Vania Hinkovska-Galcheva

Tristetraprolin (TTP)-14-3-3 Complex Formation Protects TTP from Dephosphorylation by Protein Phosphatase 2a and Stabilizes Tumor Necrosis Factor-α mRNA

Tumor necrosis factor (TNF)-α is a major cytokine produced by alveolar macrophages in response to pathogen-associated molecular patterns such as lipopolysaccharide. TNF-α secretion is regulated at both transcriptional and post-transcriptional levels. Post-transcriptional regulation occurs by modulation of TNF-α mRNA stability via the binding of tristetraprolin (TTP) to the adenosine/uridine-rich elements found in the 3′-untranslated region of the TNF-α transcript. Phosphorylation plays important roles in modulating mRNA stability, because activation of p38 MAPK by lipopolysaccharide stabilizes TNF-α mRNA. We hypothesized that the protein phosphatase 2A (PP2A) regulates this signaling pathway. Our results show that inhibition of PP2A by okadaic acid or small interference RNA significantly enhanced the stability of TNF-α mRNA. This result was associated with increased phosphorylation of p38 MAPK and MAPK-activated kinase 2 (MK-2). PP2A inhibition increased TTP phosphorylation and enhanced complex formation with chaperone protein 14-3-3. TTP physically interacted with PP2A in transfected mammalian cells. A functional consequence of TTP-14-3-3 complex formation appeared to be protection of TTP from dephosphorylation by inhibition of the binding of PP2A to phosphorylated TTP. Mutation of the MK-2 phosphorylation sites of TTP did not influence TNF-α adenosine/uridine-rich element binding and did not alter the increased TNF-α 3′-untranslated region-dependent luciferase activity induced by PP2A-small interference RNA silencing. Our data indicate that, although phosphorylation stabilizes TNF-α mRNA, PP2A regulates the mRNA stability by modulating the phosphorylation state of members of the p38/MK-2/TTP pathway.

Ceramide-dependent PP2A regulation of TNFα-induced IL-8 production in respiratory epithelial cells

IL-8 is a key mediator in the pathophysiology of acute lung injury. TNFalpha stimulates IL-8 production in respiratory epithelial cells by activating both the NF-kappaB and MAP kinase pathways. The precise mechanism by which these pathways are downregulated to terminate IL-8 production remains unclear. We studied the regulatory role of the serine/threonine phosphatase, PP2A, on the signaling pathways involved in IL-8 production from respiratory epithelial cells. Inhibition of PP2A using okadaic acid or gene knockdown using siRNA resulted in an augmentation of TNFalpha-induced IL-8 production. We also found that PP2A inhibition resulted in prolonged activation of JNK, p38, and ERK resulting in both increased transcriptional activation of the IL-8 promoter and posttranscriptional stabilization of IL-8 mRNA. Because TNFalpha had been shown to activate ceramide accumulation, and separate studies had linked ceramide with activation of PP2A, we hypothesized the pathway of TNFalpha-inducing ceramide to activate PP2A comprised an endogenous regulatory pathway. Inhibition of the immediate sphingomyelinase-dependent pathway as well as the de novo synthesis pathway of ceramide production reduced serine/threonine phosphatase activity and augmented IL-8 production. These data suggest that ceramide plays a role in activating PP2A to terminate ongoing IL-8 production. In summary, our data suggest that in respiratory epithelium, TNFalpha induces ceramide accumulation, resulting in subsequent activation of PP2A, which targets those kinases responsible for transcriptional activation of IL-8.

Transforming growth factor-β1 stimulates degranulation and oxidant release by adherent human neutrophils

The signal transduction pathways that are activated by cytokines and growth factors binding to their receptors on human neutrophils (PMN) are poorly understood. When PMN in suspension encounter many of these agonists they are not activated, but rather are primed for subsequent activation. We and others reported that when PMN are plated onto fibrinogen and stimulated with cytokines or with the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) they respond by releasing hydrogen peroxide (H2O2) and the specific granule component lactoferrin. Transforming growth factor‐β1 (TGF‐β1) is released by many cells including PMN. It has been reported that TGF‐β1 stimulates chemotaxis but not exocytosis or superoxide production by cells in suspension. We hypothesized that TGF‐β1 would activate PMN to release H2O2 when they were adherent to fibrinogen, a response mediated by β2 integrin receptors. In this study, we determined whether TGF‐β1 stimulated H2O2 and lactoferrin release by PMN adherent to fibrinogen. TGF‐β1 stimulated H2O2 and lactoferrin release from adherent PMN in a concentration‐dependent manner, with effects seen in the range of 0.1 to 100 pg/mL. Both H2O2 and lactoferrin release were detected by 60 min and continued for at least 180 min. Adhesion and spreading of PMN paralleled H2O2 and lactoferrin release. Ethanol (200 mM) blocked both H2O2 and lactoferrin release, suggesting the involvement of the phospholipase D pathway. In PMN labeled with lyso[3H]phoephatidylcholine, we observed that TGF‐β1 treatment caused an increase in [3H]phoephatidate. Propranolol (150 μM), an inhibitor of phosphatidate phosphohydrolase, blocked both H2O2 and lactoferrin release, suggesting that the conversion of phosphatidic acid to diradylglycerol is an important step in PMN activation by TGF‐β1. Overall, these results are similar to those reported for fMLP activation of adherent PMN and suggest that a common pathway is involved in both chemoattractant and cytokine activation. J. Leukoc. Biol. 60: 772–777; 1996.

Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase.

Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high resolution crystal structures of human LPLA2 and a low resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

Ceramide-1-Phosphate in Phagocytosis and Calcium Homeostasis

Sphingolipids are well established sources of important signaling molecules. For example, ceramide (Cer) has been described as a potent inhibitor of cell growth and inducer of apoptosis. In contrast, ceramide-1-phosphate (C1P) has been reported to have mitogenic properties and to inhibit apoptosis. Our understanding of the distinct biological roles of C1P in the regulation of DNA synthesis, inflammation, membrane fusion and intracellular Ca2+ increase has rapidly expanded. C1P is a bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). This chapter specifically focuses on the role of C1P in phagocytosis and Ca2+ homeostasis. Studies of the metabolism of C1P during phagocytosis, may lead to a better understanding of its role in signaling. Potentially, the inhibition of CERK and C1P formation may be a therapeutic target for inflammation.

Ceramide kinase promotes Ca2+ signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells.

Ceramide-1-phosphate (C1P) is a novel bioactive sphingolipid formed by the phosphorylation of ceramide catalyzed by ceramide kinase (CERK). In this study, we evaluated the mechanism by which increased C1P during phagocytosis enhances phagocytosis and phagolysosome formation in COS-1 cells expressing hCERK. Stable transfectants of COS-1 cells expressing FcγRIIA or both FcγRIIA/hCERK expression vectors were created. Cell fractionation studies demonstrated that hCERK and the transient receptor potential channel (TRPC-1) were enriched in caveolae fractions. Our data establish that both CERK and TRPC-1 localize to the caveolar microdomains during phagocytosis and that CERK also colocalizes with EIgG in FcγRIIA/hCERK-bearing COS-1 cells. Using high-speed fluorescence microscopy, FcγRIIA/hCERK transfected cells displayed Ca2+ sparks around the phagosome. In contrast, cells expressing FcγRIIA under identical conditions displayed little periphagosomal Ca2+ signaling. The enhanced Ca2+ signals were accompanied by enhanced phagolysosome formation. However, the addition of pharmacological reagents that inhibit store-operated channels (SOCs) reduced the phagocytic index and phagolysosomal fusion in hCERK transfected cells. The higher Ca2+ signal observed in hCERK transfected cells as well as the fact that CERK colocalized with EIgG during phagocytosis support our hypothesis that Ca2+ signaling is an important factor for increasing phagocytosis and is regulated by CERK in a manner that involves SOCs/TRPCs.

Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2

Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.