Stanley M. Tahara

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.

NANOG Metabolically Reprograms Tumor-Initiating Stem-like Cells through Tumorigenic Changes in Oxidative Phosphorylation and Fatty Acid Metabolism

Stem cell markers, including NANOG, have been implicated in various cancers; however, the functional contribution of NANOG to cancer pathogenesis has remained unclear. Here, we show that NANOG is induced by Toll-like receptor 4 (TLR4) signaling via phosphorylation of E2F1 and that downregulation of Nanog slows down hepatocellular carcinoma (HCC) progression induced by alcohol western diet and hepatitis C virus protein in mice. NANOG ChIP-seq analyses reveal that NANOG regulates the expression of genes involved in mitochondrial metabolic pathways required to maintain tumor-initiating stem-like cells (TICs). NANOG represses mitochondrial oxidative phosphorylation (OXPHOS) genes, as well as ROS generation, and activates fatty acid oxidation (FAO) to support TIC self-renewal and drug resistance. Restoration of OXPHOS activity and inhibition of FAO renders TICs susceptible to a standard care chemotherapy drug for HCC, sorafenib. This study provides insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis, and chemoresistance through reprogramming of mitochondrial metabolism.

Synthesis, Conformation and Hydrolytic Stability of p1,p3−Dinucleoside Triphosphates Related to mRNA 5′-cap, and Comparative Kinetic Studies on their Nucleoside and Nucleoside Monophosphate Analogs

P1,P3−Dinucleoside triphosphates, N(5′)G(5′)G, have been prepared in which N is 7-Me-, 7-Et-, 7-Bn, N2, 7-diMe- or N2,N2, 7-triMe-guanosine. Conformations of the nucleoside moieties have been deter...

Reduced Phagocytosis of Colloidal Carriers Using Soluble CD47

AbstractPurpose. This study was designed to illustrate the feasibility of using soluble CD47 protein to antagonize phagocytosis of colloidal drug carriers by macrophages. Methods. Expression of CD47-streptavidin (CD47-SA) fusion protein was achieved in B21CodonPlus host cells following IPTG induction. Murine macrophage cell line J774A.1, expressing high levels of SIRPα, was selected as the biologic model system for phagocytosis. FITC-labeled perfluorocarbon (PFC) emulsions were used as the colloidal carriers to trigger phagocytosis. Microscopy (inverted light and UV-fluorescence) and flow cytometry were used to qualitatively and quantitatively determine the degree of phagocytosis, respectively. Results. The bacterially expressed, purified CD47-SA had neither cytotoxic nor cytostatic effects when incubated with J774A.1 cells up to a concentration of 400 nM for 24 h. Phagocytosis of FITC-labeled PFC emulsions was significantly diminished when macrophages were pretreated with 100 nM CD47-SA for 1 h. Conclusions. We demonstrated that soluble CD47-SA antagonized phagocytosis of colloidal carriers to a significant degree by interaction with macrophage SIRPα.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-199a2 attenuates endothelin-1 expression via hypoxia-inducible factor-1α.

Background: Elevated plasma levels of PlGF are associated with increased endothelin-1 and pulmonary hypertension (PH) in SCD. Results: miR-199a2, which targets HIF-1α mRNA, located in host gene DNM3os is co-transcriptionally regulated by PPARα. Conclusion: PPARα agonist induction of miR-199a2 reduced ET-1 levels. Significance: PPARα agonist reduction of ET-1 levels via induced miR-199a2 provides an alternative strategy to ameliorate PH. Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3′-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels.

Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2

B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

MicroRNA-18a Improves Human Cerebral Arteriovenous Malformation Endothelial Cell Function

Background and Purpose— Cerebral arteriovenous malformation (AVM) is a vascular disease that disrupts normal blood flow and leads to serious neurological impairment or death. Aberrant functions of AVM-derived brain endothelial cells (AVM-BECs) are a disease hallmark. Our aim was to use microRNA-18a (miR-18a) as a therapeutic agent to improve AVM-BEC function. Methods— Human AVM-BECs were tested for growth factor production and proliferation under different shear flow conditions and evaluated for tubule formation. Thrombospondin-1, inhibitor of DNA-binding protein 1, and vascular endothelial growth factor (VEGF) isotype mRNA levels were quantified by quantitative real-time polymerase chain reaction. Thrombospondin-1, VEGF-A, and VEGF-D protein expression was measured using enzyme-linked immunosorbent assay. Proliferation and tubule formation were evaluated using bromodeoxyuridine incorporation and growth factor–reduced Matrigel assays, respectively. Results— miR-18a increased thrombospondin-1 production but decreased inhibitor of DNA-binding protein 1, a transcriptional repressor of thrombospondin-1. miR-18a reduced VEGF-A and VEGF-D levels, both overexpressed in untreated AVM-BECs. This is the first study reporting VEGF-D overexpression in AVM. These effects were most prominent under arterial shear flow conditions. miR-18a also reduced AVM-BEC proliferation, improved tubule formation, and was effectively internalized by AVM-BECs in the absence of extraneous transfection reagents. Conclusions— We report VEGF-D overexpression in AVM and the capacity of miR-18a to induce AVM-BECs to function more normally. This highlights the clinical potential of microRNA as a treatment for AVM and other vascular diseases.

MicroRNA 648 Targets ET-1 mRNA and Is Cotranscriptionally Regulated with MICAL3 by PAX5

ABSTRACT Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3′ untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3′ UTR of ET-1 mRNA. Since miR-648 is located in a 5′-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

Abstract Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease

miR-301a/miR-454, which targets the 3′-UTR of ET-1 and PAI-1, are located in the intron of the SKA2 gene. These miRNAs are transcriptionally regulated by PPAR-α. Fenofibrate, a PPAR-α agonist increases levels of miR-301a/miR-454, with potential for amelioration of pulmonary hypertension.