MaryBeth Ferris

TNF‐α induces TGF‐β1 expression in lung fibroblasts at the transcriptional level via AP‐1 activation

Tumour necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β1) are peptides with multiple biological activities that influence neoplastic, immunologic and fibroproliferative diseases. There are clear interrelationships and overlap between the actions of TNF‐α and TGF‐β1 in lung fibrosis; therefore, we postulated that TNF‐α may play a significant role in regulating TGF‐β1 expression in lungs. We recently reported that TNF‐α activates the extracellular regulated kinase (ERK)‐specific pathway in fibroblasts resulting in stabilization of TGF‐β1 mRNA and increased expression of TGF‐β1. In the current study, we further investigated the molecular mechanisms involved in TNF‐α regulation of TGF‐β1 expression. Nuclear run‐on assays showed that treatment of Swiss 3T3 fibroblasts with TNF‐α increased transcription of the TGF‐β1 gene in an ERK independent manner. Pre‐treatment with the activator protein‐1 (AP‐1) inhibitor curcumin attenuated TNF‐α induced transcription of the TGF‐β1 gene. TNF‐α induced increased levels of c‐Jun and C‐Fos in the nucleus accompanied by phosphorylation of c‐Jun. In electrophoretic mobility shift assays, AP‐1 binding to an AP‐1 binding site found within the TGF‐β1 promoter was increased in nuclear extracts from Swiss 3T3 fibroblasts treated with TNF‐α. Together, these results suggest that TNF‐α induces expression and DNA binding of AP‐1 resulting in increased transcription of the TGF‐β1 gene. It is essential to know which transcription pathways are activated because of the wide distribution of TNF‐α and TGF‐β1, the general lack of effective treatments for fibroproliferative disease and the possibility that targeting the correct transcription factors could be palliative.

Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

Human Cytomegalovirus Infection Dysregulates the Canonical Wnt/β-catenin Signaling Pathway

Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected in utero. During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/β-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/β-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of β-catenin and expression of β-catenin target genes in these cells. HCMV infection leads to β-catenin protein accumulation in a discrete juxtanuclear region. Levels of β-catenin in membrane-associated and cytosolic pools, as well as nuclear β-catenin, are reduced after infection; while transcription of the β-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/β-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.

The latent form of TGFbeta(1) is induced by TNFalpha through an ERK specific pathway and is activated by asbestos-derived reactive oxygen species in vitro and in vivo.

Tumor necrosis factor-α (TNFα) and transforming growth factor-β1 (TGFβ1) are potent peptide growth factors that are likely to play important roles in the development of interstitial pulmonary fibrosis (IPF). Previously we showed that TNFα and TGFβ1 are up-regulated in macrophages, epithelial and mesenchymal cells early after exposure to chrysotile asbestos, particularly at sites of fiber deposition in vivo. We also showed that TNFα receptor knockout mice are resistant to asbestos-induced fibrosis. Importantly, vectors that over-express TNFα cause inflammation and fibrogenesis along with increased TGFβ1 production in C57Bl/6 mice. Recently we reported that TNFα activates the extracellular regulated kinase pathway in fibroblasts leading to a 200–400% increase in TGFβ1 mRNA and protein. The mechanism of TNFα induction of TGFβ1 expression appears to be complex, involving both transcriptional and post-transcriptional mechanisms. In asbestos-exposed animals, this TGFβ1 is produced on alveolar surfaces in a latent form (controlled by binding of a latent associated peptide [LAP]) that must be activated for the TGFβ1 to bind to its receptors and induce its multiple biological effects. Thus, we recently reported that, in vitro, reactive oxygen species (ROS) derived from chrysotile and crocidolite asbestos activate TGFβ1 by oxidation of the LAP. Now, in preliminary findings, we have shown that over-expression of latent TGFβ1 prior to asbestos exposure of fibrogenic-resistant TNFα receptor knockout mice produces asbestos lesions with the same severity as seen in normal C57/Bl6 mice. This finding plus the demonstration of increased amounts of TGFβ1, increased Smad activation and amelioration of the developing disease by treating the mice with an anti-oxidant all support the concept that, in vivo, latent TGFβ1 is activated by asbestos-generated oxygen radicals and consequently mediates at least a component of the consequent fibrogenesis. Taken together, these findings support the postulate that TNFα controls fibrogenesis by regulating TGFβ1 expression and that one mechanism through which ROS induce lung fibrosis is by activating latent TGFβ 1.

Secreted Oral Epithelial Cell Membrane Vesicles Induce Epstein-Barr Virus Reactivation in Latently Infected B Cells

ABSTRACT In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts. IMPORTANCE Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agents in vitro, the mechanisms that signal reactivation in vivo are poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV-positive B cells, where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium.

Kaposi's sarcoma-associated herpesvirus G-protein coupled receptor activates the canonical Wnt/β-catenin signaling pathway.

BackgroundKSHV is a tumorigenic γ-herpesvirus that has been identified as the etiologic agent of Kaposi’s sarcoma (KS), a multifocal highly vascularized neoplasm that is the most common malignancy associated with acquired immunodeficiency syndrome (AIDS). The virus encodes a constitutively active chemokine receptor homologue, vGPCR that possesses potent angiogenic and tumorigenic properties, and is critical for KSHV pathobiology. To date, a number of signaling pathways have been identified as key in mediating vGPCR oncogenic potential.FindingsIn this study, we identify a novel pathway, the Wnt/β-catenin pathway, which is dysregulated by vGPCR expression in endothelial cells. Expression of vGPCR in endothelial cells enhances the nuclear accumulation of β-catenin, that correlates with an increase in β-catenin transcriptional activity. Activation of β-catenin signaling by vGPCR is dependent on the PI3K/Akt pathway, as treatment of vGPCR-expressing cells with a pharmacological inhibitor of PI3K, leads to a decreased activation of a β-catenin-driven reporter, a significant decrease in expression of β-catenin target genes, and reduced endothelial tube formation.ConclusionsGiven the critical role of Wnt/β-catenin signaling in angiogenesis and tumorigenesis, the findings from this study suggest a novel mechanism in KSHV-induced malignancies.

Human cytomegalovirus infection of human adipose-derived stromal/stem cells restricts differentiation along the adipogenic lineage

Abstract Human adipose-derived stromal/stem cells (ASCs) display potential to be used in regenerative stem cell therapies and as treatments for inflammatory and autoimmune disorders. Despite promising use of ASCs as therapeutics, little is known about their susceptibility to infectious agents. In this study, we demonstrate that ASCs are highly susceptible to human cytomegalovirus (HCMV) infection and permissive for replication leading to release of infectious virions. Additionally, many basic ASC functions are inhibited during HCMV infection, such as differentiation and immunomodulatory potential. To our knowledge this is the first study examining potential adverse effects of HCMV infection on ASC biology. Our results suggest, that an active HCMV infection during ASC therapy may result in a poor clinical outcome due to interference by the virus.