Bik reduces hyperplastic cells by increasing Bak and activating DAPk1 to juxtapose ER and mitochondria.

Bik reduces hyperplastic epithelial cells by releasing calcium from endoplasmic reticulum stores and causing apoptosis, but the detailed mechanisms are not known. Here we report that Bik dissociates the Bak/Bcl-2 complex to enrich for ER-associated Bak and interacts with the kinase domain of DAPk1 to form Bik-DAPk1-ERK1/2-Bak complex. Bik also disrupts the Bcl2-IP3R interaction to cause ER Ca2+ release. The ER-associated Bak interacts with the kinase and calmodulin domains of DAPk1 to increase the contact sites of ER and mitochondria, and facilitate ER Ca2+ uptake by mitochondria. Although the Bik BH3 helix was sufficient to enrich for ER-Bak and elicit ER Ca2+ release, Bik-induced mitochondrial Ca2+ uptake is blocked with reduced Bak levels. Further, the Bik-derived peptide reduces allergen- and cigarette smoke-induced mucous cell hyperplasia in mice and in differentiated primary human airway epithelial cultures. Therefore, Bik peptides may have therapeutic potential in airway diseases associated with chronic mucous hypersecretion.Bcl-2 interacting killer (Bik) decreases airway epithelial hyperplasia via apoptosis mediated by calcium release from the endoplasmic reticulum (ER), but the mechanism is unclear. Here the authors show that Bik promotes Bak enrichment at the ER to tether mitochondria for efficient calcium transfer.

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single-nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells, while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells.

Syndecan-2 exerts antifibrotic effects by promoting caveolin-1-mediated transforming growth factor-ß receptor I internalization and inhibiting transforming growth factor-ß1 signaling.

RATIONALE: Alveolar transforming growth factor (TGF)-ß1 signaling and expression of TGF-ß1 target genes are increased in patients with idiopathic pulmonary fibrosis (IPF) and in animal models of pulmonary fibrosis. Internalization and degradation of TGF-ß receptor TßRI inhibits TGF-ß signaling and could attenuate development of experimental lung fibrosis. OBJECTIVES: To demonstrate that after experimental lung injury, human syndecan-2 confers antifibrotic effects by inhibiting TGF-ß1 signaling in alveolar epithelial cells. METHODS: Microarray assays were performed to identify genes differentially expressed in alveolar macrophages of patients with IPF versus control subjects. Transgenic mice that constitutively overexpress human syndecan-2 in macrophages were developed to test the antifibrotic properties of syndecan-2. In vitro assays were performed to determine syndecan-2-dependent changes in epithelial cell TGF-ß1 signaling, TGF-ß1, and TßRI internalization and apoptosis. Wild-type mice were treated with recombinant human syndecan-2 during the fibrotic phase of bleomycin-induced lung injury. MEASUREMENTS AND MAIN RESULTS: We observed significant increases in alveolar macrophage syndecan-2 levels in patients with IPF. Macrophage-specific overexpression of human syndecan-2 in transgenic mice conferred antifibrotic effects after lung injury by inhibiting TGF-ß1 signaling and downstream expression of TGF-ß1 target genes, reducing extracellular matrix production and alveolar epithelial cell apoptosis. In vitro, syndecan-2 promoted caveolin-1-dependent internalization of TGF-ß1 and TßRI in alveolar epithelial cells, which inhibited TGF-ß1 signaling and epithelial cell apoptosis. Therapeutic administration of human syndecan-2 abrogated lung fibrosis in mice. CONCLUSIONS: Alveolar macrophage syndecan-2 exerts antifibrotic effects by promoting caveolin-1-dependent TGF-ß1 and TßRI internalization and inhibiting TGF-ß1 signaling in alveolar epithelial cells. Hence, molecules that facilitate TßRI degradation via endocytosis represent potential therapies for pulmonary fibrosis.

Identification of residues of SARS-CoV nsp1 that differentially affect inhibition of gene expression and antiviral signaling.

An epidemic of Severe Acute Respiratory Syndrome (SARS) led to the identification of an associated coronavirus, SARS-CoV. This virus evades the host innate immune response in part through the expression of its non-structural protein (nsp) 1, which inhibits both host gene expression and virus- and interferon (IFN)-dependent signaling. Thus, nsp1 is a promising target for drugs, as inhibition of nsp1 would make SARS-CoV more susceptible to the host antiviral defenses. To gain a better understanding of nsp1 mode of action, we generated and analyzed 38 mutants of the SARS-CoV nsp1, targeting 62 solvent exposed residues out of the 180 amino acid protein. From this work, we identified six classes of mutants that abolished, attenuated or increased nsp1 inhibition of host gene expression and/or antiviral signaling. Each class of mutants clustered on SARS-CoV nsp1 surface and suggested nsp1 interacts with distinct host factors to exert its inhibitory activities. Identification of the nsp1 residues critical for its activities and the pathways involved in these activities should help in the design of drugs targeting nsp1. Significantly, several point mutants increased the inhibitory activity of nsp1, suggesting that coronaviruses could evolve a greater ability to evade the host response through mutations of such residues.

New World hantaviruses activate IFNlambda production in type I IFN-deficient vero E6 cells.

BACKGROUND: Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner. METHODOLOGY/PRINCIPAL FINDINGS: We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs. CONCLUSIONS/SIGNIFICANCE: Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus propagation.

Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain.

The severe acute respiratory syndrome (SARS) epidemic was caused by the spread of a previously unrecognized infectious agent, the SARS-associated coronavirus (SARS-CoV). Here we show that SARS-CoV could inhibit both virus- and interferon (IFN)-dependent signaling, two key steps of the antiviral response. We mapped a strong inhibitory activity to SARS-CoV nonstructural protein 1 (nsp1) and show that expression of nsp1 significantly inhibited the activation of all three virus-dependent signaling pathways. We show that expression of nsp1 significantly inhibited IFN-dependent signaling by decreasing the phosphorylation levels of STAT1 while having little effect on those of STAT2, JAK1, and TYK2. We engineered an attenuated mutant of nsp1 in SARS-CoV through reverse genetics, and the resulting mutant virus was viable and replicated as efficiently as wild-type virus in cells with a defective IFN response. However, mutant virus replication was strongly attenuated in cells with an intact IFN response. Thus, nsp1 is likely a virulence factor that contributes to pathogenicity by favoring SARS-CoV replication.

Hypoxia-activated metabolic pathway stimulates phosphorylation of p300 and CBP in oxygen-sensitive cells.

Transcription co-activators and histone acetyltransferases, p300 and cyclic AMP responsive element-binding protein-binding protein (CBP), participate in hypoxic activation of hypoxia-inducible genes. Here, we show that exposure of PC12 and cells to 1-10% oxygen results in hyperphosphorylation of p300/CBP. This response is fast, long lasting and specific for hypoxia, but not for hypoxia-mimicking agents such as desferioxamine or Co2+ ions. It is also cell-type specific and occurs in pheochromocytoma PC12 cells and the carotid body of rats but not in hepatoblastoma cells. The p300 hyperphosphorylation specifically depends on the release of intracellular calcium from inositol 1,4,5-triphosphate (IP3)-sensitive stores. However, it is not inhibited by pharmacological inhibitors of any of the kinases traditionally known to be directly or indirectly calcium regulated. On the other hand, p300 hyperphosphorylation is inhibited by several different inhibitors of the glucose metabolic pathway from generation of NADH by glyceraldehyde 3-phosphate dehydrogenase, through the transfer of NADH through the glycerol phosphate shuttle to ubiquinone and complex III of the mitochondrial respiratory chain. Inhibition of IP3-sensitive calcium stores decreases generation of ATP, and this inhibition is significantly stronger in hypoxia than in normoxia. We propose that the NADH glycerol phosphate shuttle participates in generating a pool of ATP that serves either as a co-factor or a modulator of the kinases involved in the phosphorylation of p300/CBP during hypoxia.

Mechanism for transcriptional synergy between interferon regulatory factor (IRF)-3 and IRF-7 in activation of the interferon-beta gene promoter.

The interferon-beta promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-kappaB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-beta gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-beta promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-beta promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-beta gene depends on a unique promoter context and on the role played by coactivators as architectural factors.

Interferon regulatory factor-7 synergizes with other transcription factors through multiple interactions with p300/CBP coactivators.

Interferon regulatory factor (IRF)-7 is activated in response to virus infection and stimulates the transcription of a set of cellular genes involved in host antiviral defense. The mechanism by which IRF-7 is activated and cooperates with other transcription factors is not fully elucidated. Activation of IRF-7 results from a conformational change triggered by the virus-dependent phosphorylation of its C terminus. This conformational change leads to dimerization, nuclear accumulation, DNA-binding, and transcriptional transactivation. Here we show that activation of IRF-7, like that of IRF-3, is dependent on modifications of two distinct sets of Ser/Thr residues. Moreover, we show that different virus-inducible cis-acting elements display requirements for specific IRFs. In particular, the virus-responsive element of the ISG15 gene promoter can be activated by either IRF-3 or IRF-7 alone, whereas the P31 element of the interferon-beta gene is robustly activated only when IRF-3, IRF-7, and the p300/CBP coactivators are all present. Furthermore, we find that IRF-7 interacts with four distinct regions of p300/CBP. These interactions not only stimulate the intrinsic transcriptional activity of IRF-7, but they are also indispensable for its ability to strongly synergize with other transcription factors, including c-Jun and IRF-3.

Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein-protein interactions.

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3.