Mohammad Jamaluddin

Retinoic Acid-Inducible Gene I Mediates Early Antiviral Response and Toll-Like Receptor 3 Expression in Respiratory Syncytial Virus-Infected Airway Epithelial Cells

ABSTRACT Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I “knockdown” significantly inhibited early nuclear factor-κB (NF-κB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-β), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-κB binding but did inhibit IFN-β, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-κB/RelA transcription by its ability to block the activating phosphorylation of NF-κB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-β secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-κB activation.

Identification of direct genomic targets downstream of the nuclear factor-κB transcription factor mediating tumor necrosis factor signaling

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-κB (NF-κB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-κB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-κB inhibitor to systematically identify NF-κB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-κB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F) <0.001) and demonstrated a change in signal intensity of ± 3-fold relative to control. Of these, 28 were NF-κB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-κB-dependent genes and five genes not previously known to be NF-κB-dependent (Gro-β and-γ, IκBϵ, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-κB regulated. Expression of constitutively active enhanced green fluorescent·NF-κB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-κB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IκBα/ϵ, Gro-β/γ, and Naf-1 promoters directly bound NF-κB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-κB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Expression of Respiratory Syncytial Virus-Induced Chemokine Gene Networks in Lower Airway Epithelial Cells Revealed by cDNA Microarrays

ABSTRACT The Paramyxovirus respiratory syncytial virus (RSV) is the primary etiologic agent of serious epidemic lower respiratory tract disease in infants, immunosuppressed patients, and the elderly. Lower tract infection with RSV is characterized by a pronounced peribronchial mononuclear infiltrate, with eosinophilic and basophilic degranulation. Because RSV replication is restricted to airway epithelial cells, where RSV replication induces potent expression of chemokines, the epithelium is postulated to be a primary initiator of pulmonary inflammation in RSV infection. The spectrum of RSV-induced chemokines expressed by alveolar epithelial cells has not been fully investigated. In this report, we profile the kinetics and patterns of chemokine expression in RSV-infected lower airway epithelial cells (A549 and SAE). In A549 cells, membrane-based cDNA macroarrays and high-density oligonucleotide probe-based microarrays identified inducible expression of CC (I-309, Exodus-1, TARC, RANTES, MCP-1, MDC, and MIP-1α and -1β), CXC (GRO-α, -β, and -γ, ENA-78, interleukin-8 [IL-8], and I-TAC), and CX3C (Fractalkine) chemokines. Chemokines not previously known to be expressed by RSV-infected cells were independently confirmed by multiprobe RNase protection assay, Northern blotting, and reverse transcription-PCR. High-density microarrays performed on SAE cells confirmed a similar pattern of RSV-inducible expression of CC chemokines (Exodus-1, RANTES, and MIP-1α and -1β), CXC chemokines (I-TAC, GRO-α, -β, and -γ, and IL-8), and Fractalkine. In contrast, TARC, MCP-1, and MDC were not induced, suggesting the existence of distinct genetic responses for different types of airway-derived epithelial cells. Hierarchical clustering by agglomerative nesting and principal-component analyses were performed on A549-expressed chemokines; these analyses indicated that RSV-inducible chemokines are ordered into three related expression groups. These data profile the temporal changes in expression by RSV-infected lower airway epithelial cells of chemokines, chemotactic proteins which may be responsible for the complex cellular infiltrate in virus-induced respiratory inflammation.

Angiotensin II induces gene transcription through cell-type-dependent effects on the nuclear factor-κB (NF-κB) transcription factor

The vasopressor octapeptide, angiotensin II (Ang II), exerts homeostatic responses in cardiovascular tissues, including the heart, blood vessel wall, adrenal cortex and liver (a major source of circulating plasma proteins). One of the effects of Ang II is to induce expression of regulatory, structural and cytokine genes that play important roles in long-term control of blood pressure, vascular remodeling, cardiac hypertrophy and inflammation. The identification of nuclear signaling pathways and target transcription factors has provide important insight into cellular responses and the spectrum of genes controlled by Ang II. Here we will review how Ang II activates the transcription factors, Activator Protein 1 (AP-1), Signal Transducer and Activator of Transcription (STATs ), and Nuclear Factor-κB (NF-κB). NF-κB is of particular interest because it is an important mediator of resynthesis of the Ang II precursor, angiotensinogen AGT. Through this positive feedback loop, long-term changes in the activity of the renin angiotensin system occur. Although NF-κB is ubiquitously expressed, surprisingly the mechanism for Ang II-inducible NF-κB regulation differs between aortic smooth muscle cells (VSMCs) and hepatocytes. In VSMC, Ang II induces nuclear translocation of cytoplasmic transactivatory NF-κB proteins through proteolysis of its inhibitor, IkB. By contrast, in hepatocytes, Ang II induces large nuclear isoforms of NF-κB1 to bind DNA through a mechanism independent of changes in IkB turnover. NF-κB activation depends upon the activity of DAG-sensitive PKC isoforms and ROS signaling pathway. These observations indicate that significant differences exist in Ang II signaling depending upon cell-type involved and suggest the possibility that tissue-selective modulation of Ang II effects can be modulated in cardiac tissues.

A Promoter Recruitment Mechanism for Tumor Necrosis Factor-α-induced Interleukin-8 Transcription in Type II Pulmonary Epithelial Cells DEPENDENCE ON NUCLEAR ABUNDANCE OF Rel A, NF-κB1, AND c-Rel TRANSCRIPTION FACTORS

The alveolar macrophage-derived peptide tumor necrosis factor-α (TNFα) initiates pulmonary inflammation through its ability to stimulate interleukin-8 (IL-8) synthesis in alveolar epithelial cells through an incompletely described transcriptional mechanism. In this study, we use the technique of ligation-mediated polymerase chain reaction (LMPCR) to record changes in transcription factor occupancy of the IL-8 promoter after TNFα stimulation of A549 human alveolar cells. Using dimethylsulfate/LMPCR, no detectable proteins bind the TATA box in unstimulated cells. By contrast, TNFα rapidly induces protection of G residues at −79 and −80 coincident with endogenous IL-8 gene transcription. Using DNase I/LMPCR, we observe inducible protection of nucleotides −60 to −99 (the TNF response element) and nucleotides −3 to −32 (containing the TATA box). Surprisingly, extensive TATA box protection is only seen after TNFα stimulation. Using a two-step microaffinity isolation/Western immunoblot DNA binding assay, we observe that the NF-κB subunits Rel A, NF-κB1, and c-Rel inducibly bind the TNF response element; these proteins undergo rapid TNFα-inducible increases in nuclear abundance as a consequence of IκBα proteolysis. Furthermore, the peptide aldehyde N-acetyl-Leu-Leu-norleucinal, an agent that blocks both IκBα proteolysis and NF-κB subunit translocation, abrogates recombinant human TNFα-inducible IL-8 gene transcription. These studies demonstrate that IL-8 is activated by a promoter recruitment mechanism in alveolar epithelial cells, where NF-κB subunit translocation is required for (and coincident with) binding of the constitutively active TATA box-binding proteins.

RelA Ser276 Phosphorylation Is Required for Activation of a Subset of NF-κB-Dependent Genes by Recruiting Cyclin-Dependent Kinase 9/Cyclin T1 Complexes

ABSTRACT NF-κB plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-κB/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-κB-dependent genes, serine 276 (Ser276)-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-β, interleukin-8 (IL-8), and IκBα. Previous work has shown that TNF-inducible RelA Ser276 phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser276 RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser276 formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser276 RelA is required only for activation of IL-8 and Gro-β, with IκBα being unaffected. These data were confirmed in experiments using RelA−/− murine embryonic fibroblasts reconstituted with a RelA Ser276Ala mutation. In addition, we observe that phospho-Ser276 RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser276 RelA-P-TEFb complex is required for IL-8 and Gro-β gene activation but not for IκBα gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser276 RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-β genes. The second involves a phospho-Ser276 RelA-independent activation of genes preloaded with Pol II, exemplified by the IκBα gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.

Reactive oxygen species mediate virus-induced STAT activation: role of tyrosine phosphatases.

Respiratory syncytial virus (RSV) is the leading cause of epidemic respiratory tract illness in children in the United States and worldwide. RSV infection of airway epithelial cells induces formation of reactive oxygen species (ROS), whose production mediates the expression of cytokines and chemokines involved the immune/inflammatory responses of the lung. In this study, we have investigated the role of ROS in RSV-induced signal transducers and activators of transcription (STAT) activation and interferon regulatory factor (IRF) gene expression in human airway epithelial cells. Our results indicate that RSV replication induces IRF-1 and -7 gene transcription, a response abrogated by antioxidants. RSV infection induces binding of STAT to the IRF-1 γ-interferon-activated sequence (GAS) and IRF-7 interferon-stimulated responsive element (ISRE). STAT1 and STAT3 bind IRF-1 GAS, whereas STAT1, STAT2, IRF-1, and IRF-9 bind IRF-7 ISRE. Antioxidant treatment blocks RSV-induced STAT binding to both the IRF-1 GAS and IRF-7 ISRE by inhibition of inducible STAT1 and STAT3 tyrosine phosphorylation, suggesting that RSV-induced ROS formation is required for STAT activation and IRF gene expression. Although protein tyrosine phosphorylation is necessary for RSV-induced STAT activation, Janus kinase and Src kinase activation do not mediate this effect. Instead, RSV infection inhibits intracellular tyrosine phosphatase activity, which is restored by antioxidant treatment. Pharmacological inhibition of tyrosine phosphatases induces STAT activation. Together, these results suggest that modulation of phosphatases could be an important mechanism of virus-induced STAT activation. Treatment of alveolar epithelial cells with the NAD(P)H oxidase inhibitor diphenylene iodonium abolishes RSV-induced STAT activation, indicating that NAD(P)H oxidase-produced ROS are required for downstream activation of the transcription factors IRF and STAT in virus-infected airway epithelial cells.

Requirement of a Novel Upstream Response Element in Respiratory Syncytial Virus-Induced IL-8 Gene Expression

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. In this study, we compare mechanisms for induction of the CXC chemokine IL-8, in human type II alveolar (A549) cells by RSV infection and by stimulation with the cytokine TNF. Promoter deletion and mutagenesis experiments indicate that although the region from −99 to −54 nt is sufficient for TNF-induced IL-8 transcription, this region alone is not sufficient for RSV-induced IL-8 transcription. Instead, RSV requires participation of a previously unrecognized element, spanning from −162 to −132 nt, that we term the RSV response element (RSVRE), and a previously characterized element at −132 to −99 nt, containing an AP-1 binding site. RSV infection of A549 cells induces increased RSVRE- and AP-1-binding activities and increased synthesis of IFN regulatory factor-1 protein, which is present in the RSVRE-binding complex. These data confirm that the IL-8 gene enhancers are controlled in a stimulus-specific fashion and participation of distinct promoter elements is required to activate gene transcription. These observations are important for rational design of inhibitors of RSV-induced lung inflammation.

Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus.

ABSTRACT Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normally T-cell expressed and presumably secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study, we analyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenesis experiments indicate that RSV requires the presence of five different cisregulatory elements, located in the promoter fragment spanning from −220 to +55 nucleotides, corresponding to NF-κB, C/EBP, Jun/CREB/ATF, and interferon regulatory factor (IRF) binding sites. Although site mutations of the NF-κB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene transcription to 50% or less, only mutations affecting IRF binding completely abolish RANTES inducibility. Supershift and microaffinity isolation assays were used to identify the different transcription factor family members whose DNA binding activity was RSV inducible. Expression of dominant negative mutants of these transcription factors further established their central role in virus-induced RANTES promoter activation. Our finding that the presence of multiple cis regulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely dependent on binding of IRF transcription factors. The identification of regulatory mechanisms of RANTES gene expression is fundamental for rational design of inhibitors of RSV-induced lung inflammation.

Ribavirin Treatment Up-Regulates Antiviral Gene Expression via the Interferon-Stimulated Response Element in Respiratory Syncytial Virus-Infected Epithelial Cells

ABSTRACT Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX3C subclasses. Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 μg/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to ∼13% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting ∼49% of the ∼6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1α and -1β, which are important in RSV-induced pulmonary pathology, and interferon (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-β expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1α, STAT-1β, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.