Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

Type-IIA secreted phospholipase A2 is an endogenous antibiotic-like protein of the host

Type-IIA secreted phospholipase A₂ (sPLA₂-IIA) has been proposed to play a role in the development of inflammatory diseases. It has been shown to release arachidonic acid, the precursor of proinflammatory eicosanoids, to hydrolyze phospholipids of pulmonary surfactant, and to bind to specific receptors located on cell surface membranes. However, the most established biological role of sPLA₂-IIA is related to its potent bactericidal property in particular toward Gram-positive bacteria. This enzyme is present in animal and human biological fluids at concentrations sufficient to kill bacteria. Human recombinant sPLA₂-IIA is able to kill Gram-positive bacteria at concentrations as low as 1.1 ng/ml. This remarkable property is due to the unique preference of sPLA₂-IIA for anionic phospholipids such as phosphatidylglycerol, the main phospholipid component of bacterial membranes. Much higher concentrations of sPLA₂-IIA are required for its action on host cell membranes and surfactant both of which are mainly composed by phosphatidylcholine, a poor substrate for sPLA₂-IIA. Transgenic mice over-expressing human sPLA₂-IIA are resistant to infection by Staphylococcus aureus, Escherichia coli, and Bacillus anthracis, the etiological agent of anthrax. Conversely, certain bacteria, such as B. anthracis, E. coli and Bordetella pertussis are able to inhibit sPLA₂-IIA expression by host cells, thus highlighting a mechanism by which these bacteria can subvert the host immune system. Intranasal instillation of recombinant sPLA₂-IIA protects mice from mortality caused by pulmonary anthrax. Interestingly, this protective effect was obtained even with B. anthracis strains that down-regulate the expression of endogenous sPLA₂-IIA, indicating that instilled sPLA₂-IIA can overcome the subversive action of B. anthracis. We conclude that sPLA₂-IIA is an efficient endogenous antibiotic of the host and can play a role in host defense against pathogenic bacteria. It can be used as a therapeutic agent in adjunct with current therapy to treat bacteria resistant to multiple antibiotics.     

Induction of distinct sets of secretory phospholipase A2 in rodents during inflammation

Although the expression of the prototypic secretory phospholipase A₂ (sPLA₂), group IIA (sPLA₂-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA₂ enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA₂s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA₂-V, and to a lesser extent that of sPLA₂-IID, -IIE, and -IIF, were increased, whereas that of sPLA₂-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA₂-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA₂-IIA inhibitor that turned out to inhibit sPLA₂-IID, -IIE, -V and -X as well. In contrast, sPLA₂-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA₂s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation.     

Modulation of Ca2+ mobilization by protein kinase C in the submandibular duct cell line A253

The expression of protein kinase C (PKC) isoforms and the modulation of Ca²⁺ mobilization by PKC were investigated in the human submandibular duct cell line A253. Three new PKC (nPKC) isoforms (, , and ) and one atypical PKC (aPKC) isoform () are expressed in this cell line. No classical PKC (cPKC) isoforms were present. The effects of the PKC activator phorbol 12-myristate-13-acetate (PMA) and of the PKC inhibitors calphostin C (CC) and bisindolymaleimide I (BSM) on inositol 1,4,5-trisphosphate (IP₃) and Ca²⁺ responses to ATP and to thapsigargin (TG) were investigated. Pre-exposure to PMA inhibited IP₃ formation, Ca²⁺ release and Ca²⁺ influx in response to ATP. Pre-exposure to CC or BSM slightly enhanced IP₃ formation but inhibited the Ca²⁺ release and the Ca²⁺ influx induced by ATP. In contrast, pre-exposure to PMA did not modify the Ca²⁺ release induced by TG, but reduced the influx of Ca²⁺ seen in the presence of this Ca²⁺-ATPase inhibitor. These results suggest that PKC modulates elements of the IP₃/Ca²⁺ signal transduction pathway in A253 cells by (1) inhibiting phosphatidylinositol turnover and altering the sensitivity of the Ca²⁺ channels to IP₃, (2) altering the activity, the sensitivity to inhibitors, or the distribution of the TG-sensitive Ca²⁺ ATPase, and (3) modulating Ca²⁺ entry pathways.     

Roles of secreted phospholipase A2 group IIA in inflammation and host defense

Among all members of the secreted phospholipase A₂ (sPLA₂) family, group IIA sPLA₂ (sPLA₂-IIA) is possibly the most studied enzyme. Since its discovery, many names have been associated with sPLA₂-IIA, such as “non-pancreatic”, “synovial”, “platelet-type”, “inflammatory”, and “bactericidal” sPLA_2. Whereas the different designations indicate comprehensive functions or sources proposed for this enzyme, the identification of the precise roles of sPLA₂-IIA has remained a challenge. This can be attributed to: the expression of the enzyme by various cells of different lineages, its limited activity towards the membranes of immune cells despite its expression following common inflammatory stimuli, its ability to interact with certain proteins independently of its catalytic activity, and its absence from multiple commonly used mouse models. Nevertheless, elevated levels of the enzyme during inflammatory processes and associated consistent release of arachidonic acid from the membrane of extracellular vesicles suggest that sPLA₂-IIA may contribute to inflammation by using endogenous substrates in the extracellular milieu. Moreover, the remarkable potency of sPLA₂-IIA towards bacterial membranes and its induced expression during the course of infections point to a role for this enzyme in the defense of the host against invading pathogens. In this review, we present current knowledge related to mammalian sPLA₂-IIA and its roles in sterile inflammation and host defense.     

Inhibition of type 2A secretory phospholipase A2 reduces death of cardiomyocytes in acute myocardial infarction

During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A₂ (sPLA₂-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA₂-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA₂-IIA activity and reduced infarct size (reduction 73 ± 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA₂-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA₂-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA₂-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA₂-IIA independent manner. A. van Dijk and P. A. J. Krijnen have contributed equally to the study.     

Inhibition of sPLA2-IIA Prevents LPS-Induced Neuroinflammation by Suppressing ERK1/2-cPLA2α Pathway in Mice Cerebral Cortex

Neuroinflammation is involved in various central nervous system (CNS) disorders, including brain infections, ischemia, trauma, stroke, and degenerative CNS diseases. In the CNS inflammation, secretory phospholipase A₂-IIA (sPLA₂-IIA) acts as a mediator, resulting in the generation of the precursors of pro-inflammatory lipid mediators, such as prostaglandins (PGs) and leukotrienes (LTs). However, the role of sPLA₂-IIA in neuroinflammation is more complicated and remains unclear yet. In the present study, we investigated the effect of sPLA₂-IIA inhibition by specific inhibitor SC-215 on the inflammation in LPS-induced mice cerebral cortex and primary astrocytes. Our results showed that the inhibition of sPLA₂-IIA alleviated the release of PGE₂ by suppressing the activation of ERK1/2, cPLA₂α, COX-2 and mPGES-1. These findings demonstrated that sPLA₂-IIA showed the potential to regulate the neuroinflammation in vivo and in vitro, indicating that sPLA₂-IIA might be a novel target for the treatment of acute neuroinflammation.     

Impaired phospholipases A2 production by stimulated macrophages from patients with acute respiratory distress syndrome

The aim of this study was to investigate whether early phase of acute respiratory distress syndrome (ARDS) is associated with changes in immune response, either systemic or localized to the lung. ARDS and control mechanically ventilated patients, as well as healthy volunteers were studied. Alveolar macrophages (AMΦ) and blood monocytes (BM) were treated ex vivo with lipopolysaccharide (LPS), interferon-γ (IFNγ), and surfactant. Phospholipase A₂ (PLA₂) activity and TLR4 expression were evaluated as markers of cell response. AMΦ from ARDS patients did not respond upon treatment with either LPS or IFN-γ by inducing PLA₂ production. On the contrary, upon stimulation, in control patients the intracellular PLA₂, (mainly cPLA₂) levels were increased, but secretion of PLA₂ (mainly sPLA₂-IIA) was observed only after treatment with LPS. Surfactant suppressed PLA₂ production in cells from both groups of patients. Increased relative changes of total PLA₂ activity and an upregulation of TLR4 expression upon stimulation was observed in BM from primary ARDS, control patients and healthy volunteers. In BM from secondary ARDS patients, however, no PLA₂ induction was observed, with a concomitant down-regulation of TLR4 expression. Cytosolic PLA₂, its activated form, p-cPLA_2, and sPLA₂-IIA were the predominant PLA₂ types within the cells, while extracellularly only sPLA₂-IIA was identified. These results support the concept of down-regulated innate immunity in early ARDS that is compartmentalized in primary and systemic in secondary ARDS. PLA₂ isoforms could serve as markers of the immunity status in ARDS. Finally, our data highlight the role of surfactant in controlling inflammation.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Selective and transient activation of protein kinase C alpha by fumonisin B1, a ceramide synthase inhibitor mycotoxin, in cultured porcine renal cells

Fumonisin B₁ (FB₁), a potent and naturally occurring mycotoxin produced by the fungus Fusarium verticillioides, has been implicated in fatal and debilitating diseases in animals and humans. FB₁ affects a variety of cell signaling proteins including protein kinase C (PKC); a serine/threonine kinase, involved in a number of signal transduction pathways that include cytokine induction, carcinogenesis and apoptosis. The aim of this study was to investigate the short-term temporal and concentration-dependent effects of FB₁ on PKC isoforms present in LLC-PK₁ cells in relation to the FB₁-induced accumulation of sphinganine and sphingosine utilizing various inhibitors and activators. Our studies demonstrated that FB₁ (0.1-1 μM) selectively and transiently activated PKCα at 5 min, without affecting PKC-δ, -ε and -ζ isoforms. At higher FB₁ concentrations and later time points (15-120 min), PKCα membrane concentrations declined to untreated levels. The observed increase in cytosol PKCα protein expression at 15 min was not associated with an increase in its activity or protein biosynthesis. Calphostin C, a PKC inhibitor, abrogated the FB₁-induced translocation of PKCα. Pre-incubation with the PKC activator, phorbol 12-myristate 13-acetate, resulted in an additive effect on membrane translocation of PKCα. Intracellular sphinganine and sphingosine concentrations were unaltered at the time points tested. Myriocin, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, did not prevent the FB₁-induced PKCα cytosol to membrane redistribution. Altering PKCα and its signal transduction pathways may be of importance in the ability of FB₁ to exert its toxicity via apoptosis and/or carcinogenesis.     

PKCι counteracts oxidative stress by regulating Hsc70 in an esophageal cancer cell line

Using a glutathione S-transferase pull-down liquid chromatography–coupled tandem mass spectrometry approach and immunoprecipitation/immunoblot analysis, we found that heat shock cognate protein 70 (Hsc70) was involved in the complex formed by atypical protein kinase Cι (PKCι) and LC3 in the esophageal cancer cell line KYSE30. Further study indicated that Hsc70 was targeted by autophagic degradation, and knockdown of PKCι down-regulated Hsc70 by promoting autophagy. PKCι knockdown sensitized cells to oxidative stress-induced apoptosis, whereas forced PKCι expression counteracted the oxidative stress-induced apoptosis via Hsc70.     

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidines as atypical protein kinase C inhibitors to control retinal vascular permeability and cytokine-induced edema

Studies demonstrate that small molecule targeting of atypical protein kinase C (aPKC) may provide an effective means to control vascular permeability, prevent edema, and reduce inflammation providing novel and important alternatives to anti-VEGF therapies for certain blinding eye diseases. Based on a literature tricyclic thieno[2,3-d]pyrimidine lead (1), an ATP-competitive inhibitor of the aPKC iota (ι) and aPKC zeta (ζ) isoforms, we have synthesized a small series of compounds in 1–2 steps from a readily available chloro intermediate. A single pyridine congener was also made using 2D NMR to assign regiochemistry. Within the parent pyrimidine series, a range of potencies was observed against aPKCζ whereas the pyridine congener was inactive. Selected compounds were also tested for their effect toward VEGF-induced permeability in BREC cells. The most potent of these (7l) was further assayed against the aPKCι isoform and showed a favorable selectivity profile against a panel of 31 kinases, including kinases from the AGC superfamily, with a focus on PKC isoforms and kinases previously shown to affect permeability. Further testing of 7l in a luciferase assay in HEK293 cells showed an ability to prevent TNF-α induced NFκB activation while not having any effect on cell survival. Intravitreal administration of 7l to the eye yielded a complete reduction in permeability in a test to determine whether the compound could block VEGF- and TNFα-induced permeability across the retinal vasculature in a rat model. The compound in mice displayed good microsomal stability and in plasma moderate exposure (AUC and C_max), low clearance, a long half-life and high oral bioavailability. With IV dosing, higher levels were observed in the brain and eye relative to plasma, with highest levels in the eye by either IV or PO dosing. With a slow oral absorption profile, 7l accumulates in the eye to maintain a high concentration after dosing with higher levels than in plasma. Compound 7l may represent a class of aPKC inhibitors for further investigation.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²⁺ signalling. In HEK293 and Jurkat cells, the Ca²⁺ release and Ca²⁺ uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²⁺ responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²⁺ concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²⁺ flux.     

Group IID,IIE, IIF and III secreted phospholipase A2s

Among the 11 members of the secreted phospholipase A₂ (sPLA₂) family, group IID, IIE, IIF and III sPLA₂s (sPLA₂-IID, -IIE, -IIF and -III, respectively) are “new” isoforms in the history of sPLA₂ research. Relative to the better characterized sPLA₂s (sPLA₂-IB, -IIA, -V and -X), the enzymatic properties, distributions, and functions of these “new” sPLA₂s have remained obscure until recently. Our current studies using knockout and transgenic mice for a nearly full set of sPLA₂s, in combination with comprehensive lipidomics, have revealed unique and distinct roles of these “new” sPLA₂s in specific biological events. Thus, sPLA₂-IID is involved in immune suppression, sPLA₂-IIE in metabolic regulation and hair follicle homeostasis, sPLA₂-IIF in epidermal hyperplasia, and sPLA₂-III in male reproduction, anaphylaxis, colonic diseases, and possibly atherosclerosis. In this article, we overview current understanding of the properties and functions of these sPLA₂s and their underlying lipid pathways in vivo.