BBA - Molecular Cell Research (v.1642, #3)

Group V and X secretory phospholipase A2s-induced modification of high-density lipoprotein linked to the reduction of its antiatherogenic functions by Yoshikazu Ishimoto; Katsutoshi Yamada; Shigenori Yamamoto; Takashi Ono; Mitsuru Notoya; Kohji Hanasaki (129-138).
The quantitative or qualitative decline of high-density lipoprotein (HDL) is linked to the pathogenesis of atherosclerosis because of its antiatherogenic functions, including the mediation of reverse cholesterol transport from the peripheral cells to the liver. We have recently shown that group X secretory phospholipase A2 (sPLA2-X) is involved in the pathogenesis of atherosclerosis via potent lipolysis of low-density lipoprotein (LDL) leading to macrophage foam cell formation. We demonstrate here that sPLA2-X as well as group V secretory PLA2 (sPLA2-V), another group of sPLA2 that can potently hydrolyze phosphatidylcholine (PC), also possess potent hydrolytic potency for PC in HDL linked to the production of a large amount of unsaturated fatty acids and lysophosphatidylcholine (lysoPC). In contrast, the classical types of group IB and IIA secretory PLA2s evoked little, if any, lypolytic modification of HDL. Treatment with sPLA2-X or -V also caused an increase in the negative charge of HDL with no oxidation and little modification of apolipoprotein AI (apoAI). Modification with sPLA2-X or -V resulted in significant decrease in the capacity of HDL to cause cellular cholesterol efflux from lipid-loaded macrophages. Immunohistochemical analysis revealed significant expression of sPLA2-X in foam cell lesions in the arterial intima of Watanabe heritable hyperlipidemic (WHHL) rabbit. These findings suggest that lipolytic modification of HDL by sPLA2-X or -V causes drastic change of HDL in terms of the production of a large amount of unsaturated fatty acids and lysoPC linked to the reduction of its antiatherogenic functions. These sPLA2-mediated modifications of plasma lipoproteins might be relevant to the pathogenesis of atherosclerosis.
Keywords: Phospholipase A2; High-density lipoprotein; Free fatty acid; Lysophosphatidylcholine; Cholesterol efflux;

Identification of Taxreb107 as a lactogenic hormone responsive gene in mammary epithelial cells by Sergio Wittlin; Kate D. Sutherland; Jane E. Visvader; Geoffrey J. Lindeman (139-147).
Mammary gland development and differentiation is regulated by a number of growth factors and hormones. Milk protein gene expression represents a hallmark of functional mammary epithelial differentiation and is coordinated by the lactogenic hormone prolactin and glucocorticoids. To date, few ‘early-response’ genes transcriptionally activated by lactogenic hormones have been described. We have used representational difference analysis (RDA) to search for lactogenic-responsive genes in SCp2 mouse mammary epithelial cells. One of the cDNAs identified encoded the DNA-binding protein Taxreb107, originally identified as a HTLV-I Tax responsive element binding protein. Increased Taxreb107 expression was confirmed following prolactin and dexamethasone-induced differentiation of SCp2 and HC11 mammary epithelial cells. Taxreb107 RNA levels were developmentally regulated in the mouse mammary gland, where levels increased substantially during mid- and late pregnancy and persisted during lactation. Overexpression of an antisense Taxreb107 cDNA construct or antisense oligonucleotide in HC11 mammary epithelial cells attenuated milk protein gene expression following prolactin and dexamethasone treatment. These findings indicate a role for Taxreb107 as a lactogenic hormone-responsive gene during differentiation of the mammary gland.
Keywords: Glucocorticoid; Mammary gland differentiation; Lactogenic; Prolactin; Representational difference analysis; Ribosomal L6 protein; Subtractive hybridisation; Taxreb107;

Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2.
Keywords: Glucocorticoid; NMR spectroscopy; Apoptosis; Thymoma; Bcl-2; Catalase;

The association of cyclin A and cyclin kinase inhibitor p21 in response to γ-irradiation requires the CDK2 binding region, but not the Cy motif by Kunihiko Fukuchi; Kentarou Nakamura; Sachiko Ichimura; Kouichi Tatsumi; Kunihide Gomi (163-171).
The cyclin kinase inhibitor p21 associates with and inhibits cyclin–CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of γ-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to γ-irradiation requires the CDK binding region, 49–71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin–CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.
Keywords: DNA damage; p21; Cyclin A; CDK2; Cy motif;

Endothelial stress by gravitational unloading: effects on cell growth and cytoskeletal organization by Sofia I.M. Carlsson; Maria T.S. Bertilaccio; Erica Ballabio; Jeanette A.M. Maier (173-179).
All organisms on Earth have evolved to survive within the pull of gravity. Orbital space flights have clearly demonstrated that the absence or the reduction of gravity profoundly affects eukaryotic organisms, including man. Because (i) endothelial cells are crucial in the maintenance of the functional integrity of the vascular wall, and (ii) cardiovascular deconditioning has been described in astronauts, we evaluated whether microgravity affected endothelial functions. We show that microgravity reversibly stimulated endothelial cell growth. This effect correlated with an overexpression of heat shock protein 70 (hsp70) and a down-regulation of interleukin 1 alpha (IL-1α), a potent inhibitor of endothelial cell growth, also implicated in promoting senescence. In addition, gravitationally unloaded endothelial cells rapidly remodelled their cytoskeleton and, after a few days, markedly down-regulated actin through a transcriptional mechanism. We hypothesize that the reduction in the amounts of actin in response to microgravity represents an adaptative mechanism to avoid the accumulation of redundant actin fibers.
Keywords: Endothelial cell; Interleukin 1 alpha; Hsp70; Cytoskeleton; Aging;

Combination of 3′ and 5′ IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model by Laurence Guglielmi; Marc Le Bert; Isabelle Comte; Marie Laure Dessain; Mireille Drouet; Christiane Ayer-Le Lievre; Michel Cogné; Yves Denizot (181-190).
To ensure the B cell differentiation stage specificity of the intronic Eμ element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a VH promoter-GFP reporter gene linked to the 3′LCR region and the Eμ element. By flow cytometry, GFP+ lymphocytes were observed amongst pro-B cells (B220+CD43+CD117+) and at all stages of differentiation up to mature B cells (B220+IgM+IgD+). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP+Thy1,2+ cells (T lymphocytes) and GFP+B220CD117+CD43+ cells (uncommitted lymphohematopoietic progenitors). Therefore, the Eμ-GFP-3′LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3′ and 5′ IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.
Keywords: B lymphocyte; Transgenic; Gene regulation; Eμ; 3′LCR;