Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland

Therapeutic potential of a plant-made cholera toxin b subunit variant for the treatment of ulcerative colitis.

Cholera toxin B subunit (CTB) is a mucosal immunomodulatory protein that induces robust mucosal and systemic antibody responses. This well-known biological activity has been exploited in cholera prevention (as a component of Dukoral® vaccine) and vaccine development for decades. On the other hand, several studies have investigated CTB’s immunotherapeutic potential in the treatment of inflammatory diseases such as Crohn’s disease and asthma. Furthermore, we recently found that a plant-made variant of CTB (CTBp) could induce colon epithelial wound healing in mouse colitis models. In this thesis, it is revealed that the wound healing effects are unique to the plant-made variant, as it has an ER retention signal KDEL sequence that provides the protein with new functions. This was determined by investigating how the C-terminal KDEL sequence contributes to the protein’s wound healing activity in vivo, in vitro, and ex vivo. In a mouse model of dextran sodium sulfate (DSS)-induced colitis, CTBp, but not CTB, mitigated colitis as characterized by lower disease activity index and inflammation scores, colon shrinkage protection, blunted escalation of blood leukocyte levels, and observable histological epithelial restitution. In vitro, a Caco2 cell wound healing model revealed CTBp’s epithelial healing activity, intracellular retention, and unique signaling pathways that were reliant on the protein’s KDEL sequence. It was determined that, upon internalization of CTBp, the KDEL sequence enables ER colocalization and retention of the protein, leading to the activation of the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm of unfolded protein response and subsequent TGFβ signaling. Lastly, using a ulcerative colitis (UC) patient colon explants, CTBp’s therapeutic potential was evaluated, which demonstrated efficacy as manifested by the induction of TGFB gene expression, upregulation of wound healing pathways and presence of viable crypts in the mucosa. In summary, CTBp exhibits unique colon mucosal would healing effects that are mediated by its colocalization to the ER and subsequent activation of IRE1/XBP1 signaling in colon epithelial cells. Furthermore, the results presented herein provide implications for the unique therapeutic potential of CTBp that may address a significant unmet need in UC treatment

Mitochondrial Oxidative Stress Promotes Cardiac Remodeling in Myocardial Infarction through the Activation of Endoplasmic Reticulum Stress

We have evaluated cardiac function and fibrosis in infarcted male Wistar rats treated with MitoQ (50 mg/kg/day) or vehicle for 4 weeks. A cohort of patients admitted with a first episode of acute MI were also analyzed with cardiac magnetic resonance and T1 mapping during admission and at a 12-month follow-up. Infarcted animals presented cardiac hypertrophy and a reduction in the left ventricular ejection fraction (LVEF) and E- and A-waves (E/A) ratio when compared to controls. Myocardial infarction (MI) rats also showed cardiac fibrosis and endoplasmic reticulum (ER) stress activation. Binding immunoglobulin protein (BiP) levels, a marker of ER stress, were correlated with collagen I levels. MitoQ reduced oxidative stress and prevented all these changes without affecting the infarct size. The LVEF and E/A ratio in patients with MI were 57.6 ± 7.9% and 0.96 ± 0.34, respectively. No major changes in cardiac function, extracellular volume fraction (ECV), or LV mass were observed at follow-up. Interestingly, the myeloperoxidase (MPO) levels were associated with the ECV in basal conditions. BiP staining and collagen content were also higher in cardiac samples from autopsies of patients who had suffered an MI than in those who had died from other causes. These results show the interactions between mitochondrial oxidative stress and ER stress, which can result in the development of diffuse fibrosis in the context of MI

Magnesium Deprivation Potentiates Human Mesenchymal Stem Cell Transcriptional Remodeling

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications

A recombinant cholera toxin b subunit variant (CTB-KDEL) exhibits unique colon mucosal healing effects that have therapeutic implications for inflammatory bowel disease.

This dissertation uncovers the mechanism and explores the utility of a recombinant cholera toxin B subunit (CTB) variant containing a KDEL endoplasmic reticulum (ER) retention motif (CTB-KDEL) as a drug candidate for the treatment of inflammatory bowel disease (IBD). CTB is a mucosal immunomodulatory protein that induces robust mucosal and systemic antibody responses. This well-known biological activity has been exploited in cholera prevention (as a component of Dukoral® vaccine) and vaccine development for decades. On the other hand, several studies have investigated CTB’s immunotherapeutic potential in the treatment of inflammatory diseases such as Crohn’s disease and asthma. Here, we reveal that CTB-KDEL, in contrast to CTB, induces colon epithelial wound healing in colitis via the activation of an unfolded protein response in colon epithelial cells. Furthermore, we in a model of chronic DSS colitis we found that weekly oral administration of CTB-KDEL, dosed before or after the onset of chronic colitis, induced by repeated dextran sodium sulfate (DSS) exposure, could significantly reduce disease severity and signs of chronicity. To address the consequences of immunogenicity, mice (C57BL/6, C3H/HeJ, or Rag1-/- strains) were pre-exposed to CTB-KDEL then subjected to DSS colitis and CTB-KDEL treatment. Subsequently, we found that the immunogenicity of CTB-KDEL does not impede the protein’s mucosal healing efficacy in vivo. The results provide implications for a novel therapeutic approach for mucosal healing, a significant unmet need in IBD treatment

A plant-made cholera toxin B subunit enhances mucosal wound healing and protects against ulcerative colitis and colon cancer.

This dissertation describes the previously unidentified effects of a plant-produced recombinant cholera toxin B subunit (CTBp) on the gastrointestinal (GI) tract and its ability to protect against inflammation in a mouse model of colonic injury and ulcerative colitis (UC). To comprehensively analyze CTBp’s impacts on the GI tract, we employed global analysis methodologies based on multi-color flow cytometry to analyze immune cell populations in GI and systemic lymphatic compartments, gene expression microarray to decipher transcript-level changes in the colon and small intestine, and 16S rRNA sequencing to characterize fecal microbiota. Based on a drastic shift observed in the immune cell profile and gene expression pattern in the distal colon, we built a new working hypothesis that CTBp may enhance mucosal protection in the colon. To address this hypothesis, we used the Caco-2 human colonic cell line and the mouse dextran sulfate sodium (DSS) colitis model. After demonstrating the potential of CTBp as a mucosal healing and anti-colitic agent, the dissertation will be summarized and future directions discussed

Fibrosis, the Bad Actor in Cardiorenal Syndromes: Mechanisms Involved

Cardiorenal syndrome is a term that defines the complex bidirectional nature of the interaction between cardiac and renal disease. It is well established that patients with kidney disease have higher incidence of cardiovascular comorbidities and that renal dysfunction is a significant threat to the prognosis of patients with cardiac disease. Fibrosis is a common characteristic of organ injury progression that has been proposed not only as a marker but also as an important driver of the pathophysiology of cardiorenal syndromes. Due to the relevance of fibrosis, its study might give insight into the mechanisms and targets that could potentially be modulated to prevent fibrosis development. The aim of this review was to summarize some of the pathophysiological pathways involved in the fibrotic damage seen in cardiorenal syndromes, such as inflammation, oxidative stress and endoplasmic reticulum stress, which are known to be triggers and mediators of fibrosis

卵巣の病態における小胞体ストレス応答の意義の解明

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 南學 正臣, 東京大学准教授 永松 健, 東京大学講師 坊垣 昌彦, 東京大学講師 平田 陽一郎, 東京大学講師 山下 裕玄University of Tokyo(東京大学