Konstantin D. Taganov

NF-κB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses

Activation of mammalian innate and acquired immune responses must be tightly regulated by elaborate mechanisms to control their onset and termination. MicroRNAs have been implicated as negative regulators controlling diverse biological processes at the level of posttranscriptional repression. Expression profiling of 200 microRNAs in human monocytes revealed that several of them (miR-146a/b, miR-132, and miR-155) are endotoxin-responsive genes. Analysis of miR-146a and miR-146b gene expression unveiled a pattern of induction in response to a variety of microbial components and proinflammatory cytokines. By means of promoter analysis, miR-146a was found to be a NF-κB-dependent gene. Importantly, miR-146a/b were predicted to base-pair with sequences in the 3′ UTRs of the TNF receptor-associated factor 6 and IL-1 receptor-associated kinase 1 genes, and we found that these UTRs inhibit expression of a linked reporter gene. These genes encode two key adapter molecules downstream of Toll-like and cytokine receptors. Thus, we propose a role for miR-146 in control of Toll-like receptor and cytokine signaling through a negative feedback regulation loop involving down-regulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 protein levels.

MicroRNA-155 is induced during the macrophage inflammatory response

The mammalian inflammatory response to infection involves the induction of several hundred genes, a process that must be carefully regulated to achieve pathogen clearance and prevent the consequences of unregulated expression, such as cancer. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators that has also been linked to cancer. However, the relationship between inflammation, innate immunity, and miRNA expression is just beginning to be explored. In the present study, we use microarray technology to identify miRNAs induced in primary murine macrophages after exposure to polyriboinosinic:polyribocytidylic acid or the cytokine IFN-β. miR-155 was the only miRNA of those tested that was substantially up-regulated by both stimuli. It also was induced by several Toll-like receptor ligands through myeloid differentiation factor 88- or TRIF-dependent pathways, whereas up-regulation by IFNs was shown to involve TNF-α autocrine signaling. Pharmacological inhibition of the kinase JNK blocked induction of miR-155 in response to either polyriboinosinic:polyribocytidylic acid or TNF-α, suggesting that miR-155-inducing signals use the JNK pathway. Together, these findings characterize miR-155 as a common target of a broad range of inflammatory mediators. Importantly, because miR-155 is known to function as an oncogene, these observations identify a potential link between inflammation and cancer.

MicroRNAs: new regulators of immune cell development and function

Decades of research went into understanding immune cell development and function without awareness that consideration of a key element, microRNA (miRNA), was lacking. The discovery of miRNAs as regulators of developmental events in model organisms suggested to many investigators that miRNA might be involved in the immune system. In the past few years, widespread examination of this possibility has produced notable results. Results have shown that miRNAs affect mammalian immune cell differentiation, the outcome of immune responses to infection and the development of diseases of immunological origin. Some miRNAs repress expression of target proteins with well established functions in hematopoiesis. Here we bring together much of this work, which has so far only scratched the surface of this very fertile field of investigation, and show how the results illuminate many historic questions about hematopoiesis and immune function.

NF-κB dysregulation in microRNA-146a–deficient mice drives the development of myeloid malignancies

MicroRNA miR-146a has been implicated as a negative feedback regulator of NF-κB activation. Knockout of the miR-146a gene in C57BL/6 mice leads to histologically and immunophenotypically defined myeloid sarcomas and some lymphomas. The sarcomas are transplantable to immunologically compromised hosts, showing that they are true malignancies. The animals also exhibit chronic myeloproliferation in their bone marrow. Spleen and marrow cells show increased transcription of NF-κB–regulated genes and tumors have higher nuclear p65. Genetic ablation of NF-κB p50 suppresses the myeloproliferation, showing that dysregulation of NF-κB is responsible for the myeloproliferative disease.

MicroRNA-125b potentiates macrophage activation.

MicroRNA (miR)-125b expression is modulated in macrophages in response to stimulatory cues. In this study, we report a functional role of miR-125b in macrophages. We found that miR-125b is enriched in macrophages compared with lymphoid cells and whole immune tissues. Enforced expression of miR-125b drives macrophages to adapt an activated morphology that is accompanied by increased costimulatory factor expression and elevated responsiveness to IFN-γ, whereas anti–miR-125b treatment decreases CD80 surface expression. To determine whether these alterations in cell signaling, gene expression, and morphology have functional consequences, we examined the ability of macrophages with enhanced miR-125b expression to present Ags and found that they better stimulate T cell activation than control macrophages. Further indicating increased function, these macrophages were more effective at killing EL4 tumor cells in vitro and in vivo. Moreover, miR-125b repressed IFN regulatory factor 4 (IRF4), and IRF4 knockdown in macrophages mimicked the miR-125b overexpression phenotype. In summary, our evidence suggests that miR-125b is at least partly responsible for generating the activated nature of macrophages, at least partially by reducing IRF4 levels, and potentiates the functional role of macrophages in inducing immune responses.

Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites

ABSTRACT The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.

Evidence that the retroviral DNA integration process triggers an ATR-dependent DNA damage response

Caffeine is an efficient inhibitor of cellular DNA repair, likely through its effects on ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases. Here, we show that caffeine treatment causes a dose-dependent reduction in the total amount of HIV-1 and avian sarcoma virus retroviral vector DNA that is joined to host DNA in the population of infected cells and also in the number of transduced cells. These changes were observed at caffeine concentrations that had little or no effect on overall cell growth, synthesis, and nuclear import of the viral DNA, or the activities of the viral integrase in vitro. Substantial reductions in the amount of host-viral-joined DNA in the infected population, and in the number of transductants, were also observed in the presence of a dominant-negative form of the ATR protein, ATRkd. After infection, a significant fraction of these cells undergoes cell death. In contrast, retroviral transduction is not impeded in ATM-deficient cells, and addition of caffeine leads to the same reduction that was observed in ATM-proficient cells. These results suggest that activity of the ATR kinase, but not the ATM kinase, is required for successful completion of the viral DNA integration process and/or survival of transduced cells. Components of the cellular DNA damage repair response may represent potential targets for antiretroviral drug development.

STAT3 Induction of miR-146b Forms a Feedback Loop to Inhibit the NF-κB to IL-6 Signaling Axis and STAT3-Driven Cancer Phenotypes

An epigenetic modification prevents the production of a tumor-suppressing and anti-inflammatory microRNA in receptor-negative breast cancers. Micro-Mediated Feedback Chronic inflammation and interleukin-6 (IL-6), which is produced in response to nuclear factor κB (NF-κB) signaling, is a proinflammatory cytokine associated with cancer. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor stimulated in response to IL-6 and its receptor-bound kinases from the Janus kinase (JAK) family. Xiang et al. found that STAT3 stimulated expression of the gene encoding the microRNA miR-146b, which inhibited NF-κB–mediated induction of IL-6 to prevent a proinflammatory response in normal breast epithelial cells. However, promoter methylation reduced miR-146b expression in breast cancer cell lines and patient tissue, and its expression correlated with survival in patients with estrogen receptor– or triple-negative breast cancer. In addition to inhibiting STAT3 activity and cell migration and invasion, introduction of a miR-146b mimic was as cytotoxic as pharmacological inhibition of JAK to triple-negative breast cancer cells in culture, and combination therapy in cells was additive. The findings suggest that therapies reintroducing or stimulating miR-146b production may be beneficial to patients with tumors with high STAT3 activity. Interleukin-6 (IL-6)–mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor κB (NF-κB)–dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3–driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-κB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis.

Evidence that Stable Retroviral Transduction and Cell Survival following DNA Integration Depend on Components of the Nonhomologous End Joining Repair Pathway

ABSTRACT We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PKCS) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PKCS-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PKCS gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.

Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro

ABSTRACT Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.