Role of TRAIL and the pro-apoptotic Bcl-2 homolog Bim in acetaminophen-induced liver damage

Acetaminophen (N-acetyl-para-aminophenol (APAP), paracetamol) is a commonly used analgesic and antipyretic agent. Although considered safe at therapeutic doses, accidental or intentional overdose causes acute liver failure characterized by centrilobular hepatic necrosis with high morbidity and mortality. Although many molecular aspects of APAP-induced cell death have been described, no conclusive mechanism has been proposed. We recently identified TNF-related apoptosis-inducing ligand (TRAIL) and c-Jun kinase (JNK)-dependent activation of the pro-apoptotic Bcl-2 homolog Bim as an important apoptosis amplification pathway in hepatocytes. In this study, we, thus, investigated the role of TRAIL, c-JNK and Bim in APAP-induced liver damage. Our results demonstrate that TRAIL strongly synergizes with APAP in inducing cell death in hepatocyte-like cells lines and primary hepatocyte. Furthermore, we found that APAP strongly induces the expression of Bim in a c-JNK-dependent manner. Consequently, TRAIL- or Bim-deficient mice were substantially protected from APAP-induced liver damage. This study identifies the TRAIL-JNK-Bim axis as a novel target in the treatment of APAP-induced liver damage and substantiates its general role in hepatocyte death.

The ω3-polyunsaturated fatty acid derivatives AVX001 and AVX002 directly inhibit cytosolic phospholipase A(2) and suppress PGE(2) formation in mesangial cells

ω3-polyunsaturated fatty acids (ω3-PUFAs) are known to exert anti-inflammatory effects in various disease models although their direct targets are only poorly characterized.

Aggretin, a heterodimeric C-type lectin from Calloselasma rhodostoma (malayan pit viper), stimulates platelets by binding to alpha 2beta 1 integrin and glycoprotein Ib, activating Syk and phospholipase Cgamma 2, but does not involve the glycoprotein VI/Fc receptor gamma chain collagen receptor

Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.

The proapoptotic Bcl-2 family member Bim plays a central role during the development of virus-induced hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.

FTY720 suppresses interleukin-1beta-induced secretory phospholipase A2 expression in renal mesangial cells by a transcriptional mechanism

BACKGROUND AND PURPOSE: FTY720 is a potent immunomodulatory prodrug that is converted to its active phosphorylated form by a sphingosine kinase. Here we have studied whether FTY720 mimicked the action of sphingosine-1-phosphate (S1P) and exerted an anti-inflammatory potential in renal mesangial cells. EXPERIMENTAL APPROACH: Prostaglandin E(2) (PGE(2)) was quantified by an enzyme-linked immunosorbent-assay. Secretory phospholipase A(2) (sPLA(2)) protein was detected by Western blot analyses. mRNA expression was determined by Northern blot analysis and sPLA(2)-promoter activity was measured by a luciferase-reporter-gene assay. KEY RESULTS: Stimulation of cells for 24 h with interleukin-1beta (IL-1beta) is known to trigger increased PGE(2) formation which coincides with an induction of the mRNA for group-IIA-sPLA(2) and protein expression. FTY720 dose-dependently suppressed IL-1beta-induced IIA-sPLA(2) protein secretion and activity in the supernatant. This effect is due to a suppression of cytokine-induced sPLA(2) mRNA expression which results from a reduced promoter activity. As a consequence of suppressed sPLA(2) activity, PGE(2) formation is also reduced by FTY720. Mechanistically, the FTY720-suppressed sPLA(2) expression results from an activation of the TGFbeta/Smad signalling cascade since inhibition of the TGFbeta receptor type I by a specific kinase inhibitor reverses the FTY720-mediated decrease of sPLA(2) protein expression and sPLA(2) promoter activity. CONCLUSIONS AND IMPLICATIONS: In summary, our data show that FTY720 was able to mimic the anti-inflammatory activity of TGFbeta and blocked cytokine-triggered sPLA(2) expression and subsequent PGE(2) formation. Thus, FTY720 may exert additional in vivo effects besides the well reported immunomodulation and its anti-inflammatory potential should be considered.

Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase-Bim axis and reveals glutathione-S-transferase P1 as Achilles' heel

Glutathione-S-transferase of the Pi class (GSTP1) is frequently overexpressed in a variety of solid tumors and has been identified as a potential therapeutic target for cancer therapy. GSTP1 is a phase II detoxification enzyme and conjugates the tripeptide glutathione to endogenous metabolites and xenobiotics, thereby limiting the efficacy of antitumor chemotherapeutic treatments. In addition, GSTP1 regulates cellular stress responses and apoptosis by sequestering and inactivating c-Jun N-terminal kinase (JNK). Thiazolides are a novel class of antibiotics for the treatment of intestinal pathogens with no apparent side effects on the host cells and tissue. Here we show that thiazolides induce a GSTP1-dependent and glutathione-enhanced cell death in colorectal tumor cell lines. Downregulation of GSTP1 reduced the apoptotic activity of thiazolides, whereas overexpression enhanced it. Thiazolide treatment caused strong Jun kinase activation and Jun kinase-dependent apoptosis. As a critical downstream target of Jun kinase we identified the pro-apoptotic Bcl-2 homolog Bim. Thiazolides induced Bim expression and activation in a JNK-dependent manner. Downregulation of Bim in turn significantly blocked thiazolide-induced apoptosis. Whereas low concentrations of thiazolides failed to induce apoptosis directly, they potently sensitized colon cancer cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic drug-induced cell death. Although GSTP1 overexpression generally limits chemotherapy and thus antitumor treatment, our study identifies GSTP1 as Achilles' heel and thiazolides as novel interesting apoptosis sensitizer for the treatment of colorectal tumors.

Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

Occurrence of gustducin-immunoreactive cells in von Ebner's glands of guinea pigs

An immunohistochemical examination of guinea-pig taste buds in vallate papillae revealed gustducin-immunoreactive cells in the area of von Ebner’s glands, minor salivary glands. Since there have been no reports describing those cells in these locations for other species, we investigated these glands in order both to localize the cells and compare their immunoreactive characteristics with corresponding cells in the vallate taste buds. The gustducin-immunoreactive cells coincided with cells containing no secretory granules in the end portion of the glands, which was supported by the electron-microscopic immunocytochemistry. Double immunofluorescence microscopy confirmed these cells to be entirely immunopositive to type III inositol 1,4,5-triphosphate receptor (IP3R-3), phospholipase Cβ2 (PLCβ2), and villin and also partly immunopositive to neuron-specific enolase (NSE) and calbindin D-28K. The gustducin-immunoreactive cells in the vallate taste buds exhibited completely the same immunoreactivities for these five molecules. Accordingly, the present results give credence to a consideration that the gustducin-immunnoreactive cells in both locations are identical in function(s) e.g., chemo-reception.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

Clinical and immunologic effects of H1 antihistamine preventive medication during honeybee venom immunotherapy

BACKGROUND: H1 antihistamines increase safety during allergen-specific immunotherapy and might influence the outcome because of immunoregulatory effects. OBJECTIVE: We sought to analyze the influence of 5 mg of levocetirizine (LC) on the safety, efficacy, and immunologic effects of ultrarush honeybee venom immunotherapy (BVIT). METHOD: In a double-blind, placebo-controlled study 54 patients with honeybee venom allergy received LC or placebo from 2 days before BVIT to day 21. Side effects during dose increase and systemic allergic reactions (SARs) to a sting challenge after 120 days were analyzed. Allergen-specific immune response was investigated in skin, serum, and allergen-stimulated T-cell cultures. RESULTS: Side effects were significantly more frequent in patients receiving placebo. Four patients receiving placebo dropped out because of side effects. SARs to the sting challenge occurred in 8 patients (6 in the LC group and 2 in the placebo group). Seven SARs were only cutaneous, and 1 in the placebo group was also respiratory. Difference of SARs caused by the sting challenge was insignificant. Specific IgG levels increased significantly in both groups. Major allergen phospholipase A(2)-stimulated T cells from both groups showed a slightly decreased proliferation. The decrease in IFN-gamma and IL-13 levels with placebo was not prominent with LC, whereas IL-10 levels showed a significant increase in the LC group only. Decreased histamine receptor (HR)1/HR2 ratio in allergen-specific T cells on day 21 in the placebo group was prevented by LC. CONCLUSIONS: LC reduces side effects during dose increase without influencing the efficacy of BVIT. LC modulates the natural course of allergen-specific immune response and affects the expression of HRs and cytokine production by allergen-specific T cells.

Current management options for liposarcoma and challenges for the future

Liposarcoma (LS) represents one of the most common soft tissue sarcomas. There are three major subtypes, namely, well/dedifferentiated, myxoid/round cell and pleomorphic LS. In general, LS is known to be a relatively chemo-resistant sarcoma subtype with the exception of the myxoid variant. Conventional chemotherapy with doxorubicin and ifosfamide represents the mainstay of systemic treatment in the first line. Other active cytotoxic agents include gemcitabine and docetaxel and the marine-derived compounds trabectedin. Recent progress in molecular diagnostics of each single LS subtype has improved the knowledge of the molecular characteristics and has led to two recent treatment targets: the amplification of mouse double minute 2 homolog and cyclin-dependent kinase-4 in well- and dedifferentiated LS. Thus far, only early-phase trials are reported and no new drugs have been introduced in daily clinical practice. The focus of this review is on current systemic treatment options, including novel strategies.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.