Inhibition Of Caspase Activity Research Articles

Diosgenin attenuates the brain injury induced by transient focal cerebral ischemia-reperfusion in rats

The aim of the present study is to explore the potential cerebroprotection of diosgenin against the transient focal cerebral ischemia-reperfusion (I/R) injury and its possible underlying mechanisms. The diosgenin at two dose levels, namely 100 and 200mgkg−1, was intragastrically administrated once daily for 7-day period prior to the surgery. Then, the rats were subjected to middle cerebral artery occlusion (MCAO) using the intraluminal thread for 90min. After 24h reperfusion, several diagnostic indicators were evaluated and all animals were sacrificed to harvest their brains and blood for subsequent biochemical analyses. The results indicated that diosgenin treatment significantly inhibited the death rate and improved the impaired neurological functions along with neurological deficit scores and cerebral infarct size as compared with the rats exposed to I/R insult without agents administration. The increase in the number of apoptotic cells determined by TUNEL in the hippocampus CA1 and cortex was also apparently attenuated in the diosgenin treatment group, which was closely correlated with suppression of Caspase-3 activity and Bax/Bcl-2 ratio. In addition the elevated concentrations of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in blood serum of the I/R treated rats were reduced almost to their normal level. Further results obtained from the Western blotting analysis revealed that the protein expression of IκBα in the injured brain was up-regulated, while the p65 subunit of NF-κB was down-regulated in nucleus after the treatment. Collectively, this neuroprotection of diosgenin against I/R injury may be attained through its anti-apoptosis, anti-inflammation and intervening the NF-κB signal pathway properties. Due to the satisfactory findings, diosgenin might be a powerful therapeutic agent to combat the similar disease in future clinic.

Caspase inhibitor zVAD-fmk protects against acute pancreatitis-associated lung injury via inhibiting inflammation and apoptosis

Background/objectivesPulmonary apoptosis is an important pathogenic mechanism of acute lung injury induced by many factors. This study aims to investigate whether the caspase inhibitor zVAD-fmk has a protective effect against lung injury in the severe acute pancreatitis model (SAP) in rats. MethodsSeventy-two Sprague-Dawley rats were randomly divided into Sham, SAP, and SAP + zVAD-fmk groups. The SAP model was established by injection of 5% sodium taurocholate into the pancreatic duct. Animals were sacrificed at 3 h, 6 h, 12 h, and 24 h after operation and then HE staining analysis was performed to assess the lung injury. ELISA was used to detect the activity of myeloperoxidase (MPO) and the concentrations of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Western blotting was used to detect the expression of cleaved caspase-3 in the lung tissues. ResultsRats in SAP group showed obvious lung injury through pathologic examination. Pretreatment with zVAD-fmk significantly inhibited a post-SAP increase in the activation of MPO, TNF-α, IL-1β, and caspase-3, and decreased lung injury induced by SAP as determined by the pathologic score. ConclusionOur results suggest that apoptosis plays an important role in acute pancreatitis-associated lung injury (APALI), and inhibition of caspase activity may represent a new therapeutic approach for the treatment of APALI.

Infection of epithelial cells withChlamydia trachomatisinhibits TNF-induced apoptosis at the level of receptor internalization while leaving non-apoptotic TNF-signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro-apoptotic and non-apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro-apoptotic signal of TNF involves the activation of caspase-8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis-infected cells, TNF-induced apoptosis was blocked upstream of caspase-8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase-8 activation, cFLIP, was targeted by RNAi. However, when caspase-8 was directly activated by experimental over-expression of its upstream adapter Fas-associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non-apoptotic TNF-signalling, particularly the activation of NF-κB, initiates at the plasma membrane, while the activation of caspase-8 and pro-apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis-infected cells, NF-κB activation through TNF was unaffected, while the internalization of the TNF-TNF-receptor complex was blocked, explaining the lack of caspase-8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis-infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non-apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.

CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo.

Caspase-3 carries out the executioner phase of apoptosis, however under special circumstances, cells can survive its activity. To document systematically where and when cells survive caspase-3 activation in vivo, we designed a system, CasExpress, which drives fluorescent protein expression, transiently or permanently, in cells that survive caspase-3 activation in Drosophila. We discovered widespread survival of caspase-3 activity. Distinct spatial and temporal patterns emerged in different tissues. Some cells activated caspase-3 during their normal development in every cell and in every animal without evidence of apoptosis. In other tissues, such as the brain, expression was sporadic both temporally and spatially and overlapped with periods of apoptosis. In adults, reporter expression was evident in a large fraction of cells in most tissues of every animal; however the precise patterns varied. Inhibition of caspase activity in wing discs reduced wing size demonstrating functional significance. The implications of these patterns are discussed.

Fluid shear stress suppresses TNF-α-induced apoptosis in MC3T3-E1 cells: Involvement of ERK5-AKT-FoxO3a-Bim/FasL signaling pathways

TNF-α is known to induce osteoblasts apoptosis, whereas mechanical stimulation has been shown to enhance osteoblast survival. In the present study, we found that mechanical stimulation in the form of fluid shear stress (FSS) suppresses TNF-α induced apoptosis in MC3T3-E1 cells. Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family that has been implicated in cell survival. We also demonstrated that FSS imposed by flow chamber in vitro leads to a markedly activation of ERK5, which was shown to be protective against TNF-α-induced apoptosis, whereas the transfection of siRNA against ERK5 (ERK5-siRNA) reversed the FSS-medicated anti-apoptotic effects. An initial FSS-mediated activation of ERK5 that phosphorylates AKT to increase its activity, and a following forkhead box O 3a (FoxO3a) was phosphorylated by activated AKT. Phosphorylated FoxO3a is sequestered in the cytoplasm, and prevents it from translocating to nucleus where it can increase the expression of FasL and Bim. The inhibition of AKT-FoxO3a signalings by a PI3K (PI3-kinase)/AKT inhibitor (LY294002) or the transfection of ERK5-siRNA led to the nuclear translocation of non-phosphorylated FoxO3a, and increased the protein expression of FasL and Bim. In addition, the activation of caspase-3 by TNF-α was significantly inhibited by aforementioned FSS-medicated mechanisms. In brief, the activation of ERK5-AKT-FoxO3a signaling pathways by FSS resulted in a decreased expression of FasL and Bim and an inhibition of caspase-3 activation, which exerts a protective effect that prevents osteoblasts from apoptosis.

Neuroprotective Effects of Hydrogen Sulfide Against Early Brain Injury and Secondary Cognitive Deficits Following Subarachnoid Hemorrhage.

Although the neuroprotective effects of hydrogen sulfide (H2 S) have been demonstrated in several studies, whether H2 S protects against early brain injury (EBI) and secondary cognitive dysfunction in subarachnoid hemorrhage (SAH) model remains unknown. This study was undertaken to evaluate the influence of H2 S on both acute brain injury and neurobehavioral changes as well as the underlying mechanisms after SAH. The H2 S donor, NaHS, was administered via an intraperitoneal injection at a dose of 5.6 mg/kg at 2 h, 6 h, 24 h, and 46 h after SAH in rat model. The results showed that NaHS treatment significantly improved brain edema and neurobehavioral function, and attenuated neuronal cell death in the prefrontal cortex, associated with a decrease in Bax/Bcl-2 ratio and suppression of caspase-3 activation at 48 h after SAH. NaHS also promoted phospho-Akt and phospho-ERK levels. Furthermore, NaHS treatment significantly enhanced the levels of brain-derived neurotrophic factor (BDNF) and phospho-CREB. Importantly, NaHS administration improved learning and memory performance in the Morris water maze test at 7 days post-SAH in rats. These results demonstrated that NaHS, as an exogenous H2 S donor, could significantly alleviate the development of EBI and cognitive dysfunction induced by SAH via Akt/ERK-related antiapoptosis pathway, and upregulating BDNF-CREB expression.

Protective effects of aloin on oxygen and glucose deprivation-induced injury in PC12 cells.

The present study aims to determine whether aloin could protect cells from ischemic and reperfusion injury in vitro and to elucidate the related mechanisms. Oxygen and glucose deprivation model in PC12 cells was used in the present study. 2-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assay and Hoechst 33342 nuclear staining were used to evaluate the protective effects of aloin, at concentrations of 10, 20, or 40 μg/mL in PC12 cells. PCR was applied to detect fluorescence caspase-3, Bax and Bcl-2 mRNA expression in PC12 cells. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were evaluated by biochemical method. The concentration of intracellular-free calcium [Ca(2+)]i, mitochondrial membrane potential (MMP) were determined to estimate the degree of neuronal damage. It was shown that aloin (10, 20, and 40 μg/mL) significantly attenuated PC12 cells damage with characteristics of an increased injured cells absorbance of MTT and releases of LDH, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-reoxygenation. Meanwhile pretreatment with aloin significantly reduced injury-induced intracellular ROS, increased MMP (P<0.01), but it inhibited [Ca(2+)]i (P<0.01) elevation in a dose-dependent manner. Furthermore, pre-treatment with aloin significantly up-regulated Bcl-2 mRNA expression, down-regulated Bax mRNA expression and consequently activated caspase-3 mRNA expression in a dose-dependent manner. The results indicated that the protection of aloin on OGD-induced apoptosis in PC12 cells is associated with its suppression on OGD-induced oxidative stress and protection on mitochondrial function and inhibition of caspase activity. Alion could be a promising candidate in the development of a novel class of anti-ischemic agent.

Dickkopf 1 (Dkk1) Regulates Hematopoietic Stem Cell Regeneration

Bone marrow endothelial cells (BM ECs) have been shown to regulate HSC regeneration following myelosuppression. The role of osteolineage cells in regulating HSC regeneration remains less well understood. Here, we show that deletion of the pro-apoptotic genes, Bak and Bax, in osterix (Osx)-expressing osteoprogenitor cells promoted HSC regeneration and hematopoietic radioprotection of mice following total body irradiation (TBI). We identified Dkk1 to be enriched in the BM of radioprotected OsxCre;Bak1-/-;BaxFL/- mice and found that Bak /Bax-deficient osteolineage cells expressed increased levels of Dkk1 compared to Bax-expressing osteolineage cells (p=0.003). Treatment of irradiated BM HSCs with DKK1 in vitro significantly increased the recovery of phenotypic HSCs (p=0.0002), colony forming cells (CFCs)(p=0.003) and long-term repopulating HSCs compared to control cultures (p=0.009). Systemic administration of Dkk1 to lethally irradiated C57Bl6 mice accelerated the recovery of mature blood counts (p=0.008), BM HSCs (p=0.008) and progenitor cells (p=0.007). Furthermore, survival after lethal irradiation was markedly increased in Dkk1 treated mice (93%) compared to saline controls (27%; p=0.0004). Conversely, systemic administration of anti-Dkk1 antibody significantly delayed recovery of BM HSCs (p=0.002), peripheral white blood cells (p=0.0004), neutrophils (p<0.0001) and lymphocytes (p=0.002) in irradiated mice compared to irradiated, control mice. Dkk1 promoted HSC regeneration via suppression of reactive oxygen species (ROS) and inhibition of caspase activation in HSCs following irradiation. Dkk1 represents a novel, osteoprogenitor cell-derived paracrine factor which is necessary for normal hematopoietic regeneration following irradiation and can be therapeutically delivered to accelerate hematopoietic reconstitution. DisclosuresHimburg:Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA.

Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA- mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency whereas a reverse trend was observed with apicidin. Together, these finding provide a new strategy for differential mitochondrial targeting in cancer therapy.

Role of AKT and ERK pathways in controlling sensitivity to ionizing radiation and adaptive response induced by low-dose radiation in human immune cells

Despite many studies of the effect of ionizing radiation, biological mechanisms of action might differ greatly depend on dose, dose rate, and cell type. This study was performed to explore the effects of low- and high-dose radiation in human immune cell lines. We examined cell sensitivity after irradiation with 0.05, 0.1, or 2Gy in two normal cell lines and three tumor cell lines. Low-dose radiation of 0.05 and 0.1Gy had no effect on cell survival in any tested cell line, with the exception of IM-9 cells, whose viability was transiently increased. However, IM-9 and C1R-sB7 cells were very sensitive to high-dose radiation-induced cell death, whereas Jurkat and JM1 cells showed moderate sensitivity, and THP-1 cells were completely resistant. This radiosensitivity was correlated with basal AKT activation, which is induced by phosphorylation. In radiosensitive IM-9 cells, priming with chronic low-dose irradiation blocked cell death induced by high-dose radiation challenge via inhibition of caspase activation and PARP cleavage. AKT phosphorylation was not altered in IM-9 cells, but ERK phosphorylation was greatly elevated immediately after chronic low-dose irradiation. Taken together, our results suggest that the different responses of normal and tumor cells to low-dose and high-dose radiation depend on AKT activation, which is regulated by protein phosphatase 2 (PP2A). In radiosensitive normal cells lacking basal AKT activity, chronic low-dose radiation increases activation of the ERK pathway, which plays an important role in the adaptive response to radiation, providing a very important insight into understanding the effects of ionizing radiation on health.

TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila.

The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using Drosophila genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs.

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells.

BackgroundDoxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.MethodsThe present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.Results and conclusionIn the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.

The Adhesion GPCR CD97/ADGRE5 inhibits apoptosis

The Adhesion G protein-coupled receptor (GPCR) CD97/ADGRE5 is induced, upregulated, and/or biochemically modified in various malignancies, compared to the corresponding normal tissues. As tumor cells are generally more resistant to apoptosis, we here studied the ability of CD97 to regulate tumor cell survival under apoptotic conditions. Stable overexpression of wild-type CD97 reduced serum starvation- and staurosporine-induced intrinsic and tumor necrosis factor (TNF)/cycloheximide-induced extrinsic apoptosis, indicated by an increase in cell viability, a lower percentage of cells within the subG0/G1 phase, expressing annexin V, or having condensed nuclei, and a reduction of DNA laddering. Protection from cell death by CD97 was accompanied by an inhibition of caspase activation and modulation of anti- and pro-apoptotic members of the BCL-2 superfamily. shRNA-mediated knockdown of CD97 and, in part, truncation of the seven-span transmembrane (TM7) region of CD97 increased caspase-mediated apoptosis. Protection from apoptosis required not only the TM7 region but also cleavage of the receptor at its GPCR proteolysis site (GPS), whereas alternative splicing of its extracellular domain had no effect. Together, our data indicate a role of CD97 in tumor cell survival.

Astragaloside IV and cycloastragenol are equally effective in inhibition of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation in the endothelium

Ethnopharmacological relevanceAstragaloside IV and cycloastragenol are present together in Astragalus membranaceus Moench (Fabaceae) and this study aims to simultaneously investigate their regulation of endothelial homeostasis in the setting of endoplasmic reticulum stress (ER stress). Material and methodsWe stimulated endothelial cells with palmitate (PA 100μM) to evoked ROS-associated ER stress and observed the effects of astragaloside IV and cycloastragenol on thioredoxin-interacting protein (TXNIP) expression, NLRP3 inflammasome activation and mitochondrion-dependent apoptosis. ResultsAstragaloside IV and cycloastragenol inhibited ROS generation and attenuated ER stress inducer IRE1α phosphorylation, indicating the inhibition of ROS-associated ER stress. In response to ER stress, TXNIP expression increased, accompanied with NLRP3 induction and increased IL-1β and IL-6 production, but these alternations were reversed by treatment with astragaloside IV and cycloastragenol, demonstrating the inhibitory effects of astragaloside IV and cycloastragenol on TXNIP/NLRP3 inflammasome activation. Inflammasome activation led to mitochondrial cell death in endothelial cells, whereas astragaloside IV and cycloastragenol restored the loss of the mitochondrial membrane potential with inhibition of caspase-3 activity, and thereby protected cells from ER stress-induced apoptosis. Astragaloside IV and cycloastragenol enhanced AMPK phosphorylation and AMPK inhibitor compound C diminished their beneficial effects, indicative of the potential role of AMPK in their regulation. ConclusionsAstragaloside IV and cycloastragenol suppressed ROS-associated ER stress and then inhibited TXNIP/NLRP3 inflammasome activation with regulation of AMPK activity, and thereby ameliorated endothelial dysfunction by inhibiting inflammation and reducing cell apoptosis. Simultaneous investigations further showed that astragaloside IV and cycloastragenol were equally effective in regulation of endothelial homeostasis.

TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation.

Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells.

Functional analysis of free fatty acid receptor GPR120 in human eosinophils: implications in metabolic homeostasis.

Recent evidence has shown that eosinophils play an important role in metabolic homeostasis through Th2 cytokine production. GPR120 (FFA4) is a G protein-coupled receptor (GPCR) for long-chain fatty acids that functions as a regulator of physiological energy metabolism. In the present study, we aimed to investigate whether human eosinophils express GPR120 and, if present, whether it possesses a functional capacity on eosinophils. Eosinophils isolated from peripheral venous blood expressed GPR120 at both the mRNA and protein levels. Stimulation with a synthetic GPR120 agonist, GW9508, induced rapid down-regulation of cell surface expression of GPR120, suggesting ligand-dependent receptor internalization. Although GPR120 activation did not induce eosinophil chemotactic response and degranulation, we found that GW9508 inhibited eosinophil spontaneous apoptosis and Fas receptor expression. The anti-apoptotic effect was attenuated by phosphoinositide 3-kinase (PI3K) inhibitors and was associated with inhibition of caspase-3 activity. Eosinophil response investigated using ELISpot assay indicated that stimulation with a GPR120 agonist induced IL-4 secretion. These findings demonstrate the novel functional properties of fatty acid sensor GPR120 on human eosinophils and indicate the previously unrecognized link between nutrient metabolism and the immune system.

Activation of Caspase-8 and Caspase-9 are Required for PC12 Cells Differentiation

Nerve growth factor- (NGF) induced potent activation of caspase-8, and caspase-9 in PC12 cells in a time-dependent manner. Peptide inhibition of caspase activity led to a potent reduction in neurite outgrowth in NGF-treated cells. Although NGF treatment resulted in cell-cycle G0/G1 arrest as detected by FACS cell-cycle analysis, caspase inhibitors (caspase-8 and caspase-9 inhibitors) could block the G0/G1 arrest in PC12 cells. We demonstrated the unique caspase cascade, caspase-8, caspase-3, and caspase-9 to occur, in this order. In conclusion, present results confirm a unique role for the caspase-8 and caspase-9 mediated signal cascade in the differentiation of PC12 cells.

Bucillamine prevents cisplatin-induced ototoxicity through induction of glutathione and antioxidant genes.

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.

Necroptosis suppresses inflammation via termination of TNF- or LPS-induced cytokine and chemokine production.

TNF promotes a regulated form of necrosis, called necroptosis, upon inhibition of caspase activity in cells expressing RIPK3. Because necrosis is generally more pro-inflammatory than apoptosis, it is widely presumed that TNF-induced necroptosis may be detrimental in vivo due to excessive inflammation. However, because TNF is intrinsically highly pro-inflammatory, due to its ability to trigger the production of multiple cytokines and chemokines, rapid cell death via necroptosis may blunt rather than enhance TNF-induced inflammation. Here we show that TNF-induced necroptosis potently suppressed the production of multiple TNF-induced pro-inflammatory factors due to RIPK3-dependent cell death. Similarly, necroptosis also suppressed LPS-induced pro-inflammatory cytokine production. Consistent with these observations, supernatants from TNF-stimulated cells were more pro-inflammatory than those from TNF-induced necroptotic cells in vivo. Thus necroptosis attenuates TNF- and LPS-driven inflammation, which may benefit intracellular pathogens that evoke this mode of cell death by suppressing host immune responses.