Effect of RNF113A deficiency on oxidative stress-induced NRF2 pathway.

The ring finger protein 113A (RNF113A) serves as an E3 ubiquitin ligase and a subunit of the spliceosome. Mutations in the RNF113A gene are associated with X-linked trichothiodystrophy (TTD). However, the cellular roles of RNF113A remain largely unknown. In this study, we performed transcriptome profiling of RNF113A knockout (KO) HeLa cells using RNA sequencing and revealed the upregulation of NRF2 pathway-associated genes. Further analysis confirmed that the KO of RNF113A promotes nuclear localization of the NRF2 protein and elevates the mRNA levels of NRF2 target genes. RNF113A KO cells showed high levels of intracellular reactive oxygen species (ROS) and decreased resistance to cell death following H2O2 treatment. Additionally, RNF113A KO cells more sensitively formed stress granules (SGs) under arsenite-induced oxidative stress. Moreover, RNF113A KO cells exhibited a decrease in glutathione levels, which could be attributed to a reduction in GLUT1 expression levels, leading to decreased glucose uptake reactions and lower intracellular glucose levels. These alterations potentially caused a reduction in ROS scavenging activity. Taken together, our findings suggest that the loss of RNF113A promotes oxidative stress-mediated activation of the NRF2 pathway, providing novel insights into RNF113A-associated human diseases.

PAX4 loss of function increases diabetes risk by altering human pancreatic endocrine cell development.

The coding variant (p.Arg192His) in the transcription factor PAX4 is associated with an altered risk for type 2 diabetes (T2D) in East Asian populations. In mice, Pax4 is essential for beta cell formation but its role on human beta cell development and/or function is unknown. Participants carrying the PAX4 p.His192 allele exhibited decreased pancreatic beta cell function compared to homozygotes for the p.192Arg allele in a cross-sectional study in which we carried out an intravenous glucose tolerance test and an oral glucose tolerance test. In a pedigree of a patient with young onset diabetes, several members carry a newly identified p.Tyr186X allele. In the human beta cell model, EndoC-ßH1, PAX4 knockdown led to impaired insulin secretion, reduced total insulin content, and altered hormone gene expression. Deletion of PAX4 in human induced pluripotent stem cell (hiPSC)-derived islet-like cells resulted in derepression of alpha cell gene expression. In vitro differentiation of hiPSCs carrying PAX4 p.His192 and p.X186 risk alleles exhibited increased polyhormonal endocrine cell formation and reduced insulin content that can be reversed with gene correction. Together, we demonstrate the role of PAX4 in human endocrine cell development, beta cell function, and its contribution to T2D-risk.

In Vitro Expansion of Vδ1+ T Cells from Cord Blood by Using Artificial Antigen-Presenting Cells and Anti-CD3 Antibody.

γδ T cells have the potential for adoptive immunotherapy since they respond to bacteria, viruses, and tumors. However, these cells represent a small fraction of the peripheral T-cell pool and require activation and proliferation for clinical benefits. In cord blood, there are some γδ T cells, which exhibit a naïve phenotype, and mostly include Vδ1+ T cells. In this study, we investigated the effect of CD3 signaling on cord blood γδ T-cell proliferation using K562-based artificial antigen presenting cells expressing costimulatory molecules. There were significantly more Vδ1+ T cells in the group stimulated with anti-CD3 antibody than in the group without. In cultured Vδ1+ T cells, DNAM-1 and NKG2D were highly expressed, but NKp30 and NKp44 showed low expression. Among various target cells, Vδ1+ T cells showed the highest cytotoxicity against U937 cells, but Daudi and Raji cells were not susceptible to Vδ1+ T cells. The major cytokines secreted by Vδ1+ T cells responding to U937 cells were Granzyme B, IFN-γ, and sFasL. Cytotoxicity by Vδ1+ T cells correlated with the expression level of PVR and Nectin of DNAM-1 ligands on the surface of target cells. Compared to Vδ2+ T cells in peripheral blood, cord blood Vδ1+ T cells showed varying cytotoxicity patterns depending on the target cells. Here, we determined the ideal conditions for culturing cord blood Vδ1+ T cells by observing that Vδ1+ T cells were more sensitive to CD3 signals than other subtypes of γδ T cells in cord blood. Cultured cord blood Vδ1+ T cells recognized target cells through activating receptors and secreted numerous cytotoxic cytokines. These results are useful for the development of tumor immunotherapy based on γδ T cells.

Kazinol C from Broussonetia kazinoki stimulates autophagy via endoplasmic reticulum stress-mediated signaling.

Autophagy modulators are considered putative therapeutic targets because of the role of autophagy in cancer progression. Kazinol C, a 1,3-diphenylpropane from the plant Broussonetia kazinoki, has been shown to induce apoptosis in colon cancer cells through the activation of AMPK at high concentrations. In the present study, we found that Kazinol C induced autophagy through endoplasmic reticulum stress-mediated unfolded protein response signaling in several normal and cancer cell lines at low concentrations of Kazinol C that did not induce apoptosis. Kazinol C activated the transducers of unfolded protein response signaling, leading to target gene expression, LC3-II conversion, and TFEB nuclear translocation. Chemical inhibition of endoplasmic reticulum stress reduced LC3-II conversion. In addition, blockade of autophagy by knockout of Atg5 or treatment with 3-MA enhanced Kazinol C-induced apoptosis. In summary, we have uncovered Kazinol C as a novel autophagy inducer and confirmed the role of autophagy as a cellular stress protector.

Inhibition of autophagy sensitizes lignan-induced endoplasmic reticulum stress-mediated cell death.

Relationship between autophagy and endoplasmic reticulum (ER) stress and their application to treat cancer have been actively studied these days. Recently, a lignan [(-)-(2R, 3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from Alnus japonica has been found to reduce the viability of colon cancer cells. In this study, we have observed DFS-induced autophagy in a variety of cancer cell lines. In addition, DFS led to ER stress, based on the activation of unfolded protein response (UPR) transducers and an elevated expression of UPR target genes in prostate and colon cancer cells. Further investigation has shown that DFS triggered the activation of AMP-activated protein kinase (AMPK) signaling and nuclear translocation of transcription factor EB (TFEB). Furthermore, the cytotoxicity of DFS was potentiated by the co-treatment of autophagy inhibitor in these cancer cells. This study has provided a noble implication that the combination of DFS and autophagy inhibition exerts a synergistic effect in cancer treatment.