Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 µM forskolin plus 1 µM IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of 32P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ~23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
Rho guanosine 5'-triphosphate; protein kinase A inhibitor; guanine nucleotide dissociation inhibitor; endothelial resistance
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INTRODUCTION |
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INCREASED VASCULAR
PERMEABILITY is a hallmark of inflammation that occurs in acute
and chronic diseases such as atherosclerosis, acute respiratory
distress syndrome, and diabetes. Inflammatory mediators (i.e.,
thrombin, TNF-, transforming growth factor-
) and oxidants
activate a repertoire of signaling events in endothelium that results
in the development of gaps between cells, leading to barrier
dysfunction (17, 19, 25, 34, 35, 39). The cAMP-dependent
protein kinase (PKA) has significant and profound protective actions
against increases in endothelial permeability. We and others (4,
10, 32, 42, 43) have shown that the ubiquitous cellular
messenger cAMP prevents increases in endothelial permeability in
response to a wide range of inflammatory mediators, including oxidants.
Although growing evidence indicates that cAMP activates both
PKA-dependent and -independent actions (8, 11, 26), our
recent observations show that protective actions of cAMP against
barrier dysfunction are likely mediated predominantly through
PKA-dependent signaling mechanisms (32, 43).
Despite the abundant reports documenting cAMP/PKA signaling in inhibition of increases in permeability, the specific mechanisms by which this occurs remain unclear and controversial. One proposed hypothesis suggests that PKA regulates the phosphorylation of myosin light chain (MLC). The phosphorylation of MLC in endothelial cells is mediated primarily by MLC kinase (MLCK), and subsequent MLC-mediated contractions provide the critical mechanical tension in promoting intercellular gap formation that leads to increases in endothelial permeability (20, 33). Endothelial-specific MLCK has been shown to contain PKA consensus phosphorylation sites, and cAMP has been reported to inhibit in vitro MLCK activity (18). Yet, increased intracellular cAMP appears unable to prevent thrombin-induced increases in MLC phosphorylation or contraction of endothelial cells (38), suggesting that protective mechanisms of PKA may not be a direct reversal of MLC phosphorylation and, therefore, of contraction.
Another class of critical regulatory proteins in regulation of endothelial barrier function is the family of Rho GTPases (2, 9, 14, 36, 45, 47). The family consists of 20 distinct members [Rho (A, B, C); Rac (1, 2, 3); Cdc42; TC10; TCL; Chp (1, 2); RhoG; Rnd (1, 2, 3); RhoBTB (1, 2); RhoD; Rif; and TTF)] (15), of which Cdc42, Rac1, and RhoA are the most characterized (6) and are essential in the relay of signals to the actin cytoskeleton in regulation of activities such as cell adhesion, motility, cell cycle progression, and apoptosis. Current findings suggest that RhoA may regulate multiple targets that may be crucial determinants of endothelial barrier function, including adherens junctions (24, 29, 47) and myosin-associated protein phosphatases (5, 13, 41).
Rho GTPases may be targets of regulation by PKA in endothelial cells. In nonendothelial cells, PKA is shown to phosphorylate RhoA at Ser188 in cytotoxic lymphocytes, resulting in inactivation of RhoA (27). In another study, RhoA and cAMP were found to have antagonistic roles in regulating the cellular morphology of epithelial-like SH-EP cells and neuronal-like SH-SY cells (12). However, in human mast cells, stimulation of adenylate cyclase with forskolin or loading cells with the cell-permeable cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate activated Cdc42 (16). The regulation of Rho GTPases by PKA in endothelial cells has not been well studied, but these observations suggest that inhibition of RhoA may potentially be an important signaling mechanism in the prevention of increases in permeability.
A mechanism of inhibition of Rho GTPase activation is through regulation of GTP/GDP cycling. Rho GTPases serve as molecular switches whose activity is regulated by cycling the protein between a GTP-bound "on" form and a GDP-bound "off" form. The cycling is tightly controlled by accessory proteins: guanine nucleotide dissociation inhibitor (GDI) captures GDP-bound Rho and maintains it in an inactive cytosolic complex; GTPase-activating protein (GAP) stimulates hydrolysis of GTP to GDP; and guanine nucleotide exchange factor (GEF) activates GTPases by enhancing the release of bound GDP. Phosphorylation of GDI has been implicated in the regulation of its ability to bind with Rho GTPases (7, 36), suggesting that the phosphorylation profile of GDI is an important factor determining the activation (and inactivation) of Rho GTPases.
In this study, we investigated the hypothesis that PKA protects against barrier dysfunction through inhibition of RhoA activation in endothelial cells. Our key findings indicated that pretreatment of endothelial cells with cAMP-elevating agents 1) inhibited thrombin-induced RhoA activation and translocation to membrane; 2) prevented the thrombin-induced dissociation of RhoA from GDI; and 3) facilitated recovery of the thrombin-mediated resistance drop and partially inhibited the extent of resistance drop. Infection of endothelial cells with replication-deficient adenovirus containing the PKI gene (PKA inhibitor) blocked cAMP-mediated protective effects on both RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
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MATERIALS AND METHODS |
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Materials.
The following reagents were purchased as follows: DH5 competent
cells, MCDB-131 medium, penicillin-streptomycin,
L-glutamine, sodium pyruvate, phosphate-buffered saline
(PBS), MEM nonessential amino acids, and MEM vitamins from GIBCO-BRL
(Gaithersburg, MD); human epidermal growth factor (EGF),
hydrocortisone, endothelial cell growth supplement, heparin, forskolin,
IBMX, isopropyl
-D-1-thiogalactoside (IPTG), and
phenylmethylsulfonyl fluoride (PMSF) from Sigma Chemical (St. Louis,
MO); glutathione sepharose 4B, ECL kit, and horseradish peroxidase
(HRP)-conjugated anti-mouse IgG and anti-rabbit IgG antibodies from
Amersham Pharmacia Biotech (Piscataway, NJ); protein A/G plus agarose
and antibodies directed against RhoA, Cdc42, and GDI from Santa Cruz
Biotechnology (San Diego, CA); antibody directed against Rac1 from BD
Transduction Laboratories (San Jose, CA); and human
-thrombin from
Enzyme Research Laboratories (South Bend, IN). All other reagents are
indicated in the text.
Cell culture. Human dermal (foreskin) microvascular endothelial cells (HMEC) were maintained in culture in MCDB-131 medium, supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT), 10 ng/ml EGF, 1 µg/ml hydrocortisone, 1% penicillin-streptomycin, and 1% L-glutamine. HMEC is an immortalized cell line transformed by SV40 large T antigen and has been shown to retain endothelial cell phenotypic and functional characteristics (3, 32). The cell line exhibits the expected morphological and functional endothelial phenotypes: it expresses and secretes von Willebrand factor, takes up acetylated low-density lipoprotein (LDL), forms tubes when grown in Matrigel, and expresses CD31 (platelet/endothelial cell adhesion molecule-1), CD36, intercellular adhesion molecule-1, and CD44 (3). The cell line also binds purified T cells in a regulatable manner and responds to cytokines comparably to nontransformed endothelial cells. HMEC were passaged 5-7 days when confluent and used for studies at population doublings between 35 and 40.
Human pulmonary artery endothelial cells (HPAEC) were purchased from Clonetics (San Diego, CA). These cells have been characterized to be endothelial in origin by the uptake of acetylated LDL and positive staining for von Willebrand factor. The HPAEC were grown in basal medium containing EGM-2 Bulletkit growth supplement (Clonetics), 10% FBS, and 5% penicillin-streptomycin. HPAEC were cultured to 10-15 population doublings for use in studies.Rho GTPase affinity-binding assay.
The affinity-binding assay was used to determine activation of Rho
GTPases. pGEX-2T containing rhotekin-RhoA binding domain or
CD-PAK was provided by Dr. John G. Collard (The Netherlands Cancer Institute, Amsterdam, The Netherlands). Bacterial-expressed glutathione S-transferase (GST)-rhotekin (binds
RhoA-GTP) or GST-CD-PAK fusion protein (binds Cdc42-GTP and Rac1-GTP)
was purified from IPTG (0.1 mM)-induced DH5 cells transformed with
the respective cloned plasmid. Confluent HMEC grown in 100-mm dishes
were serum starved overnight and treated according to experimental
protocol. The cells were then quickly washed with ice-cold PBS and
lysed in GST-FISH buffer [50 mM Tris (pH 7.4), 10% glycerol, 100 mM NaCl, 1% Nonidet P-40, 2 mM MgCl2, 25 mM NaF, and 1 mM
EDTA] plus protease inhibitor cocktail (10 µg/ml of pepstatin A, 10 µg/ml each of aprotinin and leupeptin, and 1 mM PMSF). Cell lysates were pelleted by centrifugation at 10,000 g at 4°C for 5 min, and equal volumes of cell supernatant were incubated with
GST-rhotekin or GST-CD-PAK bound to glutathione sepharose 4B beads (15 µg) at 4°C for 1 h. The Rho GTPase-fusion protein-bead complex
was washed three times with GST-FISH buffer plus protease inhibitor cocktail, and the GTP-bound Rho GTPases were eluted by boiling each
sample in 2× Laemmli sample buffer. The eluted sample and total cell
lysate were electrophoresed on 12.5% SDS-PAGE, transferred to
nitrocellulose membrane, and blocked with 5% nonfat milk at room
temperature for 2 h. The membrane was incubated at 4°C overnight with affinity-purified antibodies directed against RhoA, Cdc42, or Rac1
for detection of the respective GTP-bound Rho GTPases and total Rho
GTPases. The membrane was incubated with the appropriate secondary
HRP-conjugated antibodies for enhanced chemiluminescence detection. The
amount of GTP-bound Rho was quantified by scanning densitometry and
normalized to total Rho (GTP-bound + GDP-bound forms) in cell lysates.
Immunofluorescent confocal microscopy. Endothelial cells were plated on glass chamber slides coated with 1 µg/ml of fibronectin and grown overnight. The cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature, incubated with the appropriate primary antibodies directed against RhoA, Cdc42, or Rac1 for 1 h at room temperature followed by an additional 1-h incubation with biotinylated secondary antibodies, and detected with streptavidin conjugated with Cy2 (Jackson ImmunoResearch Laboratory, West Grove, PA). Image analysis was performed using an Olympus Confocal Fluoroview microscope equipped with argon laser (Olympus America, Melville, NY).
Immunoprecipitation. Confluent HMEC grown in 100-mm dishes were serum starved overnight and treated according to experimental protocol. The cells were then quickly washed with ice-cold PBS and lysed in radioimmune precipitation buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P-40, 1 mM EGTA, 1 mM EDTA, 1 mM orthovanadate, 50 mM NaF] plus protease inhibitor cocktail (10 µg/ml of pepstatin A, 10 µg/ml each of aprotinin and leupeptin, and 1 mM PMSF). The cell lysate was passed through a 21-gauge needle eight times, centrifuged at 4°C at 10,000 g for 10 min, and precleared by incubation with 1 µg of normal rabbit IgG with 10 µl of protein A/G plus Agarose. The supernatant was transferred to fresh tubes, incubated with 1.6 µg of rabbit anti-GDI antibody for 1 h at 4°C, then 20 µl of protein A/G plus Agarose was added, and it incubated overnight at 4°C on a rocker platform. The immunoprecipitated protein complex was collected by centrifugation at 2,500 rpm at 4°C for 5 min, washed four times with PBS, boiled in 1× electrophoresis sample buffer, and separated by SDS-PAGE. Western blot analysis was made using anti-RhoA or anti-GDI antibodies to determine coimmunoprecipitation of RhoA with GDI. As negative control, a separate group of cells was used for immunoprecipitation without the precipitating antibody.
Phosphorylation of Rho-GDI.
Serum-starved confluent monolayers of HMEC grown in 60-mm dishes were
preloaded with 200 µCi/ml [32P]orthophosphate overnight
in phosphate-free medium, after which the HMEC were treated according
to experimental protocol. Cells were quickly rinsed twice with ice-cold
PBS and lysed for 20 min on ice with 300 µl of radioimmune
precipitation buffer plus protease inhibitor cocktail and then
immunoprecipitated with anti-GDI antibody (as described in
Immunoprecipitation). The protein complexes were transferred
to nitrocellulose membrane, and autoradiogram was made by exposure to
Kodak X-OMAT X-ray film at 80°C. Western blot was made with
anti-GDI antibody to determine equal protein loading. As negative
control, a separate group of cells was without the precipitating antibody.
Transendothelial electrical resistance.
The transendothelial electrical resistance, which provides an index of
endothelial barrier function, was measured in real time using the
electric cell-substrate impedance sensor (ECIS) system, which detects
cell impedance changes in a highly sensitive manner (Applied
BioPhysics, Troy, NY) (21, 31, 39). The system consists of
a large gold-plated electrode (0.15 cm2) and smaller
gold-plated electrodes (5 × 104 cm2)
with a 500-µl well fitted above each small electrode. The smaller electrode allows the impedance of the electrode-electrolyte interface at 4,000 Hz to predominate over the solution resistance. The small and
large counter electrodes are connected to a phase-sensitive lock-in
amplifier, and an alternating current is supplied through the 1-M
resistor. The measured electrical impedance indicates the restriction
of current flow through the cell monolayer between the electrodes and
thus provides an index of the endothelial barrier function.
Infection with adenovirus containing protein kinase A inhibitor.
An E1, E3
replication-deficient adenovirus
containing full-length protein kinase A inhibitor (PKI) cDNA (AdPKI)
was constructed and characterized as described previously
(32). Confluent monolayers of HMEC were infected with
AdPKI at 100 multiplicities of infection (MOI) (MOI = plaque-forming units/target cell) for 2 days, and the cells were
treated according to experimental protocol. The optimum infection
protocol for HMEC has been determined in previous studies (31,
32). Heat-inactivated AdPKI served as control virus as described
(32).
Statistics. Single sample data were analyzed by the two-tailed t-test. A multiple range test (Scheffé's test) was used for comparison of experimental groups with a single control group (44).
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RESULTS |
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Regulation of transendothelial resistance by PKA.
Transendothelial electrical resistance studies were made to evaluate
the regulation of the HMEC barrier function by PKA. Results indicated that thrombin (10 nM) stimulation of HMEC induced rapid decreases in resistance, which reversed to near baseline levels with
time (41.9 ± 2.8 min; Fig. 1).
Forskolin and IBMX (FI) pretreatment for 15 min resulted in an initial
baseline resistance increase and prevented ~23% of the
thrombin-induced decreased resistance (Fig. 1B). FI
pretreatment also decreased time required for the resistance drop to
recover to near baseline (16.2 ± 1.0 min; Fig. 1C).
Infection with recombinant AdPKI to overexpress PKI in HMEC prevented
the FI-mediated protective effects on the thrombin-induced resistance
changes (Fig. 1). The results confirm our previous studies that the
protective actions of cAMP against endothelial barrier dysfunction are
mediated predominantly through PKA (31, 32, 43).
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Activation responses of Rho GTPases.
The immunofluorescent intracellular distribution of Rho GTPases was
determined in two different endothelial cell types, HMEC and HPAEC, to
evaluate similarity of localization of RhoA, Cdc42, and Rac1. The
results indicated that both endothelial cell types express the three
GTPases (Fig. 2). RhoA was found
predominantly in the cytosol with some localization in the nucleolus;
Cdc42 was distributed in the cytosol and the cell periphery; and Rac1 was distributed diffusely in the cytosol with some nuclear
localization.
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Inhibition of thrombin-induced RhoA activation. To investigate regulation of RhoA activation by the cAMP/PKA pathway, HMEC were pretreated with forskolin (10 µM) and IBMX (1 µM) for 15 min to increase intracellular cAMP levels and PKA activity before activation with 10 nM thrombin as previously described (31, 32, 43). Results showed that FI pretreatment inhibited the thrombin-induced increases in the GTP-bound form of RhoA (Fig. 4), indicating inhibition of RhoA activation. In contrast, neither thrombin alone nor FI pretreatment alter levels of Cdc42-GTP (Fig. 5) or Rac1-GTP (Fig. 6), indicating selectivity of regulation of the Rho GTPases by thrombin. In separate studies, HMEC were infected with the recombinant AdPKI to overexpress PKI for specific inhibition of endogenous PKA (see MATERIALS AND METHODS). After infection, cells were pretreated with FI before stimulation with thrombin as for previous studies, and RhoA activation was determined by affinity-binding assay. Results from the studies indicated that PKI overexpression prevented the inhibitory effects of FI on thrombin-induced RhoA activation, restoring ~75% of the thrombin-activated RhoA response (Fig. 4). Infection of HMEC with control heat-inactivated AdPKI was ineffective in blocking the FI inhibitory effect on RhoA activation (data not shown).
The RhoA activation response was additionally evaluated by RhoA translocation to the membrane. The membrane translocation was quantified from randomly selected 20-25 cells/group. The cell membrane area was selected on the basis of predetermined x and y coordinates on the microscope stage at a constant magnification and fluorescent intensity. With the use of confocal system software, the membrane area was outlined and fluorescent intensity quantified. Results were presented as means ± SD from three separate studies. Stimulation of HMEC with thrombin (10 nM for 15 min) increased RhoA fluorescence at the cell membrane 65% over control, indicating RhoA translocation to membrane. In HMEC pretreated with FI, the thrombin-induced increased translocation was reduced by >50% (Fig. 7). These results are consistent with the affinity-binding assay studies showing that the thrombin-induced activation of RhoA was inhibited by cAMP.
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DISCUSSION |
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The critical findings from this study show that pretreatment of HMEC with cAMP-elevating agents 1) inhibited thrombin-induced RhoA activation and translocation to membrane; 2) prevented the thrombin-induced dissociation of RhoA from GDI; and 3) facilitated recovery of the thrombin-mediated resistance decrease and partially inhibited the extent of resistance drop. Infection of HMEC with the recombinant AdPKI blocked the cAMP-mediated protective effects on RhoA activation and resistance changes. The results provide evidence that PKA inhibited RhoA activation in endothelial cells and support a signaling mechanism of protection against vascular endothelial barrier dysfunction.
Our results indicated that thrombin stimulation of human microvascular endothelial cells increased the GTP-bound form of RhoA and RhoA translocation to the cell membrane, indicating that activation of RhoA was associated with its translocation. Activation of Rho GTPases is known to be associated with changes in subcellular localization in several cell types, and many of the responses are translocated to the membrane that is dependent on a posttranslational isoprenylation step (1, 37). This finding provides further support for previous studies in which human umbilical vein endothelial cells (HUVEC) were stimulated by thrombin, resulting in a rapid (<1 min) increase in RhoA activation (36, 45). Several pieces of evidence now implicate RhoA in the regulation of mediator-induced increases in endothelial permeability (9, 14, 36, 40, 45, 47).
Our identification of RhoA as a target for negative regulation by PKA provides a potentially critical mechanism by which the PKA signaling pathway is known to be protective against vascular endothelial barrier dysfunction in response to a wide range of inflammatory mediators. The inhibition of RhoA by the cAMP/PKA pathway has been observed in nonendothelial cells. In a mouse lymphoid cell line, the elevation of intracellular cAMP inhibited GTP/GDP exchange, resulting in inhibition of chemoattractant-induced RhoA activation and cell adhesion (28). PKA was also observed to inhibit RhoA activation in human lymphocytes by direct phosphorylation of the COOH terminus at Ser188, allowing the GTP-bound form of RhoA to be complexed with GDI, thereby preventing translocation to membrane (27). This phosphorylation did not alter the ability of RhoA to bind GTP, nor did it modify its intrinsic GTPase activity. These authors hypothesized that the PKA-mediated phosphorylation of RhoA supports an alternative pathway to terminate RhoA-GTP signaling independent of GTP/GDP cycling (27). However, in endothelial cells, despite the finding that elevation of cAMP inhibited lipopolysaccharide-induced MLC phosphorylation, RhoA was not phosphorylated (14). However, RhoA activity was not measured in this report and, therefore, it is not known whether cAMP inhibited activation of RhoA under these experimental conditions (14). Nonetheless, these observations suggest that PKA signaling may involve both direct and indirect mechanisms of regulation of Rho GTPases.
The mechanisms by which inhibition of RhoA activation prevent endothelial barrier dysfunction may be through regulation of several targets critical for the regulation of endothelial barrier function. For example, the expression of dominant negative RhoA in endothelial cells was reported to prevent mediator-induced disassembly of both adherens and tight junctions (47). In one study, inhibition of RhoA resulted in inhibition of increased permeability and phosphorylation of the tight junction protein occludin (24). These studies suggest that junctional proteins may be direct targets of regulation by Rho GTPases. Furthermore, several pieces of evidence document RhoA in the inhibition of myosin-associated protein phosphatases, leading to increased MLC phosphorylation and increased permeability (5, 13, 41). However, Moy and coworkers (38) observed that increased intracellular cAMP was unable to prevent thrombin-induced increases in MLC phosphorylation or contraction of endothelial cells, suggesting that the protective effects of the cAMP/PKA pathway may not be through regulation of MLC phosphorylation. Rho GTPases have also been implicated in the activation of protein kinase C (PKC) (23), an enzyme known to signal increases in endothelial permeability (17, 34, 35).
Because the turning on and off of Rho GTPases are tightly regulated by accessory proteins GDI, GEF, and GAP, we investigated whether PKA-mediated inhibition of RhoA activation involved regulation of one of these proteins, GDI. Our results showed that cAMP-elevating agents prevented the thrombin-induced dissociation of RhoA from GDI, suggesting enhanced stabilization of the Rho-GDI protein complex. GDI is an ubiquitously expressed protein that has been shown to form a complex with different Rho family members, such as RhoA, Rad, Rac2, or CDC42Hs, to maintain them in the inactive state in the cytoplasm (22). Our immunofluorescent observation that cAMP prevented the thrombin-induced RhoA translocation to the membrane is consistent with this thesis. This finding suggests that the RhoA-GDI complex was important in maintaining RhoA in an inactive state in the endothelial cell cytoplasm.
We found that thrombin induced phosphorylation of GDI in endothelial
cells. This finding supports the observation made by Mehta and
coworkers (36), who observed that thrombin phosphorylated GDI in HUVEC through activation of PKC, and proposes that the phosphorylation inhibited GDI function, leading to activation of RhoA.
Interestingly, in our studies, cAMP did not inhibit the thrombin-mediated phosphorylation of GDI but enhanced the
thrombin-mediated phosphorylation, suggesting that PKA-mediated
inhibition of RhoA activation (and inhibition of RhoA dissociation from
GDI) was not through inhibition of GDI phosphorylation. However, the
GDI amino acid sequence contains both PKA and PKC consensus
phosphorylation sites, and it is possible that cAMP may inhibit RhoA
activation by mediating phosphorylation of GDI at distinct residues
responsible for stabilizing GDI association with RhoA. At present, the
functional significance of the several phosphorylation sites of GDI
remains to be determined.
We found that the protective action of PKA against endothelial barrier dysfunction consisted of reducing >50% of time required for the thrombin-induced resistance decrease to return to normal baseline levels. Furthermore, PKA inhibited ~23% of the thrombin-induced resistance decrease. In an earlier report, Moy and coworkers (38) also reported that cAMP facilitated restoration of the thrombin-induced resistance decrease to baseline in HUVEC; however, cAMP did not inhibit the initial resistance decrease in thrombin-stimulated cells. Overall, these findings suggest that PKA-mediated protection may be primarily through regulation of the recovery phase of the endothelial barrier dysfunction, and these findings provide further support of our earlier observation that PKA signals the prevention of thrombin-induced increases in transendothelial transport of tracer albumin (32).
We also found that overexpression of PKI did not completely prevent the cAMP-mediated facilitation of recovery, implicating involvement of PKA-independent mechanisms in regulation of this phase of barrier dysfunction. Although the cellular functions of cAMP are presumed to occur predominantly through activation of PKA, it has become increasingly apparent that cAMP has PKA-independent actions as well. For example, cAMP can directly activate a family of guanine nucleotide exchange factors, which in turn activates Ras (8) and Rap-1 (11, 26). Ras and Rap-1 are important in mediating cellular processes such as proliferation, differentiation, and gene expression. The precise role of these potential direct targets of cAMP in regulation of endothelial barrier function remains to be determined.
We observe that thrombin activated RhoA, but not Rac1 or Cdc42, in
HMEC. Similarly, van Nieuw Amerongen and coworkers (45) reported that thrombin activated RhoA, but not Rac, in HUVEC. These
findings suggest that thrombin shows selectivity in the activation of
Rho GTPases. It is evident that endothelial cells in general
likely express the three primary Rho GTPases, since we found that the
subcellular distributions of RhoA, Rac1, and Cdc42 occurred in both
HMEC and HPAEC. At present, it is not known whether other inflammatory
mediators show similar selectivity in the activation of Rho GTPases. To
date, there have been few studies identifying which inflammatory
mediators activate Rho GTPases. Studies have mostly investigated the
functional effects of inhibition of Rho GTPases by use of bacterial
toxins or transgene expression of mutant forms of RhoA, Rac, or Cdc42.
Nonetheless, the results from such studies provide some indirect
support that other inflammatory mediators, such as TNF-
(46), lipopolysaccharide (14), bradykinin
(2), and platelet-activating factor (2), activate Rho GTPases of the vascular endothelium.
In summary, we report that treatment of endothelial cells with cAMP-elevating agents 1) inhibited thrombin-induced RhoA activation and translocation to membrane; 2) prevented the thrombin-induced dissociation of RhoA from GDI; and 3) facilitated recovery of the thrombin-mediated resistance decrease and partially inhibited the extent of resistance drop. Infection of endothelial cells with the recombinant AdPKI to overexpress PKI blocked the cAMP-mediated protective effects on both RhoA activation and resistance changes. The results provide evidence that PKA inhibited RhoA activation in endothelial cells and support a potentially critical signaling mechanism of protection against vascular endothelial barrier dysfunction.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62649 and the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. Lum, Dept. of Pharmacology, Rush Presbyterian St. Luke's Medical Center, 2242 W. Harrison St., Suite 260, Chicago, IL 60612 (hlum{at}rush.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 14, 2003;10.1152/ajplung.00429.2002
Received 16 December 2002; accepted in final form 5 February 2003.
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