Shigeru Oshima

Golgi membrane-associated degradation (GOMED) is a process that leading to the degradation of proteins that have passed through the trans-Golgi membranes upon Golgi stress. GOMED is morphologically similar to autophagy, but the substrates degraded are different, and they thus have different biological roles. Although the substrate recognition mechanism of autophagy has been clarified in detail, that of GOMED is completely unknown. Here we report that GOMED degrades its substrate proteins selectively via optineurin (OPTN), as we found that the degradation of GOMED substrates is s`uppressed by the loss of OPTN. OPTN binds to K33 polyubiquitin-tagged proteins that have passed through the Golgi, which are then incorporated into GOMED structures for eventual degradation. In vivo, GOMED is known to be involved in the removal of mitochondria from erythrocytes, and in Optn-deficient mice, mitochondria are not degraded by GOMED, resulting in the appearance of erythrocytes containing mitochondria. These findings provide insight into the substrate recognition mechanism of GOMED.

The intestinal mucosa harbors diverse lymphocyte populations, including double negative CD4^–CD8αβ^–TCRαβ⁺ T (DNT) cells, in the intraepithelial compartment and the lamina propria. Here we report that DNT cells in mouse small intestines are motile and reach across the epithelial barrier to capture luminal antigens (Ags). DNT cells then migrate to mesenteric lymph nodes (MLN) and upregulate MHC-II, as evidenced by a sizeable fraction of mouse DNT cells in Peyer’s patches (PP) and MLN expressing MHC-II but little or no co-stimulatory molecules. Functionally, the presentation of intestinal antigens by DNT cells tolerizes antigen-specific naive CD4⁺ T cells, with this tolerization reversed by conditional ablation of MHC-II in T cells, thereby rendering mutant mice hypersusceptible to intestinal inflammation. Intriguingly, intestinal T cells in patients with Crohn’s disease also express lower levels of HLA-DR than those in healthy controls. Our findings thus potentially implicate MHC-II⁺ DNT cells in intestinal immune homeostasis and pathogenesis of inflammatory bowel disease.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α‐related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2‐Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX‐2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20‐deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX‐2 cells also induced excessive chemokine expression, mimicking A20‐deficient HSCs. A20 overexpression suppressed chemokine expression in LX‐2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20‐silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1–JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1–JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1–JNK pathway for the regulation of inflammation in chronic hepatitis.

Aims/hypothesis Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. Methods We established the mouse model, ‘Gcg-Timer’, in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. Results Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. Conclusions/interpretation Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. Data availability Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090. Graphical Abstract