ERdj4 Expression Research Articles

C. sakazakii activates AIM2 pathway accompanying with excessive ER stress response in mammalian mammary gland epithelium.

Bovine mastitis is a common inflammatory disease caused by various factors. The main factor of mastitis is pathogenic microorganism infection, such as Staphylococcus aureus, Escherichia coli, and Streptococcus. Cronobacter sakazakii (C. sakazakii) is a newly discovered pathogenic bacteria in milk products, which seriously threat human health in recent years. At present, it has not been reported that the pathogenesis of mastitis is caused by C. sakazakii. This study investigated the inflammation of mammary gland epithelium, which was induced by C. sakazakii for the first time. We focused on bacterial isolation, histological observation, AIM2 inflammasome pathways, endoplasmic reticulum stress, and apoptosis. The results showed that C. sakazakii-induced inflammation caused damage of tissue, significantly increased the production of pro-inflammatory cytokines (including TNF-α, IL-1β, and IL-6), activated the AIM2 inflammasome pathway (increased the expression of AIM2 and cleaved IL-1β), and induced endoplasmic reticulum stress (increased the expression of ERdj4, Chop, Grp78) and apoptosis (increased the ratio of Bax/Bcl-2, a marker of apoptosis). In conclusion, it is suggested that it maybe inhibite AIM2 inflammasome pathways and alleviate endoplasmic reticulum stress (ER stress) against the C. sakazakii-induced inflammation.

Apoptosis induction associated with the ER stress response through up-regulation of JNK in HeLa cells by gambogic acid.

BackgroundGambogic acid (GA) was extracted from the dried yellow resin of gamboge (Garcinia hanburyi) which is traditionally used as a coloring material for painting and cloth dying. Gamboge has been also used as a folk medicine for an internal purgative and externally infected wound. We focused on the mechanisms of apoptosis induction by GA through the unfold protein response (ER stress) in HeLa cells.MethodsThe cytotoxic effect of GA against HeLa cells was determined by trypan blue exclusion assay. Markers of ER stress such as XBP-1, GRP78, CHOP, GADD34 and ERdj4 were analyzed by RT-PCR and Real-time RT-PCR. Cell morphological changes and apoptotic proteins were performed by Hoechst33342 staining and Western blotting technique.ResultsOur results indicated a time- and dose-dependent decrease of cell viability by GA. The ER stress induction is determined by the up-regulation of spliced XBP1 mRNA and activated GRP78, CHOP, GADD34 and ERdj4 expression. GA also induced cell morphological changes such as nuclear condensation, membrane blebbing and apoptotic body in Hela cells. Apoptosis cell death detected by increased DR5, caspase-8, −9, and −3 expression as well as increased cleaved-PARP, while decreased Bcl-2 upon GA treatment. In addition, phosphorylated JNK was up-regulated but phosphorylated ERK was down-regulated after exposure to GA.ConclusionsThese results suggest that GA induce apoptosis associated with the ER stress response through up-regulation of p-JNK and down-regulation of p-ERK in HeLa cells.

Endoplasmic Reticulum Stress Is Implicated in Intestinal Failure-Associated Liver Disease.

Intestinal failure-associated liver disease (IFALD) is the most serious consequence of long-term parenteral nutrition for intestinal failure. Little is known about the pathogenesis of IFALD, although many of the risk factors are also linked to endoplasmic reticulum stress (ERS). We propose that ERS may have a role in the development of IFALD. Archived liver tissue from patients with early and late IFALD, as well as from normal controls, was used for RNA extraction and immunohistochemistry to demonstrate the presence of ERS markers. Mean relative RNA levels of glucose regulatory protein 78 in normal liver (n = 3), early IFALD (n = 15), and late IFALD (n = 5) were 0.5, 37.86, and 212.11, respectively. Mean relative expression of ERDj4 (ER DnaJ homologue 4, a downstream ERS effector) in normal liver, early IFALD, and late IFALD was 5.51, 216.68, and 213.22, respectively. The degree of splicing of X-box binding protein 1 in IFALD compared with normal liver was significantly higher (mean, 0.0779 normal, 0.102 early IFALD, 0.2063 late IFALD). This is the first description of ERS in IFALD. This information may open up new therapeutic possibilities in the form of chemical chaperones known to ameliorate ERS.

The BiP cochaperone ERdj4 is required for B cell development and function.

ERdj4 is a BiP cochaperone regulated by the unfolded protein response to facilitate degradation of unfolded and/or misfolded proteins in the endoplasmic reticulum. As the unfolded protein response plays a critical role in B cell maturation and antibody production, ERdj4 gene trap mice were generated to determine if this chaperone was required for B cell homeostasis. Homozygosity for the trapped allele resulted in hypomorphic expression of ERdj4 in bone marrow cells and abnormal development of hematopoietic lineages in the bone marrow. The number of myeloid cells was increased, while the number of erythroid and B lymphoid cells was reduced in ERdj4 gene trap mice compared to controls. An intrinsic B cell defect was identified that decreased survival of B cell precursors including large and small pre-B, and immature B cells. Consistent with impaired B lymphopoiesis, the number of mature follicular B cells was reduced in both the bone marrow and spleen of ERdj4 gene trap mice. Paradoxically, unchallenged ERdj4 gene trap mice showed non-specific hypergammaglobulinemia and gene trap B cells exhibited increased proliferation, survival and isotype switching in response to LPS stimulation. Although ERdj4 gene trap mice responded normally to T cell-independent antigen, they failed to mount a specific antibody response to T cell-dependent antigen in vivo. Collectively, these findings demonstrate that the chaperone activity of ERdj4 is required for survival of B cell progenitors and normal antibody production.

Deficiency of the BiP cochaperone ERdj4 causes constitutive endoplasmic reticulum stress and metabolic defects

Endoplasmic reticulum-localized DnaJ 4 (ERdj4) is an immunoglobulin-binding protein (BiP) cochaperone and component of the endoplasmic reticulum-associated degradation (ERAD) pathway that functions to remove unfolded/misfolded substrates from the ER lumen under conditions of ER stress. To elucidate the function of ERdj4 in vivo, we disrupted the ERdj4 locus using gene trap (GT) mutagenesis, leading to hypomorphic expression of ERdj4 in mice homozygous for the trapped allele (ERdj4(GT/GT)). Approximately half of ERdj4(GT/GT) mice died perinatally associated with fetal growth restriction, reduced hepatic glycogen stores, and hypoglycemia. Surviving adult mice exhibited evidence of constitutive ER stress in multiple cells/tissues, including fibroblasts, lung, kidney, salivary gland, and pancreas. Elevated ER stress in pancreatic β cells of ERdj4(GT/GT) mice was associated with β cell loss, hypoinsulinemia, and glucose intolerance. Collectively these results suggest an important role for ERdj4 in maintaining ER homeostasis during normal fetal growth and postnatal adaptation to metabolic stress.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

Hypomorphic ERdj4 Expression Induces ER Stress and Compromises Insulin Secretion in Beta Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A Defect in the Bone Marrow Microenvironment of ERdj4GT/GT Mice Impacts B Cell Development

Abstract 3463ERdj4 is a critical component of the unfolded protein response (UPR) that facilitates the removal of unfolded/misfolded substrates from the ER lumen for proteasomal degradation. Since previous studies have linked the UPR to B lymphopoiesis [Reimold, A.M. et al., Nature, 2001; Zhang, K. et al., JCI, 2005], we hypothesized that a deficiency in ERdj4 would impact B cell development. The ERdj4 allele was disrupted by a gene trap leading to hypomorphic expression of ERdj4 in mice homozygous for the gene trap allele (ERdj4GT/GT). Pro-B, pre-B, immature, and mature B cell populations were significantly reduced in the bone marrow of ERdj4GT/GT mice, which was associated with an increase in pro-B cell death. ERdj4GT/GT donor cells transplanted into ERdj4+/+ recipients rescued all stages of B cell development, implicating a defect in the bone marrow microenvironment. Osteoblasts support B cell development through VCAM-1, SDF-1 and IL-7 signaling [Visnjic, D. et al., Blood, 2004; Zhu, J. et al., Blood, 2007]; coincidently, the UPR is essential for osteoblast differentiation [Tohmonda, T. et al., EMBO, 2011; Wei, J. et al., J Cell Physiol, 2008]. ERdj4 deficiency in primary bone cells was associated with increased ER stress. Osteoblast-specific markers and the B cell growth factor SDF-1 was also significantly reduced in primary ERdj4GT/GT bone cells. Importantly, osteoblasts were reduced in femur tissue sections of ERdj4GT/GT mice. Collectively, these data suggest that the loss of ERdj4 in osteoblasts is associated with a defect in B lymphopoiesis. This research was supported by the NHLBI (HL103923 and HL086492). Disclosures:No relevant conflicts of interest to declare.

Ankylosing spondylitis macrophage production of higher levels of interleukin-23 in response to lipopolysaccharide without induction of a significant unfolded protein response

Previous studies of the HLA-B27-transgenic rat model of ankylosing spondylitis (AS) suggested that macrophages develop an intracellular stress response called the unfolded protein response (UPR) and, as a result, secrete increased amounts of cytokines in response to Toll-like receptor agonists such as lipopolysaccharide (LPS). Our objective was to determine whether macrophages from AS patients also undergo a UPR and secrete increased cytokines/chemokines in response to LPS. Peripheral blood monocytes isolated from 10 AS patients and 10 healthy controls were differentiated in vitro with macrophage colony-stimulating factor. Select samples were treated with interferon-γ (IFNγ) to up-regulate class I major histocompatibility complex (HLA-B) expression prior to stimulation with LPS for either 3 hours (for RNA) or 8-24 hours (for supernatant). UPR induction was assessed by measuring the expression of messenger RNA for ERdj4, BiP, and CCAAT/enhancer binding protein homologous protein 10 (CHOP). Although IFNγ treatment up-regulated HLA-B expression (2-fold; P < 0.0001), neither IFNγ nor LPS substantially enhanced BiP or CHOP expression (<1.3-fold). ERdj4 expression increased weakly, but not significantly, in AS samples treated with IFNγ plus LPS (2.2-fold; P = 0.31). In response to LPS, AS macrophages secreted more CXCL9, interleukin-10 (IL-10), IL-12p70, IL-23, and tumor necrosis factor α than did control macrophages (P ≤ 0.025). The most striking difference was observed for IL-23 (median 265 pg/ml in AS patients versus 9 pg/ml in controls; P = 0.0007). We did not detect significant differences in IL-6, IL-8, or IFNβ production. The greater production of IL-23 by AS patient macrophages in response to LPS provides further support for the development of Th17/IL-23-directed therapy. Since significant UPR induction was not detected in AS patient macrophages, the relationship between UPR and inflammatory cytokine production remains unclear.

Abstract B150: Toyocamycin inhibits ER stress-induced XBP1 mRNA splicing through IRE1α activation

Abstract Poorly vascularized solid-tumor cells encounter a range of cytotoxic conditions such as hypoxia, nutrient deprivation and pH changes. These cytotoxic conditions in solid tumor are considered to cause ER stress. There is now abundant evidence to suggest that ER stress response is activated in various tumors to protect tumor cells from cytotoxic condition-induced apoptosis. XBP1 is considered to be a critical transcription factor of this process and required for tumor survival. Therefore, small molecule inhibitors of XBP1 activation would provide a new type of therapeutic strategy for cancer treatment. In this study, we screened small molecule inhibitors of ER stress-induced XBP1 activation from culture broth of microorganisms and we identified toyocamycin (H, Nishimura et al.: J. Antibiot., 9, 60 (1956)). Thus, we investigated the mechanism by which toyocamycin inhibited ER stress-induced XBP1 activation. XBP1 is activated by IRE1α-mediated unconventional splicing of XBP1 mRNA, which resulted in creating a translational frame shift to produce an active XBP1 transcription factor. In HeLa cells, toyocamycin inhibited the transcriptional activity of XBP1, since toyocamycin suppressed tunicamycin or 2-deoxy-glucose, a well known ER stressor, -induced GRP78, EDEM, and ERdj4 expression that are known to be transcriptionaly regulated by active XBP1. Furthermore, toyocamycin inhibited ER stress-induced endogenous XBP1 mRNA splicing evaluated by RT-PCR. Although toyocamycin is known as RNA synthesis inhibitor, toyocamycin could not decrease the expression level of XBP1 mRNA. Thus, the inhibitory activity of toyocamycin against XBP1 activation is not due to the RNA synthesis inhibition. On the other hand, toyocamycin was unable to suppress IRE1α-mediated XBP1 mRNA cleavage in vitro. Therefore, toyocamycin is strongly suggested to inhibit ER stress-induced IRE1 activation. Finally, we examined the effect of toyocamycin on cell viability under ER stress condition, because XBP1 inhibitor is considered to suppress ER stress-induced cytoprotective effect thereby inducing selective killing of stressed cells. Although toyocamycin had only a weak activity to induce cell death in HeLa cells, toyocamycin synergistically induced cell death when toyocamycin was incubated with 2-deoxy-glucose or tunicamycin. These results indicated that toyocamycin selectively induces cell death under ER stress condition. Taken together, our results suggested that XBP1 is a potent molecular target for developing anti-cancer drug. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B150.

ERdj4 and ERdj5 Are Required for Endoplasmic Reticulum-associated Protein Degradation of Misfolded Surfactant Protein C

Mutations in the SFTPC gene associated with interstitial lung disease in human patients result in misfolding, endoplasmic reticulum (ER) retention, and degradation of the encoded surfactant protein C (SP-C) proprotein. In this study, genes specifically induced in response to transient expression of two disease-associated mutations were identified by microarray analyses. Immunoglobulin heavy chain binding protein (BiP) and two heat shock protein 40 family members, endoplasmic reticulum-localized DnaJ homologues ERdj4 and ERdj5, were significantly elevated and exhibited prolonged and specific association with the misfolded proprotein; in contrast, ERdj3 interacted with BiP, but it did not associate with either wild-type or mutant SP-C. Misfolded SP-C, ERdj4, and ERdj5 coprecipitated with p97/VCP indicating that the cochaperones remain associated with the misfolded proprotein until it is dislocated to the cytosol. Knockdown of ERdj4 and ERdj5 expression increased ER retention and inhibited degradation of misfolded SP-C, but it had little effect on the wild-type protein. Transient expression of ERdj4 and ERdj5 in X-box binding protein 1(-/-) mouse embryonic fibroblasts substantially restored rapid degradation of mutant SP-C proprotein, whereas transfection of HPD mutants failed to rescue SP-C endoplasmic reticulum-associated protein degradation. ERdj4 and ERdj5 promote turnover of misfolded SP-C and this activity is dependent on their ability to stimulate BiP ATPase activity.