Serine 26 in Early Growth Response-1 Is Critical for Endothelial Proliferation, Migration, and Network Formation.

Background Vascular endothelial cell proliferation, migration, and network formation are key proangiogenic processes involving the prototypic immediate early gene product, Egr-1 (early growth response-1). Egr-1 undergoes phosphorylation at a conserved Ser26 but its function is completely unknown in endothelial cells or any other cell type. Methods and Results A CRISPR/Cas9 strategy was used to introduce a homozygous Ser26>Ala mutation into endogenous Egr-1 in human microvascular endothelial cells. In the course of generating mutant cells, we produced cells with homozygous deletion in Egr-1 caused by frameshift and premature termination. We found that Ser26 mutation in Egr-1, or Egr-1 deletion, perturbed endothelial cell proliferation in models of cell counting or real-time growth using the xCELLigence System. We found that Ser26 mutation or Egr-1 deletion ameliorated endothelial cell migration toward VEGF-A165 (vascular endothelial growth factor-A) in a dual-chamber model. On solubilized basement membrane preparations, Ser26 mutation or Egr-1 deletion prevented endothelial network (or tubule) formation, an in vitro model of angiogenesis. Flow cytometry further revealed that Ser26 mutation or Egr-1 deletion elevated early and late apoptosis. Finally, we demonstrated that Ser26 mutation or Egr-1 deletion increased VE-cadherin (vascular endothelial cadherin) expression, a regulator of endothelial adhesion and signaling, permeability, and angiogenesis. Conclusions These findings not only indicate that Egr-1 is essential for endothelial cell proliferation, migration, and network formation, but also show that point mutation in Ser26 is sufficient to impair each of these processes and trigger apoptosis as effectively as the absence of Egr-1. This highlights the importance of Ser26 in Egr-1 for a range of proangiogenic processes.

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

AIMS: In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent. METHODS AND RESULTS: We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B's ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner. CONCLUSION: These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative disease.

Thermostable small-molecule inhibitor of angiogenesis and vascular permeability that suppresses a pERK-FosB/ΔFosB-VCAM-1 axis.

Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months' storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Extracellular signal-regulated kinase-1 phosphorylates early growth response-1 at serine 26.

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser12 and Ser26 with the latter ∼12 times more abundant than Ser12. Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser26>Ala26 mutant Egr-1) revealed that Ser26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser26. pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1.

Developing Neolignans as Proangiogenic Agents: Stereoselective Total Syntheses and Preliminary Biological Evaluations of the Four Guaiacylglycerol 8-O-4'-Coniferyl Ethers.

Stereoselective total syntheses of the four stereoisomeric forms of guaiacylglycerol 8-O-4'-coniferyl ether, viz., compounds 1, ent-1, 2, and ent-2, have been established. The key step involves an Evans/Seebach auxiliary-controlled and syn-selective aldol process followed, in the reaction sequences leading to the anti-compounds, by a Mitsunobu reaction involving a benzylic alcohol residue. The proangiogenic properties of the synthetic materials were evaluated in a human microvascular endothelial cell tubule formation assay, thus revealing that they are all active, with the 8S-configured compounds 1 and 2 being the most potent.

DNAzyme targeting c-jun suppresses skin cancer growth.

Worldwide, one in three cancers is skin-related, with increasing incidence in many populations. Here, we demonstrate the capacity of a DNAzyme-targeting c-jun mRNA, Dz13, to inhibit growth of two common skin cancer types-basal cell and squamous cell carcinomas-in a therapeutic setting with established tumors. Dz13 inhibited tumor growth in both immunodeficient and immunocompetent syngeneic mice and reduced lung nodule formation in a model of metastasis. In addition, Dz13 suppressed neovascularization in tumor-bearing mice and zebrafish and increased apoptosis of tumor cells. Dz13 inhibition of tumor growth, which required an intact catalytic domain, was due in part to the induction of tumor immunity. In a series of good laboratory practice-compliant toxicology studies in cynomolgus monkeys, minipigs, and rodents, the DNAzyme was found to be safe and well tolerated. It also did not interfere in more than 70 physiologically relevant in vitro bioassays, suggesting a reduced propensity for off-target effects. If these findings hold true in clinical trials, Dz13 may provide a safe, effective therapy for human skin cancer.

Yin Yang-1 inhibits tumor cell growth and inhibits p21WAF1/Cip1 complex formation with cdk4 and cyclin D1.

The GLI-Krüppel zinc finger factor yin yang-1 (YY1) is a complex protein that regulates a variety of processes including transcription, proliferation, development and differentiation. YY1 inhibits cell growth in a cell type-specific manner. The role played by YY1 in its control of tumor cell growth is unclear and controversial. We show here that YY1 can suppress the growth of different tumor cell types in vitro, including human breast carcinoma cells and glioblastoma cells. YY1 also blocked the growth of 13762 MAT mammary adenocarcinoma isografts in rats. YY1 inhibited 13762 MAT tumor growth by approximately 80% compared with the GFP alone group 21 days after injection. YY1 inhibited proliferating cell nuclear antigen (PCNA) expression and pRbSer249/Thr252 phosphorylation without influencing tumor microvascular density. Moreover, YY1 inhibited p21WAF1/Cip1 complex formation with cdk4 and cyclin D1. These findings demonstrate that YY1 can negatively regulate the growth of multiple malignant cell types.

Interplay between heme oxygenase-1 and the multifunctional transcription factor yin yang 1 in the inhibition of intimal hyperplasia.

RATIONALE: induction of heme oxygenase (HO)-1 protects against experimental atherosclerotic diseases, and certain pharmacological HO-1 inducers, like probucol, inhibit the proliferation of vascular smooth muscle cells and, at the same time, promote the growth of endothelial cells in vivo and in vitro. OBJECTIVE: because such cell-specific effects are reminiscent of the action of the transcription factor Yin Yang (YY)1, we tested the hypothesis that there is a functional relationship between HO-1 and YY1. METHODS AND RESULTS: we report that probucol increases the number of YY1(+) cells in rat carotid artery following balloon injury at a time coinciding with increased HO-1 expression. The drug also induces the expression of YY1 mRNA and protein in rat aortic smooth muscle cells (RASMCs) in vitro, as do other known HO-1 inducers (tert-butylhydroquinone and hemin) and overexpression of HO-1 using a human HMOX1 cDNA plasmid. Conversely, overexpression of YY1 induces expression of HO-1 in RASMCs. Induction of YY1 expression is dependent on HO-1 enzyme activity and its reaction product CO, because pharmacological inhibition of heme oxygenase activity or CO scavenging block, whereas exposure of RASMCs to a CO-releasing molecule increases, YY1 expression. Furthermore, RNA interference knockdown of YY1 prevents probucol or adeno-HO-1 from inhibiting RASMC proliferation in vitro and neointimal formation in vivo. CONCLUSIONS: our findings show, for the first time, that HO-1 functionally interplays with the multifunctional transcription factor YY1 and that this interplay explains some of the protective activities of HO-1.

Macrophage migration inhibitory factor increases leukocyte-endothelial interactions in human endothelial cells via promotion of expression of adhesion molecules.

Macrophage migration inhibitory factor (MIF) has been shown to promote leukocyte-endothelial cell interactions, although whether this occurs via an effect on endothelial cell function remains unclear. Therefore, the aims of this study were to examine the ability of MIF expressed by endothelial cells to promote leukocyte adhesion and to investigate the effect of exogenous MIF on leukocyte-endothelial interactions. Using small interfering RNA to inhibit HUVEC MIF production, we found that MIF deficiency reduced the ability of TNF-stimulated HUVECs to support leukocyte rolling and adhesion under flow conditions. These reductions were associated with decreased expression of E-selectin, ICAM-1, VCAM-1, IL-8, and MCP-1. Inhibition of p38 MAPK had a similar effect on adhesion molecule expression, and p38 MAPK activation was reduced in MIF-deficient HUVECs, suggesting that MIF mediated these effects via promotion of p38 MAPK activation. In experiments examining the effect of exogenous MIF, application of MIF to resting HUVECs failed to induce leukocyte rolling and adhesion, whereas addition of MIF to TNF-treated HUVECs increased these interactions. This increase was independent of alterations in TNF-induced expression of E-selectin, VCAM-1, and ICAM-1. However, combined treatment with MIF and TNF induced de novo expression of P-selectin, which contributed to leukocyte rolling. In summary, these experiments reveal that endothelial cell-expressed MIF and exogenous MIF promote endothelial adhesive function via different pathways. Endogenous MIF promotes leukocyte recruitment via effects on endothelial expression of several adhesion molecules and chemokines, whereas exogenous MIF facilitates leukocyte recruitment induced by TNF by promoting endothelial P-selectin expression.

Phosphorylation and acetylation of histone H3 and autoregulation by early growth response 1 mediate interleukin 1beta induction of early growth response 1 transcription.

OBJECTIVE: The transcription factor early growth response (EGR)-1 has been implicated as a key vascular phenotypic switch through its control of inducible transcription. EGR-1 autoregulation, and histone modification in the EGR-1 promoter, represent key mechanisms in EGR-1 control, but have not been explored. METHODS AND RESULTS: We demonstrate that EGR-1 regulates its own transcription and that this involves histone H3 phosphorylation and acetylation. EGR-1 transactivates its promoter in smooth muscle cells exposed to interleukin (IL) 1beta through a novel cis-acting element (-211/-203). PD98059, which inhibits mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) attenuates IL-1beta-inducible phosphorylation of extracellular signal-regulated kinase 1/2 and mitogen and stress-activated protein kinases 1/2; and reduces levels of phosphorylated and acetylated histone H3. Histone deacetylase inhibition enhances EGR-1 transcription in response to cytokine. Conversely, suppression of histone modification with mitogen and stress-activated protein kinase 1/2 short interfering RNA, or the histone H3 acetyltransferase inhibitor Garcinol, inhibits IL-1beta-inducible EGR-1 transcription. EGR-1 interacts with the acetyltransferase p300. Acetylated H3 and phosphorylated H3 are enriched at the promoter of EGR-1; and EGR-1 is enriched at the promoters of tissue factor and plasminogen activator inhibitor 1 in response to IL-1beta, and attenuated by PD98059, Garcinol, and mitogen and stress-activated protein kinase 1/2 short interfering RNA. CONCLUSIONS: IL-1beta induction of EGR-1 transcription involves histone H3 phosphorylation, acetylation, and autoregulation by EGR-1.

Angiotensin II-inducible platelet-derived growth factor-D transcription requires specific Ser/Thr residues in the second zinc finger region of Sp1.

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-zeta. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-zeta and have generated novel anti-peptide antibodies recognizing the PKC-zeta-phosphorylated form of Sp1. Angiotensin II, which activates PKC-zeta phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II-inducible gene expression.

Yin Yang-1 inhibits vascular smooth muscle cell growth and intimal thickening by repressing p21WAF1/Cip1 transcription and p21WAF1/Cip1-Cdk4-cyclin D1 assembly.

Vascular injury initiates a cascade of phenotype-altering molecular events. Transcription factor function in this process, particularly that of negative regulators, is poorly understood. We demonstrate here that the forced expression of the injury-inducible GLI-Krüppel zinc finger protein Yin Yang-1 (YY1) inhibits neointima formation in human, rabbit and rat blood vessels. YY1 inhibits p21(WAF1/Cip1) transcription, prevents assembly of a p21(WAF1/Cip1)-cdk4-cyclin D1 complex, and blocks downstream pRb(Ser249/Thr252) phosphorylation and expression of PCNA and TK-1. Conversely, suppression of endogenous YY1 elevates levels of p21(WAF1/Cip1), PCNA, pRb(Ser249/Thr252) and TK-1, and increases intimal thickening. YY1 binds Sp1 and prevents its occupancy of a distinct element in the p21(WAF1/Cip1) promoter without YY1 itself binding the promoter. Additionally, YY1 induces ubiquitination and proteasome-dependent degradation of p53, decreasing p53 immunoreactivity in the artery wall. These findings define a new role for YY1 as both an inducer of p53 instability in smooth muscle cells, and an indirect repressor of p21(WAF1/Cip1) transcription, p21(WAF1/Cip1)-cdk4-cyclin D1 assembly and intimal thickening.

Inducible platelet-derived growth factor D-chain expression by angiotensin II and hydrogen peroxide involves transcriptional regulation by Ets-1 and Sp1.

Platelet-derived growth factor D-chain (PDGF-D) is the newest member of the PDGF family of mitogens and chemoattractants expressed in a wide variety of cell types, including vascular smooth muscle cells (SMCs). The molecular mechanisms regulating PDGF-D transcription are not known. Primer extension analysis mapped a single transcriptional start site to the ccAGCGC motif with several potential Ets motifs located upstream. Ets-1, but not Ets-1 bearing only the DNA-binding domain, activates the PDGF-D promoter and mRNA expression in SMCs. Ets site D3 ((-470)GGAT(-467)) is singly required for basal and Ets-1-inducible PDGF-D promoter-dependent expression. D3 supports the interaction of endogenous and recombinant Ets-1 and Sp1. Sp1, like Ets-1, induces PDGF-D transcription and mRNA expression, which is blocked by mutant Ets-1. H2O2 stimulates Ets-1, but not Sp1, and activates D3-dependent PDGF-D transcription. Ets-1 and Sp1 siRNA block peroxide-inducible PDGF-D expression. Angiotensin II (ATII) induction of PDGF-D and Ets-1 was blocked by prior incubation of the cells with PEG-catalase, but not BSA, indicating that ATII-inducible Ets-1 and PDGF-D expression is mediated via H2O2. Thus, 2 separate trans-acting factors regulate PDGF-D transcription, alone and in response to oxidative stress.

Ets-1 stimulates platelet-derived growth factor A-chain gene transcription and vascular smooth muscle cell growth via cooperative interactions with Sp1.

The platelet-derived growth factor (PDGF) family of ligands (composed of A-, B-, C-, and D-chains), potent mitogens, and chemoattractants for cells of mesenchymal origin has been implicated in numerous vascular pathologies involving smooth muscle cell (SMC) hyperplasia. Understanding the molecular mechanisms mediating PDGF transcription would provide new insights into strategies to control PDGF-dependent pathophysiologic processes. We demonstrated previously that PDGF-A expression is under the positive regulatory influence of Sp1, Sp3, and Egr-1 and is negatively controlled by GCF2, NF-1(X), and WT-1. In this article, we demonstrate that Ets-1 induces PDGF-A expression in primary rat aortic SMCs at the level of transcription and mRNA expression. Electrophoretic mobility shift, supershift, and mutational analyses revealed a functional role for the (-555)TTCC(-552) motif in the PDGF-A promoter that binds endogenous Ets-1. Chromatin immunoprecipitation analysis showed the interaction of endogenous and exogenous Ets-1 or glutathione S-transferase-tagged Ets-1, bearing only the DNA-binding domain with the authentic PDGF-A promoter. Conversely, dominant-negative mutant of Ets-1 blocked the promoter interaction of endogenous Ets-1. Overexpression of Ets-1 but not the mutant form of Ets-1 activates the PDGF-A promoter cooperatively with Sp1. Sp1, which interacts with Ets-1, failed to induce PDGF-A promoter-dependent expression if the promoter contained a site-specific mutation in this novel Ets-binding site. Small interfering RNA to Ets-1 and Sp1 blocked PDGF-BB- and serum-inducible PDGF-A expression. SMC growth was stimulated by Ets-1 and Sp1 separately and further increased by both factors together. Ets-1-inducible mitogenesis is blocked by antibodies neutralizing PDGF-A and involves activation of the PDGF alpha-receptor, which binds PDGF-A. These findings identify a functional cis-acting element for Ets-1 in the PDGF-A promoter and demonstrate that Sp1 and Ets-1 cooperatively activate PDGF-A transcription in vascular SMCs.

Regulation of inducible heparanase gene transcription in activated T cells by early growth response 1.

Cleavage of heparan sulfate by the beta-D-endoglucuronidase heparanase (HPSE) is a fundamental event in a number of important physiological processes including inflammation, wound healing, and angiogenesis. HPSE activity has also been directly correlated with pathological conditions such as tumor growth and metastasis and autoimmune disease. The tight regulation of HPSE expression and function is critical to ensure homeostasis of the normal physiological processes to which it contributes and to prevent imbalance toward pathological situations. Little is known about the transcriptional mechanisms that regulate HPSE expression. In this study we have shown human HPSE gene transcription in Jurkat T cells is induced upon activation. Functional analysis of the HPSE promoter has identified a 280-bp region that is highly inducible. Mutation studies together with supershift experiments have identified a 4-bp motif that binds the transcription factor early growth response-1 (Egr1) and is critical in regulating inducible HPSE gene transcription. Furthermore, the overexpression of Egr1 resulted in the enhanced activation of the HPSE promoter. By using MAPK pathway inhibitors, we have also shown that inducible expression of HPSE mRNA and the activity of the 280-bp HPSE promoter element are dependent on the ERK1/2 (MEK1/2) pathway. This pathway is critical for induction of Egr1 expression at both the mRNA and protein level in T cells, an observation that provides further support to Egr1 playing an important role as a key activator of HPSE expression. In addition, HPSE and Egr1 were shown to co-localize by immunohistochemistry to invading mononuclear leukocytes in actively induced experimental autoimmune encephalomyelitis in rats. These findings provide the first insight into the mechanisms controlling inducible transcription of the HPSE gene, and could represent an important lead into understanding how HPSE expression is deregulated in metastatic tumor cells.

Blockade of vascular smooth muscle cell proliferation and intimal thickening after balloon injury by the sulfated oligosaccharide PI-88: phosphomannopentaose sulfate directly binds FGF-2, blocks cellular signaling, and inhibits proliferation.

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

NF1/X represses PDGF A-chain transcription by interacting with Sp1 and antagonizing Sp1 occupancy of the promoter.

The regulatory mechanisms mediating basal and inducible platelet-derived growth factor (PDGF)-A expression have been the focus of intense recent investigation, but repression of PDGF-A expression is largely unexplored. Here we isolated a nuclear factor that interacts with the proximal region of the PDGF-A promoter using bulk binding assays and chromatography techniques. Peptide mass fingerprint and supershift analysis revealed this DNA-binding protein to be NF1/X. NF1/X repressed PDGF-A promoter-dependent transcription and endogenous mRNA expression, which was reversible by oligonucleotide decoys bearing an NF1/X-binding site. Mutation in the DNA-binding domain of NF1/X abolished its repression of PDGF-A promoter. NF1/X antagonized the activity of a known activator of the PDGF-A chain, Sp1, by inhibiting its occupancy of the proximal PDGF-A promoter. NF1/X physically and specifically interacts with Sp1 via its subtype-specific domain and blocks Sp1 induction of the promoter. NF1/X residues 311-416 mediated NF1/X suppression of basal PDGF-A transcription, whereas residues 243-416 were required for NF1/X repression of Sp1-inducible promoter activity. These findings demonstrate that repression of PDGF-A gene transcription is governed by interplay between NF1/X and Sp1.