ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis.

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress-induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress-activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1-dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.

Effect of Th1/Th2 cytokine pretreatment on RSV-induced gene expression in airway epithelial cells.

BACKGROUND: Respiratory syncytial virus (RSV) infection in infants with Th2 predisposition is thought to increase the risk of allergic sensitization, recurrent wheezing, and bronchial asthma during childhood. We attempted to clarify the molecular mechanisms by which Th1/Th2 predisposition in the host alters RSV infection and facilitates airway inflammation. METHODS: A549 human airway epithelial cells were inoculated with live or UV-treated RSV after pretreatment with either a combination of tumor necrosis factor (TNF)-α and interferon-γ (Th1-primed) or a combination of TNF-α and interleukin-4 (Th2-primed) for 48 h. The gene and protein expression profiles of RSV-infected A549 cells were examined. RESULTS: GeneChip analysis indicated that, at 96 h after inoculation with RSV, the expression of 62 genes was specifically enhanced (more than 2-fold by normalized data) in Th2-primed cells compared to that in unprimed or Th1-primed cells. An increase in mRNA and protein levels of monocyte chemoattractant protein (MCP)-1/CCL2 among those 62 genes was confirmed by real-time PCR and cytometric bead assay, respectively. RSV replication was markedly diminished in Th1-primed airway epithelial cells but not in Th2-primed cells, which was presumably caused at least in part by the early induction of antiviral genes. CONCLUSIONS: These results suggest that Th1/Th2 predisposition in the host prior to RSV infection critically regulates inflammatory reactions in the airways through alteration of gene expression, and that MCP-1/CCL2 plays an important role in the pathogenesis of severe RSV infection and the subsequent development of asthma in Th2-predisposed hosts.

CXCR3 binding chemokine and TNFSF14 over expression in bladder urothelium of patients with ulcerative interstitial cystitis.

PURPOSE: We investigated the genes responsible for ulcerative interstitial cystitis by DNA microarray analysis and quantitative real-time polymerase chain reaction. MATERIALS AND METHODS: Bladder urothelial tissues were taken from a site apart from the ulcerative lesion in 9 patients with ulcerative interstitial cystitis and from a normal-looking area in 9 controls, including 7 with bladder carcinoma and 2 with benign prostatic hyperplasia. Total RNA was extracted from bladder samples and gene expression was compared between these 2 groups using Whole Human Genome DNA microarray 44K (Agilent Technologies, Santa Clara, California). Microarray data were analyzed by GeneSpring GX software and Ingenuity Pathway Analysis. Chosen genes were confirmed for altered transcription by quantitative real-time polymerase chain reaction. RESULTS: We identified 564 probes that were significantly expressed in mRNA more than 4-fold vs those in controls using volcano plot analysis (p <0.001). Further network Ingenuity Pathway Analysis of these genes showed the top 3 functions, including 1) cell-to-cell signaling and interaction, and hematological system development and function, 2) inflammatory disease and 3) cellular development. Quantitative real-time polymerase chain reaction confirmed increased mRNA expression of several genes in the bladder samples of patients with ulcerative interstitial cystitis, including CXCR3 binding chemokines (CXCL9, 10 and 11) and TNFSF14 (LIGHT). CONCLUSIONS: Our study using DNA microarray analysis followed by quantitative real-time polymerase chain reaction reveals over expression of genes related to immune and inflammatory responses, including T-helper type 1 related chemokines, and cytokines such as CXCR3 binding chemokines and TNFSF14. These genes may be potential interstitial cystitis biomarkers.

Interferon-alpha/beta receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006.

BACKGROUND: Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are known to exert a strong adjuvant effect on Th1 immune responses. Although several genes have been reported, no comprehensive study of the gene expression profiles in human cells after stimulation with CpG ODN has been reported. RESULTS: This study was designed to identify a CpG-inducible gene cluster that potentially predicts for the molecular mechanisms of clinical efficacy of CpG ODN, by determining mRNA expression in human PBMC after stimulation with CpG ODN. PBMCs were obtained from the peripheral blood of healthy volunteers and cultured in the presence or absence of CpG ODN 2006 for up to 24 hours. The mRNA expression profile was evaluated using a high-density oligonucleotide probe array, GeneChip. Using hierarchical clustering-analysis, out of a total of 10,000 genes we identified a cluster containing 77 genes as having been up-regulated by CpG ODN. This cluster was further divided into two sub-clusters by means of time-kinetics. (1) Inflammatory cytokines such as IL-6 and GM-CSF were up-regulated predominantly 3 to 6 hours after stimulation with CpG ODN, presumably through activation of a transcription factor, NF-kappaB. (2) Interferon (IFN)-inducible anti-viral proteins, including IFIT1, OAS1 and Mx1, and Th1 chemoattractant IP-10, were up-regulated predominantly 6 to 24 hours after stimulation. Blocking with mAb against IFN-alpha/beta receptor strongly inhibited the induction of these IFN-inducible genes by CpG ODN. CONCLUSION: This study provides new information regarding the possible immunomodulatory effects of CpG ODN in vivo via an IFN-alpha/beta receptor-mediated paracrine pathway.

DNA microarray analysis of white adipose tissue from obese (fa/fa) Zucker rats treated with a beta3-adrenoceptor agonist, KTO-7924.

We aimed to examine the effects of KTO-7924 (beta3-adrenoceptor agonist) on lipid metabolism and mRNA expressions in retroperitoneal white adipose tissue (RP WAT) in obese (fa/fa) Zucker rats using DNA microarray. Oral KTO-7924 for 28 days significantly decreased RP WAT weight, plasma triglyceride, free fatty acid, and insulin, and improved insulin resistance in oral glucose tolerance tests. In RP WAT of KTO-7924-treated rats, DNA microarray analysis revealed specifically enhanced mRNA expressions of uncoupling protein 1 (UCP1) and cytochrome c oxidase subunit VIII-H (COX8H), which are reportedly highly expressed in brown adipose tissue (BAT). Since these mRNA expression levels in RP WAT were significantly lower in obese (fa/fa) Zucker rats than in lean Zucker rats, these genes may be important in lipid metabolism. Our results imply that in obese (fa/fa) Zucker rats, continuous stimulation of beta3-adrenoceptors by KTO-7924 causes BAT-like adipocytes to appear in RP WAT, and improves lipid metabolism.

Lipopolysaccharide-binding protein critically regulates lipopolysaccharide-induced IFN-beta signaling pathway in human monocytes.

LPS binding to Toll-like receptor 4 induces a large number of genes through activation of NF-kappaB and IFN-regulatory factor-3 (IRF-3). However, no previous reports have tested the role of serum proteins in LPS-induced gene expression profiles. To investigate how serum proteins affect LPS-induced signaling, we investigated LPS-inducible genes in PBMC using an oligonucleotide probe-array system. Approximately 120 genes up-regulated by LPS were hierarchically divided into two clusters. Induction of one cluster, containing only IFN-inducible genes, was serum dependent. Real-time PCR analysis confirmed that IFN-inducible genes were induced only in the presence of serum, whereas inflammatory genes were induced both in the presence and absence of serum. Further analysis demonstrated that addition of LPS-binding protein (LBP), but not of soluble CD14 to the serum-free medium enabled the induction of IFN-inducible genes and IFN-beta itself by LPS in human monocytes. The mRNAs for IFN-beta and IFN-inducible genes were induced by LPS only in the presence of serum from LBP(+/+) mice, and not in the presence of serum from LBP(-/-) mice. Blocking experiments also confirmed the involvement of LBP in this phenomenon. Immunoblotting analysis showed that phosphorylation of c-Jun N-terminal kinase, p38, IRF-3, tyrosine kinase 2, and STAT1 by LPS, but not of NF-kappaB and extracellular signal-regulated kinase was abrogated in the absence of LBP. This critical role for LBP implies the presence of possible mechanisms linking LBP to the intracellular signaling between Toll-like receptor 4 and IRF-3, leading to the induction of IFN-beta by LPS.

Corticosteroid and cytokines synergistically enhance toll-like receptor 2 expression in respiratory epithelial cells.

Respiratory epithelial cells play important roles not only in host defense mechanisms, but also in inflammatory responses. Inhaled corticosteroids are widely used for the treatment of patients with inflammatory lung disorders, including asthma, chronic obstructive pulmonary disease, and sarcoidosis. Corticosteroids effectively reduce the production of inflammatory mediators, such as cytokines and chemokines. Although these molecules are also essential for host defense responses, there is no convincing evidence that inhaled corticosteroids increase susceptibility to lower respiratory tract infections. To test the involvement of Toll-like receptor (TLR) family molecules in this phenomenon, we examined the effects of various cytokines and corticosteroid on the expression of TLRs in human respiratory epithelial cells. Among the TLRs tested, TLR2 expression was significantly enhanced after stimulation with a combination of tumor necrosis factor-alpha and interferon-gamma. Dexamethasone synergistically enhanced TLR2 expression in combination with tumor necrosis factor-alpha and interferon-gamma in terms of both mRNA and protein levels. Furthermore, increased cell-surface TLR2 was functional, judging from the remarkable induction of interleukin-6, interleukin-8, and beta-defensin-2 after stimulation with peptidoglycan. These results provide evidence for a novel function of corticosteroids in airway inflammatory disorders, and indicate that the use of inhaled corticosteroids in such disorders may have a beneficial role in host defense mechanisms.

CpG oligodeoxynucleotides directly induce CXCR3 chemokines in human B cells.

CpG oligodeoxynucleotides (CpG ODN) are known to elicit Th1 immune responses via TLR9. However, the precise mechanisms through which B cells are involved in this phenomenon are not fully understood. We investigated the effect of CpG ODN on the induction of Th1-chemoattractant CXCR3 chemokines, IP-10, Mig, and I-TAC, in B cells. Cells from the RPMI 8226 human B cell line and human peripheral B cells were stimulated with three distinct classes of CpG ODN. As a result, CXCR3 chemokines were strongly up-regulated by CpG-B and CpG-C, but only weakly by CpG-A. Though CXCR3 chemokines are known to be induced by IFNs, blocking mAbs against IFN receptors did not inhibit their induction by CpG-B. Induction of CXCR3 chemokines was blocked by two NF-kappaB inhibitors and a p38 inhibitor. These results strongly suggest that CXCR3 chemokines are directly induced by CpG ODN via NF-kappaB- and p38-dependent pathways in human B cells.

Eosinophil degranulation during pregnancy and after delivery by cesarean section.

BACKGROUND: The physiological roles of eosinophils that accumulate in the uterus during pregnancy and of uterus-dwelling mast cells remain unknown. In the present study, we investigated the degranulation of eosinophils and mast cells within the normal course of pregnancy and after delivery by measuring the urinary concentrations of eosinophil-derived neurotoxin (EDN) and N-methylhistamine. METHODS: Spot urine samples from 65 pregnant women and 15 nonpregnant, age-matched women were examined. Urinary EDN and N-methylhistamine concentrations were measured by radioimmunoassay and standardized with urinary creatinine concentration. RESULTS: A significant increase in the urinary EDN concentration was observed until the second trimester in the normal pregnancies. The elevated urinary EDN levels decreased after the onset of labor in the third trimester and normalized within 1 month after normal vaginal delivery. In women who underwent a cesarean section, the urinary EDN concentration was significantly higher for up to 1 month after delivery, compared to that in women who underwent a vaginal delivery. In contrast, the urinary N-methylhistamine concentration did not change until the second trimester and was significantly decreased during the third trimester. No significant correlation between the peripheral blood eosinophil count and the urinary EDN concentration was observed in these subjects. CONCLUSIONS: Eosinophils appear to play a role in the progression of pregnancy and recovery after a cesarean section through the degranulation of eosinophils. In addition, mast cell degranulation does not appear to be related to the contraction of uterine smooth muscle during labor.