Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.

Ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1/CD39) regulates neutrophil chemotaxis by hydrolyzing released ATP to adenosine

Polymorphonuclear neutrophils release ATP in response to stimulation by chemoattractants, such as the peptide N-formyl-methionyl-leucyl-phenylalanine. Released ATP and the hydrolytic product adenosine regulate chemotaxis of neutrophils by sequentially activating purinergic nucleotide and adenosine receptors, respectively. Here we show that that ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) is a critical enzyme for hydrolysis of released ATP by neutrophils and for cell migration in response to multiple agonists (N-formyl-methionyl-leucyl-phenylalanine, interleukin-8, and C5a). Upon stimulation of human neutrophils or differentiated HL-60 cells in a chemotactic gradient, E-NTPDase1 tightly associates with the leading edge of polarized cells during chemotaxis. Inhibition of E-NTPDase1 reduces the migration speed of neutrophils but not their ability to detect the orientation of the gradient field. Studies of neutrophils from E-NTPDase1 knock-out mice reveal similar impairments of chemotaxis in vitro and in vivo. Thus, E-NTPDase1 plays an important role in regulating neutrophil chemotaxis by facilitating the hydrolysis of extracellular ATP.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation.

Substrate binding tunes conformational flexibility and kinetic stability of an amino acid antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.