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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Centro de Investigaciones Biológicas,
Consejo Superior Investigaciones Cientificas (CSIC), Madrid,
Spain; Laboratory of Molecular Cell Sciences, Riken, Wako,
Saitama, Japan; Servicio Anatomía
Patológica, Hospital Gómez Ulla, Madrid,
Spain; Division of Liver Diseases, Department of Medicine,
Mount Sinai School of Medicine, New York, NY; and Maine Medical Center
Research Institute, Scarborough, ME.
Endoglin is an endothelial membrane glycoprotein involved in
cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor- Coordinated gene expression is a crucial
requirement in the response to tissue injury. Extracellular matrix
proteins,1-3 growth factors such as transforming growth
factor- Members of the TGF- Accumulating evidence suggests an important role for endoglin in
vascular remodeling and cardiovascular development. Endoglin expression
is regulated during heart development in humans and chicken;23-25 it is highly expressed at the level of the
endocardial cushion during valve formation and by the mesenchymal cells
of the atrioventricular canal during heart septation.23
Its role in morphogenesis is further underscored by the finding that
mice embryos homozygous for a mutant endoglin die at 10 to
10.5 days after coitum because of vascular and cardiac
anomalies.25-27
The gene encoding endoglin is also the target for the autosomal
dominant disorder known as hereditary hemorrhagic telangiectasia type 1 (HHT1) (Osler-Weber-Rendu syndrome).28 The most
common clinical manifestations of HHT1 are the development of
vascular telangiectases in skin and nasal mucosa with bleeding and
arteriovenous malformations in lung, liver, and
brain.29,30 Interestingly, fibrosis and cirrhosis also
develop in some patients with liver involvement, suggesting that the
hepatic injury response is also defective.31
Reduced levels of functional endoglin (haploinsufficiency), rather than
a dominant-negative effect of the mutant allele, is widely accepted as
the pathogenic mechanism of HHT1.30,32 For this reason,
studies elucidating the regulation of endoglin gene expression are essential to ultimately correct HHT1. In this regard, we
have characterized the promoter region of the human endoglin gene,33 and, more recently, we found that the proximal
upstream promoter contains a critical Sp1 site required for its basal
activity and that Sp1 is involved in the TGF- Endoglin expression is up-regulated in microvascular endothelial cells
in human and porcine models of tissue repair.35,61 However, the molecular basis for endoglin gene stimulation
in this pathologic setting is unknown. Krüppel-like factor 6 (KLF6), previously called Zf9/COPEB, is a zinc finger transcription
factor cloned from hepatic mesenchymal cells, placenta, and
leukocytes.36,37 It belongs to the family of
Krüppel-like transcription factors, which recognize a GC box
motif in responsive promoters.36,38 A role for KLF6 in
response to tissue injury is suggested by its rapid induction in
activated hepatic stellate cells, the key fibrogenic cell type in
liver injury, and by its induction in endothelial cells after vascular
injury.39 Moreover, KLF6 transactivates key genes directly
involved in the injury response, including collagen Based on the induction of endoglin35 and
KLF639 during vascular injury and the dependence
of endoglin transactivation on GC boxes, we have explored
the capacity of KLF6 to regulate endoglin gene expression.
We have colocalized KLF6 and subsequent endoglin
induction in vascular endothelial cells following carotid balloon
injury in rats. Moreover, endothelial injury in cultured human
umbilical vein endothelial cells (HUVECs) led to the immediate induction of KLF6, followed 6 hours later by the up-regulation of
endoglin. Furthermore, KLF6 stimulates endoglin
promoter activity, which is dependent on a region overlapping an Sp1
site. Finally, functional and physical cooperation between KLF6 and Sp1
leads to marked up-regulation not only of endoglin, but also of
TGF- KLF6 and endoglin detection in arterial injury
Cells
For endothelial denudation injury, 50- to 300-µm-wide wounds were systematically created with a sterile pipette tip throughout a confluent monolayer of HUVECs until only 20% of the cells remained adherent to the culture dish. Plates were washed, fresh medium was added, and cells were cultured at 37°C. Flow cytometry In endothelial denudation experiments, endoglin expression was determined in HUVECs by incubation with the mouse monoclonal antibody P4A4 against human endoglin.41 For KLF6 analysis, HUVECs were fixed in 3.5% formaldehyde and were permeabilized with 100 µg/mL lysophosphatidyl choline before incubation with the primary antibody (Zf9; Santa Cruz Biotechnology). Cells were incubated with fluorescein isothiocyanate (FITC)-Labeled rabbit anti-mouse IgG (DAKO, Glostrup, Denmark) and washed, and their fluorescence was estimated with an EPICS-XL (Coulter, Hialeah, FL) using logarithmic amplifiers.To investigate the effect of KLF6 on endogenous endoglin expression, HeLa cells were cotransfected with KLF6 (pCIneo-KLF6)39 and the green fluorescence protein (pEGFP-C2; BD Biosciences) expression vectors (1 µg/well each) using FuGENE 6 (Roche, Barcelona, Spain). After 24 hours, cells were incubated with P4A4 antibody, followed by FluoroLinkCy5-labeled goat anti-mouse IgG (Amersham Biosciences, Barcelona, Spain). Fluorescence was estimated with a FACSVantage (Becton Dickinson, San Jose, CA). Reverse transcription-polymerase chain reaction Total RNA was isolated from HUVECs and from HeLa and M1 cells using the RNAeasy kit (Qiagen, Hilden, Germany) and was reverse transcribed by avian myeloblastosis virus (AMV) reverse transcription (RT). The resultant cDNA was used as a template for polymerase chain reaction (PCR) performed with a combination of specific oligonucleotide primers for KLF6 (5'-CGGCCAAGTTTACCTCCG-3' and 5'-CATGAGCATCTGTAAGGC-3'), endoglin (5'-TCCATTGTGACCTTCAGCC-3' and 5'-GGAGATGCAGGAAGACACTG-3' for HeLa and M1 cells or 5'-TGGTACATCTACTCGCACACGC-3' and 5'-GGCTATGCCATGCTG CTGGTGG-3' for HUVECs and BAECs), actin (5'-AGGCCAACCGCGAAGATTGACC-3' and 5'-GAAGTCCAGGGCGACGTAGCAC-3') or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5'-GGCTGAGAACGGGAAGCT TGTCA-3' and 5'-CGGCCATCACGCCACACAGT-3') and AmpliTaq polymerase (Perkin-Elmer). Amplified products were analyzed in agarose gels, stained with ethidium bromide, and quantified by densitometry.Endoglin mRNA analysis by real-time PCR BAECs were grown to 70% confluence and were transiently transfected with pCIneo or pCIneo-KLF6 plasmids using lipofectamine 2000 (Life Technologies). Cells were harvested, and total cellular RNA was extracted using the RNAqueous-4PCR Kit (Ambion, Austin, TX). Synthesis of cDNA was performed on 2 µg total RNA per sample with random primers using the Reverse Transcription System (Promega, Madison, WI). For quantitative analysis of endoglin mRNA, the reverse transcriptase product was diluted 4 times in nuclease-free H2O and was loaded as a PCR volume of 10 µL for real-time PCR in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Amplifications were performed using oligonucleotide primers for bovine GAPDH (AJ000039) as a housekeeping gene (5'-CAATGACCCCTTCATTGACC-3' and 5'-GATCTCGCTCCTGGAAGATG-3') and for the conserved endoglin cytoplasmic domain (see above) and SYBR Green.Endoglin promoter plasmid construction The different constructs of the endoglin promoter were generated by PCR amplification of the 3.3-kb SacII/SacII fragment of the endoglin promoter.33 Oligonucleotides corresponding to positions 2450/2436, 1950/1936,
965/951, 450/436, 350/336, 250/236, 150/136,
50/36, and +50/+64 were used in combination with the common
oligonucleotide +336/+350. Each of these oligonucleotides contained the
HindIII site in 5' and the XhoI site in 3'. After PCR amplification, the resultant products were purified, double digested with HindIII and XhoI, and cloned at the
HindIII/XhoI sites of the pXP2
vector42 to generate the following constructs: pCD105(2450/+350), pCD105(1950/+350),
pCD105(965/+350), pCD105(450/+350), pCD105(350/+350), pCD105(250/+350),
pCD105(150/+350), pCD105(50/+350), and
pCD105(+50/+350). The Sp1 site mutant of pCD105
(50/+350) was generated by site-directed
mutagenesis.34
GAL-4 one-hybrid system constructs The KLF6-GAL4 and GAL4-Sp1 constructs and GAL4-LUC reporter were used as described.37 Drosophila expression vector encoding the 778 amino acids of full-length Sp1 (pAC-Sp1) was a generous gift from Dr Robert Tjian.43 Plasmids pAc- NSp1 (deletion of amino acids 2-257), MSp1
(deletion of amino acids 265-548), and CSp1
(deletion of amino acids 552-778) were constructed by ligating
end-filled AccI-XbaI fragments from the
corresponding pCIneo Sp1 deletion mutants into
dephosphorylated end-filled XhoI pAC. Original
pCIneo-N, M, and C
deletion mutants were constructed with PCR amplification using the Sp1
cloning vector as a template and was subcloned into the
XbaI/AccI site of the pCIneo mammalian expression vector (Promega, WI). The following primers were used: 5'-ACCTTGCTACCTGTCAACAGC-3' and 5'-CATGGGGGGATCCACTAGTT-3' for N
cDNA; 5'-AATGCCCCAGGTGATCATGG-3' and 5'-GCTGTTGACAGGTAGCAAGG-3' for
M cDNA; 5'-GCTTCTGAGATCAGGCAC-3' and 5'-CACCTGGGGCATTTGCTATAGC-3' for C cDNA.
Transient transfection Mammalian expression vectors encoding KLF6 (pCIneo-KLF6) and Sp1 (pCIneo-Sp1), Drosophila expression vectors encoding KLF6 (pAC-KLF6) and Sp1 (pAC-Sp1), and bacterial expression vector encoding GST-KLF6 fusion protein (pGEX-KLF6) have been described.39,40 pcDNA3-EGR1 expression vector encoding EGR1 was kindly provided by Dr Ward (Bath University, United Kingdom). Transient transfection was performed using SuperFect Transfection Reagent (Qiagen) in serum-free medium containing 1 µg endoglin promoter constructs, with or without KLF6-pCIneo, KLF6-pAC, or the same expression vector for Sp1. All transfections contained the same amount of total DNA (2 µg), with the balance composed of the corresponding empty expression vectors. Luciferase activity was determined in cell lysates using a TD20/20 luminometer (Promega). Correction for transfection efficiency was made by cotransfection with pCMV- -galactosidase (BD Biosciences), using
galactolight (Tropix) as a substrate. Transactivation assay results
were expressed as arbitrary units of luciferase activity or as a -fold
induction with respect to the corresponding untreated sample.
For experiments documenting functional cooperation, transient
transfection was performed in Drosophila cells using
Cellfectin reagents (GIBCO BRL, Gaithersburg, MD) in 1 mL serum-free
medium containing a combination of different amounts of
Sp1-pAC and KLF6-pAC, with or without 500 ng
reporter plasmids. These reporters were composed of luciferase cDNA
fused with either the collagen Immunoprecipitation and GST pull-down Forty hours after transfection, COS-7 cells were lysed,34 and total extracts were incubated with anti-Sp1 or anti-Zf9/KLF6 (Santa Cruz Biotechnology). Immunocomplexes were precipitated with protein-G Sepharose and were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Proteins were transferred to Hybond-C extra nitrocellulose (Amersham Biosciences) and probed with antibodies, and signals were developed using the Super Signal reagent (Pierce, Rockford, IL) for enhanced chemiluminescence. Experiments were repeated at least 3 times with similar results, and a representative experiment is shown in the corresponding figure. The glutathione S-transferase (GST) fusion protein GST-KLF6 has been described.39Direct binding of KLF6 and Sp1 was performed using recombinant Sp1 (Promega) and KLF6-GST.45 Samples were combined with either glutathione-Sepharose 4B beads or anti-Sp1 antibody-conjugated agarose (Santa Cruz Biotechnology) and were incubated overnight at 4°C on a rotating mixer. Precipitated proteins were separated by SDS-PAGE. Western blotting was performed using rabbit polyclonal anti-Sp1 or anti-Zf9/KLF6 antibodies (Santa Cruz Biotechnology), followed by peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) as before.45 Protein bands were visualized using the Amersham Biosciences enhanced chemiluminescence (ECL) system.
KLF6 and endoglin expression are increased in carotid artery after balloon injury We examined whether KLF6 colocalized with endoglin to vascular endothelial cells when arterial injury occurred based on KLF6 colocalization with uPA in these cells.39 In healthy coronary arteries, endoglin is present at low abundance and is found primarily on endothelial cells, adventitial fibroblasts, and some medial smooth muscle cells.35 To analyze the potential role of KLF6 as an activator of endoglin transcription after vascular injury in vivo, the distal half of the left carotid artery of rats was injured with a balloon catheter and was immunostained with endoglin and KLF6 antibodies at progressive intervals (Figure 1). In resting endothelium and at 3 hours after injury, endoglin and KLF6 were expressed weakly, whereas 12 hours after injury, KLF6 was clearly induced. This induction was maintained up to 48 hours and decayed afterward; at 7 days, KLF6 levels were similar to those of resting endothelium. On the other hand, the kinetics of endoglin staining revealed a time delay with respect to KLF6. Endoglin up-regulation started at 24 hours, peaked at 48 hours, and was sustained for at least 7 days after injury, consistent with the high stability of the protein.46 At 24 to 48 hours, endoglin and KLF6 levels were greatly increased in vascular endothelial cells. Weak immunoreactivity was also detected in the medial smooth muscle cells of the injured carotid artery, similar to what we previously observed for uPA.39 When the same incubations were made with an irrelevant nonimmune antibody, no signal was detected, confirming the specificity of expression. These results demonstrate that KLF6 induction precedes endoglin up-regulation in vascular endothelial cells.
Endothelial denudation of HUVECs sequentially induces KLF6 and endoglin To explore the temporal relationship between KFL6 and endoglin expression in an injury model in which expression could be quantified and clearly ascribed to endothelial cells, denudation injury was performed in HUVECs, and cells were analyzed at different intervals by RT-PCR, flow cytometry, and fluorescence microscopy.The expression of KLF6 and endoglin mRNA was analyzed by
semiquantitative RT-PCR using total RNA from denuded HUVEC monolayers. As a control, levels of the transcription factor Sp1, involved in basal
transcription of endoglin,34 were also
monitored. After 20 cycles of PCR, specific cDNA bands of 318, 179, and 300 bp
Protein expression after endothelial denudation was also measured at different times using flow cytometry. The cytometry profiles for endoglin and KLF6 are shown side by side, together with a graphic summarizing the protein dynamics during the denudation process (Figure 2B). Expression of endoglin clearly increased over the levels of unwounded HUVECs approximately 12 hours after injury, and this increase was maintained afterward (36 hours). KLF6 expression increased after 2 hours and peaked at 6 hours, whereas endoglin expression followed 6 hours after the early induction of KLF6. This figure is consistent with pulse-chase analysis of endoglin in HUVECs.46 The subcellular localization of KLF6 and endoglin after injury in HUVECs was studied by immunofluorescence microscopy (Figure 2C). At time 0, KLF6 was evenly distributed throughout the cytoplasm, whereas nuclei lacked expression. After 3 hours, the cells at the wound edge displayed increased expression of KLF6, including some nuclei expression. Nuclear localization of KLF6 peaked at 6 hours and then KLF6 returned to the cytoplasm, mimicking the behavior of KLF6 after injury in activated hepatic stellate cells.37 After 24 hours, HUVEC growth restored the integrity of the monolayer and the expression of KLF6 to that observed before the onset of injury. On the other hand, endoglin staining was only found at the plasma membrane at all time points. Given the basal high levels of endoglin expression in HUVECs48 and the limitations of this technique, no quantitative differences could be inferred. Although endoglin appeared to be evenly distributed on the cell surface, KLF6 translocated from an early, dispersed cytoplasmic distribution to a conspicuous localization in the nucleus at 3 to 6 hours after injury. After 8 hours, the process of nuclear localization was reversed, and KLF6 was only found in the cytoplasm. There was no specific staining when cells were incubated with the secondary antibody alone (data not shown). Increased endogenous endoglin mRNA expression following transient transfection of KLF6 To establish endoglin as a potential transcriptional target of KLF6, transient transfection was performed in HeLa cells, M1 fibroblasts, and BAECs, which express different levels of endogenous endoglin. Endoglin was detected by flow cytometry in nontransfected versus KLF6-transfected HeLa cells (Figure 3A). Endoglin transcripts were also quantitated by RT-PCR in HeLa and M1 cells after transient transfection of KLF6 (Figure 3B-C). Mean fluorescent intensity from endogenous endoglin was increased on KLF6 transfection (Figure 3A), whereas mock transfection with empty vector, pCIneo, did not alter endoglin levels significantly (data not shown). Moreover, the levels of endoglin RNA were much higher after KLF6 transfection in HeLa cells (Figure 3B) and in M1 fibroblasts (Figure 3C). After 20 cycles of PCR, only the endoglin-specific band was visible in KLF6-transfected HeLa and M1 cells. This and the calculated ratio of endoglin versus GAPDH RNA levels confirmed the specificity of the endoglin promoter's response to KLF6 (Figure 3B-C). As a control for KLF6 specificity, cells were separately transfected with Egr-1, a member of the same Krüppel-like transcription factor family as KLF6; Egr-1 did not alter endogenous levels of endoglin (Figure 3A). The induction of endogenous endoglin mRNA by KLF6 was also confirmed using quantitative real-time RT-PCR in BAECs. Endoglin transcription levels were increased 3.2- or 4-fold after transfection with 5 or 10 µg KLF6 plasmid, respectively, compared to transfection with the empty vector (Figure 3D). This transcriptional activity is remarkably similar to the effect of KLF6 on other gene targets.37
Transcriptional activation of the endoglin promoter by KLF6 To further establish endoglin as a transcriptional target of KLF6, transient cotransfection was performed in HeLa cells, which express low levels of endoglin and display a relatively high efficiency of transfection, using serial deletions of the endoglin promoter driving expression of the luciferase gene (Figure 4A). The basal activity of the full-length promoter construct (2450/+350) was similar to that of
smaller constructs, including 350/+350, 250/+350, and 150/+350.
Interestingly, a significant decrease in the basal promoter activity
was observed in the intermediate constructs 1950/+350, 965/+350,
and 450/+350, suggesting the presence of a repressor sequence within
the 1950/450 fragment. This finding is in agreement with the
activity found in a different series of endoglin promoter
constructs.49 When KLF6 was cotransfected with the panel
of promoter plasmids, the activity was stimulated in all constructs
(from 1.8- to 3.2-fold induction), except in the most minimal
construct, pCD105(+50/+350). As shown in Figure 4B, the KLF6
transactivation effect was also observed in M1 human fibroblasts (from
2.5- to 4.3-fold induction) and in the human endothelial cell line
HMEC-1 (from 1.7- to 3.5-fold induction), using
pCD105(2450/+350), pCD105(1950/+350),
pCD105(450/+350), and pCD105(50/+350) as
representative promoter constructs. For these cell types, the minimal
construct pCD105(+50/+350) was again not transactivated.
To determine the capacity of KLF6 to transactivate the
endoglin promoter in a cell system devoid of endogenous
KLF6, SL-2 Drosophila cells37 were used to
assess KLF6 transactivation (Figure 4B). The induction by KLF6 ranged
from 2.5- to 5-fold, and, interestingly, transactivation was preserved
even in the pCD105( Functional cooperation between KLF6 and Sp1 in transactivating
endoglin and key molecules regulating TGF- 37/29 bp of the endoglin
promoter contains an Sp1 consensus site (CCCAGCCC)33 that
is required for the basal and TGF--induced transcription of
endoglin.34 Like Sp1, KLF6 belongs to the family of
Krüppel-like transcription factors39 that recognize a
GC box motif in responsive promoters.50 To investigate
whether KLF6 acts through the GC-rich motif at 37/29 of the
endoglin promoter (Figure 5A), a reporter
containing a mutation in the consensus Sp1 site (CCC to
TTT at 37) was transfected into SL-2 cells. As shown in
Figure 5B, this mutation did not affect the basal promoter activity,
but it did abolish transactivation by KLF6, indicating that KLF6
requires this site for endoglin transactivation.
Because KLF6 and Sp1 act through the same site in the
endoglin promoter, we studied their individual and combined
contributions to endoglin transcription in SL-2 cells, which
lack endogenous KLF6 and Sp1. The cells were transfected with the
proximal endoglin promoter reporter construct pCD105
( Because the promoters of other key molecules regulating TGF- Physical interaction between Sp1 and KLF6 Transcriptional cooperation between Sp1 and KLF6 at the proximal endoglin promoter (Figure 5) raised the possibility that KLF6 and Sp1 are present within the same transcriptional complex. To test this directly in mammalian cells, transfections of KLF6 were carried out in COS-7 cells, which are a suitable system to overexpress exogenous proteins with high efficiency, and were followed by coimmunoprecipitation experiments. As shown in Figure 6A, transfected KLF6 coprecipitated with endogenous Sp1. Conversely, using an antibody against Sp1, KLF6 could be detected in the immunoprecipitate (Figure 6B).
These findings identified KLF6 and Sp1 within the same immunoprecipitate but did not establish their direct interaction. The latter was examined by performing direct in vitro GST pull-down experiments using recombinant GST-KLF6 and recombinant Sp1. As shown in Figure 6C, KLF6 and Sp1 bound directly to each other, whether using glutathione-Sepharose affinity with or without Sp1 followed by Sp1 Western blot analysis or using the reverse combination of anti-Sp1 agarose followed by KLF6 Western blot analysis. GST alone did not bind Sp1 protein (data not shown). We used the GAL4-LUC one-hybrid reporter system as another means of demonstrating a direct and functional interaction between KLF6 and Sp1. In this system, a fusion protein was generated in which either KLF6, Sp1, or Sp1-deletion mutants were expressed in frame 5' of the GAL4 DNA-binding domain, which was then cotransfected with a GAL4-responsive reporter. Transient transfections in HeLa cells showed that when KLF6-GAL4 interacted with the promoter through its GAL4 DNA-binding domain, there was no additive effect on transactivation of either wild-type KLF6 or Sp1 (Figure 6D). However, when Sp1-GAL4 interacted with the GAL4-responsive promoter, marked cooperation was observed if either Sp1 or KLF6 was cotransfected. Interestingly, in contrast to the results in HeLa cells, Sp1 markedly cooperated with KLF6-GAL4 transactivation in Drosophila Schneider cells (Figure 6E). Therefore, we used this system to map the domain(s) of Sp1 required to cooperate with KLF6 transactivation. The C-terminal domain of Sp1 contains the DNA-binding domain through the Zn+2 fingers, whereas M and N domains (middle and N-terminal) contain 2 glutamine-rich domains involved in transactivation.43 Deletion constructs of Sp1 in the pAC vector were generated for expression in Drosophila that lacked either the N or the M region, or the C-terminal of the protein, and were assessed for their ability to cooperate with KLF6-GAL4-mediated transactivation. As shown in Figure 6E, some cooperativity was preserved when either the N or the M region was deleted, albeit less than that observed with full-length Sp1 and KLF6-GAL4. However, a complete loss of cooperation with KLF6 occurred after deletion of the C-terminal domain (amino acids 552-778), representing the DNA binding domain. This interaction between the C-terminal domains of 2 Krüppel-like factors has been reported previously for erythroid Krüppel-like factor (EKLF) and others.52,53 Furthermore, our data suggest the interaction does not require that both factors be bound to DNA. In the GAL4 system we used, none of the Sp1 constructs was capable of binding directly to the GAL4-responsive reporter DNA.
This study emphasizes the potential of KLF6 to respond to vascular
injury by stimulating endoglin gene expression. Furthermore, coexpression of Sp1 creates the potential for a cooperative
transactivation of endoglin and of other key molecules that
regulate TGF- Transcriptional induction of endoglin during vascular repair
therefore reflects several possible activities of KLF6. First, TGF- KLF6 is induced as an immediate-early gene in hepatic stellate cells, the key cell regulating extracellular matrix production during tissue repair.37 In general, KLF6 is a labile factor in vitro that disappears quickly after withdrawal of the appropriate stimulus, which may include PMA and serum,39 or, as in the experiments described herein, after mechanical injury in cultured endothelial cells. Egr-1, another zinc finger early-response gene in vitro, is induced in endothelial cells in a similar pattern after injury.59 However, our data suggest that the induction of endoglin by KLF6 is not generalized to all zinc finger proteins because Egr-1 does not promote endoglin expression. KLF6 and Sp1 are dependent on the GC-rich consensus motif at Our data suggest a model whereby Sp1 and KLF6 have similar DNA-binding
properties but different biologic roles in vascular injury. Both
proteins may potentially bind DNA at the same Sp1 consensus in the
endoglin promoter, CCCAGCCC ( In the absence of endogenous Sp1, as in Drosophila SL-2
cells, KLF6 can replace Sp1 for basal transactivation of the
endoglin promoter. This result is in agreement with the
transactivation of TGF-
We thank Dr Angel Corbí for stimulating discussions, Dr Carlos Rius for plasmids, Drs S. Hayashi and Y. Suzuki for technical assistance, and Dr Pedro Lastres for flow cytometry analysis.
Submitted August 13, 2001; accepted June 5, 2002.
Supported by grants from Ministerio de Ciencia y Tecnología (SAF2000-0132) (C.B.), Comunidad Autónoma de Madrid, the National Institutes of Health (DK37340) (S.L.F), and the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan (S.K.). T.S.-E. and F.S.-R. are recipients of fellowships from Comunidad Autónoma de Madrid. M.P.C. is supported by a grant from the Dutch Cancer Society Koningin Wilhelmina Fonds.
L.M.B. and T.S.-E. contributed equally to this paper.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Luisa M. Botella, Centro de Investigaciones Biológicas, CSIC, Velázquez, 144, 28006 Madrid, Spain; e-mail: cibluisa{at}cib.csic.es.
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  B. Rockx, T. Baas, G. A. Zornetzer, B. Haagmans, T. Sheahan, M. Frieman, M. D. Dyer, T. H. Teal, S. Proll, J. van den Brand, et al. Early Upregulation of Acute Respiratory Distress Syndrome-Associated Cytokines Promotes Lethal Disease in an Aged-Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Infection J. Virol., July 15, 2009; 83(14): 7062 - 7074. [Abstract] [Full Text] [PDF]
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M. Rubinstein, G. Idelman, S. R. Plymate, G. Narla, S. L. Friedman, and H. Werner Transcriptional Activation of the Insulin-Like Growth Factor I Receptor Gene by the Kruppel-Like Factor 6 (KLF6) Tumor Suppressor Protein: Potential Interactions between KLF6 and p53 Endocrinology, August 1, 2004; 145(8): 3769 - 3777. [Abstract] [Full Text] [PDF]
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J. A. Potian, H. Aviv, N. M. Ponzio, J. S. Harrison, and P. Rameshwar Veto-Like Activity of Mesenchymal Stem Cells: Functional Discrimination Between Cellular Responses to Alloantigens and Recall Antigens J. Immunol., October 1, 2003; 171(7): 3426 - 3434. [Abstract] [Full Text] [PDF]
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 G. Narla, S. L. Friedman, and J. A. Martignetti Kruppel Cripples Prostate Cancer: KLF6 Progress and Prospects Am. J. Pathol., April 1, 2003; 162(4): 1047 - 1052. [Full Text] [PDF]
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signaling components by cooperative
interaction between Sp1 and KLF6: their potential role in the
response to vascular injury
Top
Abstract
Introduction
1(I),
urokinase-type plasminogen activator, TGF-
corresponding to KLF6, endoglin,
) or
pCIneo-KLF6 (
) are shown on the right. (D) Induction of
endogenous endoglin expression after transfection with KLF6. BAECs were
grown on 10-cm plastic plates and transiently transfected with
pCIneo empty vector (control) or pCIneo-KLF6
plasmid (5 or 10 µg). Cells were harvested 24 hours later, total RNA
was extracted, and synthesis of cDNA was performed. Comparative
quantitation of endoglin mRNA to GAPDH was analyzed with real-time
RT-PCR. Fluorescence signals were analyzed during each of 40 cycles
(denaturation 15 seconds at 95°C, annealing 15 seconds at 56°C, and
extension 40 seconds at 72°C). Relative expression was calculated
using the comparative threshold cycle (CT) method.
CT indicates the fractional cycle number at which the
amplified gene amounts to a fixed threshold within the linear phase of
amplification. Median CT of triplicate measurements was
used to calculate 
(CT). Shown is 1 of 2 representative
experiments.
