Biosynthesis of Prostaglandins D2 and E2 in Chick Dorsal Root Ganglion During Development

Newly formed prostaglandins (PGs), which are assumed to act as modulators of afferent sensory messages, were studied in chick dorsal root ganglia (DRG) during development. [1-14C]Arachidonic acid was converted by DRG homogenates from 1-week-old chickens into two major 14C-PGs: PGE2 and PGD2. The enzymatic conversion of arachidonic acid was characterized as follows: (a) Boiled preparations were inactivated; (b) synthesis of PGs was inhibited by pretreatment with aspirin or indomethacin and enhanced by esculetin, a protector of cyclooxygenase; and (c) [14C]PGE2 and [14C]PGD2 accumulation was a protein dose-dependent process. Further fractionation of crude homogenates indicated that PG endoperoxide synthetase (EC 1.14.99.1) and PGE2 synthetase (EC 5.3.99.3) were membrane-bound enzymes, whereas PGD2 synthetase (EC 5.3.99.2) was recovered in the cytosol. During development, from embryonic day 10 to day 14 after hatching, PGD2 synthetase activity remained constant; in contrast, a sharp rise in [14C]PGE2 synthesis was observed from embryonic day 14 to 18. The time curves of PGD2 and PGE2 synthetase specific activity may be related to changes taking place in the cell population of developing DRG. It is therefore suggested that arachidonic acid would be enzymatically converted early into PGD2 by maturing ganglion cells and then later into PGE2 by proliferating fibroblasts.     

Prostaglandin E(2) and stem cell factor can deliver opposing signals to B lymphocyte precursors

The synthetic prostanoid, 16,16-dimethyl PGE(2), suppressed B lymphopoiesis in mice and proliferation of normal B cell precursors or the F10 pro-B cell line to interleukin 7 in culture. This was not the case with two other prostanoids, PGD(2) and PGF(2alpha), or agonists for PGI(2) agonist and thromboxane A(2) agonist receptors. PGE(2), but not the related prostanoids or agonists, induced apoptosis in F10 cells. The apoptotic response was mediated by the EP2 class of PGE(2) receptors and required an increase in intracellular cyclic adenosine 3',5'-monophosphate, activation of protein kinase A, and protein synthesis. The influence of PGE(2) on F10 cells was diminished in the presence of a cloned stromal cell line or stem cell factor. These findings describe another potential regulatory circuit in bone marrow which might influence B lymphopoiesis under disease or steady-state conditions.     

New insights into PKC family affairs: three novel phosphorylation sites in PKCepsilon and at least one is regulated by PKCalpha

The question of how Bax is activated during apoptosis to perform its role in permeabilization of mitochondrial membranes is intriguing for investigators in the wide field of cell death research. In their paper published in the Biochemical Journal in 2006, Capano and Crompton presented their discovery that simulated ischaemia causes rapid activation of AMPK (AMP-activated protein kinase) which phosphorylates and activates p38 MAPK (mitogen-activated protein kinase) leading to Bax activation and translocation to mitochondria in isolated cardiac myocytes. This was the first report on the molecular mechanism of Bax activation and migration during ischaemia-induced apoptosis in cardiomyocytes.     

An improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) production in biological fluids

We report an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that accurately measures prostaglandins D(2) (PGD(2)) and E(2) (PGE(2)) in cell culture supernatants and other biological fluids. The limit of detection for each prostaglandin was 20 pg/ml (0.20 pg, 0.55 fmol on-column), and the interday and intraday coefficients of variation were less than 5%. Both d(4)-PGE(2) and d(4)-PGD(2) were used as surrogate standards to control for differential loss and degradation of the analytes. Stability studies indicated that sample preparation time should be less than 8h to measure PGD(2) accurately, whereas preparation time did not affect PGE(2) measurement due to its greater stability in biological samples. As an application of the method, PGD(2) and PGE(2) were measured in culture supernatants from A549 cells and RAW 264.7 cells. The human lung alveolar cell line A549 was found to produce PGE(2) but no PGD(2), whereas the murine macrophage cell line RAW 264.7 produced PGD(2) and only trace amounts of PGE(2). This direct comparison showed that COX-2 gene expression can lead to differential production of PGD(2) and PGE(2) by epithelial cells and macrophages. Because PGE(2) is antiasthmatic and PGD(2) is proasthmatic, we speculate that the balance of production of these eicosanoids by epithelial cells and macrophages in the lung contributes to the pathogenesis of chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and lung cancer.     

Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis

Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.     

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein Bim(S). Regulated expression of Bim(S) in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of Bim(S) showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, Bim(S) enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting.     

Decreased sensitivity of atopic mononuclear cells to prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2)

The effect of PGE2 and PGD2 on several lymphocyte functions in vitro was evaluated in nonatopic and atopic subjects. Both PGE2 and PGD2 inhibited phytohemagglutinin-induced protein synthesis ([3H] leucine uptake) by nonatopic mononuclear cells and T cells in a dose-dependent manner (10(-6) to 10(-12) M). Protein synthesis by atopic mononuclear cells was not significantly suppressed by the above concentration of PGE2. Although PGD2 effectively suppressed protein synthesis by atopic mononuclear cells and T cells at 10(-6) M, lower concentrations were ineffective. Kinetic studies revealed significant differences in the suppressive effects of PGE2 and PGD2 on atopic and nonatopic mononuclear cells at 24 and 48 h, but not at 72 or 96 hr. Protein synthesis by T helper-enriched populations (suppressor cell depletion by anti-Leu-2b + complement) obtained from nonatopics was significantly reduced by PGE2 and PGD2, suggesting that these mediators may be directly inhibiting the responding population. By contrast, protein synthesis by T suppressor-enriched populations (helper cell depletion by OKT4 + complement) obtained from nonatopics was enhanced by PGE2 and PGD2, suggesting that the PG were activating these cells. Atopic T helper and T suppressor cells exhibited decreased responsiveness to PGE2 and PGD2 compared with nonatopic cells. PGE2 and PGD2 inhibited the phytohemagglutinin-stimulated proliferative response ([3H]thymidine uptake) by both atopic and nonatopic mononuclear cells in a dose-dependent manner and to the same extent. However, although PGE2 and PGD2 generated functional suppressor activity (when using a coculture technique) in nonatopic mononuclear cells, these mediators failed to activate atopic suppressor cells. These results suggest that reduced responses by atopic T cells to signals provided by PGE2 and PGD2 are not solely restricted to suppressor cell function, and could indicate an impaired ability to regulate immune and/or inflammatory reactions.     

CUGBP2 downregulation by prostaglandin E2 protects colon cancer cells from radiation-induced mitotic catastrophe

Prostaglandin E(2) (PGE(2)) is a potent inhibitor of ionizing radiation (IR)-induced cell death. Exposure of colon cancer cells to IR leads to increased CUGBP2 expression. Therefore, we tested the hypothesis that PGE(2) radioprotects colon cancer cells by inhibiting CUGBP2 expression. Exposure of HCT-116 cells to gamma-IR (0-12 Gy) resulted in a dose-dependent reduction in cell growth and an increase in the G(2)-M phase of the cell cycle. Western blot analyses demonstrated increased levels of activated caspase 9 and caspase 3. In addition, whereas Bax expression is increased, that of Bcl-2 and Bcl-x(L) was reduced. Further analyses demonstrated increased activation of Chk1 and Chk2 kinases, coupled with higher levels of nuclear cyclin B1 and Cdc2. Pretreatment with PGE(2) suppressed the activation of caspase 3 and caspase 7 and inhibited Bax expression. In addition, PGE(2) treatment restored growth and colony formation to control levels. IR significantly upregulated the expression of CUGBP2 in the cells, which was suppressed when cells were pretreated with PGE(2). Ectopic overexpression of CUGBP2 also induced apoptosis. Furthermore, it reversed the PGE(2)-mediated protection from IR-induced mitotic catastrophe. Furthermore, there was an increase in nuclear localization of cyclin B1 and Cdc2 coupled with increased phosphorylation of p53, Chk1, Chk2, and Cdc25c proteins. Cell cycle analysis also demonstrated increased G(2)-M transition. In contrast, siRNA-mediated suppression of CUGBP2 expression restored normal cell cycle progression and decreased IR-induced apoptosis. Taken together, these data demonstrate that PGE(2) protects colon cancer cells from IR-induced mitotic catastrophe in part through suppression of CUGBP2 expression.     

Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis.

Apoptosis is a physiological counterbalance to mitosis and plays important roles in tissue development and homeostasis. Cytosolic Ca(2+) has been implicated as a proapoptotic second messenger involved in both triggering apoptosis and regulating cell death-specific enzymes. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and endoplasmic reticulum (ER) membranes; however, the molecular mechanism of Bax translocation and its relationship to Ca(2+) is largely unknown. Here we provide functional evidence for a synergistic interaction between the movements of intracellular Ca(2+) and cytosolic Bax in the induction of apoptosis. Overexpression of Bax in cultured cells causes a loss of ER Ca(2+) content. Depletion of ER Ca(2+) through activation of the ryanodine receptor enhances the participation of Bax into the mitochondrial membrane. Neither Bax translocation nor Bax-induced apoptosis is affected by buffering of cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, suggesting that depletion of ER Ca(2+) rather than elevation of cytosolic Ca(2+) is the signal for cell apoptosis. This dynamic interplay of Ca(2+) and Bax movements may serve as an amplifying factor in the initial signaling steps of apoptosis.     

Prostaglandin D2 mediates neuronal protection via the DP1 receptor

Cyclo-oxygenases (COXs) catalyze the first committed step in the synthesis of the prostaglandins PGE(2), PGD(2), PGF(2alpha), PGI(2) and thomboxane A(2). Expression and enzymatic activity of COX-2, the inducible isoform of COX, are observed in several neurological diseases and result in significant neuronal injury. The neurotoxic effect of COX-2 is believed to occur through downstream effects of its prostaglandin products. In this study, we examined the function of PGD(2) and its two receptors DP1 and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) (DP2) in neuronal survival. PGD(2) is the most abundant prostaglandin in brain and regulates sleep, temperature and nociception. It signals through two distinct G protein-coupled receptors, DP1 and DP2, that have opposing effects on cyclic AMP (cAMP) production. Physiological concentrations of PGD(2) potently and unexpectedly rescued neurons in paradigms of glutamate toxicity in cultured hippocampal neurons and organotypic slices. This effect was mimicked by the DP1-selective agonist BW245C but not by the PGD(2) metabolite 15d-PGJ(2), suggesting that neuroprotection was mediated by the DP1 receptor. Conversely, activation of the DP2 receptor promoted neuronal loss. The protein kinase A inhibitors H89 and KT5720 reversed the protective effect of PGD(2), indicating that PGD(2)-mediated neuroprotection was dependent on cAMP signaling. These studies indicate that activation of the PGD(2) DP1 receptor protects against excitotoxic injury in a cAMP-dependent manner, consistent with recent studies of PGE(2) receptors that also suggest a neuroprotective effect of prostaglandin receptors. Taken together, these data support an emerging and paradoxical neuroprotective role of prostaglandins in the CNS.     

Caspase-mediated Bak activation and cytochrome c release during intrinsic apoptotic cell death in Jurkat cells

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.     

Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

P53 inactivation is often observed in Burkitt''s lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection.     

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.     

Astragaloside IV, a novel antioxidant, prevents glucose-induced podocyte apoptosis in vitro and in vivo

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 μg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg(-1)·d(-1)) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation.     

Resveratrol induces p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated Bax protein oligomerization on mitochondria to initiate cytochrome c release and caspase activation

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.     

Acetic acid triggers cytochrome c release in yeast heterologously expressing human Bax

Proteins of the Bcl-2 protein family, including pro-apoptotic Bax and anti-apoptotic Bcl-xL, are critical for mitochondrial-mediated apoptosis regulation. Since yeast lacks obvious orthologs of Bcl-2 family members, heterologous expression of these proteins has been used to investigate their molecular and functional aspects. Active Bax is involved in the formation of mitochondrial outer membrane pores, through which cytochrome c (cyt c) is released, triggering a cascade of downstream apoptotic events. However, when in its inactive form, Bax is largely cytosolic or weakly bound to mitochondria. Given the central role of Bax in apoptosis, studies aiming to understand its regulation are of paramount importance towards its exploitation as a therapeutic target. So far, studies taking advantage of heterologous expression of human Bax in yeast to unveil regulation of Bax activation have relied on the use of artificial mutated or mitochondrial tagged Bax for its activation, rather than the wild type Bax (Bax α). Here, we found that cell death could be triggered in yeast cells heterologoulsy expressing Bax α with concentrations of acetic acid that are not lethal to wild type cells. This was associated with Bax mitochondrial translocation and cyt c release, closely resembling the natural Bax function in the cellular context. This regulated cell death process was reverted by co-expression with Bcl-xL, but not with Bcl-xLΔC, and in the absence of Rim11p, the yeast ortholog of mammalian GSK3β. This novel system mimics human Bax α regulation by GSK3β and can therefore be used as a platform to uncover novel Bax regulators and explore its therapeutic modulation.

     

Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha.

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.     

Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation

Members of the Bcl-2 family of proteins control the cellular commitment to apoptosis, although their role in Fas-induced apoptosis is ill-defined. In this report we demonstrate that activation of the Fas receptor present on a human breast epithelial cell line resulted in a conformational change in the N terminus of the pro-apoptotic protein Bax. This conformational change appeared to occur in the cytosol and precede Bax translocation to the mitochondria. Overexpression of the anti-apoptotic protein Bcl-2 inhibited both the conformational change of Bax as well as its relocalization to the mitochondria. Bcl-2 overexpression did not, however, inhibit Fas-induced cleavage of both procaspase-8 and the pro-apoptotic protein Bid, indicating that Bcl-2 functions downstream of these events. These results suggest that the mechanism by which Bcl-2 inhibits Bax mitochondrial translocation and subsequent amplification of the apoptotic cascade is not by providing a physical barrier to Bax, but rather by inhibiting an upstream event necessary for Bax conformational change.     

Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP-Bax-stable H9c2 cell line for apoptosis analysis

The proapoptotic protein Bax plays an important role in cardiomyocytic cell death. Ablation of this protein has been shown to diminish cardiac damage in Bax-knockout mice during ischemia-reperfusion. Presently, studies of Bax-mediated cardiac cell death examined primarily the expression levels of Bax and its prosurvival factor Bcl-2 rather than the localization of this protein, which dictates its function. Using immunofluorescence labeling, we have shown that in neonatal rat cardiomyocytes and in H9c2 cardiomyoblasts, Bax translocates from cytosol to mitochondria upon the induction of apoptosis by hypoxia-reoxygenation-serum withdrawal and by the presence of the free-radical inducer menadione. Also, we found that Bax translocation to mitochondria was associated with the exposure of an NH2-terminal epitope, and that this translocation could be partially blocked by the prosurvival factors Bcl-2 and Bcl-XL. To visualize the translocation of Bax in living cells, we have developed an H9c2 cell line that stably expresses green fluorescent protein (GFP)-tagged Bax. This cell line has GFP-Bax localized primarily in the cytosol in the absence of apoptotic inducers. Upon induction of apoptosis by a number of stimuli, including menadione, staurosporine, sodium nitroprusside, and hypoxia-reoxygenation-serum withdrawal, we could observe the translocation of Bax from cytosol to mitochondria. This translocation was not affected by retinoic acid-induced differentiation of H9c2 cells. Additionally, this translocation was associated with loss of mitochondrial membrane potential, release of cytochrome c, and fragmentation of nuclei. Finally, using a tetramethylrhodamine-based dye, we have shown that a rapid screening process based on the loss of mitochondrial membrane potential could be developed to monitor GFP-Bax translocation to mitochondria. Overall, the GFP-Bax-stable H9c2 cell line that we have developed represents a unique tool for examining Bax-mediated apoptosis, and it could be of great importance in screening therapeutic compounds that could block Bax translocation to mitochondria to attenuate apoptosis.