Nuclear Factor-κB-Independent Anti-Inflammatory Action of Salicylate in Human Endothelial Cells

In contrast to aspirin, salicylate, its active metabolite, possesses profound anti-inflammatory properties without blocking cyclooxygenase. Inhibition of the transcription factor nuclear factor-κB (NF-κB) has been discussed to play a role in the anti-inflammatory profile of salicylate. However, NF-κB-independent effects of salicylate have been assumed but have up to now been poorly investigated. Therefore, the aim of the present study was to investigate NF-κB-independent anti-inflammatory mechanisms of salicylate in human umbilical vein endothelial cells using interleukin-4 (IL-4) as NF-κB-independent proinflammatory stimulus and P-selectin as inflammatory read-out parameter. Using quantitative real-time reverse transcriptionpolymerase chain reaction, we found that salicylate decreases IL-4-induced P-selectin expression. As judged by Western blot analysis, salicylate increased endothelial heme oxygenase-1 (HO-1) protein levels. Using both the HO-1 inhibitor tin(II) protoporphyrin IX and HO-1 antisense oligonucleotides, we causally linked the induction of HO-1 to the decrease of P-selectin. Moreover, we were interested in the signaling mechanisms leading to the up-regulation of HO-1 by salicylate. c-Jun NH2-terminal kinase (JNK) was found to be activated by salicylate, and we could causally link this activation to the induction of HO-1 by using the JNK inhibitor 1,9-pyrazoloanthrone. By applying activator protein-1 (AP-1) decoys, it was shown that the transcription factor AP-1 is crucially involved in the up-regulation of HO-1 downstream of JNK. In summary, our study introduces HO-1 as novel NF-κB-independent anti-inflammatory target of salicylate in human endothelial cells. Moreover, we elucidated the JNK/AP-1 pathway as crucial for the induction of HO-1 by salicylate

Control of CAR-T cell activity in space and time: the next level of anti-tumor action

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Atrial Natriuretic Peptide, a Regulator of Nuclear Factor-κB Activation in Vivo

Natriuretic peptides (NPs) comprise a family of vasoactive hormones that play important roles in the regulation of cardiovascular and renal homeostasis. Along this line, atrial NP (ANP) (international non-proprietary name: carperitide, HANP) is an approved drug for the treatment of acute heart failure. In recent years, evidence has been given that the NP system possesses a far broader biological spectrum than the regulation of blood pressure and volume homeostasis. In fact, a substantial amount of in vitro work indicates that ANP affects important inflammatory processes and signaling pathways. Quite surprisingly, however, no information exists on the in vivo antiinflammatory potential and signaling of ANP. We show here that pretreatment of lipopolysaccharide (Salmonella abortus equi, 2.5 mg/kg)-challenged mice with ANP (5μg/kg iv, 15 min) rapidly inhibits nuclear factor-κB activation via inhibition of phosphorylation and degradation of the IκB-α protein. ANP also reduces Akt activation upon lipopolysaccharide injection. In ANP-pretreated mice, the increase of TNF-α serum concentration is markedly prevented; most importantly, the survival of these animals improved. These findings demonstrate both in vitro and in vivo an antiinflammatory profile of ANP that deserves to be further investigated in a therapeutic perspective

MAPK phosphatase-1 represents a novel antiinflammatory target of glucocorticoids in the human endothelium

Glucocorticoids are well-established anti- inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NF-κB. In recent years, however, transcription factorindependent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase-1 (MKP-1) as a pivotal mediator of the anti-inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF-α-activated human endothelial cells and used the adhesion molecule E-selectin as inflammatory read-out parameter. Dex is known to reduce the expression of E-selectin, which is largely regulated by NF-κB. Here, we communicate that Dex at low concentrations (1–100 nM) markedly attenuates E-selectin expression without affecting NF-κB. Importantly, Dex is able to increase the expression of MKP-1, which causes an inactivation of TNF-α-induced p38 MAPK and mediates inhibition of E-selectin expression. In endothelial MKP-1ˉ/ˉ cells differentiated from MKP-1ˉ/ˉ embryonic stem cells and in MKP-1-silenced human endothelial cells, Dex did not inhibit TNF-α-evoked E-selectin expression. Thus, our findings introduce MKP-1 as a novel and crucial mediator of the anti-inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP-1 as an important and promising antiinflammatory drug target

Coronavirus 3CL(pro )proteinase cleavage sites: Possible relevance to SARS virus pathology

BACKGROUND: Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. RESULTS: We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR), transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. CONCLUSIONS: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website:

Dexamethasone-Induced Expression of Endothelial Mitogen-Activated Protein Kinase Phosphatase-1 Involves Activation of the Transcription Factors Activator Protein-1 and 3',5'-Cyclic Adenosine 5'-Monophosphate Response Element-Binding Protein and the Generation of Reactive Oxygen Species

We have recently identified the MAPK phosphatase (MKP)-1 as a novel mediator of the antiinflammatory properties of glucocorticoids (dexamethasone) in the human endothelium. However, nothing is as yet known about the signaling pathways responsible for the up-regulation of MKP-1 by dexamethasone in endothelial cells. Knowledge of the molecular basis of this new alternative way of glucocorticoid action could facilitate the identification of new antiinflammatory drug targets. Thus, the aim of our study was to elucidate the underlying molecular mechanisms. Using Western blot analysis, we found that dexamethasone rapidly activates ERK, c-jun Nterminal kinase (JNK), and p38 MAPK in human umbilical vein endothelial cells. By applying the kinase inhibitors PD98059 (MAPK kinase-1) and SP600125 (JNK), ERK and JNK were shown to be crucial for the induction of MKP-1. Using EMSA and a decoy oligonucleotide approach, the transcription factors activator protein-1 (activated by ERK and JNK) and cAMP response element-binding protein (activated by ERK) were found to be involved in the up-regulation of MKP-1 by dexamethasone. Interestingly, dexamethasone induces the generation of reactive oxygen species (measured by dihydrofluorescein assay), which participate in the signaling process by triggering JNK activation. Our work elucidates a novel alternative mechanism for transducing antiinflammatory effects of glucocorticoids in the human endothelium. Thus, our study adds valuable information to the efforts made to find new antiinflammatory principles utilized by glucocorticoids. This might help to gain new therapeutic options to limit glucocorticoid side effects and to overcome resistance

Non-classical protein secretion in bacteria

BACKGROUND: We present an overview of bacterial non-classical secretion and a prediction method for identification of proteins following signal peptide independent secretion pathways. We have compiled a list of proteins found extracellularly despite the absence of a signal peptide. Some of these proteins also have known roles in the cytoplasm, which means they could be so-called "moon-lightning" proteins having more than one function. RESULTS: A thorough literature search was conducted to compile a list of currently known bacterial non-classically secreted proteins. Pattern finding methods were applied to the sequences in order to identify putative signal sequences or motifs responsible for their secretion. We have found no signal or motif characteristic to any majority of the proteins in the compiled list of non-classically secreted proteins, and conclude that these proteins, indeed, seem to be secreted in a novel fashion. However, we also show that the apparently non-classically secreted proteins are still distinguished from cellular proteins by properties such as amino acid composition, secondary structure and disordered regions. Specifically, prediction of disorder reveals that bacterial secretory proteins are more structurally disordered than their cytoplasmic counterparts. Finally, artificial neural networks were used to construct protein feature based methods for identification of non-classically secreted proteins in both Gram-positive and Gram-negative bacteria. CONCLUSION: We present a publicly available prediction method capable of discriminating between this group of proteins and other proteins, thus allowing for the identification of novel non-classically secreted proteins. We suggest candidates for non-classically secreted proteins in Escherichia coli and Bacillus subtilis. The prediction method is available online

Metalloporphyrins inactivate caspase-3 and -8

Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1