APP induces neuronal apoptosis through APP-BP1-mediated downregulation of β-catenin

Alzheimer's disease (AD) is a neurodegenerative disease associated with progressive dementia. This mini-review focuses on how the amyloid precursor protein (APP) plays a central role in AD and Down syndrome as the regulator of the APP-BP1/hUba3 activated neddylation pathway. It is argued that the physiological function of APP is to downregulate the level of beta-catenin. However, this APP function is abnormally amplified in patients with familial AD (FAD) mutations in APP and presenilins, resulting in the hyperactivation of neddylation and the decrease of beta-catenin below a threshold level. Evidence in the literature is summarized to show that dysfunction of APP in downregulating beta-catenin may underlie the mechanism of neuronal death in AD and Down syndrome.     

NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α

Natriuretic peptide receptor 3 (NPR3) is a clearance receptor by binding and internalizing natriuretic peptides (NPs) for ultimate degradation. Patients with cardiac failure show elevated NPs. NPs are linked to poor long-term survival because of their apoptotic effects. However, the underling mechanisms have not been identified yet. Here we report the role of NPR3 in anti-apoptosis via the breast cancer type 1 susceptibility protein (BRCA1) and tumor necrosis factor α (TNF-α ). To demonstrate a role for NPR3 in apoptosis, stable H9C2 cardiomyocyte cell lines using shRNA to knockdown NPR3 were generated. The activities of caspase-3, 8, and 9 were significantly increased in NPR3 knockdown H9C2 cardiomyocytes. Knockdown of NPR3 increased the expression of BRCA1. Also NPR3 knockdown remarkably increased the activity of cAMP response element-binding protein (CREB), a positive regulatory element for BRCA1 expression. BRCA1 showed dispersed nuclear localization in non-cardiomyocytes while predominantly cytoplasmic localization in H9C2 cells. Meanwhile, NPR3 knockdown significantly increased TNF-α gene expression. These data show that NPR3 knockdown in H9C2 cells triggered both extrinsic and intrinsic apoptotic pathways. NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α, which are regulators of apoptosis. Our studies demonstrate anti-apoptosis role of NPR3 in protecting cardiomyocytes and establish the first molecular link between NP system and programmed cell death.     

Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppression of caspases-9 and -3 activation but not of caspase-8 activation, suggesting that radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis additionally requires changes upstream of caspase-8 activation. Herein, upregulation of FLIP may play a crucial role. Cleavage of bid, upregulation of bax, downregulation of bcl-2 and release of cytochrome c after TNF-α-administration depend on radiation-induced upregulation of IκB, thus demonstrating an apoptosis permitting effect of IκB. H. Gürleyen and H. Christiansen contributed equally to this work.     

Disruption of group IVA cytosolic phospholipase A(2) attenuates myocardial ischemia-reperfusion injury partly through inhibition of TNF-α-mediated pathway

Group IVA cytosolic phospholipase A(2) (cPLA(2)α), which preferentially cleaves arachidonic acid from phospholipids, plays a role in apoptosis and tissue injury. Downstream signals in response to tumor necrosis factor (TNF)-α, a mediator of myocardial ischemia-reperfusion (I/R) injury, involve cPLA(2)α activation. This study examined the potential role of cPLA(2)α and its mechanistic link with TNF-α in myocardial I/R injury using cPLA(2)α knockout (cPLA(2)α(-/-)) mice. Myocardial I/R was created with 10-wk-old male mice by 1 h ligation of the left anterior descending coronary artery, followed by 24 h of reperfusion. As a result, compared with wild-type (cPLA(2)α(+/+)) mice, cPLA(2)α(-/-) mice had a 47% decrease in myocardial infarct size, preservation of echocardiographic left ventricle (LV) function (%fractional shortening: 14 vs. 21%, respectively), and lower content of leukotriene B(4) and thromboxane B(2) (62 and 50% lower, respectively) in the ischemic myocardium after I/R. Treatment with the TNF-α inhibitor (soluble TNF receptor II/IgG1 Fc fusion protein, sTNFR:Fc) decreased myocardial I/R injury and LV dysfunction in cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) mice. sTNFR:Fc also suppressed cPLA(2)α phosphorylation in the ischemic myocardium after I/R of cPLA(2)α(+/+) mice. Similarly, sTNFR:Fc exerted protective effects against hypoxia-reoxygenation (H/R)-induced injury in the cultured cardiomyocytes from cPLA(2)α(+/+) mice but not cPLA(2)α(-/-) cardiomyocytes. H/R and TNF-α induced cPLA(2)α phosphorylation in cPLA(2)α(+/+) cardiomyocytes, which was reversible by sTNFR:Fc. In cPLA(2)α(-/-) cardiomyocytes, TNF-α induced apoptosis and release of arachidonic acid to a lesser extent than in cPLA(2)α(+/+) cardiomyocytes. In conclusion, disruption of cPLA(2)α attenuates myocardial I/R injury partly through inhibition of TNF-α-mediated pathways.     

Resolvin D1 prevents TNF-α-mediated disruption of salivary epithelial formation

Sj?gren's syndrome is a chronic autoimmune disorder characterized by inflammation of salivary glands resulting in impaired secretory function. Our present studies indicate that chronic exposure of salivary epithelium to TNF-α and/or IFN-γ alters tight junction integrity, leading to secretory dysfunction. Resolvins of the D-series (RvDs) are endogenous lipid mediators derived from DHA that regulate excessive inflammatory responses leading to resolution and tissue homeostasis. In this study, we addressed the hypothesis that activation of the RvD1 receptor ALX/FPR2 in salivary epithelium prevents and/or resolves the TNF-α-mediated disruption of acinar organization and enhances monolayer formation. Our results indicate that 1) the RvD1 receptor ALX/FPR2 is present in fresh, isolated cells from mouse salivary glands and in cell lines of salivary origin; and 2) the agonist RvD1 (100 ng/ml) abolished tight junction and cytoskeletal disruption caused by TNF-α and enhanced cell migration and polarity in salivary epithelium. These effects were blocked by the ALX/FPR2 antagonist butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe. The ALX/FPR2 receptor signals via modulation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways since, in our study, blocking PI3K activation with LY294002, a potent and selective PI3K inhibitor, prevented RvD1-induced cell migration. Furthermore, Akt gene silencing with the corresponding siRNA almost completely blocked the ability of Par-C10 cells to migrate. Our findings suggest that RvD1 receptor activation promotes resolution of inflammation and tissue repair in salivary epithelium, which may have relevance in the restoration of salivary gland dysfunction associated with Sj?gren's syndrome.     

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-α-mediated apoptosis

Tumour necrosis factor-α(TNF-α) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-α increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-α induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-α (50ng/ml), a slow rise in intracellular free Ca²⁺ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3h after the addition of TNF-α) were observed in fluo-3 and furared or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-α in the presence of BAPTA/AM, an intracellular Ca²⁺ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-α-mediated Ca²⁺ rise. Moreover, when cells were exposed to TNF-α with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca²⁺ are two cross-talk messengers important in TNF-α-mediated apoptosis.     

Gsdma3 is a new factor needed for TNF-α-mediated apoptosis signal pathway in mouse skin keratinocytes

Gsdma3, a newly found gene, is expressed restrictedly in mouse skin keratinocytes and gastrointestinal tract. But until now, there is little information on the regulation and the function of Gsdma3 in skin keratinocytes. In our previous study, we found that Gsdma3 mutation resulted in a decrease in catagen-associated apoptosis of hair follicle keratinocytes. Apoptosis of skin keratinocytes is strictly regulated by a series of signal pathways, among of which, tumor necrosis factor (TNF)-α-induced signal pathway has been extensively studied. To further investigate the role and the pathway of Gsdma3 involved in skin keratinocyte apoptosis, using immunofluorescence, RT-PCR, western blot and TUNEL analysis, we showed here that accompanying TNF-α-induced apoptosis and Caspase-3 expression in mouse skin keratinocytes in vivo and in vitro, Gsdma3 expression was significantly upregulated. After Gsdma3 gene mutation, TNF-α-induced apoptosis and Caspase-3 expression in skin keratinocytes were reduced. The injection of Gsdma3 expression plasmid could directly enhance the apoptosis and Caspase-3 expression in skin keratinocytes. These results, taken together, indicated that in mouse skin keratinocytes, Gsdma3 expression could be regulated by TNF-α. Gsdma3 was not only involved in but also necessary for the TNF-α-induced apoptosis pathway by directly enhancing the Caspase3 expression as well as the apoptosis induction.     

Effect of simvastatin on endothelial cell apoptosis mediated by Fas and TNF-α

Although there is evidence suggesting that statins may exert an endothelial protecting effect, recent in vitro data have shown that these compounds may induce endothelial cells (EC) apoptosis. We previously reported that the Fas-death receptor may induce apoptosis of the liver sinusoid endothelial cells (LSEC), and that TNF-α increases the susceptibility of these cells to suffer Fas-mediated apoptosis. Based on this evidence, in this study, we investigated the effect of simvastatin on Fas-mediated LSEC apoptosis. Simvastatin induced a significant reduction in LSEC viability, in a dose dependent manner, under serum-containing or serum-free conditions. This effect was prevented by mevalonate and GGPP, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. The simvastatin effect on LSEC death was not associated with increased activation of caspase-3. We found that simvastatin increased the susceptibility of LSEC death mediated by Fas. Further, simvastatin increased LSEC-apoptosis induced by Fas and TNF-α. Mevalonate and GGPP partially prevented simvastatin-induced sensitization to LSEC death mediated by Jo2 and TNF-α, but not Jo2 alone. Simvastatin did not induce up-regulation of the Fas on the LSEC. Our results provide evidence of simvastatin in modulating Fas-mediated apoptosis in endothelial cells. These results may have clinical implications in those clinical conditions associated with high levels of FasL and TNF-α.     

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease.     

Mitochonic acid-5 attenuates TNF-α-mediated neuronal inflammation via activating Parkin-related mitophagy and augmenting the AMPK–Sirt3 pathways

Mitochondrial dysfunction has been found to be associated with neuronal inflammation; however, no effective drug is available to attenuate neuroinflammation via sustaining mitochondrial function. In the current study, experiments were performed to understand the beneficial effects of mitochonic acid 5 (MA-5) on tumor necrosis factor-α (TNF-α)-mediated neuronal injury and mitochondrial damage. Our data illustrated that MA-5 pretreatment reduced inflammation response induced by TNF-α in CATH.a cells. Molecular investigations demonstrated that MA-5 pretreatment repressed oxidative stress, inhibited endoplasmic reticulum stress, sustained cellular energy metabolism, and blocked cell apoptosis induced by TNF-α stress. Further, we found that MA-5 treatment elevated the expression of Sirtuin 3 (Sirt3) and this effect was dependent on the activation of AMP-activated protein kinase (AMPK) pathway. Blockade of AMPK abolished the promotive action of MA-5 on Sirt3 and thus mediated mitochondrial damage and cell death. Besides, we also found that MA-5 treatment augmented Parkin-related mitophagy and increased mitophagy promoted CATH.a cells survival via improving mitochondrial function. Knockdown of Parkin abolished the beneficial action of MA-5 on mitochondrial homeostasis and CATH.a cell survival. Altogether, our results confirm that MA-5 is an effective drug to attenuate neuroinflammation via sustaining mitochondrial damage and promoting CATH.a cell survival. The protective action of MA-5 on neuronal damage is associated with Parkin-related mitophagy and the activation of AMPK–Sirt3 pathways.     

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/β-mediated apoptosis in Ubp43-deficient hematopoietic cells

Stress associated proteins (SAPs) in plants contain A20-type zinc finger (A20_ZF) domains and are involved with abiotic stress response. A20-type zinc finger domains in animals reportedly recognize ubiquitin as a regulatory signal in cell. However, it remains unclear whether A20_ZF domains in plants perform similar roles. AtSAP5, a SAP from Arabidopsis thaliana, exhibits a unique sequence feature among 10 AtSAPs harboring A20_ZF domains. The highly conserved diaromatic patch is replaced by the dialipathic patch. Here we investigated whether AtSAP5 recognizes ubiquitin and the roles of the dialipathic patch in ubiquitin binding in vitro. GST pulldown assay reveals that AtSAP5 binds polyubiquitin rather than monoubiquitin. AtSAP5 shows preferences for linear and K63-linked polyubiquitin chains to K48-linked one. The A20_ZF domain of AtSAP5 is sufficient for linkage-specific polyubiquitin recognition. The dialipathic patch in AtSAP5 plays an important role in K48-linked polyubiquitin recognition. Taken together, our results suggest that AtSAP5 participates in polyubiquitin recognition in plants and that the dialipathic patch in AtSAP5 is critical in binding K48-linked polyubiquitn chains.     

Involvement of both tumor necrosis factor-α-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin

We previously reported that necrosis occurs predominantly in porcine renal tubular LLC-PK₁ cells, when the cells were exposed transiently to a high concentration of cisplatin. Moreover, we demonstrated that generation of reactive oxygen species and subsequent production of tumor necrosis factor-α (TNF-α) through phosphorylation of p38 MAPK are implicated in the pathogenesis of cisplatin-induced renal cell injury. However, some TUNEL-positive cells appeared in renal proximal tubules of rats after systemic injection of cisplatin, suggesting an involvement of apoptosis. In the present study, we found in LLC-PK₁ cells that both apoptosis and necrosis were elicited when the cells were exposed to 200 μM cisplatin for 1 h followed by incubation for 24 h in the presence of 20 μM cisplatin. The cisplatin-induced necrosis was largely attenuated by the antioxidant N-acetylcysteine, while apoptosis was prevented by the specific inhibitors for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-α but not by the p38 MAPK inhibitor SB203580. On the other hand, SB203580 attenuated the cisplatin-induced increase in TNF-α production. These findings suggest that p53-mediated activations of caspases-2, -8 and -3 play a key role in cisplatin-induced renal cell apoptosis, while oxidative stress-induced TNF-α synthesis via p38 MAPK phosphorylation contributed to the necrosis.     

Inhibition of TNF-α-mediated inflammatory responses by a benzodioxolylacetylamino-linked benzothiazole analog in human fibroblast-like synoviocytes

The pathologic processes of rheumatoid arthritis are mediated by a number of cytokines, chemokines, and matrix metalloproteinases, the expressions of which are controlled by NF-κB. This study was performed to explore the effects of a benzothiazole analog, SPA0537, on the control of the NF-κB activation pathway. We also investigated whether SPA0537 had any anti-inflammatory effects in human rheumatoid fibroblast-like synoviocytes (FLS). SPA0537 inhibited the nuclear translocation and the DNA binding of NF-κB subunits, which correlated with the inhibitory effects on IKK phosphorylation and IκBα degradation in TNF-α-stimulated rheumatoid FLS. These events further suppressed chemokine production, matrix metalloproteinase secretion, and TNF-α-induced cell proliferation. In addition, SPA0537 inhibited the osteoclast differentiation induced by macrophage colony-stimulating factor (MCSF) and receptor activator of the NF-κB ligand (RANKL) in bone marrow macrophages. These findings suggest that SPA0537 exerts anti-inflammatory effects in rheumatoid FLS through the inhibition of the NF-κB pathway. Therefore, it may have therapeutic value for the treatment of rheumatoid arthritis.     

EVER2 protein binds TRADD to promote TNF-α-induced apoptosis

EVER1 and 2 confer resistance to cutaneous oncogenic human papillomavirus infections by downregulating the activating protein 1 (AP-1) signaling pathway. Defects in their expression are associated with susceptibility to epidermodysplasia verruciformis, which is characterized by persistent β-HPV infection, tumor necrosis factor alpha (TNF-α) overproduction in keratinocytes and the development of skin cancers. TNF-α-induced apoptosis is a key defense strategy, preventing the persistence of the virus within cells, but the role of EVER proteins in this cell death mechanism triggered by extrinsic stimuli is unknown. We show here that EVER2 induces TNF-α- and TRAIL-dependant apoptosis. It interacts with the N-terminal domain of TRADD, impairs the recruitment of TRAF2 and RIPK1 and promotes apoptosis. The skin cancer-associated EVER2 I306 allele results in an impaired TRADD–EVER2 interaction, with lower levels of cell death following treatment with TNF-α. These data highlight a new, critical function of EVER2 in controlling cell survival in response to death stimuli.     

Propionibacterium acnes induces acute TNFα-mediated apoptosis of hepatocytes followed by inflammatory T-cell-mediated granulomatous hepatitis in mice

The CD3+/TCR+ T-cell-mediated hepatic inflammation induced byPropionibacterium acnes could be divided into an acute and a chronic phase. The acute phase occurred within 72 h after injection and displayed hepatic apoptosis. Anti-TNF antibody inhibited both theP. acnes-induced hepatic apoptosis and lymphocyte infiltration seen in this phase, indicating the involvement of this cytokine. Thereafter, a chronic phase was manifested from days 7 to 14 after injection. It was characterized as granulomatous inflammation admixed with apoptosis of infiltrating lymphocytes and some hepatocytes. Immunohistochemical staining showed that the infiltrating lymphocytes displayed TNF, TNF type I receptor and a variety of cytokines including IL-1, IL-4, IL-6, IL-10, IFN or IL-12. Interestingly, in naive mice, the arteries in the liver constitutively expressed IFN. Its expression appeared to be substantially increased at 48 h, decreased at 72 h, and increased again on day 14 afterP. acnes injection. Furthermore, Fas or FasL was only detected on the lymphocytes within the granuloma. We conclude thatP. acnes can induce a TNF-mediated acute hepatic apoptosis which subsequently progress to a T-cell-mediated granulomatous hepatitis with increased expression of multiple cytokines and Fas/FasL.     

Chemical inhibition of HSP90 inhibits TNF-α mediated proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLS) proliferate abnormally and resist apoptosis. Geldanamycin (GA) and other HSP90 inhibitors have emerged as promising therapeutic agents that inhibited cancer cell growth. In this study, we explored the effects of HSP90 inhibitor, GA, on tumor necrosis factor (TNF)-α-induced proliferation and apoptosis of RAFLS, and the underlying mechanism. Human RAFLS was isolated from the knee joints of patients with RA and subjected to TNF-α treatment in combination of various concentration of GA. We found that GA dose-dependently inhibited TNF-α-induced RAFLS proliferation as measured, but promoted RAFLS apoptosis. Further mechanistic study identified that GA dose-dependently attenuated TNF-α-mediated activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways, both of which are involved in TNF-α-mediated RAFLS proliferation. Moreover, GA-induced apoptosis and mitochondrial damage of RAFLS, as evidenced by increased Bax/Bcl-2 ratio and mitochondrial cytochrome c release, and enhanced cleavages of caspase-3, caspase-9, and poly-(ADP-ribose) polymerase. Collectively, our results revealed that chemical inhibition of HSP90 by GA suppressed TNF-α-induced proliferation of RAFLSs through the MAPK and NF-κB signaling pathways and induces RAFLS apoptosis via mitochondria-dependent pathway. These findings demonstrated for the first time that HSP90 inhibition in RAFLS could be therapeutic beneficial for RA.