Regulation of the selenoprotein SelS by glucose deprivation and endoplasmic reticulum stress - SelS is a novel glucose-regulated protein

SelS is a newly identified selenoprotein and its gene expression is up-regulated in the liver of Psammomys obesus after fasting. We have examined whether SelS is regulated by glucose deprivation and endoplasmic reticulum (ER) stress in HepG2 cells. Glucose deprivation and the ER stress inducers tunicamycin and thapsigargin increased SelS gene expression and protein content several-fold in parallel with glucose-regulated protein 78. The overexpression of SelS increased Min6 cell resistance to oxidative stress-induced toxicity. These results indicate that SelS is a novel member of the glucose-regulated protein family and its function is related to the regulation of cellular redox balance.     

Pro178 and Pro183 of Selenoprotein S Are Essential Residues for Interaction with p97(VCP) during Endoplasmic Reticulum-associated Degradation

During endoplasmic reticulum (ER)-associated degradation, p97(VCP) is recruited to the ER membrane through interactions with transmembrane proteins, such as selenoprotein S (SelS), selenoprotein K (SelK), hrd1, and gp78. SelS has a single-spanning transmembrane domain and protects cells from ER stress-induced apoptosis through interaction with p97(VCP). The cytosolic tail of SelS consists of a coiled-coil domain, a putative VCP-interacting motif (VIM), and an unpronounced glycine- and proline-rich secondary structure. To understand the regulatory mechanism of SelS during ER stress, we investigated the interaction of the protein with p97(VCP) using mouse neuroblastoma cells and human embryonic kidney 293 cells. The SelS expression level increased when ER stress was induced. In addition, the effect of ER stress was enhanced, and recruitment of p97(VCP) to the ER membrane was inhibited in SelS knockdown cells. The effect of SelS knockdown was rescued by ectopic expression of SelS U188C. p97(VCP) interacted with SelS U188C and was recruited to the ER membrane. The expression of SelS[ΔVIM], which is a VIM deletion mutant of SelS, also showed both a recovery effect and an interaction with p97(VCP) in cells. However, mutants in which the proline residue positions 178 or 183 of SelS were changed to alanine or were deleted did not interact with p97(VCP). The proline mutants did not rescue ER stress in SelS knockdown cells. These results suggest that both Pro¹⁷⁸ and Pro¹⁸³ of SelS play important roles in the translocation of p97(VCP) to the ER membrane and protect cells from ER stress.     

Influence of SelS gene silence on β-Mercaptoethanol-mediated endoplasmic reticulum stress and cell apoptosis in HepG2 cells

Selenoprotein S (SelS), a transmembrane selenoprotein, may be related to the response of endoplasmic reticulum (ER) stress. In this report, the influence of selenite supplementation and SelS gene silence on β-mercaptoethanol (β-ME)-mediated ER stress and cell apoptosis in HepG2 cells were examined. The results showed that SelS protein expression was markedly increased by 10 mM β-ME and 100 nM sodium selenite in HepG2 cells. GRP78 protein level was significantly increased after treatment with 10 mM β-ME in HepG2 cells, which suggested that β-ME was also an ER stress inducer. Meanwhile, β-ME (10 mM) was found to induce cell apoptosis, which was alleviated obviously when cells were pretreated with 100 nM selenite before exposure to β-ME. Moreover, the suppression of SelS gene by siRNA could aggravate HepG2 cell apoptosis induced by β-ME significantly. In conclusion, these results suggested that β-ME, also an ER stress agent, could induce cell apoptosis, and SelS may play an important role in protecting cells from apoptosis induced by ER stress in HepG2 cells.     

Selenoprotein S-dependent Selenoprotein K Binding to p97(VCP) Protein Is Essential for Endoplasmic Reticulum-associated Degradation

Cytosolic valosin-containing protein (p97(VCP)) is translocated to the ER membrane by binding to selenoprotein S (SelS), which is an ER membrane protein, during endoplasmic reticulum-associated degradation (ERAD). Selenoprotein K (SelK) is another known p97(VCP)-binding selenoprotein, and the expression of both SelS and SelK is increased under ER stress. To understand the regulatory mechanisms of SelS, SelK, and p97(VCP) during ERAD, the interaction of the selenoproteins with p97(VCP) was investigated using N2a cells and HEK293 cells. Both SelS and SelK co-precipitated with p97(VCP). However, the association between SelS and SelK did not occur in the absence of p97(VCP). SelS had the ability to recruit p97(VCP) to the ER membrane but SelK did not. The interaction between SelK and p97(VCP) did not occur in SelS knockdown cells, whereas SelS interacted with p97(VCP) in the presence or absence of SelK. These results suggest that p97(VCP) is first translocated to the ER membrane via its interaction with SelS, and then SelK associates with the complex on the ER membrane. Therefore, the interaction between SelK and p97(VCP) is SelS-dependent, and the resulting ERAD complex (SelS-p97(VCP)-SelK) plays an important role in ERAD and ER stress.     

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.     

Paneth cell degranulation and lysozyme secretion during acute equine alimentary laminitis

Summary The equine Paneth cell response to a shift in the microbial balance of the intestinal tract was studied by inducing an acute episode of alimentary laminitis in 6 mature ponies. The normal bacterial population of the gut was modified by administration of a carbohy-drate-rich ration. During acute laminitis a dramatic degranulation of the Paneth cells occurred in the intestinal glands throughout the duodenum, jejunum, and ileum. Bacteriocidal lysozyme, which was immunohistochemically identified as a component of the Paneth cell secretory granule, was evident in the glandular lumina and in degranulated Paneth cells. These results indicate that lysozyme is secreted by the equine Paneth cell in an apparent attempt to regulate the changing microbial population induced by carbohydrate overload of the gut. From these observations, it is suggested that the Paneth cell plays a role in the mucosal defense system of the equine intestinal tract.     

Paneth cell degranulation and lysozyme secretion during acute equine alimentary laminitis

The equine Paneth cell response to a shift in the microbial balance of the intestinal tract was studied by inducing an acute episode of alimentary laminitis in 6 mature ponies. The normal bacterial population of the gut was modified by administration of a carbohydrate-rich ration. During acute laminitis a dramatic degranulation of the Paneth cells occurred in the intestinal glands throughout the duodenum, jejunum, and ileum. Bacteriocidal lysozyme, which was immunohistochemically identified as a component of the Paneth cell secretory granule, was evident in the glandular lumina and in degranulated Paneth cells. These results indicate that lysozyme is secreted by the equine Paneth cell in an apparent attempt to regulate the changing microbial population induced by carbohydrate overload of the gut. From these observations, it is suggested that the Paneth cell plays a role in the mucosal defense system of the equine intestinal tract.     

Events at the host-microbial interface of the gastrointestinal tract. V. Paneth cell alpha-defensins in intestinal host defense

Host defense of the small intestine is mediated, in part, by antimicrobial peptides, including alpha-defensins. In the small intestine, Paneth cells, specialized secretory epithelial cells located at the base of the crypt invaginations lining the intestinal wall, produce alpha-defensins. The alpha-defensins are cysteine-rich cationic peptides with antibiotic activity against a wide range of bacteria and other microbes. Studies of transgenic and knockout mice have supported a pivotal role of Paneth cell alpha-defensins in protection from bacterial pathogens. New data suggest that deficient expression of Paneth cell alpha-defensins may contribute to the pathophysiology of Crohn's disease, a chronic inflammatory bowel disease.     

Gut commensal bacteria,Paneth cells and their relations to radiation enteropathy

In steady state, the intestinal epithelium forms an important part of the gut barrier to defend against luminal bacterial attack. However, the intestinal epithelium is compromised by ionizing irradiation due to its inherent self-renewing capacity. In this process, small intestinal bacterial overgrowth is a critical event that reciprocally alters the immune milieu. In other words, intestinal bacterial dysbiosis induces inflammation in response to intestinal injuries, thus influencing the repair process of irradiated lesions. In fact, it is accepted that commensal bacteria can generally enhance the host radiation sensitivity. To address the determination of radiation sensitivity, we hypothesize that Paneth cells press a critical “button” because these cells are central to intestinal health and disease by using their peptides, which are responsible for controlling stem cell development in the small intestine and luminal bacterial diversity. Herein, the most important question is whether Paneth cells alter their secretion profiles in the situation of ionizing irradiation. On this basis, the tolerance of Paneth cells to ionizing radiation and related mechanisms by which radiation affects Paneth cell survival and death will be discussed in this review. We hope that the relevant results will be helpful in developing new approaches against radiation enteropathy.     

Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.     

Microbial-host interactions specifically control the glycosylation pattern in intestinal mouse mucosa

The glycosylation of the intestinal cell layer is thought to control several key functions of the gut such as vectorial transports, defence against microbial agents or immunological processes. It has been assumed that the gut microflora may modulate the glycosylation pattern of the intestinal cell layer. However, there is no direct evidence for this regulatory process. The first goal of this work was to establish the germ-free mice intestinal glycosylation baseline using a histochemical approach and a panel of ten lectins with defined glycan specificities to tissue sections prepared from various cellular compartments of the small and large intestine. Using this baseline, we have studied the contribution of the gut microflora on the carbohydrate composition of glycoconjugates of intestinal cells by comparing the germ-free and conventional mice glycosylation patterns. Analysis of the germ-free mice intestinal glycosylation baseline revealed that the expression of glycans depends on the proximodistal gradient (small to large intestine) and on the cell lineage (absorptive, goblet, crypt, and Paneth cells), indicating that mice are able to create and maintain a strict topological and cell lineage-specific regulation of glycosyltransferase expression. By comparing germ-free and conventional mice, we find that the gut microflora specifically modulates the gut glycosylation pattern, quantitatively as well as qualitatively by changing the cellular and subcellular distribution of glycans. This is the first report in mice to directly demonstrate the critical contribution of microflora to intestinal glycosylation, a key characteristic of the gut.     

A key role for E-cadherin in intestinal homeostasis and Paneth cell maturation

Background

E-cadherin is a major component of adherens junctions. Impaired expression of E-cadherin in the small intestine and colon has been linked to a disturbed intestinal homeostasis and barrier function. Down-regulation of E-cadherin is associated with the pathogenesis of infections with enteropathogenic bacteria and Crohn''s disease.

Methods and Findings

To genetically clarify the function of E-cadherin in intestinal homeostasis and maintenance of the epithelial defense line, the Cdh1 gene was conditionally inactivated in the mouse intestinal epithelium. Inactivation of the Cdh1 gene in the small intestine and colon resulted in bloody diarrhea associated with enhanced apoptosis and cell shedding, causing life-threatening disease within 6 days. Loss of E-cadherin led cells migrate faster along the crypt-villus axis and perturbed cellular differentiation. Maturation and positioning of goblet cells and Paneth cells, the main cell lineage of the intestinal innate immune system, was severely disturbed. The expression of anti-bacterial cryptidins was reduced and mice showed a deficiency in clearing enteropathogenic bacteria from the intestinal lumen.

Conclusion

These results highlight the central function of E-cadherin in the maintenance of two components of the intestinal epithelial defense: E-cadherin is required for the proper function of the intestinal epithelial lining by providing mechanical integrity and is a prerequisite for the proper maturation of Paneth and goblet cells.     

Shiga toxin binding in normal and inflamed human intestinal mucosa

Shiga toxins are associated with haemolytic uraemic syndrome but human intestinal epithelium does not express the Gb3 receptor. We describe Gb3 expression and Shiga toxin binding in histologically normal intestine and demonstrate that the pattern is unaltered in inflammatory disease states. Gb3 expression and Shiga toxin binding were identified in Paneth cells in both normal and inflamed mucosae.     

Regulated production of the chemokine CCL28 in human colon epithelium

The chemokine CCL28 is constitutively expressed by epithelial cells at several mucosal sites and is thought to function as a homeostatic chemoattractant of subpopulations of T cells and IgA B cells and to mediate antimicrobial activity. We report herein on the regulation of CCL28 in human colon epithelium by the proinflammatory cytokine IL-1, bacterial flagellin, and n-butyrate, a product of microbial metabolism. In vivo, CCL28 was markedly increased in the epithelium of pathologically inflamed compared with normal human colon. Human colon and small intestinal xenografts were used to model human intestinal epithelium in vivo. Xenografts constitutively expressed little, if any, CCL28 mRNA or protein. After stimulation with the proinflammatory cytokine IL-1, CCL28 mRNA and protein were significantly increased in the epithelium of colon but not small intestinal xenografts, although both upregulated the expression of another prototypic chemokine, CXCL8, in response to the identical stimulus. In studies of CCL28 regulation using human colon epithelial cell lines, proinflammatory stimuli, including IL-1, bacterial flagellin, and bacterial infection, significantly upregulated CCL28 mRNA expression and protein production. In addition, CCL28 mRNA expression and protein secretion by those cells were significantly increased by the short-chain fatty acid n-butyrate, and IL-1- or flagellin-stimulated upregulation of CCL28 by colon epithelial cells was synergistically increased by pretreatment of cells with n-butyrate. Consistent with its upregulated expression by proinflammatory stimuli, CCL28 mRNA expression was attenuated by pharmacological inhibitors of NF-kappaB activation. These findings indicate that CCL28 functions as an "inflammatory" chemokine in human colon epithelium and suggest the notion that CCL28 may act to counterregulate colonic inflammation.     

Early Mucosal Sensing of SIV Infection by Paneth Cells Induces IL-1β Production and Initiates Gut Epithelial Disruption

HIV causes rapid CD4+ T cell depletion in the gut mucosa, resulting in immune deficiency and defects in the intestinal epithelial barrier. Breakdown in gut barrier integrity is linked to chronic inflammation and disease progression. However, the early effects of HIV on the gut epithelium, prior to the CD4+ T cell depletion, are not known. Further, the impact of early viral infection on mucosal responses to pathogenic and commensal microbes has not been investigated. We utilized the SIV model of AIDS to assess the earliest host-virus interactions and mechanisms of inflammation and dysfunction in the gut, prior to CD4+ T cell depletion. An intestinal loop model was used to interrogate the effects of SIV infection on gut mucosal immune sensing and response to pathogens and commensal bacteria in vivo. At 2.5 days post-SIV infection, low viral loads were detected in peripheral blood and gut mucosa without CD4+ T cell loss. However, immunohistological analysis revealed the disruption of the gut epithelium manifested by decreased expression and mislocalization of tight junction proteins. Correlating with epithelial disruption was a significant induction of IL-1β expression by Paneth cells, which were in close proximity to SIV-infected cells in the intestinal crypts. The IL-1β response preceded the induction of the antiviral interferon response. Despite the disruption of the gut epithelium, no aberrant responses to pathogenic or commensal bacteria were observed. In fact, inoculation of commensal Lactobacillus plantarum in intestinal loops led to rapid anti-inflammatory response and epithelial tight junction repair in SIV infected macaques. Thus, intestinal Paneth cells are the earliest responders to viral infection and induce gut inflammation through IL-1β signaling. Reversal of the IL-1β induced gut epithelial damage by Lactobacillus plantarum suggests synergistic host-commensal interactions during early viral infection and identify these mechanisms as potential targets for therapeutic intervention.     

Hepatic deficiency of selenoprotein S exacerbates hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), which are all complex metabolic disorders. Selenoprotein S (SelS) is an endoplasmic reticulum (ER) resident selenoprotein involved in regulating ER stress and has been found to participate in the occurrence and development of IR and T2DM. However, the potential role and mechanism of SelS in NAFLD remains unclear. Here, we analyzed SelS expression in the liver of high-fat diet (HFD)-fed mice and obese T2DM model (db/db) mice and generated hepatocyte-specific SelS knockout (SelS^H-KO) mice using the Cre-loxP system. We showed that hepatic SelS expression levels were significantly downregulated in HFD-fed mice and db/db mice. Hepatic SelS deficiency markedly increased ER stress markers in the liver and caused hepatic steatosis via increased fatty acid uptake and reduced fatty acid oxidation. Impaired insulin signaling was detected in the liver of SelS^H-KO mice with decreased phosphorylation levels of insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/Akt), which ultimately led to disturbed glucose homeostasis. Meanwhile, our results showed hepatic protein kinase Cɛ (PKCɛ) activation participated in the negative regulation of insulin signaling in SelS^H-KO mice. Moreover, the inhibitory effect of SelS on hepatic steatosis and IR was confirmed by SelS overexpression in primary hepatocytes in vitro. Thus, we conclude that hepatic SelS plays a key role in regulating hepatic lipid accumulation and insulin action, suggesting that SelS may be a potential intervention target for the prevention and treatment of NAFLD and T2DM.Subject terms: Metabolic syndrome, Obesity