Yoshinori Katsuragi

p62/SQSTM1 functions as a signaling hub and an autophagy adaptor.

p62/SQSTM1 is a stress‐inducible cellular protein that is conserved among metazoans but not in plants and fungi. p62/SQSTM1 has multiple domains that mediate its interactions with various binding partners and it serves as a signaling hub for diverse cellular events such as amino acid sensing and the oxidative stress response. In addition, p62/SQSTM1 functions as a selective autophagy receptor for degradation of ubiqutinated substrates. In the present review, we describe the current knowledge about p62 with regard to mammalian target of rapamycin complex 1 activation, the Keap1–Nrf2 pathway and selective autophagy.

IFN-γ–Producing and IL-17–Producing γδ T Cells Differentiate at Distinct Developmental Stages in Murine Fetal Thymus

γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ–producing and IL-17–producing γδ T cells developed from DN2 cells, only IFN-γ–producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17–producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17–producing γδ T cells, although a unique subset of IFN-γ–producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.

Meis1 Regulates Epidermal Stem Cells and Is Required for Skin Tumorigenesis

Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.

Bcl11b heterozygosity promotes clonal expansion and differentiation arrest of thymocytes in γ‐irradiated mice

Bcl11b encodes a zinc‐finger transcription factor and functions as a haploinsufficient tumor suppressor gene. Bcl11bKO/KO mice exhibit differentiation arrest of thymocytes during β‐selection as has been observed with other mouse models involving knockouts of genes in the Wnt/β‐catenin signaling pathway. Recurrent chromosomal rearrangement at the BCL11B locus occurs in human T‐cell leukemias, but it is not clear how such rearrangement would contribute to lymphomagenesis. To address this issue, we studied clonal cell growth, cell number, and differentiation of thymocytes in Bcl11bKO/+ mice at different time points following γ‐irradiation. Analysis of D‐J rearrangement at the T cell receptor β‐chain (TCRβ) locus and cell surface markers by flow cytometry revealed two distinct populations of clonally growing thymocytes. In one population, thymocytes share a common D‐J rearrangement but retain the capacity to differentiate. In contrast, thymocytes in the second population have lost their ability to differentiate. Since the capacity to self renew and differentiate into multiple cell lineages are fundamental properties of adult stem cells, the differentiation competent population of thymocytes that we have isolated could potentially function as cancer stem cells. We also demonstrate increased expression of β‐catenin, a well‐known oncogenic protein, in Bcl11bKO/+ thymocytes. Collectively, the Bcl11bKO/+ genotype contributes to clonal expansion and differentiation arrest in part through an increase in the level of β‐catenin. (Cancer Sci 2010)

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/β-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of β-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of β-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of β-catenin pathway.

Bcl11b prevents the intrathymic development of innate CD8 T cells in a cell intrinsic manner

If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαβ(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

Priming of lineage-specifying genes by Bcl11b is required for lineage choice in post-selection thymocytes

T-lineage committed precursor thymocytes are screened by a fate-determination process mediated via T cell receptor (TCR) signals for differentiation into distinct lineages. However, it remains unclear whether any antecedent event is required to couple TCR signals with the transcriptional program governing lineage decisions. Here we show that Bcl11b, known as a T-lineage commitment factor, is essential for proper expression of ThPOK and Runx3, central regulators for the CD4-helper/CD8-cytotoxic lineage choice. Loss of Bcl11b results in random expression of these factors and, thereby, lineage scrambling that is disconnected from TCR restriction by MHC. Initial Thpok repression by Bcl11b prior to the pre-selection stage is independent of a known silencer for Thpok, and requires the last zinc-finger motif in Bcl11b protein, which by contrast is dispensable for T-lineage commitment. Collectively, our findings shed new light on the function of Bcl11b in priming lineage-specifying genes to integrate TCR signals into subsequent transcriptional regulatory mechanisms.CD4 and CD8 T cells develop in the thymus with their transcription programs controlled by ThPOK and Runx3, respectively. Here the authors show that a pre-commitment event modulated by the transcription factor, Bcl11b, is required for the proper expression of ThPOK and Runx3 and correct CD4/CD8 lineage commitment.

Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10–16% of T‐cell acute lymphoblastic leukemias. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas, a model of T‐cell acute lymphoblastic leukemia, when γ‐irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation‐induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11bflox/+;Lck‐Cre and Bcl11bflox/+;CD4‐Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage, whereas in the latter the loss occurs in the more differentiated CD4+CD8+ double‐positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60 days after 3 Gy γ‐irradiation. Half (9/18) of the thymuses in the Bcl11bflox/+;Lck‐Cre group showed limited rearrangement sites at the T‐cell receptor‐β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11bflox/+;CD4‐Cre group did. This indicates that the origin of the premalignant thymocytes is not in double‐positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

Clonally Expanding Thymocytes Having Lineage Capability in Gamma-Ray–Induced Mouse Atrophic Thymus

PURPOSE To characterize, in the setting of gamma-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. METHODS AND MATERIALS A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after gamma-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRbeta locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. RESULTS Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4(+)CD8(+) double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as gammaH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. CONCLUSION The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.