The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.     

Fate of surface immunoglobulin during induction of lymphocyte proliferation

The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose-linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose-linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.     

Evidence for proteolytic processing and stimulated organelle redistribution of iPLA2β

Over the past decade, important roles for the 84–88 kDa Group VIA Ca²⁺-independent phospholipase A₂ (iPLA₂β) in various organs have been described. We demonstrated that iPLA₂β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA₂β, and certain stimuli promote perinuclear localization of iPLA₂β. To gain a better understanding of its mobilization, iPLA₂β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA₂β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA₂β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA₂β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA₂β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA₂β in β-cells and we find here that these stimuli promote differential localization of iPLA₂β in subcellular organelles. Further, mass spectrometric analyses identified iPLA₂β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA₂β and redistribution of iPLA₂β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA₂β facilitates its participation in multiple biological processes.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A(2)alpha blocks arachidonic acid release in a variety of mammalian cells

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.     

Enhanced phosphorylation of endogenous membrane proteins during induction of B lymphocyte proliferation

Phosphorylation of endogenous proteins was assessed employing membrane preparations derived from splenocytes induced to proliferate in response to Sepharose linked anti-immunoglobulins. Time course studies revealed that enhanced protein phosphorylation was preceded by cell enlargement and was either followed by or closely related in time to the onset of DNA synthesis. Thus maximal enhancement of phosphorylation was initially observed at 24 h whereas cell enlargement was optimal at 16 h at a time when there was no enhancement in protein phosphorylation. Furthermore thymidine incorporation was maximal at 32 h and low at 24 h when phosphoprotein synthesis was maximally enhanced. Taken together, these results suggest that phosphorylation of endogenous membrane proteins may be involved in signalling entry of cells into S phase of the cell cycle.     

Nonresponsiveness of immature B lymphocytes to anti-immunoglobulin is reversed by pronase

The glutathione (GSH) conjugation reaction of the active metabolite of a potent protein-pyrolysate carcinogen, 2-nitroso-6-methyldipyrido[1,2-a:3',2'-d]imidazole (NO-Glu-P-1), occurred in glycerol matrix inside a fast atom bombardment (FAB) mass spectrometer. The short lived GSH-conjugates were detected by in situ FAB analysis. The precursor-product relationship between the conjugates was indicated by following the reaction by measuring [M + H]+ ions of the conjugates.     

‘Schmidt's Antonovka’ is identical to ‘Common Antonovka’, an apple cultivar widely used in Russia in breeding for biotic and abiotic stresses

Progenies of ‘Schmidt's Antonovka’ (SA) have been widely used in Western breeding programs as a source of scab resistance. The identity of SA has remained obscure, especially due to the existence of a series of ‘Antonovka’ cultivars with different origins. In this paper we show Schmidt's Antonovka to be identical to Анто?новка обыкновенный or ‘Common Antonovka’ (CA), an old Russian cultivar of unknown origin, by comparing simple sequence repeat (SSR) and SNP genotyping data from several first-generation descendants of SA from two European collections and a CA accession from the germplasm collection held at VNIISPK (The All-Russian Research Institute of Horticultural Breeding, Orel, Russia). The use of CA in Russian breeding programs is also briefly reviewed.