Department of Surgery (Vascular), Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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We have previously reported that exposure of cultured bovine
aortic endothelial cells (EC) to 10% average strain resulted in an
increase in tissue plasminogen activator (tPA) mRNA, immunoreactive tPA
protein, and tPA activity in the medium. The present study was designed
to examine the regulation of tPA gene expression in EC by cyclic
strain. We performed a functional analysis of the tPA promoter by
transfecting bovine aortic EC with a 1.4-kilobase (kb) construct of the
human tPA promoter coupled to chloramphenicol acetyltransferase. We
found that subjecting the EC to 10% average strain (and not 6%
average strain) resulted in a 2.6-fold increase in activity of the
1.4-kb tPA promoter by 4 h. Analysis of deletion mutants of the
promoter transfected into EC demonstrated a 60% drop-off in activity
between position 145 and
105. Deoxyribonuclease I
protection analysis of the segment downstream of position
196 suggested involvement of activator protein-2 (AP-2) and adenosine 3',5'-cyclic monophosphate-responsive element (CRE)-like
binding sites, which was confirmed by electrophoretic mobility shift
assays. Site-directed mutants of either the AP-2 or CRE-like regions
resulted in a 65% decrease in activity compared with the wild type.
Double mutations abolished basal transcription and any strain-induced activity. A shear stress responsive element (SSRE) binding site is
present at
945, but site-directed mutants did not show any drop
in activity compared with wild type by cyclic strain. These studies
demonstrate that cyclic strain regulates tPA gene transcription in
bovine aortic EC and that this transcriptional activation is dependent
on factors that are similar to those activated with phorbol ester.
endothelium; hemodynamics; tissue plasminogen activator gene expression
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INTRODUCTION |
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THE CELLULAR AND FLUID milieu of the blood vessel wall in vivo is a complex interactive system, with the endothelial lining assuming a major role in hemostasis. The relative nonthrombogenicity of the endothelium in vivo results from several properties of endothelial cells (EC), including their ability to synthesize and secrete anticoagulant molecules such as prostacyclin and heparin-like molecules and the expression of thrombomodulin on cell surfaces. In addition, EC participate in fibrinolysis by secreting enzymes with fibrinolytic activity, including urokinase and tissue-type plasminogen activator (tPA) (6).
Because the endothelium of blood vessels is exposed not only to the continually changing chemical composition of blood but also to rhythmic pulsatile blood flow, we postulate that cyclic strain, analogous to the repetitive, pressure-induced vascular wall distension, can play an important role in regulating the structure and function of EC in vitro. Our laboratory has developed and characterized an instrument capable of exerting repetitive tensional deformation on attached monolayers of cells (5, 46). Our previous work demonstrated that EC respond to cyclic strain with an increase in proliferation (30, 46) and with a change in morphology (22, 45) and migration (52, 53). Furthermore, we have also reported that EC exposed to cyclic strain express and release significant amounts of vasoactive compounds, including tPA, compared with the stationary EC (2, 3, 21, 23, 44, 48). tPA stimulation in EC subjected to cyclic strain was dependent on the amplitude of the deformation, since exposure to 6% average strain did not alter tPA expression. In addition, this response was specific for tPA, since plasminogen activator inhibitor-1 production was similar in both groups (21, 23).
The mechanism of increased tPA expression by EC in response to cyclic strain remains undetermined. The objective of this study was to define the nuclear events involved in the transcriptional regulation of tPA gene expression in EC exposed to cyclic strain by a functional analysis of the tPA promoter.
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METHODS |
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Cell culture. Bovine aortic EC were obtained by gently scraping the intimal surface of bovine thoracic aorta and were maintained in Dulbecco's modified Eagle's medium-F-12 containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 5 µg/ml deoxycytidine, 5 µg/ml thymidine, and 1% penicillin-streptomycin and Fungizone in culture flasks. EC were grown at 37°C in a humidified 5% CO2 incubator and used from passages 2 to 10. EC were identified by their typical morphology and by positive staining for 1,1-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine (di-I)-acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, MA) (22, 30, 46).
Experimental protocol. EC were grown to confluence on flexible-bottomed culture plates coated with type I collagen (Flex I plate, Flexcell, McKeesport, PA). The plates were positioned on a vacuum manifold, which was situated in an incubator. The applied vacuum was regulated by solenoid valves controlled by a computer program. The strain apparatus (Flexercel, Flexcell) has been previously described and characterized in detail (4, 5, 16, 17). When vacuum is applied to the culture plates, the bottoms are deformed to a known percent elongation. When vacuum is released, the plate bottoms return to their original conformation. For these experiments, the flexible membranes were deformed with a 150-mmHg vacuum (10% average strain) or a 37.5-mmHg vacuum (6% average strain) at 60 cycles/min (i.e., 0.5-s elongation alternating with 0.5-s relaxation) for up to 48 h.
Nuclear runoff transcription assays.
Nuclear transcription assays were performed as described previously (3,
19). Stationary EC and EC that were exposed to 10% average strain for
4 h were lysed in buffer containing 10 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl (pH
7.4), 10 mM NaCl, 3 mM MgCl2, and
0.5% Nonidet P-40, and the nuclei were recovered by centrifugation at
500 g for 5 min at 4°C. The nuclei
were then resuspended at 26°C for 30 min in runoff buffer
[35% glycerol, 10 mM Tris · HCl (pH 8.0), 5 mM
MgCl2, 80 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.8 units RNasin, 4 mM dATP, 4 mM dGTP, and 4 mM
dCTP] and 200 µCi
[-32P]UTP (3,000 Ci/mmol, Amersham). The nuclei were next digested with 10 µg
deoxyribonuclease (DNase) 1 (ribonuclease free) at 26°C for 5 min
followed by incubation with 10-20 mg of proteinase K in buffer
containing 5% sodium dodecyl sulfate, 50 mM EDTA, and 100 mM
Tris · HCl (pH 8.0) for 30 min at 37°C. Nascent
elongated transcripts of radiolabeled RNA molecules were extracted by
the guanidinium isothiocyanate method, precipitated with isopropanol, and dissolved in buffer containing 50 mM Tris · HCl
(pH 8.0), 150 mM NaCl, and 1 mM EDTA.
Transfection procedures and chloramphenicol acetyltransferase
assay.
A group of deletion mutants of the human tPA promoter (1452,
408,
332,
196,
145, and
105 to
position +308) fused to the chloramphenicol acetyltransferase (CAT)
reporter gene was kindly provided by Dr. Robert Medcalf (32). The
longest fragment was obtained from a tPA cosmid clone, pPA cos3, by
cleavage with EcoR I and
BamH I followed by cloning into
pBLCAT3. Deletion mutants were
prepared from digestion with various restriction enzymes. The plasmids
were prepared by cesium chloride gradient centrifugation. Transfection
of EC with the tPA promoter-CAT gene constructs was performed by
employing the calcium phosphate precipitation method, incorporating a
glycerol shock (31). After 24 h, the transfected cells were seeded on
collagen-coated wells of stretch plates and incubated overnight. After
exposure to cyclic strain for 2, 4, 8, 24, and 48 h, cells were
harvested and relative changes in CAT activity were quantitated by
determining the percentage of [14C]chloramphenicol
converted to its acetylated products by thin-layer chromatography and
liquid scintillation counting (see Fig.
1B) (31). The
transfection efficiency of the various constructs was evaluated by
cotransfecting EC with cytomegalovirus promoter and
lac Z gene construct.
-Galactosidase assay (31) was taken as a direct index of the
efficiency of transfection and used to normalize CAT activities among
various experiments. Cell viability was maintained throughout the
incubation period, and transfected cells showed typical responses to
exposure to cyclic strain (e.g., shape change and axial alignment with
60 cycles/min, 24% strain).
DNase I protection experiments of tPA promoter.
The DNA containing the tPA gene, including the 196
5'-flanking region to 308 (in first intron), was obtained by
double digestion with Hind III and
EcoR I from the deletion mutant
provided by Dr. Medcalf. The Hind III
end was labeled with
[32P]dCTP by Klenow
fragment of DNA polymerase (31). Nuclear protein extract was prepared
from EC as previously described (13, 14). The reaction was performed
with the DNase I footprinting system according to manufacturer's
instruction (GIBCO BRL, Gaithersburg, MD). Briefly, 10 µg nuclear
extract were incubated with nonspecific competitor poly(dI-dC) at room
temperature for 20 min. One nanogram of 5' end-labeled fragment
of the
196 tPA promoter-CAT construct was then added, and the
samples were left at room temperature for a further 10 min. DNase I (10 µg/ml) was then added, the samples were incubated at room temperature
for 1 min, and the reaction was terminated by adding STOP
buffer. DNA was separated in sequencing gel and an A+G sequencing
reaction by the Maxam-Gilbert method was performed (18, 31).
Electrophoretic mobility shift assays. Nuclear extracts were isolated from bovine aortic EC after being subjected to cyclic strain as described previously (13, 14). Gel shift assays were performed with the DNA binding protein assay system (GIBCO BRL). On the basis of previously reported data on the nucleotide sequence of the adenosine 3',5'-cyclic monophosphate (cAMP)-responsive element (CRE)-like and activator protein-2 (AP-2) transcription sites in the tPA promoter (10, 32), oligomers were synthesized and annealed with the complementary strand.
The oligonucleotides were end labeled with [32P]ATP by T4 kinase (31). Nuclear extracts were incubated in buffer containing poly(dI-dC) at room temperature for 15 min and then incubated with the radioactive oligonucleotide at room temperature for 10 min. The mixture was separated by electrophoresis on a 6% (wt/vol) nondenaturing polyacrylamide gel in 0.25× Tris-borate-EDTA, and the dried gels were exposed to film.Mutagenesis of shear stress responsive element, AP-2, and CRE-like
regions.
Previously established site-directed mutants of the AP-2 and CRE-like
binding regions on the 440 fragment (32) were utilized. To
create shear stress responsive element (SSRE) mutants at the
945
region, site-directed mutagenesis was performed by using the altered
sites in vitro mutagenesis system (Promega, Madison, WI). Briefly, a
tPA subclone in vector pBLCAT3,
supplied by Dr. Medcalf, was double digested with
Xba I and
Kpn I. This 3.2-kilobase (kb) fragment
was subcloned into Kpn I and
Xba I sites of the polylinker of
pAlter-1 phagemid. The vector DNA was transformed into competent cells
of JM109. A single-stranded template for mutagenesis reaction was
prepared by growing individual colonies and infecting the culture with
helper phage R408. A mutant oligonucleotide was designed and
synthesized, which was 33 base pairs (bp) long with the SSRE mismatch
located in the center. The SSRE sequence GAGACC was changed to GAATTC,
which can be cleaved by EcoR I for easy verification. This oligonucleotide was then annealed to the single-stranded DNA template, and the mutant strand was synthesized and
ligated. The DNA was transformed into a repair minus strain of
E. coli (BMH71-18 mut S), and the
amplified DNA was transformed into JM109 to ensure proper segregation
of mutant and wild-type plasmids. EcoR
I cleavage and direct sequencing were used in analysis of
transformants.
Statistical analysis. Data are presented as mean ± SE. Student's paired or unpaired t-test and analysis of variance with post hoc testing were used as needed to determine the significance of differences between means. P < 0.05 was considered significant.
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RESULTS |
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Cyclic strain can directly influence tPA transcription. An earlier study demonstrated an increase in tPA mRNA by reverse transcriptase-polymerase chain reaction when EC were exposed to cyclic strain (23). Figure 1A is a representative nuclear runoff transcription assay and confirms that there was significant induction of new tPA transcripts in nuclei isolated from EC exposed to 4 h of 10% average strain compared with nuclei from control stationary cells. The increase in tPA transcription was specific, since only minimal induction of GAPDH transcripts was observed with cyclic strain.
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DNase I protection and electrophoretic mobility shift assay
experiments.
Because the drop-off in activity occurred downstream of position
145, we performed a DNase I protection analysis of the
196 to +308 fragment. Figure
3A
shows that there were two major protein-binding sites
("footprints"): one position between bp
102 and
115
(5'-AATGACATCACGGCT-3') and the other in the first exon
between bp +60 and 74 (5'-GACCCCACCCCCTGC-3'). These
correspond exactly to the CRE-like and AP-2 consensus sequences, respectively, which have been previously shown to be important in
PMA-mediated tPA activity (32). This result indicates that exposure of
EC to cyclic strain in culture results in the production of nuclear
proteins that can bind to specific regions in the promoter of a gene
that is responsive to strain.
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Role of AP-2, CRE-like, and SSRE binding regions in tPA regulation
by strain.
To directly test the functional role of these binding sites on the tPA
promoter, site-directed mutants of these regions were obtained from Dr.
Medcalf and utilized in the transfection experiments. Figure
4A shows
the effect of transfection of EC with the 440 promoter fragment
containing either CRE-like or AP-2 mutants. There was a significant
60% decrease in induction of promoter activity with both mutant
constructs compared with the wild-type promoter, indicating the
importance of these binding sites. The construct containing the double
mutation did not respond to strain, although it is important to note
that the basal, constitutive CAT levels for this construct were at
background levels.
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DISCUSSION |
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One of the most significant advances in vascular research has been the realization that external physical forces can influence the biology of vascular cells. The exposure of EC to a flowing culture medium or to repetitive elongation has been demonstrated to result in changes in morphology, proliferation, and secretion of macromolecules (3, 9, 11, 12, 21-23, 30, 37, 44, 48, 50). Now, the major impetus in the field has been to define the "mechanosensor(s)" on the cells that are sensitive to the different external forces, as well as the coupling intracellular pathways and the subsequent nuclear events that precede the cell response (8, 24, 43). This study was designed to investigate the latter issue.
We have previously reported (21, 23) that bovine aortic EC exposed to cyclic strain had a significant increase in tPA gene expression. A strain amplitude dependence was supported by greater tPA-specific staining in the high-strain membrane periphery (7-24% strain) in comparison to the low-strain center (0-7% strain). Diamond and co-workers (11, 12) have also shown that exposure of cultured EC to 25 dynes/cm2 shear stress for 24 h stimulated a 3-fold increase in tPA secretion and a 10-fold increase in tPA mRNA. Both sets of in vitro studies are consistent with animal experiments that indicate that different perfusion regimens of hindlimb can alter tPA secretion (49). Together, these data strongly suggest that tPA production can be regulated by changes in pulsatile flow.
The mechanism for the regulation of tPA by cyclic strain is unknown. tPA activity and mRNA levels have been shown to be increased in HeLa cells and EC exposed to phorbol esters and other stimulators of the protein kinase C (PKC) pathway (51). Activation of the phosphoinositide pathway may play multiple roles, since elevated intracellular calcium levels are required for the acute release of tPA (49) and the production of diacylglycerol (DAG) and PKC regulates the synthesis of tPA (29). This response is potentiated fivefold with simultaneous administration of cAMP. Stimulation of tPA was not seen with stimulation by cAMP alone but occurs only in conjunction with phorbol ester (27). Levin et al. (26) reported that changes in mRNA levels in response to PMA and PMA-forskolin precede and determine tPA antigen secretion. They confirmed that this response was modulated by a cAMP-sensitive pathway. Subsequent reports by others have led to the conclusion that both pathways interacted using a PKC-tyrosine kinase- and cAMP-dependent signal transduction pathway (33).
Furthermore, alternate signaling pathways may be utilized by different mechanical stimuli. For example, Levin and co-workers (28) reported an increase in tPA production in cells exposed to a hyperosmotic environment. EC that were incubated in hyperosmotic medium showed a dose-dependent decrease in cell volume and a 1.9- and 3.7-fold induction of tPA secretion at 425 and 485 mosmol/kgH2O, respectively. However, they postulated that this increase occurs through a different signal pathway, as inositol trisphosphate (IP3) and DAG levels were unchanged in the hyperosmotic cells.
Previous studies in our laboratory have demonstrated that cyclic strain alters the second messenger metabolic pathways that may potentially play a role in the regulation of tPA. For example, cyclic strain leads to activation of protein kinase A (PKA)-adenylate cyclase (25, 34), thereby increasing intracellular cAMP, which is also accompanied by an increased binding of the nuclear factor CRE (14). We have also reported that the phosphoinositide pathway is activated in EC subjected to cyclic strain. EC exposed to 60 cycles/min, 24% strain, had an enhanced phosphatidylinositol turnover with the production of IP3 and DAG (15, 40) and activation of PKC (41) within seconds of the initiation of cyclic strain. Furthermore, the induction of certain fos and jun mRNA occurred by 30 min, and levels of the transcription factor AP-1 increased by 4 h after cyclic strain (14, 47).
In this study, we demonstrate that exposure of EC to cyclic strain can
result in a two- to threefold increase in tPA promoter activity and the
strain-mediated induction requires sequences downstream of position
145. DNase I protection analysis indicates binding of at least
two nuclear proteins to the promoter region of the tPA gene. The
temporal nature of this binding, as documented by EMSA, indicates that
they occur at different intervals. The AP-2 binding peaks at 4 h, and
the CRE-like factor appears at 1 h. These results are consistent with a
complex but interactive activation of different intracellular pathways,
such as the PKA and PKC cascade. The functional nature of these nuclear
proteins is confirmed by the promoter analyses that demonstrate
diminution of promoter activity when these areas are deleted or mutated
(Fig. 4). These two binding sites have already been shown to be crucial for basal expression of tPA (32), and the responses to cAMP, forskolin,
and PMA are well characterized. Both DNA binding sites have been
reported to convey the effects of activators of the PKA- and
PKC-dependent pathways (1, 36). The apparent synergy between AP-2 and
the CRE sites in transcription modulation has also been reported in
other genes (35, 42). Binding of transcription factors to these
promoter elements has been posited to result in similar conformational
changes in different genes (7, 20).
Resnick and co-workers (37, 38) recently demonstrated that, by transfecting bovine aortic EC with a reporter gene consisting of a 1.3-kb fragment, or a series of 5'-deletion mutants, of human platelet-derived growth factor-B (PDGF-B) gene promoter coupled to CAT reporter gene, they were able to identify an SSRE necessary for the increased PDGF-B activity in response to applied shear force. With the use of EMSA, these authors were able to show that nuclear extracts from transfected cells formed distinct protein-DNA complexes when probed with oligonucleotides from the SSRE region of the PDGF-B chain promoter. This was the first description of the activation of a "mechanosensitive" nuclear transcription, and this sequence (GAGACC) and its complementary sequence have been noted in other genes that are responsive to shear stress (37).
In collaboration with Resnick and co-workers (37, 38), we
have recently demonstrated that the SSRE may also play a significant role in the regulation of cyclic strain-sensitive genes. Using similar
gel shift assays with the SSRE nucleotide and nuclear extracts from
bovine aortic EC exposed to an average of 10% cyclic strain at a
frequency of 60 cycles/min for up to 24 h, we showed transient but
specific binding of the SSRE probe as early as 30 min, suggesting that
cyclic strain induced activation of SSRE (39). However, as made evident
by the present studies, although binding is demonstrated, this does not
necessarily imply functionality. The SSRE site that is located at 945 bp upstream of the tPA promoter start site did not seem to be necessary
for tPA induction by cyclic strain. First, there was no diminution in
activity between the 1452 and
440 deletion constructs
(Fig. 2). Second, direct testing of the site with an SSRE-directed
mutant showed no difference in activity compared with the wild type
(Fig. 4B).
In conclusion, EC exposed to cyclic strain express and release significant amounts of tPA compared with the stationary EC. The mechanism of this effect seems to occur through activation of multiple intracellular coupling pathways, including PKA and PKC. These in turn result in binding of different nuclear factors to the promoter region of the tPA gene, resulting in new tPA gene transcripts. Thus external physical forces, including cyclic strain, may play a significant role in modulating the biosynthesis of tPA and may have important implications in physiological processes.
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ACKNOWLEDGEMENTS |
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We acknowledge the gift of the tPA/CAT promoter constructs from Dr. Robert Medcalf.
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FOOTNOTES |
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This work was funded in part by grants to B. E. Sumpio [National Heart, Lung, and Blood Institute Grant R01-HL-47345, Veterans Affairs Merit Award, and American Heart Association (National Affiliation) grants]. W. Du is a Council for Tobacco Research Scholar.
Address for reprint requests: B. E. Sumpio, Dept. of Surgery (Vascular), Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06510.
Received 23 January 1997; accepted in final form 3 July 1997.
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