TNF-alpha Converting Enzyme Research Articles

Cell-cell interaction promotes rat marrow stromal cell differentiation into endothelial cell via activation of TACE/TNF-alpha signaling.

Marrow stromal cells (MSCs) are capable of differentiating into various cell types including endothelial cells. Microenvironment is important in cell fate determination. Tumor necrosis factor-alpha converting enzyme (TACE), a well-characterized "sheddase," participates in the differentiation process of multiple lineages by the proteolytic release of membrane-bound proteins such as tumor necrosis factor-alpha (TNF-alpha). We investigated the endothelial differentiation of MSCs under two coculture conditions: 1) direct MSCs-rat brain microvascular endothelial cells (rBMECs) contact coculture; and 2) indirect coculture of MSCs and rBMECs. Also, we examined the role of TACE/TNF-alpha signaling in the process of differentiation under direct coculture condition. We found that endothelial differentiation of MSCs was substantially enhanced in MSCs-rBMECs direct contact coculture, but not in indirect transwell coculture condition. Transcript levels of TACE and TNF-alpha as well as TACE protein expression were significantly upregulated in direct, but not in indirect, coculture condition. Addition of human recombinant TACE promoted gene expression of endothelial specific markers including vWF, CD31, VE-cadherin, Flk-1, and Flt-1 in the differentiating MSCs. Furthermore, inhibition of TACE with TAPI-2 or inhibition of TNF-alpha with Etanercept attenuated endothelial differentiation of MSCs in the direct coculture condition. We demonstrated for the first time that direct MSCs-rBMECs interaction stimulated the endothelial differentiation of MSCs via TACE/TNFalpha signaling.

The Study of the Inhibition of the Recombinant TACE Prodomain to Endotoxemia in Mice

Objective:To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-α (TNF-α) converting enzyme (TACE) on TACE activity and to develop an approach to interfere with inflammation processes.Methods:The cDNA encoding the full-length ectodomain (T1300) and prodomain (T591) of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+)-T1300 and pET-28a (+)-T591) were constructed and transformed into E. coli BL21. After Ni2+-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning.Results:The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-α, which mediated the accumulation of TNF-α on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice.Conclusions:The recombinant prodomain of TACE has the ability to inhibit sTNF-α release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.

TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds

Because outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) might reemerge, identifying antiviral compounds is of key importance. Previously, we showed that the cellular factor TNF-α converting enzyme (TACE), activated by the spike protein of SARS-CoV (SARS-S protein), was positively involved in viral entry, implying that TACE is a possible target for developing antiviral compounds. To demonstrate this possibility, we here tested the effects of TACE inhibitors on viral entry. In vitro and in vivo data revealed that the TACE inhibitor TAPI-2 attenuated entry of both pseudotyped virus expressing the SARS-S protein in a lentiviral vector backbone and infectious SARS-CoV. TAPI-2 blocked both the SARS-S protein-induced shedding of angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV, and TNF-α production in lung tissues. Since the downregulation of ACE2 by SARS-S protein was proposed as an etiological event in the severe clinical manifestations, our data suggest that TACE antagonists block SARS-CoV infection and also attenuate its severe clinical outcome.

Effects of biological drug adalimumab on tumour necrosis factor‐α‐converting enzyme activation

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE) is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the TNF family. No investigations into the regulation of this enzyme by autoantibodies have been reported. In this study, we tested the hypothesis that anti-Ro/SSA autoantibodies, purified from IgG fractions of patients with primary Sjögren's syndrome, are capable to regulate TACE expression and activation in human salivary gland epithelial cells (SGEC). We also evaluated the potential physiological and therapeutic consequences of TNF-alpha blocking by the biological agent adalimumab, the first fully human (100% human peptide sequences) therapeutic anti-TNF-alpha antibody, on post-translational regulation of TACE. Taken together, our results show a dose-dependent increase in TACE expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein. Adalimumab treatment brought TACE expression to levels than those observed in untreated SGEC. These findings, showing the presence of autoantibodies-dependent mechanisms by which TACE levels are regulated in human SGECs, may have implications in the context of current investigations on the pathological role of autoantibodies.

Abstract 5308: Role of Vascular Endothelial Growth Factor-A in the Development of Mice CaCl 2 Induced Abdominal Aortic Aneurysm

Background: Abdominal aortic aneurysm (AAA) is histologically characterized by increased angiogenesis, chronic inflammation, and extracellular matrix degradation. We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, which is a potent angiogenic and proinflammatory factor, in the development of AAA. Methods and Results: We obtained human aortic wall samples (n=10) during surgical repair of AAA. Autopsy samples from normal aortic wall were served as control (n=6). Immunohistochemistry showed increased CD31 positive vessels, VEGF-A expression, and CD68 positive macrophage infiltration in the aortic wall of AAA compared with control samples. Next, we induced AAA in mice by periaortic application of CaCl 2 . Mice were treated with intraperitoneal injection of soluble VEGF-A receptor-1 (sFlt-1) 1mg/g (FLT, n=8) or PBS 1 μ l (CON, n=8) on alternative day and sacrificed 28 days after the operation. Blood pressure, heart rate, and body weight on day 28 were comparable between FLT and CON. Treatment with sFlt-1 significantly reduced aneurismal size (1.7±0.9 vs. 1.3±0.2 mm, p< 0.01) in association with lowering serum VEGF-A level (p< 0.01). Elastica von Gieson staining showed that wavy morphology of the elastic lamellae was destroyed in CON, but was preserved in FLT. Administration of sFlt-1 reduced zymographic active form of matrix metalloproteinase (MMP)-9 abundance and decreased MMP-9 positive cell count in the aortic wall compared with CON (both p<0.05). Immunohistochemistry showed significantly fewer Mac-3 positive monocytes/macrophages, CD31 and VEGF-A positive cells in periaortic tissues in FLT compared with CON (all p<0.05). Aortic wall mRNA expression of MCP-1, TNF-alpha, and TNF-alpha converting enzyme (TACE) in FLT was lower than that in CON (all p< 0.05). Immunoblotting showed decreased expression of p-JNK in FLT compared with CON (p< 0.05). Conclusions: VEGF-A was overexpressed in arterial wall of human AAA and experimental model of AAA. Treatment with sFlt-1 in mice prevented AAA development, in association with reduced neoangiogenesis, MMPs activity, infiltration of inflammatory cells and JNK activity, and preserved ECM structure. These findings suggest a crucial role of VEGF-A in the development of AAA.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögren's syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription-PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.

Expression of tumor necrosis factor-alpha converting enzyme and matrix metalloproteinase-3 in proliferated synovium in a patient with synovitis-acne-pustulosis-hyperostosis-osteitis syndrome: a case report

IntroductionSynovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome is a rare disorder. The etiology remains unknown and the treatment is still empirical. Synovitis is one of the major manifestations, but information on histopathological features is still lacking. In this case, we investigated the histopathological features of SAPHO syndrome synovitis.Case presentationWe present the case of a 53-year-old Japanese woman with SAPHO syndrome accompanied by marked knee synovitis and palmoplantar pustulosis. We found abundant sterile joint fluid in the right knee, and a blood test showed abnormally high values of C-reactive protein (17.26 mg/dl) and matrix metalloproteinase-3 (800 ng/ml). Arthroscopic surgery revealed marked proliferation of villous synovial tissues similar to rheumatoid arthritis and standard microscopic findings were also similar to rheumatoid arthritis. Furthermore, for the first time, we demonstrated by immunohistochemistry the expression of tumor necrosis factor-alpha (TNF-α) converting enzyme, TNF-α and matrix metalloproteinase-3 in the proliferated synovial lining cells. After arthroscopic synovectomy, her knee symptoms immediately diminished and laboratory data (matrix metalloproteinase-3 and C-reactive protein) normalized within 2 weeks of surgery.ConclusionWe demonstrate the expression of TNF-α converting enzyme, TNF-α and matrix metalloproteinase-3 in SAPHO syndrome synovitis for the first time and also show, both macro- and microscopically, the similarity between SAPHO syndrome and rheumatoid arthritis synovitis. These new findings support the recently reported successful treatment of SAPHO syndrome with antirheumatic drugs, especially with anti-TNF-α agents.

Prediction of novel and selective TNF-alpha converting enzyme (TACE) inhibitors and characterization of correlative molecular descriptors by machine learning approaches

The inhibition of TNF-α converting enzyme (TACE) has been explored as a feasible therapy for the treatment of rheumatoid arthritis (RA) and Crohn's disease (CD). Recently, large numbers of novel and selective TACE inhibitors have been reported. It is desirable to develop machine learning (ML) models for identifying the inhibitors of TACE in the early drug design phase and test the prediction capabilities of these ML models. This work evaluated four ML methods, support vector machine (SVM), k-nearest neighbor ( k-NN), back-propagation neural network (BPNN) and C4.5 decision tree (C4.5 DT), which were trained and tested by using a diverse set of 443 TACE inhibitors and 759 non-inhibitors. A well-established feature selection method, the recursive feature elimination (RFE) method, was used to select the most appropriate descriptors for classification from a large pool of descriptors, and two evaluation methods, 5-fold cross-validation and independent evaluation, were used to assess the performances of these developed models. In this study, all these ML models have already achieved promising prediction accuracies. By using the RFE method, the prediction accuracies are further improved. In k-NN, the model gives the best prediction for TACE inhibitors (98.32%), and the SVM bears the best prediction for non-inhibitors (99.51%). Both the k-NN and SVM model give the best overall prediction accuracy (98.45%). To the best of our knowledge, the SVM model developed in this work is the first one for the classification prediction of TACE inhibitors with a broad applicability domain. Our study suggests that ML methods, particularly SVM, are potentially useful for facilitating the discovery of TACE inhibitors and for exhibiting the molecular descriptors associated with TACE inhibitors.

SLITRK1 Binds 14-3-3 and Regulates Neurite Outgrowth in a Phosphorylation-Dependent Manner

Rare genetic variants of SLITRK1 have been previously associated with Tourette syndrome (TS), attention-deficit/hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD) symptoms. We studied SLITRK1 processing and phosphorylation. To explore potential signaling pathways of the cytoplasmic domain of SLITRK1, we made use of the yeast two-hybrid screen. We observed that the extracellular domain of SLITRK1 is secreted in vitro and in vivo and that this process is activated by protein kinase C and inhibited by an inhibitor of tumor necrosis factor-alpha converting enzyme (TACE). We observed that SLITRK1 undergoes gamma-secretase cleavage to release a SLITRK1 intracellular domain (SICD). We identified an interaction between SLITRK1 and 14-3-3 proteins and observed that these proteins co-localized in cortical neuronal cultures and were coprecipitated from rat brain lysates, consistent with an interaction in vivo. We mapped the binding site to the very COOH-terminus of SLITRK1, as deletion of the last six amino acids of SLITRK1 abolished the interaction. We demonstrated phosphorylation of SLITRK1 by protein kinase A (PKA), protein kinase C (PKC), and casein kinase II (CK2) and observed that CK2 phosphorylates SLITRK1 in the 14-3-3 binding site. Mutating the CK2 phosphorylation site of SLITRK1 decreased binding to 14-3-3 and inhibited SLITRK1-mediated neurite outgrowth. Our results shed light on the cell biology of SLITRK1, including its protein phosphorylation and potential molecular pathways for SLITRK1 function, and should contribute to further understanding the role of SLIRTK1 in developmental neuropsychiatric conditions such TS, OCD, and ADHD.

Cellular prion protein coupling to TACE‐dependent TNF‐α shedding controls neurotransmitter catabolism in neuronal cells

Despite considerable efforts to unravel the role of cellular prion protein (PrP(C)) in neuronal functions, the mechanisms by which PrP(C) takes part in the homeostasis of a defined neuronal phenotype remain poorly characterized. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, we assessed the contribution of PrP(C) to bioaminergic cell functions. We established that in 1C11-derived neuronal cells antibody-mediated PrP(C) ligation triggered tumor necrosis factor (TNF)-alpha release, through recruitment of the metalloproteinase TNF-alpha converting enzyme (TACE). TNF-alpha shed in response to PrP(C) acts as a second message signal, eliciting serotonin (5-HT) or norepinephrine (NE) degradation in 1C11(5-HT) or 1C11(NE) cells, respectively. Our data thus introduced TNF-alpha as a PrP(C)-dependent modulator of neuronal metabolism. Of note, we previously reported on a control of neurotransmitter catabolism by 5-HT(2B) or alpha(1D) autoreceptors in 1C11 bioaminergic neurons, via the same TACE/TNF-alpha pathway (Ann. N Y Acad. Sci. 1091, 123). Here, we show that combined stimulation of PrP(C) and these two bioaminergic receptors add their effects on neurotransmitter degradation. Overall, these observations unveil a novel contribution of PrP(C) to the control of neuronal functions and may have implications regarding dysfunction of the bioaminergic systems in prion diseases.

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.

ADAM17 as a Therapeutic Target in Multiple Diseases

As a metalloproteinase specialized in releasing membrane-tethered proteins, A Disintegrin and A Metalloproteinase 17 (ADAM17), also known as Tumor necrosis factor-alpha Converting Enzyme (TACE) or less commonly CD156q, has received more than its share of attention. This is mainly because major contemporary pathologies like cancer, inflammatory and vascular diseases seem to be connected to its cleavage abilities. The involvement in such a broad spectrum of diseases is due to the large variety of substrates that ADAM17 is able to cut. ADAM17 can activate growth factors or inactivate receptors by shedding their extracellular domain from the cell membrane. Similarly, it can detach cells by cleaving cell adhesion molecules. Some of these proteolytic events are part of cleavage cascades known as Regulated Intramembrane Proteolysis and lead to intracellular signaling. It is therefore clear that ADAM17 literally fulfills a key role in diverse processes and pathologies, making it a prime target for developing therapies. Here we review the role of ADAM17 in health and disease and highlight the problems to overcome for ADAM17 to mature towards a therapeutically valuable target.

Minireview: Pref-1: Role in Adipogenesis and Mesenchymal Cell Fate

Preadipocyte factor-1 [Pref-1; also called Dlk1 (Delta-like protein 1)] is made as an epidermal growth factor-repeat containing transmembrane protein that produces a biologically active soluble form by TNF-alpha-converting enzyme (TACE)-mediated cleavage. Soluble Pref-1 activates the MAPK kinase/ERK pathway. In adipose tissue, Pref-1 is specifically expressed in preadipocytes but not in adipocytes and thus is used as a preadipocyte marker. Inhibition of adipogenesis by Pref-1 has been well established in vitro as well as in vivo by ablation and overexpression of Pref-1. SRY (sex determining region Y)-box 9 (Sox9), a transcription factor expressed in preadipocytes to suppress CCAAT enhancer binding protein beta and (C/EBP) delta expression, is required to be down-regulated before adipocyte differentiation can proceed. By activating MAPK kinase/ERK, Pref-1 prevents down-regulation of Sox9, resulting in inhibition of adipogenesis. Furthermore, by inducing Sox9, Pref-1 promotes chondrogenic induction of mesenchymal cells but prevents chondrocyte maturation as well as osteoblast differentiation. Thus, Pref-1 directs multipotent mesenchymal cells toward the chondrogenic lineage but inhibits differentiation into adipocytes as well as osteoblasts and chondrocytes. Pref-1, encoded by an imprinted gene, has also been detected in progenitor cells in various tissues during regeneration and therefore may have a more general role in maintaining cells in an undifferentiated state.

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion

Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.

Discovery of Potent, Selective, and Orally Active Carboxylic Acid Based Inhibitors of Matrix Metalloproteinase-13

The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would therefore be a novel disease modifying therapy for the treatment of arthritis. Our efforts have resulted in the discovery of a series of carboxylic acid inhibitors of MMP-13 that do not significantly inhibit the related MMP-1 (collagenase-1) or tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE). It has previously been suggested (but not proven) that inhibition of the latter two enzymes could lead to side effects. A promising carboxylic acid lead 9 was identified and a convergent synthesis developed. This paper describes the optimization of 9 and the identification of a compound 24f for further development. Compound 24f is a subnanomolar inhibitor of MMP-13 (IC(50) value 0.5 nM and K(i) of 0.19 nM) having no activity against MMP-1 or TACE (IC(50) of >10000 nM). Furthermore, in a rat model of MMP-13-induced cartilage degradation, 24f significantly reduced proteoglycan release following oral dosing at 30 mg/kg (75% inhibition, p < 0.05) and at 10 mg/kg (40% inhibition, p < 0.05).

TACE‐induced cleavage of Neogenin disinhibits CNS axon/neurite growth by RGM in rat primary cortical neuron

Neogenin is multifunctional receptor implicated in axon navigation, neuronal differentiation, morphogenesis, and cell death. Recently, neogenin appears to be a high‐affinity receptor for the repulsive guidance molecule (RGM), and RGMa‐neogenin interaction inhibits axon regeneration. While performing a two‐hybrid screen using the intracellular domain of neogenin, we observed that neogenin bound to ADAM17, also called TACE (TNF‐alpha converting enzyme). TACE is a 70‐kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases, and plays a role in proteolytic processing of some kinds of receptors, ligands, and enzymes. This process is of physiological importance. We show here that the cleavage of neogenin by TACE attenuates RGMa‐dependent neurite outgrowth inhibition in rat primary cortical neuron. Thus, TACE can be used as a molecular target for clinical conditions characterized by a failure of CNS regeneration

Mechanisms and consequences of macrophage apoptosis in atherosclerosis

Macrophage apoptosis is an important feature of atherosclerotic plaque development. Research directed at understanding the functional consequences of macrophage death in atherosclerosis has revealed opposing roles for apoptosis in atherosclerotic plaque progression. In early lesions, macrophage apoptosis limits lesion cellularity and suppresses plaque progression. In advanced lesions, macrophages apoptosis promotes the development of the necrotic core, a key factor in rendering plaques vulnerable to disruption and in acute lumenal thrombosis. The first section of this review will examine the role of phagocytic clearance of apoptotic macrophages, a process known as efferocytosis, in the dichotomous roles of macrophage apoptosis in early vs. advanced lesions. The second section will focus on the molecular and cellular mechanisms that are thought to govern macrophage death during atherosclerosis. Of particular interest is the complex and coordinated role that the endoplasmic reticulum (ER) stress pathway and pattern recognition receptors (PRRs) may play in triggering macrophage apoptosis.

A025 P38 MAPK inhibition prevents TACE-mediated receptor shedding and improves the hemostatic function of stored platelets

Platelet transfusion is a lifesaving procedure widely used during surgery and chemotherapy. During storage platelets undergo several modifications that reduce their postransfusion survival and functionality. One important feature of this platelet storage lesion (PSL) is the shedding of surface glycoproteins such as GPIbα and GPV. We have recently demonstaretd that metalloproteinase inhibitors prevent storage-induced shedding of adhesion receptors, resulting in markedly improved postransfusion recovery and hemostatic function of platelets in mice. We now demonstrate that TNF-alpha converting enzyme (TACE/ADAM17) mediates receptor shedding during platelet storage for 16 to 18 hours at 37oC or 22oC. Using pharmacological inhibitors, we demonstrate that TACE/ADAM17-dependent shedding of GPIbα and GPV from both mouse and human platelets during storage required p38 MAP kinase signaling. In contrast, protein kinase C, Erk MAPK, and caspases were not involved. Inhibition of p38 MAPK during platelets storage also markedly improved the posttransfusion recovery and hemostatic function of stored platelets in vivo. Moreover, p38 MAPK inhibition during storage of human platelets improved the adhesion of these cells to collagen under flow. Inhibition of p38 MAPK also prevented TACE-dependent shedding of GPIbα from platelets undergoing mitochondrial injury, a model for PSL. Phosphorylation of p38 MAPK was observed upon platelet storage and mitochondrial injury. In summary, our data suggest that inhibition of p38 MAPK or TACE during storage may significantly improve the quality of stored platelets.

Anti‐inflammatory Effect of Palmitoylethanolamide on Human Adipocytes

Obesity leads to the appearance of an inflammatory process, which can be initiated even with a moderate weight gain. Palmitoylethanolamide (PEA) is an endogenous lipid, secreted by human adipocytes, that possesses numerous anti-inflammatory properties. The main purpose of this study was to investigate the anti-inflammatory effect of PEA on human adipocytes, as well as in a murine model. The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-treated human subcutaneous adipocytes in primary culture and CF-1 mice was investigated by enzyme-linked immunosorbent assay. The effects of PEA on adipocyte TNF-alpha secretion were explored as well as some suspected PEA anti-inflammatory pathways: nuclear factor-kappaB (NF-kappaB) pathway, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) gene expression, and TNF-alpha-converting enzyme (TACE) activity. The effects of PEA on the TNF-alpha serum concentration in intraperitoneally LPS-treated mice were also studied. We demonstrate that the LPS induced secretion of TNF-alpha by human adipocytes is inhibited by PEA. This action is neither linked to a reduction in TNF-alpha gene transcription nor to the inhibition of TACE activity. Moreover, PPAR-alpha is not implicated in this anti-inflammatory activity. Lastly, PEA exhibits a wide-reaching anti-inflammatory action as the molecule is able to completely inhibit the strong increase in TNF-alpha levels in the serum of mice treated with high doses of LPS. In view of its virtual lack of toxicity, PEA might become a potentially interesting candidate molecule in the prevention of obesity-associated insulin resistance.

Porphyromonas gingivalis stimulates TACE production by T cells

Tumour necrosis factor-alpha converting enzyme (TACE), also known as ADAM17, is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the tumour necrosis factor family. The aim of this study was to investigate the effect of Porphyromonas gingivalis on TACE production by a human T-cell line, to identify putative virulence factors involved in this process, and to investigate the effect of doxycycline. P. gingivalis 6-day culture supernatants were used to challenge Jurkat T cells for 6 h. Secreted and cell-associated TACE levels were measured by enzyme-linked immunosorbent assay, whereas messenger RNA expression was investigated by quantitative real-time polymerase chain reaction. To investigate the involvement of cysteine proteases or proteinaceous components in general, P. gingivalis culture supernatants were treated with the specific chemical inhibitor TLCK or heat-inactivated, respectively. The effect of doxycycline on the regulation of TACE secretion by P. gingivalis was also investigated. P. gingivalis challenge resulted in a concentration-dependent enhancement of TACE messenger RNA expression and protein release by Jurkat cells. TLCK treatment or heat treatment of P. gingivalis culture supernatants decreased TACE release to control levels. Doxycycline inhibited TACE secretion dose dependently. The induction of TACE by T cells in response to P. gingivalis may in turn favour the shedding of host cell-bound cytokines into the local microenvironment, potentially amplifying the inflammatory response. In the present experimental system, P. gingivalis cysteine proteases are involved in TACE release by T cells.