A critical role for the protein phosphatase 2A B'α regulatory subunit in dephosphorylation of sphingosine kinase 1

Sphingosine kinase 1 (SK1) is an important regulator of cellular signalling that has gained recent attention as a potential target for anti-cancer therapies. SK1 activity, subcellular localization and oncogenic function are regulated by phosphorylation and dephosphorylation at Ser225. ERK1/2 have been identified as the protein kinases responsible for phosphorylation and activation of SK1. Conversely, dephosphorylation and deactivation of SK1 occurs by protein phosphatase 2A (PP2A). Active PP2A, however, is a heterotrimer, composed of tightly associated catalytic and structural subunits that can interact with an array of regulatory subunits, which are critical for determining holoenzyme substrate specificity and subcellular localization. Thus, PP2A represents a large family of holoenzyme complexes with different activities and diverse substrate specificities. To date the regulatory subunit essential for targeting PP2A to SK1 has remained undefined. Here, we demonstrate a critical role for the B'α (B56α/PR61α/PPP2R5A) regulatory subunit of PP2A in SK1 dephosphorylation. B'α was found to interact with the c-terminus of SK1, and reduce SK1 phosphorylation when overexpressed, while having no effect on upstream ERK1/2 activation. siRNA-mediated knockdown of B'α increased SK1 phosphorylation, activity and membrane localization of endogenous SK1. Furthermore, overexpression of B'α blocked agonist-induced translocation of SK1 to the plasma membrane and abrogated SK1-induced neoplastic transformation of NIH3T3 fibroblasts. Thus, the PP2A-B'α holoenzyme appears to function as an important endogenous regulator of SK1.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

Enteroaggregative Escherichia coli infection induces IL-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-κB and AP-1 in INT-407 cells

Multiple mucosal immune factors, such as TNF-α and IL-1β, are thought to be key mediators involved in inflammatory bowel disease. We evaluated the role of the pro-inflammatory cytokine TNF-α on nitric oxide synthase (NOS) expression in indomethacin-induced jejunoileitis in rats. Jejunoileitis was induced in rats with subcutaneous injections of indomethacin (7.5 mg/kg) 24 h apart for two consecutive days, and animals were randomized into four groups. Group 1 received only indomethacin. Group 2 was treated with a daily dose of phosphodiesterase (PDE) inhibitor (theophylline or pentoxifylline) by oral gavage for 2 days before and 4 days after indomethacin. Group 3 received a single dose of anti-TNF-α monoclonal antibody (TNF-Ab, IP) 30 min before indomethacin. Group 4 was treated with 1 h hyperbaric oxygenation (HBO₂) for 5 days after indomethacin. Rats were sacrificed at 12 h or 4 days after final indomethacin injection. PDE inhibitor, TNF-Ab, or HBO₂ treatment significantly decreased indomethacin-induced ulceration, myeloperoxidase activity, and disease activity index. Although indomethacin significantly increased serum TNF-α and nitrate/nitrite (NOx) concentrations above control values at 12 h, inducible NOS (iNOS) expression was detected only at day 4. Serum IL-1β levels did not change at 12 h but increased 4-fold after 4 days. Indomethacin had no effect on constitutive NOS. Treatment with PDE inhibitor, TNF-Ab, or HBO₂ significantly reduced serum/tissue TNF-α, IL-1β, NOx, and iNOS expression. Our data show TNF-α plays an early pro-inflammatory role in indomethacin-induced jejunoileitis. Additionally, down-regulation of NOx by PDE inhibitors, TNF-Ab, or HBO₂ suggests that TNF-α modulates iNOS expression.     

Protein phosphatase 1α mediates ceramide-induced ERM protein dephosphorylation: a novel mechanism independent of phosphatidylinositol 4, 5-biphosphate (PIP2) and myosin/ERM phosphatase

ERM (ezrin, radixin, and moesin) proteins are cytoskeletal interacting proteins that bind cortical actin, the plasma membrane, and membrane proteins, which are found in specialized plasma membrane structures such as microvilli and filopodia. ERM proteins are regulated by phosphatidylinositol 4, 5-biphosphate (PIP(2)) and by phosphorylation of a C-terminal threonine, and its inactivation involves PIP(2) hydrolysis and/or myosin phosphatase (MP). Recently, we demonstrated that ERM proteins are also subject to counter regulation by the bioactive sphingolipids ceramide and sphingosine 1-phosphate. Plasma membrane ceramide induces ERM dephosphorylation whereas sphingosine 1-phosphate induces their phosphorylation. In this work, we pursue the mechanisms by which ceramide regulates dephosphorylation. We found that this dephosphorylation was independent of hydrolysis and localization of PIP(2) and MP. However, the results show that ERM dephosphorylation was blocked by treatment with protein phosphatase 1 (PP1) pharmacological inhibitors and specifically by siRNA to PP1α, whereas okadaic acid, a PP2A inhibitor, failed. Moreover, a catalytic inactive mutant of PP1α acted as dominant negative of the endogenous PP1α. Additional results showed that the ceramide mechanism of PP1α activation is largely independent of PIP(2) hydrolysis and MP. Taken together, these results demonstrate a novel, acute mechanism of ERM regulation dependent on PP1α and plasma membrane ceramide.     

Palmitate induces SHIP2 expression via the ceramide-mediated activation of NF-κB,and JNK in skeletal muscle cells

Aims

Elevated plasma free fatty acids impair the insulin signaling by induction of the expression of protein phosphatases. However, the effect of palmitate on SH2-containing inositol 5′-phosphatase 2 (SHIP2) expression has not been investigated. Here we investigated the effects of palmitate on SHIP2 expression and elucidated the underlying mechanisms in skeletal muscle cells.

Main methods

SHIP2 mRNA and protein levels were measured in C2C12 myotubes exposed to palmitate. Specific inhibitors were used to identify the signaling pathways involved in SHIP2 expression.

Key findings

The results showed that 0.5 mM palmitate significantly upregulates the mRNA and protein levels of SHIP2 in C2C12 cells. To address the role of palmitate intracellular metabolites in SHIP2 expression, the myotubes were treated with palmitate in the presence of ceramide and diacylglycerol synthesis inhibitors. The results demonstrated that only ceramide synthesis inhibition could prevent palmitate-induced SHIP2 expression in these cells. In addition, the incubation of muscle cells with different concentrations of C2-ceramide dose-dependently enhanced SHIP2 expression. Furthermore, the inhibition of both JNK and NF-κB pathways could prevent ceramide-induced SHIP2 expression in myotubes.

Significance

These findings suggest that palmitate contributes to SHIP2 overexpression in skeletal muscle via the mechanisms involving the activation of ceramide-JNK and NF-κB pathways.     

Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARα in mice

Calorie restriction (CR) extends lifespans in a wide variety of species. CR induces an increase in the NAD⁺/NADH ratio in cells and results in activation of SIRT1, an NAD⁺-dependent protein deacetylase that is thought to be a metabolic master switch linked to the modulation of lifespans. CR also affects the expression of peroxisome proliferator-activated receptors (PPARs). The three subtypes, PPARα, PPARγ, and PPARβ/δ, are expressed in multiple organs. They regulate different physiological functions such as energy metabolism, insulin action and inflammation, and apparently act as important regulators of longevity and aging. SIRT1 has been reported to repress the PPARγ by docking with its co-factors and to promote fat mobilization. However, the correlation between SIRT1 and other PPARs is not fully understood. CR initially induces a fasting-like response. In this study, we investigated how SIRT1 and PPARα correlate in the fasting-induced anti-aging pathways. A 24-h fasting in mice increased mRNA and protein expression of both SIRT1 and PPARα in the livers, where the NAD⁺ levels increased with increasing nicotinamide phosphoribosyltransferase (NAMPT) activity in the NAD⁺ salvage pathway. Treatment of Hepa1-6 cells in a low glucose medium conditions with NAD⁺ or NADH showed that the mRNA expression of both SIRT1 and PPARα can be enhanced by addition of NAD⁺, and decreased by increasing NADH levels. The cell experiments using SIRT1 antagonists and a PPARα agonist suggested that PPARα is a key molecule located upstream from SIRT1, and has a role in regulating SIRT1 gene expression in fasting-induced anti-aging pathways.     

Oxidative stress induces inactivation of protein phosphatase 2A,promoting proinflammatory NF-κB in aged rat kidney

The molecular inflammation hypothesis of aging proposes that redox dysregulation causes an age-related activation of NF-κB and its signaling to upregulate various proinflammatory genes. In the present study, we focused on the inactive form of the protein phosphastase 2 A (PP2A). More specifically, we aimed to define the correlation between PP2A inactivation and NF-κB activation by age-related oxidative stress. Experimentations were designed to determine the effect of oxidative stress-induced PP2A inactivation on NF-κB activity, utilizing prooxidants t-BHP and AAPH, the PTP inhibitor Na₃VO₄, and the PP2A inhibitor Calyculin A and PP2A siRNA, in HEK293T cells. We also assessed the phosphorylation of PP2A catalytic subunit (PP2Ac) and the activities of PP2A and NF-κB in aged rat kidney, utilizing aging-retarding 40% calorie restriction (CR) −60% of food intake and inflammation-triggering LPS paradigms. Results revealed that an oxidative stress-induced PTK/PTP imbalance led to phosphorylation of PP2Ac, following exposures to t-BHP, AAPH, and Na₃VO₄ in HEK293T cells. Subsequently, we found that Calyculin A and PP2A siRNA activates NIK/IKK and MAPKs, leading to upregulation of NF-κB and its dependent oxidative stress. Also, the contrasting relation between PP2A inactivation and NF-κB activation was confirmed by AAPH-induced oxidative status in mice, and non-induced normal status or LPS-induced inflammatory status in aged rats while the antioxidative, anti-inflammatory, anti-aging effects of CR significantly blunted these actions. Thus, we present evidence that PP2A inactivation via PTK/PTP imbalance provoked by oxidative stress causes NF-κB activation, which contributes to the accumulation of oxidative stress in aged rat kidney.     

Chronic social isolation induces NF-κB activation and upregulation of iNOS protein expression in rat prefrontal cortex

Exposure of an organism to stress, results in oxidative stress and increased nitric oxide (NO) production in the brain. The role of the processes caused by chronic stress in the prefrontal cortex has not been fully investigated. Considering that chronic stress increases NO production by the enzyme nitric oxide synthase (NOS), we examined the cytosolic neuronal (nNOS) or inducible (iNOS) protein levels in the prefrontal cortex of rats exposed to 21 d of chronic social isolation stress, an animal model of depression, alone or in combination with 2 h of acute immobilization or cold (4 °C) stress (combined stress). Antioxidative status via cytosolic CuZnSOD and mitochondrial MnSOD activity, cytosolic redox status via reduced glutathione (GSH) concentration were determined. Furthermore, cytosolic inducible heat shock protein 70 (Hsp70i), cytosolic/nuclear distributions of NF-κB and serum corticosterone (CORT) were also investigated to elucidate the possible mechanism involved in the cellular NOS pathway. Our results showed that both acute stressors led to increases of CORT and nNOS protein while iNOS protein expression was unaffected. In contrast to the acute stress, chronic social isolation compromised hypothalamic–pituitary-adrenal axis functioning such that the normal stress response was impaired following subsequent acute stressors. Downregulated redox GSH status as well as decreased activity of CuZnSOD and MnSOD suggests the existence of oxidative stress which remained as such following combined stressors. Changes in redox-status associated with decreased Hsp70i protein expression enabled NF-κB translocation into the nucleus, causing increased cytosolic nNOS and iNOS protein expression. Results suggest that NOS signaling pathway plays a differential role between acute and chronic stress whereby state of oxidative/nitrosative stress after chronic social isolation is caused, at least in part, by NF-κB activation and increased iNOS protein expression.     

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.     

Cannabinoid receptor 1 induces a biphasic ERK activation via multiprotein signaling complex formation of proximal kinases PKCε, Src, and Fyn in primary neurons

Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKC? at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKC?, selectively, and that the CB1R-activated PKC? acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.     

Gambogenic acid induces Noxa-mediated apoptosis in colorectal cancer through ROS-dependent activation of IRE1α/JNK

BackgroundGambogenic acid (GNA), an active component of Garcinia hanburyi Hook.f. (Clusiaceae) (common name gamboge), exerts anti-inflammatory and antitumor properties. However, the underlying mechanism of GNA in colorectal cancer (CRC) is still not well understood.PurposeThis study aimed to investigate the antitumor effects and mechanisms of GNA on CRC in vitro and in vivo.MethodsCell viability, colony formation and cell apoptosis assays were performed to determine the antitumor effects of GNA. qRT-PCR and Western blotting were performed to evaluate the expression of genes or proteins affected by GNA in vitro and in vivo. HCT116 colon cancer xenografts and the APC^min/+ mice model were used to confirm the antitumor effects of GNA on CRC in vivo.ResultsGNA induced Noxa-mediated apoptosis by inducing reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation. Moreover, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme-1α (IRE1α) leading to JNK phosphorylation. ROS scavenger attenuated GNA-induced IRE1α activation and JNK phosphorylation. Knockdown of IRE1α also prevented GNA-induced JNK phosphorylation. In vivo, GNA suppressed tumor growth and progression in HCT116 colon cancer xenografts and the APC^min/+ mices model.ConclusionThese findings revealed that GNA induced Noxa-mediated apoptosis by activating the ROS/IRE1α/JNK signaling pathway in CRC both in vitro and in vivo. GNA is therefore a promising antitumor agent for CRC treatment.     

N-Acetylcysteine and Allopurinol Confer Synergy in Attenuating Myocardial Ischemia Injury via Restoring HIF-1α/HO-1 Signaling in Diabetic Rats

Objectives

To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1α (HIF-1α)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium.

Methods

Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1α inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1α and HO-1 achieved by gene knock-down with related siRNAs.

Results

Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1α, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1α protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1α with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1α gene knock-down in vitro.

Conclusion

NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1α and HO-1 signaling.     

Zerumbone suppresses IKKα, Akt,and FOXO1 activation,resulting in apoptosis of GBM 8401 cells

Background

Zerumbone, a sesquiterpene compound isolated from subtropical ginger, Zingiber zerumbet Smith, has been documented to exert antitumoral and anti- inflammatory activities. In this study, we demonstrate that zerumbone induces apoptosis in human glioblastoma multiforme (GBM8401) cells and investigate the apoptotic mechanism.

Methods

We added a caspase inhibitor and transfected wild-type (WT) IKK and Akt into GBM 8401 cells, and measured cell viability and apoptosis by MTT assay and flow cytometry. By western blotting, we evaluated activation of caspase-3, dephosphorylation of IKK, Akt, FOXO1 with time, and change of IKK, Akt, and FOXO1 phosphorylation after transfection of WT IKK and Akt.

Results

Zerumbone (10∽50 μM) induced death of GBM8401 cells in a dose-dependent manner. Flow cytometry studies showed that zerumbone increased the percentage of apoptotic GBM cells. Zerumbone also caused caspase-3 activation and poly (ADP-ribose) polymerase (PARP) production. N-benzyloxycarbonyl -Val-Ala-Asp- fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, hindered zerumbone-induced cell death. Transfection of GBM 8401 cells with WT IKKα inhibited zerumbone-induced apoptosis, and zerumbone significantly decreased IKKα phosphorylation levels in a time-dependent manner. Similarly, transfection of GBM8401 cells with Akt suppressed zerumbone-induced apoptosis, and zerumbone also diminished Akt phosphorylation levels remarkably and time-dependently. Moreover, transfection of GBM8401 cells with WT IKKα reduced the zerumbone-induced decrease in Akt and FOXO1 phosphorylation. However, transfection with WT Akt decreased FOXO1, but not IKKα, phosphorylation.

Conclusion

The results suggest that inactivation of IKKα, followed by Akt and FOXO1 phosphorylation and caspase-3 activation, contributes to zerumbone-induced GBM cell apoptosis.     

Agonistic antibody to the α1-adrenergic receptor mobilizes intracellular calcium and induces phosphorylation of a cardiac 15-kDa protein

Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the α₁-adrenergic receptor (α₁-AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (anti-α₁) was generated against the second extracellular loop of the α₁-AR that bound to its peptide epitope with high affinity (K _D ~50 nM). We studied anti-α₁ effects on intracellular calcium (Ca_i), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-α₁ induced pronounced but transient increases in Ca_i in CHO cells expressing the human α₁-AR (CHO-α₁) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-α₁ on Ca_i. However, preincubation with the antibody attenuated the effect of the α₁-AR antagonist prazosin. In neonatal cardiomyocytes anti-α₁ induced a robust phosphorylation of a 15-kDa protein that is involved in α₁-AR signalling. Our data support the notion that elevation of Ca_i is a general feature of agonistic antibodies’ action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the α₁-AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action.     

Induction of growth cessation by acacetin via β-catenin pathway and apoptosis by apoptosis inducing factor activation in colorectal carcinoma cells

Molecular Biology Reports - Acacetin, a bioflavanoid, contains anti-inflammatory and anti-cancer activities as shown in different experimental models. However, its anticancer potential and...     

Matrix-m™ adjuvant induces local recruitment, activation and maturation of central immune cells in absence of antigen

Saponin-based adjuvants are widely used to enhance humoral and cellular immune responses towards vaccine antigens, although it is not yet completely known how they mediate their stimulatory effects. The aim of this study was to elucidate the mechanism of action of adjuvant Matrix-M™ without antigen and Alum was used as reference adjuvant. Adjuvant Matrix-M™ is comprised of 40 nm nanoparticles composed of Quillaja saponins, cholesterol and phospholipid. BALB/c mice were subcutaneously injected once with, 3, 12 or 30 µg of Matrix-M™, resulting in recruitment of leukocytes to draining lymph nodes (dLNs) and spleen 48 h post treatment. Flow cytometry analysis identified CD11b⁺ Gr-1^high granulocytes as the cell population increasing most in dLNs and spleen. Additionally, dendritic cells, F4/80^int cells, T-, B- and NK-cells were recruited to dLNs and in spleen the number of F4/80^int cells, and to some extent, B cells and dendritic cells, increased. Elevated levels of early activation marker CD69 were detected on T-, B- and NK-cells, CD11b⁺ Gr-1^high cells, F4/80^int cells and dendritic cells in dLNs. In spleen CD69 was mainly up-regulated on NK cells. B cells and dendritic cells in dLNs and spleen showed an increased expression of the co-stimulatory molecule CD86 and dendritic cells in dLNs expressed elevated levels of MHC class II. The high-dose (30 µg) of Matrix-M™ induced detectable serum levels of IL-6 and MIP-1β 4 h post administration, most likely representing spillover of locally produced cytokines. A lesser increase of IL-6 in serum after administration of 12 µg Matrix-M™ was also observed. In conclusion, early immunostimulatory properties were demonstrated by Matrix-M™ alone, as therapeutic doses resulted in a local transient immune response with recruitment and activation of central immune cells to dLNs. These effects may play a role in enhancing uptake and presentation of vaccine antigens to elicit a competent immune response.     

The antimicrobial peptide,human β-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases

Previous studies have demonstrated increased expression and raised levels of human β-defensin (hBD)-1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri-implantitis, oral bone-resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD-1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD-1 and cell viability was assayed by MTT. PBMCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) in the presence or absence of non-toxic doses of hBD-1. In vitro osteoclastogenesis was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, osteoclast-specific gene expression, and a resorption pit assay. Involvement of mitogen-activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD-1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP-positive multinuclear cells and resorption areas on the dentin slices, and further up-regulated expressions of osteoclast-specific genes compared to those by RANKL treatment (p < 0.05). However, hBD-1 treatment without RANKL failed to induce formation of osteoclast-like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD-1 co-treatment (p < 0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD-1-enhanced osteoclastogenesis. Collectively, hBD-1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs.     

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.