Setsuko Mise-Omata

CD28 as a molecular amplifier extending TCR ligation and signaling capabilities.

Evidence has gathered that CD28 costimulation facilitates T cell activation by potentiating TCR intrinsic-signaling. However, the underlying molecular mechanism is largely unknown. Here we show that, by enhancing T cell/APC close contacts, CD28 facilitates TCR signal transduction. Moreover, the signal supplied by CD28 does not lead to increased Zap-70 and Lat phosphorylation, but amplifies PLCgamma1 activation and Ca(2+) response. We provide evidence that the PTK Itk controls the latter function. Our data suggest that CD28 binding to B7 contributes to setting the level of TCR-induced phosphorylated Lat for recruiting signaling complexes, whereas the CD28 signal boosts multiple pathways by facilitating PLCgamma1 activation. These results should provide a conceptual framework for understanding quantitative and qualitative aspects of CD28-mediated costimulation.

A Proximal κB Site in the IL-23 p19 Promoter Is Responsible for RelA- and c-Rel-Dependent Transcription

IL-23 is a heterodimeric cytokine composed of a unique p19 subunit and a common p40 subunit is shared with IL-12. IL-23 promotes the inflammatory response by inducing the expansion of CD4+ cells producing IL-17. The regulation of p19 gene expression has been less studied than that of p40 subunit expression, which in macrophages is well known to be dependent on NF-κB. To clarify the role of NF-κB in expression of the p19 gene, we analyzed mRNA levels in NF-κB-deficient macrophages. As reported to occur in dendritic cells, p19 expression was dramatically reduced in c-rel-deficient macrophages. Moreover, we found that p19 expression was halved in rela-deficient macrophages, but it was enhanced in p52-deficient macrophages. The p19 promoter contains three putative κB sites, located at nt −642 to −632 (κB–642), nt −513 to −503 (κB–513), and nt −105 to −96 (κB–105), between the transcription start site and −937 bp upstream in the p19 promoter region. Although EMSA analysis indicated that both κB–105 and κB–642, but not κB–513, bound to NF-κB in vitro, luciferase-based reporter assays showed that the most proximal κB site, κB–105, was uniquely indispensable to the induction of p19 transcription. Chromatin immunoprecipitation demonstrated in vivo association of RelA, c-Rel, and p50 with κB–105 of the p19 promoter. These results provide the evidence that the association of RelA and c-Rel with the proximal κB site in the p19 promoter is required to induce of p19 expression.

Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor

CD28 was thought to represent a prototypic membrane receptor responsible for delivering the classically defined ‘second signal’ needed to avoid T cell paralysis when recognizing antigen presented by appropriate antigen presenting cells (APCs). Almost two decades after its molecular identification, the mechanism by which this ‘second receptor’ facilitates clonal expansion and differentiation upon antigen encounter is still not fully elucidated. There may be at least two reasons for this partially gray picture: the use of nonphysiological experimental conditions to study it and the fact that the action of CD28 may be partly masked by the presence of additional T cell surface receptors that also provide some costimulatory signals, although not equivalent to the one delivered through CD28. Thus, instead of aging, the study of CD28 is still a topical subject. What is appearing through work of recent years is that far from being purely qualitative, the CD28 signal provides a key quantitative contribution to potently boost the T cell antigen receptor (TCR) signal. In other words, CD28 is in part a signaling ‘sosia’ of the TCR. Also, it is clear now that CD28 operates via multiple molecular effects. Still, what we do not understand is the ‘qualitative’ part of this signal, perhaps due to lack of identification of unique signaling components and/or pathways activated by CD28 only. Here we review a series of recent findings pointing towards novel avenues to better understand the molecular basis of CD28 function.

Mammalian actin binding protein 1 is essential for endocytosis but not lamellipodia formation: functional analysis by RNA interference

Mammalian actin binding protein 1 (mAbp1, also called SH3P7/Hip55) is structurally and functionally related to yeast Abp1 and to cortactin, both of which have been implicated in endocytotic processes. mAbp1 associates through its SH3 domain with dynamin, a large GTPase essential for vesicle fission. To clarify the function of mAbp1, we specifically knocked down its expression in human embryonic kidney 293T cells, using RNA interference (RNAi). Co-transfection of a short interfering RNA (siRNA) together with a plasmid coding for a surface marker, followed by purification of transfected cells, enabled us to obtain a cell population having up to 90% inhibition of mAbp1 expression. In mAbp1-knocked down cells, transferrin (Tf) receptor endocytosis was significantly inhibited and intracellular distribution of the early endosomal compartment was modified. In contrast, in these cells actin and microtubule filaments appeared normal, and formation of lamellipodia induced by active Rac was not inhibited. This study provides definitive evidence that mAbp1 is indispensable for receptor-mediated endocytosis.

The NF-κB RelA Subunit Confers Resistance to Leishmania major by Inducing Nitric Oxide Synthase 2 and Fas Expression but Not Th1 Differentiation

Although the NF-κB transcription factors participate in both innate and adaptive immune responses, little is known about the role of the RelA subunit because mice lacking the rela gene die at embryonic day 14. To elucidate the role of RelA in Leishmania major infection, we prepared fetal liver chimeric mice by adoptively transferring embryonic day 13.5 rela−/− or rela+/+ fetal liver into lethally irradiated host mice. About 90% of the peripheral lymphocytes of the chimeric mice had differentiated from rela fetal liver cells. The rela−/− fetal liver chimeric mice were highly sensitive to infection with L. major and died within 11 wk after infection. Despite the severity of the disease, parasite Ag-reactive Th1 cells developed normally. The rela−/− macrophages were less able to control intracellular parasite replication than rela+/+ macrophages, despite showing equally efficient phagocytosis. Both in vitro NO production of macrophages and in vivo expression of NO synthase 2 in the lesions and draining lymph nodes was reduced in rela−/− fetal liver chimeric mice. Moreover, up-regulation of Fas in rela−/− macrophages was impaired both after in vitro stimulation with LPS and after in vivo infection with L. major, implying a defect in their ability to eliminate infected cells. Thus, RelA is necessary for macrophages to be resistant to intracellular parasite infection.

The inhibitory effects of a RANKL-binding peptide on articular and periarticular bone loss in a murine model of collagen-induced arthritis: a bone histomorphometric study

IntroductionWe designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis.MethodsTwenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed.ResultsThe OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites.ConclusionThe peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.

Peptide drugs accelerate BMP-2-induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling.

Both W9 and OP3‐4 were known to bind the receptor activator of NF‐κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide‐induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3‐4 accelerated BMP‐2‐induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL‐binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP‐2‐induced bone regeneration by the RANKL‐binding peptides.

NF-κB RELA-deficient bone marrow macrophages fail to support bone formation and to maintain the hematopoietic niche after lethal irradiation and stem cell transplantation

Bone remodeling and hematopoiesis are interrelated and bone marrow (BM) macrophages are considered to be important for both bone remodeling and maintenance of the hematopoietic niche. We found that NF-κB Rela-deficient chimeric mice, generated by transplanting Rela (-/-) fetal liver cells into lethally irradiated hosts, developed severe osteopenia, reduced lymphopoiesis and enhanced mobilization of hematopoietic stem and progenitor cells when BM cells were completely substituted by Rela-deficient cells. Rela (-/-) hematopoietic stem cells from fetal liver had normal hematopoietic ability, but those harvested from the BM of osteopenic Rela (-/-) chimeric mice had reduced repopulation ability, indicating impairment of the microenvironment for the hematopoietic niche. Osteopenia in Rela (-/-) chimeric mice was due to reduced bone formation, even though osteoblasts differentiated from host cells. This finding indicates impaired functional coupling between osteoblasts and hematopoietic stem cell-derived cells. Rela-deficient BM macrophages exhibited an aberrant inflammatory phenotype, and transplantation with wild-type F4/80(+) BM macrophages recovered bone formation and ameliorated lymphopoiesis in Rela (-/-) chimeric mice. Therefore, RELA in F4/80(+) macrophages is important both for bone homeostasis and for maintaining the hematopoietic niche after lethal irradiation and hematopoietic stem cell transplantation.

p100, a precursor of NF-κB2, inhibits c-Rel and reduces the expression of IL-23 in dendritic cells.

Nuclear factor κB regulates various genes involved in the immune response, inflammation, cell survival, and development. NF-κB activation is controlled by proteins possessing ankyrin repeats, such as IκBs. A precursor of the NF-κB2 (p52) subunit, p100, contains ankyrin repeats in its C-terminal portion and has been found to act as a cytoplasmic inhibitor of RelA in the canonical pathway of NF-κB activation. Here, we demonstrate that p100 also suppresses c-Rel function in dendritic cells. Expression of the p19 and p40 subunits of IL-23, a c-Rel-dependent cytokine, was enhanced in p100-deficient cells, although expression of a RelA-dependent cytokine, TNF-α, was reduced. Nuclear translocation of c-Rel was enhanced in p100-deficient cells. p100, and not the processed p52 form, associated with c-Rel in the steady state and dissociated immediately after lipopolysaccharide stimulation in wild-type dendritic cells. Four hours after the stimulation, p100 was newly synthesized and associated with c-Rel again. In cells expressing both c-Rel and RelA, c-Rel is preferentially suppressed by p100.

Egress of Mature Murine Regulatory T Cells from the Thymus Requires RelA

The mechanism of egress of mature regulatory T cells (Tregs) from the thymus to the periphery remains enigmatic, as does the nature of those factors expressed in the thymic environment. In this study, we examined the fate of thymic Tregs in TNF-α/RelA double-knockout (TA-KO) mice, because TA-KO mice retain a Treg population in the thymus but have only a small Treg population at the periphery. Transplantation of whole TA-KO thymus to under the kidney capsule of Rag1-null mice failed to induce the production of donor-derived splenic Tregs expressing neuropilin-1, which is reported to be a marker of naturally occurring Tregs, indicating that TA-KO thymic Tregs either do not leave the thymus or are lost at the periphery. We next transplanted enriched TA-KO thymic Tregs to the peripheries of TA-KO mice and traced mouse survival. Transplantation of TA-KO thymic Tregs rescued the lethality in TA-KO mice, demonstrating that TA-KO thymic Tregs remained functional at the periphery. The TA-KO thymic Treg population had highly demethylated CpG motifs in the foxp3 locus, indicating that the cells were arrested at a late mature stage. Also, the population included a large subpopulation of Tregs expressing IL-7Rα, which is a possible marker of late-stage mature Tregs. Finally, TA-KO fetal liver chimeric mice developed a neuropilin-1+ splenic Treg population from TA-KO cells, suggesting that Treg arrest was caused by a lack of RelA in the thymic environment. Taken together, these results suggest that egress of mature Tregs from the thymus depends on RelA in the thymic environment.