Specific triggering of the Fas signal transduction pathway in normal human keratinocytes

The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.     

Specific inhibition of granzyme B by parainfluenza virus type 3

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.     

Activation of signal transduction kinases by tamoxifen

PURPOSE: To study the signal transduction mechanisms of tamoxifen via the activation of MAPKs, JNK and ERK in order to understand its regulation of gene expression. METHODS: The effects of tamoxifen (TAM) on the activation of serine/threonine mitogen-activated protein kinase (MAPK, p42/ERK2) and the stress-activated protein kinases (p46 SAPK or c-Jun N-terminal kinase, JNK1) were evaluated using a human cervical epitheloid carcinoma HeLa cell line. RESULTS: TAM activated both JNK1 and ERK2 activities in a time- and dose-dependent manner in HeLa cells. The activation of JNK1 was enhanced when the cells were pretreated with prooxidant H2O2. CONCLUSIONS: These studies show that TAM activates the signal transduction kinases, JNK1 and ERK2, which may play important roles in the regulation of gene expression by TAM.     

Anti-inflammatory action of dapsone: inhibition of neutrophil adherence is associated with inhibition of chemoattractant-induced signal transduction

Dapsone has clinical utility as an anti-inflammatory agent but the mechanism of this action remains unknown. We have previously reported that dapsone inhibits beta2 integrin (CD11b/CD18)-mediated adherence of human neutrophils in vitro and now describe studies designed to discover how dapsone-mediated inhibition of this neutrophil function occurs. Results indicate that dapsone interferes with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli. They also show that dapsone-mediated suppression of this pathway inhibits the generation of second messengers essential to the activation of beta2 integrin molecules, as well as respiratory and secretory functions of neutrophils exposed to chemoattractants. We propose that the inhibition of chemoattractant-induced signal transduction by dapsone suppresses neutrophil recruitment and local production of toxic respiratory and secretory products in the affected skin of dermatitis herpetiformis and other neutrophilic dermatoses.     

Oncogenes and signal transduction

Cancer biologists now have a unified concept to guide their search for specific genetic abnormalities and new therapeutic approaches: The genes and proteins that participate in the conversion of normal into malignant cells are also involved in the key process that converts extracellular signals into intracellular events that culminate in division and growth.     

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.     

Lactogenic hormone signal transduction

Dialyzers are reused in approximately three quarters of the dialysis units in the United States, but the effect of reprocessing on dialyzer performance has not been extensively evaluated. In a crossover study of six chronic hemodialysis patients, we determined urea, creatinine, phosphate, and beta2-microglobulin clearances and dialysate protein loss for two types of low-flux and two types of high-flux dialyzers during use numbers 1, 2, 5, and 15. Dialyzers were reprocessed by an automated machine using Renalin (Renal Systems, Plymouth, MN) as the germicide. Dialyzer arterial and venous blood and dialysate outflow samples were obtained at 5 and 180 minutes of each dialysis session to evaluate solute clearances. Urea, creatinine, and phosphate clearances were calculated using dialysate concentrations, whereas beta2-microglobulin clearance was calculated using plasma concentrations to include its removal by adsorption to the dialysis membrane. There was a trend for urea, creatinine, and phosphate clearances to decrease with reuse for both low-flux and high-flux dialyzers, but these differences were not statistically significant. The clearance of beta2-microglobulin and dialysate total protein concentration was small for low-flux dialyzers; these values were not dependent on reuse. There was a trend for beta2-microglobulin clearance and dialysate total protein concentration to decrease during a dialysis treatment using high-flux dialyzers. More significantly, beta2-microglobulin clearance and dialysate total protein concentration decreased substantially with the reuse of high-flux dialyzers. These observations show that the maintenance of small solute clearances during reuse of high-flux dialyzers does not ensure the maintenance of large solute clearances.     

Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

Phosphatidate phosphohydrolase and signal transduction

A Mg(2+)-independent and N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) has been identified in the plasma membrane of cells and it has been purified. The enzyme is a multi-functional phosphohydrolase that can dephosphorylate phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate and these substrates are competitive inhibitors of the reaction. The action of PAP-2 could terminate signalling by these bioactive lipids and at the same time generates compounds such as diacylglycerol, sphingosine and ceramide which are also potent signalling molecules. In relation to phosphatidate metabolism, sphingosine (or sphingosine 1-phosphate) stimulates phospholipase D and thus the formation of phosphatidate. At the same time sphingosine inhibits PAP-2 activity thus further increasing phosphatidate concentrations. By contrast, ceramides inhibit the activation of phospholipase D by a wide variety of agonists and increase the dephosphorylation of phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. These actions demonstrate "cross-talk' between the glycerolipid and sphingolipid signalling pathways and the involvement of PAP-2 in modifying the balance of the bioactive lipids generated by these pathways during cell activation.     

Angiotensin II signal transduction pathways

It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin-angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.     

Endotoxin signal transduction in macrophages

Through its action on macrophages, bacterial lipopolysaccharide (LPS) or endotoxin can trigger responses that are protective or injurious to the host. This review examines the effects of LPS on macrophages by following events from the cell surface to the nucleus. The involvement of protein tyrosine kinases, mitogen-activated protein kinases, protein kinase C, G proteins, protein kinase A, ceramide-activated protein kinase, and microtubules in this process are reviewed. At the nuclear level, rel, C/EBP, Ets, Egr, fos, and jun family members have been implicated in activation of LPS-inducible gene expression.     

Sphingomyelin pathway in signal transduction

The purpose of this study was to evaluate the effect of high-dose oral calcium on biochemical indices of bone formation, bone bisphosphonate clearance (BBC) and bone mineral content (BMC) of the distal forearm in patients undergoing hemodialysis. Eighteen patients agreed to participate and were randomized in a double-blind manner to receive either 2 g elemental calcium/day (n = 9) or placebo (n = 9) for 6 months. Previous treatment with aluminum-containing phosphate binders was continued unchanged throughout the study. In the placebo group, serum alkaline phosphatase and osteocalcin tended to increase by 8.0 and 10.2%, respectively, while BBC changed significantly by 49.5% (p < 0.05). In the calcium group the opposite was observed with small decreases in alakline phosphatase and osteocalcin by 8.2 and 11.0%, respectively, and no change in BBC. BMC decreased by 5.0% in the placebo group, but increased by 5.2% in the calcium group, resulting in a difference of 10.2% (p < 0.05). The present study demonstrates that high-dose oral calcium tends to reduce bone turnover and seems able to prevent bone loss in hemodialyzed patients.     

Regulation of antigen receptor signal transduction by protein tyrosine kinases

The purpose of this study was to assess the value of electron beam computed tomography in the detection of cardiac calcifications in coronaries and valves of dialysis patients and to determine the rate at which calcification progresses. Forty-nine chronic hemodialysis patients aged 28 to 74 years were compared with 102 non-dialysis patients aged 32 to 73 years with documented or suspected coronary artery disease, all of whom underwent coronary angiography. We used high-resolution electron beam computed tomography scanning to make 30 axial slices with a distance of 3 mm between each slice. The number of calcifications, the surface area, and the average and highest density values were measured. We calculated a quantitative coronary artery calcium score and assessed calcification of mitral and aortic valves. In dialysis patients, the measurements were repeated after 12 months. The coronary artery calcium score was from 2.5-fold to fivefold higher in the dialysis patients than in the non-dialysis patients. Hypertensive dialysis patients had higher calcium scores than non-hypertensive dialysis patients (P < 0.05). A stepwise, multiple regression analysis confirmed the importance of age and hypertension. No correlation between calcium, phosphate, or parathyroid hormone values and the coronary calcium score was identified; however, the calcium score was inversely correlated with bone mass in the dialysis patients (r = 0.47, P < 0.05). The mitral valve was calcified in 59% of dialysis patients, while the aortic valve was calcified in 55%. The coronary artery calcium score was correlated with aortic valvular, but not mitral valvular calcification. A repeat examination of the dialysis patients at an interval of 1 year showed a disturbing tendency for progression. Our data under-score the frequency and severity of coronary and valvular calcifications in dialysis patients, and illustrate the rapid progression of this calcification. Finally, they draw attention to hypertension as an important risk factor in this process.