Research Service, Stratton Veterans Affairs Medical Center; and the Center for Cardiovascular Science, The Albany Medical College, Albany, New York 12208
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ABSTRACT |
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The intracellular
serine/threonine kinase protein kinase C (PKC) has an important role in
the genesis of pulmonary edema. This review discusses the PKC-mediated
mechanisms that participate in the pulmonary endothelial response to
agents involved in lung injury characteristic of the respiratory
distress syndrome. Thus the paradigms of PKC-induced lung injury are
discussed within the context of pulmonary transvascular fluid exchange.
We focus on the signal transduction pathways that are modulated by PKC and their effect on lung endothelial permeability. Specifically, -thrombin, tumor necrosis factor (TNF)-
, and reactive oxygen species are discussed because of their well-established roles in both
human and experimental lung injury. We conclude that PKC, most likely
PKC-
, is a primary supporter for lung endothelial injury in response
to
-thrombin, TNF-
, and reactive oxygen species.
antisense; edema; reactive oxygen species; transcription
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INTRODUCTION |
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THE VESSEL INTIMA AND CAPILLARIES of the pulmonary and systemic circulation are lined by a monolayer of endothelial cells that serve to control and restrict the luminal to abluminal movement of water and protein. Specifically, the pulmonary endothelium constitutes the interface between the blood and extravascular tissue of the lungs. This homeostatic barrier function of the endothelium is ultimately maintained by the dynamic regulation of the endothelial cell shape, endothelial cell-to-cell adherence, and endothelial-extracellular matrix adherence (101). The homeostasis of the lung, as indicated by the ventilation-to-perfusion ratio, exchange of blood gases, metabolism, and transit of blood from the right to left side of the circulation, is invariably influenced by changes in the endothelial barrier function (101, 110, 112). Thus compromise of systemic and pulmonary vascular endothelial function is a main component of the pathophysiology of inflammation and its associated pernicious syndrome sepsis (18, 44, 112, 116, 177). In the lungs, the endothelial response is characterized by a decrease in its restrictive barrier function, resulting in an increase in endothelial permeability and transvascular fluid and protein flux into the interstitial space (i.e., edema), which ultimately contributes to the pathogenesis of multiple organ failure (MOF) associated with sepsis (18, 44, 116, 177).
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PULMONARY EDEMA |
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The movement of water and macromolecules into the lung
interstitium, airways, and alveoli is characteristic of acute lung injury, which is pathogenetic for the respiratory distress syndrome (RDS) (110, 112, 181). The pulmonary endothelium has a
paramount role in the pathophysiology of acute lung injury because an
alteration in its function as a restrictive barrier contributes to the
changes in the forces that modulate edema (101, 110, 112, 140,
181). The forces that govern the movement of water and protein
leading to edema formation are described by the Starling equation for fluid exchange (172). A brief review of the Starling
equation for fluid exchange is presented here to facilitate
understanding of the role of PKC in lung edema.
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In human studies and in experimental models of acute lung injury,
pulmonary edema is induced by increases in Pmv and
Kfc and by a decrease in (18, 44, 76,
81, 83, 110, 112, 141, 172). During RDS, the compromised
pulmonary endothelium maintains, at least in part, a decrease in
protein selectivity (i.e., decrease in
), an increase in
transvascular fluid flux (i.e., increase in
Kfc), and an altered metabolism of inflammatory mediators [e.g., angiotensin I, bradykinin, endothelin, prostacyclin, thromboxane A2, superoxide
( · O
)], and
reactive oxygen species [e.g., hydrogen peroxide
(H2O2,), · O
are due,
in part, to the response to inflammatory mediators such as · NO (77),
H2O2 (80), thrombin (83,
101, 110), and tumor necrosis factor (TNF)-
(Fig. 1)
(81, 159). Isolated lung studies indicate that the
increase in Kfc is primarily due to an
increase in permeability to water, in addition to heterogeneous
increases in surface area. In lung injury that progresses toward
alveolar flooding, the extra-alveolar and alveolar epithelium also
exhibits a decrease in barrier function and an increased generation of inflammatory mediators [e.g., inducible nitric oxide synthase (iNOS)
· NO].
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DETERMINATION OF ENDOTHELIAL BARRIER FUNCTION |
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The early in vivo study of transvascular fluid flux used
time-dependent lung weight measurements during different
Pmv and mv (172). Then,
with the advent of the chronic sheep lung lymph fistula model using the
online determination of fluid and protein flux, Ppm,
pm, and
i permitted the estimation of
(110, 172). In addition, by using the isolated lung in
situ and ex vivo, investigators were able to determine
Kfc, in addition to Pmv and
.
Thus the forces that control pulmonary edema were becoming understood
but with the caveat that each technique had its limitation. Ultimately, the pursuit of a focus on the endothelium and the requirement for
specificity of experimental manipulation on a single cell type prompted
the evaluation of the transendothelial movement of molecules within an
isolated endothelial cell monolayer, in the absence of underlying
tissues. The successful isolation and culture of endothelial cells from
lung macrovessels (e.g., artery and vein) and microvessels enabled the
study of the pulmonary endothelial cell's metabolism and response to
inflammatory mediators that ultimately determine the value of
Pmv, Kfc, and
(21, 38). Thus the barrier function of the endothelium is now
characterized by the microscopic visualization of tracer molecules
(e.g., horseradish peroxidase, see Ref. 180), measurement
of transendothelial clearance of molecules (e.g., albumin and dextran),
and transendothelial electrical resistance (TEER) (29, 38, 42,
86, 107, 115, 163, 173, 180).
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THE BASICS OF PKC |
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PKC is a family of serine/threonine kinases characterized by
at least eleven different isotypes. PKC isotypes are differentially regulated by calcium (Ca2+), diacylglycerol, and
phospholipids and differ in structure, expression, intracellular
localization, substrate utilization, and mechanisms of activation
(31, 129, 130, 192). PKC is composed of four conserved
(C1-C4) and five variable (V1-V5)
domains (Fig. 2). C1 and C2 constitute
the regulatory domain and contain binding sites for phospholipids
(e.g., phosphatidylserine), Ca2+, diacylglycerol, and
phorbol esters (Fig. 2). The C3 and C4 regions contain the catalytic
domain that has binding sites for ATP and different PKC substrates
(Fig. 2). The conserved region of the regulatory domain, within
residues 19-36, has structural features of a pseudosubstrate;
therefore, it maintains PKC in the inactive form during the absence of
phospholipid activators. The activation of PKC begins with the release
of membrane phospholipids in response to phospholipase activity [e.g.,
phospholipase C (PLC)]. The phospholipids interact with the C1 and C2
domains, which provides the free energy required for the dissociation
of the NH2-terminal pseudosubstrate from the active site,
which allows substrate binding (72) (Fig. 2). The C1 and
C2 domains each have their own determinants for membrane recognition,
and the C1 domain is present in most PKC isotypes (82).
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PKC isotypes are involved in signal transduction pathways that govern a
wide range of physiological processes, such as differentiation, proliferation, gene expression, brain function, membrane transport, and
the organization of cytoskeletal and extracellular matrix proteins
(22). The PKC isotypes are subdivided into three groups: the classical, novel, and atypical. This subdivision is based on the
structural and functional differences in the conserved domains
C1-C4 (Fig. 2) (128). The classical PKC-,
PKC-
1/2, and PKC-
isotypes are Ca2+ and
diacylglycerol dependent. These PKC isotypes have the conserved diacylglycerol-binding C1 domain and the Ca2+-binding C2
domain. The C1 domains consist of a tandem C1A and C1B arrangement that
can bind the endogenous diacylglycerol and exogenous phorbol esters
(Fig. 2) (167). The novel PKC-
, PKC-
, PKC-
,
PKC-
, and PKC-µ isotypes contain C2 domains that lack Ca2+-binding ability but still retain functional C1A and
C1B domains that can bind the endogenous diacylglycerol and exogenous
phorbol esters (Fig. 2). The atypical PKC-
, PKC-
, and PKC-
isotypes (31, 99, 123, 129, 137) lack a functional C2
domain and contain a single C1 domain that lacks the ability to bind
diacylglycerol and phorbol esters (25, 149). Therefore,
the mechanism of activation of the atypical PKC isotypes is both
Ca2+ and diacylglycerol independent and involves other
lipid-dependent pathways (123). Thus for example the
phorbol ester 2-O-tetradecanoylphorbol-3-acetate or the
lipid diacylglycerol would activate the classical and novel PKC
isotypes but not the atypical PKC isotypes. The activation of the novel
PKC isotypes will persist in the presence of EDTA, but the classic PKC
isotypes would not exhibit activity (22, 151).
In addition to the classic activation mechanisms indicated above, other
PKC activation mechanisms have been proposed. Slater et al.
(167) have shown that PKC- activation is also dependent on lack of a C1-C2 domain interaction, corresponding to a
transition of PKC-
from a closed inactive state to an open active
state (Fig. 2). They showed that PKC-
isotype bound specifically and with high affinity to an
C1A-C1B fusion protein of PKC-
. The
C1A-C1B domain activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner comparable with activation
resulting from membrane-phosphatidylserine association. Interestingly,
the
C1A-C1B domain also activated the classical PKC-
1/2, and
PKC-
isotypes, but not the novel PKC-
or PKC-
isotypes that
were each activated by their own C1 domains. PKC-
, PKC-
1/2, and
PKC-
isotypes were unaffected by the C1 domain of the PKC-
isotype and only slightly activated by that of PKC-
isotypes. Thus
the activation mechanism of the novel PKC isotypes may be similar to
that of the classic isotypes. PKC-
isotype activity was unaffected by its own C1 domain and those of the other PKC isotypes. Another key
determinant of PKC activity is the phosphorylation of the PKC molecule,
its intracellular localization, and proteolytic degradation.
Phosphorylation is controlled by PKC-mediated autophosphorylation and
the phosphorylation mediated by other kinases such as
3-phosphoinositide-dependent kinase-1 and tyrosine kinases (40,
94).
The distribution of PKC activity is regulated by its direct interaction with accessory proteins (e.g., receptor for activated C protein) that target the movement of the PKC molecule to different intracellular compartments, which confers selectivity by associating individual isotypes with specific substrates (149, 156, 169). PKC activity is also determined by its degradation. The literature indicates that the calcium-lipid-dependent protease calpain-µ can degrade PKC to a catalytically active PKM by cleaving off the regulatory domain (43, 46, 154, 161). PKM may be a constitutively active enzyme that mediates long-term phosphorylation activity of PKC. Yet the regulation of PKC activity is maintained because of further degradation into inactive degradation products by calpain-m (43, 46, 154, 161).
Identification and characterization of the PKC isotypes were restricted by the availability of PKC isotype-specific pharmacological inhibitors and activators. However, recent technology offers better isotype specificity, which includes oligonucleotide antisense (AS)-induced inhibition of expression, expression of wild-type and dominant-negative PKC vectors, PKC isotype knockout mice, and peptide fragments to either inhibit or promote translocation of PKC isotypes to specific anchoring proteins (9, 12, 22, 184, 189).
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PKC IN LUNG INJURY |
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PKC is implicated in many cellular responses associated with lung
injury, including endothelial permeability (104, 107, 165,
164), cell contraction (100, 196), migration
(40), proliferation (40, 197),
apoptosis (162), mucous secretion (84), gene expression (147), and the
organization of cytoskeletal and extracellular matrix proteins
(4, 195, 196). Throughout the last five decades,
investigation into the pathogenesis of the increased endothelial
permeability associated with RDS has indicated a role for many
mediators, such as cytokines (e.g., IL-1, TNF-) (52, 93,
146), growth factors [e.g., vascular endothelial growth factor
(VEGF)] (93), peptides (substance P, bradykinin)
(7, 150, 183), proteases (e.g., elastase) (19), complement activation (e.g., C5a) (125,
188), intravascular coagulation (e.g., thrombin) (59, 102,
103, 110, 170), reactive oxygen and nitrogen species (e.g.,
H2O2,
· O
) (20, 80, 89, 164, 165), and lung
sequestration of neutrophils (95) (Fig. 1). The
intracellular signal pathways that cause an increase in endothelial
permeability are still not completely defined despite extensive study
of the effect of the above mediators of endothelial dysfunction.
Importantly, however, PKC is now known to be a necessary part in the
regulation of endothelial permeability and edema formation induced by
three known mediators of RDS: H2O2, thrombin,
and TNF-
(7, 23, 50, 54, 87, 107, 164, 165). This
review highlights the role of PKC in the endothelial barrier alteration
induced by H2O2, thrombin, and TNF-
. We
discuss the role of PKC in the vascular permeability of acute lung
injury as assessed in in vivo, ex vivo, and in vitro studies using
cultured endothelial cells of pulmonary origin.
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PKC IN PHORBOL ESTER-INDUCED LUNG INJURY |
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The involvement of PKC in lung injury began with studies using the phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12-myristate 13-acetate (PMA). TPA and PMA bind strongly to the C1 diacylglycerol regulatory domain, thereby activating the classic and novel PKC isotypes (Fig. 2) (130, 151). Conversely, long-term activation of the PKC by phorbol esters results in degradation of PKC, which depletes the PKC activity (130, 151). Thus the use of PMA and TPA holds the caveat that each isotype of PKC is differentially activated and/or depleted, which will give an indication only to the class of PKC involved in the response (9, 15, 99, 117, 151). Despite these disadvantages, phorbol esters are still used as a "screen" for indicating a role for PKC function in lung endothelial injury.
In studies using whole animals, PMA induces a route-, time-, and dose-dependent pathophysiology (e.g., edema, increased vascular/endothelial permeability, hypertension, and hypoxia) similar to acute lung injury and RDS (179). Alveolar instillation of PMA results in alveolar edema supposedly due to intense activation of alveolar macrophages and pulmonary epithelium in anesthetized rats and sheep (65). Vascular PMA infusion causes an early decrease in endothelial cell angiotensin converting-enzyme activity before and independently of changes in capillary surface area and edema (45, 118). The early functional change induced by PMA is time dependent because it is followed by frank pulmonary edema due to activation of the many cell types in the lung's vasculature (45, 118).
The presence of many cell types in the lung and edema formation in vivo
confounded accurate study of endothelial barrier function; therefore,
investigators began focusing on ex vivo techniques and cell culture. In
isolated perfused guinea pig lungs without polymorphonuclear leukocytes
(PMN), PMA increased pulmonary arterial pressure (Ppa),
pulmonary capillary pressure (Ppc), and lung weight; decreased the ratio of arterial-to-venous vascular resistance; but had
no effect on Kfc (76). A caveat to
this study is that the severe increase in vascular resistance did not
undermine the measurement in Kfc
(76). Thus experiments using cultured endothelial cells
from various species and vascular sites were performed (63, 66,
160, 185). The results indicated that, in bovine pulmonary arterial endothelial cell monolayers, short-term treatment with PMA
(108-10
6 M) increased endothelial
permeability to albumin (107). The negative controls
4-
-phorbol-didecanoate and 1-mono-oleoyl glycerol, which did
not activate PKC, had no effect on endothelial permeability to albumin
(107). The early use of PKC inhibitors indicated that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) (68)
reduced the PMA-induced increase in endothelial permeability,
whereas the isoquinoline
N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride
(HA1004) (68), which had no affect on PKC activity, also
had no effect on endothelial permeability in response to PMA.
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ISOTYPES OF PKC IN PHORBOL ESTER-INDUCED LUNG INJURY |
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The specific PKC isotypes involved in the PMA-induced barrier
dysfunction have also been studied. In the human dermal microvascular endothelial cell line HMEC-1, the overexpression of PKC-1 augmented the PMA-induced increase in endothelial permeability. PMA stimulated the translocation of PKC-
1 from the cytosol to the membrane, indicating PKC-
1 activation (126). In another study, a
decrease in PKC-
1 expression reduced the effect of PMA on barrier
function (187). Interestingly, downregulation of PKC with
PMA can delay the normalization of endothelial permeability that
follows the altered barrier function in response to thrombin (see
PKC IN THROMBIN-INDUCED LUNG ENDOTHELIAL INJURY),
indicating that PKC isotypes have disparate roles in changing
endothelial permeability that are dependent on the agonist used to
induce the inflammatory response (105).
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LEUKOCYTES IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS |
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There are many potential mechanisms for PMA-induced, PKC-dependent lung injury due to the myriad of signal transduction pathways affected by PKC. A role for PMN is noted because in studies using isolated rat lungs not depleted of PMN, PMA added to the perfusate increased microvascular permeability as expressed by increases in Ppa, Kfc, lung weight gain, lung wt/body wt ratio, and the protein concentration of the bronchoalveolar lavage fluid (27). The PMA-induced effect on endothelial permeability was attributed to activated PMN and their release of inflammatory mediators, including reactive oxygen species and proteases (Fig. 1) (5, 83, 134, 141). However, other leukocytes can mediate acute lung injury, as observed in PMA instillation of PMN-depleted lungs in the presence of mononuclear leukocytes (142).
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REACTIVE OXYGEN SPECIES IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS |
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In animal studies, reactive oxygen species mediate, at least in
part, the response to PMA because the reactive oxygen species scavenger
dimethylthiourea (26) prevents pulmonary hypertension, and
the enzyme CuZn superoxide dismutase (SOD) (121)
attenuates the increase in vascular permeability (26).
Similarly, in cultured endothelial cells, PMA is used as a model for
reactive oxygen species-induced activation of nuclear transcription
factors such as NF-B and activator protein (AP)-1 (73).
The importance of the effect of reactive oxygen species on NF-
B and
AP-1 is dictated by DNA promoter-driven expression of many substances
that have a role in endothelial barrier dysfunction such as the
intercellular adhesion molecules (ICAM), prostaglandin E2
(PGE2), TNF-
, and IL-8 (1, 49, 50, 97,
138). PMA causes depletion of glutathione in pulmonary arterial
endothelial cells that is inhibited by CuZn-SOD, supporting the notion
that reactive oxygen species participate in the effect of PKC
activation (144). Glutathione depletion markedly enhances
PMA-induced expression of ICAM and PGE2 in human umbilical
vein endothelial cells, which supports the notion of reactive oxygen
species-driven expression for mediators of inflammation. The PKC
inhibitor H7 prevents most of PMA-induced ICAM-1 expression, PGE2 production, and the effect of glutathione depletion.
The glutathione effect is also inhibited by the antioxidant quercetin (73). Thus PKC-induced glutathione depletion enhances
susceptibility of vascular endothelial cells to the effects of reactive
oxygen species generation and its downstream effect on gene expression (73). The increase in reactive oxygen species
concentration is probably due to the activation of NADPH oxidase,
because PKC phosphorylates p22phox and p47phox
(2, 10, 32, 34). It is well established that reactive oxygen species mediate endothelial barrier dysfunction indirectly via
downstream signal molecules and/or by direct effects on the cell cytoskeleton.
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INTRACELLULAR SIGNAL PATHWAYS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS |
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The downstream signaling pathways by which PKC activation
increases endothelial permeability have been studied using cultured endothelial cells (185) (Fig.
3). PMA induces rapid phosphorylation of
the Rho-GDP guanine nucleotide dissociation inhibitor (GDI) in human
umbilical vein endothelial cells. Phosphorylated GDI permits
Rho-GDP/GTP exchange resulting in Rho-GTP as a co-actor for Rho kinase
(ROCK). ROCK is implicated in modulation of myosin-actin-ATPase activity (24, 108, 180) and therefore in cell contraction (Fig. 3). Bovine microvessel endothelial cells grown on a flexible substrate contract on addition of agents that cause increased endothelial permeability in vivo or in vitro, including
angiotensin II, thrombin, bradykinin, and the stable analog of
thromboxane A2 U-44069 (122). PKC depletion by
preincubation with PMA prevented the contraction by angiotensin II. The
inactive analog 4--phorbol 12,13-didecanoate did not inhibit
contraction, providing direct evidence that contraction of microvessel
endothelial cells may mediate the increase in endothelial permeability
in response to activation of PKC (122). Similarly,
PMA-induced depletion of PKC prevents thrombin-induced GDI
phosphorylation, Rho activation, and thrombin-induced decrease in TEER
(119), supporting a PKC
GDI
Rho-GDP/GTP
Rho-GTP
ROCK
myosin-actin-ATPase pathway (Fig. 3). Evidence for
another signaling pathway indicates that PMA triggers a Ras-dependent
signal transduction in primary human umbilical vein endothelial cells
and in the permanent endothelial cell line ECV304 (119)
(Fig. 3). Selective inhibition of the mitogen-activated protein
kinase (MAPK) and extracellular signal-regulated kinases (ERK)
significantly attenuates the PMA-induced reduction in TEER, consistent
with PKC- and ERK-mediated endothelial cell barrier regulation
(185). PMA also produced a time-dependent increase in the
activity of Raf-1, a serine/threonine kinase known to activate MAPK
kinase (MEK), and increased the activity of Ras, which binds
and activates Raf-1. Inhibition of Ras completely abolished PMA-induced
Raf-1 activation, suggesting that the sequential activation of PKC
Ras
Raf-1
MEK
ERK is also involved in endothelial barrier
regulation by PMA (185) (Fig. 3).
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INTRACELLULAR TARGETS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS |
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In bovine pulmonary artery endothelial cell monolayers, PMA and
-thrombin induce rapid and concentration-dependent activation and
translocation of PKC that are temporarily associated with agonist-mediated endothelial cell contraction and increased in endothelial permeability. PMA and
-thrombin induce
phosphorylation of the cytoskeletal protein actinin, the
calmodulin-binding protein caldesmon77, and the
intermediate filament protein vimentin. The inhibition of PKC
prevents the
-thrombin- and PMA-induced phosphorylation of the above
cytoskeletal proteins, attenuates the cell contraction, and reduces the
increase in endothelial permeability (57, 168). In human
umbilical vein endothelial cells, thrombin increases intracellular Ca2+ concentration
([Ca2+]i), endothelial permeability, and
activation of PKC-
and causes alterations in vascular
endothelial (VE)-cadherin junctions (153). The
thrombin-induced alteration in VE-cadherin junctions occurred in
association with actin stress fiber formation and 20-kDa myosin light
chain (MLC20) phosphorylation. Inhibition of PKC prevented the disruption of VE-cadherin and the increase in endothelial permeability caused by the thrombin. This supports the notion that the
permeability response to thrombin is mediated by PKC-induced cell
contraction (via actin/myosin) activity and cell-cell adherence (via
VE-cadherins) (Fig. 3). However, in human umbilical vein endothelial
cells, thapsigargin-induced discontinuities in VE-cadherin junctions
occurred without formation of actin stress fibers and phosphorylation of the MLC20 (153).
The inhibition of PKC prevented the disruption of
VE-cadherin and the increase in endothelial permeability in response to
thapsigargin. These results suggest that PKC-mediated barrier
dysfunction occurs via MLC-dependent and -independent mechanisms that
depend on the agonist used (Fig. 3).
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PARACELLULAR TARGETS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS |
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The increase in endothelial permeability that occurs in response
to PMA-induced PKC activation is associated with disruption of
endothelial cell monolayer integrity manifested as endothelial contraction and intercellular gap formation (63, 185). The endothelial transport of solutes and fluids occurs via transcellular and paracellular pathways. The paracellular pathways are regulated by
intercellular junctional proteins [i.e., junctional adhesion molecules
(JAM)] (11, 36, 37, 135), claudins (178),
occludins (127), and the transmembrane adhesion junction
proteins cadherins (e.g., VE-cadherin) (36, 37).
JAM regulates tight junctions and is also implicated in the processes
of neutrophil and monocyte transendothelial migration, because it is
constitutively expressed on circulating monocytes, neutrophils,
lymphocytes subsets, and platelets (36, 37, 135). In
epithelial cells from mucosal tissue sampled from patients with
inflammatory bowel disease, there was downregulation of occludin,
zonula occludens (ZO)-1, claudin-1, JAM, -catenin, and E-cadherin
primarily in regions of actively transmigrating PMN
(92). Thus adherent/migrating leukocytes can
directly modify junctional proteins, which may contribute to
endothelial barrier dysfunction. The vascular endothelial junction-associated molecule is prominently expressed on high endothelial venules and is present on the endothelia of other vessels
(135). Claudins are components of the tight junctional complex in endothelial cells. In endothelial cells, claudin-5 is
expressed more than claudin-1 and -2. A role for PKC in the expression
of tight junction proteins is noted in the choroid plexus, because PMA
increased immunoreactivity of claudin-1 and reduced immunoreactivity of
claudin-2 and -5 (98). The phosphorylation state of
occludin is thought to be important in both tight junction assembly and
regulation. Clarke et al. (28) have shown that PKC
activation leads to dephosphorylation of occludin and increases in
permeability in the epithelial cell line LLC-PK1. In epithelial cells,
PMA-induced PKC activation results in decreases in threonine phosphorylation of occludin, which correlated closely with the rapid
decreases in transepithelial electrical resistance, indicating a role
of a serine/threonine phosphatase in response to PKC activation (Fig.
3).
The adhesion of cells at adherence junctions is also achieved through the calcium-dependent homotypic interaction of cadherins (Fig. 3). The cadherins are associated with the cytoplasmic catenins 120(cas)/p120(ctn) and the splice variant p100. Endothelial cells have PKC-dependent and -independent pathways that regulate the serine/threonine phosphorylation of p120/p100, further demonstrating a connection between PKC and cadherins in endothelial cells (148). In human dermal microvascular endothelial cells exposed to hypoxia/aglycemia, increases in endothelial permeability were associated with significant decreases in the concentrations of occludin and cadherin. The hypoxia/aglycemia-mediated permeability changes and decreases in junctional proteins were blocked by chelation of intracellular Ca2+ and by inhibition of PKC, PKG, and p38 MAPK. Thus the altered permeability may occur through PKC-, PKG-, MAPK-, and Ca2+-mediated dissociation of cadherin-actin and occludin-actin junctional bonds (136). Angiopoietin-1, a ligand for the endothelium-specific tyrosine kinase receptor Tie-2 (35), supports junctional localization of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreases the phosphorylation of PECAM-1 and VE-cadherin. Angiopoietin-1 induces tightening of the endothelial junctions, thereby reducing endothelial permeability. Angiopoietin-1 inhibits thrombin- and VEGF-induced increases in endothelial permeability (56). Thus PKC-mediated phosphorylation of tight and adherence-junctional proteins can contribute to increased endothelial permeability (Fig. 3).
Cell-matrix adherence associated with focal adhesion plaques and
protease activity is also influenced by PKC-mediated events (Fig. 3).
Stimulation of coronary venular endothelial cells with PMA enhanced
tyrosine phosphorylation of paxillin and focal adhesion kinase
(p125FAK), suggesting a possible involvement of protein
tyrosine kinases and their associated focal adhesion plaques in the
control of PMA-induced endothelial barrier dysfunction (Fig. 3)
(193). PMA treatment of primary human umbilical vein
endothelial cells and the transformed endothelial cell line ECV304
induces increased expression (at the level of the promoter, mRNA, and
protein activity) of matrix metalloproteinase-9 (MMP-9) (Fig.
4). MMP-9 degrades native type IV
collagen and is implicated in barrier dysfunction, decreased
cell-matrix adhesion, and angiogensesis (60).
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PKC IN THROMBIN-INDUCED LUNG ENDOTHELIAL INJURY |
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Thrombin is a serine protease that was initially described as a procoagulant because it activates platelets and catalyzes the conversion of fibrinogen to fibrin (51), yet thrombin also has direct effects on endothelial cells because of its specific binding to the protease-activated receptor (PAR) on the endothelium (185). The thrombin receptor has seven transmembrane domains composed of an extracellular NH2 terminus, three extracellular loops, three intracellular loops, and an intracellular COOH terminus. The thrombin receptor is coupled to the pertussis toxin-sensitive and -insensitive heterotrimeric G proteins Gi and Gq. PAR is activated by thrombin-mediated cleavage of the NH2 terminus between Arg41 and Ser42, generating a new NH2 terminus beginning at Ser42 that functions as a "tethered ligand" as a result of its binding to specific sites on the PAR receptor (185).
The thrombin-PAR paradigm is implicated in endothelial injury and the
formation of pulmonary edema in diseases such as sepsis (70,
174), RDS (109, 110, 112, 139), and pulmonary
emboli (78, 109, 110, 112). Thrombin challenge in vivo
causes lung edema by increasing Pmv and i
with a decreased
. In the isolated lung, studies confirm the in vivo
data and also indicate an increase in Kfc. In
morphological studies of the dog and sheep, thrombin produces focal
disruption of the microvascular endothelium associated with
intravascular fibrin and PMN sequestration and increased pulmonary
vascular permeability to proteins (110-112, 120). In
bovine pulmonary arterial endothelial cell monolayers,
-thrombin
causes a dose-dependent increase in endothelial permeability to albumin (6, 7, 58, 59, 102, 145, 153). Interestingly,
-thrombin, which lacks the fibrinogen recognition site, also causes
a dose-dependent increase in endothelial permeability to albumin
(6, 39, 174). In bovine pulmonary arterial endothelial
cell monolayers, the
-thrombin-active catalytic site is required for
the increase in transendothelial permeability to albumin
(7).
There are many postulated mechanisms for the thrombin-induced effect on lung edema (111, 114). With regard to our discussion specific to endothelial function, thrombin causes an increase in cytosolic [Ca2+]i, which is a critical participant in intracellular signaling (102, 103). The increase in [Ca2+]i is connected to important downstream effects implicated in regulation of endothelial permeability to protein such as: 1) phospholipase activation, 2) generation of prostaglandin, leukotrienes, reactive oxygen species, and reactive nitrogen species, and 3) cell contraction (Fig. 2). Specifically, thrombin activates endothelial cell phosphatidylinositol-specific PLC that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 hydrolysis results in generation of the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol. 1,4,5-Triphosphate regulates intracellular [Ca2+]i by mobilizing the release of Ca2+ from internal cellular stores. In addition, the phosphorylated metabolite of IP3 inositol 1,3,4,5-tetrakisphosphate may stimulate external Ca2+ entry into the cell. The increase in [Ca2+]i and diacylglycerol are known to activate PKC (Fig. 1) (130).
PKC participates in -thrombin-induced increases in endothelial
permeability (7, 105, 119, 153, 168, 187).
-Thrombin induces a rapid and dose-dependent translocation of PKC activity from
the cytosol to the membrane, as assessed by
-[32P]ATP
phosphorylation of H1 histone in bovine pulmonary arterial endothelial
cell monolayers. Thrombin-induced PKC activation is temporally
associated with endothelial cell contraction demonstrated by changes in
cell morphology, similar at least in part to the effects of PMA
described above (168). It is also possible that the
-thrombin-induced increase in endothelial permeability occurs independently of PLC activation and increased
[Ca2+]i, despite the fact that the
-thrombin-induced increase in endothelial permeability requires a
PKC-dependent pathway (7). In similar studies,
inhibition of PKC activity prevents
-thrombin-induced phosphorylation of the cytoskeletal protein caldesmon77 and
the intermediate filament vimentin and attenuates the endothelial cell
contraction as indicated above (168). In other studies,
-thrombin induced a PKC-
-dependent increase in stress fibers, a
disruption of junctional VE-cadherin, and a decrease in TEER (153). Again, these results demonstrate that
-thrombin-induced PKC activity results in alteration of the
cytoskeleton, an event resulting in endothelial barrier dysfunction
(168) (Fig. 3). The phosphorylation of GDI and
Rho-dependent endothelial barrier dysfunction is a potential mechanism
for the effect of
-thrombin-induced activation of PKC-
as seen
with PMA (Fig. 3) (119). In the same study
(119), PKC-
did not have a role in the
-thrombin-induced increase in endothelial permeability, indicating a
PKC isotype-specific role in response to
-thrombin. Interestingly, a
role for another PKC isotype is indicated in the response to
-thrombin in endothelial cells. In a human dermal microvascular
endothelial cell line (HMEC-1), PKC-
1 downregulates the
-thrombin
receptor and suppresses the increase in endothelial permeability in
response to
-thrombin (187). HMEC-1 transduced with
full-length PKC-
1 AS cDNA or control pLNCX vector created the stable
cell lines HMEC-AS and HMEC-pLNCX, respectively. In the HMEC-AS, there
was expression of the AS-PKC-
1 transcript and a decreased PKC-
1
protein level without a change in PKC-
or PKC-
. The baseline
endothelial permeability of the HMEC-1, HMEC-pLNCX, and HMEC-AS were
comparable.
-Thrombin induced a similar increase in permeability in
HMEC-1 and HMEC-pLNCX. In contrast,
-thrombin stimulation of HMEC-AS
enhanced the increase in endothelial permeability compared with HMEC-1
and HMEC-pLNCX (187). Thus PKC-
1, via a negative
feedback loop, modulates endothelial monolayer injury in response to
-thrombin (187). However, the role of PKC-
1 changes
with the mediator used to alter the endothelial function, because
overexpression of the PKC-
1 isotype augments the increase in
endothelial permeability in response to PMA (as indicated above), as
opposed to the suppressive role of PKC-
1 during the response to
-thrombin (126).
In addition to phosphorylation events, the action of phosphatases has
an impact on the response to PKC activation. Specifically, the
serine/threonine protein phosphatases (PPs), including PP1, PP2A, and
PP2B, are implicated in PKC-mediated endothelial injury (105). Bovine pulmonary microvessel endothelial cells
express three major PPs: PP1, PP2A, and PP2B (105).
Inhibition of PP2B, but not of PP1 and PP2A, potentiated
-thrombin-induced increases in PKC-
activity but not PKC-
activity. The inhibition of PP2B prevented normalization of the
thrombin-induced decrease in TEER; therefore, PP2B, via its effect
on PKC-
activity, has a role in restoring thrombin-induced
endothelial barrier dysfunction, i.e., thrombin
PP2a activity
PKC-
activity (Fig. 3) (105).
![]() |
PKC IN TNF-![]() |
---|
TNF- is a mediator of sepsis syndrome and RDS (13, 81,
144). In in vivo models of pulmonary injury, TNF-
causes an increase in pulmonary vascular resistance (PVR), Ppa,
Ppc, and Kfc and a decrease in
(54, 69, 81). Notably, TNF-
causes an increase in
pulmonary vascular permeability in vivo (81), in the
isolated lung (69, 159), and in bovine pulmonary arterial endothelial cell (52) and pulmonary microvessel
endothelial cell monolayers (53, 62).
Earlier studies of TNF--induced modulation of signaling pathways
indicate that TNF-
induces activation of PKC-
and/or PKC-
isotypes in pulmonary artery endothelium in vitro (55). In
vivo treatment of guinea pigs with TNF-
induces pulmonary edema,
increases PVR, Ppc, and
· O
in regulating endothelial
permeability, because TNF-
-induced activity of PKC-
(i.e., assay
of translocation and phosphorylation) and AS to PKC-
prevented the
effects of TNF-
. PKC-
and the catalytically active degradation
product of PKC-
, PKM-
, were associated with the endothelial cell
cytoskeletal fraction, supporting the notion that prolonged endothelial
barrier dysfunction in response to TNF-
is dependent on the
interaction of PKC-
with its cytoskeletal targets (52).
It is not clear what the mechanisms are for activation of PKC- nor
the identity of the targets for activated PKC-
and PKM-
during
increased endothelial permeability in response to TNF-
. TNF-
has
been shown to bind to the TNF receptor p55 and activate phospholipase C
(PLC) via the activity of the TNF receptor-associated death domain
(52). Theoretically, activation of PLC will
generate diacylglycerol and IP3, resulting in release of
calcium from the sarcoplasmic reticulum and activation of PKC-
by
binding of diacylglycerol and calcium to the regulatory domains of
PKC-
, similar to
-thrombin. As previously mentioned, PKC is known
to phosphorylate substrates critical for maintenance of the endothelial
cytoskeleton. Goldblum et al. (62) demonstrated that
TNF-
-induced endothelial barrier dysfunction was prevented by
stabilization of actin polymers using phallacidin, supporting a role
for MLC kinase and actin-myosin in TNF-
-induced barrier dysfunction
(62). Petrache et al. (143) showed that
TNF-
significantly increased MLC phosphorylation, stress fiber and
paracellular gap formation, endothelial permeability, and
apoptosis. Yet, the specific pharmacological reduction in MLC
phosphorylation reduced the TNF-
-induced stress fiber formation and
apoptosis but had no effect on TNF-
-induced barrier
dysfunction. These results indicate that TNF-
-induced barrier
dysfunction occurs independently of actin-myosin-mediated contraction,
similar to the studies using thapsigargin. The integration of the
studies of Ferro et al. (52) and Goldblum et al.
(62) would indicate that stabilization of actin fibers
affects junctional proteins and thus the permeability response to
TNF-
-induced PKC-
activation (TNF-
PKC-
activation
actin dynamics
altered junctional proteins
altered cell-cell
adherence
barrier dysfunction) (Fig. 3) (36, 37).
TNF- also causes an increase in the association of PKC-
and
PKM-
with the peripheral membrane (159), similar to the
effect of
-thrombin. PKC phosphorylates membrane-bound
substrates such as p22phox and p47phox
(1, 32, 34, 71). PKC activation mediates TNF-
-induced alterations in the generation of · NO,
ONOO
, and glutathione (79, 144, 159) in the
isolated guinea pig lung and in pulmonary artery and/or microvessel
endothelial cells. Huang and Yuan (74) have shown that
increases in microvascular permeability in response to PMA are mediated
by · NO, and PKC-
/-
isotypes have been
shown to induce transcription of endothelial nitric oxide synthase
(eNOS) in human umbilical vein endothelial cells (96).
Thus, in addition to a direct effect on the cytoskeleton, downstream
targets for PKC-
and PKM-
activation are pathways leading to
generation of reactive nitrogen and oxygen species via membrane-bound
eNOS and NADPH oxidase, respectively. In human pulmonary artery endothelial cells, the PKC isotype needed for TNF-
-induced oxidant generation is probably PKC-
because the AS
oligonucleotide to PKC-
prevents oxidant generation in response to
TNF-
(Fig. 3) (147).
![]() |
PKC IN REACTIVE OXYGEN SPECIES-INDUCED LUNG ENDOTHELIAL INJURY |
---|
Reactive oxygen species are known to affect practically all areas
of cell homeostasis, such as cell division, cell death, DNA chemistry,
lipid membranes, protein oxidation, and expression of mRNA
(41). Thus reactive oxygen species are implicated in the
pathogenesis of cancer, diabetes mellitus, arteriosclerosis, neurological degenerative diseases, rheumatoid arthritis,
ischemia/reperfusion injury, inflammatory bowel disease, and
the MOF associated with shock and trauma (41). However,
reactive oxygen species are also important regulators of constitutive
signaling processes that maintain normal cell function (41,
155). The source of reactive oxygen species in endothelium is
not completely defined and is an area of much interest and debate
(106). The multiple cell types inherent in the lung
provide a multitude of reactive oxygen species generating pathways such
as NAD(P)H oxidase, xanthine oxidase, and oxidative phosphorylation in
mitochondria (17, 30, 62a, 67). Yet in endothelium from
systemic and pulmonary origin, an important role for NADPH oxidase in
· O
The characterization of NAD(P)H oxidase activation in response to
activation of PKC has been mostly studied using PMA, the potent
nonspecific activator of the classic and novel PKC isotypes (2,
10, 30, 32, 34, 97). It is clear that PKC- activates the
NADPH oxidase in PMN (2, 10,30, 32, 34, 97). Surprisingly, there is a lack of data on the isotype(s) of PKC that activate NAD(P)H
oxidase in endothelium. An earlier report by Murphy et al.
(125) showed that TNF-stimulated generation of
· O
vs. PKC-
) and compartmentalization of PKC with NAD(P)H oxidase (e.g., assembly of subcomponents in membrane vs. actin-cytoskeleton) (2, 10, 32, 34, 48, 62a, 71, 97). Finally, the role of PKC isotypes during NAD(P)H oxidase activation will vary with the type of challenge used to cause · O
vs.
-thrombin) (2, 10, 32, 34, 48, 62a, 71, 97).
In addition to endothelium-derived reactive oxygen species, lung injury has been traditionally attributed to reactive oxygen species derived from activated PMN and macrophages (Fig. 1). An important feature of acute inflammation is the increased attachment and emigration of PMN from the blood across postcapillary venules and small veins into the extravascular space (124). The adhesion of PMN to the endothelium is the initial step in the transendothelial emigration of PMN and an important factor contributing to increased endothelial permeability (14, 146, 170, 198). The endothelium expresses PMN adhesion molecules such as selectins and ICAM (61, 176, 199).
PMA has been used increase the endothelial adherence of PMN and
macrophages and to induce activation and generation of
· O
A role for PMN-derived oxidants in lung injury is verified because
H2O2 increases Ppa,
Ppc, lung weight, and Kfc (76,
80) in isolated guinea pig lungs. In isolated rat lungs,
H2O2-induced edema is associated with a
decreased , but with no effect on the permeability-surface area
product, indicating no change in the permeability to water
(190). In isolated perfused rabbit lungs, low
concentrations of H2O2 causes pulmonary edema
by elevating Kfc and Ppc
(31), similar to the isolated guinea pig lung.
In addition to PKC mediating the generation of reactive oxygen species,
the lung injury that occurs in response to reactive oxygen species is
also mediated by PKC (Fig. 4). In isolated guinea pig lungs, perfusion
with low concentrations of H2O2 causes
increases in Ppa, Ppc, lung weight, and
Kfc that are prevented by the PKC inhibitor H7
(68), but not by HA1004 (68, 80). PKC is a primary mediator for reactive oxygen species-induced endothelial barrier dysfunction because H2O2-induced
increases in endothelial permeability to protein are prevented by
inhibitors of PKC activity (87, 164, 165). In human
saphenous vein endothelial cells, during exposure to high
concentrations of H2O2 (10 mM), the expression and regulation of PKC-, PKC-
, and PKC-
isotypes were
investigated. PKC-
and PKC-
but not PKC-
were detected in the
saphenous vein endothelial cell. H2O2 induced
the translocation of PKC-
from the cytosol to the cell membrane and
increased PKC-
content in both cytosol and membrane. In bovine
pulmonary endothelium, H2O2 increases
diacylglycerol and causes PKC activation (171). In bovine
pulmonary microvessel endothelium, H2O2 caused
the translocation of PKC-
isotype from the cytosol to the cell
membrane, indicative of PKC-
activation (164). The PKC
inhibitors H7 (68) and calphostin C (91)
prevented the PKC translocation (164), whereas the
inactive isoquinoline HA1004 had no effect. However, the inhibition of PKC activation only partially reduced the
H2O2-induced increase in endothelial
permeability to 125I-albumin (164), indicating
that PKC activation plays an important role but is not the only
requirement for the H2O2-induced barrier alteration. Thus the downstream effects of H2O2
on barrier function are cell type dependent via PKC-
, PKC-
, and
PKC-
(23).
Similar to -thrombin and TNF-
, a role for the cytoskeleton in the
effect of H2O2-induced PKC activation is
indicated because the endothelial barrier dysfunction is associated
with increased MLC phosphorylation and cell contraction that is blocked
by the PKC inhibitors H7 and staurosporin and by PMA-induced depletion (100). Moreover, in bovine pulmonary arterial
endothelial cell monolayers, PKC inhibitors are able to prevent
H2O2-induced increases in actin stress fibers
and disruption of the peripheral actin band (79).
H2O2 caused the endothelium to enlarge and
become "rounded" compared with the normal cobblestone pattern
(164). Peripheral actin bands are denser in
H2O2-treated monolayers, and interendothelial
gaps are even more prominent (164) than in
-thrombin-induced cytoskeletal changes (145).
Reactive oxygen species have been shown to regulate the behavior of the tight junction proteins occludin and ZO-1 (90). H2O2 caused redistribution of occludin and dissociation of occludin from ZO-1 (90). In cultured mouse lung microvascular endothelial cells, the H2O2-induced loss of vascular integrity is associated with disruption of the endothelial adherence junction VE-cadherin (3, 194). Endothelial cells exposed to H2O2 displayed decreased amounts of VE-cadherin on the cell surface; however, VE-cadherin binding to the cytoskeleton was not diminished by H2O2. Thus H2O2 may decrease adhesive bonds between apposed endothelial cells by sequestration of junctional cadherins contributing to increases in endothelial permeability to protein (3). Rhodamine phalloidin staining reveals that the reactive oxygen species-induced increase in endothelial permeability is also mediated by endothelial cell-substrate adhesion. Exposure of bovine pulmonary arterial endothelial cell monolayers to high concentrations of H2O2 increased the association of the focal adhesive proteins paxillin, talin, and vinculin with the cytoskeleton (4). H2O2 also increased the phosphorylation of an 80-kDa polypeptide, which is probably the cytoskeletal protein myristoylated alanine-rich C kinase substrate, a calmodulin- and actin-binding protein and prominent PKC substrate (64, 195). However, the 80-kDa phosphorylation returned to below basal levels by 30 min after H2O2, indicating that other mechanisms are needed to maintain the endothelial barrier dysfunction and the altered cytoskeleton in response to H2O2 (195).
Other kinases are involved in the effect of reactive oxygen species on endothelial permeability, because H2O2-induced increases in endothelial monolayer permeability are attenuated by inhibition of the nonreceptor tyrosine kinase Src-kinase. H2O2 also caused a rapid decrease in total cellular phosphatase activity that facilitated a compensatory increase in cellular phosphotyrosine residues (88). In bovine pulmonary arterial endothelial cell monolayers, MAPK-p38 but not MAPK-ERK is essential for H2O2-induced increase in endothelial permeability (131). PKC activation induces activation of the MAPK pathway (Fig. 3). In human umbilical vein endothelial cells, MAPK-p38 is involved in the H2O2-mediated increase in endothelial permeability, and this occurs concomitant with stress fiber and intracellular gap formation (89). In the same cell type, H2O2 causes a time-dependent increase in endothelial permeability by MAPK-ERK1/ERK2 signaling pathways (90) (Fig. 3).
The PKC- and MAPK-linked pathways, via the activities of the
transcription factors NF-B and AP-1, have an important role in
regulation of redox-sensitive genes important for endothelial barrier
regulation (Fig. 3) (49). PKC mediates the activation of
NF-
B by · O
B
activation. Moreover, the downregulation of PKC by PMA decreases · O
B
activation (132). In porcine aortic endothelial cells,
H2O2 increases nuclear levels of NF-
B and
AP-1 via the activities of both tyrosine kinases and PKC, because
calphostin C and the tyrosine kinase activity inhibitor
herbimycin A decrease the response to H2O2
(H2O2
PKC and tyrosine kinase
activation
NF-
B and AP-1
gene regulation) (Fig. 4)
(8).
The other important reactive oxygen species, namely · NO, is implicated in PKC-mediated regulation of endothelial barrier function (133). Three different NOS isotypes that generate · NO, which differ in mechanisms of activation, have been characterized (75). The constitutive NOS group includes neuronal NOS and eNOS. The activity of the constitutive NOS isotypes is primarily dependent on Ca2+-calmodulin and the availability of tetrahydrobiopterin, NADPH, L-arginine, and O2 (75). The activation of the constitutive NOS isotypes is classically induced by increases in intracellular Ca2+ levels (75). The activation of pulmonary eNOS is primarily dependent on the calcium-dependent increased binding of calmodulin to the regulatory domains in eNOS (75). In addition, eNOS via its myristoylated sites, is associated with the cell membrane and caveolae where it generates · NO available to extracellular targets (75). The inducible NOS group is iNOS. Activation of iNOS is independent of increased intracellular Ca2+ because of the increased affinity of the cofactor calmodulin for iNOS (75, 175).
· NO has been shown to have diverse effects on
vascular function (75). The classic role of eNOS-derived
· NO is to modulate vascular diameter in
response to vasoactive substances such as acetylcholine
(75) and to decrease the endothelial adherence of
blood-derived formed elements (54). Other studies indicate that · NO (derived from eNOS and/or iNOS) may
mediate autoregulation in response to pressure- and flow-induced
changes in shear stress (75). Also, TNF--induced lung
arterial and microvessel endothelial injury is mediated by eNOS-derived
· NO (53). It has been shown that
selective iNOS inhibitors ameliorate the hemodynamic instability characteristic of experimental sepsis, suggesting that iNOS-derived · NO mediates vascular failure (75, 157,
175). Yet literature reviews generally indicate that during
sepsis the long-term benefits of iNOS inhibition are uncertain and that
further study of eNOS and iNOS is needed (75, 157, 175).
The PKC-dependent mechanisms for eNOS activation in response to TNF-
are not well described. The PKC-mediated phosphorylation of
myristolated arginine-rich C kinase substrate may induce the release of
calmodulin, resulting in increased activity of eNOS (74, 96,
197). eNOS is located in the caveolar membrane microdomain and
is associated with the protein caveolin (74, 96).
Caveolin is known to inhibit eNOS, an effect prevented by
phosphorylation of caveolin by PKC (74, 96). Huang and
Yuan (74) have shown that increases in microvascular
permeability in response to PMA are mediated by
· NO. PKC-
/-
have been shown to induce
transcription of eNOS in HUVEC (96).
![]() |
PERSPECTIVE: THE PKC-POSITIVE FEEDBACK LOOP FOR INJURY |
---|
Thrombin, TNF, and H2O2, our mediator-axis
of lung injury, all cause their effects on the cytoskeletal-adhesive
barrier function via the activity of the PKC paradigm. Yet, as alluded
to above, thrombin, TNF, and H2O2 are all
linked to each other via PKC-dependent pathways. A typical scenario
that probably mediates, in part, lung endothelial injury is depicted in
Fig. 4. TNF increases tissue factor, decreases plasminogen activator,
and increases plasminogen activator inhibitor expression, resulting in
increased thrombin activity (122, 171, 191). Thrombin and
TNF increase PMN activation/endothelial adherence by expression of
ICAM, favoring the activity of reactive oxygen species and proteases
and the release of cytokines. Reactive oxygen species modulate
transcription factors such as NF-B and AP-1, which affect the
expression of many molecules such as TNF, ICAM, and vascular cell
adhesion molecule. Thus the intriguing possibility exists that
there is a PKC-dependent "feed-forward" (positive feedback)
mechanism for pulmonary endothelial dysfunction because, as described
above, thrombin, TNF, and H2O2 all cause PKC-dependent endothelial injury characterized by increased endothelial permeability to protein.
There are targets regulated by PKC that highlight the controversy about
the critical pathways that maintain permeability. It is probable that
the mechanism for PKC-induced barrier dysfunction depends on the
inflammatory ligand used, because MLCK mediates the response to
thrombin but not the barrier dysfunction induced by TNF. It is also
probable that the role for PKC varies with the targeted response,
because PKC- mediates barrier function, and PKC-
mediates the
release of reactive oxygen species. This indicates there is
isotype-specific PKC activation by upstream pathways and specific
targeting mechanisms that are not well understood and need to be
defined. The consequent mediators of lung injury (i.e., proteases,
reactive oxygen/nitrogen species) generated in response to PKC
activation have a prominent role in the response to TNF and thrombin.
It is well established that proteases and reactive oxygen/nitrogen
species can injure the endothelium, but do they also contribute to the
feedback regulation of PKC? Finally, it is known that PKC activation
within the PMN and macrophage causes the generation of proteases and
reactive oxygen/nitrogen species. The important PKC isotypes in the PMN
and macrophage are different compared with the PKC isotypes in the
endothelial cell. It is important to know how to target the PKC
isotypes among the leukocytes and in the endothelial cell. Moreover, we
need to know the specific effects of leukocyte-PKC modulation because of the potential effects on the immune response, in addition to the
modulation of lung injury.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by the Department of Veterans Affairs Medical Research Service Merit Review and National Heart, Lung, and Blood Institute Grant HL-59901-02.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. Johnson, 151 Research Service, 113 Holland Ave., Stratton V. A. Medical Center, Albany, NY 12208 (E-mail: jmurd{at}msn.com).
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.
10.1152/ajplung.00106.2002
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