Shin Yonehara

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.

Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity

The cellular protein retinoic acid-inducible gene I (RIG-I) senses intracellular viral infection and triggers a signal for innate antiviral responses including the production of type I IFN. RIG-I contains a domain that belongs to a DExD/H-box helicase family and exhibits an N-terminal caspase recruitment domain (CARD) homology. There are three genes encoding RIG-I-related proteins in human and mouse genomes. Melanoma differentiation associated gene 5 (MDA5), which consists of CARD and a helicase domain, functions as a positive regulator, similarly to RIG-I. Both proteins sense viral RNA with a helicase domain and transmit a signal downstream by CARD; thus, these proteins share overlapping functions. Another protein, LGP2, lacks the CARD homology and functions as a negative regulator by interfering with the recognition of viral RNA by RIG-I and MDA5. The nonstructural protein 3/4A protein of hepatitis C virus blocks the signaling by RIG-I and MDA5; however, the V protein of the Sendai virus selectively abrogates the MDA5 function. These results highlight ingenious mechanisms for initiating antiviral innate immune responses and the action of virus-encoded inhibitors.

Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes

To study the contribution of putative perforin-independent mechanism in the antigen-specific target destruction by cytotoxic T lymphocytes CD8+ CTL lines were established from spleen cells of chimeric mice produced by injecting perforin (-/-) embryonic stem cells into blastocysts of RAG-2(-/-) mice. When tested on normal concanavalin A blasts, these perforin-deficient cytotoxic T lymphocyte lines were found to be capable of inducing antigen-specific target cell lysis accompanied by DNA degradation. In contrast, with target cells carrying a mutation in Fas molecule, perforin-independent cytotoxicity was not detectable. These data not only confirmed the primary role of perforin but simultaneously revealed a major contribution of a perforin-independent Fas-mediated pathway in antigen-specific cytolysis.

Molecular Cloning of a Novel Scavenger Receptor for Oxidized Low Density Lipoprotein, SR-PSOX, on Macrophages

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) by macrophages has been implicated in foam cell transformation in the process of atherogenesis. Although several scavenger receptor molecules, including class A scavenger receptors and CD36, have been identified as OxLDL receptors on macrophages, additional molecules on macrophages may also be involved in the recognition of OxLDL. From a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, we isolated a cDNA encoding a novel protein designated SR-PSOX (scavenger receptor that bindsphosphatidylserine and oxidized lipoprotein), which acts as a receptor for OxLDL. SR-PSOX was a type I membrane protein consisting of 254 amino acids, expression of which was shown on human and murine macrophages with a molecular mass of 30 kDa. SR-PSOX could specifically bind with high affinity, internalize, and degrade OxLDL. The recognition of OxLDL was blocked by polyinosinic acid and dextran sulfate but not by acetylated low density lipoprotein. Taken together, SR-PSOX is a novel class of molecule belonging to the scavenger receptor family, which may play important roles in pathophysiology including atherogenesis.

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.

RPA-like Mammalian Ctc1-Stn1-Ten1 Complex Binds to Single-Stranded DNA and Protects Telomeres Independently of the Pot1 Pathway

Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

Dclk1 distinguishes between tumor and normal stem cells in the intestine

There is great interest in tumor stem cells (TSCs) as potential therapeutic targets; however, cancer therapies targeting TSCs are limited. A drawback is that TSC markers are often shared by normal stem cells (NSCs); thus, therapies that target these markers may cause severe injury to normal tissues. To identify a potential TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was reported as a candidate NSC marker in the gut, but recent reports have implicated it as a marker of differentiated cells (for example, Tuft cells). Using lineage-tracing experiments, we show here that Dclk1 does not mark NSCs in the intestine but instead marks TSCs that continuously produce tumor progeny in the polyps of ApcMin/+ mice. Specific ablation of Dclk1-positive TSCs resulted in a marked regression of polyps without apparent damage to the normal intestine. Our data suggest the potential for developing a therapy for colorectal cancer based on targeting Dclk1-positive TSCs.

Cell surface‐anchored SR‐PSOX/CXC chemokine ligand 16 mediates firm adhesion of CXC chemokine receptor 6‐expressing cells

Direct contacts between dendritic cells (DCs) and T cells or natural killer T (NKT) cells play important roles in primary and secondary immune responses. SR‐PSOX/CXC chemokine ligand 16 (CXCL16), which is selectively expressed on DCs and macrophages, is a scavenger receptor for oxidized low‐density lipoprotein and also the chemokine ligand for a G protein‐coupled receptor CXC chemokine receptor 6 (CXCR6), expressed on activated T cells and NKT cells. SR‐PSOX/CXCL16 is the second transmembrane‐type chemokine with a chemokine domain fused to a mucin‐like stalk, a structure very similar to that of fractalkine (FNK). Here, we demonstrate that SR‐PSOX/CXCL16 functions as a cell adhesion molecule for cells expressing CXCR6 in the same manner that FNK functions as a cell adhesion molecule for cells expressing CX3C chemokine receptor 1 (CX3CR1) without requiring CX3CR1‐mediated signal transduction or integrin activation. The chemokine domain of SR‐PSOX/CXCL16 mediated the adhesion of CXCR6‐expressing cells, which was not impaired by treatment with pertussis toxin, a Gαi protein blocker, which inhibited chemotaxis of CXCR6‐expressing cells induced by SR‐PSOX/CXCL16. Furthermore, the adhesion activity was up‐regulated by treatment of SR‐PSOX/CXCL16‐expressing cells with a metalloprotease inhibitor, which increased surface expression levels of SR‐PSOX/CXCL16. Thus, SR‐PSOX/CXCL16 is a unique molecule that not only attracts T cells and NKT cells toward DCs but also supports their firm adhesion to DCs.

Continuous ERK Activation Downregulates Antiproliferative Genes throughout G1 Phase to Allow Cell-Cycle Progression

BACKGROUND The ERK family of MAP kinase plays a critical role in growth factor-stimulated cell-cycle progression from G0/G1 to S phase. It has been suggested that sustained activation, but not transient activation, of ERK is necessary for inducing S phase entry. Although the essential role of ERK MAP kinase in growth factor-stimulated gene expression, especially expression of immediate-early genes, is well established, it has remained unclear how ERK activity duration affects the promotion of G1 phase progression to S phase. RESULTS We have found that inhibition of ERK activation by the MEK inhibitor or dominant-negative MEK1 even immediately before the onset of S phase leads to the cessation of S phase entry. Our analyses reveal that there are ERK-dependent downregulated genes, whose expression levels return to their original levels rapidly after ERK inactivation, and that their downregulation mostly requires AP-1 activity. Remarkably, microinjection experiments demonstrate that many of the downregulated genes act as antiproliferative genes during G1 phase and that their forced expression to the levels before growth factor stimulation even in late G1 phase blocks S phase entry. CONCLUSIONS Thus, continuous ERK activation downregulates antiproliferative genes until the onset of S phase to allow successful G1 phase progression. This mechanism may also work as a fail-safe mechanism, which prevents inappropriate stimuli that induce transient ERK activation from causing S phase entry.

Roles of caspase-8 and caspase-10 in innate immune responses to double-stranded RNA.

Upon viral infection, host cells trigger antiviral immune responses by inducing type I IFN and inflammatory cytokines. dsRNA generated during viral replication is recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5, which interact with an adaptor, IFN-β promoter stimulator-1, to activate the transcription factors NF-κB and IFN regulatory factor 3. In this article we demonstrate that caspase-8 and caspase-10 are involved in these pathways. Both caspases were cleaved during dsRNA stimulation, and overexpression of a cleaved form of these caspases activated NF-κB. Knockdown of caspase-10 or caspase-8 in a human cell line resulted in the reduction of inflammatory cytokine production. Cells derived from caspase-8-deficient mice also showed reduced expression of inflammatory cytokines as well as NF-κB activation. Furthermore, the Fas-associated death domain protein interacted with these two caspases and IFN-β promoter stimulator 1. These results indicate that caspase-8 and caspase-10 are essential components that mediate NF-κB-dependent inflammatory responses in antiviral signaling.