Etsuko Yasugi

Cell membrane dynamics and the induction of apoptosis by lipid compounds

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well‐controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.

Lipids in Harderian Glands and Their Significance

The Harderian gland, discovered by Harder (1694), is well developed in rodents (rat, mouse, golden hamster, Mongolian gerbil, guinea pig, etc.), lagomorphs (rabbit, pika) and cetaceans, and may have been lost in primates, terrestrial carnivores, and some other species. The Harderian gland occupies a considerable part of the orbit and is located around the posterior half of the eyeball. Histologically, a single layer of columnar epithelial cells surrounds the lumen, with the single excretory duct directly opening onto the eyelid. The surface of the gland is smooth and is covered with endothelium of the orbital venous sinus (Sakai and Yohro 1981).

Phospholipid turnover in the inflamed intestinal mucosa : Arachidonic acid-rich phosphatidyl/plasmenyl-ethanolamine in the mucosa in inflammatory bowel disease

Abstract: Cytosolic phospholipase A2 (PLase A2) is activated by low Ca2+ concentrations and translocates from the cytosol to the cell membrane, releasing arachidonic acid; the arachidonic acid cascade then leads to the production of many inflammatory mediators. The aim of this study, accordingly, was to investigate the role of phospholipid metabolism in the intestinal mucosa in inflammatory bowel disease (IBD). Surgically resected specimens from patients with Crohns disease (CD), ulcerative colitis (UC), and colrectal cancer (non-cancerous tissue; as a control) were submitted to phospholipid analysis and a PLase A2 assay, which measures the degradation of endogenous mucosal phospholipids. A high percentage of plasmenylethanolamine (plas.E) was detected in the glycerophospholipid fraction of CD mucosa. The arachidonic acid content of the phosphatidylethanolamine plus plas.E subfraction was higher in inflamed than in intact mucosa in CD. PLaseA2 activity, resulting in lysophosphatidyl ethanolamine production, was detected only in inflamed mucosa from CD and UC patients, but not in normal mucosa from controls. PLaseA2 activity was highest in moderately inflamed mucosa adjacent to a severely ulcerated area. The PLaseA2 that reacts with endogenous phosphatidylcholine (PC) to form lysoPC was found irrespective of the presence of inflammation. The PLaseA2 that reacts with ethanolamine-containing phospholipids is more closely related to inflammation than other PLaseA2 isoenzymes in IBD mucosa.

Peroxisome proliferator-activated receptor γ ligands stimulate myeloid differentiation and lipogenensis in human leukemia NB4 cells

Peroxisome proliferator‐activated receptor γ (PPARγ) plays a central role in adipocyte and macrophage differentiation. Pioglitazone (Actos, AD4833), an antidiabetic drug, and 15‐deoxy‐Δ12,14‐prostaglandin J2 (PGJ2) have recently been identified as synthetic and natural ligands for PPARγ, respectively. In this study, we examined the effects of PPARγ ligands on differentiation and lipogenesis in promyelocytic leukemia NB4 cells, in which PPARγ protein was expressed and ligand‐stimulated PPARγ‐specific transcription of adipocyte fatty‐acid binding protein was confirmed. Treatment with PPARγ ligand (AD4833 or PGJ2) alone markedly suppressed proliferation but did not induce differentiation. The combined treatment of the cells with PPARγ ligand and all‐trans retinoic acid (ATRA) synergistically induced myelocytic differentiation, as determined by nitroblue tetrazolium reducing ability and cell morphology. During these processes of differentiation, we observed marked accumulation of lipid droplets in the cytoplasm. The cellular triacylglycerol levels increased 2.7‐fold after treatment with the inducers. Simultaneously, BODIPY‐fatty acid was incorporated into the cytosol and concentrated in lipid droplets. The biosynthesis of triacylglycerol‐containing BODIPY‐fatty acids was increased twofold in differentiated cells. These findings clearly demonstrate that treatment with PPARγ ligands not only induced differentiation but also stimulated lipogenesis in NB4 cells, indicating a close association between differentiation and lipogenesis in PPARγ‐stimulated human myeloid cells.

Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells

Dolichyl monophosphate (Dol‐P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5–20 min), caspase‐3‐like protease activation (2–4 h), chromatin condensation and DNA ladder formation (3–4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis‐inducing factor (AIF) are early events (1–3 h) in the apoptotic process induced by Dol‐P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti‐AIF antibody. Both caspase‐8 and caspase‐3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol‐P and AIF is one of the important factors which induce chromatin condensation in nuclei.

Dihydroheptaprenyl and dihydrodecaprenyl monophosphates induce apoptosis mediated by activation of caspase-3-like protease

Dolichyl phosphate, an essential carrier lipid in the biosynthesis of N-linked glycoprotein, has been found to induce apoptosis in rat glioma C6 cells and human monoblastic leukemia U937 cells. In the present study, dolichyl phosphate and structurally related compounds were examined regarding their apoptosis-inducing activities in U937 cells. Dihydroheptaprenyl and dihydrodecaprenyl phosphates, of which isoprene units are shorter than that of dolichyl phosphate, induced apoptosis in U937 cells. This phenomenon occurred in a dose- and time-dependent manner, as seen with dolichyl phosphate-induced apoptosis. Derivatives of the same isoprene units of dolichyl phosphate, such as dolichol, dolichal or dolichoic acid, did not induce DNA fragmentation. Farnesyl phosphate and geranylgeranyl phosphate also failed to induce apoptosis. During apoptosis, the caspase family of cysteine proteases play important roles. We observed that apoptosis induced by dihydroprenyl phosphate was mediated by caspase-3-like (CPP32-like) activation but not by caspase-1-like (ICE-like) activation. This caspase-3-like activation was inhibited by a specific inhibitor of caspase-3, DEVD-CHO, but not by an caspase-1 inhibitor YVAD-CHO. We interpret these results to mean that dihydroprenyl phosphates with more than seven isoprene units have apoptosis-inducing activity and that their signal is mediated by caspase-3-like activation.

CPP32 ACTIVATION DURING DOLICHYL PHOSPHATE-INDUCED APOPTOSIS IN U937 LEUKEMIA CELLS

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre‐CPP32 to generate p17. Peptide inhibitors YVAD‐cmk and Z‐Asp‐CH2‐DCB (specific to ICE) and DEVD‐cho (specific to CPP32) blocked the dolichyl phosphate‐induced apoptosis. The dolichyl phosphate‐induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2′,5′‐dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.