BBA - Molecular and Cell Biology of Lipids (v.1584, #1)

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents by E Chiarpotto; C Allasia; F Biasi; G Leonarduzzi; F Ghezzo; G Berta; G Bellomo; G Waeg; G Poli (1-8).
Among the oxidative breakdown products of ω-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor β1 (TGFβ1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFβ1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFβ1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.
Keywords: 4-Hydroxy-2,3-nonenal (HNE); Transforming growth factor β1; Gene expression; Macrophage; Fibrosis;

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism by Prathima Acharya; Mark L Segall; Mohamed Zaiou; Julie Morrow; Karl H Weisgraber; Michael C Phillips; Sissel Lund-Katz; Julian Snow (9-19).
Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4<apoE3<apoE2; T max≈52<56<59 °C, respectively). Helical contents at the onset and completion of denaturation (as monitored calorimetrically) were about 51% and 35%, respectively, for 22-kDa apoE3. As much as 70% of the unfolding enthalpy for 22-kDa apoE3 could be due to disruption of α-helix. The monomeric 22-kDa fragments unfold differently, with both apoE3 and apoE4 exhibiting unfolding intermediates, whereas apoE2 unfolds according to a two-state mechanism. Interactions with the C-terminal domain appear to destabilize the N-terminal domains in full-length apoE2 and apoE3, but less so for full-length apoE4. Self-association of full-length apoE at higher concentrations (1.5–2.5 mg/ml), as well as interaction of full-length apoE with phospholipid, enhances thermal stability. Cooperative changes in secondary structure, as monitored by circular dichroism, begin near 37 °C for full-length apoE3 and apoE4, indicating that full-length apoE3 and apoE4, but not apoE2, may be partially unfolded in vivo. The differences in stability and unfolding behavior are likely to contribute to the molecular basis for defects in lipid transport and neurological function induced by apoE polymorphism.
Keywords: Apolipoprotein E; Differential scanning calorimetry; Circular dichroism;

Spermine increases phosphatidylinositol 4,5-bisphosphate content in permeabilized and nonpermeabilized HL60 cells by Ronald F Coburn; David H Jones; Clive P Morgan; Carl B Baron; Shamshad Cockcroft (20-30).
The polyamine spermine (N,N′bis[3-aminopropyl]-1,4-butanediamine) activates phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P5K) and phosphatidylinositol 4-kinase (PtdIns4K) in vitro. Spermine concentration increases that occur in proliferating cells were approximated in streptolysin O-permeabilized HL60 cells. When phospholipase C was activated by GTPγS in the presence of PITPα, 0.1–1.2 mM spermine evoked increases in PtdIns(4,5)P2 contents in a dose-dependent manner to 110–170% of control and concomitantly decreased inositol phosphate formation by 10–50%. Spermine-induced increases in PtdIns(4,5)P2 content in permeabilized cells also occurred during GTPγS stimulation in the absence of PITPα, were augmented in the presence of PITPα, occurred in unstimulated cells and were additive to PtdIns(4,5)P2 formation evoked by ARF1, another activator of phosphoinositide kinases. Slowly developing spermine-evoked increases in PtdIns(4,5)P2 contents occurred in nonpermeabilized cells that were abolished in the presence of a spermine transport inhibitor. Data are consistent with spermine at physiological concentrations evoking a PITPα-dependent shift in formation of PtdIns(4,5)P2 from compartments that contained an active phospholipase C to compartments that were separated from an active PLC and from PtdIns(4,5)P2 formed by ARF1.
Keywords: Phosphatidylinositol 4,5-bisphosphate; Phosphatidylinositol 4-kinase; Phosphatidylinositol-4-phosphate 5-kinase; Spermine; Lipid domain; Cell proliferation;

Active-dimeric form of lipoprotein lipase increases in the adipose tissue of patients with rheumatoid arthritis treated with prednisolone by Teruki Kidani; Kenshi Sakayama; Hiroshi Masuno; Nobuo Takubo; Yoshiro Matsuda; Hiromichi Okuda; Haruyasu Yamamoto (31-36).
The synthesis, activity and mass of LPL in adipose tissue were studied in patients with rheumatoid arthritis (RA) treated with prednisolone (PSL) (PSL-treated group) and untreated patients with osteoarthritis (untreated group). LPL activity and mass in the extracts of acetone/ether powder of adipose tissue were 2.4 and 1.6 times, respectively, higher in the PSL-treated group than in the untreated group. There were no differences in the amount of 35S incorporated into LPL during the 2-h incubation of adipose tissue with [35S]methionine between PSL-treated and untreated groups. These results indicate that degradation of LPL was inhibited in the adipose tissue of the PSL-treated group. In the adipose tissue of the untreated group, 72% of the LPL was the inactive-monomeric form, which was eluted with 0.4–0.75 M NaCl from the heparin–Sepharose column, and 28% was the active-dimeric form, which was eluted with 0.8–1.2 M NaCl. In the adipose tissue of the PSL-treated group, 40% was inactive-monomeric, and 60% was active-dimeric. Thus, the relative amount of the active-dimeric form of LPL was increased in the adipose tissue of the PSL-treated group. Taken together, our present results indicate that the higher level of LPL activity in the PSL-treated group was a result of the inhibition of the degradation of the active-dimeric form.
Keywords: Lipoprotein lipase; Dimer; Monomer; Human adipose tissue; Prednisolone; Rheumatoid arthritis;

Human colorectal cancer cells efficiently conjugate the cyclopentenone prostaglandin, prostaglandin J2, to glutathione by Brian Cox; Laine J Murphey; William E Zackert; Rebecca Chinery; Ramona Graves-Deal; Olivier Boutaud; John A Oates; Robert J Coffey; Jason D Morrow (37-45).
Cyclopentenone prostaglandins (PGs), particularly those of the J-series, affect proliferation and differentiation in a number of cell lines. J-ring PGs have been shown to be ligands for the peroxisome proliferator-activated receptor (PPAR)-γ and to modulate NF-κB-mediated gene transcription. We have previously reported that large quantities of eicosanoids, including PGJ2, are produced by the human colorectal cancer cell line HCA-7 while lesser amounts of Δ12-PGJ2 and 15-deoxy-Δ12,14-PGJ2 are formed. In this and other cell lines, cyclopentenone PGs have been shown to increase cell proliferation, but factors that influence their formation and metabolism are poorly understood. Unlike other PGs, cyclopentenone PGs contain α,β-unsaturated carbonyl groups that readily adduct various biomolecules such as glutathione (GSH) in vitro. We now report that in HCA-7 cells, PGJ2 is largely metabolized by conjugation to GSH. Characterization of the adducts by liquid chromatography (LC)–mass spectrometry (MS) revealed two major metabolites consisting of (1) a novel GSH conjugate in which the carbonyl at C-11 of PGJ2 is reduced and (2) intact PGJ2 conjugated to GSH. Approximately 70% of the PGJ2 added to HCA-7 cells was esterifed to GSH after 2 h of incubation, suggesting this pathway represents the major route of metabolic disposition of PGJ2 in HCA-7 cells.
Keywords: Prostaglandin; Cyclopentenone; Colorectal cancer; Glutathione adduct; Eicosanoid; Lipid;

Molecular characterization of the human PLC β1 gene by Daniela Peruzzi; Michela Aluigi; Lucia Manzoli; Anna Maria Billi; Francesco Paolo Di Giorgio; Manuela Morleo; Alberto M Martelli; Lucio Cocco (46-54).
Inositide-specific phospholipase C (PLC) signaling constitutes a central intermediate in a number of cellular functions among which the control of cell growth raises a particular interest. Indeed, we have previously shown that nuclear phospholipase C β1 (PLC β1) is central for the regulation of mitogen-induced cell growth. We have also assigned by fluorescence in situ hybridization (FISH) analysis the PLC β1 to human chromosome 20p12. In this study, we have carried out a detailed analysis of the human gene, showing the existence of alternative splicing, which gives rise, besides the two forms (1a and 1b) already shown in rodents, to a new 600 bp smaller form coding for a 110 kDa protein. We have also identified a new exon at the 5′, showing no homology with the rodent sequence. Here we provide the complete determination of the exon/intron structure of the gene spanning 250 kb of DNA. We found that the exons are quite small, ranging from 49 to 222 bp, while the introns vary between 108 bp and 34,400 bp. The availability of the understanding of the genome organization of this inositide-specific PLC, which represents a key step of the cell cycle related signaling, could actually pave the way for further genetic analysis of p12 region of human chromosome 20 in diseases involving alterations of the control of cell growth such as malignancies.
Keywords: Phospholipase C; Human PLC β1; Inositol phospholipid;

Oxylipin profiling in pathogen-infected potato leaves by Cornelia Göbel; Ivo Feussner; Mats Hamberg; Sabine Rosahl (55-64).
Plants respond to pathogen attack with a multicomponent defense response. Synthesis of oxylipins via the lipoxygenase (LOX) pathway appears to be an important factor for establishment of resistance in a number of pathosystems. In potato cells, pathogen-derived elicitors preferentially stimulate the 9-LOX-dependent metabolism of polyunsaturated fatty acids (PUFAs). Here we show by oxylipin profiling that potato plants react to pathogen infection with increases in the amounts of the 9-LOX-derived 9,10,11- and 9,12,13-trihydroxy derivatives of linolenic acid (LnA), the divinyl ethers colnelenic acid (CnA) and colneleic acid (CA) as well as 9-hydroxy linolenic acid. Accumulation of these compounds is faster and more pronounced during the interaction of potato with the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, which does not lead to disease, compared to the infection of potato with Phytophthora infestans, the causal agent of late blight disease. Jasmonic acid (JA), a 13-LOX-derived oxylipin, accumulates in potato leaves after infiltration with P. syringae pv. maculicola, but not after infection with P. infestans.
Keywords: Jasmonic acid; Lipoxygenase pathway; Phytophthora infestans; Pseudomonas syringae pv. maculicola; Solanum tuberosum;

Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure by Chang-Hua Yu; Vincenzo Panagia; Paramjit S Tappia; Song-Yan Liu; Nobuakira Takeda; Naranjan S Dhalla (65-72).
Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca2+ movements and the association of abnormal Ca2+ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.
Keywords: Sarcolemma; Phospholipase D; Phosphatidate phosphohydrolase; Phosphatidic acid; Diacylglycerol; Congestive heart failure;