Reactive oxygen species and p38 mitogen-activated protein kinase mediate tumor necrosis factor α-converting enzyme (TACE/ADAM-17) activation in primary human monocytes

Tumor necrosis factor α-converting enzyme (TACE) is responsible for the shedding of cell surface TNF. Studies suggest that reactive oxygen species (ROS) mediate up-regulation of TACE activity by direct oxidization or modification of the protein. However, these investigations have been largely based upon nonphysiological stimulation of promonocytic cell lines which may respond and process TACE differently from primary cells. Furthermore, investigators have relied upon TACE substrate shedding as a surrogate for activity quantification. We addressed these concerns, employing a direct, cell-based fluorometric assay to investigate the regulation of TACE catalytic activity on freshly isolated primary human monocytes during LPS stimulation. We hypothesized that ROS mediate up-regulation of TACE activity indirectly, by activation of intracellular signaling pathways. LPS up-regulated TACE activity rapidly (within 30 min) without changing cell surface TACE expression. Scavenging of ROS or inhibiting their production by flavoprotein oxidoreductases significantly attenuated LPS-induced TACE activity up-regulation. Exogenous ROS (H(2)O(2)) also up-regulated TACE activity with similar kinetics and magnitude as LPS. H(2)O(2)- and LPS-induced TACE activity up-regulation were effectively abolished by a variety of selective p38 MAPK inhibitors. Activation of p38 was redox-sensitive as H(2)O(2) caused p38 phosphorylation, and ROS scavenging significantly reduced LPS-induced phospho-p38 expression. Inhibition of the p38 substrate, MAPK-activated protein kinase 2, completely attenuated TACE activity up-regulation, whereas inhibition of ERK had little effect. Lastly, inhibition of cell surface oxidoreductases prevented TACE activity up-regulation distal to p38 activation. In conclusion, our data indicate that in primary human monocytes, ROS mediate LPS-induced up-regulation of TACE activity indirectly through activation of the p38 signaling pathway.     

Citrullination, a possible functional link between susceptibility genes and rheumatoid arthritis

Antibodies directed to citrullinated proteins (anti-cyclic citrullinated peptide) are highly specific for rheumatoid arthritis (RA). Recent data suggest that the antibodies may be involved in the disease process of RA and that several RA-associated genetic factors might be functionally linked to RA via modulation of the production of anti-cyclic citrullinated peptide antibodies or citrullinated antigens.     

Insertion of leader peptidase into the thylakoid membrane during synthesis in a chloroplast translation system

The mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins are poorly understood. In this study, we have used a translation system isolated from chloroplasts to begin to investigate these mechanisms. The bacterial membrane protein leader peptidase (Lep) was used as a model protein because its targeting and insertion mechanisms are well understood for Escherichia coli and for the endoplasmic reticulum. Lep could thus provide insight into the functional homologies between the different membrane systems. Lep was efficiently expressed in the chloroplast translation system, and the protein could be inserted into thylakoid membranes with the same topology as in E. coli cytoplasmic membranes, following the positive-inside rule. Insertion of Lep into the thylakoid membrane was stimulated by the trans-thylakoid proton gradient and was strongly inhibited by azide, suggesting a requirement for SecA activity. Insertion most likely occurred in a cotranslational manner, because insertion could only be observed if thylakoid membranes were present during translation reactions but not when thylakoid membranes were added after translation reactions were terminated. To halt the elongation process at different stages, we translated truncated Lep mRNAs without a stop codon, resulting in the formation of stable ribosome nascent chain complexes. These complexes showed a strong, salt-resistant affinity for the thylakoid membrane, implying a functional interaction of the ribosome with the membrane and supporting a cotranslational insertion mechanism for Lep. Our study supports a functional homology for the insertion of Lep into the thylakoid membrane and the E. coli cytoplasmic membrane.     

Increasing Glucose 6-Phosphate Dehydrogenase Activity Restores Redox Balance in Vascular Endothelial Cells Exposed to High Glucose

Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Human T lymphocytes synthesize the 92 kDa type IV collagenase (gelatinase B)

In order for T cells to exit the circulatory system, traverse the endothelial basement membrane, and arrive in target tissues, these cells must attach to and degrade basement membrane proteins. 12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to stimulate lymphoid cell adhesion to basement membrane components. We have used TPA to study the ability of human lymphoid cells to secrete type IV collagenases, enzymes capable of degrading basement membrane proteins. Here, we found that human primary T cells and H-9 lymphoid cells synthesize the 92 kDa type IV collagenase (gelatinase B) and TPA stimulates the synthesis and secretion of this protease. Peak TPA-stimulated gelatinase B secretion and mRNA accumulation were observed 9 hours after TPA treatment, while the peak adhesion to type IV collagen was observed only 3 hours after TPA treatment. The protein kinase C inhibitor, H-7, inhibited TPA-stimulated gelatinase B secretion. Both the primary T cells and H-9 lymphoid cells also expressed the mRNA for the tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that TPA - stimulated lymphoid cells adhere to type IV collagen and subsequently synthesize and secrete gelatinase B and TIMP-1. We conclude that lymphoid cell extravasation may involve cellular employment of adhesion mechanisms prior to degradation of the matrix, which is similar to the process of extravasation used by metastatic cells. © 1993 Wiley-Liss, Inc.     

Essential Role of PACT-Mediated PKR Activation in Tunicamycin-Induced Apoptosis

Cellular stresses such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds result in accumulation of misfolded proteins in the endoplasmic reticulum (ER) and lead to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double-stranded RNA (dsRNA)-activated protein kinase (PKR) and its protein activator PACT in tunicamycin-induced apoptosis. We demonstrate for the first time that PACT is phosphorylated in response to tunicamycin and is responsible for PKR activation by direct interaction. Furthermore, PACT-induced PKR activation is essential for tunicamycin-induced apoptosis, since PACT as well as PKR null cells are markedly resistant to tunicamycin and show defective eIF2α phosphorylation and C/EBP homologous protein (CHOP, also known as GADD153) induction especially at low concentrations of tunicamycin. Reconstitution of PKR and PACT expression in the null cells renders them sensitive to tunicamycin, thus demonstrating that PACT-induced PKR activation plays an essential function in induction of apoptosis.