Action of Akt Pathway on La-Induced Hippocampal Neuron Apoptosis of Rats in the Growth Stage.

This study investigated the influences of lanthanum (La) exposure on learning and memory and the expression of apoptosis-related proteins in offspring rats. Wistar female rats were randomly divided into a control group (NC) and 0.25%, 0.5% and 1.0% LaCl3 treatment groups, with eight per group. La dye was transmitted to offspring rats through parental blood circulation and breast milk before delactation and through water drinking after delectation. Offspring rats were killed at 14, 28 and 42 days after birth. Hippocampal neurons were observed by microscope, and apoptosis and necrosis were tested. The expression levels of apoptosis-related proteins were detected by Western blot, and Morris water maze experiments were used to measure learning and memory abilities. LaCl3 groups showed longer escape latency periods and swimming distances than the NC group (p < 0.05). The 1.0% LaCl3 group passed across the target quadrants and platforms more times and stayed in the target quadrants for less time, than the NC group (p < 0.05). At 42 days, the apoptosis rate and necrosis in the hippocampus of the 1.0% LaCl3 group were significantly higher than those of other groups. There was a significant difference among LaCl3 groups in terms of protein expressions measured in the hippocampus. In LaCl3 groups, caspase-3 and caspase-9 were significantly higher than in the NC group (p < 0.05). Therefore, La exposure can promote neuronal apoptosis by regulating the protein expressions of Akt, Bcl-2, Bcl-xl, Bax, Bad, caspase-3 and caspase-9, thus damaging learning and memory and the hippocampal neurons of offspring rats.

Interferon γ is a STAT1-dependent direct inducer of BCL6 expression in imatinib-treated chronic myeloid leukemia cells.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Personalized prostate cancer therapy based on systems analysis of the apoptosis regulatory network.

Targeting the androgen receptor axis provides only temporary relief for advanced prostate cancer, which often evolves into androgen-independent disease. The wide variety of signaling mechanisms connected with the pathophysiology of androgen-independent prostate cancer poses both conceptual and practical challenges for the design of efficient therapies. Analysis of apoptosis regulation in prostate cancer suggests the potential value of a systems approach that integrates information on the topology of the antiapoptotic signaling network, the signal transduction pathways that inhibit apoptosis, and the expression of proteins of the Bcl2 family. This approach could be used to identify patients most likely to respond to treatments with drugs that inhibit the signaling pathways controlling apoptosis.

Baicalin induces apoptosis in leukemia HL-60/ADR cells via possible down-regulation of the PI3K/Akt signaling pathway.

BACKGROUND: The effect and possible mechanism of traditional Chinese medicine, baicalin, on the PI3K/ Akt signaling pathway in drug-resistant human myeloid leukemia HL-60/ADR cells have been investigated in this current study. METHODS: HL-60/ADR cells were treated by 20, 40, 80 µmol/L baicalin followed by cell cycle analysis at 24h. The mRNA expression level of the apoptosis related gene, Bcl-2 and bad, were measured by RT-PCR on cells treated with 80 µmol/L baicalin at 12, 24 and 48hr. Western blot was performed to detect the changes in the expression of the proteins related to HL-60/ADR cell apoptosis and the signaling pathway before and after baicalin treatment, including Bcl-2, PARP, Bad, Caspase 3, Akt, p-Akt, NF-κB, p-NF-κB, mTOR and p-mTOR. RESULTS: Sub-G1 peak of HL-60/ADR cells appeared 24 h after 20 µmol/L baicalin treatment, and the ratio increased as baicalin concentration increased. Cell cycle analysis showed 44.9% G0/G1 phase cells 24 h after baicalin treatment compared to 39.6% in the control group. Cells treated with 80 µmol/L baicalin displayed a trend in decreasing of Bcl-2 mRNA expression over time. Expression level of the Bcl-2 and PARP proteins decreased significantly while that of the PARP, Caspase-3, and Bad proteins gradually increased. No significant difference in Akt expression was observed between treated and the control groups. However, the expression levels of p-Akt, NF-κB, p-NF-κB, mTOR and p-mTOR decreased significantly in a time-dependent manner. CONCLUSIONS: We conclude that baicalin may induce HL-60/ADR cell apoptosis through the PI3K/AKT signaling pathway.

Activation of mitochondrial apoptosis pathways in cutaneous squamous cell carcinoma cells by diclofenac/hyaluronic acid is related to upregulation of Bad as well as downregulation of Mcl-1 and Bcl-w.

Actinic keratosis (AK) is characterized by high prevalence and the risk to proceed to squamous cell carcinoma (SCC). Cyclooxygenase-2 (COX-2)-mediated prostaglandin E2 (PGE (2) ) synthesis has been reported in AK and SCC, and the COX inhibitor diclofenac in hyaluronic acid (diclofenac/HA) was approved for AK therapy. Its mode of action, however, remained to be unravelled. In the present study, diclofenac resulted in reduced PGE (2) levels in apoptosis-sensitive cutaneous SCC cell lines (SCL-II, SCC-12, SCC-13) whereas no PGE (2) and no COX-2 expression was detectable in a SCC cell line resistant to apoptosis induction (SCL-I). Activation of mitochondrial apoptosis pathways was evident in SCC cells owing to loss of the mitochondrial membrane potential and release of the mitochondrial factors cytochrome c and apoptosis-inducing factor. Characteristic proapoptotic changes at the level of Bcl-2 proteins occurred in sensitive cells, as upregulation of Bad and downregulation of Mcl-1 and Bcl-w. In contrast, Bad was already high, and Mcl-1 and Bcl-w were already low in resistant SCL-I, even without treatment, which may be explained by the lack of PGE (2) . An antiapoptotic downregulation of proapoptotic Bcl-2 proteins Noxa and Puma was, however, also seen in SCL-I, suggesting here pathways independent of COX-2. The regulations of Mcl-1 and Bad were also reproduced in SCC cells by the more selective COX-2 inhibitor celecoxib, thus further underlining the specific role of COX-2. The findings illuminate the mode of action of diclofenac/HA in SCC cells as well as principles of their resistance, which may allow further adaptation and improvement of the new therapy.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells.

OBJECTIVES: Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. METHODS: Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. RESULTS: Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. CONCLUSIONS: These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

[Apoptosis-modulating effects of heat shock proteins: the influence of Hsp27 chaperone on TBA Bcl-2 family proteins in Jurkat cell line].

The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD.

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.

Reduction in lipopolysaccharide-induced apoptosis of fibroblasts obtained from a patient with gingival overgrowth during nifedipine-treatment.

OBJECTIVE: We have previously demonstrated that the mechanism of nifedipine (NIF)-induced gingival overgrowth is related to the observation that proliferation and cell cycle progression of gingival fibroblasts derived from NIF reactive patient (NIFr) are greater than those from NIF non-reactive patient (NIFn). Gingival overgrowth has also been reported to be a result of inhibited apoptosis of gingival fibroblasts. Apoptosis in fibroblasts is induced by lipopolysaccharide (LPS). Thus, we focused upon evaluating whether there is a difference in LPS-induced apoptosis between NIFn and NIFr. METHODS: Both NIFn and NIFr were arrested in DMEM containing 0.5% FBS, stimulated by LPS, and assayed for apoptosis, cell cycle analysis, Western blotting, and caspase activity. RESULTS: Compared to NIFn, the number of apoptotic cells was significantly decreased and the percentage of cells in S and G(2)/M phase was significantly increased in NIFr. The levels of Bax and cytochrome c proteins in NIFr were not up-regulated by LPS compared with NIFn. Both NIFn and NIFr displayed the following changes in protein expression: increased Bad, decreased Bcl-xL, and unchanged Bcl-2 and p53. Caspase-3 and -9 activities were significantly increased by LPS in NIFn but were unchanged in NIFr. Caspase-2 activity remained constant whilst caspase-8 activity significantly increased upon LPS treatment in both NIFn and NIFr. CONCLUSION: Bad, Bax, cytochrome c, p53, and caspases-2, -3, -8, and -9 are pro-apoptotic proteins. Bcl-2 and Bcl-xL are anti-apoptotic proteins. Thus, the mechanism of NIF-induced gingival overgrowth might be related to decreased apoptosis in NIFr through a reduction of Bax, cytochrome c, and caspase-3 and -9.

Butyric acid induces apoptosis via oxidative stress in Jurkat T-cells.

Reactive oxygen species (ROS) are essential for the induction of T-cell apoptosis by butyric acid, an extracellular metabolite of periodontopathic bacteria. To determine the involvement of oxidative stress in apoptosis pathways, we investigated the contribution of ROS in mitochondrial signaling pathways, death-receptor-initiated signaling pathway, and endoplasmic reticulum stress in butyric-acid-induced T-cell apoptosis. N-acetyl-L-Cysteine (NAC) abrogated mitochondrial injury, cytochrome c, AIF, and Smac release, and Bcl-2 and Bcl-xL suppression and Bax and Bad activation induced by butyric acid. However, the decrease in cFLIP expression by butyric acid was not restored by treatment with NAC; increases in caspase-4 and -10 activities by butyric acid were completely abrogated by NAC. NAC also affected the elevation of GRP78 and CHOP/GADD153 expression by butyric acid. These results suggest that butyric acid is involved in mitochondrial-dysfunction- and endoplasmic reticulum stress-mediated apoptosis in human Jurkat T-cells via a ROS-dependent mechanism.

alpha3beta1 integrin promotes cell survival via multiple interactions between 14-3-3 isoforms and proapoptotic proteins.

Laminin-5 and alpha3beta1 integrin promote keratinocyte survival; however, the downstream signaling pathways for laminin-5/alpha3beta1 integrin-mediated cell survival had not been fully established. We report the unexpected finding of multiple interactions between 14-3-3 isoforms and proapoptotic proteins in the survival signaling pathway. Ln5-P4 motif within human laminin-5 alpha3 chain promotes cell survival and anti-apoptosis by inactivating Bad and YAP. This effect is achieved through the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes, which is initiated by alpha3beta1 integrin and FAK/PI3K/Akt signaling. These complexes result in cytoplasmic sequestration of Bad and YAP and their subsequent inactivation. An increase in Akt1 activity in cells induces 14-3-3zeta and sigma, p-Bad, and p-YAP, promoting cell survival, whereas decreasing Akt activity suppresses the same proteins and inhibits cell survival. Suppression of 14-3-3zeta with RNA-interference inhibits cell viability and promotes apoptosis. These results reveal a new mechanism of cell survival whereby the formation of 14-3-3zeta/p-Bad and 14-3-3sigma/p-YAP complexes is initiated by laminin-5 stimulation via the alpha3beta1 integrin and FAK/PI3K/Akt signaling pathways, thereby resulting in cell survival and anti-apoptosis.

Antiapoptotic effects of vasopressin in the neuronal cell line H32 involve protein kinase Calpha and beta.

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway.

Sodium selenite induces apoptosis in acute promyelocytic leukemia-derived NB4 cells through mitochondria-dependent pathway.

Our previous study has shown that sodium selenite can cause apoptosis in acute promyelocytic leukemia-derived NB4 cells in a caspase-dependent manner involving Deltapsim disruption and cleavage of Bcl-2, but more detailed mechanism(s) remain unclear. Here we showed that mitochondrial apoptosis signaling pathway played a vital role in apoptosis induced by sodium selenite based on the following findings: 1) cytochrome c release, activation of caspase 9, mitochondrial targeting, and oligermerization of Bax; 2) caspase 9, but not caspase 8, inhibitor could attenuate apoptosis; 3) downregulation of Bax and Bad by siRNA could delay sodium selenite-induced apoptosis. Further investigation showed that ROS was an essential inducer of deltapsim disruption and apoptosis by sodium selenite. Our findings here demonstrate that sodium selenite can induce apoptosis in NB4 cells through a mechanism involving ROS, activation of proapoptotic proteins Bad and Bax, Deltapsim disruption, release of cytochrome c, and consequent initiation of caspase cascade.

Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia.

UNLABELLED: Insulin is neuroprotective following transient global brain ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear. OBJECTIVE: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a. METHODS: We utilized rats subjected to 10 minutes of global brain ischemia, with or without insulin administered at the onset of reperfusion. RESULTS: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusion nor insulin induced any change in the phosphorylation or subcellular localization of FoxO3a, Bim or Bad. A single bolus of high-dose insulin reduced CA1 neuronal cell death and thus represents a potential therapeutic intervention for global brain ischemia. DISCUSSION: These results reveal that proximal elements of a known cell-survival pathway are triggered by high-dose insulin during early reperfusion. Insulin induces robust PI3K-dependent phosphorylation of Akt by 30 minute reperfusion and results in improvement of hippocampal structure and function. However, the Akt substrates FoxO3a, Bim and Bad do not undergo corresponding changes in phosphorylation or subcellular localization in this model of global brain ischemia. The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified.

Involvement of BH3-only proteins in hematologic malignancies.

The interaction between anti-apoptotic and pro-apoptotic members of the Bcl-2 family proteins determines life or death for cancer cells. In this context, BH3-only proteins (such as Bim), members of the pro-apoptotic Bcl-2 family proteins, act as key initiators of apoptosis by activating Bax and Bak through liberating them from anti-apoptotic Bcl-2 members. This then leads to the disruption of mitochondrial outer membrane, and eventually promotes proteolytic cascades for cellular dismantling. We here review the growing evidence of how BH3-only proteins are involved in tumorigenesis and in apoptosis induced by anti-cancer agents in hematologic malignancies. A deeper understanding of the roles of BH3-only proteins in cell death regulation may yield crucial insights for the further development of more effective and rational cell killing strategies. Recent developments in the direct therapeutic manipulation of Bcl-2 proteins using BH3-mimicking agents, such as ABT-737 or GX15-070, for hematologic malignancies are also summarized.

Mechanisms of platelet-derived growth factor-mediated neuroprotection--implications in HIV dementia.

Platelet-derived growth factor (PDGF) has been implicated in promoting survival and proliferation of immature neurons, and even protecting neurons from gp120-induced cytotoxicity. However, the mechanisms involved in neuroprotection are not well understood. In the present study we demonstrate the role of phosphatidylinositol 3-kinase (PI3K)/Akt signaling in PDGF-mediated neuroprotection. Pharmacological inhibition of PI3K greatly reduced the ability of PDGF-BB to block gp120 IIIB-mediated apoptosis and cell death in human neuroblastoma cells. The role of Akt in PDGF-mediated protection was further corroborated using a dominant-negative mutant of Akt, which was able to block the protective effect of PDGF. We next sequentially examined the signals downstream of Akt in PDGF-mediated protection in human neuroblastoma cells. In cells pretreated with PDGF prior to gp120 there was increased phosphorylation of both GSK-3beta and Bad, an effect that was inhibited by PI3-kinase inhibitor. Nuclear translocation of NF-kappaB, which lies downstream of GSK-3beta, however, remained unaffected in cells treated with PDGF. In addition to inducing phosphorylation of Bad, PDGF-mediated protection also involved down-regulation of the proapoptotic protein Bax. Furthermore, PDGF-mediated protection also involved the inhibition of gp120-induced release of mitochondrial cytochrome C. Our findings thus underscore the roles of both PI3K/Akt and Bcl family pathways in PDGF-mediated neuroprotection.

Propofol protects hepatic L02 cells from hydrogen peroxide-induced apoptosis via activation of extracellular signal-regulated kinases pathway.

BACKGROUND: Propofol protects cells against ischemia/reperfusion injury in several organs, but there are few reports of its effect on liver epithelial cells. We investigated the effect of propofol preconditioning on human hepatic L02 cells under hydrogen peroxide (H2O2)-induced oxidative stress and attempted to determine whether the extracellular signal-regulated kinases (ERK) pathway is involved in this process. METHODS: Preconditioned or nonpreconditioned human hepatic L02 cells were exposed to H2O2 and the changes of apoptosis were evaluated by TUNEL assay, Caspase-3 and poly ADP-ribose polymerase (PARP) cleavage. Activation of ERK1/2 and mitogen-activated protein kinase//ERK Kinase 1/2 (MEK1/2) was measured by Western blot analysis. The mRNA expression of Bcl-2, Bcl-x(L), Bad, and Bax was quantified by real-time quantitative reverse transcriptase polymerase chain reaction. RESULTS: Propofol preconditioning reduced the population of apoptotic cells and Caspase-3 and PARP cleavage induced by H2O2 inhepatic L02 cells. L02 cells treated with propofol (0.01-0.3 mM) alone, led to a dose-dependent activation of ERK and MEK, and such activation was detected within 0.5 h and eventually declined to <50% at 4 h. The addition of the specific inhibitor PD98059 completely abolished the activation of ERK and aggravated the extent of apoptosis. Moreover, propofol treatment repressed the mRNA expression of proapoptotic genes Bad and Bax, and this repression could be partly reversed by PD98059. CONCLUSIONS: These findings demonstrate that propofol protects hepatic L02 cells from H2O2-induced apoptosis, partly through activating the MEK-ERK pathway and further suppressing Bad and Bax expression.

Transplacental exposure to the vacuolar-ATPase inhibitor bafilomycin disrupts survival signaling in beta cells and delays neonatal remodeling of the endocrine pancreas.

A wave of beta cell apoptosis occurs around 2 weeks of age in rats and mice. We have previously reported that exposure in utero to bafilomycin, a plecomacrolide antibiotic that inhibits the vacuolar (v)ATPase enzyme and contaminates the human diet, delays this neonatal wave and accelerates diabetes in non-obese diabetic (NOD) mice. Here we exposed C57BL/6J mice in utero to bafilomycin and assessed the effects on islet morphology, apoptosis and activation of cell survival signaling in beta cells. The neonatal wave of beta cell apoptosis was associated with high expression and low phosphorylation of the pro-apoptotic Bcl-2 family protein Bad, whereas after weaning (3 weeks of age) Bad was down-regulated and beta cell apoptosis was low. In contrast, in bafilomycin-exposed mice the frequency of apoptotic beta cells and the expression of Bad remained high after weaning. Bafilomycin exposure also inactivated the insulin/IGF signaling pathway intermediate, FoxO1, and increased the insulin content in neonatal islets. Thus, exposure in utero to bafilomycin disrupts the regulation of Bad in neonatal beta cells, increases cell survival signaling and delays the neonatal wave of apoptosis. Increased expression of Bad in adult beta cells provides an explanation for accelerated diabetes in bafilomycin-exposed NOD mice, whereby disruption of neonatal islet-cell turnover may render the adult beta cells more susceptible to induced cell death.

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells.

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca2+]c) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca2+) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca2+ transients ([Ca2+]c) and changes of mitochondrial Ca2+ concentrations ([Ca2+]m) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca2+]c and [Ca2+]m elevations elicited by K+ pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca2+]m was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca2+]c transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca2+ channel agonist Bay K 8644 enhanced K(+)-evoked [Ca2+]m peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K+ generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca2+]c and [Ca2+]m transients elicited by K+, in PC12 cells overexpressing Bcl2, is related to the reduction of Ca2+ entry through L-type Ca2+ channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca2+ channels, the subsequent Ca2+ entry, and mitochondrial Ca2+ overload.

Overactivation of calcineurin induced by amyloid-beta and prion proteins.

Amyloid-beta protein (A beta) and the scrapie isoform of prion protein (PrPSs) have a central role in the pathogenesis of Alzheimer's disease (AD) and prion-related encephalopathies (PRE), respectively. In both disorders, the deposition of these misfolded proteins is accompanied by apoptotic neuronal loss. However, the pathogenesis and molecular basis of A beta- and PrPSc-neurotoxic effects are not completely understood. The Ca2+/calmodulin-dependent phosphatase calcineurin (CaN), through the dephosphorylation of the proapoptotic protein BAD, may be the link between Ca2+homeostasis deregulation and apoptotic neuronal death. In this study we used primary cultures of rat brain cortical neurons in order to investigate whether A beta and PrP affect CaN activity. We observed that synthetic peptides of A beta (A beta 25-35 and A beta 1-40) and PrP (PrP106-126) increased CaN activity, but did not affect the levels of this protein phosphatase. Moreover, we found that these peptides reduced the levels of BAD phosphorylated at serine residue 112, and this effect was prevented by the CaN inhibitor FK506. Since dephosphorylated BAD translocates to mitochondria, where it triggers cytochrome c release, we determined the levels of BAD in mitochondrial and cytosolic fractions. The data obtained showed that A beta- and PrP-treated neurons had higher levels of BAD in mitochondria than control neurons. This increase in mitochondrial BAD levels was matched by a decrease in cytochrome c. FK506 prevented the alterations of mitochondrial BAD and cytochrome c levels induced by A beta and PrP peptides. Taken together the data suggest that A beta and PrP increased CaN activity, inducing BAD dephosphorylation and translocation to mitochondria and, subsequently, cytochrome c release that may trigger an apoptotic cascade. Therefore, therapeutic strategies targeting CaN might be valuable for these neurodegenerative disorders.