Kee Hun Do

Endoplasmic Reticulum Stress-activated C/EBP Homologous Protein Enhances Nuclear Factor-κB Signals via Repression of Peroxisome Proliferator-activated Receptor γ

Endoplasmic reticulum (ER) stress is a causative factor of inflammatory bowel diseases. ER stress mediators, including CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), are elevated in intestinal epithelia from patients with inflammatory bowel diseases. The present study arose from the question of how chemical ER stress and CHOP protein were associated with nuclear factor-κB (NF-κB)-mediated epithelial inflammatory response. In a human intestinal epithelial cell culture model, chemical ER stresses induced proinflammatory cytokine interleukin-8 (IL-8) expression and the nuclear translocation of CHOP protein. CHOP was positively involved in ER-activated IL-8 production and was negatively associated with expression of peroxisome proliferator-activated receptor γ (PPARγ). ER stress-induced IL-8 production was enhanced by NF-κB activation that was negatively regulated by PPARγ. Mechanistically, ER stress-induced CHOP suppressed PPARγ transcription by sequestering C/EBPβ and limiting availability of C/EBPβ binding to the PPARγ promoter. Due to the CHOP-mediated regulation of PPARγ action, ER stress can enhance proinflammatory NF-κB activation and maintain an increased level of IL-8 production in human intestinal epithelial cells. In contrast, PPARγ was a counteracting regulator of gut inflammatory response through attenuation of NF-κB activation. The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

Repression of Peroxisome Proliferator-Activated Receptor γ by Mucosal Ribotoxic Insult-Activated CCAAT/Enhancer-Binding Protein Homologous Protein

CCAAT/enhancer-binding protein homologous protein (CHOP) is a crucial stress-responsive factor in various mucosal injuries, including cellular translational stress conditions. In this study, chemical ribosome-inactivating stresses were assessed for their effects on stress-inducible CHOP expression and its association with epithelial inflammatory cytokine production. Several representative ribotoxic agents (deoxynivalenol, anisomycin, and 15-acetyldeoxynivalenol) enhanced CHOP expression and its nuclear translocation in human intestinal epithelial cells. Moreover, CHOP was a strong positive regulator of IL-8 production, but CHOP-mediated IL-8 production was inversely associated with expression of the mucosal regulatory factor peroxisome proliferator-activated receptor γ (PPARγ). Based on our recent report that PPARγ is a negative regulator of mRNA stability of IL-8, PPARγ was linked to a notable mRNA stabilizing protein, HuR, since ribotoxin-induced IL-8 mRNA is stabilized by HuR protein. Expression of exogenous PPARγ suppressed ribotoxin-triggered cytoplasmic translocation of HuR. In contrast, PPARγ-regulating CHOP was a positive modulator of HuR protein export from nuclei. Taken together, the results indicate that ribotoxin-induced CHOP protein is positively associated with production of proinflammatory cytokine IL-8, but it downregulates PPARγ action, subsequently allowing the cytosolic translocation of HuR protein and stabilization of IL-8 mRNA in gut epithelial cells. CHOP and PPARγ may represent critical mechanistic links between ribotoxic stress and proinflammatory cytokine production, and they may have a broader functional significance with regard to gastrointestinal stresses by toxic mucosal insults.

Pro-inflammatory NF-κB and early growth response gene 1 regulate epithelial barrier disruption by food additive carrageenan in human intestinal epithelial cells

The widely used food additive carrageenan (CGN) has been shown to induce intestinal inflammation, ulcerative colitis-like symptoms, or neoplasm in the gut epithelia in animal models, which are also clinical features of human inflammatory bowel disease. In this study, the effects of CGN on pro-inflammatory transcription factors NF-κB and early growth response gene 1 product (EGR-1) were evaluated in terms of human intestinal epithelial barrier integrity. Both pro-inflammatory transcription factors were elevated by CGN and only NF-κB activation was shown to be involved in the induction of pro-inflammatory cytokine interleukin-8. Moreover, the integrity of the in vitro epithelial monolayer under the CGN insult was maintained by both activated pro-inflammatory transcription factors NF-κB and EGR-1. Suppression of NF-κB or EGR-1 aggravated barrier disruption by CGN, which was associated with the reduced gene expression of tight junction component zonula occludens 1 and its irregular localization in the epithelial monolayer.

Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

ABSTRACT The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probiotic E. coli Nissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using the in vitro physically wounded monolayer model, ABC transporter-mediated EGF secretion by probiotic E. coli Nissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle.

Two In-and-out Modulation Strategies for Endoplasmic Reticulum Stress-linked Gene Expression of Pro-apoptotic Macrophage-inhibitory Cytokine 1

Background: Modulation of a pro-apoptotic MIC-1 was addressed under ER stress-linked alterations of intracellular components. Results: Transporting of transcript-associated complex across cellular compartments is critical for MIC-1 mRNA stabilization. Conclusion: Stabilization is due to complex formation with pumped-out RNA-binding protein and their timely exit from the stress granule. Significance: Harmonized in-and-out modulation implicates key regulatory processes of MIC-1 expression in ER stress-linked therapy and biology. Excessive and persistent insults during endoplasmic reticulum (ER) stress lead to apoptotic cell death that is implicated in a range of chronic inflammatory diseases and cancers. Macrophage inhibitory cytokine 1 (MIC-1), a member of the transforming growth factor-β superfamily, is diversely linked to the pathogenesis of cancer. To investigate the precise molecular mechanisms of MIC-1 gene regulation, ER stress and its related signals were studied in human colon cancer cells. Functionally, MIC-1 played pivotal roles in ER stress-linked apoptotic death, which was also influenced by C/EBP homologous protein, a well known apoptotic mediator of ER stress. ER stress enhanced MIC-1 mRNA stability instead of transcriptional activation, and there were two mechanistic translocations critical for mRNA stabilization. First, C/EBP homologous protein triggered protein kinase C-linked cytosolic translocation of the HuR/ELAVL1 (Elav-like RNA-binding protein 1) RNA-binding protein, which bound to and stabilized MIC-1 transcript. As the second critical in-and-out regulation, ER stress-activated ERK1/2 signals contributed to enhanced stabilization of MIC-1 transcript by controlling the extended holding of the nucleated mRNA in the stress granules fusing with the mRNA-decaying processing body. We propose that these two sequential in-and-out modulations can account for stabilized transcription and subsequent translation of pro-apoptotic MIC-1 gene in human cancer cells under ER stress.

Involvement of Epidermal Growth Factor Receptor-Linked Signaling Responses in Pseudomonas fluorescens-Infected Alveolar Epithelial Cells

ABSTRACT Pseudomonas fluorescens is an opportunistic indoor pathogen that can cause severe airway proinflammatory responses. Pulmonary epithelium, like other mucosal epithelial linings of the body, constitutes the first line of defense against airway microbial pathogens. Mucosal epithelial cells can be a sentinel of pathogenic bacteria via stimulation of specific cell surface receptors, including the epidermal growth factor receptor (EGFR) and Toll-like receptor (TLR). This study addressed the involvement of EGFR in airway epithelial pathogenesis by P. fluorescens. Human A549 pneumocytes showed prolonged production of proinflammatory interleukin-8 (IL-8) in response to infection with P. fluorescens, which was via the nuclear factor-kappa B (NF-κB) signaling pathway. Production of proinflammatory cytokine IL-8 was not mediated by P. fluorescens lipopolysaccharide, a representative TLR4 agonist, but was mediated through EGFR-linked signals activated by the opportunistic bacteria. Moreover, EGFR signals were involved in NF-κB signal-mediated production of proinflammatory cytokines. Along with persistent NF-κB activation, P. fluorescens enhanced the EGFR phosphorylation and subsequent activation of downstream mediators, including protein kinase B or extracellular-signal-regulated kinases 1/2. Blocking of EGFR-linked signals increased epithelial susceptibility to pathogen-induced epithelial cell death, suggesting protective roles of EGFR signals. Thus, airway epithelial exposure to P. fluorescens can trigger antiapoptotic responses via EGFR and proinflammatory responses via TLR4-independent NF-κB signaling pathway in human pneumocytes.

Activating Transcription Factor 3-mediated Chemo-intervention with Cancer Chemokines in a Noncanonical Pathway under Endoplasmic Reticulum Stress

Background: Cancer-favoring ER stress responses drive proinflammatory programs including cancer chemokine production. Results: ER stress-induced cancer chemokines are regulated by preventive exposure to a natural flavone apigenin via ATF3. Conclusion: ATF3 epigenetically suppresses expression of EGR-1, a noncanonical proinflammatory transcriptional modulator crucial to cancer chemokine induction. Significance: ATF3-mediated chemokine suppression implicates a novel chemo-intervention with ER stress response-related tumorigenesis. The cell-protective features of the endoplasmic reticulum (ER) stress response are chronically activated in vigorously growing malignant tumor cells, which provide cellular growth advantages over the adverse microenvironment including chemotherapy. As an intervention with ER stress responses in the intestinal cancer cells, preventive exposure to flavone apigenin potentiated superinduction of a regulatory transcription factor, activating transcription factor 3 (ATF3), which is also known to be an integral player coordinating ER stress response-related gene expression. ATF3 superinduction was due to increased turnover of ATF3 transcript via stabilization with HuR protein in the cancer cells under ER stress. Moreover, enhanced ATF3 caused inhibitory action against ER stress-induced cancer chemokines that are potent mediators determining the survival and metastatic potential of epithelial cancer cells. Although enhanced ATF3 was a negative regulator of the well known proinflammatory transcription factor NF-κB, blocking of NF-κB signaling did not affect ER stress-induced chemokine expression. Instead, immediately expressed transcription factor early growth response protein 1 (EGR-1) was positively involved in cancer chemokine induction by ER stressors. ER stress-induced EGR-1 and subsequent chemokine production were repressed by ATF3. Mechanistically, ATF3 directly interacted with and recruited HDAC1 protein, which led to epigenetic suppression of EGR-1 expression and subsequent chemokine production. Conclusively, superinduced ATF3 attenuated ER stress-induced cancer chemokine expression by epigenetically interfering with induction of EGR-1, a transcriptional modulator crucial to cancer chemokine production. Thus, these results suggest a potent therapeutic intervention of ER stress response-related cancer-favoring events by ATF3.

Novel regulatory action of ribosomal inactivation on epithelial Nod2-linked proinflammatory signals in two convergent ATF3-associated pathways.

In response to excessive nucleotide-binding oligomerization domain–containing protein 2 (Nod2) stimulation caused by mucosal bacterial components, gut epithelia need to activate regulatory machinery to maintain epithelial homeostasis. Activating transcription factor 3 (ATF3) is a representative regulator in the negative feedback loop that modulates TLR-associated inflammatory responses. In the current study, the regulatory effects of ribosomal stress-induced ATF3 on Nod2-stimulated proinflammatory signals were assessed. Ribosomal inactivation caused persistent ATF3 expression that in turn suppressed proinflammatory chemokine production facilitated by Nod2. Decreased chemokine production was due to attenuation of Nod2-activated NF-κB and early growth response protein 1 (EGR-1) signals by ATF3. However, the underlying molecular mechanisms involve two convergent regulatory pathways. Although ATF3 induced by ribosomal inactivation regulated Nod2-induced EGR-1 expression epigenetically through the recruitment of histone deacetylase 1, NF-κB regulation was associated with posttranscriptional regulation by ATF3 rather than epigenetic modification. ATF3 induced by ribosomal inactivation led to the destabilization of p65 mRNA caused by nuclear entrapment of transcript-stabilizing human Ag R protein via direct interaction with ATF3. These findings demonstrate that ribosomal stress-induced ATF3 is a critical regulator in the convergent pathways between EGR-1 and NF-κB, which contributes to the suppression of Nod2-activated proinflammatory gene expression.

Nation-Based Occurrence and Endogenous Biological Reduction of Mycotoxins in Medicinal Herbs and Spices

Medicinal herbs have been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. Moreover, the health benefits of spices have been extensively recognized in recent studies. However, inevitable contaminants, including mycotoxins, in medicinal herbs and spices can cause serious problems for humans in spite of their health benefits. Along with the different nation-based occurrences of mycotoxins, the ultimate exposure and toxicities can be diversely influenced by the endogenous food components in different commodities of the medicinal herbs and spices. The phytochemicals in these food stuffs can influence mold growth, mycotoxin production and biological action of the mycotoxins in exposed crops, as well as in animal and human bodies. The present review focuses on the occurrence of mycotoxins in medicinal herbs and spices and the biological interaction between mold, mycotoxin and herbal components. These networks will provide insights into the methods of mycotoxin reduction and toxicological risk assessment of mycotoxin-contaminated medicinal food components in the environment and biological organisms.

Mechanism-based alternative monitoring of endoplasmic reticulum stress by 8-keto-trichothecene mycotoxins using human intestinal epithelial cell line.

Type B 8-keto trichothecenes are muco-active mycotoxins that are notable contaminants in cereal-based food stuffs. Epithelial responses are of primary concern during gastrointestinal exposure in human and animal intoxications. Therefore, optimized biomarkers to assess the specific action of trichothecenes on the human epithelial barrier are needed. In the present study, 8-keto trichothecenes were tested to trigger endoplasmic reticulum (ER) stresses that are evident as alteration of eukaryotic initiation factors 2α phosphorylation and other ER stress markers in human intestinal epithelial cells. Based on the ER stress-inducing activity of 8-keto trichothecenes, we developed a bio-monitoring method using intestinal epithelial cells constitutively expressing secretory alkaline phosphatase (SEAP) reporter, which can be used as a sensitive and selective indicator for monitoring ER stress in the epithelial barrier. Type B 8-keto trichothecenes suppressed the release of SEAP in a dose-dependent manner, showing a negative correlation between logarithm of toxin dose and SEAP activity. However, tested toxins did not affect SEAP enzymatic activity and mRNA levels, but reduced the cellular release of SEAP. Moreover, in order to correct the monitoring of 8-keto trichothecene in crop matrix, the bioassay was proven to work in standard cereal extract of corn and rice. The mechanism-based monitoring of 8-keto trichothecenes is promising as a supplementary analysis means of the presence of bioactive 8-keto trichothecenes, which are potentially exposed in human gut epithelia.