INVITED REVIEW
Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury

Alma Siflinger-Birnboim and Arnold Johnson

Research Service, Stratton Veterans Affairs Medical Center; and the Center for Cardiovascular Science, The Albany Medical College, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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, alpha -thrombin, tumor necrosis factor (TNF)-alpha , 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-alpha , is a primary supporter for lung endothelial injury in response to alpha -thrombin, TNF-alpha , and reactive oxygen species.

antisense; edema; reactive oxygen species; transcription


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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).


    PULMONARY EDEMA
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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.
Transvascular fluid flux 

= <IT>K</IT><SUB>fc</SUB> [(P<SUB>mv</SUB> − P<SUB>i</SUB>) + &sfgr;(&pgr;<SUB>i</SUB> − &pgr;<SUB>mv</SUB>)]
where Kfc is the capillary filtration coefficient, Pmv is the microvascular hydrostatic pressure, Pi is the interstitial hydrostatic pressure, sigma  is the protein reflection coefficient, pi i is the interstitial oncotic capillary pressure, and pi mv is the microvascular oncotic pressure.

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 sigma  (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 sigma ), 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<UP><SUB>2</SUB><SUP>−</SUP></UP>) and nitric oxide ( · NO)] (Fig. 1) (47, 49, 54, 112, 113, 152, 172, 182). The increase in Pmv is caused by a decrease in the ratio of the precapillary to postcapillary resistance arising from the altered levels of a number of endothelium-dependent mediators that have been implicated in RDS, such as angiotensin II, endothelin, thromboxane A2, prostacyclin, reactive nitrogen species [e.g., · NO, peroxynitrite (ONOO-)], and reactive oxygen species [e.g., hydrogen peroxide (H2O2,), · O<UP><SUB>2</SUB><SUP>−</SUP></UP>, hydroxyl radical ( · OH)] (Fig. 1) (110, 112). The increase in Kfc and the decrease in sigma  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)-alpha (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) right-arrow · NO].


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Fig. 1.   The cell types and mediators involved in the protein kinase C (PKC) paradigm of lung injury. PMN, neutrophils; · NO, nitric oxide; ONOO-, peroxynitrite; · OH, hydroxyl radical.


    DETERMINATION OF ENDOTHELIAL BARRIER FUNCTION
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

The early in vivo study of transvascular fluid flux used time-dependent lung weight measurements during different Pmv and pi mv (172). Then, with the advent of the chronic sheep lung lymph fistula model using the online determination of fluid and protein flux, Ppm, pi pm, and pi i permitted the estimation of sigma  (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 sigma . 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 sigma  (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).


    THE BASICS OF PKC
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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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Fig. 2.   A molecular model for PKC isotypes and PKC activation. C1-C4, conserved domains; V1-V5, variable domains; DAG, diacylglycerol; PSS, NH2-terminal pseudosubstrate; PS, permeability-surface area product; S, substrate.

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-alpha , PKC-beta 1/2, and PKC-gamma 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-epsilon , PKC-delta , PKC-eta , PKC-theta , 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-iota , PKC-lambda , and PKC-zeta 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-alpha activation is also dependent on lack of a C1-C2 domain interaction, corresponding to a transition of PKC-alpha from a closed inactive state to an open active state (Fig. 2). They showed that PKC-alpha isotype bound specifically and with high affinity to an alpha C1A-C1B fusion protein of PKC-alpha . The alpha C1A-C1B domain activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner comparable with activation resulting from membrane-phosphatidylserine association. Interestingly, the alpha C1A-C1B domain also activated the classical PKC-beta 1/2, and PKC-gamma isotypes, but not the novel PKC-delta or PKC-epsilon isotypes that were each activated by their own C1 domains. PKC-alpha , PKC-beta 1/2, and PKC-gamma isotypes were unaffected by the C1 domain of the PKC-delta isotype and only slightly activated by that of PKC-epsilon isotypes. Thus the activation mechanism of the novel PKC isotypes may be similar to that of the classic isotypes. PKC-zeta 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).


    PKC IN LUNG INJURY
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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-alpha ) (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<UP><SUB>2</SUB><SUP>−</SUP></UP>, · OH, · NO, ONOO-) (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-alpha (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-alpha . 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.


    PKC IN PHORBOL ESTER-INDUCED LUNG INJURY
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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 (10-8-10-6 M) increased endothelial permeability to albumin (107). The negative controls 4-alpha -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.


    ISOTYPES OF PKC IN PHORBOL ESTER-INDUCED LUNG INJURY
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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-beta 1 augmented the PMA-induced increase in endothelial permeability. PMA stimulated the translocation of PKC-beta 1 from the cytosol to the membrane, indicating PKC-beta 1 activation (126). In another study, a decrease in PKC-beta 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).


    LEUKOCYTES IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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).


    REACTIVE OXYGEN SPECIES IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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-kappa B and activator protein (AP)-1 (73). The importance of the effect of reactive oxygen species on NF-kappa 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-alpha , 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.


    INTRACELLULAR SIGNAL PATHWAYS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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-alpha -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 right-arrow GDI right-arrow Rho-GDP/GTP right-arrow Rho-GTP right-arrow ROCK right-arrow 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 right-arrow Ras right-arrow Raf-1 right-arrow MEK right-arrow ERK is also involved in endothelial barrier regulation by PMA (185) (Fig. 3).


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Fig. 3.   PKC-mediated activation of signaling pathways that target critical proteins that impact on endothelial barrier function. GDI, guanine nucleotide dissociation inhibitor; ROCK, rho kinase; VE, vascular endothelial; JAM, junctional adhesion molecule; FAK, focal adhesion kinase.


    INTRACELLULAR TARGETS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS
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ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
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ISOTYPES OF PKC IN...
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PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

In bovine pulmonary artery endothelial cell monolayers, PMA and alpha -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 alpha -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 alpha -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-alpha 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).


    PARACELLULAR TARGETS IN PKC-MEDIATED LUNG INJURY IN RESPONSE TO PHORBOL ESTERS
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ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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, beta -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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Fig. 4.   A model for PKC-mediated endothelial cell barrier dysfunction during respiratory distress syndrome (RDS). Sepsis via endotoxin causes the release of TNF-alpha and H2O2, primarily from macrophages and PMN, and the generation of thrombin. The data indicate that PKC-alpha is a major isotype that promotes the vascular derangements associated with the inflammatory mediators known to cause RDS. PKC-beta has a repressive role at least with respect to alpha -thrombin-induced endothelial injury. TNF-alpha , H2O2, and alpha -thrombin activate endothelial PKC that leads to at least 4 downstream pathways as indicated: 1) PKC causes generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), probably via the activities of NADPH oxidase and nitric oxide synthase (NOS), 2) PKC indirectly/directly affects the phosphorylation of cytoskeletal targets, 3) PKC modifies gene expression by altering the activity of transcription factors such as NF-kappa B and activator protein (AP)-1, and 4) PKC can increase activity of matrix metalloproteases such as MMP-9. The "positive-feedback" loop results from increased expression of intercellular adhesion molecules (ICAM), tissue factor (TF), and plasminogen activator inhibitor (PAI) and decreased activity of plasminogen activator (PA). The loop promotes leukocyte sequestration, the generation of alpha -thrombin and TNF-alpha , and formation of ROS and RNS.


    PKC IN THROMBIN-INDUCED LUNG ENDOTHELIAL INJURY
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ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
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ISOTYPES OF PKC IN...
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REACTIVE OXYGEN SPECIES IN...
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PKC IN THROMBIN-INDUCED LUNG...
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PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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 pi i with a decreased sigma . 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, alpha -thrombin causes a dose-dependent increase in endothelial permeability to albumin (6, 7, 58, 59, 102, 145, 153). Interestingly, gamma -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 alpha -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 alpha -thrombin-induced increases in endothelial permeability (7, 105, 119, 153, 168, 187). alpha -Thrombin induces a rapid and dose-dependent translocation of PKC activity from the cytosol to the membrane, as assessed by gamma -[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 alpha -thrombin-induced increase in endothelial permeability occurs independently of PLC activation and increased [Ca2+]i, despite the fact that the alpha -thrombin-induced increase in endothelial permeability requires a PKC-dependent pathway (7). In similar studies, inhibition of PKC activity prevents alpha -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, alpha -thrombin induced a PKC-alpha -dependent increase in stress fibers, a disruption of junctional VE-cadherin, and a decrease in TEER (153). Again, these results demonstrate that alpha -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 alpha -thrombin-induced activation of PKC-alpha as seen with PMA (Fig. 3) (119). In the same study (119), PKC-epsilon did not have a role in the alpha -thrombin-induced increase in endothelial permeability, indicating a PKC isotype-specific role in response to alpha -thrombin. Interestingly, a role for another PKC isotype is indicated in the response to alpha -thrombin in endothelial cells. In a human dermal microvascular endothelial cell line (HMEC-1), PKC-beta 1 downregulates the alpha -thrombin receptor and suppresses the increase in endothelial permeability in response to alpha -thrombin (187). HMEC-1 transduced with full-length PKC-beta 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-beta 1 transcript and a decreased PKC-beta 1 protein level without a change in PKC-alpha or PKC-epsilon . The baseline endothelial permeability of the HMEC-1, HMEC-pLNCX, and HMEC-AS were comparable. alpha -Thrombin induced a similar increase in permeability in HMEC-1 and HMEC-pLNCX. In contrast, alpha -thrombin stimulation of HMEC-AS enhanced the increase in endothelial permeability compared with HMEC-1 and HMEC-pLNCX (187). Thus PKC-beta 1, via a negative feedback loop, modulates endothelial monolayer injury in response to alpha -thrombin (187). However, the role of PKC-beta 1 changes with the mediator used to alter the endothelial function, because overexpression of the PKC-beta 1 isotype augments the increase in endothelial permeability in response to PMA (as indicated above), as opposed to the suppressive role of PKC-beta 1 during the response to alpha -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 alpha -thrombin-induced increases in PKC-alpha activity but not PKC-beta activity. The inhibition of PP2B prevented normalization of the thrombin-induced decrease in TEER; therefore, PP2B, via its effect on PKC-alpha activity, has a role in restoring thrombin-induced endothelial barrier dysfunction, i.e., thrombin right-arrow black-trianglePP2a activity right-arrow black-down-triangle PKC-alpha activity (Fig. 3) (105).


    PKC IN TNF-alpha -INDUCED LUNG ENDOTHELIAL INJURY
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ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
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ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
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INTRACELLULAR TARGETS IN PKC-...
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PKC IN THROMBIN-INDUCED LUNG...
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PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

TNF-alpha is a mediator of sepsis syndrome and RDS (13, 81, 144). In in vivo models of pulmonary injury, TNF-alpha causes an increase in pulmonary vascular resistance (PVR), Ppa, Ppc, and Kfc and a decrease in sigma  (54, 69, 81). Notably, TNF-alpha 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-alpha -induced modulation of signaling pathways indicate that TNF-alpha induces activation of PKC-alpha and/or PKC-beta isotypes in pulmonary artery endothelium in vitro (55). In vivo treatment of guinea pigs with TNF-alpha induces pulmonary edema, increases PVR, Ppc, and · O<UP><SUB>2</SUB><SUP>−</SUP></UP> concentration, and decreases · NO concentration, all of which are prevented by the PKC inhibitor calphostin (159). Moreover, the PKC activation noted in an isolated pulmonary artery segment was also inhibited by calphostin C (159). In bovine pulmonary microvessel endothelial cells, Ferro et al. (52) showed a specific role for PKC-alpha in regulating endothelial permeability, because TNF-alpha -induced activity of PKC-alpha (i.e., assay of translocation and phosphorylation) and AS to PKC-alpha prevented the effects of TNF-alpha . PKC-alpha and the catalytically active degradation product of PKC-alpha , PKM-alpha , were associated with the endothelial cell cytoskeletal fraction, supporting the notion that prolonged endothelial barrier dysfunction in response to TNF-alpha is dependent on the interaction of PKC-alpha with its cytoskeletal targets (52).

It is not clear what the mechanisms are for activation of PKC-alpha nor the identity of the targets for activated PKC-alpha and PKM-alpha during increased endothelial permeability in response to TNF-alpha . TNF-alpha 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-alpha by binding of diacylglycerol and calcium to the regulatory domains of PKC-alpha , similar to alpha -thrombin. As previously mentioned, PKC is known to phosphorylate substrates critical for maintenance of the endothelial cytoskeleton. Goldblum et al. (62) demonstrated that TNF-alpha -induced endothelial barrier dysfunction was prevented by stabilization of actin polymers using phallacidin, supporting a role for MLC kinase and actin-myosin in TNF-alpha -induced barrier dysfunction (62). Petrache et al. (143) showed that TNF-alpha 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-alpha -induced stress fiber formation and apoptosis but had no effect on TNF-alpha -induced barrier dysfunction. These results indicate that TNF-alpha -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-alpha -induced PKC-alpha activation (TNF-alpha right-arrow PKC-alpha activation right-arrow actin dynamics right-arrow altered junctional proteins right-arrow altered cell-cell adherence right-arrow barrier dysfunction) (Fig. 3) (36, 37).

TNF-alpha also causes an increase in the association of PKC-alpha and PKM-alpha with the peripheral membrane (159), similar to the effect of alpha -thrombin. PKC phosphorylates membrane-bound substrates such as p22phox and p47phox (1, 32, 34, 71). PKC activation mediates TNF-alpha -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-alpha /-epsilon 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-alpha and PKM-alpha 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-alpha -induced oxidant generation is probably PKC-zeta because the AS oligonucleotide to PKC-zeta prevents oxidant generation in response to TNF-alpha (Fig. 3) (147).


    PKC IN REACTIVE OXYGEN SPECIES-INDUCED LUNG ENDOTHELIAL INJURY
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ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
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ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP> generation is indicated (5, 10, 48, 62a, 69, 71, 97, 200). Endothelial oxidase can produce substantial · O<UP><SUB>2</SUB><SUP>−</SUP></UP> using either NADPH or NADH [i.e., NAD(P)H oxidase], as opposed to PMN oxidase, which uses primarily NADPH (10, 71).

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-beta 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<UP><SUB>2</SUB><SUP>−</SUP></UP> was not prevented by the PKC inhibitor staurosporin in rat pulmonary arterial endothelial cells. The activation of NAD(P)H oxidase and generation of · O<UP><SUB>2</SUB><SUP>−</SUP></UP> must be strictly regulated because of the myriad effects of reactive oxygen species; therefore, the interaction of PKC with NAD(P)H oxidase should also be strictly controlled. As an example, mechanisms that govern the interaction of PKC with NAD(P)H oxidase include isotype-specific activation of PKC (e.g., PKC-alpha vs. PKC-zeta ) 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<UP><SUB>2</SUB><SUP>−</SUP></UP> generation (e.g., PMA vs. TNF-alpha vs. alpha -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<UP><SUB>2</SUB><SUP>−</SUP></UP>, · NO, and H2O2 in models of leukocyte-derived, oxidant-dependent lung injury. In rats, PMA-induced lung injury is inhibited by catalase, but not SOD, indicating that H2O2 mediates the effect rather than · O<UP><SUB>2</SUB><SUP>−</SUP></UP> (83). The reduction of PMN-endothelium adherence protects against PMN-dependent edema in isolated lungs, because experiments indicate that the stimulated PMN needs close apposition to the endothelial cell to cause reactive oxygen species-dependent endothelial barrier dysfunction (160). Interestingly, to verify whether PMN-derived oxidants are responsible for increases in endothelial permeability, studies were done using PMN from normal donors (nl-PMN) and donors with X chromosome-linked chronic granulomatous disease (CGD-PMN) (85, 160). CGD-PMN lack the membrane-bound NADPH oxidase activity needed to initiate the respiratory burst response and release of reactive oxygen species (16, 33, 158). PMA induced activation of endothelium-adherent nl-PMN and increased endothelial permeability to albumin. However, PMA treatment of CGD-PMN does not increase endothelial permeability to albumin, demonstrating that endothelial barrier dysfunction is mediated by PMN-derived, PKC-induced, NADPH oxidase-generated reactive oxygen species (85, 160). In PMN-bovine pulmonary microvessel endothelial cell coculture studies, H2O2 mediates the increase in endothelial permeability because catalase and glutathione prevent the barrier dysfunction in response to PMN activation (166).

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 sigma , 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-alpha , PKC-epsilon , and PKC-zeta isotypes were investigated. PKC-alpha and PKC-epsilon but not PKC-zeta were detected in the saphenous vein endothelial cell. H2O2 induced the translocation of PKC-alpha from the cytosol to the cell membrane and increased PKC-epsilon 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-beta isotype from the cytosol to the cell membrane, indicative of PKC-beta 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-alpha , PKC-beta , and PKC-epsilon (23).

Similar to alpha -thrombin and TNF-alpha , 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 alpha -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-kappa 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-kappa B by · O<UP><SUB>2</SUB><SUP>−</SUP></UP> because the PKC inhibitors calphostin C and chelerythrine chloride inhibit · O<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced NF-kappa B activation. Moreover, the downregulation of PKC by PMA decreases · O<UP><SUB>2</SUB><SUP>−</SUP></UP>-induced NF-kappa B activation (132). In porcine aortic endothelial cells, H2O2 increases nuclear levels of NF-kappa 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 right-arrow PKC and tyrosine kinase activation right-arrow NF-kappa B and AP-1 right-arrow 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-alpha -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-alpha 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-alpha /-epsilon have been shown to induce transcription of eNOS in HUVEC (96).


    PERSPECTIVE: THE PKC-POSITIVE FEEDBACK LOOP FOR INJURY
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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-kappa 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-alpha mediates barrier function, and PKC-zeta 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


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PULMONARY EDEMA
DETERMINATION OF ENDOTHELIAL...
THE BASICS OF PKC
PKC IN LUNG INJURY
PKC IN PHORBOL ESTER-INDUCED...
ISOTYPES OF PKC IN...
LEUKOCYTES IN PKC-MEDIATED LUNG...
REACTIVE OXYGEN SPECIES IN...
INTRACELLULAR SIGNAL PATHWAYS...
INTRACELLULAR TARGETS IN PKC-...
PARACELLULAR TARGETS IN PKC-...
PKC IN THROMBIN-INDUCED LUNG...
PKC IN TNF-alpha -INDUCED LUNG...
PKC IN REACTIVE OXYGEN...
PERSPECTIVE: THE PKC-POSITIVE...
REFERENCES

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