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Published as doi: 10.1096/fj.07-8573com.
 (The FASEB Journal. 2008;22:225-235.)
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(The FASEB Journal. 2008;22:225-235.)
© 2008 FASEB

GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression

Martin F. Crook*, Michelle Olive*, Hai-Hui Xue{dagger},1, Thomas H. Langenickel*, Manfred Boehm*, Warren J. Leonard{dagger} and Elizabeth G. Nabel*,2

* Cardiovascular Branch and

{dagger} Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

2Correspondence: National Heart, Lung, and Blood Institute, Bldg. 31/Rm. 5A48, 31 Center Dr., Bethesda, MD 20892, USA; E-mail: nabele{at}nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyclin-dependent kinase inhibitor p27Kip1 arrests cell cycle progression through G1/S phases and is regulated by phosphorylation of serine/threonine residues. Recently, we identified the serine/threonine kinase, KIS, which phosphorylates p27Kip1 on serine 10 leading to nuclear export of p27Kip1 and protein degradation. However, the molecular mechanisms of transcriptional activation of the human KIS gene and its biological activity are not known. We mapped the transcription initiation site ~116 bp 5' to the translation start site, and sequences extending to –141 were sufficient for maximal promoter activity. Mutation in either of two Ets-binding sites in this region resulted in an approximately 75–80% decrease in promoter activity. These sites form at least 3 specific complexes, which contained GA-binding protein (GABP). Knocking down GABP{alpha} by siRNA in vascular smooth muscle cells (VSMCs) diminished KIS gene expression and reduced cell migration. Correspondingly, in serum stimulated GABP{alpha}-deficient mouse embryonic fibroblasts (MEFs), KIS gene expression was also significantly reduced, which was associated with an increase in p27Kip1 protein levels and a decreased percentage of cells in S-phase. Consistent with these findings, following vascular injury in vivo, GABP{alpha}-heterozygous mice demonstrated reduced KIS gene expression within arterial lesions and these lesions were significantly smaller compared to GABP+/+ mice. In summary, serum-responsive GABP binding to Ets-binding sites activates the KIS promoter, leading to KIS gene expression, cell migration, and cell cycle progression.—Crook, M. F., Olive, M., Xue, H.-H., Langenickel, T. H., Boehm, M., Leonard, W. J., Nabel, E. G. GA-binding protein regulates KIS gene expression, cell migration, and cell cycle progression.


Key Words: kinase interacting strathmin • p27Kip1 • vascular smooth muscle cells • vascular biology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE PROTEIN p27Kip1 IS A MEMBER of the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) (1) and plays a critical role in regulating the progression through the G1/S phases of the cell cycle (1) . Inactivation of p27Kip1 is observed in diseases characterized by cell proliferation, including cancers and cardiovascular diseases (2 3 4) . Phosphorylation on Ser-10 of p27Kip1 triggers changes in the abundance of the protein and its subcellular localization by nuclear export to the cytoplasm (5 6 7) . Recently, we identified a serine/threonine kinase, KIS, that phosphorylates Ser-10 and leads to nuclear export of p27Kip1 and cell cycle progression (6) . KIS contains a putative tyrosine kinase catalytic domain as well as a serine/threonine kinase catalytic domain and a novel RNA recognition binding motif (RRM) that is highly homologous to the RNA splicing factor U2AF (8) . KIS expression is activated by mitogens during G0/G1 phase of the cell cycle, providing a possible mechanism by which extracellular signals lead to a reduction in nuclear p27Kip1, thus promoting cellular proliferation.

Tissue remodeling following injury is a regulated balance between pro- and antiproliferative molecules. Injury to the heart and blood vessels, through hemodynamic or oxidative stresses, leads to a remodeling process that is characterized by excessive cellular hypertrophy or proliferation, respectively. In vascular diseases, such as atherosclerosis, instent restenosis or transplant arteriopathy, maladaptive or excessive VSMC proliferation leads to arterial lesion formation and clinical symptoms (9) . Previous studies suggest that p27Kip1 regulates VSMC proliferation after vascular injury through its CKI activity (10) . In healthy, noninjured arteries, p27Kip1 is highly expressed in VSMCs of the artery media. However, following vascular injury, p27Kip1 levels rapidly fall, concomitant with an increase in intimal and medial VSMC proliferation. During the later phases of arterial wound repair, p27Kip1 expression increases, leading to a cessation of proliferating intimal and medial VSMCs. Overexpression of p27Kip1 in injured arteries inhibits VSMC proliferation and lesion formation (3) , while homozygous deletion of p27Kip1 in mice directly increases proliferation and arterial lesions (11) . p27Kip1 also regulates cell proliferation and wound repair in other organ systems, including the kidney (12) , the skin (13) , cancers (14) , and inflammatory diseases (15) .

Ets-domain proteins comprise a large family of DNA-binding proteins that are found in organisms ranging from fruit flies to humans. Ets-family members regulate signaling in development, oncogenesis, and viral gene expression (16) . Moreover, these proteins play critical roles in vascular development, angiogenesis, and smooth muscle cell proliferation (17 18 19) . Previous studies have demonstrated that Ets-domain proteins are highly expressed in VSMCs after mitogenic stimulation and in vascular proliferative diseases, such as atherosclerosis (20) . One member of the Ets-family is the GA-binding protein, GABP{alpha}, which recognizes a consensus GGAA/T motif. The GABPs are cellular heteromeric DNA-binding proteins that regulate activation of nuclear genes encoding mitochondrial proteins (21), adenovirus early genes (22) , and herpes simplex virus immediate-early genes (23) . GABPs are composed of a DNA binding {alpha} subunit and a transactivation {alpha}β subunit (24) . GABP{alpha} is activated in a cell cycle-dependent manner and regulates the expression of genes involved cell cycle progression, including the CKIs (25) .

Given the role of p27Kip1 in tissue remodeling and repair, we hypothesized that KIS inhibition may be an important target to stabilize nuclear p27Kip1, promoting cell cycle arrest. We now find that monomeric GABP{alpha} and heterodimeric GABP{alpha}/β1/2 bind Ets sites in the KIS promoter and GABP binding to Ets-domain sites are essential for transactivation of the KIS gene. Treatment of VSMCs with siRNAs silencing GABP{alpha} expression inhibits KIS expression. In GABP{alpha}-deficient MEFs, KIS gene expression decreases, and p27Kip1 protein levels increase. GABP{alpha}-heterozygous mice undergoing vascular injury in vivo develop significantly smaller arterial proliferative lesions compared with wild-type mice. These results suggest that GABP has a key role in transactivation of the KIS promoter and contributes to the molecular regulation of cell cycle progression and cell migration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture, transfection, and luciferase assays
Human VSMCs (Cambrex Bioproduct, East Rutherford, NJ, USA) were cultured in SmG2 bullet kit media (Cambrex) or HEPES buffered DMEM supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) and 10% FBS at 37°C with 5% CO2. For transient transfections, cells were seeded onto 6 cm dishes, grown to 60% confluency, and transfected with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) and 500 ng of the indicated plasmid (pGL3 backbone) and a control pRL-SV40 plasmid. Luciferase activity was quantified 24 h after transfection using the dual luciferase assay system (Promega, Madison, WI, USA), and luciferase activity was normalized to expression of Renilla luciferase. For the serum stimulation studies, human VSMCs were grown in 6-well plates, serum-starved for 72 h, and stimulated for 4, 8, 16 or 24 h in 10% FBS medium. For the promoter studies under serum stimulation conditions, human VSMCs were serum-starved for 72 h, transfected using the Amaxa Nucleofactor Ket (Amaxa, Gaithersburg, MD, USA), and stimulated for 24 h. Experiments were performed in triplicate.

Plasmids
A BAC clone (Research Genetics, Carlsbad, CA, USA) containing human genomic DNA corresponding to the 5' flanking region of the human KIS gene was identified in a Blast search using human KIS cDNA sequence on the NCBI database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Human KIS promoter fragments were amplified by PCR and directionally cloned into the HindIII site of the Firefly luciferase reporter vector pGL3-basic (Promega). Site-directed mutagenesis was performed using a Quikchange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Mouse GABP{alpha}, GABPβ1, and GABPβ2 were cloned into pCR2.1 using standard methods to generate in vitro translated proteins.

Quantitative PCR using Taqman probes
Total RNA (1 µg) isolated from human VSMCs or mouse femoral arteries was reversed-transcribed using the Taqman Reverse Transcription Reagents Kit (Applied Biosystems, Foster City, CA, USA) and subjected to automated fluorescent RT-PCR on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). All Taqman probes were labeled with the reporter fluorescein at the 5'-end and with the quencher tetramethylrhodamine at the 3'-end. The KIS mRNA was measured using the TaqMan® Gene Expression Assay at exon boundary 4–5 (Ref: Hs01111762_m1). The following nucleotides derived from mouse KIS were synthesized and used as primers and probes for real-time PCR: mouse KIS forward primer 5'-GAGTATGGTTTCCGCAAAGAG-3'; mouse KIS reverse primer 5'-TGGGAGAGAAGTGTATGGTAAAGAC-3'; mouse KIS probe 5'-6FAM-TGTGACCCTGCAACTGCTCCAGTGTAMRA-3'; Taqman rodent GAPDH control reagents (Applied Biosystems) were used as control primers and probe for normalizing measurements of mouse mRNA. Assays-on-Demand GAPD Endogenous Control Reagents (Applied Biosystems) were used for normalizing measurements of human mRNA. Each predicted RT-PCR product spanned an intron-exon boundary, and each RT-PCR reaction was performed in triplicate in a final volume of 50 µl and assessed by the comparative Ct ({Delta}{Delta}Ct) method (Applied Biosystems, ABI Prism 7700 User Bulletin #2).

In vitro transcription and translation
All in vitro translated protein was generated using the TNT Coupled Reticulolysate System (Promega).

Electrophoretic mobility shift assay (EMSA)
Nuclear extracts (1 µg) of human VSMCs were generated via the NE-PER kit (Pierce Biotechnology, Rockford, IL, USA) and incubated with 32P-labeled double-stranded oligonucleotides and 1 µg of poly(dI:dC) in a total volume of 10 µl containing 20 mM HEPES-KOH (pH 7.9), 5% glycerol, 65 mM KCl, 1 mM DTT, 7.5 mM MgCl2, 0.1 mM EDTA, and 0.3 mM PMSF. In competition assays, nuclear extracts were incubated with unlabeled double-stranded oligonucleotides or GABP{alpha} (26) , Ets-1, Ets-2, or Sap1{alpha} antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 20 min at 4°C prior to incubation with the 32P-labeled probe. The mixture was incubated for 30 min at room temperature. The samples were resolved by 6% nondenaturing polyacrylamide gel electrophoresis and analyzed by phosphoimaging (Bio-Rad, Hercules, CA, USA).

Chromatin immunoprecipitation (ChIP)
ChIP studies in human VSMCs were performed using GABP{alpha} antibodies (H-180; Santa Cruz Biotechnology) as described previously (26) . Input and immunoprecipitated DNA samples were analyzed by PCR (36 cycles) with primers amplifying the –390/+205 KIS promoter region containing the GGAA motifs (Forward: 5'-gtaaactctctttttgttcctggggt; Reverse: 5'-tacccagacggctctgtacct). PCR products were separated by electrophoresis on a 1% agarose/TBE gel containing ethidium bromide (1 ng/mL) and visualized using uv transilluminator.

siRNA transfections
RNA interference was performed by transfection of human VSMCs with double stranded RNA oligos (Dharmacon, Chicago, IL, USA) using the Amaxa Nucleofector Kit (Amaxa). GABP{alpha} was targeted with SMART pool siRNA against GABP{alpha} (Dharmacon). The nonhomologous siRNA 5'-AAGCTGACCCTGAAGTTCATC-3' was used as a control. Total RNA was extracted from human VSMCs 48 h after transfection using an RNeasy kit (Qiagen, Valencia, CA, USA). Nuclear extract were isolated 24 h after transfection as described before. Each transfections were performed in triplicate.

GABP{alpha}tp/tp mouse embryonic fibroblasts (MEFs)
GABP{alpha}+/tp mice were generated by a gene trap method (26) . MEFs were generated from 12.5 day post-coitum mouse embryos of wild-type and GABP{alpha}tp/tp genotypes. Embryos were harvested and placed in sterile PBS. The heads and internal organs were removed and carcasses were disrupted in 1 ml of PBS by passing them five times through an 18-gauge hypodermic needed connected to a 3 ml syringe. Cells from each individual embryo were incubated in a 6 cm dish containing 5 ml of DMEM and 10% FBS for 2 days at 37°C with 5% CO2. Cells were maintained by continual passage (1:4 split ratio using 0.25x trypsin/EDTA). Analyses were performed between passages 2 and 4. Western blotting was performed with anti-p27Kip1 antibodies (Santa Cruz Biotechnology) as described previously (6) .

Migration studies
Migration experiments were carried out using a multiwell cell culture insert system employing transwell filters with a pore size of 8 µm (BD Biosciences, San Jose, CA, USA). Human VSMCs were transfected with SMART pool siRNA and serum-starved for 72 h, followed by serum stimulation for 8 h. A total of 50,000 cells were plated into each transwell and allowed to migrate for 2 h toward a gradient of FBS and recombinant PDGF-BB (50 ng/ml, Cell Signaling Technology, Danvers, MA, USA). Migration was stopped by washing and formalin fixation of the cells. Remaining nonmigrating cells were removed from each transwell using a cotton swab. Migrated cells were stained with DAPI, and one representative field of view was counted under a fluorescence microscope. Data were calculated from 6 independent experiments.

Vascular wire injury in mice
GABP{alpha}tp/+ and GABP{alpha}+/+ mice were investigated using an established model of vascular wire injury (11) . This procedure was performed by one surgeon (H. San) who was blinded to genotype. This injury leads to complete endothelial denudation and medial VSMC apoptosis; the cellularity of the media decreases and then becomes repopulated by proliferating VSMCs. Each group consisted of at least 5 mice and 10 arteries. Intima and media cross-section areas were measured by two independent observers blinded to genotype, using a computerized measuring system.

Statistical analysis
Data are expressed as a mean ± SEM. Comparisons between experimental groups were performed using unpaired Student’s t test or ANOVA as appropriate. Differences between groups were considered significant at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We previously observed that KIS protein levels increase in NIH 3T3 cells after serum stimulation, suggesting that KIS gene expression is regulated by mitogens (6) . To determine whether KIS gene expression is stimulated by mitogens in a primary cell line, low passage human VSMCs were serum-starved for 72 h and then incubated with 10% FBS for 4, 8, 16, or 24 h. Total RNA was extracted from cells and quantified for KIS gene expression by Taqman quantitative PCR. We found low basal levels of KIS gene expression in serum-starved human VSMCs, but KIS expression increased ~20-fold 8 h and 40-fold 16 h after serum stimulation and remained elevated 24 h after serum induction (Fig. 1 ), indicating that KIS gene expression is inducible in primary cells relevant to human vascular disease. Accordingly, we hypothesized that transcription of the KIS gene can be activated by mitogen-responsive transcription factors.


Figure 1
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  		Figure 1. KIS gene expression is serum-inducible in VSMCs. Total RNA was isolated from human VSMCs serum-starved for 72 h or from cells stimulated with 10% FBS for 4, 8, 16, or 24 h. KIS mRNA levels were measured by Taqman qPCR, normalized to GAPDH gene expression and expressed relative to the 0 h time point set to 1. n = 3 experiments in each group. ***P < 0.0001.

To define the proximal promoter region, we first identified the KIS transcription initiation site(s) using 5'RACE-PCR analysis and RNase protection assays. Reverse gene-specific primers were designed corresponding to 94 bp downstream of the translation start codon in human KIS exon 1. We amplified two DNA fragments (324 bp and 245 bp) from a human testis RACE-ready cDNA library (Fig. 2 A, B), suggesting that at least two different transcription initiation sites exist. Since the 5'-RACE PCR incorporated a 35 bp "5'-RACE adapter" primer, effectively both 289 and 210 bp fragments of the KIS cDNA were amplified (Fig. 2A, B ). We next employed RNase protection assays using total RNA extracted from human VSMCs and RNA probes, corresponding to the RACE PCR products, to determine which transcription initiation start site was preferentially used in these human VSMCs. Two cRNA probes of 289 and 210 nucleotides, complementary to –79 to + 210 and + 1 to + 210 fragments in the KIS gene, respectively, were used in RNase mapping. Both probes gave two protected bands of ~180 bp and 210 bp (Fig. 2C ), consistent with the transcription initiation sites that had been identified by 5'-RACE (Fig. 2B ). No consensus TATA or CCAAT boxes were found within 500 bp of the transcription initiation site. Furthermore, this region possesses the characteristics of a TATA-less promoter, including GC-rich sequences (Fig. 2D ).


Figure 2
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  		Figure 2. Identification of the start-site of transcription. A, B) 5' RACE PCR products were generated from a human testes cDNA library, separated by electrophoresis on a 2% agarose gel, visualized by ethidium bromide staining (A), and subjected to Southern blotting (B) using a region corresponding to +88/+108 of the human KIS gene as a probe. C) RNase protection assays were performed on total RNA obtained from serum-starved and serum-stimulated (24 h) human VSMCs. Total RNA (10 µg) was hybridized to either a probe spanning –79/+210 or + 1/+210 of the human KIS gene, and protected fragments were electrophoresed on a 6% polyacrylamide/8 M urea denaturing gel. Yeast total RNA (10 µg) was included as a negative control for both probes. D) Nucleotide sequence of the 5' end of the human KIS gene illustrating the positions of the transcription initiation start-sites, a putative GC box, and several putative Ets-binding sites (bold and underlined).

To delineate the region of the KIS promoter responsible for maximal basal activity, we analyzed a series of 5' deletion mutants using transient transfection assays. Reporter gene expression did not significantly differ in cells transfected with constructs containing truncated mutants of the KIS promoter ranging from 1 kb to 141 bp upstream of the transcriptional start site (Fig. 3 A). However, further deletions of the promoter from –141 bp to –41 bp essentially abrogated activity, suggesting that the core promoter resided in this region. Similar results were observed when transfections were performed in primary cultures of human VSMCs (Fig. 3B ). A more refined deletion analysis in this region was performed in human VSMCs to elucidate putative transcription factor binding sites. Significant reductions in promoter activity were observed in asynchronous human VSMCs following deletions of regions spanning –141/–128 (36%), –128/–112 (49%), –86/–68 (83%) and –52/–41 (55%) upstream of the translational start-site. These data suggest that a region spanning –141 bp to –41 bp of the KIS promoter is required for maximal basal expression.


Figure 3
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  		Figure 3. Reporter gene analysis of the KIS promoter (A, B) Progressively truncated regions of the human KIS promoter were subcloned into the firefly luciferase reporter vector pGL3 and transfected into 293 cells (A) and human VSMCs (B). Constructs were cotransfected with a SV40-Renilla luciferase vector in cells to normalize for variations in transfection efficiency. Cell lysates were harvested 24 h after transfection and both Firefly and Renilla luciferase activity were measured. C, D) An Ets-binding site is required for maximal basal level of human KIS reporter gene expression. Substitution mutations were made in several putative transcription binding sites (including NFAT and Ets-binding sites) of the 235 bp upstream region of the human KIS promoter and cloned into Firefly luciferase reporter constructs. Both wild-type and mutant constructs were cotransfected with a SV40-Renilla luciferase vector in human VSMCs to normalize for variations in transfection efficiency. Cell lysates were harvested 24 h as described in Fig. 3A, B. Data are expressed as a percentage of luciferase activity compared with pGL3–141 ± SEM. E) Luciferase activity (Firefly:Renilla) demonstrating that the –141 bp to –41 bp region of the KIS promoter, which contain the Ets binding sites, is required for activation of the KIS promoter in the presence of serum. Investigations were carried out in triplicate in at least 3 independent experiments in Fig. 3A–E. ***P < 0.001.

To identify the transcription factors that bind to the –141 to –41 region of the KIS promoter, we searched a transcription factor database (Transfac- http://www.gene-regulation.com/pub/databases.html#transfac) and compared the DNA sequence in the KIS promoter with consensus binding motifs (27) . The putative transcription binding sites most homologous with consensus binding motifs in these regions included a GC-box positioned in the –141/–131 region and three putative Ets-binding sites (EBSs) (Fig. 2C ). We noted that the EBS-3 site has high homology to the consensus binding motif for GABP{alpha} (16) . To further characterize which of these putative EBSs may be important in regulating KIS gene expression, we generated mutants and measured reporter gene activity in human VSMCs. Interestingly, the substitution mutation (TTCC to TCTC) in either EBS-2 or EBS-3 led to a 75–80% reduction in promoter activity (Fig. 3C, D ). A single mutation of EBS-1 reduced the promoter activity only modestly, but simultaneous mutation of both EBS-1 and EBS-2 sites had a stronger effect. In contrast, substitution mutations in other regions of KIS promoter, such as a putative NFAT site, had minimal effects on reporter gene activity. In addition, luciferase reporter gene studies demonstrated that the –141 bp to –41 bp regions of the KIS promoter, which contain the Ets binding sites, are required for activation of the KIS promoter in the presence of serum (Fig. 3E ). These data suggest that the Ets-binding sites contribute to maximal basal expression of KIS in human VSMCs.

To determine which transcription factors bind to the –103/–73 (spanning EBS-1 and EBS-2) and the –52/–42 (spanning EBS-3) regions of KIS promoter, we performed EMSAs using nuclear extracts from human VSMCs cultured in 10% FBS and oligonucleotides corresponding to the region –103/–73 or –55/–34. We first used –55/–34 oligonucleotide spanning EBS-3 as a probe, and approximately four protein-DNA complexes of different migration were observed (Fig. 4 A, lane 1). Interestingly, mutation of EBS-3 abrogated complex I, suggesting these complexes were formed with this Ets binding motif (Fig. 4A , lane 2). We next used an antibody recognizing the conserved Ets domain in the EMSA and found that this antibody can supershift complex I (Fig. 4B , lane 2 vs. 1). This result suggests that an Ets factor is contained in complex I. We then tested an array of antibodies against Ets factors in supershift assays. Whereas Ets-1, Ets-2, and Sap1{alpha} antibodies did not have an effect on complex I, antibodies against GABP{alpha} or GABPβ decreased the intensity of parental complex I with a concurrent appearance of a band with slower migration (Fig. 4B ). Thus, GABP formed a major complex with EBS-3.


Figure 4
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  		Figure 4. EMSA competition experiments using an oligonucleotide probe corresponding to the –55 to –34 region spanning EBS-3. A) Nuclear extracts were prepared from primary cultures of human VSMCs. EMSA assays were conducted by incubating 1 µg of nuclear extracts with 32P-labeled wild-type or mutant –55 to –34 region probes. 10- to 250-fold molar excess of unlabeled competitor –55 to –34 oligonucleotides were preincubated with the nuclear extracts for 15 min at 4°C prior to addition of the probe to demonstrate the specificity of the complexes. B) GABP{alpha} containing complexes bind to the human KIS promoter in vitro. EMSA supershift experiments for GABP{alpha} were conducted by preincubating human VSMC nuclear extracts with GABP{alpha} antisera or control antisera prior to addition of the –55/–34 probe. Similar binding reactions using antibodies to the Ets domain (4–123), GABP{alpha}, GABPβ1, Ets-1, Ets-2, and Sap1{alpha} were also included in the experiment. EMSA competition experiments using oligonucleotides corresponding to the –103 to –73 EBS-1/EBS-2 region as probes (C, D). Nuclear extracts were prepared from primary cultures of human VSMCs. EMSA assays were conducted by incubating 1 µg of nuclear extracts with 32P-labeled double-stranded oligonucleotides corresponding to either wild-type or mutant –103 to –73 region probes. C) EMSA assays were conducted by incubating 1 µg of nuclear extracts with 32P-labeled double-stranded wild-type or mutant probes corresponding to the –103 to –73 region of the human KIS promoter. D) EMSA supershift experiments for GABP{alpha} were conducted by preincubating nuclear lysates with GABP{alpha} or control antisera prior to addition of the probe. Similar binding reactions using antibodies raised against Ets-domain (4–123), GABP{alpha}, GABPβ1, Ets-1, Ets-2, and Sap1{alpha} were included.

We then tested –103/–73 oligonucleotide as a probe in EMSA. A number of complexes appeared, and mutation of EBS-1 did not affect the formation of these complexes. However, mutation of EBS-2 abrogated complexes II and III (Fig. 4C ). We also tested a panel of antibodies as in Fig. 4B and found that anti-Ets domain, GABP{alpha}, and GABPβ antibodies all supershifted complex II, while only the former two antibodies supershifted complex III (Fig. 4D , lanes 2–4), suggesting the complex II contains both GABP{alpha} and GABPβ, whereas complex III is formed from GABP{alpha} alone. In contrast, antibodies against other Ets factors including Ets-1, Ets-2, and Sap1{alpha} did not affect either complex (Fig. 4D , lanes 5–7). Taken together, these results showed that both EBS-2 and EBS-3 can bind to GABP heterodimers.

We next investigated the effects of cell cycle progression on the binding of GABP to the EBSs of the KIS promoter. We performed EMSAs using nuclear extracts derived from serum-starved human VSMCs and human VSMCs that were stimulated to enter the cell cycle by the addition of serum and probes that corresponded to the EBS-1/EBS-2 and EBS-3 regions of the human KIS promoter. The addition of serum resulted in a marked increase in the formation of GABP/probe complexes when using probes corresponding to either region of the KIS promoter (Fig. 5 A, B), suggesting that the increased binding of GABP to either region may underlie the mechanism of serum-induced KIS gene expression. To determine whether GABP{alpha} binds to the KIS promoter at an endogenous level in vivo, we analyzed chromatin of human VSMCs by chromatin immunoprecipitation using primers that flank the –390/+205 region of the KIS promoter containing the Ets-binding sites. PCR analysis of chromatin immunoprecipitates, isolated with GABP{alpha} antibodies, resulted in the amplification of a 595 bp product that is consistent with the –390/+205 region of the KIS promoter (Fig. 5C ). No such PCR product was detectable from control immunoprecipitates. Taken together, these data suggest that in human VSMCs, endogenous GABP{alpha} binds to –390/+205 region of the KIS promoter in vivo.


Figure 5
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  		Figure 5. Serum stimulation increases the binding of GABP to the –103/–73 or –55/–34 regions of the KIS promoter. A, B) EMSA analyses were performed using 32P-labeled double-stranded oligonucleotides corresponding to either the –103/–73 (A) or –55/–34 (B) region of the human KIS promoter and nuclear extracts derived from human VSMCs that were serum-starved for 48 h or from cells stimulated with 10% FBS at the indicated time points. C) Chromatin immunoprecipitation studies using primers that flank the –390/+205 region of the KIS promoter containing the Ets-binding sites identifies endogenous GABP{alpha} binds to –390/+205 region of the KIS promoter in vivo. Lane 1: 10% input. Lane 2: control IgG. Lane 3: GABP{alpha} antibodies. Arrow denotes the –390/+205 product.

To further substantiate the direct regulation of KIS by GABP in vivo, we used small interference RNAs (siRNAs) to knockdown the expression of GABP{alpha} in human VSMCs. We used a SMART-pool of siRNA that targets four regions of the GABP{alpha} transcript with a nonhomologous siRNA as a negative control. Transfection of human VSMCs with the SMART-pool siRNA targeting GABP{alpha} effectively silenced the expression of GABP{alpha} (Fig. 6 A), and KIS expression in these transfected cells was also greatly diminished (Fig. 6B ). Knock-down of GABP{alpha} in serum-stimulated cells also reduced the DNA-protein complex formation seen by EMSA (Fig. 6C , lanes 3 and 4 compared to lanes 1 and 2). These data further corroborate that GABP directly regulates KIS gene expression.


Figure 6
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  		Figure 6. GABP{alpha}-depleted cells demonstrate reduced KIS gene expression, cell migration and percent of cells in S-phase entry. A–C) Human VSMCs were transfected with 200 µM of each siRNA. Total RNA was isolated from cells 24 h after transfection and analyzed for GABP{alpha} (A) and KIS (B) mRNA by Taqman qPCR (data were normalized to GAPDH) or for DNA-protein complex formation by EMSA (C). A non-specific band (NS) was not affected by the GABP{alpha} si RNA. D) Reduced migration in human VSMCs depleted of GABP{alpha}, using a Boyden Chamber assay. E) Reduced S-phase entry in human VSMCs depleted of GABP{alpha}. Serum-stimulated cells were stained with propidium iodide and analyzed by FACS. n = 3 experiments in each group. *P < 0.05. **P < 0.01; ***P < 0.001.

Previous data has suggested that p27Kip1 mediates cell migration through the Rho pathway (44) . To examine the role of GABP{alpha} on cell migration, we compared properties of GABP{alpha}-deficient VSMCs with VSMCs transfected with control siRNA. Interestingly, following knock-down of GABP{alpha} in human VSMCs, we found a 25% reduction in cell migration, using a Boyden Chamber assay, compared to control-treated VSMCs (166±11 vs. 221±11 cells/hour, respectively, P<0.01) (Fig. 6D ). In addition, knock-down of GABP{alpha} in these human VSMCs resulted in a significant reduction in the percent of cells entering S-phase (Fig. 6E , P<0.001). Taken together, these data suggest that GABP regulates cell migration, in addition to cell proliferation, possibly through its effects on KIS, which in turn modulates p27Kip1 function.

Given the important role of KIS in promoting cell cycle progression, we next examined whether GABP{alpha} deficiency could negatively affect cell cycle. Embryos homozygous for the null GABP{alpha} allele die prior to implantation, suggesting a direct role for GABP{alpha} throughout embryogenesis and in embryonic stem cells (30) . Previously, we generated GABP{alpha}+/tp mice, which have hypomorphic expression of GABP{alpha} using a gene trap strategy. While GABP{alpha}tp/tp mice exhibited embryonic lethality, these embryos can develop up to day E14.5 (26) . Although this precluded us from examining KIS expression and cell cycle status in GABP{alpha}–/– or GABP{alpha}tp/tp VSMCs, we investigated GABP{alpha}tp/tp MEFs. Both GABP{alpha}+/+ and GABP{alpha}tp/tp MEFs were serum-starved for 48 h and then stimulated to enter the cell cycle by the addition of culture media containing 20% FBS. Under serum-starved conditions, KIS gene expression was ~50% lower in GABP{alpha}tp/tp MEFs compared to GABP{alpha}+/+controls. Following serum stimulation, KIS gene expression was induced two-fold in GABP{alpha}+/+ MEFs; however, up-regulation of KIS in GABP{alpha}tp/tp MEFs was essentially absent (Fig. 7 A). In agreement with previously published data, p27Kip1 was expressed in serum-starved GABP{alpha}+/+ and GABP{alpha}tp/tp cells (41) . This is consistent with the observation that GABP expression and activity is low in the absence of mitogen stimulation. The lack of an effect on p27Kip1 protein levels may suggest that KIS requires additional stimuli to alter p27Kip1 levels, including Skp2 or other factors (5 6 7) . In response to serum, GABP{alpha}+/+ MEFs rapidly down-regulated p27Kip1 expression to an undetectable level, whereas GABP{alpha}tp/tp MEFs retained substantial expression of p27Kip1 (Fig. 7B ). These results indicate that under mitogenic conditions, GABP{alpha} deficiency resulted in defective expression of KIS, leading to accumulation of p27Kip1.


Figure 7
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  		Figure 7. A) KIS gene expression is reduced in serum stimulated GABP{alpha}tp/tp MEFs, which are associated with reduced percentage of cells in S-phase entry. GABP{alpha}tp/tp and GABP{alpha}+/+ MEFs were serum-starved for 48 h and then stimulated with 20% FBS for 9 h. Total RNA was isolated from cells, and endogenous KIS mRNA was quantified by Taqman qPCR and normalized to levels of GAPDH. Levels of KIS mRNA are expressed relative to that measured in serum-starved GABP{alpha}+/+ MEFs. The data represent 3 independent experiments *P < 0.05. B) p27Kip1 protein levels are increased in GABP{alpha}tp/tp MEFs. GABP{alpha}tp/tp and GABP{alpha}+/+ MEFs were serum-starved for 48 h and then stimulated with 20% FBS for 20 h. p27Kip1 protein was measured in cell lysates by Western blotting with p27Kip1 antibodies. C) Cell cycle progression is reduced in GABP{alpha}tp/tp MEFS. GABP{alpha}tp/tp and GABP{alpha}+/+ MEFs were serum-starved for 48 h and then stimulated with 20% FBS for 20 h. Both serum-starved and serum-stimulated cells were stained with propidium iodide and analyzed by FACS. The regions corresponding to G0/G1, S, and G2/M phases of the cell cycle are indicated. n = 3 experiments in each group.

Serum stimulation was associated with an increase in the percentage of S-phase cells in GABP{alpha}+/+ MEFs; however, we observed a smaller percentage of S-phase cells in GABP{alpha}tp/tp MEFs than in the wild-type MEFs (Fig. 7C ). Taken together, these data demonstrated that GABP{alpha}-deficient cells exhibit reduced KIS gene expression. Furthermore, KIS expression fails to increase in these cells in response to mitogenic factors such as serum, thereby leading to increased p27Kip1 protein levels and reduced S phase entry.

To determine the biological activity following GABP{alpha} activation of KIS in VSMCs, we examined the VSMC proliferative response to arterial injury in GABP{alpha}+/+ and GABP{alpha}tp/+ mice, using an established model of vascular injury (11) . Because GABP{alpha}tp/tp mice are not viable, we were unable to perform the vascular injury experiments in adult homozygous mice. We, therefore, tested GABP{alpha}tp/+ mice, since previous experiments in our lab have confirmed that the heterozygous p27Kip1 mice develop an intermediate phenotype between the wild-type and homozygous genotypes in this biological assay (11) . Two weeks following an intralumenal wire injury to the femoral artery, GABP{alpha}+/+ arteries developed significantly larger intimal VSMC lesions compared to GABP{alpha}tp/+ mice, measured as the ratio of the intima to the media layers of the artery (P=0.03) (Fig. 8 A, B). To examine the correlation between vascular lesion formation in GABP{alpha}tp/+ and GABP{alpha}+/+ mice and KIS and p27Kip1 regulation, we measured KIS gene expression in the respective GABP{alpha} arteries. We found that levels of KIS mRNA are low in uninjured arteries, rise significantly 7 days following vascular injury, and remain elevated at 14 days (Fig. 8C ) (P<0.0001). We found that KIS mRNA was significantly lower in arteries of GABP{alpha}-deficient mice 7 days after injury in contrast to wild-type mice. This difference in KIS gene expression is even more apparent 14 days after injury. Previous work from our lab has demonstrated that p27Kip1 protein is expressed in arterial medial of uninjured VSMCs, and following injury, p27Kip1 protein levels fall, consistent with VSMC proliferation in the arterial media and migration of VSMCs from the arterial media to the intima where cell proliferation continues (3 , 9 10 11) . At ~14 days, p27Kip1 protein levels rise, coincident with a decrease in VSMC proliferation, and the vascular lesion is formed. The time course of KIS gene expression is consistent with these observations in that KIS RNA levels are low in uninjured arteries, and rise following vascular injury at a time when p27Kip1 levels are declining. In addition, levels of KIS RNA were higher in GABP{alpha}+/+ arteries compared with GABP{alpha}tp/+ arteries, consistent with the larger lesions observed in GABP{alpha}+/+ vs. GABP{alpha}tp/+ arteries (Fig. 8C ). Taken together, these in vivo findings are consistent with the siRNA and GABP{alpha}tp/tp MEF studies, suggesting that GABP{alpha} regulates KIS gene expression in primary human VSMCs and the vascular repair process that accompanies vascular diseases.


Figure 8
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  		Figure 8. GABP{alpha}tp/+ arteries demonstrate reduced KIS gene expression and smaller vascular lesions in vivo. A) Representative photomicrographs of GABP{alpha}+/+ and GABP{alpha}tp/+ arteries two weeks after vascular injury, demonstrating marked cellular hyperplasia in the GABP{alpha}+/+ artery (left) and markedly reduced cellular neointima in GABP{alpha}tp/+ artery (right) (H+E staining). B) Intima to media area ratio in GABP{alpha}+/+ (left) and GABP{alpha}tp/+ (right) mice 2 weeks following vascular injury. C) mKIS gene expression in GABP{alpha}+/+ arteries and GABP{alpha}tp/+ arteries without vascular injury (0 day) and 7 or 14 days following vascular injury. mKIS mRNA levels are normalized to GAPDH gene expression and expressed relative to the 0 day time point for each genotype. n = 4 arteries in each group. *P = 0.03. ***P < 0.0001.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The serine/threonine kinase KIS regulates cell cycle progression by mediating p27Kip1 subcellular localization (6) . On exposure to mitogens, KIS phosphorylates nuclear p27Kip1 on Ser-10, leading to nuclear export and p27Kip1 degradation. Accordingly, the molecular mechanisms that govern KIS gene expression also regulate cell cycle progression and cell proliferation. In this study, we investigated the molecular mechanisms that regulate KIS gene expression in human VSMCs and find that KIS gene expression is regulated by the Ets family transcription factor, GABP.

Ets-family members share a conserved DNA-binding domain whose structure has an overall topology similar to that of the "winged helix-turn-helix" family of proteins (16) . Ets-domains bind DNA as monomers and recognize a consensus sequence that contains a core GGA(A/T) motif (31 , 32) . GABP is a cellular heteromeric DNA-binding protein that is composed of a DNA binding {alpha} subunit and a transactivating β subunit (reviewed in 33 ) that form heterotetramers (34 , 35) . Interestingly, we observed three Ets-binding sites situated in close proximity (Fig. 2) . Substitution mutations in either the EBS-2 or EBS-3 sites led to a significant reduction in reporter gene activity. We show that GABP binds to both sites in vitro, suggesting that a GABP heterotetrameric interaction may also form in the KIS promoter and activate gene expression.

Note that in agreement with previously published data (25 , 41) , our EMSA analysis of serum-stimulated cells shows that GABP DNA binding activity continues to increase up to 24 h after serum stimulation, despite our observation that induction of KIS mRNA is maximal after 18 h of serum stimulation. This discordance could reflect the increase in GABP{alpha} expression that occurs in cells after serum stimulation in addition to the initial activation of GABP (41) . In contrast, maximal occupancy/activation of the regulatory elements in KIS promoter may occur after only 16 h. The increase in active GABP that occurs after 16 h stimulation may be more physiologically relevant to other GABP-responsive genes that play important roles later in the cell cycle.

We have previously shown that p27Kip1 plays an important role in regulating VSMC proliferation and vascular disease (3 , 9 10 11) . Cell migration is also a critical component in the pathogenesis of vascular diseases. A recent study suggests that cytoplasmic p27Kip1 may alter cell migration by modulation of the Rho pathway (44) . Our observation that VSMC migration is reduced following GABP{alpha} knockdown suggests that GABP may also be an important regulator of cell migration, particularly in vascular disease. Indeed, the reduced migration in GABP{alpha}-depleted cells is consistent with the smaller lesions observed in GABP{alpha} tp/+ arteries in vivo. Since KIS phosphorylates p27Kip1 on serine 10, leading to nuclear to cytoplasmic redistribution of p27Kip1, GABP may regulate cell migration through its effects on KIS gene expression (6) .

GABP{alpha} regulates the expression of several cell cycle genes, including Rb (36 , 37) , DNA polymerase alpha (38 , 41) , thymidylate synthase (39 , 41) , and immunologically important genes such as IL-7R{alpha} (26) . Moreover, Yang et al. have provided convincing evidence that GABP is necessary and sufficient for cell cycle reentry (41) . Indeed, these authors found that genetic disruption of GABP{alpha} selectively prevents entry into the cell cycle and reduces expression of genes required for degradation of cyclin-dependent kinase inhibitors, concluding that GABP{alpha} regulates a pathway distinct from the cyclins and CDKs. Our data add to these findings and provides evidence for a new GABP{alpha}-regulated gene, KIS. We would propose that GABP{alpha} binding to Ets sites in the KIS promoter leads to KIS activation, which in turn, results in phosphorylation and degradation of p27Kip1. Furthermore, we find that KIS gene expression is significantly reduced in serum-stimulated GABP{alpha}-deficient MEFS and in injured GABP{alpha}tp/+ arteries, leading to reductions in cell migration and proliferation and smaller vascular lesions. We would offer that GABP{alpha} regulation of KIS gene expression in addition to other modes of p27Kip1 regulation might represent one of the pathways by which GABP{alpha} regulates reentry into the cell cycle, independent of the cyclins and CDKs. In addition, our data extend GABP’s role in vascular biology to the regulation of cell migration. However, the model of GABP{alpha} regulation of cell cycle reentry and cell migration is complex with the likelihood of multiple GABP{alpha} responsive genes.

GABP{alpha} has been described as the critical regulator of Skp2 gene expression in response to mitogenic stimulation (25 , 41) . Like KIS, Skp2 is an important regulator of p27Kip1 and functions as the F-box component of an SCF-type ubiquitin ligase complex that is important for at least one mechanism of p27Kip1 ubiquitination and degradation via the proteasome (40) . Likewise, as stated above, GABP is also critical for re-entry into the cell cycle by regulating a pathway(s) distinct from the D-type cyclins and CDKs (41) . Previous studies have suggested that the DNA binding activity of GABP{alpha} is regulated by serum stimulation (25 , 41) , as is KIS gene expression (6) . Furthermore, activation of the Ras-Raf-Erk signaling pathway has been shown to activate promoters in a GABP-dependent manner (42 , 43) . Whether KIS gene expression is also regulated via Ras-Raf-MAPK pathway remains to be determined. Given that GABP modulates the expression of regulators of cell cycle progression, including those that modulate p27Kip1 function, such as Skp2, the regulation of KIS gene expression by GABP is most likely part of a larger complex molecular mechanism that tightly coordinates cell cycle progression and cell migration.

In summary, we have delineated the molecular mechanisms by which GABP binding to Ets sites in the KIS promoter activates KIS gene expression, leading to cell proliferation and cell cycle progression. Knockdown of GABP{alpha} mRNA reduces KIS expression and cell migration; GABP{alpha}tp/tp MEFs exhibit increased p27Kip1 expression and reduced S-phase entry on serum stimulation in contrast to wild-type MEFS. GABP{alpha}tp/+ mice in vivo also display reduced vascular proliferation and arterial lesion formation after vascular injury, as compared with wild-type mice. These molecular mechanisms provide insight into our understanding of cell cycle regulation and cell migration in primary cell lines, findings with application to vascular diseases and the identification of new therapeutic targets for these diseases.


   ACKNOWLEDGMENTS
 
We thank Jim Hagman for generously providing Ets domain antisera 4–123 and members of the Nabel and Leonard labs for critical review of the manuscript. We thank LAMS staff and Hong San and Robin Schwartzbeck for their assistance with the transgenic mice. This work was supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute.


   FOOTNOTES
 
1 Current address: Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA

Received for publication March 15, 2007. Accepted for publication July 26, 2007.


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