Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 microm N-acetylsphingosine (C(2)-ceramide) or 20 microm H(2)O(2) alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 microm H(2)O(2) enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 microm C(2)-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H(2)O(2), and in contrast, purified catalase inhibited the enhancement of oxidative damage by H(2)O(2) in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C(2)-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C(2)-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H(2)O(2) enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.     

Activation of Akt Kinase Inhibits Apoptosis and Changes in Bcl-2 and Bax Expression Induced by Nitric Oxide in Primary Hippocampal Neurons

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.     

Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

Oxidative stress plays a critical role in cardiac injuries during ischemia/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts. H2O2 treatment induced apoptosis in H9c2 cells, and pretreatment of cells with IGF-1 suppressed apoptotic cell death. The antiapoptotic effect of IGF-1 was blocked by LY294002 (an inhibitor of phosphatidylinositol 3-kinase) and by PD98059 (an inhibitor of extracellular signal-regulated kinase (ERK)). The protective effect of IGF-1 was also blocked by rapamycin (an inhibitor of p70 S6 kinase). Furthermore, H9c2 cells stably transfected with constitutively active PI 3-kinase (H9c2-p110*) and Akt (H9c2-Gag-Akt) constructs were more resistant to H2O2 cytotoxicity than control cells. Although H2O2 activates both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), IGF-1 inhibited only JNK activation. Activated PI 3-kinase (H9c2-p110*) and pretreatment of cells with IGF-1 down-regulated Bax protein levels compared to control cells. Taken together, our results suggest that IGF-1 transmits a survival signal against oxidative stress-induced apoptosis in H9c2 cells via PI 3-kinase and ERK-dependent pathways and the protective effect of IGF-1 is associated with the inhibition of JNK activation and Bax expression.     

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.     

Potential sites of PI-3 kinase function in the endocytic pathway revealed by the PI-3 kinase inhibitor, wortmannin

Previously we have shown that PDGF receptor mutants that do not bind PI- 3 kinase internalize after ligand binding, but fail to downregulate and degrade. To define further the role of PI-3 kinase in trafficking processes in mammalian cells, we have investigated the effects of a potent inhibitor of PI-3 kinase activity, wortmannin. At nanomolar concentrations, wortmannin inhibited both the transfer of PDGF receptors from peripheral compartments to juxtanuclear vesicles, and their subsequent degradation. In contrast, the delivery of soluble phase markers to lysosomes, assessed by the accumulation of Lucifer yellow (LY) in perinuclear vesicles after 120 min of incubation, was not blocked by wortmannin. Furthermore, wortmannin did not affect the rate of transferrin uptake, and caused only a small decrease in its rate of recycling. Thus, the effects of wortmannin on PDGFr trafficking are much more pronounced than its effects on other endocytic events. Unexpectedly, wortmannin also caused a striking effect on the morphology of endosomal compartments, marked by tubulation and enlargement of endosomes containing transferrin or LY. This effect was somewhat similar to that produced by brefeldin A, and was also blocked by pre-treatment of cells with aluminum fluoride (AlF4-). These results suggest two sites in the endocytic pathway where PI-3 kinase activity may be required: (a) to sort PDGF receptors from peripheral compartments to the lysosomal degradative pathway; and (b) to regulate the structure of endosomes containing lysosomally directed and recycling molecules. This latter function could be mediated through the activation of AlFt4-)-sensitive GTP-binding proteins downstream of PI-3 kinase.     

Insulin-like growth factor-1 protects peroxynitrite-induced cell death by preventing cytochrome c-induced caspase-3 activation

We investigated the effect of IGF-1 on cell death induced by peroxynitrite in human neuroblastoma SH-SY5Y cells. Exposure of the cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, caused cytochrome c release from the mitochondria, caspase-3-like activation, and cell death. Pre-incubation of the cells with the caspase-3 inhibitor partially prevented SIN-1-induced cell death. Simultaneous addition of IGF-1 reduced SIN-1-induced caspase-3-like activation and cell death, whereas IGF-1 failed to reduce the release of cytochrome c. IGF-1 increased Akt phosphorylation, and Akt phosphorylation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. In addition, wortmannin prevented IGF-1-evoked inhibition of cell death and caspase-3-like activation. In a cell-free system, addition of cytochrome c to cytosolic fraction resulted in caspase-3-like activation. The activation was reduced when the cytosolic fraction prepared from IGF-1-treated cells was used. These results suggest that IGF-1 protects peroxynitrite-induced cell death downstream of cytochrome c release through the inhibition of caspase-3-like activation.     

Wortmannin enhances activation of CPP32 (Caspase-3) induced by TNF or anti-Fas

CPP32/apopain (Caspase-3), a protease of the Ced-3/ICE family, is a central mediator in the apoptosis induced by TNF or anti-Fas. In this study we demonstrate that wortmannin, an inhibitor of PI-3K, enhances the activation of CPP32 (Caspase-3) and DNA fragmentation in TNF-treated U937 cells and anti-Fas-treated Jurkat cells. Caspase-3-like activity, Ac-DEVD-MCA cleavage activity, is enhanced by wortmannin in the range of the concentration (1 - 100 nM) specifically inhibiting PI-3K. LY294002, another PI-3K inhibitor, also enhances Caspase-3-like activity, but inhibitors for myosin light chain kinase and calmodulin dependent kinase do not have any effect on the Caspase-3-like activity. Wortmannin (1 - 100 nM) enhances the processing of Caspase-3 (32K) into active form (17K) in TNF- or anti-Fas-treated cells, but not in untreated cells. These observations suggest that inhibition of PI-3K induces the activation of processing enzyme of Caspase-3 or increases the susceptibility of Caspase-3 to the processing enzyme. PI-3K seems to protect the cells from apoptosis by suppressing the activation of Caspase-3.     

Erythropoietin attenuated high glucose-induced apoptosis in cultured human aortic endothelial cells

High glucose-induced apoptosis in vascular endothelial cells may contribute to the acceleration of atherosclerosis associated with diabetes. Here, we show that erythropoietin attenuates high glucose-induced apoptosis in cultured human aortic endothelial cells (HAECs). Exposure of HAECs to high glucose level for 72h significantly increased the number of apoptotic cells compared with normal glucose level, as evaluated by TUNEL assay. Simultaneous addition of erythropoietin (100 U/ml) significantly attenuated high glucose-induced apoptosis. In parallel, exposure to high glucose level induced caspase-3 activation and erythropoietin also prevented it. Erythropoietin stimulated Akt phosphorylation in a dose-dependent manner (1-100 U/ml). PI3 kinase inhibitor, wortmannin or LY294002 eliminated erythropoietin's inhibitory effect on caspase-3 activity. In conclusion, erythropoietin may attenuate high glucose-induced endothelial cell apoptosis via PI-3 kinase pathway. Replacing therapy with erythropoietin is often used for correction of renal anemia, but may have potential in preventing atherosclerosis in diabetic patients with end-stage renal failure.     

Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3beta signaling pathways and changes in protein expression

Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin-(IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3beta (GSK-3beta) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3beta may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of "candidate" proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3beta signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases.     

Identification of ASK1, MKK4, JNK, c-Jun, and caspase-3 as a signaling cascade involved in cadmium-induced neuronal cell apoptosis

Cd induces oxidative stress and apoptosis in various cells by activating mitogen-activated protein kinases (MAPKs), but the precise signaling components of the MAPK cascade and their role in neuronal apoptosis are still unclear. Here, we report that Cd treatment of SH-SY5Y cells caused apoptosis through sequential phosphorylation of the apoptosis signal regulating kinase 1, MAPK kinase 4, c-Jun N-terminal kinase (JNK), and c-Jun as determined by overexpression of dominant negative (DN) constructs of these genes or using a specific JNK inhibitor SP600125. Both Cd-induced JNK and c-Jun phosphorylation and apoptosis were inhibited dramatically by N-acetyl-L-cysteine, a free radical scavenger. In addition, caspase inhibitors, zDEVD and zVAD, reduced apoptosis but not JNK and c-Jun phosphorylation induced by Cd, while overexpression of DN JNK1 inhibited caspase-3 activity. Taken together, our data suggested that the JNK/c-Jun signaling cascade plays a crucial role in Cd-induced neuronal cell apoptosis and provides a molecular linkage between oxidative stress and neuronal apoptosis.     

Pro-apoptotic signaling in neuronal cells following iron and amyloid beta peptide neurotoxicity

In a previous report, we characterized several oxidative stress parameters during the course of amyloid beta (Abeta) peptide/Fe2+-induced apoptotic death in neuronal cells. In extending these findings, we now report a marked decrease in protein kinase C (PKC) isoforms, reduced Akt serine/threonine kinase activity, Bcl 2-associated death promoter (BAD) phosphorylation and enhanced p38 mitogen-activated protein kinase (MAPK) and caspase-9 and -3 activation, 12 h after addition of both 5 micro m Abeta and 5 micro m Fe2+. These activities reminiscent for a pro-apoptotic cellular course were blocked in the presence of the iron chelator deferroxamine. Abeta alone, increased PKC isoform levels between three- and four-fold after 12 h, enhanced Akt activity approximately eight-fold and Ser136 BAD phosphorylation two-fold, suggesting that by itself is not toxic. Fe2+ alone transiently enhanced p38 MAPK and caspase-9 and -3 enzymes indicative for cell damage, but was not sufficient to cause cell death as previously indicated. GF, a PKC inhibitor or wortmannin, a blocker of the Akt pathway enhanced Abeta/Fe2+-induced toxicity, while SB, a p38 MAPK inhibitor, prevented cell damage and apoptosis. These findings further support the hypothesis that metal ion chelation and inhibitors of pro-apoptotic kinase cascades may be beneficial for Alzheimer's disease therapy.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis

The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Apelin suppresses apoptosis of human osteoblasts

Objectives: Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. Osteoblast can express apelin and APJ. The aim of this study was to investigate the action of apelin on apoptosis of human osteoblasts. Results: Apelin inhibited human osteoblasts apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Our study also showed an increased Bcl-2 protein expression and decreased Bax protein expression under the treatment of apelin. Apelin decreased cytochrome c release and caspase-3 activation in human osteoblasts. Apelin activated phosphatidylinositol-3 kinase (PI-3 kinase) and Akt. The apelin-induced activation of Akt was blocked by suppression of APJ with siRNA. LY294002 (a PI-3 kinase inhibitor) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO; an Akt inhibitor) abolished apelin induced activation of Akt, and, LY294002 or HIMO abolished the anti-apoptotic activity of apelin. Furthermore, apelin protects against apoptosis induced by the glucocorticoid dexamethasone. Conclusions: Apelin suppresses serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway. Hui Xie and Ling-Qing Yuan both authors contributed equally to this work.     

Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.     

Cell Adhesion Induces the Plasminogen Activator Inhibitor-1 Gene Expression through Phosphatidylinositol 3-Kinase/Akt Activation in Anchorage Dependent Cells

Type-I plasminogen activator inhibitor (PAI-1) is the primary inhibitor of both tissue- and urokinase-type plasminogen activators (t-PA, u-PA) and is thus a primary regulator of plasminogen activation and possibly of extracellular proteolysis. In anchorage-dependent cells, the PAI-1 gene was regulated by cell adhesion. PAI-1 gene expression was induced more evidently in cells adhered to the culture plate than in nonadherent cells. In this study, we investigated the signal pathway of the PAI-1 gene expression regulated by cell adhesion. We found the induction of both PAI-1 mRNA and protein, when cells adhered to culture dish, was inhibited by the PI-3 kinase specific inhibitors (Ly294002 and wortmannin). The cells seeded on collagen-1 coated plate with low serum further demonstrated that the PAI-1 gene expression was prolonged by the cell adhesion. The above-mentioned PI-3 kinase specific inhibitors also blocked the PAI-1 maintenance when cell adhered to collagen-1 coated plate. In addition, we found that both PI-3 kinase and its downstream molecule, Akt, were activated more evidently in adherent cells than in nonadherent cells. Furthermore, we transfected antisense oligodeoxynucleotides of Akt (AS-ODN-Akt) into cells to block the expression of Akt and found that the induction of PAI-1 mRNA was also inhibited. Hence, we conclude that the induction of PAI-1 gene expression is cell adhesion dependent and is through PI-3 kinase and Akt activation.