The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization.

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

Activity of phospholipases A and lysophospholipase in turkey semen and oviducal fluid.

Changes in lipid composition of turkey semen have previously been reported to occur during in vitro storage and may be mediated by endogenous hydrolysis of phospholipids. To investigate the presence of phospholipases able to initiate such degradation, phospholipaseA2 (PLA2), phospholipase A1 (PLA1), and lysophospholipase (LPLase) activities were measured in turkey spermatozoa and seminal plasma. These enzymes were also measured in the oviductal fluid because they may be involved in the process prior to fertilization in the female. In spermatozoa and seminal plasma, the major PLA2 was a calcium-dependent and sodium deoxycholate (DOC) stimulated enzyme. However, calcium-independent PLA2 activities were also detected with different characteristics in spermatozoa (DOC inhibited enzyme) and seminal plasma (DOC stimulated enzyme). Additionally, PLA1 activity and high LPLase activity were present in spermatozoa and seminal plasma. In vitro storage of semen for 48 h did not affect PLA2 and LPLase activities. By contrast, PLA1 was the major phospholipase activity detected in oviductal fluid. A PLA2 activity stimulated by calcium or DOC and LPLase activity were also detected, but both were low relative to PLA1. These results showed that turkey semen had several enzymatic activities able to hydrolyze phospholipids. In addition, the phospholipase activities described here in the oviductal fluid could be involved in membrane destabilization prior to fertilization.

Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation.

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.

New developments in phospholipase A2.

Some of the most recent data concerning various phospholipases A2, with special emphasis on secretory, cytosolic, and calcium-independent phospholipases A2 are summarized. Besides their contribution to the production of proinflammatory lipid mediators, the involvement of these enzymes in key cell responses such as apoptosis or tumor cell metastatic potential is also discussed, taking advantage of transgenic models based on gene invalidation by homologous recombination. The possible role of secretory and cytosolic platelet-activating factor acetyl hydrolases is also briefly mentioned. Finally, the ectopic expression in epididymis of an intestinal phospholipase B opens some novel issues as to the possible function of phospholipases in reproduction.

Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.

From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.

Inhibition of platelet arachidonic acid liberation by endothelium-derived relaxing factor (EDRF) as studied with sin-1, a nitric oxide generating drug. Evidence for calcium-dependent and calcium-independent mechanisms.

In order to investigate possible effects of endothelium-derived relaxing factor (EDRF or NO.) on platelet phospholipase A2 activity, human platelets labelled with [3H]arachidonic acid ([3H]AA) were stimulated with thrombin (0.5 IU/ml) in the absence or in the presence of sin-1, a vasodilator and platelet inhibitor releasing NO. by spontaneous decomposition at physiological pH. Sin-1 promoted a dose-dependent inhibition of [3H]AA liberation, which was identical in the presence or in the absence of 1 mM Ca2+ in the external medium, suggesting that a reduction of Ca2+ influx was not responsible for this metabolic effect. Using fura-2 as a fluorescent Ca2+ indicator, sin-1 was found to inhibit similarly both Ca2+ influx and Ca2+ mobilization, the latter effect being directly related to a reduction of inositol 1,4,5-tris phosphate production by phospholipase C. However, comparison of cytoplasmic free calcium concentrations ([Ca2+]i) and of [3H]AA liberation attained by platelets treated under various experimental conditions indicated the lack of a direct relationship between [Ca2+]i and platelet phospholipase A2 activity. The effects of sin-1 on [3H]AA liberation could be reproduced by a membrane-permeant analogue of cGMP (8-bromo cyclic GMP), with no evidence of additional effects of sin-1 under these conditions. These data bring further support to the view that Ca2+, although being a necessary cofactor of intracellular phospholipase A2, is not the only regulator of the enzyme. Owing to the multiple effects of this drug on various events involved in membrane-signal transduction (Ca2+ influx, phospholipase C and phospholipase A2 activation), it is suggested that sin-1 inhibits platelet function at an early step of signal transduction, probably by elevating cGMP through a direct effect of NO. on cytosolic guanylate cyclase.

Calcium-independent phospholipases from guinea pig digestive tract as probes to study the mechanism of lipocortin.

Two calcium-independent phospholipases isolated from guinea pig pancreas (lipase Ia, 37 kDa) and from guinea pig intestine (phospholipase B, 97 kDa) have been used to probe the mechanism of phospholipase inhibition by lipocortin. In the presence of calcium, both enzymes were inhibited by lipocortin I in a manner very similar to the inhibition of pig pancreas phospholipase A2. By using phospholipases that lack a requirement for calcium, we have for the first time been able to dissociate enzymatic activity from the role of calcium in the inhibitory process. It was found that lipocortin was without effect against phospholipase A1 and phospholipase B in the absence of calcium, under which conditions the inhibitory protein is unable to interact with anionic phospholipid surfaces. The same behavior toward phospholipase A1 was observed with two other related proteins, endonexin II or lipocortin V, and p68/67-kDa calelectrin or lipocortin VI. Together with the observation that lipocortins are active only in the presence of limited amounts of substrate, these data give further support to the "surface depletion model" of lipocortin inhibition, rather than to a mechanism involving a direct interaction between enzyme and inhibitor.

Purification of a new, calcium-independent, high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush-border membrane.

A phospholipase A2 activity directed against phosphatidylcholine was previously described in brush-border membrane from guinea pig intestine (Diagne, A., Mitjavila, S., Fauvel, J., Chap, H., and Douste-Blazy, L. (1987) Lipids 22, 33-40). In the present study, this enzyme was solubilized either with Triton X-100 or upon papain treatment, suggesting a structural similarity with other intestinal hydrolases such as leucine aminopeptidase, sucrase, or trehalase. The papain-solubilized form, which is thought to lack the short hydrophobic tail responsible for membrane anchoring, was purified 1800-fold to about 90% purity by ion exchange chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA44, and hydrophobic chromatography on phenyl-Sepharose. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a main band with an apparent molecular mass of 97 kDa was detected under reducing and nonreducing conditions. In the latter case, phospholipase A2 activity could be recovered from the gel and was shown to coincide with the 97-kDa protein detected by silver staining. The enzyme activity was unaffected by EGTA and slightly inhibited by CaCl2. The purified enzyme displayed a similar activity against phosphatidylcholine and phosphatidylethanolamine, whereas 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 50% compared to diacylglycerophospholipids. Using phosphatidylcholine labeled with either [3H]palmitic acid or [14C]linoleic acid in the 1- or 2-positions, respectively, the purified enzyme catalyzed the removal of [3H]palmitic acid, although at a lower rate compared to [14C]linoleic acid. This resulted in the formation of sn-glycero-3-phosphocholine, but only 1-[3H]palmitoyl-sn-glycero-3-phosphocholine was detected as an intermediary product. In agreement with this, 1-acyl-2-lyso-sn-[14C]glycero-3-phosphocholine was deacylated at almost the same rate as the sn-2-position of phosphatidylcholine. Since upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the two hydrolytic activities were detected at the same position as 97-kDa protein, the enzyme is thus considered as a phospholipase A2 with lysophospholipase activity (phospholipase B), which might be involved in phospholipid digestion.