Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224-6801
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ABSTRACT |
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To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dependent endothelial cell barrier regulation, we investigated the impact of the cAMP/PKA inhibitors Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery and bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stress fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotype evolves in an MLC-independent fashion. We next examined the role of extracellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Raf-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf-1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI-induced ERK activation, caldesmon phosphorylation, and stress fiber formation. In summary, our data elucidate the involvement of the p42/44 ERK pathway in cytoskeletal rearrangement evoked by reductions in PKA activity and suggest the involvement of significant cross talk between cAMP- and ERK-dependent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.
Raf-1; mitogen-activated protein kinase; caldesmon
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INTRODUCTION |
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PULMONARY VASCULAR ENDOTHELIUM functions as a semiselective tissue barrier between circulating blood components and the interstitium. During acute lung injuries, increases in circulating cytokines, bioactive agents, and biophysical forces such as stretch or shear stress lead to increased endothelial cell permeability, formation of intercellular gaps, and life-threatening edema. Much less is known regarding mechanisms of barrier restoration; however, studies using whole animals, isolated perfused and ventilated lungs, and cultured endothelial cell models have shown that increased concentrations of intracellular cAMP and augmented cAMP-dependent protein kinase A (PKA) activity enhance and restore endothelial barrier function (1, 2, 12, 14, 15, 28, 31, 33). cAMP is generated from intracellular ATP by adenylyl cyclase, a family of membrane-bound enzymes containing at least nine isoforms that vary in their sensitivities to activation by the stimulatory heterotrimeric GTP regulatory protein Gs, to inhibition by Gi (6, 43), and to Ca2+ and protein kinase C (PKC) (42). A key cellular target for cAMP is PKA, whose two regulatory subunits bind cAMP and induce a conformational change that produces subunit dissociation from the catalytic subunits, resulting in enzymatic activation (23, 44). Cholera holotoxin, via ADP ribosylation of Gs, increases adenylyl cyclase activity and the synthesis of cAMP. These events dramatically reduce basal endothelial cell permeability, prevent thrombin-, phorbol 12-myristate 13-acetate (PMA)-, and pertussis toxin-induced permeability and gap formation (14, 31, 33) and reverse thrombin-induced permeability and gap formation (31). Both forskolin, a well-known direct adenylyl cyclase activator, and the cAMP analog dibutyryl cAMP also provide barrier protection, indicating that the cAMP barrier protective effects are accomplished specifically via its target, PKA activation (4, 11). Although cGMP analogs were ineffective in these studies of barrier protection, indicating that cross-activation of cGMP-dependent kinase was unlikely (31), the role of cGMP in barrier protection remains controversial (50).
We and others have demonstrated that endothelial cell barrier regulation is highly dependent on the actomyosin cytoskeleton, regulated through both myosin light chain kinase (MLCK)-dependent and -independent signaling pathways (12, 37). For example, thrombin-induced endothelial cell contraction and permeability involve the phosphorylation of myosin light chains (MLC) catalyzed by a novel nonmuscle MLCK isoform (12, 13, 45, 47). In contrast, endothelial cell barrier dysfunction produced by PKC activation is not correlated with MLCK activities but evolves via a signaling pathway that includes the activation of extracellular signal-regulated kinases (ERKs) and phosphorylation of endothelial cell cytoskeletal targets such as caldesmon (31, 40, 46). PKA phosphorylates endothelial MLCK, thereby reducing MLCK activity, leading to decreased basal level MLC phosphorylation (12, 13), and directly phosphorylates many actin-binding proteins such as adducin, dematin, and filamin, resulting in reduced actin bundling (3, 20, 26, 48).
Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and Rp diastereomer of 8-bromoadenosine 3',5'-cyclic monophosphorothioate (Rp-8-BrcAMPS) are nonhydrolyzable diasteromers of cAMP, which competitively bind to the regulatory subunits of PKA to effectively prevent cAMP-induced dissociation and activation of the enzyme (36). Both agents inhibit PKA activity in human microvascular and macrovascular endothelial cells and other cell types in vitro and in vivo at concentrations ranging from 10 to 500 µM (22, 35). PKA inhibitor (PKI) is a NH2-terminal myristoylated peptide that specifically and directly inhibits PKA catalytic activity (29). Although it is established that elevated cAMP and PKA activity enhances endothelial cell barrier function, the effect of reduced intracellular PKA on endothelial cell signaling mechanisms involved in barrier regulation remain unclear. In this study, we used the specific PKA inhibitors Rp-cAMPS, Rp-8-BrcAMPS, and PKI to determine the effect of decreased PKA activity on the endothelial cell cytoskeleton and further investigated the involvement of mitogen-activated protein kinase (MAPK) signaling pathways in Rp-cAMPS- and PKI-induced endothelial cell cytoskeletal rearrangement.
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MATERIAL AND METHODS |
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Reagents. Endothelial cells were cultured in medium 199 (M199; Life Technologies, Rockville, MD) supplemented with 20% (vol/vol) colostrum-free bovine serum (Irvine Scientific, Santa Ana, CA), 15 µg/ml endothelial cell growth supplement (Collaborative Research, Bedford, MA), 1% antibiotic and antimycotic (10,000 U/ml penicillin, 10 mg/ml streptomycin, and 25 µg/ml amphotericin B; Life Technologies), and 0.1 mM nonessential amino acids (Life Technologies). Rp-cAMPS, Rp-8-BrcAMPS, and PKI were purchased from Calbiochem (La Jolla, CA). MLC antibody was produced in rabbit against baculovirus-expressed and purified smooth muscle MLC by Biodesign International (Kennebunk, ME), and rabbit anti-phospho-p44/42 ERK was purchased from New England Biolabs (Beverly, MA). Phospho-caldesmon Ser789 antibody was kindly provided by Dr. Leonard P. Adam (Bristol-Myers Squibb, Princeton, NJ). Unless specified, all other reagents were obtained from Sigma (St. Louis, MO).
Bovine endothelial cell culture. Bovine pulmonary artery endothelial cells were purchased from American Type Culture Collection (Manassas, VA) and utilized at passages 19-24. Bovine lung microvascular endothelial cells were purchased from Cell Systems (Kirkland, WA) and used at passages 3-9. Cells were cultured and maintained in complete medium at 37°C in a humidified atmosphere of 5% CO2-95% air. Endothelial cells grew to contact-inhibited monolayers with the typical cobblestone morphology. Cells from each primary flask were detached with 0.05% trypsin and resuspended in fresh culture medium and placed in appropriate size flasks or dishes.
MLC phosphorylation assay. Assays were performed as we previously described in detail (12, 38). Briefly, after endothelial cell monolayers were incubated with either PKA inhibitors or vehicle controls, cells were lysed in 10% trichloroacetic acid, and the precipitates were homogenized and subjected to urea-polyacrylamide gel electrophosphoresis followed by immunoblotting with anti-MLC antibody. Immunoreactive proteins were detected using the enhanced chemiluminescence detection system (ECL; Amersham, Little Chalfront, UK), and the separated unphosphorylated, monophosphorylated, and diphosphorylated forms of MLC were quantified by laser scanning densitometry.
Western immunoblotting. After treatment, endothelial cell monolayers grown in 35-mm dishes were rinsed with ice-cold PBS, lysed with 100 µl of 2× SDS sample buffer (25), scraped into 1.5-ml microcentrifuge tubes, and boiled immediately for 5 min. Extracts (10 µl) were separated on 12% SDS-PAGE and transferred to nitrocellulose (30 V, 18 h). After being blocked with PBS-T (PBS with 0.1% Tween 20) containing 5% nonfat milk for 1 h, nitrocellulose blots were reacted with primary antibodies diluted in PBS-T containing 5% BSA for 1 h, washed with PBS-T (3 × 10 min), incubated with peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:10,000 dilution, Sigma; or goat anti-mouse IgG, 1:10,000 dilution, Bio-Rad Laboratories, Richmond, CA) diluted in PBS-T with 5% nonfat milk for 1 h, and again washed with PBS-T (3 × 10 min). Finally, immunoreactive proteins were detected using ECL. The relative intensities of the protein bands were quantified by scanning densitometry.
Raf-1 kinase activity assay.
Raf-1 kinase activity was determined using a commercially available kit
(Upstate Biotechnology, Lake Placid, NY) according to manufacturer's
recommendations with minor modifications. Confluent endothelial cells
grown in 60-mm dishes were treated with either Rp-8-BrcAMPS
(200 µM) or vehicle control for 10 min after 18 h of serum
starvation in M199. The cells were lysed on ice in lysis buffer (500 µl; 50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM
Na3VO4, 50 mM NaF, 5 mM sodium pyrophosphate,
10 mM sodium glycerophosphate, 0.1% -mercaptoethanol, and 0.1%
Triton X-100) including a 1:500 diluted protease-inhibitory cocktail
[200 µM 4-(2-aminoethyl)benzenesulfonyl fluoride, 160 nM
aprotinin, 10 µM bestatin, 3 µM E-64, 4 µM leupeptin, and 2 µM
pepstatin A; Calbiochem, La Jolla, CA] for 30 min. Cell lysates were
transferred into 1.5-ml Eppendorf tubes, debris was removed by a 10-min
centrifugation at 16,000 g (4°C), and the supernatants
were incubated with 4 µg of sheep anti-human c-Raf kinase
COOH-terminal antibodies on ice for 2 h, followed by incubation
with 100 µl of PBS-prewashed and lysis buffer-equilibrated protein G
Sepharose slurry (containing 30% protein G Sepharose 4 fast flow;
Amersham Pharmacia Biotech, Piscataway, NJ) for 2 h at 4°C with
gentle agitation. Protein G Sepharose with immunoprecipitaed Raf-1 was
washed and incubated with inactive glutathione S-transferase
(GST)-mitogen-activated protein (MAP) kinase kinase 1 (MEK1), inactive
GST-p42 MAP kinase in the kinase assay buffer containing magnesium,
ATP, and an inhibitor cocktail to inhibit other serine/threonine
kinases. The activated p42 MAP kinase was then used to
phosphorylate myelin basic protein (MBP) in the presence of
[
-32P]ATP. The radiolabeled substrate was allowed to
bind to P81 phosphocellulose paper, and the radioactivity per minute
was measured in a scintillation counter. In parallel, sheep IgG was
used in separate immunoprecipitation reactions to control nonspecific
binding of cellular proteins with the primary antibody. This
nonspecific radioactive count (in counts/min) was subtracted from the
counts per minute generated by anti-c-Raf antibody, resulting in the
relative Raf-1 kinase activities (in counts/min).
Detection of ERK activation. Endothelium grown in 35-mm dishes was rinsed three times with M199 and incubated with Rp-cAMPS, Rp-8-BrcAMPS (200 µM), or PKI (20 µM) for the indicated periods of time in M199 (700 µl) in a 37°C incubator with 5% CO2. After being rinsed with ice-cold PBS, cells were lysed with 2× SDS sample buffer (100 µl), scraped into 1.5-ml Eppendorf tubes, and boiled for 5 min. The cell lysates (10 µl) were subjected to 12% SDS-PAGE, followed by Western blotting. ERK1/2 phosphorylation was detected with 1 µg/ml of rabbit anti-phospho-p44/42 MAPK; total ERKs were detected with monoclonal anti-pan ERK antibody (50 ng/ml, Transduction Laboratories, Lexington, KY). In some experiments, cells were preincubated with 50 µM PD-98059 or 10 µM UO-126 for 30 min before Rp-cAMPS or PKI treatment.
Measurement of caldesmon phosphorylation.
PKA inhibition-induced caldesmon phosphorylation was assessed using two
complementary methods. First, the composite phosphorylation of
caldesmon was measured in caldesmon immunoprecipitates obtained from
32P-labeled cells. Endothelial cell monolayers in 60-mm
dishes were serum starved in PO-32P]orthophosphate for 4 h. After being rinsed
with PO
Detection of actin stress fiber formation by fluorescent staining. Endothelial cells grown on gelatinized coverslips were rinsed with M199 and incubated with Rp-cAMPS, Rp-8-BrcAMPS (200 µM), or PKI (20 µM) in M199 for 15 min. Monolayers were then rinsed with PBS, fixed in 3.7% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were then washed briefly with PBS (2 min each × 3), blocked with PBS-T containing 2% BSA for 30 min, and incubated with 1 U/ml of Texas Red-X phalloidin (Molecular Probes, Eugene, OR) diluted in PBS with 2% BSA for 30 min. After being washed three times with PBS-T (2 min each), coverslips were mounted on slides with SlowFade mounting medium (Molecular Probes). Cells were analyzed using a ×60 oil objective with a Nikon Eclipse TE 300 microscope. Images were captured by Sony Digital Photo camera DKC 5000. The same exposure time was applied to all samples within one experiment.
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RESULTS |
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cAMP-dependent PKA inhibition induces
actin stress fiber and intercellular gap formation in endothelium.
We have recently reported that decreased PKA activity by
Rp-cAMPS treatment resulted in significant decreases in
electrical resistance across bovine pulmonary artery endothelial cell
monolayers, indicating an increase in endothelial cell permeability
(32). To study the mechanisms involved in PKA-mediated
barrier regulation, we first investigated the effect of reduced PKA
activity on endothelial cell cytoskeletal organization. Endothelial
cells were incubated with Rp-cAMPS or its highly
cell-permeable form Rp-8-BrcAMPS and stained with Texas
Red-X phalloidin to detect filamentous actin (F-actin). Whereas stress
fiber and gap formation were not present in vehicle-treated cells (Fig.
1A), Rp-cAMPS
treatment (200 µM, 15 min) significantly increased the intensity of
F-actin staining, the number of actin stress fibers, and the number of
paracellular gaps in endothelial cell monolayers (Fig. 1B).
These findings are similar to thrombin treatment (100 nM, 10 min, Fig.
1C), which also increased both stress fiber formation and
the number of gaps. However, whereas thrombin reduced F-actin staining
at the cortical ring, Rp-cAMPS induced strong actin
polymerization of the peripheral band at this site. These results
indicate that reductions in PKA activity with Rp-cAMPS and
with Rp-8-BrcAMPS (data not shown) produce actin
reorganization via a mechanism that likely differs from that evoked by
thrombin and are highly consistent with an important role for PKA in
endothelial barrier regulation.
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MLC phosphorylation is not significantly increased
by Rp-cAMPS.
Our laboratory has previously shown that the phosphorylation status of
MLC is a key determinant of endothelial cell contraction and
intercellular gap formation (12, 47). Increased PKA
activity evoked by cholera toxin decreases the level in MLC
phosphorylation by attenuating MLCK activity, leading to marked
reductions in endothelial cell permeability (12, 31) and
transendothelial migration of neutrophilic leukocytes
(15). Therefore, to study the signaling pathways involved
in Rp-cAMPS-induced cytoskeleton remodeling, we examined the
phosphorylation status of MLC after Rp-cAMPS treatment in
endothelial cell monolayers. Interestingly, despite significant actin
stress fiber formation, inhibition of endothelial cell PKA activity by
Rp-cAMPS (200 µM, 10 and 60 min) or by
Rp-8-BrcAMPS (data not shown) failed to significantly alter MLC phosphorylation, whereas thrombin (100 nM, 10 min) as a positive control induced dramatic increases in diphosphorylated MLC (Fig. 2). Furthermore, pretreatment with
Rp-cAMPS (200 µM, 30 min) did not affect thrombin-induced
MLC phosphorylation, suggesting that the endothelial cell cytoskeleton
reorganization mediated by reductions in PKA activity occurs
independently of MLCK activity.
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Role of ERK signaling pathway in endothelial cell
stress fiber formation induced by decreased PKA
activity.
We have recently shown that ERK1/2 activation is a key event in
PKC-induced endothelial cell barrier dysfunction (46),
which, similar to Rp-cAMPS, evolves via a MLCK-independent
mechanism. We next examined the role of the ERK signaling cascade in
Rp-cAMPS-induced actin reorganization. Rp-cAMPS
and Rp-8-BrcAMPS (200 µM) induced significant ERK
phosphorylation in a time-dependent manner (Fig. 3), reaching maximum ERK activation at 10 min and falling thereafter to diminished levels after 30 min.
Pretreatment with PD-98059 (50 µM, 30 min), a specific ERK kinase
(MEK-1) inhibitor, completely abolished Rp-cAMPS-induced ERK
activation without affecting the total amount of ERK protein (Fig.
4A), indicating that
activation of ERKs by Rp-cAMPS occurred specifically via
MEK-1 activity. Furthermore, a NH2-terminal myristoylated
PKA-inhibitory peptide, PKI, which specifically and directly inhibits
PKA catalytic activity (29), was used to confirm that the
effects of Rp-cAMPS are exclusively attributed to PKA
inhibition. Consistent with Rp-cAMPS effects in bovine
macrovascular endothelial cells, PKI (20 µM, 10 min) dramatically
enhanced ERK activation in bovine microvascular endothelial cells (Fig.
4B, top), which was distinguished by anti-pan ERK antibody because phosphorylated ERK migrates slower on acrylamide gels
(Fig. 4B, bottom). Like Rp-cAMPS,
PKI-induced ERK activation also was abolished by specific MEK-1
inhibition (UO-126; Fig. 4B). PKI (20 µM) also produced
stress fiber and intercellular gap formation in endothelial cell
monolayers, which were significantly attenuated by UO-126 (Fig.
4C). Similarly, inhibition of MEK-1 by PD-98059 partially
blocked Rp-cAMPS-induced stress fiber formation in
endothelial cells (data not shown). These data suggest that the actin
polymerization avidly enhanced by inhibition of PKA activity is ERK
dependent. Reduced PKA activity also increased the activity of the
upstream MEK-activating Raf-1 kinase as measured by the phosphorylation
of MBP through the Raf-1-MEK1-ERK2 kinase cascade. As shown in Fig.
5, the incubation of endothelial cells with Rp-8-BrcAMPS (200 µM, 10 min) produced a 2.6-fold
increase in Raf-1 activity, consistent with previous reports that the
elevation of cellular cAMP levels downregulates Raf-1 kinase activity
(8, 27). These data provide further confirmation that
inhibition of PKA activity activates the ERK pathway. Consistent with
these data, elevation of cAMP level and PKA activity by forskolin,
which increases cellular cAMP by activating adenylyl cyclase, reduced basal ERK phosphorylation (16) and caused F-actin
dissolution (data not shown). These findings confirm an important
association between PKA and ERK activities and cytoskeletal regulation.
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Role of caldesmon phosphorylation in
Rp-cAMPS- and PKI-induced
endothelial cell cytoskeletal reorganization.
Although our findings suggest that ERK may be involved in endothelial
cell barrier regulation by PKA, the mechanisms of ERK-mediated cytoskeletal rearrangement remain unclear. However, phosphorylation of
the thin filament-associated cytoskeletal protein caldesmon, a known
ERK target, has been postulated as an important event in the regulation
of smooth muscle contraction (34, 49). Furthermore, thrombin-, PMA- and pertussis toxin-mediated endothelial cell barrier
dysfunction are associated with caldesmon phosphorylation and
redistribution to new actin filaments (16, 40, 46). Therefore, to further study the mechanisms of PKA inhibition-induced endothelial cell cytoskeletal rearrangement and cell contraction, we
investigated the effect of reduced PKA activity on caldesmon phosphorylation. First, 32P-labeled endothelial cells were
treated with Rp-8-BrcAMPS (200 µM, 10 and 30 min), and the
composite caldesmon phosphorylation in immunoprecipitates from
cell lysates was determined by autoradiography. Inhibition of PKA
significantly enhanced caldesmon phosphorylation both at 10 min (80%
increase) and at 30 min (120% increase, Fig. 6A). To define ERK involvement
in caldesmon phosphorylation, Rp-cAMPS-treated endothelial
cell lysates were immunoblotted with an antibody generated against the
phosphopeptide
781CQSVDKVTS*PTKV793, which
contains the specific ERK phosphorylation site Ser789
present in mammalian h-caldesmon (21). As shown in Fig.
6B, caldesmon phosphorylation, induced by PKA inhibition and
detected by the ERK-specific antibody, was significantly increased
after 10 min (70% increase) and was sustained through 60 min.
Furthermore, inhibition of PKA activity by PKI induced a twofold
increase in caldesmon phosphorylation, which was completely abolished
by UO-126 pretreatment (Fig. 6C). These data confirm that
the reduction of PKA activity results in significant caldesmon
phosphorylation, which is mediated at least partially by ERK and may
contribute to the cytoskeletal reorganization induced by the decrease
in PKA activity in endothelium.
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DISCUSSION |
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There is substantial evidence that activation of cAMP-dependent PKA enhances vascular barrier integrity, whereas decreased cAMP is correlated with impaired barrier function (12, 31, 41, 42). This implies that PKA has an important role in vascular barrier regulation by modulating the balance between endothelial cell contractile and tethering forces in some manner, although the precise signaling pathways involved remain unclear. Our data support this central role of PKA in barrier regulation and strongly implicate MAPK activity as a relevant signaling paradigm. Three specific PKA antagonists (Rp-cAMPS, Rp-8-BrcAMPS, and PKI) were utilized to explore the potential mechanisms of PKA in endothelial cell permeability regulation and were found to produce dramatic actin cytoskeletal rearrangement and intercellular gap formation, indicating cell contraction and barrier dysfunction. The actin rearrangement after PKI and Rp-cAMPS is neither reminiscent of PMA, which produces cortical actin dissolution without stress fiber formation (46), nor thrombin, where the stress fibers are thick and accompanied by cortical actin disappearance (12). Previous studies in our laboratory have shown that increased PKA activity by cholera toxin significantly reduces MLCK activity via enhanced MLCK phosphorylation (13). PKA inhibition by Rp-cAMPS, however, failed to affect basal or thrombin-induced MLC phosphorylation. These results suggest that the endothelial cell barrier dysfunction mediated by reductions in PKA activity does not involve a direct effect on MLCK activity, findings that are similar to other specific models of vascular permeability that evolve in a MLC-independent fashion (16, 46). Consistent with two PKC-dependent models of endothelial cell barrier dysfunction produced by pertussis toxin and PMA, where the activation of MAPK signaling pathways has been implicated (16, 46), we found that inhibition of PKA activity transiently increased MAPK phosphorylation, especially ERK1/2 MAPK, and facilitated subsequent actin rearrangement and stress fiber formation. The mechanism by which inhibition of PKA increases ERK activity is unclear but involves an increase in the activity of Raf-1 kinase, the enzyme immediately upstream of MAPK kinase (MEK). This is consistent with the notion that basal cAMP production and PKA activity inhibit the MAPK signaling cascade by phosphorylating Raf-1 and, presumably, reducing Raf-1 binding affinity to Ras-GTP and directly decreasing Raf-1 catalytic activity (5, 8, 19, 27, 51). It is most likely that reduction in this basal PKA activity allows the accumulation of active Raf-1 and sequential increases in MEK and ERK MAPK activities. Our results are similar to the findings of D'Angelo et al. (8), who found that basal PKA activity constitutively suppresses activation of Raf-1/ERK in bovine brain capillary endothelial cells. Our data extend this observation in several ways but most importantly suggest that basal PKA activity in bovine lung endothelium is not only sufficient to suppress the ERK signaling pathway but is also necessary for the maintenance of endothelial cell barrier integrity (32, 41).
One potentially important endothelial cytoskeletal target for ERK phosphorylation is nonmuscle caldesmon, a 77-kDa actin-, myosin- and calmodulin-binding protein hypothesized to function as a molecular switch in the regulation of nonmuscle and smooth muscle contraction (39) by modulating the dynamics of actin filament organization (34, 52). Studies in bovine endothelium have shown that thrombin- and PMA-mediated endothelial barrier dysfunction is temporally linked and directly associated with caldesmon phosphorylation and redistribution (40). Furthermore, MAPK-induced smooth muscle caldesmon phosphorylation has been suggested to reverse the inhibitory effects of caldesmon on cross-bridge cycling, thereby allowing actomyosin contraction (7, 9, 17, 18). Although this remains controversial (24, 30), antisense oligodeoxynucleotide strategies to deplete caldesmon from smooth muscle significantly attenuated agonist-induced tension development (10). In the present study, using antibodies specifically immunoreactive with ERK-mediated caldesmon phosphorylation sites, we demonstrated that reduction of PKA activity stimulated caldesmon phosphorylation, implicating a role of ERK-dependent caldesmon phosphorylation in PKA barrier regulation. Our data also indicate, however, that additional pathways exist that link PKA and actin stress fiber formation since complete MEK inhibition attenuated but did not abolish PKI- or Rp-cAMPS-induced F-actin rearrangement. Further experiments to explore these pathways are currently in progress.
In summary, our study demonstrates the critical role of cAMP and PKA activity in the regulation of endothelial cell cytoskeleton reorganization. Pharmacological reduction in PKA activity is sufficient to induce actin stress fiber formation, leading to cell contraction mediated, at least in part, by the activation of a Raf-1/ERK signaling pathway. Although the exact ERK targets that produce endothelial cell cytoskeletal rearrangement resulting in paracellular gap formation remain unclear, our data support an active role of caldesmon in this process.
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ACKNOWLEDGEMENTS |
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We thank Drs. William T. Gerthoffer and Leonard P. Adam for providing important reagents and Lakshmi Natarajan for superb technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-58064 and HL-50533 to J. G. N. Garcia and HL-67307 to A. D. Verin.
Address for reprint requests and other correspondence: J. G. N. Garcia, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, 4B.77, Baltimore, MD 21224-6801 (E-mail:drgarcia{at}jhmi.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.
Received 10 August 2000; accepted in final form 8 January 2001.
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