1 Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612; 2 Department of Biological Chemistry, University of California, Davis, California 95616; 3 Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 4 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104-1687
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ABSTRACT |
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First published September 5, 2001;
10.1152/ ajpcell.00256.2001.The expression and function of the
endogenous inhibitor of cAMP-dependent protein kinase (PKI) in
endothelial cells are unknown. In this study, overexpression of rabbit
muscle PKI gene into endothelial cells inhibited the cAMP-mediated
increase and exacerbated thrombin-induced decrease in endothelial
barrier function. We investigated PKI expression in human pulmonary
artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel
endothelial cells (HBMECs). RT-PCR using specific primers for human
PKI
, human PKI
, and mouse PKI
sequences detected
PKI
and PKI
mRNA in all three cell types. Sequencing and BLAST
analysis indicated that forward and reverse DNA strands for PKI
and
PKI
were of >96% identity with database sequences. RNase
protection assays showed protection of the 542 nucleotides in HBMEC and
HPAEC PKI
mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI
mRNA. Western blot analysis indicated that PKI
protein was detected
in all three cell types, whereas PKI
was found in HBMECs. In
summary, endothelial cells from three different vascular beds express
PKI
and PKI
, which may be physiologically important in
endothelial barrier function.
cAMP; protein kinase inhibitor; endothelial resistance; barrier function
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INTRODUCTION |
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VASCULAR-BASED DISORDERS such as ischemia-reperfusion injury, atherosclerosis, diabetes, and stroke are associated with significant endothelial cell barrier dysfunction. One signaling system, the cAMP-dependent serine/threonine protein kinase (PKA), has a significant and profound effect in the protection against endothelial barrier dysfunction. There is substantial evidence documenting the protective effects of PKA against endothelial barrier dysfunction in a variety of in vitro and in vivo experimental systems in which intracellular cAMP levels were elevated with either cAMP analogs, reagents that stimulate cAMP production (i.e., forskolin), or agents that inhibit its metabolism (i.e., phosphodiesterase inhibitors) (2-4, 14, 16, 18, 20, 21, 24). We showed that overexpression of the highly specific PKA inhibitor (PKI) gene in endothelial cells was effective in fully reversing the barrier-enhancing effects of increased cAMP (16), providing direct evidence for the protective role of PKA. However, the cellular and molecular mechanisms by which PKA inhibits endothelial barrier dysfunction remain undefined. Further, the upstream regulation of endothelial PKA in this function is entirely not known.
Several observations indicate that full activation of PKA is under multiple modulatory mechanisms. In the absence of cAMP, PKA exists as an inactive tetramer consisting of two regulatory and two catalytic subunits. Binding of cAMP to the regulatory subunit lowers the affinity by four orders of magnitude, which in turn causes dissociation of the tetramer into a dimer of regulatory subunit and two active monomers of the catalytic subunit (27). Further, full activation of PKA appears to require phosphorylation of its catalytic subunit by a phosphoinositide-dependent protein kinase (5, 6). This latter observation raises the interesting possibility that PKA is regulated by its phosphorylation state, which in turn determines the cell's responsiveness to cAMP.
Another level of modulation of PKA is by endogenous PKI. PKI is a family of isoforms of distinct genes and has widespread tissue distribution (7, 15). This heat-stable protein has high affinity (0.2 nM) and specific binding for the substrate binding site (as pseudosubstrate inhibitor) on the catalytic subunit of PKA (23, 27, 30). The physiological functions of PKI have not been fully established, but PKI has been shown to contain a nuclear export signal for the PKA catalytic subunit (29). Because of its differential isoform distribution in adult tissues and its distinct interactions with the PKA catalytic subunit, PKI is hypothesized to be critical in the modulation of basal PKA activity in general cell activities (7, 9). Although endogenous PKI has not been studied in endothelial cells, it is possible that PKI may be a key modulator of PKA in endothelial cell activities, such as the permeability response. In support of this thesis, we observed that inhibition of PKA resulted in alterations in endothelial cell-cell junctions and actin filament organization, which were accompanied by impairment of the barrier (22).
The goal of this study was to investigate the involvement of PKI in
regulation of endothelial permeability and PKI isoform expression in
endothelial cells. Results indicated that 1) overexpression of rabbit muscle PKI decreased endothelial barrier restrictiveness, and
2) endothelial cells derived from the human brain
microvessel, pulmonary artery, and foreskin microvessel expressed
predominantly PKI and PKI
isoforms. We conclude that PKI
and
PKI
may be important in modulating PKA in the regulation of vascular
endothelial barrier function.
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MATERIALS AND METHODS |
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Cell Culture
Human dermal (foreskin) microvascular endothelial cells (HMECs) (1) were maintained in culture in MCDB 131 medium, supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT), 10 ng/ml human epidermal growth factor (EGF), 1 µg/ml hydrocortisone, 5% penicillin-streptomycin, and 5% L-glutamine. HMECs are an immortalized cell line transformed by simian virus 40 (SV40) large T antigen and have been shown to retain endothelial cell phenotypic and functional characteristics. They exhibit the expected morphological and functional endothelial phenotypes: express and secrete von Willebrand factor, take up acetylated low density lipoprotein (LDL), form tubes when grown in Matrigel, and express CD31 (platelet endothelial cell adhesion molecule-1), CD36, intercellular adhesion molecule-1 (ICAM-1), and CD44 (1). They also bind purified T cells in a regulatable manner and respond to cytokines in a similar way to nontransformed endothelial cells. HMECs were passaged 5-7 days when confluent and were used for studies at population doublings between 25 and 40.Human brain microvascular endothelial cells (HBMECs) were provided by
Dr. K. S. Kim (Children's Hospital, Los Angeles, CA). HBMECs were
cultured in RPMI 1640 supplemented with 10% FBS, 10% NuSerum (Becton
Dickinson, Bedford, MA), endothelial cell growth supplement (30 µg/ml), heparin (5 U/ml), 1 mM sodium pyruvate, 1 mM MEM nonessential
amino acids, 1 mM MEM vitamins, 5% L-glutamine, and 5%
penicillin-streptomycin. HBMECs were grown and used at population
doublings between 30 and 40. These cells were immortalized by SV40
large T antigen and have been shown to retain endothelial cell
phenotypic and functional characteristics (25, 26). The cells were positive for von Willebrand factor, carbonic anhydrase IV,
and Ulex Europeus agglutinin I, took up fluorescently labeled acetylated LDL, and expressed -glutamyl transpeptidase,
demonstrating their brain endothelial cell characteristics. When
treated with tumor necrosis factor-
, vascular cell adhesion
molecule and ICAM-1 were expressed.
Human pulmonary artery endothelial cells (HPAECs) were purchased from Clonetics (San Diego, CA). These cells have been characterized to be endothelial in origin by the uptake of acetylated LDL and by positive staining for von Willebrand factor. The HPAECs were grown in basal medium containing EGM-2 Bulletkit growth supplement (Clonetics), 10% FBS, and 5% penicillin-streptomycin. HPAECs were cultured to 10-15 population doubling for use in studies.
The transformed renal embryonic kidney cell line, 293 cells (CRL 1573; American Type Culture Collection, Manassas, VA) (13), were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/ml penicillin, 100 U/ml streptomycin, and 10 mg/ml amphotericin B.
RT-PCR
The endothelial expression of PKI mRNA was determined from HMECs, HBMECs, and HPAECs by RT-PCR. Total RNA was extracted using an RNA STAT-60 isolation kit (Tel-Test, Friendswood, TX) according to the manufacturer's protocol. RNA concentration was determined by spectrophotometry at an absorbance of 260 nm. RNA integrity was assessed by agarose gel electrophoresis and ethidium bromide staining. The RT-PCR was made using the GeneAmp RNA PCR kit (Perkin Elmer, Branchburg, NJ), and thermal cycling reactions were run in the GeneAmp 2400 PCR System (PE Biosystems, Foster City, CA). Isolated total RNA was subjected to reverse transcription with oligo(dT) primers, generating cDNA copies of the RNA sequence. The subsequent PCR was performed using specific primer sets based on sequences for PKI isoforms, human PKI
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RNase Protection Assay
RNase protection assay (RPA) was performed as previously described (10). Probes were allowed to hybridize to target RNA in reactions containing 10.0 µg of total cell RNA and 2.5 × 104 cpm of PKIRiboprobe templates.
Purified and sequenced RT-PCR products for human PKI and human
PKI
(see RT-PCR) were ligated into pGEM-T-Easy
vector, chemically transformed into DH5
competent cells, and plated
onto Luria-Bertani (LB)/ampicillin agar plates at 37°C. A
successfully transformed colony (screen by restriction digests) was
grown in LB overnight at 37°C, and the plasmid-DNA insert was
isolated using the Wizard Plus Minipreps DNA purification system. The
plasmid-DNA insert was linearized with PstI (PKI
) or
BamHI (PKI
) and transcribed with T7 RNA polymerase to
produce radiolabeled antisense probes.
In vitro transcription of radiolabeled probes.
Probe synthesis was conducted with 1 mM each of CTP, ATP, and UTP,
respectively, 9.38 µM of [32P]GTP (800 Ci/mmol), and 25 µM of unlabeled GTP. One microgram of template DNA with appropriate
RNA polymerase (T7 RNA polymerase) was added to each reaction and was
allowed to transcribe at 37°C for 40 min, and template was digested
with DNase (Ambion) at 37°C for 15 min. The [32P]GTP
not incorporated into the probe was removed using Centri-Spin 40 purification columns (Princeton Separation, Adelphia, NJ). Radiolabeled
probes were used in RNase protection assays to detect mRNA levels of
PKI and PKI
from total cell RNA isolated from HMECs, HBMECs, and HPAECs.
Protein Expression of PKI Isoforms
Cell collection and preparation. HMECs, HBMECs, and HPAECs were grown to confluency in 60-mm culture dishes. The cells were placed on ice, washed two times with ice-cold Ca2+- and Mg2+-free PBS, and collected by scraping in RIPA buffer [in mM: 150 NaCl, 1 EDTA, 1 EGTA, 50 Tris · HCl (pH 7.4), 1% Nonidet P-40, 1 NaF, and 1 sodium vanadate, as well as 0.25% sodium deoxycholate, 1 pepstatin A, 1 phenylmethylsulfonyl fluoride, 25 µg/ml leupeptin, and 25 µg/ml aprotinin]. The cell extract was sonicated with 10 pulses using the sonifier (Branson Ultrasonics, Danbury, CT) and was heated to 92°C for 10 min. After being cooled to 4°C, the endothelial cell lysate was centrifuged at 14,000 rpm for 15 min, and the supernatant was collected for protein concentration, which was determined using the bichinchoninic acid protein assay kit with BSA as standard (Pierce, Rockford, IL).
Antibody preparation.
Affinity-purified polyclonal antibody to full-length murine PKI was
prepared as described previously (7). In brief, purified histidine-tagged PKI
protein was conjugated to keyhole
limpet hemocyanin and used for immunization of rabbits for antibody
production. The antiserum raised against PKI
was affinity purified
on nitrocellulose blots bound with the purified fusion protein. In
addition, polyclonal anti-peptide antibodies were prepared against
peptides for rat PKI
-(5-22) (TTYADFIASGRTGRRNAI)
and rat PKI
-(5-22) (SVISSFASSARAGRRNAL) as
previously described (12, 15).
Western blot analysis. The heat-treated cell lysates were loaded at constant protein concentrations, and the proteins were separated by SDS-polyacrylamide gradient gel electrophoresis. The separated proteins were electrotransferred to nitrocellulose or polyvinylidene difluoride membranes. Nonspecific binding of antibody to membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST). The blocked membrane was then incubated with primary antibodies, diluted in TBST with 1% nonfat dry milk, overnight at 4°C in a rocker. The blot was washed five times with TBST and incubated with anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase, and the bands were detected using the enhanced chemiluminescence kit.
Transendothelial Electrical Resistance
The transendothelial electrical resistance, an index of endothelial barrier function, was determined in real time using the electric cell-substrate impedance sensor (ECIS) system (Applied BioPhysics, Troy, NY) (11, 19). The system consists of one large gold-plated electrode (10For resistance measurement, endothelial cells (105 cells) were plated onto a sterile, fibronectin-coated gold-plated electrode and grown to confluency. The electrode was mounted onto the ECIS system housed within an incubator (maintained at 37°C, 5% CO2, and 100% humidity) and connected to the lock-in amplifier. After a period of 15 min of equilibration, the cells were challenged with reagents according to experimental protocol and resistances were recorded continuously in real time.
Construction of Rabbit Muscle PKI in Endothelial Cells
An E1Materials
The following were purchased from GIBCO BRL (Gaithersburg, MD): DH5 ![]() |
RESULTS |
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Overexpression of PKI Decreases Endothelial Barrier Restrictiveness
The role of PKI in the regulation of endogenous PKA in endothelial barrier function was investigated by overexpression of endothelial cells with AdPKI. HMECs were infected at 100 multiplicity of infection (MOI) of AdPKI for 2 days for the study. We have demonstrated that the use of this protocol for adenovirus infection of HMECs was highly efficient (>95%) for gene transfer (16). The cells were treated with 10 µM forskolin (direct activator of adenylyl cyclase) plus 1 µM IBMX (phosphodiesterase inhibitor) to increase intracellular cAMP levels, and the transendothelial electrical resistance was measured in real time. In control noninfected HMECs, the combination of forskolin plus IBMX significantly increased resistance above baseline (Fig. 1), indicating increased restrictiveness of the endothelial monolayer. However, this increased resistance was abolished in the PKI-overexpressing HMECs (Fig. 1).
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We also determined the effects of PKI on mediator-induced changes in
endothelial barrier function. HMEC were infected at 100 MOI with AdPKI
or control AdNull for 2 days, and the transendothelial electrical
resistance, an index of barrier function, was determined in response to
1.0 nM thrombin. Stimulation of HMECs with thrombin caused transient
decreases in resistance in which the AdPKI-infected HMECs showed
greater maximal decreases in resistance relative to the control
AdNull-infected HMECs (Fig.
2A). Figure 2B
summarizes the maximal thrombin-mediated resistance decrease from
baseline in the controls (noninfected wild-type HMECs and HMECs
infected with AdNull) and HMECs infected with AdPKI. The results
indicated that, in HMECs infected with AdPKI, the thrombin-mediated
resistance decrease was significantly greater (6,526 ± 494
)
than in either control wild-type HMECs or AdNull groups (
4,137 ± 123 and
3,837 ± 163
, respectively).
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PKI, PKI
, and PKI
mRNA Expression in Endothelial Cells
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We next used RNase protection assay to confirm the expression of PKI
and PKI
in endothelial cells. Riboprobe templates were generated
from purified RT-PCR products for PKI
and PKI
(see MATERIALS AND METHODS). Total RNA isolated from
HBMECs, HPAECs, and HMECs was reacted with the antisense probes.
Results indicated that the PKI
antisense probe protected the 542 nucleotides of PKI
mRNA from HPAECs and HBMECs (Fig.
4, top). The PKI
antisense probe protected the 240 nt of PKI
mRNA from HBMECs, HPAECs, and HMECs (Fig. 4, bottom).
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PKI protein expression was investigated using Western blot analyses.
Anti-peptide polyclonal antibodies directed against PKI and PKI
(15) and affinity-purified polyclonal anti-PKI
antibody (7) were used for Western blot analysis of HBMEC, HPAEC,
and HMEC cell lysates. A strong band at ~20 kDa was observed for
PKI
from the three endothelial cell types (Fig.
5A). PKI
was detected as a
band of ~12 kDa in HBMECs only; PKI
was not detected in any of the
three cell types (Fig. 5B). Positive control using testis
tissue lysate (15) is shown for PKI
and PKI
(Fig.
5B).
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DISCUSSION |
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Expression of the exogenous rabbit muscle PKI gene into endothelial cells inhibited the increase in basal endothelial resistance induced by the cAMP-elevating agents, forskolin and IBMX, providing strong evidence that PKA was responsible for mediating the increase in barrier function. This observation was consistent with our earlier finding that PKI overexpression abolished the protective effects of cAMP-elevating agents (i.e., forskolin, IBMX, and cholera toxin) against a thrombin-induced increase in endothelial permeability (16, 22). Interestingly, PKI overexpression also exacerbated the decrease in thrombin-induced transendothelial resistance. This result suggests that PKA normally functioned to maintain the extent of basal endothelial barrier restrictiveness; thus PKI may be a physiological modulator of PKA activity in the endothelial barrier response. Inhibition of PKA also promotes actin stress fiber formation and loss of catenin (22), supporting the notion that endogenous PKA may be important in regulation of basal cell barrier restrictiveness.
The results from the current study indicate that endothelial cells
derived from human brain, pulmonary artery, and foreskin expressed
predominantly PKI and PKI
. PKI
mRNA and protein were not
detected in any of the endothelial cells. PKI
mRNA was expressed by
all three endothelial cell types as determined by both RT-PCR and RNase
protection assays. This message expression corresponded with PKI
protein expression from the three endothelial cell types. Further,
PKI
mRNA expression was observed in the three endothelial cell types
when determined by RT-PCR; however, RNase protection assay detected
PKI
mRNA for only HPAECs and HBMECs, but not HMECs. This finding may
possibly be attributed to relatively low levels of the PKI
message
expressed by HMECs, which was detectable with amplification by RT-PCR.
PKI
protein expression was detected in HBMECs, but not from HMECs
or HPAECs, suggesting low levels or lack of the protein expression by
these cells. PKI
has been reported to be predominantly expressed in
the cerebral cortex and muscle, whereas PKI
is more limited to the
testis (28). Overall, the results are consistent with
observations made by Collins and Uhler (7) who reported
that PKI
was abundant and more widely expressed than either PKI
and PKI
in tissues, such as heart, skeletal muscle, testis, spleen,
lung, liver, and kidney. The more prevalent expression of PKI
in
endothelial cells suggests that this isoform may be important in
modulating a broad range of basic cellular activities, including the
regulation of basal and mediator-induced changes in endothelial barrier function.
A comparison of the sequences between human PKI and PKI
coding
regions indicated ~53% homology, whereas the rabbit muscle PKI used
for the functional studies showed 74% and 53% homology with human
PKI
and PKI
, respectively. The amino acid sequences in PKI
important for binding to and inhibition of the PKA catalytic subunit
have been shown to be relatively conserved among PKI isoforms (7). PKI inhibits PKA through interactions within the
substrate binding site of the PKA catalytic subunit, functioning as a
competitive inhibitor. Although the overexpression of rabbit muscle PKI
into endothelial cells likely inhibited PKA in a similar way to PKI
and PKI
, the relative potency and efficacy may be different. PKI
is known to have greater binding affinity to the PKA catalytic subunit
than PKI
(inhibition constant Ki = 0.073 and 0.44 nm, respectively) (7, 9). The sixfold difference
in affinity may be physiologically significant, since the basal level
of the catalytic subunit is normally maintained quite low in cells.
It is now known that, in addition to its inhibition potential of PKA,
PKI functions to shuttle the PKA catalytic subunit out of the nucleus
via a nuclear export signal (NES) (29, 31). All members of
the PKI family possess an amino-terminal inhibitory region and a
central region containing the NES (7). In general, the
least conserved region occurs in the carboxy terminus of the molecule
(7). These differences among the isoforms and their differential expressions suggest possible isoform-specific
physiological function(s). It remains to be determined whether PKI
and PKI
have differential functions in endothelial cells.
PKI was not detected in the endothelial cells by RT-PCR. One reason
may be the use of primer-based mouse DNA sequences, which may not be
highly conserved with human PKI
. However, the human PKI
gene has
not been cloned. Nonetheless, the result was not surprising, since
PKI
expression is mostly limited to testis (28).
In summary, the main findings are that 1) overexpression of
rabbit muscle PKI decreased endothelial barrier restrictiveness, and
2) endothelial cells derived from human brain microvessel, pulmonary artery, and foreskin microvessel expressed predominantly PKI and PKI
isoforms. We conclude that PKI
and PKI
may be important in modulating PKA in the regulation of vascular endothelial barrier function.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-62649 (H. Lum), the American Heart Association, National (H. Lum), and a Veterans Affairs Grant (C. E. Patterson).
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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 8 June 2001; accepted in final form 28 August 2001.
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