Epithelial recognition and elimination against aberrant cells.

Epithelial cells, which are non-immune cells, not only function as a physical defence barrier but also continuously monitor and eliminate aberrant epithelial cells in their vicinity. In other words, it has become evident that epithelial cells possess immune cell-like functions. In fact, recent research has revealed that epithelial cells recognise the Major Histocompatibility Complex I (MHC-I) of aberrant cells as a mechanism for surveillance. This cellular defence mechanism of epithelial cells probably detects aberrant cells more promptly than the conventional immune response, making it a novel and primary biological defence. Furthermore, there is the potential for this new immune-like biological defence mechanism to establish innovative treatment for disease prevention, leading to increasing anticipation for its future medical applications. In this review, we aim to summarise the recognition and attack mechanisms of aberrant cells by epithelial cells in mammals, with a particular focus on the field of cancer. Additionally, we discuss the potential therapeutic applications of epithelial cell-based defence against cancer, including novel prophylactic treatment methods based on molecular mechanisms.

Cell competition in vertebrates - a key machinery for tissue homeostasis.

Cell competition is a process by which cells with different properties compete with each other for survival and space, and consequently suboptimal/abnormal cells are often eliminated from, in particular, epithelial tissues. In the last few years, cell competition studies have been developing at an explosive speed, and the molecular mechanisms of cell competition have been considerably revealed. For instance, upon cell competition, loser cells are eliminated from tissues via a variety of loser phenotypes, including apoptosis, cell differentiation, and cell death-independent extrusion. In addition, upstream regulatory mechanisms for the induction of these phenotypes have been elucidated. Furthermore, it has become evident that cell competition is involved in various physiological and pathological processes and thus is a crucial and indispensable homeostatic machinery that is required for embryonic development and prevention of diseases and ageing. Moreover, cell competition now has a profound impact on other research fields such as regenerative medicine. In this review, we will summarize the development of these recent studies, especially focusing on cell competition in vertebrates.

Revisiting the Mechanism of Electric Field Sensing in Graphene Devices.

Electric field sensing has various real-life applications, such as early prediction of lightning. In this study, we effectively used graphene as an electric field sensor that can detect both positive and negative electric fields. The response of the sensor is recorded as the change in drain current under the application of an electric field. In addition, by systematic analysis, we established the mechanism of the graphene electric field sensor, and it is found to be different from the previously proposed one. The mechanism relies on the transfer of electrons between graphene and the traps at the SiO2/graphene interface. While the direction of charge transfer depends on the polarity of the applied electric field, the amount of charge transferred depends on the magnitude of the electric field. Such a charge transfer changes the carrier concentration in the graphene channel, which is reflected as the change in drain current.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

Epithelial cells remove precancerous cells by cell competition via MHC class I-LILRB3 interaction.

Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.

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.

Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

[Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 1)].

Quantitative NMR (qNMR) has been developed as an absolute quantitation method to determine the purity or content of organic compounds including marker compounds in crude drugs. The "qNMR test" has been introduced into the crude-drug section of the Japanese Pharmacopoeia (JP) for determining the purity of reagents used for the assay in the JP. In Supplement II to the JP 17th edition published in June 2019, fifteen compounds adopted qNMR test were listed as the reagents for the assay. To establish the "qNMR test" in the crude drug section of the JP, there were several problems to be solved. Previously, we reported that the handling impurity signals from reference substances and targeted marker compounds, chemical shifts of reference substances, and peak unity of signals of targeted marker compounds are important factors to conduct qNMR measurements with intended accuracy. In this study, we investigated that the hygroscopicity of reagents could cause the changes in the compounds' purity depending on increasing their water content. Twenty-one standard products used for the crude-drug test in JP were examined by water sorption-desorption analysis, and ginsenosides and saikosaponins were found to be hygroscopic. To prepare a sample solution of saikosaponin b2 for qNMR analysis, samples need to be maintained for 18 h at 25°C and 76% relative humidity; further, samples need to be weighed at the same humidity for the qNMR analysis.

ZAK Inhibitor PLX4720 Promotes Extrusion of Transformed Cells via Cell Competition.

Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine.

The COX-2/PGE2 pathway suppresses apical elimination of RasV12-transformed cells from epithelia.

At the initial stage of carcinogenesis, when RasV12-transformed cells are surrounded by normal epithelial cells, RasV12 cells are apically extruded from epithelia through cell competition with the surrounding normal cells. In this study, we demonstrate that expression of cyclooxygenase (COX)-2 is upregulated in normal cells surrounding RasV12-transformed cells. Addition of COX inhibitor or COX-2-knockout promotes apical extrusion of RasV12 cells. Furthermore, production of Prostaglandin (PG) E2, a downstream prostanoid of COX-2, is elevated in normal cells surrounding RasV12 cells, and addition of PGE2 suppresses apical extrusion of RasV12 cells. In a cell competition mouse model, expression of COX-2 is elevated in pancreatic epithelia harbouring RasV12-exressing cells, and the COX inhibitor ibuprofen promotes apical extrusion of RasV12 cells. Moreover, caerulein-induced chronic inflammation substantially suppresses apical elimination of RasV12 cells. These results indicate that intrinsically or extrinsically mediated inflammation can promote tumour initiation by diminishing cell competition between normal and transformed cells.

Determination of perillaldehyde in perilla herbs using relative molar sensitivity to single-reference diphenyl sulfone.

Perillaldehyde (PRL) is one of the essential oil components derived from perilla plants (Perilla frutescens Britton) and is a characteristic compound of the traditional medicine "perilla herb ()" listed in the The Japanese Pharmacopoeia, 17th edition (JP17). HPLC using an analytical standard of PRL has been used to quantitatively determine the PRL content in perilla herb. However, PRL reagents have been reported to decompose easily. In this study, we utilized an alternative quantitative method using on a single reference with relative molar sensitivity (RMS) based on the results of experiments performed in two laboratories. It was possible to calculate the exact RMS using an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/photodiode array (PDA) detector (or an HPLC/variable-wavelength detector [VWD]). Using the RMS of PRL to the single-reference compound diphenyl sulfone (DFS), which is an inexpensive and stable compound, the PRL content in the perilla herb could be determined using HPLC/PDA or HPLC/VWD without the need for the analytical standard of PRL. There was no significant difference between the PRL contents of perilla herb determined using the method employing the single-reference DFS with RMS and using the JP17 assay, the calibration curve of which was generated using the analytical standard of PRL with adjusted purity measured by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative assays of perilla herb and can be an alternative method for the current assay method defined in the JP17.

Accumulation of the myosin-II-spectrin complex plays a positive role in apical extrusion of Src-transformed epithelial cells.

At the initial stage of carcinogenesis, transformation occurs in single cells within the epithelium. Recent studies have revealed that the newly emerging transformed cells are often apically eliminated from epithelial tissues. However, the underlying molecular mechanisms of this cancer preventive phenomenon still remain elusive. In this study, we first demonstrate that myosin-II accumulates in Src-transformed cells when they are surrounded by normal epithelial cells. Knock-down of the heavy chains of myosin-II substantially diminishes apical extrusion of Src cells, suggesting that accumulated myosin-II positively regulates the apical elimination of transformed cells. Furthermore, we have identified ß-spectrin as a myosin-II-binding protein under the coculture of normal and Src-transformed epithelial cells. ß-spectrin is also accumulated in Src cells that are surrounded by normal cells, and the ß-spectrin accumulation is regulated by myosin-II. Moreover, knock-down of ß-spectrin significantly suppresses apical extrusion of Src cells. Collectively, these results indicate that accumulation of the myosin-II-spectrin complex plays a positive role in apical extrusion of Src-transformed epithelial cells. Further elucidation of the molecular mechanisms of apical extrusion would lead to the establishment of a novel type of cancer preventive medicine.

The paxillin-plectin-EPLIN complex promotes apical elimination of RasV12-transformed cells by modulating HDAC6-regulated tubulin acetylation.

Recent studies have revealed that newly emerging RasV12-transformed cells are often apically extruded from the epithelial layer. During this cancer preventive process, cytoskeletal proteins plectin and Epithelial Protein Lost In Neoplasm (EPLIN) are accumulated in RasV12 cells that are surrounded by normal cells, which positively regulate the apical elimination of transformed cells. However, the downstream regulators of the plectin-EPLIN complex remain to be identified. In this study, we have found that paxillin binds to EPLIN specifically in the mix culture of normal and RasV12-transformed cells. In addition, paxillin is accumulated in RasV12 cells surrounded by normal cells. Paxillin, plectin and EPLIN mutually influence their non-cell-autonomous accumulation, and paxillin plays a crucial role in apical extrusion of RasV12 cells. We also demonstrate that in RasV12 cells surrounded by normal cells, acetylated tubulin is accumulated. Furthermore, acetylation of tubulin is promoted by paxillin that suppresses the activity of histone deacetylase (HDAC) 6. Collectively, these results indicate that in concert with plectin and EPLIN, paxillin positively regulates apical extrusion of RasV12-transformed cells by promoting microtubule acetylation. This study shed light on the unexplored events occurring at the initial stage of carcinogenesis and would potentially lead to a novel type of cancer preventive medicine.

Cell competition in mammals - novel homeostatic machinery for embryonic development and cancer prevention.

In the multi-cellular community, cells with different properties often compete with each other for survival and space. This process is named cell competition and was originally discovered in Drosophila. Recent studies have revealed that comparable phenomena also occur in mammals under various physiological and pathological conditions. Within the epithelium, normal cells often recognize the presence of the neighboring transformed cells and actively eliminate them from the epithelium; a process termed EDAC (Epithelial Defense Against Cancer). Furthermore, physical force can play a crucial role in the intercellular recognition and elimination of loser cells during cell competition. Further studies are expected to reveal a variety of roles of cell competition in embryonic development and human diseases.

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease.

Current therapies for autoimmune diseases rely on traditional immunosuppressive medications that expose patients to an increased risk of opportunistic infections and other complications. Immunoregulatory interventions that act prophylactically or therapeutically to induce antigen-specific tolerance might overcome these obstacles. Here we use the transpeptidase sortase to covalently attach disease-associated autoantigens to genetically engineered and to unmodified red blood cells as a means of inducing antigen-specific tolerance. This approach blunts the contribution to immunity of major subsets of immune effector cells (B cells, CD4+ and CD8+ T cells) in an antigen-specific manner. Transfusion of red blood cells expressing self-antigen epitopes can alleviate and even prevent signs of disease in experimental autoimmune encephalomyelitis, as well as maintain normoglycemia in a mouse model of type 1 diabetes.

Plectin is a novel regulator for apical extrusion of RasV12-transformed cells.

Several lines of evidence have revealed that newly emerging transformed cells are often eliminated from the epithelium, though the underlying molecular mechanisms of this cancer preventive phenomenon still remain elusive. In this study, using mammalian cell culture systems we have identified plectin, a versatile cytoskeletal linker protein, as a novel regulator for apical extrusion of RasV12-transformed cells. Plectin is accumulated in RasV12 cells when they are surrounded by normal epithelial cells. Similarly, cytoskeletal proteins tubulin, keratin, and Epithelial Protein Lost In Neoplasm (EPLIN) are also accumulated in the transformed cells surrounded by normal cells. Knockdown or functional disruption of one of these molecules diminishes the accumulation of the others, indicating that the accumulation process of the individual protein mutually depends on each other. Furthermore, plectin-knockdown attenuates caveolin-1 (Cav-1) enrichment and PKA activity in RasV12 cells and profoundly suppresses the apical extrusion. These results indicate that the plectin-microtubules-EPLIN complex positively regulates apical elimination of RasV12-transformed cells from the epithelium in a coordinated fashion. Further development of this study would open a new avenue for cancer preventive medicine.