing complex with p65 and IRF-1 (Fig. 6E).

DISCUSSION
In this study, we have shown that proinflammatory cytokine IL-1␤ down-regulates PDGF-D expression in a time-and dose-dependent manner. This contrasts with levels of VCAM-1, which, as expected, are induced by the cytokine. p65, induced by IL-1␤, interacts with a novel element in the PDGF-D promoter and inhibits PDGF-D transcription. IRF-1 is also induced by IL-1␤ and binds to a second novel element in the PDGF-D promoter. ChIP experiments with two separate sets of primers, one amplicon ( Ϫ689 PDGF-D Ϫ445 ) spanning the NF-B recognition motif ( Ϫ471 GGGATTCTAC Ϫ462 ), with the other ( Ϫ863 PDGF-D Ϫ674 ) spanning the IRF-1 binding site further upstream ( Ϫ840 GAAAAGTGAAA Ϫ830 ). p65, IRF-1, and HDAC-1 are enriched at each region of the PDGF-D promoter in cells exposed to IL-1␤ (Fig. 6E). Co-immunoprecipitation studies demonstrate that IRF-1 and p65 form a complex containing HDAC-1 within 1 h of exposure to IL-1␤ (Figs. 4A, left and 6A,  right). Moreover, HDAC-1 siRNA ablates complex formation with IRF-1 and p65 (Fig. 4A, right) and in ChIP analysis, abrogates IRF-1 and p65 occupancy of the PDGF-D promoter (Fig. 6B). NF-B and IRF-1 are transcription factors activated by a variety of proinflammatory A, left, SMCs were transfected with 10 g of promoter plasmid construct p1168D-luciferase (luc), p1168DmKBluc (mutation in the NF-B binding site), p1168DmIRF1-luciferase (mutation in the IRF-1 binding site), 1 g of pRL-null, together with 3 g pcDNA3, p65-pcDNA3, or IRF-1-pcDNA3. Luciferase activity was determined after 24 h. Right, SMCs 24 h after being transfected with 10 g of promoter construct and 1 g of pRL-null were treated with IL-1␤ for 8 h prior to assessment of luciferase activity in the lysates. B, upper, SMCs were transfected with 10 g of p1168D-luciferase and IRF-1 siRNA or nonspecific siRNA. After 16 h, the cells were exposed to IL-1␤ for a further 8 h. Luciferase activity in the lysates was determined after 8 h. Error bars represent S.E. performed in triplicate. An asterisk denotes p Ͻ 0.05 compared with no treatment control. Lower panel, Western blot analysis for IRF-1 in cells transfected with IRF-1 siRNA and exposed to 10 ng/ml IL-1␤ for 1 h. No Addition refers to samples that did not receive IL-1␤. Data are representative of at least two independent experiments. siRNAns, nonspecific siRNA.
cytokines including IL-1␤ and play a positive regulatory role in many genes implicated in the pathogenesis of vascular disease. These include VEGF (37), VCAM-1 (36), and iNOS (38) in SMCs. Cooperativity between NF-B and IRF-1 has been reported previously. For example, recent studies demonstrate that IRF-1 and NF-B form a functional complex at the LTR-B sites in the human immunodeficiency virus type 1 enhancer, which is abrogated by specific mutations in NF-B sites in the enhancer region (39). Similarly, cooperation between IRF-1 and NF-B p65 positively regulates IL-6 (40) and iNOS (41) transcription. In the present study, IL-1␤ simulates p65⅐IRF-1 complex formation, but rather than activating PDGF-D transcription, p65 and IRF-1 bind to and repress the PDGF-D promoter. HDAC-1 is enriched at the PDGF-D promoter in cells exposed  -1 and p65. A, left, SMCs were arrested overnight, pretreated with 1 M trichostatin A for 18 h before incubation with 10 ng/ml of IL-1␤ for another 4 h. Total RNA was extracted and real-time quantitative PCR was performed for PDGF-D mRNA. An asterisk denotes p Ͻ 0.05 compared with no treatment control. Right, SMCs were arrested for 24 h and treated with 10 ng/ml of IL-1␤ for 1 h, and total protein was extracted. 500 g of protein was used for the co-immunoprecipitation (IP) with 5 g of anti-IRF-1 or anti-p65 antibody, prior to immunoblot (IB) for HDAC-1. B, ChIP analysis was performed using two separate PDGF-D promoter amplicons, Ϫ689 PDGF-D Ϫ445 and Ϫ863 PDGF-D Ϫ674 with cells transfected with 100 nM siRNA and exposed to 10 ng/ml IL-1␤ for 1 h. Total input (TI) and IgG serve as positive and negative controls, respectively. C, time-dependent enrichment of HDAC-1 at the PDGF-D promoter in response to IL-1␤. Total input and IgG serve as positive and negative controls. No Addition refers to samples that did not receive IL-1␤. D, HDAC-1 siRNA rescues the PDGF-D promoter from IL-1␤ repression. SMCs were transfected with p1168D-luciferase (luc) and 100 nM siRNA, and after 24 h, incubated with 10 ng/ml IL-1␤ for 8 h prior to harvest, and assessment of luciferase activity (upper). Alternatively, Western blot showing that HDAC-1 siRNA reduces HDAC-1 expression (lower). Data are representative of at least two independent experiments. No Addition refers to samples that did not receive IL-1␤. E, schematic depicting the formation of a cytokine-inducible gene silencing complex involving HDAC-1, p65, and IRF-1. The ChIP amplicon Ϫ689 PDGF-D Ϫ445 is indicated in blue, whereas amplicon Ϫ863 PDGF-D Ϫ674 is indicated in red.