| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, and
Division of Hematology and Oncology, Beth Israel-Deaconess Medical Center and Harvard Medical School, Boston, MA 02115
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The primary function attributed to endothelial CD73 has been catabolism of extracellular nucleotides, although CD73 may also mediate lymphocyte binding under some circumstances (8). Recent studies have revealed that when coincubated with endothelia or epithelia, neutrophils release micromolar concentrations of 5'-AMP (4, 7). 5'-AMP is rapidly converted to adenosine (via surface-expressed CD73), and adenosine liberated in this fashion directly activates surface adenosine A2B receptors (4, 7, 10) and promotes endothelial barrier function through elevation of intracellular cAMP (4, 11). Rather little is known about the regulation of endothelial CD73 and whether this molecule contributes to endothelial permeability. The cloned CD73 gene promoter bears a cAMP response element (CRE)3 (12), which is one of the consensus DNA motifs that regulate transcription through the cAMP-dependent coactivator CRE binding protein (CREB) (13). Adenosine activation of endothelia elevates intracellular cAMP (4, 14), providing the possibility that the enzymatic product of CD73 (adenosine) could transcriptionally regulate surface enzyme (CD73). To date, no direct evidence exists for this pathway. In these studies, we explored the regulation of endothelial CD73 bioactivity; surface protein expression; and transcriptional activation by adenosine, adenosine analogues, and other cAMP agonists. The results define a paracrine pathway of CD73 induction by adenosine in vascular endothelial cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human microvascular endothelial cells (HMVEC), an endothelial
primary culture isolated from adult dermis, were used throughout these
studies. HMVEC were obtained from Cascade Biologics (Portland, OR) and
cultured as previously described (4). Culture medium was
supplemented with heat-inactivated calf serum, penicillin,
streptomycin, HEPES, heparin, L-glutamine, and endothelial
mitogen factor. For preparation of experimental HMVEC monolayers,
confluent endothelial cells (passages 1, 2, or 3) were seeded at
2 x 105 cells/cm2
onto either polycarbonate permeable supports or six-well plates
precoated with 0.1% gelatin. Endothelial cell purity was assessed by
phase microscopic "cobblestone" appearance and uptake of
fluorescent acetylated low density lipoprotein.
Measurement of 5-ectonucleotidase activity
Endothelial CD73 surface activity was assessed as before
(4) by measuring the conversion of etheno-AMP (E-AMP) to
ethenoadenosine (E-ADO) (15). Briefly, HBSS with or
without 
,ß-methylene ADP (APCP) was added to HMVEC monolayers on
six-well plates. After 10 min, E-AMP (final concentration 10 µM) was
added for an additional 10 min, removed, acidified to pH 3.5 with HCl,
spun (10,000 x g for 20 s, 4°C), filtered (0.45
µm), and frozen (–80°C) until analysis via HPLC. A Hewlett-Packard
(Palo Alto, CA) HPLC (model 1050) with a HP 1100 diode array detector
was used with a reverse-phase HPLC column (Luna 5 µm C18
(2), 150 x 4.60 mm, Phenomenex, Torrance, CA). E-AMP
and E-ADO were measured with a 0–50%
methanol/H2O gradient (10 min) mobile phase (2
ml/min). Absorbance was measured at 260 nm and UV absorption spectra
were obtained at chromatographic peaks. CD73 activity was expressed as
percentage E-AMP conversion in this time frame.
CD73 immunoprecipitation
Confluent endothelial cells exposed to indicated experimental conditions (150 cm (2) confluent cells per condition) were surface labeled with biotin and lysed, and cell debris was removed by centrifugation as described previously (4). Lysates were precleared with 50 µl pre-equilibrated protein G-Sepharose (Pharmacia, Uppsala, Sweden). Immunoprecipitation of CD73 was performed with mAb 1E9 (a kind gift from Dr. Linda Thompson, Oklahoma Medical Research Foundation, Oklahoma City, OK) followed by addition of 50 µl preequilibrated protein G-Sepharose and overnight incubation. Washed immunoprecipitates were boiled in nonreducing sample buffer (2.5% SDS, 0.38 M Tris (pH 6.8), 20% glycerol, and 0.1% bromphenol blue), resolved by SDS-PAGE, electroblotted to nitrocellulose, and blocked overnight in blocking buffer. Biotinylated proteins were labeled with streptavidin-peroxidase and visualized by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Analysis of mRNA levels by PCR
RT-PCR was used to define endothelial CD73 mRNA regulation. Briefly, 1 µg of DNase I (GenHunter, Nashville, TN)-treated total RNA-derived HMVEC exposed to experimental conditions was reverse transcribed into cDNA using a reaction mixture consisting of 4 µl of 25 mM MgCl2, 2 µl of 10x reverse transcriptase buffer, 2 µl of 10 mM concentrations of each dNTP, 0.5 µl of rRNase in RNase inhibitor (20 U total), 15 U of avian myeloblastosis virus reverse transcriptase, and 0.5 µg of oligo(dT)15 primer in a total volume of 20 µl. Thermostable Tfl DNA polymerase (Promega, Madison, WI) from Thermus flavus was used for second-strand cDNA synthesis and DNA amplification. The CD73 PCR reaction contained 1 µM concentrations each of the sense primer (5'-CAC CAA GGT TCA GCA GAT CCG C-3') and the antisense primer (5'-GTT CAT CAA TGG GCG ACC GG-3'), 10 µl of 5x PCR buffer, 1 mM MgSO4, 0.2 mM dNTP, and 5 U of Tfl enzyme mix in a total volume of 50 µl. Each primer set was amplified using 25 cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 4 min, followed by a final extension of 72°C for 7 min. The PCR fragments were visualized on a 1.5% agarose gel containing 5 µg/ml ethidium bromide. To ensure that an equal amount of template was used in each amplification reaction, 5 µl of reverse transcriptase reaction was used as template with 1 µM concentrations each of human ß-actin sense primer (5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3') and antisense primer (5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3') in identical reactions; a 661-bp amplified fragment with equal intensity was observed in all samples.
EMSA
Nuclear extracts of cells exposed to indicated experimental conditions were obtained as described before (16). Briefly, confluent monolayers of HMVEC in 100-mm petri dishes were washed in ice-cold PBS and lysed by incubation in 500 µl of buffer A (10 mM HEPES (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 200 mM sucrose, 0.5 mM PMSF, 1 µg of both leupeptin and aprotinin per ml, and 0.5% Nonidet P-40) for 5 min at 4°C. The crude nuclei released by lysis were collected by microcentrifugation (15 s). Nuclei were rinsed once in buffer A and resuspended in 100 µl of buffer C (20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM PMSF, 1.0 mM DTT, and 1 µg/ml of both leupeptin and aprotinin). Nuclei were incubated on a rocking platform at 4°C for 30 min and clarified by microcentrifugation for 5 min.
The following synthetic oligonucleotide probes were synthesized (Genosys Biotechnologies, The Woodlands, TX) and used as probes in EMSAs. The CRE motif (underlined) lies at positions –87 to –81 relative to the transcription start site in the CD73 promoter; sense probe, 5'-TCG GAT CGG GTG ACG TCG CGA ACT TGC-3'. Double-stranded oligonucleotide probes for EMSA were labeled with [32P]ATP using T4 polynucleotide kinase (1 µl with specific activity of 15, 300 cpm/µl), incubated with 0.65 µg nuclear lysates for 20 min at room temperature in 26 µl annealing buffer (20 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM EDTA, 1 mM DTT, 3 mM MgCl2, 3.9 µg poly(dI-dC), and 2.5% glycerol), and separated by electrophoresis on a 4% nondenaturing polyacrylamide gel. For supershift experiments, the reaction mixtures were incubated on ice for 5 min in the presence of 3 µl specific polyclonal antisera either against CREB-1 (Santa Cruz Biotechnology, Santa Cruz, CA) or CREB-2 (also called ATF-4, Santa Cruz Biotechnology) before addition of labeled probe. The gel was dried and autoradiographed at –80°C. As a control for specificity, a CRE site mutated probe (sense sequence 5'-TCG GAT CGG GTG AAA TCG CGA ACT TGC-3') was used.
Endothelial macromolecule permeability assay
Endothelial paracellular permeability was assessed exactly as described previously (4) using HMVEC grown on polycarbonate permeable inserts (0.4 µm pore, 6.5 mm diameter; Costar, Cambridge, MA). Permeability of FITC-labeled dextran (70 kDa, Molecular Probes, Eugene, OR) was assessed on washed monolayers (HBSS) by sampling serosal fluid (50 µl at 5, 10, 15, 20, 30, and 60 min); sample volume was replaced with HBSS. Fluorescence intensity of each sample was measured (excitation, 485 nm; emission, 530 nm) (Cytofluor 2300, Millipore, Bedford, MA), and FITC-dextran concentrations were determined from standard curves generated by serial dilution of FITC-dextran. Paracellular flux rates were calculated by linear regression.
Other materials
Adenosine was obtained from Calbiochem (La Jolla, CA). Adenosine amine congener (ADAC), 8-(3-chlorostyryl) caffeine (CSC), alloxazine, and 5'-(N-cyclopropyl)-carboxamidoadenosine were obtained from Research Biochemical International (Natick, MA). E-AMP, E-Ado, APCP, 5'-(N-ethylcarboxiamido)adenosine (NECA), and AMP were obtained from Sigma (St. Louis, MO). FITC-dextran was obtained from Molecular Probes. Anti-CD73 Ab (mAb 1E9) was a generous gift from Dr. Linda Thompson (Oklahoma Medical Research Foundation). Transwell inserts were obtained from Corning Costar (Cambridge, MA).
Data analysis
CD73 bioactivity and paracellular permeability data were compared by two-factor ANOVA or by Student’s t test, where appropriate. Values are expressed as the mean and SEM of n monolayers from at least three separate experiments.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We recently demonstrated that activated polymorphonuclear
leukocyte supernatants promote endothelial barrier function
(4). Characterization of this bioactivity revealed that
polymorphonuclear leukocyte-derived 5'-AMP contributed to this action
and required 5'-AMP conversion to adenosine via endothelial surface
ecto-5'-nucleotidase (CD73). Here, we determined whether endothelial
CD73 expression might be regulated by activation of adenosine
A2 receptors, thus defining a paracrine pathway
of adenosine metabolism. As shown in Fig. 1
A, HMVEC pretreatment with
the adenosine A2 receptor agonist NECA at
concentrations that approximate measured adenosine plasma levels (1
µM) (17) induced a time-dependent increase in functional
CD73 (ANOVA, p < 0.01) as determined by HPLC analysis
of E-AMP conversion to E-adenosine (15), with a 5.4- ±
1.1-fold increase at 48 h following NECA exposure
(p < 0.001). Shorter periods of incubation
with NECA (i.e., <8 h) revealed no significant change in CD73 activity
(data not shown). These results of increased CD73 activity by NECA were
specific, since addition of the CD73 inhibitor APCP (3 µM)
significantly diminished CD73 activity in both control and 48-h
NECA-treated monolayers (Fig. 1A, filled bars). Similarly,
as shown in Fig. 1A (open bars), the addition of antagonists
(18) to adenosine A2A receptor (CSC,
10 µM) or A2B receptor (alloxazine, 10 µM) in
coincubation with NECA revealed a predominant role for the
A2B receptor (p < 0.025
compared with NECA alone), since A2A receptor
antagonists did not influence functional CD73 induction
(p = not significant compared with NECA alone).
Fig. 1B depicts a concentration-dependent increase in CD73
activity (ANOVA, p < 0.01), with a maximal increase of
4.7- ± 0.6-fold at 3 µM and an apparent EC50
of 0.8 µM. Addition of APCP revealed these differences to be
specific for CD73 (p < 0.001 in the presence
and absence of APCP). Similar results of increased CD73 activity were
observed with endothelial exposure to 5'-AMP (2.8- ± 0.3-fold increase
at 100 µM for 36 h, p < 0.05) as well as the
adenosine A2 receptor agonist
5'-(N-cyclopropyl)carboxamidoadenosine (3.6- ± 0.6-fold
increase at 10 µM for 36 h, p < 0.01). Pulsing
endothelial cells with NECA (i.e., 1-h exposure followed by removal)
was found to be as effective as continuous exposure to NECA (data not
shown), suggesting that continuous adenosine receptor activation is not
required for this response. Previous studies have indicated that
endothelial cells express adenosine A1,
A2, and A3 receptors.
However, a time-course and concentration-response curve using the
adenosine A1 receptor agonist ADAC did not
increase surface CD73 functional activity (maximal 5.5 ± 2.1%
increase in CD73 activity with 3 µM ADAC at 36 h,
p = not significant compared with no ADAC), suggesting
that the increase in CD73 activity is specific for activation of the
A2 receptor.
|
, a time course of NECA
pretreatment (5 µM NECA) revealed increased CD73 with increasing time
of exposure (maximal induction of 17.2- ± 3.8-fold at 36 h by
densitometry, p < 0.001). Increases in both surface
activity (85.0 ± 6.2% inhibition) and surface protein levels
(72.2 ± 10.5% inhibition) were inhibited by coincubation of
HMVEC with NECA (3 µM) in the presence of cycloheximide (2 µg/ml,
data not shown), suggesting the requirement for new protein
synthesis.
|
We next extended these studies to examine CD73 mRNA. Adenosine
receptor coupling is well characterized and indicates that
A1 and A3 receptor
activation decreases intracellular cAMP while A2
receptor ligation increases cAMP (18). Moreover, we have
specifically determined that adenosine A2B
receptor activation is coupled to increased intracellular cAMP
(4). Since the CD73 promoter bears a consensus CRE
(12), RT-PCR analysis was used to determine whether
adenosine A2 receptor activation induces CD73
mRNA. As shown in Fig. 3, 25 cycles of
PCR revealed no detectable CD73 message in control HMVEC, but exposure
to the adenosine A2 receptor agonist NECA (5
µM) induced a time-dependent increase in CD73 mRNA (comparison of
CD73-to-ß-actin ratios by ANOVA, p < 0.01).
Additionally, this NECA-induced response was inhibited by the
relatively selective adenosine A2B receptor
antagonist aloxazine (Fig. 3B). These data suggest that
adenosine A2B receptor activation induces
CD73 mRNA.
|
To define the relationship of intracellular cAMP to CD73
induction, other cAMP agonists were used to examine HMVEC CD73
induction. Fig. 4 demonstrates the
influence of the cAMP agonists forskolin and PGE2
on CD73 bioactivity (examined by HPLC) and revealed that both forskolin
and PGE2 induced a concentration-dependent
increase in surface CD73 activity (ANOVA, p < 0.01 for
both forskolin and PGE2). As shown in Fig. 5, forskolin (10 µM) and
PGE2 (1 µM) readily induced CD73 mRNA. As a
control for specificity, endothelial exposure to the selective
adenosine A3 receptor agonist
N6-(3-iodobenzyl)-5'-N-methylcarboxoamidoadenosine,
which decreases intracellular cAMP (19), did not influence
CD73 mRNA levels (data not shown). These data indicate that elevation
of intracellular cAMP induces CD73 mRNA and suggest that
adenosine-mediated induction of CD73 is coupled to intracellular cAMP
levels.
|
|
, induction of CD73
DNA-binding activity was evident in monolayers pre-exposed to NECA (10
µM) for 4 or 16 h at 37°C. A similar DNA-binding activity was
evident in controls not exposed to NECA, but to a far lesser extent.
Forskolin (1 µM, 4 h) served as a positive control for this
DNA-binding activity. Inclusion of anti-CREB-1 but not
anti-CREB-2 Ab during these incubations resulted in a clear
supershift, indicating the likelihood that specific DNA-binding
activity represents CREB-1.
|
Our previous studies revealed that adenosine
A2B receptor activation (via 5'-AMP) promotes
endothelial barrier function, measured as a decrease in paracellular
flux of labeled tracer (4). This activity required
endothelial surface CD73 conversion of 5'-AMP to adenosine. It is not
known whether increased CD73 (via induction by cAMP agonists)
contributes to increased 5'-AMP responses. Thus, we defined whether
forskolin-induced CD73 influenced endpoint functional responses in
intact HMVEC monolayers. As shown in Fig. 7, endothelial pre-exposure to forskolin
(5 µM for 36 h, conditions that induce CD73; see Figs. 4
and 5)
resulted in a nearly 10-fold enhanced response to exogenous
administration of 5'-AMP (apparent EC50 2
µM) compared with HMVEC not pre-exposed to forskolin (apparent
EC50 22 µM). This response was specific for
CD73, since addition of APCP blocked this response by 77.3 ±
5.6% at 30 µM 5'-AMP (p < 0.001 compared
with no APCP). These data indicate that CD73 induced by elevations in
intracellular cAMP manifests as an endpoint functional response
(increased endothelial barrier function).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CD73 has been studied widely at the functional and genomic level (2, 20). Cloning of the promoter region of human CD73 identified a CRE upstream from the CD73 start codon (21). Since genes containing such elements can be regulated by changes in intracellular cAMP (22), and we have previously demonstrated that adenosine elevates endothelial intracellular cAMP (4), we examined the impact of adenosine on CD73 transcriptional responses and cell surface expression. Our results of CRE-dependent induction of CD73 confirm previous work alluding to differences in CD73 regulation between endothelial and myeloid cell types (23). For instance, original studies suggested that CD73 promoter constructs lacking the consensus CRE were still functional and that CD73 production in myeloid cell lines was not influenced by agents that elevate intracellular cAMP (12). It has been reported, however, that agents that activate protein kinase C (i.e., PMA) strongly induce CD73 mRNA and activity in myeloid leukocytes (HL-60 cells) (24). Similarly, in Jurkat T cells, PMA induces CD73 gel shift using oligonucleotides corresponding to the CRE site (25). These latter results may be explained by the fact that induction of CRE-bearing genes can occur through many pathways, including activation both of protein kinase A and C (13). Similarly, our EMSA results demonstrating CREB-1 binding emphasize differences between endothelial cells and myeloid cells. For instance, in Jurkat T cell extracts, neither CREB-1 nor CREB-2 was found to bind to the CD73 promoter CRE site (25), suggesting some degree of cell specificity for this response. Taken together, these observations suggest that while myeloid and endothelial CD73 lack structural variability (23), significantly different regulatory properties exist.
Following activation, neutrophils release a number of biologically active mediators. Two such mediators are 5'-AMP and its metabolite, adenosine (4, 7), which can exist in human plasma at micromolar concentrations (17). Measured concentrations of 5'-AMP and adenosine are in a range sufficient to influence functions such as epithelial electrogenic Cl- secretion and endothelial permeability (4, 7). Metabolism of 5'-AMP to adenosine requires 5'-nucleotidase; both intracellular (cytosolic 5'-nucleotidase) and cell surface (5'-ectonucleotidase/CD73) forms are expressed in most cell types (2). In the present studies, we have not addressed whether both forms are similarly regulated by adenosine. Together with other ectonucleotidases, 5'-ectonucleotidase completely hydrolyzes ATP to adenosine (1, 26). Through inhibition of endothelial 5'-ectonucleotidase with APCP, our data demonstrate that the 5'-AMP-mediated decrease in endothelial permeability requires conversion of 5'-AMP to adenosine and that CD73 may be rate limiting (i.e., increased CD73 results in parallel increases in 5'-AMP-mediated bioactivity). The competitive inhibitor APCP abolishes the influence of 5'-AMP, whereas the less potent, noncompetitive inhibitor mAb 1E9 substantially diminishes the influence of 5'-AMP (4), indicating that CD73 may influence endothelial cell permeability in addition to its previously characterized role as a cell adhesion molecule (8). These results may have broad implications. For instance, it is not presently known how CD73 expression is regulated in the healthy vasculature. A number of physiologically relevant mediators flow across the healthy endothelium at any point in time (e.g., nucleotides, hormones, chemokines, and cytokines). Many of these mediators bind and activate signal transduction pathways in the endothelium (i.e., elevation in cAMP, Ca2+, etc.). Thus, it is possible that basal, low level activation of the healthy endothelium regulates CD73 expression as a bystander process and, in turn, regulates basal vascular permeability. In this regard, adenosine may serve as a feedback mechanism to directly regulate CD73.
Once liberated in the extracellular space, adenosine either is taken up into the cell (through dipyridamole-sensitive carriers) or interacts with cell surface adenosine receptors (5). Presently, four subtypes of G protein-coupled adenosine receptors exist, designated A1, A2A, A2B, and A3. These receptors are classified according to use of pertussis toxin-sensitive pathways (A1 and A3) or adenylate cyclase activation pathways (A2A and A2B) (5). Endothelial cells of many origins express constitutive adenosine receptors (5), primarily of the A2A and A2B subtypes (27, 28, 29, 30). The present studies define a transcriptional pathway mediated by adenosine receptor activation, including but not limited to CD73. In fact, many gene promoters bear response elements for cAMP (13), and, therefore, it is quite likely that adenosine also activates a number of other genes. Intriguingly, it is possible that cellular hypoxia might induce endothelial CD73. While we have not studied this aspect directly, recent work has defined a role for CRE and CRE-binding protein (CREB) in hypoxia-elicited induction of a number of proteins (31, 32), and this pathway may be particularly relevant to a number of cardiovascular diseases in vivo. For instance, adenosine production in the ischemic myocardium is attributable to activity of CD73 (33), and both CD73 activity and adenosine metabolism have been implicated in cardiac preconditioning by brief periods of ischemia (34, 35). Importantly in this regard, it was recently shown that adenosine (via activation of A1 receptors) can directly regulate extracellular adenosine levels in rat cardiac fibroblasts (36), providing a potential feed-forward loop in this metabolic process. Thus, whether adenosine-mediated CD73 induction occurs at the level of the ischemic myocardium is not known at the present time.
We show in these studies that cAMP agonist-induced CD73 enhances
5'AMP-mediated promotion of endothelial barrier function. Our recent
studies revealed that 5'-AMP promotes endothelial barrier in at least
two different endothelial sources (umbilical vein and microvascular
cells) (4), primarily through use of the
A2B receptor. At present, the mechanism(s) of
cAMP-mediated regulation of endothelial barrier are not clear.
Endothelial cells, like other eukaryotic nonmuscle cells, contain
myosin, actin, and a number of associated proteins that function in
cell motility (37, 38, 39). It is now generally accepted that
phosphorylation of the 20-kDa light chain of myosin is
required to activate the myosin ATPase essential for endothelial shape
changes (40). This process is catalyzed by myosin light
chain kinase, a Ca2+-calmodulin-dependent enzyme
system that produces a transfer of the 
-phosphate of ATP onto myosin
light chain. Moreover, it has been demonstrated that intracellular
levels of cAMP are tightly coupled to myosin light chain activity
(41). Pharmacologic maneuvers that increase intracellular
levels of cAMP (42) or cGMP (43) result in a
dephosphorylation of myosin, a redistribution of actin and myosin II,
and a decrease in endothelial isometric tension, indicating that
endothelial permeability is likely coupled to myosin light chain kinase
activity. Finally, recent in vivo work has suggested that
endotoxin-induced increases in vascular permeability are associated
with accumulation of neutrophils and that dysfunction may be coupled to
elevations in cGMP (44).
An important point of consideration is whether this adenosine-mediated CD73 induction pathway is relevant to basal and/or acute responses in vivo, particularly since the components may take up to 36–48 h for development. First, the kinetics of this response in an in vivo system is not known. Based on previous studies of other regulated endothelial surface molecules (e.g., leukocyte adhesion molecules) (45), the in vivo kinetics tend to be more rapid than those in vitro, and thus, one would predict that these responses with CD73 would occur more rapidly than in our model (24–36 h). Nonetheless, as a second point, it is important to note that endothelial cells have long been known to express functional CD73 under "basal conditions" (1) and that our work sheds light onto pathways to regulate CD73 expression. Under conditions of both acute and chronic inflammation, adenosine is a demonstrated mediator (46), and thus, it is reasonable to assume that adenosine may be an endogenous regulator of CD73 expression. Thirdly, these adenosine-regulated CD73 responses may well function in a chronic inflammatory setting. For example, CD73 has been shown to function as an adhesion molecule for lymphocytes (8, 23), and thus, cells that characteristically traffic later in the inflammatory response (i.e., lymphocytes and monocytes) may be orchestrated by endothelial expressed CD73. Taken together, it is not difficult to hypothesize a role for adenosine regulation of basal as well as acute induction of endothelial CD73.
These results confirm previous studies defining the function of CD73 on vascular endothelial cells and reveal a previously unappreciated mechanism of cAMP-mediated regulation of CD73 on vascular endothelia. This regulatory pathway extends to the level of transcriptional induction and unveils a potential role for CD73 regulation of vascular permeability.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Sean P. Colgan, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Thorn Building 704, 75 Francis Street, Boston, MA 02115.
3 Abbreviations used in this paper: CRE, cAMP response element; CREB, response element binding protein; HMVEC, human microvascular endothelial cells; E-ADO, ethenoadenosine; E-AMP, etheno-AMP; APCP, ß-methylene ADP; ADAC, adenosine amine congener; CSC, 8-(3-chlorostyryl) caffeine; NECA, 5'-(N-ethylcarboxoamido)adenosine.
Received for publication February 11, 2000. Accepted for publication August 10, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Biol. Chem. 274:19447.This article has been cited by other articles:
|
|
 |
|
  X. Sun, A. Cardenas, Y. Wu, K. Enjyoji, and S. C. Robson Vascular stasis, intestinal hemorrhage, and heightened vascular permeability complicate acute portal hypertension in cd39-null mice Am J Physiol Gastrointest Liver Physiol, August 1, 2009; 297(2): G306 - G311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Katebi, M. Soleimani, and B. N. Cronstein Adenosine A2A receptors play an active role in mouse bone marrow-derived mesenchymal stem cell development J. Leukoc. Biol., March 1, 2009; 85(3): 438 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Takedachi, D. Qu, Y. Ebisuno, H. Oohara, M. L. Joachims, S. T. McGee, E. Maeda, R. P. McEver, T. Tanaka, M. Miyasaka, et al. CD73-Generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes J. Immunol., May 1, 2008; 180(9): 6288 - 6296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Kobie, P. R. Shah, L. Yang, J. A. Rebhahn, D. J. Fowell, and T. R. Mosmann T Regulatory and Primed Uncommitted CD4 T Cells Express CD73, Which Suppresses Effector CD4 T Cells by Converting 5'-Adenosine Monophosphate to Adenosine J. Immunol., November 15, 2006; 177(10): 6780 - 6786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. H. Nemeth, C. S. Lutz, B. Csoka, E. A. Deitch, S. J. Leibovich, W. C. Gause, M. Tone, P. Pacher, E. S. Vizi, and G. Hasko Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism J. Immunol., December 15, 2005; 175(12): 8260 - 8270. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. F. Thompson, H. K. Eltzschig, J. C. Ibla, C. J. Van De Wiele, R. Resta, J. C. Morote-Garcia, and S. P. Colgan Crucial Role for Ecto-5'-Nucleotidase (CD73) in Vascular Leakage during Hypoxia J. Exp. Med., December 6, 2004; 200(11): 1395 - 1405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. L. Tsikitis, N. A. Morin, E. O. Harrington, J. E. Albina, and J. S. Reichner The Lectin-Like Domain of Complement Receptor 3 Protects Endothelial Barrier Function from Activated Neutrophils J. Immunol., July 15, 2004; 173(2): 1284 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ahmad, A. Ahmad, M. Ghosh, C. C. Leslie, and C. W. White Extracellular ATP-mediated Signaling for Survival in Hyperoxia-induced Oxidative Stress J. Biol. Chem., April 16, 2004; 279(16): 16317 - 16325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Niemela, T. Henttinen, G. G. Yegutkin, L. Airas, A.-M. Kujari, P. Rajala, and S. Jalkanen IFN-{alpha} Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5'-Nucleotidase) Up-Regulation J. Immunol., February 1, 2004; 172(3): 1646 - 1653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Headrick, B. Hack, and K. J. Ashton Acute adenosinergic cardioprotection in ischemic-reperfused hearts Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H1797 - H1818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. K. Eltzschig, J. C. Ibla, G. T. Furuta, M. O. Leonard, K. A. Jacobson, K. Enjyoji, S. C. Robson, and S. P. Colgan Coordinated Adenine Nucleotide Phosphohydrolysis and Nucleoside Signaling in Posthypoxic Endothelium: Role of Ectonucleotidases and Adenosine A2B Receptors J. Exp. Med., September 2, 2003; 198(5): 783 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. H. Neemeth, S. J. Leibovich, E. A. Deitch, E. S. Vizi, C. Szabo, and G. Hasko cDNA Microarray Analysis Reveals a Nuclear Factor-{kappa}B-Independent Regulation of Macrophage Function by Adenosine J. Pharmacol. Exp. Ther., September 1, 2003; 306(3): 1042 - 1049. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Lawrence, K. M. Comerford, and S. P. Colgan Role of VASP in reestablishment of epithelial tight junction assembly after Ca2+ switch Am J Physiol Cell Physiol, June 1, 2002; 282(6): C1235 - C1245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ledoux, D. Laouari, M. Essig, I. Runembert, G. Trugnan, J.B. Michel, and G. Friedlander Lovastatin Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Implication of Rho-Family GTPases Circ. Res., March 8, 2002; 90(4): 420 - 427. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). To determine specificity, a similar
analysis was performed in the presence of the CD73 inhibitor APCP (
)
or in the presence of adenosine receptor antagonists (
) to adenosine
A2A receptor (CSC, 10 µM) or A2B receptor
(alloxazine, 10 µM). Data are derived from six to eight
monolayers in each condition, and results are expressed as
mean ± SEM percentage E-AMP conversion. *, Statistically
different from untreated controls (p < 0.025).
**, Statistically different from 48-h NECA treatment
(p < 0.025).
) or medium
containing forskolin (FSK, 5 µM, •) for 36 h. Monolayers were
washed, and the influence of indicated concentrations of 5'-AMP on
monolayer permeability to 70-kDa FITC-dextran was determined. To define
specificity, a similar analysis was performed in the presence of the
CD73 inhibitor APCP (