Biomedical subjects

Yoshifumi Yokota

Publications and source records attributed to Yoshifumi Yokota.

At least 19 recordsLinked to original sources

PubMed2006

Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid.

Ectopic expression of CC chemokine ligand 21 (CCL21) in the thyroid leads to development of lymphoid structures that resemble those observed in Hashimoto thyroiditis. Deletion of the inhibitor of differentiation 2 (Id2) gene, essential for generation of CD3-CD4+ lymphoid tissue-inducer (LTi) cells and development of secondary lymphoid organs, did not affect formation of tertiary lymphoid structures. Rather, mature CD3+CD4+ T cells were critical for the development of tertiary lymphoid structures. The initial stages of this process involved interaction of CD3+CD4+ T cells with DCs, the appearance of peripheral-node addressin-positive (PNAd+) vessels, and production of chemokines that recruit lymphocytes and DCs. These findings indicate that the formation of tertiary lymphoid structures does not require Id2-dependent conventional LTis but depends on a program initiated by mature CD3+CD4+ T cells.

Adoptive Transfer↗

PubMed2006

Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.

Microarray gene expression profiling is a powerful tool for generating molecular cancer classifications. However, elucidating biological insights from these large data sets has been challenging. Previously, we identified a gene expression-based classification of primary uveal melanomas that accurately predicts metastatic death. Class 1 tumors have a low risk and class 2 tumors a high risk for metastatic death. Here, we used genes that discriminate these tumor classes to identify biological correlates of the aggressive class 2 signature. A search for Gene Ontology categories enriched in our class-discriminating gene list revealed a global down-regulation of neural crest and melanocyte-specific genes and an up-regulation of epithelial genes in class 2 tumors. Correspondingly, class 2 tumors exhibited epithelial features, such as polygonal cell morphology, up-regulation of the epithelial adhesion molecule E-cadherin, colocalization of E-cadherin and beta-catenin to the plasma membrane, and formation of cell-cell adhesions and acinar structures. One of our top class-discriminating genes was the helix-loop-helix inhibitor ID2, which was strongly down-regulated in class 2 tumors. The class 2 phenotype could be recapitulated by eliminating Id2 in cultured class 1 human uveal melanoma cells and in a mouse ocular melanoma model. Id2 seemed to suppress the epithelial-like class 2 phenotype by inhibiting an activator of the E-cadherin promoter. Consequently, Id2 loss triggered up-regulation of E-cadherin, which in turn promoted anchorage-independent cell growth, a likely antecedent to metastasis. These findings reveal new roles for Id2 and E-cadherin in uveal melanoma progression, and they identify potential targets for therapeutic intervention.

Animals↗

PubMed2006

Identification of the nuclear export signal in the helix-loop-helix inhibitor Id1.

Id proteins play important roles in cellular differentiation and proliferation by negatively regulating basic helix-loop-helix transcription factors. Although their intracellular localization may change depending on the biological situation, little is known about the molecular determinants underlying such changes. Here we report the identification of a nuclear export signal (NES) in Id1. The identified NES was different from that of Id2, but had the ability to confine heterologous green fluorescent protein to the cytoplasm. Thus, our results indicate that the intracellular localization of Id1 is regulated differently from that of Id2.

Amino Acid Sequence↗

PubMed2006

Localization of Id2 mRNA in the adult mouse brain.

Id proteins are negative regulators of basic helix-loop-helix transcription factors and are involved in cellular differentiation and proliferation. Four members of the Id gene family exhibit closely related but distinct expression patterns in various mammalian organs of not only embryos but also adults. Among them, Id2 is known to be expressed in Purkinje cells and neurons in the cortical layers of the adult mouse brain, suggesting that Id2 is involved in some neural functions in the adult. To get insight into the role of Id2 in the nervous system, we investigated the localization of Id2 mRNA-expressing cells in the adult mouse brain in detail by in situ hybridization with the radiolabeled antisense probe and compared it with the localization of other Id gene family members. The results indicated that Id2 mRNA is detected in more varied brain regions than previously reported. These regions include the amygdaloid complex, caudate putamen, globus pallidus, substantia nigra pars reticulata, suprachiasmatic nucleus, and the anterior part of the subventricular zone. These results suggest the possibility that Id2 plays a role in the neural activity and cognitive functions. On the other hand, Id1 was barely detectable. Although moderate or low expression of Id3 was observed diffusely, high expression was observed in some specific regions including the molecular layer of the dentate gyrus and the external capsule. Id4 mRNA was detected in the regions such as the caudate putamen and the lateral amygdaloid nucleus. Thus, the expression pattern of Id2 is distinct from those of other Id gene family members.

Animals↗

PubMed2006

Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells via Id2.

Receptor activator of NF-kappaB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl-/- mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2-/- mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.

Animals↗

PubMed2005

Genetic and biochemical properties of streptococcal NAD-glycohydrolase inhibitor.

The gene encoding streptolysin O (slo), a cytolysin of hemolytic streptococci, is transcribed polycistronically from the promoter of the preceding NAD-glycohydrolase (NADase) gene (nga). Between nga and slo, a putative open reading frame (orf1) is located whose function has been totally unknown. Present investigation demonstrated that the orf1 encodes a protein designated as streptococcal NADase inhibitor (SNI). From its nucleotide sequence, SNI was inferred to comprise 161 amino acid residues and the deduced molecular weight was 18,800. This protein was detectable only within cells. Coexpression of SNI was essential for production of streptococcal NADase, and NADase precursor existed as an inactive complex with SNI, in recombinant Escherichia coli. Monomeric NADase and SNI rapidly formed in vitro a stable heterodimer complex in the ratio 1:1, resulting in complete suppression of the hydrolase activity. Unlike other bacterial NADase inhibitors, SNI was thermostable. This protein, coexpressed and complexed with NADase, may protect the producer cocci from exhaustion of NAD.

Amino Acid Sequence↗

PubMed2005

Id helix-loop-helix proteins negatively regulate TRANCE-mediated osteoclast differentiation.

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE) induces osteoclast formation from monocyte/macrophage lineage cells via various transcription factors, including the Mi transcription factor (Mitf). Here, we show that inhibitors of differentiation/DNA binding (Ids), helix-loop-helix (HLH) transcription factors, negatively regulate TRANCE-induced osteoclast differentiation. Expression levels of Id1, Id2, and Id3 genes are significantly reduced by TRANCE during osteoclastogenesis. Interestingly, overexpression of the 3 Id genes in bone marrow-derived monocyte/macrophage lineage cells (BMMs) inhibits the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts, but it does not alter the ability of BMMs to either phagocytose or differentiate into dendritic cells (DCs). Overexpression of Id2 in BMMs attenuates the gene induction of nuclear factor of activated T cells c1 (NFATc1) and osteoclast-associated receptor (OSCAR) during TRANCE-mediated osteoclastogenesis. Furthermore, Id proteins interact with Mitf, a basic HLH (bHLH) transcription factor, and inhibit its transactivation of OSCAR, which is a costimulatory receptor expressed by osteoclast precursors, by attenuating the DNA binding ability of Mitf to the E-box site of the OSCAR promoter. Taken together, our results reveal both a new facet of negative regulation, mediated by Id proteins, as well as the mechanism whereby TRANCE signaling overcomes it, allowing osteoclastogenesis to proceed.

Animals↗

PubMed2005

Regulation of Id2 expression by CCAAT/enhancer binding protein beta.

Mice deficient for Id2, a negative regulator of basic helix-loop-helix (bHLH) transcription factors, exhibit a defect in lactation due to impaired lobuloalveolar development during pregnancy, similar to the mice lacking the CCAAT enhancer binding protein (C/EBP) beta. Here, we show that Id2 is a direct target of C/EBPbeta. Translocation of C/EBPbeta into the nucleus, which was achieved by using a system utilizing the fusion protein between C/EBPbeta and the ligand-binding domain of the human estrogen receptor (C/EBPbeta-ERT), demonstrated the rapid induction of endogenous Id2 expression. In reporter assays, transactivation of the Id2 promoter by C/EBPbeta was observed and, among three potential C/EBPbeta binding sites found in the 2.3 kb Id2 promoter region, the most proximal element was responsible for the transactivation. Electrophoretic mobility shift assay (EMSA) identified this element as a core sequence to which C/EBPbeta binds. Chromatin immunoprecipitation (ChIP) furthermore confirmed the presence of C/EBPbeta in the Id2 promoter region. Northern blotting showed that Id2 expression in C/EBPbeta-deficient mammary glands was reduced at 10 days post coitus (d.p.c.), compared with that in wild-type mammary glands. Thus, our data demonstrate that Id2 is a direct target of C/EBPbeta and provide insight into molecular mechanisms underlying mammary gland development during pregnancy.

Animals↗

PubMed2005

CpG inhibits IgE class switch recombination through suppression of NF kappa B activity, but not through Id2 or Bcl6.

The CpG motif in DNA plays a critical role in immunity via modulating the Th1/Th2 balance. In B cells, CpG-containing oligodeoxynucleotides (CpG ODNs) inhibit IL-4-mediated class switch recombination (CSR) to IgG1 and IgE through inhibition of the germline transcription (GLT) of these isotypes. However, the molecular mechanism of this inhibitory effect remains elusive. We showed here that Id2 and Bcl6, both of which inhibit IgE GLT and CSR, are not involved in this inhibitory pathway. We demonstrated that there is reduced activity of NF kappa B binding to the IgE promoter and a reduction of Irf4 protein in CpG ODN-treated B cells. These data indicate the critical role of NF kappa B and Irf4 in the regulation of IgE CSR through actions downstream of CpG signaling.

Animals↗

PubMed2005

Synergistic effect of hypoxia and TNF-alpha on production of PAI-1 in human proximal renal tubular cells.

BACKGROUND: Chronic hypoxia has been newly proposed as a common mechanism of tubulointerstitial fibrosis in the progression of various chronic inflammatory renal diseases, where plasminogen activator inhibitor-1 (PAI-1) plays an important role in the accumulation of extracellular matrix (ECM) through inhibition of plasmin-dependent ECM degradation. In the present study, we investigated the presence of PAI-1 in renal tubular cells by immunostaining renal biopsy samples. We also closely examined the effects of hypoxia and tumor necrosis factor-alpha (TNF-alpha) on PAI-1 expression in cultured human proximal renal tubular cells (HPTECs). METHODS: Confluent cells growth-arrested in Dulbecco's modified Eagle's medium (DMEM) for 24 hours were exposed to hypoxia (1% O(2)) and/or TNF-alpha at 10 ng/mL for up to 48 hours. Amounts of PAI-1 protein and mRNA after stimulation were measured by enzyme-linked immunosorbent assay (ELISA) and TaqMan quantitative polymerase chain reaction (PCR) or cDNA array analysis, respectively, and compared to those in cells incubated under control conditions (18% O(2) without TNF-alpha). Hypoxia-inducible factor-1alpha (HIF-1alpha) was demonstrated by immunoblot and immunofluorescence analyses. Human PAI-1 promoter activity was estimated by luciferase reporter gene assay. RESULTS: In crescentic glomerulonephritis, clusters of proximal tubules were specifically stained for PAI-1. cDNA array analysis identified PAI-1 as a major gene highly induced by hypoxia in HPTECs. Treatment of 24 hours with hypoxia, TNF-alpha, and their combination induced a 2.8-fold, a 1.8-fold, and a 4.6-fold increase in PAI-1 protein secretion, and produced a 3.6-fold, a 3.3-fold, and a 12.1-fold increase at the PAI-1 mRNA level, respectively. Immunoblot analysis and immunocytochemistry revealed that hypoxia-inducible factor-1alpha (HIF-1alpha) was markedly accumulated in the cell lysates and exclusively translocated to nuclei after 16 hours' exposure of HPTECs to hypoxia but not to TNF-alpha. Luciferase reporter gene assay showed that hypoxia, TNF-alpha, and their combination increased PAI-1 transcription activity by 1.8-fold, 1.4-fold, and 2.2-fold, respectively. A dominant-negative form of HIF-1alpha significantly suppressed PAI-1 transcription activity induced by hypoxia. Inhibition of nuclear factor-kappaB (NF-kappaB) caused a moderate decrease in PAI-1 production under hypoxia. CONCLUSION: Hypoxia induces PAI-1 expression via remarkable nuclear accumulation of HIF-1alpha and partially via NF-kappaB activation in HPTECs. TNF-alpha can synergistically enhance this hypoxia-induced PAI-1 expression.

Antineoplastic Agents↗

PubMed2005

Id2 mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice.

The inhibitor of differentiation Id2 is a target of the retinoblastoma (Rb) protein during mouse embryogenesis. In Rb(+/-) mice, LOH at the wild-type Rb allele initiates pituitary adenocarcinoma, a tumor derived from embryonic melanotropes. Here we identify a critical role for Id2 in initiation, growth, and angiogenesis of pituitary tumors from Rb(+/-) mice. We show that proliferation and differentiation are intimately coupled in Rb(+/-) pituitary cells before tumor initiation. In Id2-null pituitaries, premature activation of basic helix-loop-helix-mediated transcription and expression of the cdk inhibitor p27(Kip1) impairs the proliferation of melanotropes and tumor initiation. Without Id2, Rb(+/-) mice have fewer early tumor lesions and a markedly decreased proliferation rate of the tumor foci. Expression of Id2 by pituitary tumor cells promotes growth and angiogenesis by functioning as a master regulator of vascular endothelial growth factor (VEGF). In human neuroblastoma, the N-Myc-driven expression of Id2 is sufficient and necessary for expression of VEGF. These results establish that aberrant Id2 activity directs initiation and progression of embryonal cancer.

Animals↗

PubMed2005

Expression of recombinant Streptolysin O and specific antibody production.

Streptolysin O (SLO), an oxygen-labile cytolysin, is the cholesterol-binding exotoxin of hemolytic streptococci. Besides microbiological and pathological interests, this cytolysin has been used as a tool for permeabilization of biomembranes. SLO serves as a diagnostic reagent for determination of anti-SLO antibody titer in streptococcal infection. Availability of highly purified SLO, however, has been limited by low yield in streptococcal culture and purification process. Present subcloning of mature-type full-length SLO gene into an expression vector having strictly controllable araBAD promoter enabled efficient production of the cytolysin. Further, anti-SLO antibody with high specificity was obtained by immunizing with purified SLO protein.

Animals↗

PubMed2004

Wnt1 is epistatic to Id2 in inducing mammary hyperplasia, ductal side-branching, and tumors in the mouse.

BACKGROUND: During pregnancy, the mammary glands from Id2 mutant animals are deficient in lobulo-alveolar development. This failure of development is believed to be due to a proliferation defect. METHODS: We have asked whether functional Id2 expression is necessary for Wnt induced mammary hyperplasia, side branching, and cancer, by generating mice expressing a Wnt1 transgene in an Id2 mutant background. RESULTS: We show in this work that forced expression of Wnt1 in the mammary gland is capable of overcoming the block to proliferation caused by the absence of Id2. We also show that Wnt1 expression is able to cause mammary tumors in an Id2 mutant background. CONCLUSIONS: We conclude that functional Id2 expression is not required for Wnt1 to induce mammary hyperplasia and mammary tumors.

Animals↗

PubMed2004

Molecular characterization of NADase-streptolysin O operon of hemolytic streptococci.

Whether slo, the gene encoding streptolysin O (SLO), a streptococcal cytolysin, has its own promoter or not is unsettled as yet. Present analyses demonstrate that slo is a member of an operon covering the upper-stream nusG and nga (NADase) genes, from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter. Mutational conversion of the sixth nucleotide T at the putative -10 region of chromosomal nga gene into C caused a drastic decrease in both NADase and SLO activities and the disappearance of the two corresponding mRNA bands from the Northern blot profile. The initiation site of the transcription was determined at 56 bp upstream (NusG gene) and 25 bp upstream (NADase gene) of each initiation codon. Although the promoter region of slo gene is highly conserved between group A and C streptococci, the proper slo promoter is nonfunctional in group C strain H46A. Moreover, commonly conserved arrangement was limited to the nusG-nga-orf1-slo region. These results indicate an intimate relationship between NADase and SLO in the regulation of their biosynthesis. Additional results suggest that NADase, synthesized as precursor with feeble activity, is activated by removing the carboxyl terminal region during or after secretion into culture medium.

3' Flanking Region↗

PubMed2004

Nucleo-cytoplasmic shuttling of Id2, a negative regulator of basic helix-loop-helix transcription factors.

Id proteins function as negative regulators for basic helix-loop-helix transcriptional factors that play important roles in cell fate determination. They preferentially associate with ubiquitously expressed E proteins of the basic helix-loop-helix family and prevent them from binding to DNA and activating transcription. Although their small size suggests that Id proteins enter and exit the nucleus by passive diffusion, several studies have indicated that other pathways may regulate their subcellular localization. In this study, we obtained evidence that Id2 has the ability to shuttle between the nucleus and the cytoplasm. When passive diffusion was prevented by fusion with green fluorescent protein (GFP), Id2 was predominantly localized in the cytoplasm. Using GFP fusion constructs, we demonstrated that the C-terminal region is required for cytoplasmic localization. Nuclear accumulation of GFP-Id2 in cells treated with the nuclear export inhibitor leptomycin B suggests that the nuclear export receptor chromosome region maintenance protein 1 mediates the cytoplasmic localization of Id2. Id2 contains two putative leucine-rich nuclear export signals, and the nuclear export signal in the C-terminal region is essential for nuclear export. On the other hand, the helix-loop-helix domain is important for nuclear localization. Finally, experiments using reporter assays revealed an inverse correlation between nuclear export and transcriptional repression via the E-box sequence. Based on all these findings, we propose that nucleo-cytoplasmic shuttling is a novel mechanism for the regulation of Id2 function.

Active Transport, Cell Nucleus↗

PubMed2004

Id2 is dispensable for myc-induced lymphomagenesis.

The Emu-Myc transgenic mouse appears to be an accurate model of human Burkitt's lymphoma that bears MYC/Immunoglobulin gene translocations. Id2, a negative regulator of basic helix-loop-helix transcription factors, has also been proposed as a Myc target gene that drives the proliferative response of Myc by binding to and overriding the checkpoint functions of the retinoblastoma tumor suppressor protein. Targeted deletion of Id2 in mice results in defects in B-cell development and prevents the development of peripheral lymphoid nodes. In precancerous B cells and lymphomas that arise in Emu-Myc transgenic mice and in Burkitt's lymphomas, Id2 is overexpressed, suggesting that it plays a regulatory role in lymphoma development. Surprisingly, despite these connections, Emu-Myc mice lacking Id2 succumb to lethal B-cell lymphoma at rates comparable with wild-type Emu-Myc transgenics. Furthermore, precancerous splenic B cells lacking Id2 do not exhibit any significant defects in Myc-induced target gene transactivation and proliferation. However, due to their lack of secondary lymph nodes, Emu-Myc mice lacking Id2 rather succumb to disseminated lymphoma with an associated leukemia, with pronounced infiltrates of the bone marrow and other major organs. Collectively these findings argue that targeting Id2 functions may be ineffective in preventing Myc-associated malignancies.

Animals↗

PubMed2004

Impaired IgG production in mice deficient for heat shock transcription factor 1.

Heat shock factor 1 (HSF1) is a major transactivator of heat shock proteins in response to heat shock, and it is also involved in oogenesis, spermatogenesis, and placental development. However, we do not know the molecular mechanisms controlling developmental processes. In this study, we found that HSF1-null mice exhibited a significant decrease in the T cell-dependent B cell response. When mice were immunized intraperitoneally with sheep red blood cells, the sheep red blood cell-specific IgG production, especially IgG2a production, in HSF1-null mice was about 50% lower than that in wild-type mice at 6 days after the immunization, whereas IgM production was normal. The number of bromodeoxyuridine-incorporated spleen cells in immunized HSF1-null mice was one-third that in immunized wild-type mice, indicating reduced proliferation of the spleen cells. We analyzed levels of cytokines and chemokines in spleen cells and in peritoneal macrophages stimulated with lipopolysaccharide and interferon-gamma and found that expression levels of interleukin-6 and CCL5 were significantly lower in HSF1-null cells than those in wild-type cells. Furthermore, we demonstrated that the IL-6 gene is a direct target gene of HSF1. These results revealed a novel molecular link between HSF1 and a gene related to immune response and inflammation.

Animals↗

PubMed2004

Role of Id proteins in B lymphocyte activation: new insights from knockout mouse studies.

Id (inhibitor of differentiation) proteins play important roles in cell differentiation, cell cycle control, and apoptosis. They act as negative regulators of basic helix-loop-helix-type transcription factors, which positively regulate differentiation of various cell types. Id proteins work to block B lymphocyte (B cell) maturation at an early differentiation step, as demonstrated by gain-of-function studies. In recent years a series of gene-targeted mice lacking different Ids have been generated. Analyses of these gene-targeted mice provide information useful for understanding the physiological roles of Ids in B cell biology. Id3 is required for proper B cell functions and acts by controlling the cell cycle. Upon B cell activation, Id2 acts as a negative regulator to prevent potentially harmful effects brought about by excessive immunological reactions; one of its special roles is to maintain low serum concentrations of immunoglobulin E (IgE). The Id2 protein does this by antagonizing E2A and Pax5 activities, both of which are required for proper B cell activation. This review presents several new insights into B cell differentiation and activation programs and the physiological role of Id proteins in B cell activation.

Animals↗