1 Department of Pharmacology, Rush-Presbyterian-St. Luke's Medical Center, and 2 Division of Gastroenterology, Cook County Hospital, Chicago, Illinois 60612
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ABSTRACT |
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Serine/threonine (Ser/Thr) protein
phosphatases (PPs) are implicated in the recovery from endothelial
barrier dysfunction caused by inflammatory mediators. We hypothesized
that Ser/Thr PPs may regulate protein kinase C (PKC), a critical
signaling molecule in barrier dysfunction, in the promotion of barrier
recovery. Western analysis indicated that bovine pulmonary
microvascular endothelial cells (BPMECs) expressed the three major
Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506
(a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A
or okadaic acid potentiated the thrombin-induced increase in PKC
phosphotransferase activity. FK506 also potentiated thrombin-induced PKC- but not PKC-
phosphorylation. FK506 but not calyculin A or
okadaic acid inhibited recovery from the thrombin-induced decrease in
transendothelial resistance. Neither FK506 nor okadaic acid altered the
thrombin-induced resistance decrease, whereas calyculin A potentiated
the decrease. Downregulation of PKC with phorbol 12-myristate
13-acetate rescued the FK506-mediated inhibition of recovery, which was
consistent with the finding that the thrombin-induced phosphorylation
of PKC-
was reduced during the recovery phase. These results
indicated that PP2B may play a physiologically important role in
returning endothelial barrier dysfunction to normal through the
regulation of PKC.
protein kinase C-; transendothelial resistance; FK506; endothelial permeability
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INTRODUCTION |
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SERINE / THREONINE (Ser/Thr) protein phosphatases (PPs) are important in the regulation of a range of cellular activities including cell growth and transformation and are known to have profound effects on actin microfilament organization (8). It has become increasingly apparent that the Ser/Thr PPs are regulated in a sophisticated manner by a combination of targeting and regulatory subunits and specific endogenous inhibitors. The predominant Ser/Thr PP types PP1, PP2A, and PP2B are distinguished based on substrate specificity, sensitivity to specific inhibitors, and cation requirements (39).
The Ser/Thr PPs in endothelial cells are implicated in the regulation
of vascular endothelial permeability (4, 38, 44, 45),
although the precise cellular and molecular mechanisms are not well
understood. Treatment of endothelial cells with inhibitors of PP1 and
PP2A (i.e., okadaic acid and calyculin A) resulted in increases in
endothelial permeability that were accompanied by increased myosin
light chain (MLC) phosphorylation (44). The increased MLC
phosphorylation is believed to promote endothelial contraction, which
provides the increased centripetal tension required to decrease
endothelial cell-cell adhesion, resulting in increased permeability.
However, in one study (4), the okadaic acid- or calyculin
A-mediated increased endothelial permeability occurred without a
concomitant increase in MLC phosphorylation, suggesting that the
Ser/Thr PPs may also regulate endothelial barrier function independent
of MLC phosphorylation. Furthermore, in human epidermal cells, these
Ser/Thr PP inhibitors induced hyperphosphorylation of -catenin, an
adherens junction-associated protein, which was accompanied by loss of
cell-cell contact (37). Although these studies suggest
that Ser/Thr PPs promote endothelial barrier function, it remains
unclear whether Ser/Thr PPs play a physiological role in limiting
endothelial barrier dysfunction caused by inflammatory mediators.
A potential target of Ser/Thr PPs that may be important in
endothelial barrier function regulation is the family of protein kinase
C (PKC) isoforms. It is known that at least for the classic PKC- and
PKC-
(6, 15, 22), and possibly for the atypical PKC-
(41), phosphorylation controls their intrinsic catalytic activity such that their dephosphorylation results in loss of kinase
activity. Purified Ser/Thr PPs (i.e., PP1 and PP2A) have been shown to
inhibit recombinant PKC-
activity (34). In various experimental models with intact cells, okadaic acid inhibited dephosphorylation of the PKC-
, PKC-
, and PKC-
isoforms,
resulting in increased phosphotransferase activity of the isoforms
(14, 43). At present, little is known regarding the
regulation of PKC by Ser/Thr PPs in endothelial cells.
The PKC signaling pathway is a well-documented mechanism in the
regulation of mediator-induced increases in vascular endothelial permeability as demonstrated in several experimental models (19, 24, 25, 28). The PKC family consists of at least 12 known Ser/Thr protein kinases, which are classified primarily by structure, function, and cofactor requirements for activation. These isoforms are
distributed to different intracellular sites, are selectively sensitive
to PKC inhibitors and downregulation, and have different selectivity
for substrates, suggesting that the isoforms are function specific
(20, 31). Our laboratory (29, 48) and
others (2, 9) have shown that the classic PKC- and -
isoforms, which are the primary Ca2+-dependent isoforms
expressed by endothelial cells, may comprise the more important
isoforms in the regulation of endothelial permeability. The novel
PKC-
and the atypical PKC-
and -
isoforms are also present in
endothelial cells (Lum, unpublished observations); however,
their role in barrier function regulation has not been defined.
In this study, we investigated the role of Ser/Thr PPs in the
regulation of PKC in endothelial cells and the relationship to
mediator-induced endothelial barrier dysfunction. PKC activation was
determined by phosphotransferase activity and phosphorylation of
PKC- and -
isoforms, and transendothelial resistance was measured
as an index of barrier function. The results indicate that the PP2B
inhibitor FK506 (but not okadaic acid and calyculin A) potentiated the
thrombin-induced increase in PKC phosphotransferase activity and
phosphorylation of the PKC-
isoform in BPMECs. FK506 also inhibited
the recovery of the thrombin-mediated decrease in transendothelial
resistance. Downregulation of PKC rescued the FK506-mediated inhibition
of the recovery. The findings support the thesis that PP2B may play an
important physiological role in the recovery of endothelial barrier
dysfunction to normal, which may occur through the regulation of PKC in
endothelial cells.
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METHODS |
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Cell Culture
BPMECs (Vec Technologies, Rensselaer, NY) were maintained in culture in MCDB 131 medium supplemented with 10% fetal bovine serum, 10 ng/ml of human epidermal growth factor, 1 µg/ml of hydrocortisone, 90 ng/ml of heparin, and antibiotics. BPMECs, obtained at a population doubling of 10, were grown and subcultured to population doublings between 18 and 22 for study. The cells incorporated acetylated low-density lipoprotein and exhibited the expected morphological endothelial phenotype.PKC Activity
Cell collection. BPMECs were grown in six-well dishes and treated according to the experimental protocol. All subsequent steps were carried out on ice with ice-cold reagents. The cells were washed with phosphate-buffered saline (PBS) and collected by scraping in extraction buffer (0.02 M Tris, 0.5 mM EDTA, and 0.5 mM EGTA) containing protease inhibitors (2.5 mM phenylmethylsulfonyl fluoride, 25 µg/ml of leupeptin, and 25 µg/ml of aprotinin) and 10 mM 2-mercaptoethanol. The collected samples were frozen in liquid N2 until the assay was performed. Samples were lysed by two repeated freeze-thaw cycles and centrifuged for 20 min at 20,000 g at 4°C, and the supernatant was collected as the cytosolic (soluble) fraction. The pellet was resuspended in extraction buffer plus 0.3% Triton X-100, homogenized with a microfuge pestle, and incubated on ice for 30 min with periodic vortexing. The samples were centrifuged for 10 min at 20,000 g at 4°C, and the supernatant was collected as the membrane (particulate) fraction.
Phosphotransferase activity.
Phosphotransferase activity of the cell fractions was determined by
incorporation of 32P into the PKC consensus peptide
substrate (16). The reaction was performed by adding 33 µM unlabeled ATP plus [-32P]ATP to the samples in
the presence of the specific PKC peptide substrate
[Ser25]PKC19-31 (0.1 mg/ml), 0.01%
phosphatidylserine, 0.01% diacylglycerol, 5.4 mM MgCl2, 20 mM Tris base, and 20 mM CaCl2. The mixture was incubated
for 5 min before being quenched with ice-cold 75 mM H3PO4. Basal levels of activity were determined
by adding the reaction mixture to identical samples with
H3PO4 already present. Samples were vacuum
filtered through ion-exchange cellulose disks and counted in a
scintillation counter. Values were calculated as specific PKC activity
(in pmol 32P
incorporated · min
1 · mg
protein
1).
Phosphorylation of PKC
BPMECs were grown to confluence in 60-mm culture dishes and washed two times with warm phosphate-free DMEM. The cells were preloaded with [32P]orthophosphoric acid (0.3 mCi/ml) in the same medium for 3 h at 37°C and treated according to experimental protocol. The reaction was stopped by placing the dishes on ice and immediately washing them two times with ice-cold Ca2+- and Mg2+-free PBS, and the cells were collected by scraping in extraction buffer [in mM: 150 NaCl, 1 EDTA, 1 EGTA, 50 Tris-Cl (pH 7.4), 1 NaF, 1 sodium vanadate, 1 pepstatin A, and 1 phenylmethylsulfonyl fluoride; 1% Nonidet P-40; 0.25% sodium deoxycholate; 25 µg/ml of leupeptin; and 25 µg/ml of aprotinin]. The cell extract was aspirated through a 21-gauge needle to further disrupt the cells and centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was precleared with 1.0 µg of mouse IgG and protein A/G PLUS-agarose for 1 h at 4°C. The precleared lysate (400 µg) was incubated with 0.5 µg of affinity-purified anti-PKC-Western Blot Analysis
BPMECs were grown to confluence in 60-mm culture dishes. After the experimental treatment, the cells were washed two times with PBS and collected in extraction buffer as described in PKC Activity, and the protein concentration was determined with the bicinchoninic acid (BCA) protein assay kit with bovine serum albumin as a standard (Pierce, Rockford, IL). The cell lysates were loaded at constant protein concentrations, separated by SDS-PAGE in 10% acrylamide, and electrotransferred to a nitrocellulose or polyvinylidene difluoride membrane. The nonspecific binding of antibodies to the membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBS-T). The blocked membrane was then incubated with an affinity-purified MAb diluted in TBS-T with 1% nonfat dry milk overnight at 4°C in a rocker. The blot was washed five times with TBS-T and incubated with goat anti-mouse IgG secondary antibody conjugated with horseradish peroxidase. The bands were detected with the enhanced chemiluminescence kit. Specificity of the protein bands was confirmed with protein standards, control cell lysate, or the peptide immunogen as a negative control.Transendothelial Electrical Resistance
Transendothelial electrical resistance, an index of endothelial barrier function, was determined in real time with the electric cell-substrate impedance sensor (ECIS) system (Applied BioPhysics, Troy, NY) (11, 23, 27). The system consisted of a large gold-plated electrode (2 cm2) and five smaller gold-plated electrodes (10For the resistance measurement, BPMECs (105 cells) were plated onto a sterile, fibronectin-coated small gold-plated electrode and grown to confluence. The electrode was mounted onto the ECIS system within an incubator (maintained at 37°C, 5% CO2, and 100% humidity) and connected to the lock-in amplifier. After 15 min of equilibration, the cells were challenged with reagents according to experimental protocol, and resistance was recorded continuously.
Materials
Reagents.
Unless indicated, reagents were purchased from Sigma (St. Louis, MO).
MCDB 131 medium, DMEM, penicillin-streptomycin, PBS, and Hanks'
balanced salt solution were purchased from GIBCO BRL (Life
Technologies, Gaithersburg, MD); fetal bovine serum was from HyClone
(Logan, UT); the enhanced chemiluminescence kit and [32P]orthophosphoric acid were from Amersham
Life Sciences (Arlington Heights, IL); and [-32P]ATP
was purchased from NEN-DuPont (Wilmington, DE). The BCA kit was from
Pierce (Rockford, IL), and protein A/G PLUS-agarose was obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). FK506 was purchased from
BIOMOL (Plymouth Meeting, PA), and calyculin A and okadaic acid were
from Calbiochem (San Diego, CA).
Antibodies.
Anti-human PKC-, PKC-
, PP1
catalytic subunit, PP2A catalytic
subunit
, and PP2B catalytic subunit A MAbs were purchased from
Transduction Laboratories (Lexington, KY); and goat anti-mouse IgG-horseradish peroxidase MAb was purchased from Amersham Life Sciences.
Statistics
Single-sample data were analyzed by two-tailed t-test; a multiple range test (Scheffé's test) was used for comparisons of experimental groups with a single control group (40). ![]() |
RESULTS |
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Expression of PP1, PP2A, and PP2B
The expression of Ser/Thr PPs in BPMECs was determined by Western blot analysis. Results indicated that PP1, PP2A, and PP2B were expressed by these endothelial cells (Fig. 1). PP1 consistently showed bands at ~40 and 50 kDa, which were similar to the positive control A431 cells. PP2A showed a band at ~40 kDa. A band for PP2B was observed at ~60 kDa, as also indicated by the positive control rat brain tissue.
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For study, the Ser/Thr PPs in BPMECs were inhibited pharmacologically
to evaluate their role in the regulation of thrombin-induced endothelial barrier dysfunction. A range of concentrations of the
inhibitors of PP1 and PP2A (calyculin A and okadaic acid) and PP2B
(FK506) were evaluated to determine the concentrations that alone did
not alter baseline endothelial barrier function. Both calyculin A
(IC50: PP1 = 0.40 nM; PP2A = 0.25 nM) and okadaic acid (IC50: PP1 = 49 nM; PP2A = 0.28 nM) are
highly selective for PP1 and PP2A, with minimal inhibition of PP2B
(26). FK506 selectively inhibits PP2B at nanomolar
affinities by forming a complex with FK506-binding protein and does not
inhibit PP1, PP2A, or other PPs (21). Based on this
initial concentration evaluation, 200 nM okadaic acid, 2 nM calyculin
A, and 100 ng/ml of FK506 were shown to not alter baseline
transendothelial electrical resistance (Fig.
2A) nor PKC activity (Fig.
2B). Both FK506 and okadaic acid appeared to increase PKC
activity slightly, but it was not significant. These concentrations of
inhibitors were used for subsequent investigation of the involvement of
Ser/Thr PPs in thrombin-mediated endothelial barrier dysfunction.
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Effects of Ser/Thr PP Inhibitors on Thrombin-Induced PKC Activity and Phosphorylation
The proinflammatory mediator thrombin (100 nM) increased PKC phosphotransferase activity (>3-fold) in the membrane cell fraction of BPMECs within 2 min and was sustained for up to 30 min (Fig. 3A). Pretreatment of the cells with 100 ng/ml of FK506 for 15 min potentiated this increased PKC activity (12-fold over control value; Fig. 3B). Pretreatment with 2 nM calyculin A or 200 nM okadaic acid did not potentiate the thrombin-induced increase in PKC activity but tended to lower (not significantly) the increased activity (Fig. 3B).
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Because the phosphorylation state of PKC is directly correlated to its
intrinsic kinase activity (5, 6, 15, 32), we next
investigated whether FK506-mediated potentiation of thrombin-activated PKC activity was related to increased phosphorylation of the PKC enzyme. We determined phosphorylation of the classic PKC isoforms (i.e., PKC- and -
) in response to thrombin or thrombin plus FK506
because these isoforms have been reported to be important in the
regulation of endothelial permeability. Immunoprecipitation results
showed that treatment of BPMECs with 100 nM thrombin (5 min)
phosphorylated a band corresponding to PKC-
and that pretreatment with FK506 potentiated the phosphorylation of PKC-
(Fig.
4, A and B).
However, neither thrombin treatment alone nor in combination with FK506
pretreatment resulted in a phosphorylated band corresponding to PKC-
(data not shown). Negative controls (absence of the anti-PKC-
or
PKC-
MAbs) lacked the specific phosphorylated band.
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Effects of Ser/Thr PP Inhibitors on Thrombin-Induced Transendothelial Resistance
We determined whether Ser/Thr PP inhibitors alter the transendothelial electrical resistance across BPMECs in response to thrombin. Stimulation of BPMEC monolayers with 100 nM thrombin rapidly decreased the resistance, which varied between 15 and 25% below baseline (Fig. 5A). The resistance drop returned toward baseline within ~30 min (Fig. 5A). When BPMECs were pretreated with 100 ng/ml of FK506, the thrombin-induced decrease in resistance was not reversed within the period of study (>1 h; Fig. 5B). In contrast, pretreatment with 200 nM okadaic acid or 2 nM calyculin A did not significantly alter the time of recovery as evaluated by the time needed to return to 50% of maximal resistance decrease after thrombin stimulation (Fig. 5C). The effects of the Ser/Thr PP inhibitors on the initial resistance decrease in response to thrombin are shown in Fig. 6. The results indicate that neither FK506 nor okadaic acid pretreatment altered the initial resistance decrease in response to thrombin. However, pretreatment with 2 nM calyculin A significantly enhanced the thrombin-mediated decrease in resistance (Fig. 6).
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Relationship of PKC to Recovery From Endothelial Barrier Dysfunction
A continuous prolonged stimulation of cells with submaximal or maximal concentrations of phorbol esters is known to reduce or abolish PKC activity, phorbol ester binding, and PKC immunoreactivity (22). Therefore, phorbol 12-myristate 13-acetate (PMA) was used for the downregulation of PKC to test the involvement of PKC in the FK506-mediated inhibition of recovery of the thrombin-induced resistance decrease. BPMECs were treated with 1.0 µM PMA for 16 h, resulting in the loss of PKC-
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PKC downregulation rescued the FK-506 effects on the thrombin response;
that is, it protected against the FK506-mediated inhibition of recovery
(Fig. 8A). Additionally,
downregulation did not alter the thrombin-mediated initial resistance
decrease but significantly inhibited (~70%) the thrombin-mediated
resistance decrease when combined with FK506 pretreatment (Fig.
8B). Because these findings suggest a possible relationship
of recovery from barrier dysfunction with decreased PKC activity, we
next investigated whether the thrombin-mediated phosphorylation of
PKC- was decreased during the recovery period of the
thrombin-mediated resistance decrease. Results indicated that PKC-
phosphorylation was reduced to near control levels by 10 min of
thrombin treatment (Fig. 9).
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DISCUSSION |
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Findings from the present study indicated that one mechanism by which Ser/Thr PPs may reverse endothelial barrier dysfunction is through the regulation of PKC. The major supportive observations are that 1) the thrombin-induced increase in PKC activity was potentiated by the PP2B inhibitor FK506 but not by inhibitors of PP1 and PP2A, 2) FK506 also inhibited recovery of the normally reversible decrease in transendothelial resistance in response to thrombin, and 3) PKC downregulation rescued the FK506-mediated inhibition of recovery.
The results indicated that FK506 but not calyculin A or okadaic acid potentiated the thrombin-induced increased PKC activity. Because the Ser/Thr PP inhibitors are known to be potent and selective inhibitors of the classes of Ser/Thr PPs (26), the findings suggest that primarily PP2B (also known as calcineurin or Ca2+- and calmodulin-dependent PP) regulated PKC activity in the control of endothelial barrier function. The lack of potentiation by calyculin A and okadaic acid on PKC activity was likely not related to insufficient inhibitor concentrations because the studies used inhibitor concentrations that were greater than fourfold over the IC50. This observation provides novel information demonstrating the selective regulation of PKC by PP2B in endothelial cells. Importantly, the finding that FK506 alone had minimal effects on PKC activity supports the notion that the thrombin response included engagement of PP2B for the regulation of PKC. Although specific functions of PP2B remain relatively unknown, PP2B has been shown to be highly expressed in the brain and a key enzyme in the signal transduction cascade in T-cell activation responses (50). PP2B also has a narrower substrate specificity than the other Ser/Thr PPs, the majority of its most effective substrates being regulators of other protein kinases and phosphatases (39). It will be important to fully understand the specific mechanisms by which PP2B regulates PKC.
Although specific mechanisms by which Ser/Thr PPs regulate PKC are
unclear, both direct and indirect pathways may exist to limit
activation of PKC. It has been shown that the phosphorylation of PKC by
phosphoinositide-dependent kinase-1 (PDK-1) on serine and threonine
residues of the activation loop appears to be required for optimal
kinase activity (5, 15, 32). Furthermore, the upstream
regulator of PDK-1 is phosphatidylinositol (PI) 3-kinase (10). Therefore, it is conceivable that Ser/Thr PPs may
regulate at the level of PDK-1 or PI 3-kinase, which, in turn,
determines the phosphorylation state of PKC. However, findings obtained
with in vitro PKC assays indicate that both PP1 and PP2A reversibly inhibited PKC activity, implicating direct regulation of PKC by these
Ser/Thr PPs (34). In the present study, it remains to be
resolved whether PP2B in endothelial cells regulates PKC- directly
or through PDK-1 and PI 3-kinase.
The FK506-mediated increase in PKC activity and phosphorylation
of PKC- appear to be selective for the endothelium. In monkey kidney
CV-1 and mouse NIH/3T3 fibroblasts, PKC-
activation occurred after
okadaic acid treatment (41). Similarly, okadaic acid
caused activation of PKC-
and PKC-
in rat adipocytes
(43). These studies indicate that several PKC isoforms are
possible regulatory target molecules of Ser/Thr PPs.
Our results showed that FK506 predominantly inhibited the reversal phase and not the initial drop in resistance after thrombin stimulation, suggesting a possible physiological role of PP2B in the regulation of the return of endothelial barrier dysfunction to normal. The finding that FK506 also potentiated the increased PKC activity provides support that this recovery from barrier dysfunction may be mediated through the regulation of PKC. Conceivably, endothelial cells activated by an inflammatory stimulus would present with increased endothelial permeability, which is also associated with PP2B activation, resulting in the limitation of PKC activation responses and the consequent reversal of the increased permeability. Further support for this notion is provided by Verin et al. (44), who reported that thrombin treatment of bovine pulmonary artery endothelial cells resulted in increased PP2B activity that became maximal by 20 min, a time period in which barrier dysfunction was recovering. In this same study, treatment of endothelial cells with deltamethrin, a PP2B inhibitor, inhibited ~50% of the thrombin-mediated dephosphorylation of MLC induced at 60 min, which was accompanied by significant enhancement of the thrombin-mediated increased endothelial permeability (44). These results in combination with findings from the present study indicate that PP2B may have multiple targets in the regulation of the reversal of increases in endothelial permeability.
We found that PKC downregulation effectively rescued the FK506-mediated
inhibition of recovery from barrier dysfunction. These results provide
evidence that the FK506-mediated potentiation of PKC activity was
functionally correlated to inhibition of recovery from endothelial
barrier dysfunction. Furthermore, the finding that FK506 also
potentiated the phosphorylation of PKC- in response to thrombin
implicates this PKC isoform in the regulation of the recovery phase.
Additional support for this thesis is the observation that PKC-
phosphorylation had decreased to near basal level by 10 min of thrombin
activation, a period when the decreased resistance was returning toward
baseline. It is also possible that both novel and atypical classes of
PKC regulate endothelial barrier function, and future studies are
needed to address whether PPs regulate their activity as well.
The results indicate that the downregulation had a minimal effect in
inhibiting the initial decrease in resistance in response to thrombin,
suggesting that PKC isoforms resistant to PMA-mediated downregulation
and other signaling pathways are involved in this phase of the barrier
dysfunction response. However, the finding that PKC downregulation
combined with PP2B inhibition prevented ~70% of the
thrombin-mediated resistance decrease was surprising, and the
underlying basis is not apparent. We speculate that PP2B may have other
interactions with the PKC signaling pathway besides PKC-. From
previous work by Vuong et al. (48) and others
(49), selective PKC isoforms have negative feedback
regulation. It is possible that PP2B regulates other PKC isoforms that
have a negative feedback function, which would contribute to the
inhibition of the resistance decrease in response to thrombin. This
possibility has yet to be tested in our endothelial cell model.
Downregulation by PMA resulted in the complete loss of the PKC-
protein (but not PKC-
protein). Furthermore, PMA-mediated downregulation appeared to be selective for PKC because expression of
PP1, PP2A, and the thrombin receptor was unaltered and PP2B was
slightly decreased. Our results are consistent with the idea that PKC
isoforms possess different susceptibilities to downregulation (1,
13, 17, 35). Different sensitivities to downregulation have been
documented for PKC isoforms belonging to the same class (e.g., PKC-
vs. PKC-
) or between different classes of isoforms (e.g., PKC-
vs. PKC-
) (1, 13, 35). Although downregulation is
generally attributed to proteolytic degradation by endogenous proteases
such as calpain, the basis of differential sensitivity has been
hypothesized to be related to the presence of Ca2+ and
various phospholipids such as phosphatidylcholine and
phosphatidylserine (18). Furthermore, it is thought
that phosphorylation of PKC targets the enzyme for
proteolysis. Our finding that PKC-
was phosphorylated
and downregulated but that PKC-
was neither phosphorylated nor
downregulated supports this thesis.
The observation that calyculin A (equally selective for PP1 and PP2A)
enhanced the extent of the thrombin-mediated drop in resistance but
that okadaic acid (>100 times more selective for PP2A than PP1) had no
effect suggests that PP1 and not PP2A was involved in the regulation of
the barrier dysfunction. Because neither calyculin A nor okadaic acid
significantly altered the thrombin-induced PKC activity, the present
results suggest that the calyculin A-mediated response was likely
independent of PKC. PP1, in particular myosin-associated PP1, has been
proposed as a key endothelial phosphatase that dephosphorylates MLC,
promoting decreased actomyosin contraction in endothelial cells
(7, 38, 45, 46). Alternatively, calyculin A has been shown
to regulate the phosphorylation state of junctional proteins. In human
epidermal cells, both okadaic acid and calyculin A have been shown to
induce hyperphosphorylation of -catenin, a junctional protein
associated with cadherins, which was accompanied by the loss of
cell-cell contacts (37). In another study, PP2A was shown
to regulate
-catenin signaling (36). Thus these
observations suggest that PP1 and PP2A are implicated in the regulation
of the phosphorylation state of MLC and junctional proteins,
contributing to the overall regulation of endothelial barrier
dysfunction (3, 12, 33, 42, 47).
There has been little information regarding expression of Ser/Thr PPs in endothelial cells. We observed that BPMECs expressed PP1, PP2A, and PP2B, which is consistent with reports that bovine pulmonary artery endothelial cells also express these three Ser/Thr PPs (44, 45). Together, the findings indicate that in the lung, both conduit and microvascular endothelial cells express Ser/Thr PPs, suggesting their relatively conserved expression across different vascular beds.
In summary, the major findings in the present study demonstrate
that 1) the thrombin-induced activation of PKC was
potentiated by the PP2B inhibitor FK506 but not by inhibitors of PP1
and PP2A, 2) FK506 also inhibited the recovery phase of the
thrombin-induced decrease in transendothelial resistance, and
3) PKC downregulation rescued the FK506-mediated inhibition
of recovery. These results provide evidence for the notion that PP2B
may play an important physiological role in the regulation of the
return of endothelial barrier dysfunction to normal. Furthermore, PKC
isoforms, in particular PKC-, may be critical targets regulated by
PP2B for this function.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62649 (to H. Lum); the American Heart Association, National (H. Lum); the American Lung Association, National and Metropolitan Chicago (J. L. Podolski and M. E. Gurnck); and the Rush University Committee for Research (J. L. Podolski).
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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 (E-mail: 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.
Received 27 October 2000; accepted in final form 26 February 2001.
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