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The thymus has the ability to regenerate from acute injury caused by radiation, infection, and stressors. In addition to thymocytes, thymic epithelial cells in the medulla (mTECs), which are crucial for T cell self-tolerance by ectopically expressing and presenting thousands of tissue-specific antigens (TSAs), are damaged by these insults and recover thereafter. However, given recent discoveries on the high heterogeneity of mTECs, it remains to be determined whether the frequency and properties of mTEC subsets are restored during thymic recovery from radiation damage. Here we demonstrate that acute total body irradiation with a sublethal dose induces aftereffects on heterogeneity and gene expression of mTECs. Single-cell RNA-sequencing (scRNA-seq) analysis showed that irradiation reduces the frequency of mTECs expressing AIRE, which is a critical regulator of TSA expression, 15 days after irradiation. In contrast, transit-amplifying mTECs (TA-mTECs), which are progenitors of AIRE-expressing mTECs, and Ccl21a-expressing mTECs, were less affected. Interestingly, a detailed analysis of scRNA-seq data suggested that the proportion of a unique mTEC cluster expressing Ccl25 and a high level of TSAs was severely decreased by irradiation. In sum, we propose that the effects of acute irradiation disrupt the heterogeneity and properties of mTECs over an extended period, which potentially leads to an impairment of thymic T cell selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Horie, Namiki, Kinoshita, Miyauchi, Ishikawa, Hayama, Maruyama, Hagiwara, Miyao, Murata, Kobayashi, Akiyama and Akiyama.)

The deficiency of Aire, a transcriptional regulator whose defect results in the development of autoimmunity, is associated with reduced expression of tissue-restricted self-Ags (TRAs) in medullary thymic epithelial cells (mTECs). Although the mechanisms underlying Aire-dependent expression of TRAs need to be explored, the physical identification of the target(s) of Aire has been hampered by the low and promiscuous expression of TRAs. We have tackled this issue by engineering mice with augmented Aire expression. Integration of the transcriptomic data from Aire-augmented and Aire-deficient mTECs revealed that a large proportion of so-called Aire-dependent genes, including those of TRAs, may not be direct transcriptional targets downstream of Aire. Rather, Aire induces TRA expression indirectly through controlling the heterogeneity of mTECs, as revealed by single-cell analyses. In contrast, Ccl25 emerged as a canonical target of Aire, and we verified this both in vitro and in vivo. Our approach has illuminated the Aire's primary targets while distinguishing them from the secondary targets., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Background: Compared to two-dimensional cultures, three-dimensional (3D) cultures have many advantages in cancer studies. Nevertheless, their implementation is unsatisfactory. This study aimed to develop an anchorage-dependent 3D culture model for colorectal cancer research., Materials and Methods: Human HCT116, DLD-1 and SW620 colorectal cell lines were cultured in a gelatin sponge, and its applicability for morphological examination was studied., Results: The resulting specimens were suitable for scanning electron microscopy, transmission electron microscopy, and immunohistochemical examination. HCT116 formed smaller structures and migrated through the pores of the sponge. DLD-1 formed larger structures with tight cell-to-cell adhesion. SW620 also formed large structures but small clustered cells tended to attach to the anchorage more favorably. Immunohistochemical staining demonstrated phosphorylated yes-associated protein (YAP) localized near the attachment site in HCT116 cells., Conclusion: Because the gelatin sponge provided suitable anchorage and the cultured cells formed distinguishable 3D structures, this method may be useful for further colorectal cancer research., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose of Review: Middle ear cholesteatoma is an epithelial lesion that expands into the middle ear, resulting in bone destruction. However, the pathogenesis of this has been unknown. The purpose of this review is to understand the role of keratinocyte growth factor (KGF) during epithelial stem and/or progenitor cell proliferation in middle ear cholesteatoma., Recent Findings: Many researchers have investigated the molecular mechanism of middle ear cholesteatoma to establish a conservative treatment. Recently, some studies have focused on the stem cells of middle ear cholesteatoma and their detection, but the key molecules for stem cell formation were not shown., Summary: We established an animal model for middle ear cholesteatoma and are showing the results of our studies. KGF expression accelerates the proliferation of stem/progenitor cells through the induction of transcription factor p63 expression in the epithelium of the tympanic membrane and mucosal epithelium overlying the promontory of the cochlea and within the attic. This is typical in middle ear cholesteatoma. Moreover, the partial epithelial-mesenchymal transition under the p63 signaling pathway plays an essential role in epithelial cell growth in middle ear cholesteatoma formation. Understanding p63 expression following KGF expression and associated signaling events can improve therapeutic outcomes in patients with middle ear cholesteatoma.

Introduction: Partial epithelial-mesenchymal transition (p-EMT) is a process by which epithelial cells partially lose their intercellular adhesion and change to obtain migration ability. The transcription factor p63 regulates the expression of cadherin family and induces epithelial cell proliferation. In this study, we hypothesized that p-EMT under p63 expression may be a key factor in epithelial cell growth in middle ear cholesteatoma., Methods: Specimens were surgically excised from patients with congenital cholesteatoma (CC) (n = 48), acquired middle ear cholesteatoma (AC) (n = 120), and normal skin tissue (n = 34). We analyzed immunohistochemically for the EMT marker (N-cadherin), adherence junction marker (E-cadherin), and tight junction marker (claudin-1, claudin-4, occludin). We also examined the labeling index (LI) of p63 and Proliferating cell nuclear antigen (PCNA) (late S phase marker), and Snail expression as a mobility marker., Results: The expression of p63 (CC 51.0 ± 7.4%, AC 50.0 ± 5.9%) was significantly higher in the thickened epithelium of CC and AC compared with normal skin tissue (p < 0.0001). The loss of E-cadherin was observed (CC 50.0%, AC 55.8%) but the expression patterns in the tight junction were almost normal. N-cadherin was partially detected in the basal and upper layer of epithelium in CC and AC. In contrast to that of normal skin tissue, the LI of PCNA was significantly higher in AC (p < 0.0001). The positive rate of Snail was significantly higher in CC (p < 0.0001)., Conclusion: This study indicates that p-EMT via the p63 signaling pathway might plays an essential role in epithelial growth in AC and CC formation, although tight junction formation and terminal differentiation were not affected in those processes.

Hindlimb unloading (HU) of rodents has been used as a ground-based model of spaceflight. In this study, we investigated the detailed impact of 14-day HU on the murine thymus. Thymic mass and cell number were significantly reduced after 14 days of hindlimb unloading, which was accompanied by an increment of plasma corticosterone. Although corticosterone reportedly causes selective apoptosis of CD4 + CD8 + thymocytes (CD4 + CD8 + DPs) in mice treated with short-term HU, the reduction of thymocyte cellularity after the 14-day HU was not selective for CD4 + CD8 + DPs. In addition to the thymocyte reduction, the cellularity of thymic epithelial cells (TECs) was also reduced by the 14-day HU. Flow cytometric and RNA-sequencing analysis suggested that medullary TECs (mTECs) were preferentially reduced after HU. Moreover, immunohistochemical staining suggested that the 14-day HU caused a reduction of the mTECs expressing autoimmune regulator (Aire). Our data suggested that HU impacts both thymocytes and TECs. Consequently, these data imply that thymic T cell repertoire formation could be disturbed during spaceflight-like stress., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs., (© 2016 Akiyama et al.)

Background: Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model., Results: We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire(-/-) mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire(-/-) mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire(+/+) mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire(+/+) and Aire(-/-) mTECs but distinct from that of athymic cells and tissues., Conclusions: These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

Using an inducible gene expression system (Tet-ON system), the role of NGFI-A gene during the neuronal differentiation of PCI2 cells was examined. When NGFI-A was transiently over-expressed, no obvious effects on cell proliferation or neurite outgrowth were observed. Interestingly, however, NGFI-A over-expression resulted in significant retardation in NGF-induced neurite outgrowth. Similar suppressive effects were observed also on the v-K-ras-induced neurite outgrowth. These results raise the possibility that NGFI-A protein may play some negative role in NGF signaling.

Purpose: Selective gene expression in response to tumor hypoxia may provide new avenues, not only for radiotherapy and chemotherapy, but also for gene therapy. In this study, we have assessed the extent of hypoxia responsiveness of various DNA constructs by the luciferase assay to help design vectors suitable for cancer therapy., Materials and Methods: Reporter plasmids were constructed with fragments of the human vascular endothelial growth factor (VEGF) and the erythropoietin (Epo) genes encompassing the putative hypoxia-responsive elements (HRE) and the pGL3 promoter vector. Test plasmids and the control pRL-CMV plasmid were cotransfected into tumor cells by the calcium phosphate method. After 6 h hypoxic treatment, the reporter assay was performed., Results: The construct pGL3/VEGF containing the 385 bp fragment of the 5' flanking region in human VEGF gene showed significant increases in luciferase activity in response to hypoxia. The hypoxic/aerobic ratios were about 3-4, and 8-12 for murine and human tumor cells, respectively. Despite the very high degree of conservation among the HREs of mammalian VEGF genes, murine cells showed lower responsiveness than human cells. We next tested the construct pGL3/Epo containing the 150 bp fragment of the 3' flanking region in the Epo gene. Luciferase activity of pGL3/Epo was increased with hypoxia only in human cell lines. The insertion of 5 copies of the 35-bp fragments derived from the VEGF HREs and 32 bp of the E1b minimal promoter resulted in maximal enhancement of hypoxia responsiveness., Conclusions: The constructs with VEGF or Epo fragments containing HRE may be useful for inducing specific gene expression in hypoxic cells. Especially, the application of multiple copies of the HREs and an E1b minimal promoter appears to have the advantage of great improvement in hypoxia responsiveness.