Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor through effects on Bcl-3 and STAT3

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF-kappaB. CTAR2 activates the canonical NF-kappaB pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF-kappaB dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF-kappaB consensus motifs within the egfr promoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF-kappaB through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF-kappaB pathways.     

Synergistic upregulation of interleukin-8 secretion from pulmonary epithelial cells by direct and monocyte-dependent effects of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) infection is the major cause of severe bronchiolitis in infants. Pathology of this infection is partly due to excessive proinflammatory leukocyte influx mediated by chemokines. Although direct infection of the respiratory epithelium by RSV may induce chemokine secretion, little is known about the role of cytokine networks. We investigated the effects of conditioned medium (CM) from RSV-infected monocytes (RSV-CM) on respiratory epithelial (A549) cell chemokine release. RSV-CM, but not control CM (both at a 1:5 dilution), stimulated interleukin-8 (IL-8) secretion from A549 cells within 2 h, and secretion increased over 72 h to 11,360 +/- 1,090 pg/ml without affecting cell viability. In contrast, RSV-CM had only a small effect on RANTES secretion. RSV-CM interacted with direct RSV infection to synergistically amplify IL-8 secretion from respiratory epithelial cells (levels of secretion at 48 h were as follows: RSV-CM alone, 8,140 +/- 2,160 pg/ml; RSV alone, 12,170 +/- 300 pg/ml; RSV-CM plus RSV, 27,040 +/- 5,260 pg/ml; P < 0.05). RSV-CM induced degradation of IkappaBalpha within 5 min but did not affect IkappaBbeta. RSV-CM activated transient nuclear binding of NF-kappaB within 1 h, while activation of NF-IL6 was delayed until 8 h and was still detectable at 24 h. Promoter-reporter analysis demonstrated that NF-kappaB binding was essential and that NF-IL6 was important for IL-8 promoter activity in RSV-CM-activated cells. Blocking experiments revealed that the effects of RSV-CM depended on monocyte-derived IL-1 but that tumor necrosis factor alpha was not involved in this network. In summary, RSV infection of monocytes results in and amplifies direct RSV-mediated IL-8 secretion from respiratory epithelial cells by an NF-kappaB-dependent, NF-IL6-requiring mechanism.     

Interleukin-1beta-induced iNOS expression in human lung carcinoma A549 cells: involvement of STAT and MAPK pathways

For understanding of signaling molecules important in lung cancer growth and progression, IL-1beta effect was analyzed on iNOS expression and key signaling molecules in human lung carcinoma A549 cells and established the role of specific signaling molecules by using specific chemical inhibitors. IL-1beta exposure (10 ng/ml) induced strong iNOS expression in serum starved A549 cells. Detailed molecular analyses showed that IL-1beta increased expression of phosphorylated STAT1 (Tyr701 and Ser727) and STAT3 (Tyr705 and Ser727) both in total cell lysates and nuclear lysates. Further, IL-1beta exposure strongly activated MAPKs (ERK1/2, JNK1/2 and p38) and Akt as well as increased nuclear levels of NF-kappaB and HIF-1alpha in A549 cells. Use of specific chemical inhibitors for JAK1 kinase (piceatannol), JAK2 kinase (AG-490), MEK1/2 (PD98059) and JNK1/2 (SP600125) revealed that IL-1beta-induced iNOS expression involved signaling pathways in addition to JAK-STAT and ERK1/2-JNK1/2 activation. Overall, these results suggested that instead of specific pharmacological inhibitors, use of chemopreventive agents with broad spectrum efficacy to inhibit IL-1beta-induced signaling cascades and iNOS expression would be a better strategy towards lung cancer prevention and/or treatment.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.