Kazuki Okuyama

A Notch Ligand, Delta-Like 1 Functions As an Adhesion Molecule for Mast Cells

Mast cells (MCs) accumulate in chronic inflammatory sites; however, it is not clear which adhesion molecules are involved in this process. Recently, the expression of Notch ligands was reported to be upregulated in inflammatory sites. Although Notch receptors are known as signaling molecules that can activate integrins, their contributions to the adhesion of MCs have not been studied. In this study, we demonstrated that mouse MCs efficiently adhered to stromal cells forced to express a Notch ligand, Delta-like 1 (Dll1). Surprisingly, the adhesion was a consequence of direct cell–cell interaction between MCs and Dll1-expressing stromal cells rather than activation of downstream effectors of Notch receptor(s)-Dll1. The adhesion of MCs to Dll1-expressing stromal cells remained even when the cell metabolism was arrested. The recognition was blocked only by inhibition of Notch receptor(s)–Dll1 interaction by addition of soluble DLL1, or mAbs against Dll1 or Notch2. Taken together, these results indicate that Notch receptor(s) and Dll1 directly promote the adhesion of MCs to stromal cells by acting as adhesion molecules. This appreciation that Notch receptor–ligand interactions have an adhesion function will provide an important clue to molecular basis of accumulation of MCs to inflammatory sites.

Novel functional small RNAs are selectively loaded onto mammalian Ago1

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1–4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein–Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1–4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.

MicroRNA-126–mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit+Sca1+Lineage− cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion.

Small RNA as a regulator of hematopoietic development, immune response in infection and tumorigenesis

Abstract Posttranscriptional gene regulation by small RNAs (15–40-nucleotide noncoding RNAs) is now established as an important branch of the gene regulatory system. It has recently been revealed that noncoding RNAs can be categorized into different types and that they work through novel mechanisms. In addition, it has been shown that noncoding RNAs mediate intercellular communication and, importantly, that cross talk between coding and noncoding RNAs occurs. In this review, we discuss the recent findings concerning small RNAs. It was originally proposed that microRNAs (miRNAs) work to “fine tune” the determination of cell fate. However, critical functions beyond fine tuning have been revealed. In addition to miRNAs, next-generation sequencing has revealed the existence of various species of non-canonical small RNAs: mirtrons, piRNAs, 21U-RNA, endo-siRNAs, snoRNAs, usRNAs, and Y-RNA-derived small RNAs. Some of these species are involved in response to viral infection. Finally, we highlight the intracellular functions of small RNAs, which involve the exosomes.

CCR7‐independent transport of skin antigens occurs in the dermis

Under homeostatic conditions, skin DCs migrate to regional LNs transporting self‐antigens (self‐Ags). The transport of self‐Ags is considered to be critical for maintaining peripheral tolerance. Although the chemokine receptor CCR7 potently induces the migration of skin DCs to regional LNs, Ccr7−/− (Ccr7‐KO) mice do not show skin auto‐immune diseases. To resolve this inconsistency, we examined Ccr7‐KO epidermis‐ or dermis‐hyperpigmented transgenic (Tg) mice, in which the transport of skin self‐Ags is traceable by melanin granules (MGs). Under CCR7‐deficient conditions, the transport of epidermal MGs to regional LNs was impaired at 7 weeks of age. However, epidermal MGs could be transported when they had accumulated in the dermis. Ccr7‐KO‐dermis‐pigmented Tg mice confirmed the presence of CCR7‐independent transport from the dermis. Compared with WT‐dermis‐pigmented Tg mice, the amount of transported melanin and number of MG‐laden CD11c+ cells were both approximately 40% of the WT levels, while the number of MG‐laden CD205+ or CD207+ cells decreased to about 10% in skin regional LNs of Ccr7‐KO‐dermis‐pigmented Tg mice. Cell sorting highlighted the involvement of CD11c+ cells in the CCR7‐independent transport. Here, we show that CCR7‐independent transport of skin self‐Ags occurs in the dermis. This system might contribute to the continuous transport of self‐Ags, and maintain peripheral tolerance.

Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkins lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

A checkpoint in B-lymphopoiesis related to Notch resistance.

While murine B- and T-lymphopoiesis require overlapping molecules, they occur in separate organs: the bone marrow (BM) and the thymus, respectively. The BM microenvironment is incapable of supporting T-lymphopoiesis because of insufficient interactions of Notch1 with delta-like ligand (Dll). Notch1/Dll interactions also play a role in the suppression of B-lymphopoiesis in the thymus. However, it is still unclear whether the Notch1/Dll interaction alone explains why the thymus does not support B-lymphopoiesis. In this study, we compared the precursor population colonizing the thymus with that in the BM by culturing them on stromal cells expressing abundant Dll1. We demonstrated that Flt3(+) Il7r(+) B220(+) Cd19(+) BM cells gave rise to B cells under this condition. We defined them as resistant to Dll1. In the thymus, Dll1-resistant cells were undetectable. This suggested that the absence of Dll1-resistant cells might explain the absence of B-lymphopoiesis in the thymus.