MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway.

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Laquinimod decreases Bax expression and reduces caspase-6 activation in neurons.

Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.

Nicotine-Mediated Ca(2+)-Influx Induces IL-8 Secretion in Oral Squamous Cell Carcinoma Cell.

Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin-8 (IL-8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine-mediated IL-8 induction in OSCC was investigated. Augmented IL-8 secretion by Ca9-22 cells was blocked by the NF-κB inhibitor L-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)-specific inhibitor α-bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre-incubating the cells with inhibitors against mitogen-activated protein kinase (MEK), protein kinase C (PKC), and Ca(2+)/calmodulin-dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine-mediated IL-8 secretion. Pre-treatment of the Ca9-22 cells with the Ca(2+) chelator BAPTA-AM drastically inhibited IL-8 secretion. Although nicotine stimulation induced the phosphorylation of the NF-κB p65 subunit, pre-treatment with BAPTA-AM was found to inhibit this activity significantly. CaMK II-dependent p65 phosphorylation was confirmed by pre-incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca(2+) influx, which results in the activation and phosphorylation of CaMK II and NF-κB p65, respectively. Nicotine-mediated IL-8 induction should be a trigger for the initiation of various diseases.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

Fine-tuning of NFκB by glycogen synthase kinase 3ß directs the fate of glomerular podocytes upon injury.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3ß and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide (LPS) or adriamycin (ADR) elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L, and B7-1. Broad-range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3ß by lithium or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3ß was sufficient and essential for RelA/p65 phosphorylation, specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with LPS or ADR, concomitant with the suppression of podocytopathic mediators, but retained Bcl-xL in glomerulus. Broad-range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3ß-dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.

Serine protease inhibitors interact with IFN-γ through up-regulation of FasR; a novel therapeutic strategy against cancer.

Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.

Epinephrine evokes renalase secretion via α-adrenoceptor/NF-κB pathways in renal proximal tubular epithelial cells.

BACKGROUND/AIMS: Renalase is a recently discovered, kidney-specific monoamine oxidase that metabolizes circulating catecholamines. These findings present new insights into hypertension and chronic kidney diseases. Previous data demonstrated that renalase was mainly secreted from proximal tubules which could be evoked by catecholamines. The purpose of this study is to investigate whether renalase expression is induced by epinephrine via α-adrenoceptor/NFκB pathways. METHODS: HK2 cells were utilized to explore renalase expression in response to epinephrine in vitro. Phentolamine, an α-adrenoceptor antagonist, and Tosyl Phenylalanyl Chloromethyl Ketone (TPCK) were used to block α-adrenoceptor and to knock down the transcription factor NFκB, respectively. Renalase expression was analyzed using Western blot and quantitative PCR. RESULTS: Both protein and mRNA levels of renalase in HK2 cells increased in response to epinephrine (P<0.05). Epinephrine-evoked renalase expression was attenuated by phentolamine and TPCK separately (P<0.05). CONCLUSION: Epinephrine evokes renalase secretion via α-adrenoceptor/NF-κB pathways in renal proximal tubular epithelial cells.

Regulation of autophagy by α1-antitrypsin: "a foe of a foe is a friend".

Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was "fished out" (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.

The cellular inhibitor of apoptosis protein-2 is a possible target of novel treatment for endometriosis.

PROBLEM: How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? METHOD OF STUDY: Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. RESULTS: Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. CONCLUSIONS: TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.

A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development.

The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.

Angiogenic changes in co-cultures of mast cells and myocardial microvascular endothelial cells under hyperglycemic conditions.

The aim of the present study was to determine the correlation between angiogenesis and the differential expression of growth factors and their receptors when myocardial microvascular endothelial cells (MMVECs) were co-cultured with mast cell granules (MCGs) under hyperglycemic conditions. MMVECs and mast cells (MCs) were isolated from Wistar rats. An in vitro angiogenesis assay was used to observe any differences when MMVECs were co-cultured with MCGs in normal or hyperglycemic medium. The mRNA and protein expression of growth factors and their receptors were analyzed by real-time reverse transcription (RT)-PCR and western blot analysis. Real-time RT-PCR analysis demonstrated the upregulated mRNA and protein expression of vascular endothelial growth factor (VEGF) in the MMVECs; however, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk­1), decreased significantly, and the angiogenic ability of the MMVECs decreased under hyperglycemic conditions. The angiogenic ability of the MMVECs cultured under hyperglycemic conditions (even after the addition of MCGs) was inferior to that of the MMVECs cultured under normal glucose conditions. The specific inhibitor of tryptase, N-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed angiogenesis regardless of the glucose concentration, and the specific inhibitor of chymase, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), was not as effective as TLCK, which was mainly detected under hyperglycemic conditions. High glucose levels have a profound effect on angiogenesis; this effect may be more pronounced than the effects of MCGs on angiogenesis.

CpG ODN-induced matrix metalloproteinase-13 expression is mediated via activation of the ERK and NF-κB signalling pathways in odontoblast cells.

AIM: To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. METHODOLOGY: Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. RESULTS: MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. CONCLUSION: The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.

TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues.

N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.

Characterization of proteolytic activities during intestinal regeneration of the sea cucumber, Holothuria glaberrima.

Proteolysis carried out by different proteases control cellular processes during development and regeneration. Here we investigated the function of the proteasome and other proteases in the process of intestinal regeneration using as a model the sea cucumber Holothuria glaberrima. This echinoderm possesses the ability to regenerate its viscera after a process of evisceration. Enzymatic activity assays showed that intestinal extracts at different stages of regeneration possessed chymotrypsin-like activity. This activity was inhibited by i) MG132, a reversible inhibitor of chymotrypsin and peptidylglutamyl peptidase hydrolase (PGPH) activities of the proteasome, ii) E64d, a permeable inhibitor of cysteine proteases and iii) TPCK, a serine chymotrypsin inhibitor, but not by epoxomicin, an irreversible and potent inhibitor of all enzymatic activities of the proteasome. To elucidate the role which these proteases might play during intestinal regeneration, we carried out in vivo experiments injecting MG132, E64d and TPCK into regenerating animals. The results showed effects on the size of the regenerating intestine, cell proliferation and collagen degradation. These findings suggest that proteolysis by several proteases is important in the regulation of intestinal regeneration in H. glaberrima.

HMGB-1 induces IL-6 production in human synovial fibroblasts through c-Src, Akt and NF-κB pathways.

High mobility group box chromosomal protein 1 (HMGB-1) is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in HMGB-1-induced IL-6 production in human synovial fibroblast cells. HMGB-1 caused concentration- and time-dependent increases in IL-6 production. HMGB-1-mediated IL-6 production was attenuated by receptor for advanced glycation end products (RAGE) monoclonal antibody (Ab) or siRNA. Pretreatment with c-Src inhibitor (PP2), Akt inhibitor and NF-κB inhibitor (pyrrolidine dithiocarbamate and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone) also inhibited the potentiating action of HMGB-1. Stimulation of cells with HMGB-1 increased the c-Src and Akt phosphorylation. HMGB-1 increased the accumulation of p-p65 in the nucleus, as well as NF-κB luciferase activity. HMGB-1-mediated increase of NF-κB luciferase activity was inhibited by RAGE Ab, PP2 and Akt inhibitor or RAGE siRNA, or c-Src and Akt mutant. Our results suggest that HMGB-1-increased IL-6 production in human synovial fibroblasts via the RAGE receptor, c-Src, Akt, p65, and NF-κB signaling pathways.

Collagenolytic activity in sonic extracts of Tannerella forsythia.

OBJECTIVE: The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. METHODS: The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. RESULTS: The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. CONCLUSION: The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.

Caspase-3 triggers a TPCK-sensitive protease pathway leading to degradation of the BH3-only protein puma.

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.

Inflammatory responses in embryonal cardiomyocytes exposed to LPS challenge: an in vitro model of deciphering the effects of LPS on the heart.

This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.

The classical NFkappaB pathway is required for phloroglucinol-induced activation of murine lymphocytes.

BACKGROUND: Phloroglucinol (PG), a polyphenolic compound, has been proposed to show free radical scavenging, anti-tumor, and immunomodulation effects on immune cells. In this study, we investigated PG for its immunological activity and their molecular mechanisms, specifically focusing on the functional activation of nuclear factor-kappaB (NFkappaB), an important transcription factor involved in the activation and differentiation of lymphocytes, and subsequent recruitment of murine lymphocytes. METHODS: We tested whether PG may contribute to the activation of immune response through the NFkappaB pathway by (3)H-thymidine incorporation assay, Western blot, intracellular cytokine assays and Electrophoretic mobility shift assay (EMSA) in murine lymphocytes. RESULTS: PG markedly enhanced the proliferation of lymphocytes by inducing the degradation and phosphorylation of IkappaB, which leads to the activation of NFkappaB p65. Also, PG induced the activation of mitogen-activated protein kinases (MAPKs) such as ERK1/2, JNK, and p38, upstream molecules of NFkappaB pathway. In addition, PG augmented the secretion of interleukin (IL)-2 in mature T lymphocytes and their production of IL-2. Furthermore, the application of TPCK significantly reduced the activation of lymphocytes by PG via inhibiting the NFkappaB activation. CONCLUSION: These results suggest that PG induces the activation of lymphocytes by inducing the proliferative activity and secretion of IL-2 through the classical NFkappaB. GENERAL SIGNIFICANCE: The effect of PG on lymphocyte activation was assayed for the first time. These results would also elucidate its underlying mechanism.