Antiapoptotic factor humanin is expressed in normal and tumoral pituitary cells and protects them from TNF-α-induced apoptosis.
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ABSTRACT:
SUBMITTER: Gottardo MF
PROVIDER: S-EPMC4216097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-? in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-?B in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-?-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-?B pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-?-induced apoptosis of GH3 cells, suggesting that the NF-?B pathway is involved in HN action in tumor pituitary cells. HN also induced NF-?B-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.
| S-EPMC5704037 | biostudies-literature
Project description:Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I-VI) and late stages (IX-XIV) but not at middle stages (VII-VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII-VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low.
| S-EPMC5034733 | biostudies-literature
Project description:Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1-20 ?g/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.
| S-EPMC5081188 | biostudies-literature
Project description:BackgroundOsteoarthritis (OA) is a common degenerative disease, and tumor necrosis factor (TNF-α) is known to play a critical role in OA. Cortistatin (CST) is a neuropeptide discovered over 20 years ago, which plays a vital role in inflammatory reactions. However, it is unknown whether CST is involved in cartilage degeneration and OA development.MethodsThe interaction between CST and TNF-α receptors was investigated through Coimmunoprecipitation and Biotin-based solid-phase binding assay. Western blot, Real-time PCR, ELISA, immunofluorescence staining, nitrite production assay and DMMB assay of GAG were performed for the primary chondrocyte experiments. Surgically induced and spontaneous OA models were established and western blot, flow cytometry, Real-time PCR, ELISA, immunohistochemistry and fluorescence in vivo imaging were performed for in vivo experiments.FindingsCST competitively bound to TNFR1 as well as TNFR2. CST suppressed proinflammatory function of TNF-α. Both spontaneous and surgically induced OA models indicated that deficiency of CST led to an accelerated OA-like phenotype, while exogenous CST attenuated OA development in vivo. Additionally, TNFR1- and TNFR2-knockout mice were used for analysis and indicated that TNFRs might be involved in the protective role of CST in OA. CST inhibited activation of the NF-κB signaling pathway in OA.InterpretationThis study provides new insight into the pathogenesis and therapeutic strategy of cartilage degenerative diseases, including OA. FUND: The National Natural Science Foundation of China, the Natural Science Foundation of Shandong Province, Key Research and Development Projects of Shandong Province and the Cross-disciplinary Fund of Shandong University.
| S-EPMC6443028 | biostudies-literature
Project description:Several microbial pathogens can modulate the host apoptotic response to infection, which may contribute to immune evasion. Various studies have reported that infection with the sexually transmitted disease pathogen Neisseria gonorrhoeae can either inhibit or induce apoptosis. N. gonorrhoeae infection initiates at the mucosal epithelium, and in women, cells from the ectocervix and endocervix are among the first host cells encountered by this pathogen. In this study, we defined the antiapoptotic effect of N. gonorrhoeae infection in human endocervical epithelial cells (End/E6E7 cells). We first established that N. gonorrhoeae strain FA1090B failed to induce cell death in End/E6E7 cells. Subsequently, we demonstrated that stimulation with N. gonorrhoeae protected these cells from staurosporine (STS)-induced apoptosis. Importantly, only End/E6E7 cells incubated with live bacteria and in direct association with N. gonorrhoeae were protected from STS-induced apoptosis, while heat-killed and antibiotic-killed bacteria failed to induce protection. Stimulation of End/E6E7 cells with live N. gonorrhoeae induced NF-kappaB activation and resulted in increased gene expression of the NF-kappaB-regulated antiapoptotic genes bfl-1, cIAP-2, and c-FLIP. Furthermore, cIAP-2 protein levels also increased in End/E6E7 cells incubated with gonococci. Collectively, our results indicate that the antiapoptotic effect of N. gonorrhoeae in human endocervical epithelial cells results from live infection via expression of host antiapoptotic proteins. Securing an intracellular niche through the inhibition of apoptosis may be an important mechanism utilized by N. gonorrhoeae for microbial survival and immune evasion in cervical epithelial cells.
| S-EPMC2738021 | biostudies-literature
Project description:Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell-bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-alpha is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-alpha receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-alpha. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.
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Project description: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.
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Project description:Phagocytosis induced cell death (PICD) is crucial for controlling phagocyte effector cells, such as monocytes, at sites of infection, and essentially contributes to termination of inflammation. Here we tested the hypothesis, that during PICD bystander apoptosis of non-phagocyting monocytes occurs, that apoptosis induction is mediated via tumor necrosis factor-alpha (TNF-α and that TNF-α secretion and -signalling is causal. Monocytes were infected with Escherichia coli (E. coli), expressing green fluorescent protein (GFP), or a pH-sensitive Eos-fluorescent protein (EOS-FP). Monocyte phenotype, phagocytic activity, apoptosis, TNF-receptor (TNFR)-1, -2-expression and TNF-α production were analyzed. Apoptosis occured in phagocyting and non-phagocyting, bystander monocytes. Bacterial transport to the phagolysosome was no prerequisite for apoptosis induction, and desensitized monocytes from PICD, as confirmed by EOS-FP expressing E. coli. Co-cultivation with non-infected carboxyfluorescein-succinimidyl-ester- (CFSE-) labelled monocytes resulted in significant apoptotic cell death of non-infected bystander monocytes. This process required protein de-novo synthesis and still occurred in a diminished way in the absence of cell-cell contact. E. coli induced a robust TNF-α production, leading to TNF-mediated apoptosis in monocytes. Neutralization with an anti-TNF-α antibody reduced monocyte bystander apoptosis significantly. In contrast to TNFR2, the pro-apoptotic TNFR1 was down-regulated on the monocyte surface, internalized 30 min. p.i. and led to apoptosis predominantly in monocytes without phagocyting bacteria by themselves. Our results suggest, that apoptosis of bystander monocytes occurs after infection with E. coli via internalization of TNFR1, and indicate a relevant role for TNF-α. Modifying monocyte apoptosis in sepsis may be a future therapeutic option.
| S-EPMC3547953 | biostudies-literature
Project description:Ceramide regulates several different cellular responses including mechanisms leading to apoptosis. Serum- and glucocorticoid-inducible protein kinase (SGK)-1 is a serine threonine kinase, which activates survival pathways in response to stress stimuli. Recently, we demonstrated an anti-apoptotic role of SGK-1 in human umbilical endothelial cells treated with high glucose. In the present study, since ceramide induces apoptosis by multiple mechanisms in diabetes and its complication such as nephropathy, we aimed to investigate whether SGK-1 may protect even against apoptosis induced by ceramide in kidney cells. Human embryonic kidney (HEK)-293 cells stable transfected with SGK-1 wild type (SGK-1wt) and its dominant negative gene (SGK-1dn) have been used in this study. Apoptotic stimuli were induced by C2-ceramide and TNF-? to increase endogenous synthesis of ceramide. Upon activation with these stimuli, SGK-1wt transfected cells have a statistically significant reduction of apoptosis compared with SGK-1dn cells (P<0.001). This protection was dependent on activation of caspase-3 and Poly-ADP-ribose-polymerase-1 (PARP-1) cleavage. SGK-1 and AKT-1 two highly homologous kinases differently reacted to ceramide treatment, since SGK-1 increases in response to apoptotic stimulus while AKT-1 decreases. This enhancement of SGK-1 was dependent on p38-mitogen-activated-protein kinases (p38MAPK), cyclic-adenosine-monophosphate/protein kinase A (cAMP/PKA) and phosphoinositide-3-kinase (PI3K) pathways. Especially, by using selective LY294002 inhibitor, we demonstrated that the most involved pathway in the SGK-1 mediated process of protection was PI3K. Treatment with inhibitor of SGK-1 (GSK650394) significantly enhanced TNF-?-dependent apoptosis in HEK-293 cells overexpressing SGK-1wt. Caspase-3, -8 and -9 selective inhibitors confirmed that SGK-1 reduced the activation of caspase-dependent apoptosis, probably by both intrinsic and extrinsic pathways. In conclusion, we demonstrated that in kidney cells, overexpression of SGK-1 is protective against ceramide-induced apoptosis and the role of SGK-1 can be potentially explored as a therapeutic target in conditions like diabetes, where ceramide levels are increased.
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Project description:Sepsis is a systemic inflammatory disorder with organ dysfunction and represents the leading cause of mortality in non-coronary intensive care units. A key player in septic shock is Tumor Necrosis Factor-alpha (TNF-?). Phospholipase (PL)D1 is involved in the regulation of TNF-? upon ischemia/reperfusion injury in mice. In this study we analyzed the impact of PLD1 in the regulation of TNF-?, inflammation and organ damage in experimental sepsis. PLD1 deficiency increased survival of mice and decreased vital organ damage after LPS injections. Decreased TNF-? plasma levels and reduced migration of leukocytes and platelets into lungs was associated with reduced apoptosis in lung and liver tissue of PLD1 deficient mice. PLD1 deficient platelets contribute to preserved outcome after LPS-induced sepsis because platelets exhibit an integrin activation defect suggesting reduced platelet activation in PLD1 deficient mice. Furthermore, reduced thrombin generation of PLD1 deficient platelets might be responsible for reduced fibrin formation in lungs suggesting reduced disseminated intravascular coagulation (DIC). The analysis of Pld1fl/fl-PF4-Cre mice revealed that migration of neutrophils and cell apoptosis in septic animals is not due to platelet-mediated processes. The present study has identified PLD1 as a regulator of innate immunity that may be a new target to modulate sepsis.
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