Takeshi Kuwata

ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages.

During hematopoiesis, myeloid progenitor cells give rise to granulocytes and macrophages. To study the role for ICSBP, a hematopoietic cell-specific transcription factor in myeloid cell development, the gene was introduced into myeloid progenitor cells established from ICSBP-/- mice. ICSBP retrovirus-transduced cells differentiated into mature macrophages with phagocytic activity, which coincided with the induction of specific target DNA binding activity. Similar to macrophages in vivo, ICSBP-transduced cells were growth arrested, expressed many macrophage-specific genes, and responded to macrophage activation signals. Contrary to this, ICSBP transducion led to repression of granulocyte-specific genes and inhibited G-CSF-mediated granulocytic differentiation in these and other myeloid progenitor cells. Together, ICSBP has a key role in the myeloid cell lineage selection and macrophage maturation.

Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin beta gene.

We have investigated ligand‐dependent negative regulation of the thyroid‐stimulating hormone β (TSHβ) gene. Thyroid hormone (T3) markedly repressed activity of the TSHβ promoter that had been stably integrated into GH3 pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH3 cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRβ) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3‐dependent promoter repression. We suggest that ligand‐dependent histone deacetylase recruitment is a mechanism of the negative‐feedback regulation, a critical function of the pituitary–thyroid axis.

β-Catenin Simultaneously Induces Activation of the p53-p21WAF1 Pathway and Overexpression of Cyclin D1 during Squamous Differentiation of Endometrial Carcinoma Cells

The functional consequences of up-regulation of β-catenin as a transcription factor are complex in different tumors. To clarify roles during squamous differentiation (SqD) of endometrial carcinoma (Em Ca) cells, we investigated expression of β-catenin, as well as cyclin D1, p53, p21WAF1, and PML (promyelocytic leukemia) in 80 cases of Em Ca with SqD areas, in comparison with cell proliferation determined with reference to Ki-67 antigen positivity. The impact of β-catenin-T-cell factor (TCF)-mediated transcription was also examined using Em Ca cells. In clinical cases, nuclear β-catenin accumulation was more frequent in SqD areas, being positively linked with expression of cyclin D1, p53, and p21WAF1, and inversely with Ki-67 and PML immunoreactivity. Significant correlations of nuclear β-catenin, cyclin D1, p53, and p21WAF1 were noted between SqD and the surrounding carcinoma lesions. The Ishikawa cell line, with stable or tetracycline-regulated expression of mutant β-catenin, showed an increase in expression levels of cyclin D1, p14ARF, p53, and p21WAF1 but not PML, and activation of β-catenin-TCF4-mediated transcription determined with TOP/FOP constructs. The cell morphology was senescence-like rather than squamoid in appearance. Moreover, overexpressed β-catenin could activate transcription from p14ARF and cyclin D1 promoters, in a TCF4-dependent manner. These findings indicate that in Em Cas, nuclear β-catenin can simultaneously induce activation of the p53-p21WAF1 pathway and overexpression of cyclin D1, leading to suppression of cell proliferation or induction of cell senescence. However, overexpression of β-catenin alone is not sufficient for development of a squamoid phenotype in Em Ca cells, suggesting that nuclear accumulation is an initial signal for trans-differentiation.

Gamma interferon triggers interaction between ICSBP (IRF-8) and TEL, recruiting the histone deacetylase HDAC3 to the interferon-responsive element.

ABSTRACT ICSBP (IRF-8) is a transcription factor of the IRF family expressed only in the immune system. It is induced in macrophages by gamma interferon (IFN-γ) and contributes to macrophage functions. By interacting with Ets family protein PU.1, ICSBP binds to the IRF/Ets composite element and stimulates transcription. ICSBP binds to another DNA element, the IFN-stimulated response element (ISRE), a common target of the IRF family. Limited knowledge as to how ICSBP and other IRF proteins regulate ISRE-dependent transcription in IFN-γ-activated macrophages is available. By mass-spectrometric analysis of ISRE-bound proteins in macrophages, we identified TEL, another Ets member, as a factor recruited to the element in an IFN-γ-dependent manner. In vitro analysis with recombinant proteins indicated that this recruitment is due to a direct interaction between ICSBP and TEL, which is enhanced by the presence of ISRE. Significantly, the interaction with TEL in turn resulted in the recruitment of the histone deacetytase HDAC3 to the ISRE, causing increased repression of IFN-γ-mediated reporter activity through the ISRE. This repression may provide a negative-feedback mechanism operating after the initial transcriptional activation by IFN-γ. By associating with two different Ets family proteins, ICSBP exerts a dual function in IFN-γ-dependent gene regulation in an immune system-specific manner.

Upregulation of TCF4 expression as a transcriptional target of β -catenin/p300 complexes during trans -differentiation of endometrial carcinoma cells

Nuclear stabilization of β-catenin and its interaction with TCF/LEF factors are key events in transduction of the Wnt/β-catenin signal pathway. Our previous study indicated that nuclear β-catenin accumulation provides an initial signal for trans-differentiation toward the squamoid phenotype of endometrial carcinoma (Em Ca) cells in a TCF4-dependent manner, which makes this a possible factor for a positive prognosis. However, little is known about regulation of TCF4 expression in Em Cas. We show here that β-catenin can directly induce transcription from the TCF4 promoter, the effect being enhanced by the p300 coactivator. In clinical cases, nuclear β-catenin accumulation was found to frequently overlap with TCF4 immunoreactivity in morules and surrounding glandular carcinoma lesions, showing a significant positive correlation (r=0.82, P<0.0001), in contrast to areas of squamous metaplasia (SqM) within Em Cas. In cases with coexistence of two squamoid features in trans-differentiated areas, loss of nuclear β-catenin and TCF4 immunoreactivity was closely related to change in the morphology from the morular to the SqM phenotype. The TCF4 promoter contains a single consensus TCF-binding site that is critical for activation by β-catenin. The p300 coactivator, in particular N-terminal residues 1 to 670, appears sufficient to enhance β-catenin-dependent transcription, again with TCF4-dependence. These findings indicate that a positive feedback loop of TCF4 expression mediated by β-catenin/p300 may be important for initial steps during trans-differentiation of Em Ca cells. In addition, its downregulation is associated with induction of a more-differentiated squamoid phenotype.

Crosstalk between NF‐κB/p65 and β‐catenin/TCF4/p300 signalling pathways through alterations in GSK‐3β expression during trans‐differentiation of endometrial carcinoma cells

β‐Catenin/TCF4/p300 signalling loops play an important role in trans‐differentiation towards the morular phenotype of endometrial carcinomas. Crosstalk between NF‐κB and β‐catenin pathways has been proposed and we focused here on associations between these two pathways during trans‐differentiation. In normal endometrium, nuclear phosphorylated p65 (pp65), the active form NF‐κB subunit, was found to be significantly increased in the secretory phase, correlating positively with vimentin and E‐cadherin and inversely with Snail mRNA expression. On transfection of p65, vimentin, E‐cadherin, and Snail were transcriptionally altered, indicating possible roles in establishment and maintenance of the secretory phenotype. In endometrial carcinomas with morules, levels of nuclear pp65, Snail mRNA, vimentin, and cytoplasmic TNF‐α were reduced during trans‐differentiation, correlating inversely with nuclear β‐catenin. Nuclear accumulation of GSK‐3β, along with β‐catenin, was observed in morules. In cell lines, overexpression of p65 inhibited β‐catenin/TCF4‐mediated transcription, while transfection of GSK‐3β resulted in repression of TNF‐α‐induced NF‐κB activity. Moreover, nuclear GSK‐3β was increased by overexpression of β‐catenin, as well as induction of G1‐cell cycle arrest. These findings provide evidence that a shift from NF‐κB to β‐catenin signalling pathways through alterations in GSK‐3β expression may be essential for the induction of trans‐differentiation of endometrial carcinoma cells, leading to a shut‐down of mesenchymal markers. Copyright

Transcription factor Egr1 acts as an upstream regulator of β‐catenin signalling through up‐regulation of TCF4 and p300 expression during trans‐differentiation of endometrial carcinoma cells

The β‐catenin/TCF4/p300 pathway is involved in early signalling for trans‐differentiation towards the morular phenotype of endometrial carcinoma cells, but little is known about the upstream regulators. Here we show that transcription factor early growth response 1 (Egr1) acts as an initial mediator through up‐regulating the expression of TCF4 and p300. In an endometrial carcinoma cell line with abundant oestrogen receptor α, Egr1 expression at both mRNA and protein levels was significantly increased by serum and 17β‐oestradiol stimuli. Serum‐stimulated cells also showed increased expression of TCF4 and p300, while inhibition of Egr1 by specific siRNAs resulted in decreased expression. Transfection of Egr1 led to transactivation of TCF4 as well as p300 genes, through specific binding to a promoter region, and thus in turn resulted in nuclear accumulation of β‐catenin mediated by the up‐regulating TCF4. The overexpression also caused inhibition of β‐catenin/TCF4/p300‐mediated transcription, probably through sequestration of p300. Egr1 promoter activity was increased by serum but not 17β‐oestradiol, in contrast to the marked repression associated with TCF4, p300, and Egr1 itself, indicating that the regulation involves several feedback loops. In clinical samples, cells immunopositive for nuclear Egr1, as well as β‐catenin and TCF4, were found to be sporadically distributed in glandular components of endometrial carcinoma with morules. A significant positive correlation between nuclear β‐catenin and TCF4 was observed, but no such link was evident for Egr1, probably due to the existence of negative feedback regulation. Together, these data indicate that Egr1 may participate in modulation of the β‐catenin/TCF4/p300 signalling pathway as an initial event during trans‐differentiation of endometrial carcinoma cells, through its impact on several signalling networks. Copyright

Induction of p16INK4A mediated by β‐catenin in a TCF4‐independent manner: Implications for alterations in p16INK4A and pRb expression during trans‐differentiation of endometrial carcinoma cells

Excessive β‐catenin is considered to contribute to tumor progression by inducing transcription of cell cycle‐related genes such as cyclin D1 and c‐myc. In contrast, our recent studies demonstrated that β‐catenin could inhibit cell proliferation through activation of p14ARF/p53/p21WAF1 pathway during trans‐differentiation toward morular phenotype of endometrial carcinoma (Em Ca) cells. Here, we focused on associations with alterations in p16INK4A and pRb expression during this process. In clinical cases, p16INK4A immunoreactivity was found to frequently overlap with nuclear β‐catenin accumulation in small‐sized morules and surrounding glandular carcinomas (Sur‐Ca), demonstrating a significant positive correlation (r = 0.447, p < 0.0001) overall, while the immunoreactions showed stepwise decrease in enlarged morules, despite persistent accumulation of β‐catenin and p21WAF1 in nuclei. Immunoreactivity for both total pRb and its phosphorylated form was apparently decreased in all morules as compared to Sur‐Ca lesions, with a significantly positive correlation. In cell lines, transcriptional activation of p16INK4A promoter by active form β‐catenin, as well as p21WAF1, occurred through the region from −385 to −280 bp relative to the translation start site, in a TCF4‐independent manner. Moreover, cell proliferation was accompanied with phosphorylation of pRb and increased p16INK4A expression, while its inhibition by serum starvation caused decreased expression of total pRb but not p16INK4A, resulting in high relative amounts of the latter. These findings indicate that induction of p16INK4A mediated by nuclear β‐catenin and p21WAF1, along with loss of pRb expression, may be important for initial steps during trans‐differentiation of Em Ca cells. In addition, its down‐regulation is associated with progression of lesions.

Enhanced expression of p210BCR/ABL and aberrant expression of Zfp423/ZNF423 induce blast crisis of chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematopoietic disorder originating from p210BCR/ABL-transformed stem cells, which begins as indolent chronic phase (CP) but progresses into fatal blast crisis (BC). To investigate molecular mechanism(s) underlying disease evolution, CML-exhibiting p210BCR/ABL transgenic mice were crossed with BXH2 mice that transmit a replication-competent retrovirus. Whereas nontransgenic mice in the BXH2 background exclusively developed acute myeloid leukemia, p210BCR/ABL transgenic littermates developed nonmyeloid leukemias, in which inverse polymerase chain reaction detected 2 common viral integration sites (CISs). Interestingly, one CIS was transgenes own promoter, which up-regulated p210BCR/ABL expression. The other was the 5 noncoding region of a transcription factor, Zfp423, which induced aberrant Zfp423 expression. The cooperative activities of Zfp423 and p210BCR/ABL were demonstrated as follows: (1) introduction of Zfp423 in p210BCR/ABL transgenic bone marrow (BM) cells increased colony-forming ability, (2) suppression of ZNF423 (human homologue of Zfp423) in ZNF423-expressing, p210BCR/ABL-positive hematopoietic cells retarded cell growth, (3) mice that received a transplant of BM cells transduced with Zfp423 and p210BCR/ABL developed acute leukemia, and (4) expression of ZNF423 was found in human BCR/ABL-positive cell lines and CML BC samples. These results demonstrate that enhanced expression of p210BCR/ABL and deregulated expression of Zfp423/ZNF423 contribute to CML BC.

BCL11A is a SUMOylated protein and recruits SUMO‐conjugation enzymes in its nuclear body

BCL11A/EVI9 is a zinc‐finger protein predominantly expressed in brain and hematopoietic cells. Previous studies show that BCL11A is involved in acute myelomonocytic leukemia and chronic lymphoid leukemia in mouse and human, respectively. Moreover, BCL11A is localized in the characteristic nuclear body in which BCL6 is co‐localized. However, the significance of BCL11A in leukemogenesis and nuclear function remains unknown. In this study we show that BCL11A interacts with UBC9, a small ubiquitin‐like modifier (SUMO) E2 conjugating enzyme, and recruits SUMO1 into the nuclear body. A lysine residue at amino acid 634 of BCL11A is SUMOylated but not required for the SUMO1 recruitment. The N‐terminal region of BCL11A is responsible for SUMO1 recruitment as well as its nuclear body formation. We also show that SENP2, a SUMO specific peptidase, is co‐localized in the nuclear body. These results suggest that BCL11A could be involved in the SUMO conjugation system, and that BCL11A might play an important role in protein modification.