Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

A microRNA Encoded by Kaposi Sarcoma-Associated Herpesvirus Promotes B-Cell Expansion In Vivo

The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.     

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) Induces the Oncogenic miR-17-92 Cluster and Down-Regulates TGF-β Signaling

KSHV is a DNA tumor virus that causes Kaposi’s sarcoma. Upon KSHV infection, only a limited number of latent genes are expressed. We know that KSHV infection regulates host gene expression, and hypothesized that latent genes also modulate the expression of host miRNAs. Aberrant miRNA expression contributes to the development of many types of cancer. Array-based miRNA profiling revealed that all six miRNAs of the oncogenic miR-17-92 cluster are up-regulated in KSHV infected endothelial cells. Among candidate KSHV latent genes, we found that vFLIP and vCyclin were shown to activate the miR-17-92 promoter, using luciferase assay and western blot analysis. The miR-17-92 cluster was previously shown to target TGF-β signaling. We demonstrate that vFLIP and vCyclin induce the expression of the miR-17-92 cluster to strongly inhibit the TGF-β signaling pathway by down-regulating SMAD2. Moreover, TGF-β activity and SMAD2 expression were fully restored when antagomirs (inhibitors) of miR-17-92 cluster were transfected into cells expressing either vFLIP or vCyclin. In addition, we utilized viral genetics to produce vFLIP or vCyclin knock-out viruses, and studied the effects in infected TIVE cells. Infection with wildtype KSHV abolished expression of SMAD2 protein in these endothelial cells. While single-knockout mutants still showed a marked reduction in SMAD2 expression, TIVE cells infected by a double-knockout mutant virus were fully restored for SMAD2 expression, compared to non-infected TIVE cells. Expression of either vFLIP or vCycIin was sufficient to downregulate SMAD2. In summary, our data demonstrate that vFLIP and vCyclin induce the oncogenic miR-17-92 cluster in endothelial cells and thereby interfere with the TGF-β signaling pathway. Manipulation of the TGF-β pathway via host miRNAs represents a novel mechanism that may be important for KSHV tumorigenesis and angiogenesis, a hallmark of KS.     

Kaposi's sarcoma herpesvirus microRNAs target caspase 3 and regulate apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis.     

TGF-β isoforms inhibit hepatitis C virus propagation in transforming growth factor beta/SMAD protein signalling pathway dependent and independent manners

Transforming growth factor beta (TGF-β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation-associated responses. However, the antiviral activities and mechanisms of TGF-β isoforms, including TGF-β1, TGF-β2 and TGF-β3, remain unclear. Here, we demonstrated that all of the three TGF-β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF-β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF-β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF-β/SMAD signalling pathway–dependent and TGF-β/SMAD signalling pathway–independent manners. TGF-β isoforms showed additional anti-HCV activities when combined with each other. However, the elevated TGF-β1 and TGF-β2, not TGF-β3, could also induce liver fibrosis with a high expression of type I collagen alpha-1 and α-smooth muscle actin in LX-2 cells. Our results showed a new insight into TGF-β isoforms in the HCV-related liver disease progression.     

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-β signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1-155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-β-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA(12)LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-β by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3'-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-β with possible implications in several human diseases where homeostasis of TGF-β might be altered.     

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

Engulfment protein GULP is regulator of transforming growth factor-β response in ovarian cells

Transforming growth factor β (TGF-β) is a key regulatory molecule with pleiotropic effects on cell growth, migration, and invasion. As a result, impairment of proper TGF-β signaling is central to tumorigenesis and metastasis. The TGF-β receptor V (TGFBRV or LRP1) has been shown to be responsible for TGF-β-mediated cell growth inhibition in Chinese hamster ovary (CHO) cells. The LRP1 adapter protein GULP mediates internalization of the various LRP1-specific ligands, and we hypothesize that GULP acts as a novel regulator of TGF-β signaling in ovarian cells. CHO cells that overexpress exogenous GULP (FL) demonstrate enhancement in growth inhibition, migration, and invasion from TGF-β treatment, whereas cells that lack GULP (AS) show impairment of growth inhibition and decreased migration and invasion. The enhanced TGF-β response in FL cells was confirmed by a prolonged TGF-β-induced SMAD3 phosphorylation, whereas a shortening of the phosphorylation event is observed in AS cells. Mechanistically, the presence of GULP retains the TGF-β in a signaling-competent early endosome for enhanced signaling. To address this mechanism in a physiological setting, TGF-β insensitive ovarian adenocarcinoma cells (HEY) have a very low GULP expression level, similar to the observation made in a wide selection of human ovarian adenocarcinomas. Transfection of GULP into the HEY cells restored the TGF-β responsiveness, as measured by SMAD3 phosphorylation and impairment of cell growth. Because GULP expression positively regulates TGF-β signaling leading to growth inhibition, this may represent an attractive target to achieve TGF-β responsiveness in ovarian cells.