PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction

Jing Qiao, Fei Huang, and Hazel Lum

Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , transforming growth factor-beta ) 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. The following reagents were purchased as follows: DH5alpha 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 beta -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 alpha -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 DH5alpha 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 × 10-4 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-MOmega 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.

For resistance measurement, HMEC (2.5 × 105 cells/cm2) were plated onto a sterile, fibronectin-coated, eight-well, gold-plated electrode array and grown to confluence. After being changed to fresh medium, the electrode array was mounted onto holders of the ECIS system housed within an incubator (maintained at 37°C, 5% CO2, and 100% humidity) and connected to the lock-in amplifier. Voltage and phase data were stored and processed in a computer, which also controlled the output of the amplifier and relay switches to different electrodes. After equilibration for 15-30 min within the incubator, baseline resistance was recorded for another 15-30 min. HMEC monolayers typically show baseline resistance >7,000 Omega  (31); therefore, monolayers with lower resistance were rejected from study. We used the minimum baseline electrical resistance of 7,000 Omega  (corresponds to the calculated value of 3.5 Omega  · cm2) to screen for relatively restrictive monolayers since this resistance is within the reported range of values of 3-6 Omega  · cm2 for endothelial cell monolayers. The HMEC were challenged with reagents according to experimental protocol, and resistance was recorded continuously in real time for up to 4 h. Values are reported as normalized to initial baseline resistance.

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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Protective effects of cAMP-dependent protein kinase on transendothelial electrical resistance. Human microvascular endothelial cells (HMEC; 2.5 × 105 cells/cm2) were grown to confluency on fibronectin-coated gold electrodes. The cells were pretreated with forskolin (10 µM) and IBMX (1 µM) (FI; arrow) for 15 min to increase intracellular cAMP levels and were then stimulated by thrombin (Thr; 10 nM for 15 min; arrow). Control (C) received buffer challenge. In a separate group, HMEC were infected with 100 multiplicities of infection (MOI) cAMP-dependent protein kinase inhibitor cDNA (AdPKI) for 2 days before FI and thrombin treatment (PKI+FI+Thr). A: representative graph showing resistance changes in real time. B: summary graph showing the maximal resistance decrease from baseline. C: summary graph showing time needed for resistance decrease to return to near baseline; n = 8-10 separate determinations; *P < 0.05, **P < 0.001 vs. FI+Thr group. Norm., normal; Resist., resistance; PKI, protein kinase A inhibitor.

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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Fig. 2.   Subcellular localization of RhoA, Cdc42, and Rac1 in endothelial cells. The intracellular distribution of Rho GTPases in HMEC and human pulmonary artery endothelial cells (HPAEC) was evaluated by immunofluorescent laser confocal microscopy. Both endothelial cell types (HMEC: top; HPAEC: bottom) express RhoA, Cdc42, and Rac1. RhoA was found predominantly in the cytosol with some localization in the nucleolus. Cdc42 was distributed in the cytosol and at cell periphery. Rac1 was distributed diffusely in the cytosol and in the nucleus. Original magnification, ×40. Scale bar = 25 µm; n = 3.

The activation responses of the Rho GTPases in HMEC were investigated by stimulation with thrombin, a mediator known to increase vascular endothelial permeability to albumin (19, 30). Activated RhoA, Cdc42, and Rac1 were determined by affinity-binding assay for GST-fusion protein targets of the Rho GTPases. We found that the amount of activated RhoA (RhoA-GTP) in the pull-down fraction was significantly increased by 1 min of thrombin stimulation (10 nM) (Fig. 3), and the increase was sustained for up to 15 min (Fig. 4). Thrombin stimulation of HMEC for up to 15 min did not increase Cdc42-GTP (Figs. 3 and 5) or Rac1-GTP (Figs. 3 and 6) over the control cells. Total Rho (GTP-bound + GDP-bound forms) was determined from cell lysates and observed to be similar among experimental groups, indicating that experimental treatment did not increase de novo generation of Rho GTPases.


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Fig. 3.   Effects of thrombin on activation of Rho GTPases. Rho GTPase activation was determined by affinity binding of Rho target fusion proteins (see MATERIALS AND METHODS). HMEC were serum starved and treated with thrombin (10 nM) at 0, 1, or 5 min. The activated (GTP-bound form) and total Rho (Rho-GTP + Rho-GDP) are shown for RhoA (top), Rac1 (middle), and Cdc42 (bottom). Three representative determinations are shown.



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Fig. 4.   Effects of PKI overexpression on RhoA activation. HMEC were pretreated with forskolin (10 µM) and IBMX (1 µM) for 15 min to increase intracellular cAMP levels and were then stimulated by thrombin (10 nM for 15 min). Control received buffer challenge. In a separate group, HMEC were infected with 100 MOI AdPKI or control heat-inactivated AdPKI for 2 days before FI and thrombin treatment. RhoA activation was determined by affinity-binding assay as described in MATERIALS AND METHODS. Bar graph summarizes results from 4-6 separate determinations. The Western blot of the affinity binding shows a representative result. RhoA-GTP, activated RhoA; RhoA Total, RhoA-GTP + RhoA-GDP. *P < 0.05 and **P < 0.01 vs. control group.



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Fig. 5.   Effects of FI on Cdc42 activation. HMEC were pretreated with forskolin (10 µM) and IBMX (1 µM) for 15 min to increase intracellular cAMP levels and then stimulated with thrombin (10 nM) for 15 min. Cdc42 activation was determined by affinity binding as described in MATERIALS AND METHODS. Representative experiment shows activated Cdc42 (Cdc42-GTP) and Total Cdc42 (Cdc42-GTP + Cdc42-GDP). Densitometric quantification shows bands from 3 separate determinations; n = 3.



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Fig. 6.   Effects of FI on Rac1 activation. HMEC were pretreated with forskolin (10 µM) and IBMX (1 µM) for 15 min to increase intracellular cAMP levels and then stimulated with thrombin (10 nM) for 15 min. Rac1 activation was determined by affinity binding as described in MATERIALS AND METHODS. Representative experiment shows activated Rac1 (Rac1-GTP) and Total Rac1 (Rac1-GTP and Rac1-GDP). Densitometric quantification shows bands from 3 separate determinations; n = 3.

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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Fig. 7.   Effects of FI on RhoA translocation. Confluent monolayers of HMEC, grown on chamber slides, were stimulated with thrombin (10 nM) alone for 15 min or after pretreatment with forskolin (10 µM) plus IBMX (1 µM) for 15 min. The cells were prepared for immunofluorescent confocal microscopy evaluation of RhoA localization (see MATERIALS AND METHODS). A: representative immunofluorescent image showing subcellular localization of RhoA in control, thrombin, and FI plus thrombin groups. Arrows indicate localization of RhoA at membrane. Original magnification ×40; scale bar = 25 µm. B: immunofluorescent intensity at the membrane was quantified from 20 individual cell images/group; n = 3; *P < 0.01 and **P > 0.05 vs. control group.

We investigated possible mechanisms by which PKA may signal the inhibition of RhoA activation. One possible mechanism is through modulation of GDI, an important regulator of GTP/GDP cycling. GDI inhibits Rho GTPases by binding to the GDP-bound form of Rho to maintain it in a cytoplasmic inactive state. We determined the effects of FI pretreatment of HMEC on GDI binding with RhoA by coprecipitation studies. Results indicated that HMEC stimulated by thrombin (10 nM for 15 min) showed decreased coprecipitation of RhoA with GDI (Fig. 8). Pretreatment with FI prevented this decrease of the coprecipitated complex (Fig. 8), suggesting inhibition of the thrombin-induced dissociation of RhoA from GDI. FI treatment alone showed no change of RhoA coprecipitation with GDI relative to control, whereas the negative control showed absence of bands. The subsequent stripping of the membrane and detection with anti-GDI antibody indicated equal loading of proteins among groups. We also determined whether the FI-mediated inhibition of RhoA activation was associated with changes in the phosphorylation of GDI. The autoradiographic determination of in vivo incorporation of 32P by GDI in HMEC indicated that thrombin stimulation alone (10 nM, 15 min) phosphorylated a band at ~30 kDa, corresponding to the molecular weight of GDI (Fig. 9). FI pretreatment enhanced this thrombin-induced phosphorylation of GDI (Fig. 9). The negative control showed absence of phosphorylated bands.


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Fig. 8.   Coprecipitation of guanine nucleotide dissociation inhibitor (GDI) with RhoA. HMEC were serum starved, stimulated with thrombin alone (10 nM) for 15 min, or pretreated with forskolin (10 µM) and IBMX (1 µM) for 15 min and then stimulated with thrombin. Affinity-purified anti-GDI antibody was used for immunoprecipitation, and separated proteins were detected by Western blot analysis using anti-GDI or anti-RhoA antibodies. As negative control (Neg. C), a separate group of cells was without the precipitating antibody; n = 3.



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Fig. 9.   Detection of in vivo GDI phosphorylation. HMEC were preloaded with [32P]orthophosphate and treated with forskolin (10 µM) and IBMX (1 µM) for 15 min, followed by thrombin stimulation (10 nM) for 15 min. Cell lysates were prepared for immunoprecipitation with anti-GDI antibody, the immunocomplex was separated by SDS-PAGE, and autoradiograms were prepared (top). Western blot was made to detect for GDI to check for equal loading of samples (bottom). As negative control, a separate group of cells was without the precipitating antibody; n = 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PKCalpha , 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-alpha (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.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-62649 and the American Heart Association.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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