Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

Basal extrusion of single-oncogenic mutant cells induces dome-like structures with altered microenvironments.

At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

Clonogenic Culture of Mouse Thymic Epithelial Cells.

The thymus plays an essential role in the development and selection of T cells by providing a unique microenvironment that is mainly composed of thymic epithelial cells (TECs). We previously identified stem cells of medullary TECs (mTECs) that are crucial for central tolerance induction using a novel clonogenic culture system. We also found that medullary thymic epithelial stem cells (mTESCs) maintain life-long mTECs regeneration and central T cell self-tolerance in mouse models. The clonogenic efficiency of TECs in vitro is highly correlated to the TEC reconstitution activity in vivo. Here, we describe the clonogenic culture system to evaluate the self-renewing activity of TESCs. The colonies are derived from TESCs, are visualized and quantified by rhodamine-B staining on a feeder layer, and can be passaged in vitro. Thus, our system enables quantitative evaluation of TESC activity and is useful for dissecting the mechanisms that regulate TESC activity in physiological aging as well as in various clinical settings.

An improved clonogenic culture method for thymic epithelial cells.

A clonogenic assay system for thymic epithelial cells (TECs) is of crucial importance for identifying thymic epithelial stem and/or progenitor cells, evaluating their activities, and understanding the mechanisms of thymic involution. However, current systems are not sufficiently sensitive at detecting and quantifying TEC colonies from the adult thymus. Here, we optimized the culture condition to detect visible colonies from adult TECs by modifying our previous culture methods. Epidermal growth factor and leukemia inhibitory factor significantly enhanced the colony-forming efficiency of total TECs from embryo as well as adult mice when added 3 days after plating. Importantly, characteristics of the TEC colonies formed by the improved condition were almost equivalent to those by the original culture condition with respect to self-renewal and the expression of cell surface markers and intracellular keratins. Furthermore, the colonies derived from total TECs showed immature phenotypes and generated both mature cortical TECs and medullary TECs upon implantation in vivo. These data indicate a more sensitive clonogenic assay system for TECs was established and suggest the improved culture condition supports the colony formation of stem/progenitor cells for cTECs, mTECs and/or bipotent TECs.

Physiologic Thymic Involution Underlies Age-Dependent Accumulation of Senescence-Associated CD4+ T Cells.

Immune aging may underlie various aging-related disorders, including diminished resistance to infection, chronic inflammatory disorders, and autoimmunity. PD-1+ and CD153+ CD44high CD4+ T cells with features of cellular senescence, termed senescence-associated T (SA-T) cells, increasingly accumulate with age and may play a role in the immune aging phenotype. In this article, we demonstrate that, compared with young mice, the aged mouse environment is highly permissive for spontaneous proliferation of transferred naive CD4+ T cells, and it drives their transition to PD-1+ and CD153+ CD44high CD4+ T cells after extensive cell divisions. CD4+ T cells with essentially the same features as SA-T cells in aged mice are also generated from naive CD4+ T cells after extensive cell divisions under severe T-lymphopenic conditions by gamma irradiation or in developmental T cell defect, often in association with spontaneous germinal centers, as seen in aged mice. The increase in SA-T cells is significantly enhanced after thymectomy at the young adult stage, along with accelerated T cell homeostatic proliferation, whereas embryonic thymus implantation in the late adult stage markedly restricts the homeostatic proliferation of naive CD4+ T cells in the host and delays the increase in SA-T cells. Our results suggest that reduced T cell output due to physiologic thymic involution underlies the age-dependent accumulation of SA-T cells as a result of increasing homeostatic proliferation of naive CD4+ T cells. SA-T cells may provide a suitable biomarker of immune aging, as well as a potential target for controlling aging-related disorders.

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution.

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.

Relb acts downstream of medullary thymic epithelial stem cells and is essential for the emergence of RANK(+) medullary epithelial progenitors.

Thymic epithelial cells (TECs) provide essential signals for αßT-cell development, and medullary TECs (mTECs) control T-cell tolerance through both negative selection and Foxp3(+) regulatory T (Treg) cell development. Although heterogeneity within the mTEC compartment is well studied, the molecular regulators of specific stages of mTEC development are still poorly understood. Given the importance of the RANK-RANKL axis in thymus medulla formation, we have used RANK Venus reporter mice to analyze the ontogeny of RANK(+) TECs during development and correlated RANK expression with mTEC stem cells defined by SSEA-1. In addition, we have investigated how requirements for the key regulators Foxn1 and Relb map to specific stages of mTEC development. Here, we show SSEA-1(+) mTEC stem cells emerge prior to RANK expression and are present in both nude and Relb(-/-) mice, providing direct evidence that mTEC lineage specification occurs independently of Foxn1 and Relb. In contrast, we show that Relb is necessary for the effective production of downstream RANK(+) mTEC progenitors. Collectively, our work defines stage-specific requirements for critical TEC regulators during medulla development, including the timing of Relb dependency, and provides new information on mechanisms controlling mTEC specification.

Medullary thymic epithelial stem cells maintain a functional thymus to ensure lifelong central T cell tolerance.

Medullary thymic epithelial cells (mTECs) are crucial for central T cell self-tolerance. Although progenitors of mTECs have been demonstrated in thymic organogenesis, the mechanism for postnatal mTEC maintenance remains elusive. We demonstrate that implantation of embryonic TECs expressing claudin-3 and claudin-4 (Cld3,4) in a medulla-defective thymic microenvironment restores medulla formation and suppresses multiorgan autoimmunity throughout life. A minor SSEA-1(+) fraction within the embryonic Cld3,4(hi) TECs contained self-renewable clonogenic TECs, capable of preferentially generating mature mTECs in vivo. Adult SSEA-1(+)Cld3,4(hi) TECs retained mTEC reconstitution potential, although the activity decreased. The clonogenicity of TECs also declined rapidly after birth in wild-type mice, whereas it persisted in Rag2(?/?) adult mice with defective thymopoiesis. The results suggest that unipotent mTEC-restricted stem cells that develop in the embryo have the capacity to functionally reconstitute the thymic medulla long-term, thus ensuring lifelong central T cell self-tolerance.

Lymphocyte-stromal cell interaction induces IL-7 expression by interferon regulatory factors.

The interaction between lymphocytes and stromal cells plays important roles in coordinated development of early lymphocytes. IL-7 is an essential cytokine for early lymphocyte development produced by stromal cells in the thymus and bone marrow. Although IL-7 is induced by interaction of early lymphocytes and stromal cells, its molecular basis is still unknown. To address this question, we employed co-culture system with an IL-7-dependent pre-B cell line, DW34, and a thymic stromal cell line, TSt-4. Co-culture with DW34 cells enhanced the levels of IL-7 transcripts in TSt-4 cells. Interestingly, the co-culture also induced transcripts of IFN-α and IFN-ß but not of IFN-γ. In addition, exogenous IFN-ß stimulation increased the levels of IL-7 transcripts in TSt-4 cells. Next, to elucidate the molecular mechanism of IL-7 induction, we analyzed the IL-7 promoter activity by reporter assay. The IL-7 promoter showed specific transcriptional activity in TSt-4 cells. An interferon-stimulated response element (ISRE) in the IL-7 promoter was essential for the induction of IL-7 transcription by both co-culture and IFN-ß stimulation. Finally, overexpression of wild-type and dominant-negative forms of interferon regulatory factors (IRFs) activated and repressed, respectively, the IL-7 promoter in TSt-4 cells. Collectively, these results suggested that IRFs activated by lymphocyte adhesion induce IL-7 transcription through ISRE in stromal cells and that type I IFNs may be involved in the activation of IRFs. Thus, this study implied a physiological function of the IFN/IRF signal during lymphocyte development.

Increased c-Myc activity and DNA damage in hematopoietic progenitors precede myeloproliferative disease in Spa-1-deficiency.

Mice deficient for Spa-1 encoding Rap GTPase-activating protein develop myeloproliferative disorder (MPD) of late onset with frequent blast crises. The mechanisms for MPD development as well as the reasons for long latency, however, remain elusive. We demonstrate here that preleukemic, disease-free Spa-1(-/-) mice show reduced steady-state hematopoiesis and attenuated resistance to whole body γ-ray irradiation, which are attributable to the sustained p53 response in hematopoietic progenitor cells (HPCs). Preleukemic Spa-1(-/-) HPCs show c-Myc overexpression with increased p19Arf as well as enhanced γH2AX expression with activation of Atm/Chk pathway. We also show that deregulated Rap signaling in the absence of Spa-1 enhances post-transcriptional c-Myc stability and induces DNA damage in a p38MAPK-dependent manner, leading to p53 activation. Genetic studies indicate that the introduction of p53(+/-) and p53(-/-) mutations in Spa-1(-/-) mice results in the acceleration of typical MPD and rapid development of blastic leukemia, respectively. These results suggest that increased c-Myc expression and DNA damage in HPCs precede MPD development in Spa-1(-/-) mice, and the resulting p53 response functions as a barrier for the onset of MPD and blast crises progression.