Berit Johansen

Localization of Nonpancreatic Secretory Phospholipase A2 in Normal and Atherosclerotic Arteries: Activity of the Isolated Enzyme on Low-Density Lipoproteins

Secretory nonpancreatic type II phospholipase A2 (snpPLA2) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA2 in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA2 in a variety of normal tissues suggests that snpPLA2 may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA2 localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA2 immunoreactivity was observed in the arterial media that was colocalized with alpha-actin-positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA2 was observed colocalized with CD68-positive macrophages and HHF-35-positive SMCs and extracellularly in the lipid core. snpPLA2 was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA2 had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA2: (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA2 was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA2 is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA2 can release proinflammatory lipids at places of LDL deposition in the arterial wall.

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate‐limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short‐chain ceramide analogs for 30 and 60 min leads to a concentration‐dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen‐activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide‐induced arachidonic acid release. By use of photoactivatable ceramide analogs, D‐ and L‐ [125I]3‐trifluoromethyl‐3‐(m‐iodophenyl)diazirineceramides (TID‐ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D‐ and L‐TID‐ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting ‘hinge’ domain of cPLA2, efficiently bound D‐ and L‐TID‐ceramides, whereas the catalytic domain did not interact with TID‐ceramides. In vitro binding assays reveal that stearoyl‐arachidonyl‐phosphatidylcholine (SAPC)‐liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC‐liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane‐docking device that facilitates cPLA2 action in inflammatory diseases.

Molecular Basis for the Association of Group IIA Phospholipase A2 and Decorin in Human Atherosclerotic Lesions

Group IIA secretory nonpancreatic phospholipase A(2) (snpPLA(2)) is associated with collagen fibers in the extracellular matrix of human atherosclerotic plaques. Decorin, a small proteoglycan (PG) carrying chondroitin/dermatan sulfate glycosaminoglycans (GAGs), forms part of the collagen network in human arteries. To explore whether snpPLA(2) may be associated with collagen fibers via interaction with decorin, we performed (1) immunohistochemistry to compare the relative in vivo localization of snpPLA(2) and decorin in human atherosclerotic tissue and (2) in vitro experiments to study the interaction between snpPLA(2) and decorin. In atherosclerotic lesions, decorin was detected within the snpPLA(2)-positive part of the intima close to the media. Electrophoretic mobility shift assay showed that snpPLA(2) binds to decorin synthesized by human fibroblasts. Native and GAG-depleted decorin enhanced the association of snpPLA(2) to collagen types I and VI in a solid-phase binding assay. Furthermore, snpPLA(2) bound efficiently to a recombinant decorin core protein fragment B/E (Asp45-Lys359). This binding was competed with soluble decorin and inhibited at NaCl concentrations >150 mmol/L. The decorin core protein fragment B/E competed better than dermatan sulfate for binding of snpPLA(2) to decorin-coated microtiter wells. The enzymatic activity of snpPLA(2) increased 2- to 3-fold in the presence of decorin or GAG-depleted decorin. The results show that snpPLA(2) binds preferentially to the decorin protein core rather than to the GAG chain and that this interaction enhances snpPLA(2) activity. As a consequence, this active extracellular enzyme may contribute to the pathogenesis of atherosclerosis by modifying lipoproteins and releasing inflammatory lipid mediators at places of lipoprotein retention in the arterial wall.

Elevated expression of human nonpancreatic phospholipase A2 in psoriatic tissue.

In involved psoriatic tissue, which is characterized by chronic inflammation in both epidermis and dermis, elevated levels of arachidonic acid and eicosanoids have been measured. This implies that a phospholipase A2 (PLA2) may be involved in the pathogenesis of psoriasis. The PLA2s are a group of enzymes that release unsaturated fatty acids from thesn2-position of membrane phospholipids. Once released, the fatty acids are converted by various enzymes into biologically very important signaling molecules. Release of arachidonate initiates the arachidonate cascade, leading to the synthesis of eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and lipoxines. Eicosanoids are important in a variety of physiological processes and play a central role in inflammatory reactions and in intracellular signal transduction processes. Other important inflammatory mediators, such as lyso-PAF (a precursor for PAF) and other lysophospholipids, may also be formed through the action of a PLA2. We report for the first time the detection of transcripts of nonpancreatic phospholipase A2 (npPLA2, type II) and cytosolic (c) PLA2 in human skin, and overexpression of npPLA2 in involved skin from patients with psoriasis (plaque psoriasis and pustular psoriasis). Limited amounts of npPLA2 enzyme are detected immunologically in the uppermost layers of epidermis from healthy persons. Both involved and uninvolved psoriatic epidermis contain higher levels of npPLA2 than normal skin. Positive cells in dermis showed significantly higher levels of npPLA2 than epidermal cells. In dermis from healthy persons, only weak staining of a few cells could be detected. The two PLA2 enzymes detected in psoriatic skin (cytosolic and nonpancreatic) may both be involved in eicosanoid overproduction in psoriatic tissue, and the npPLA2 may also be involved in potentiating cell activation, especially T cells.

Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A2 in Human Monocyte–Derived Macrophages

Phospholipase A(2)s (PLA(2)s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA(2)s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA(2) has been shown to specifically hydrolyze low density lipoprotein (LDL)-associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA(2)s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA(2) (snpPLA(2)) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription-polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA(2) was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA(2) was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA(2) was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA(2) in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA(2) expression in human monocyte-derived macrophages. The mRNA levels of cytosolic PLA(2) group IV and snpPLA(2) group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA(2) enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA(2) enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.

Atypical /PKC Conveys 5-Lipoxygenase/Leukotriene B 4 -mediated Cross-talk between Phospholipase A 2 s Regulating NF-B Activation in Response to Tumor Necrosis Factor- and Interleukin-1*

The transcription factor nuclear factor κB (NF-κB) plays crucial roles in a wide variety of biological functions such as inflammation, stress, and immune responses. We have shown previously that secretory nonpancreatic (snp) and cytosolic (c) phospholipase A2 (PLA2) regulate NF-κB activation in response to tumor necrosis factor (TNF)-α or interleukin (IL)-1β activation and that a functional coupling mediated by the 5-lipoxygenase (5-LO) metabolite leukotriene B4 (LTB4) exists between snpPLA2and cPLA2 in human keratinocytes. In this study, we have further investigated the mechanisms of PLA2-modulated NF-κB activation with respect to specific kinases involved in TNF-α/IL-1β-stimulated cPLA2phosphorylation and NF-κB activation. The protein kinase C (PKC) inhibitors RO 31-8220, Gö 6976, and a pseudosubstrate peptide inhibitor of atypical PKCs attenuated arachidonic acid release, cPLA2 phosphorylation, and NF-κB activation induced by TNF-α or IL-1β, thus indicating atypical PKCs in cPLA2regulation and transcription factor activation. Transfection of a kinase-inactive mutant of λ/ιPKC in NIH-3T3 fibroblasts completely abolished TNF-α/IL-1β-stimulated cellular arachidonic acid release and cPLA2 activation assayed in vitro, confirming the role of λ/ιPKC in cPLA2 regulation. Furthermore, λ/ιPKC and cPLA2 phosphorylation was attenuated by phosphatidyinositol 3-kinase (PI3-kinase) inhibitors, which also reduced NF-κB activation in response to TNF-α and IL-1β, indicating a role for PI3-kinase in these processes in human keratinocytes. TNF-α- and IL-1β-induced phosphorylation of λ/ιPKC was attenuated by inhibitors toward snpPLA2 and 5-LO and by an LTB4 receptor antagonist, suggesting λ/ιPKC as a downstream effector of snpPLA2 and 5-LO/LTB4 the LTB4 receptor. Hence, λ/ιPKC regulates snpPLA2/LTB4-mediated cPLA2 activation, cellular arachidonic acid release, and NF-κB activation induced by TNF-α and IL-1β. In addition, our results demonstrate that PI3-kinase and λ/ιPKC are involved in cytokine-induced cPLA2 and NF-κB activation, thus identifying λ/ιPKC as a novel regulator of cPLA2.

Modification of LDL with human secretory phospholipase A(2) or sphingomyelinase promotes its arachidonic acid-releasing propensity.

Oxidation and lipolytic remodeling of LDL are believed to stimulate LDL entrapment in the arterial wall, expanding the inflammatory response and promoting atherosclerosis. However, the cellular responses and molecular mechanisms underlying the atherogenic effects of lipolytically modified LDL are incompletely understood. Human THP-1 monocytes were prelabeled with [3H]arachidonic acid (AA) before incubation with LDL or LDL lipolytically modified by secretory PLA2 (sPLA2) or bacterial sphingomyelinase (SMase). LDL elicited rapid and dose-dependent extracellular release of AA in monocytes. Interestingly, LDL modified by sPLA2 or SMase displayed a marked increase in AA mobilization relative to native LDL, and this increase correlated with enhanced activity of cytosolic PLA2 (cPLA2) assayed in vitro as well as increased monocyte tumor necrosis factor-α secretion. The AA liberation was attenuated by inhibitors toward cPLA2 and sPLA2, indicating that both PLA2 enzymes participate in LDL-induced AA release. In conclusion, these results demonstrate that LDL lipolytically modified by sPLA2 or SMase potentiates cellular AA release and cPLA2 activation in human monocytes. From our results, we suggest novel atherogenic properties for LDL modified by sPLA2 and SMase in AA release and signaling, which could contribute to the inflammatory gene expression observed in atherosclerosis.

Role of secretory and cytosolic phospholipase A2 enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA2) activity, has been analyzed. LysoPC increased AA release in THP‐1 and Mono Mac6 cells in a time‐ and concentration‐dependent manner. The monocytes expressed both secretory and cytosolic PLA2 enzymes and AA release was strongly reduced by cellular pretreatment with different PLA2 inhibitors and by pertussis toxin, an inhibitor of Gi‐protein activation. This indicates that both cytosolic and secretory PLA2 enzymes regulate specific lysoPC receptor‐induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.

Expression, purofocation and biochemical comparison of natural and recombinant human non-pancreatic phospholipase A2

The gene coding for human non-pancreatic phospholipase A2 (npPLA2) was cloned in a eukaryotic expression vector and transfected into chinese hamster ovary (CHO) cells. A number of cell lines stably expressing npPLA2 were obtained. Northern analysis of these cell lines showed an abundant transcript of expected size 1200 nt. The recombinant enzyme was efficiently secreted in quantities up to 400 micrograms npPLA2 per liter culture medium in the most productive cell lines. npPLA2 was purified to homogeneity from conditioned medium as previously described (1). The recombinant npPLA2 migrated by SDS--PAGE as a single band with an apparent mass of 14,000. The recombinant enzyme displayed the pH-optimum, calcium dependence and substrate preference that were characteristic of the human platelet and synovial fluid enzymes.

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