Toxicological effects of iron on intestinal cells

The aim of the present study was to investigate whether iron, which is involved in the formation of free radicals in vitro, can initiate cellular injury in human intestinal cells. The effects of various concentrations of iron were studied in preconfluent, colonic-cancerogenous cells, and also in postconfluent, differentiating cells. Cellular damage was assessed using cell proliferation (serial cell counting), tetrazolium dye (MTT) uptake, lactate dehydrogenase (LDH) release and apoptosis studies based on caspase-3 activities. Also the activities of the major antioxidative enzymes, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were measured after the cells had been exposed to iron. Our results indicated that preconfluent cells were more susceptible to iron toxicity, as assessed by a significant reduction in cell proliferation and MTT uptake in a concentration-dependent manner compared to the control. However, no evidence for MTT uptake was observed in postconfluent cells. Caspase-3 activity, an indicator of cell apoptosis, considerably increased in preconfluent cells at high iron levels compared to the control (p < 0.05), whereas postconfluent cells were not significantly affected. LDH release was similar for both groups and was significantly higher than the control at 900 microM iron and above. SOD activities were not affected by iron in either group, whereas GPx was considerably higher in iron-treated cells in both groups compared with the control (because of relatively high standard deviations this effect was not significant). In conclusion we suggest that iron exerts its toxic effects intracellularly especially in preconfluent Caco-2 cells, whereas only high iron doses were able to alter the viability of differentiating, enterocyte-like cells.     

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.     

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.     

The early phase of programmed cell death in Caco-2 intestinal cells exposed to PTH

The regulation of apoptosis is critical for ensuring the homeostasis of an organism. As such, the cell has derived various mechanisms to precisely control the balance between survival and apoptotic signaling. Parathyroid hormone (PTH) function as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. Depending on the cell type involved, PTH also inhibits or promotes the apoptosis. In a previous work we found that PTH promotes the apoptosis of human Caco-2 intestinal cells. In the current study, we demonstrate, for the first time, that stimulation of Caco-2 cells with PTH (10(-8) M) results in the dephosphorylation and translocation of pro-apoptotic protein Bad from the cytosol to mitochondria and release of cytochrome c and Smac/Diablo. The hormone also triggers mitochondria cellular distribution to the perinuclear region, morphological features consistent with apoptosis. PTH increases the enzymatic activity of caspase-3 (48 h) that is also evidenced from the appearance of its cleaved fragments in western blot experiments. Moreover, active caspase-3 is present in nucleus after PTH treatment. In addition, a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP), is degraded by 48 h of PTH treatment. Taken together, our results suggest that, in Caco-2 cells, the induction of apoptosis in response to PTH is mediated by translocation of mitochondria to the perinuclear region, dephosphorylation of Akt, dephosphorylation of Bad and its movement to the mitochondria and subsequent release of cytochrome c and Smac/Diablo which result in activation of downstream caspase-3.     

Perfusion cultures of human tumor cells: a scalable production platform for oncolytic adenoviral vectors

This study demonstrates the novel use of the HeLaS3 human tumor cell line for propagating ONYX-411, a recombinant oncolytic adenoviral vector. HeLaS3 cells enabled high levels of vector production without the risk of generating vector recombinants, which is possible with HEK293 cells. The development of a high-cell-density perfusion process using ATF technology yielded production levels as high as 6 x 10(11) vp/mL, which was approximately sevenfold greater than the titers achieved in fed-batch bioreactors. Several experiments were performed at the bench (15 L) and pilot (70 L) scales to demonstrate the robust and scalable nature of this industrially relevant technology.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.     

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.     

Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation

1. Download : Download high-res image (200KB)
2. Download : Download full-size image

     

Intestinal epithelial cells related lncRNA and mRNA expression profiles in dextran sulphate sodium-induced colitis

Intestinal epithelial barrier damage caused by intestinal epithelial cells (IECs) dysfunction plays a crucial role in the pathogenesis and development of inflammatory bowel disease (IBD). Recently, some studies have suggested the emerging role of long non-coding RNAs (lncRNAs) in IBD. The aim of this study was to reveal lncRNAs and mRNA expression profiles in IECs from a mouse model of colitis and to expand our understanding in the intestinal epithelial barrier regulation. IECs from the colons of wild-type mice and dextran sulphate sodium (DSS)-induced mice were isolated for high-throughput RNA-sequencing. A total of 254 up-regulated and 1013 down-regulated mRNAs and 542 up-regulated and 766 down-regulated lncRNAs were detected in the DSS group compared with the Control group. Four mRNAs and six lncRNAs were validated by real-time quantitative PCR. Function analysis showed that dysregulated mRNAs participated in TLR7 signalling pathway, IL-1 receptor activity, BMP receptor binding and IL-17 signalling pathway. Furthermore, the possibility of indirect interactions between differentially expressed mRNAs and lncRNAs was illustrated by the competing endogenous RNA (ceRNA) network. LncRNA ENSMUST00000128026 was predicted to bind to mmu-miR-6899-3p, regulating Dnmbp expression. LncRNA NONMMUT143162.1 was predicted to competitively bind to mmu-miR-6899-3p, regulating Tnip3 expression. Finally, the protein-protein interaction (PPI) network analysis was constructed with 311 nodes and 563 edges. And the highest connectivity degrees were Mmp9, Fpr2 and Ccl3. These results provide novel insights into the functions of lncRNAs and mRNAs involved in the regulation of the intestinal epithelial barrier.     

Outer membrane protein OmpV mediates Salmonella enterica serovar typhimurium adhesion to intestinal epithelial cells via fibronectin and α1β1 integrin

Salmonella typhimurium is an invasive Gram‐negative enteric bacterium, which causes salmonellosis, a type of gastroenteritis in humans and typhoid‐like symptoms in mice. Upon entering through the contaminated food and water, S. typhimurium adheres, colonises, and invades intestinal epithelial cells (IECs) of the small intestine. In this study, we have shown that upon deletion of the outer membrane protein OmpV, there is a significant decrease in adherence of S. typhimurium to the IECs, indicating that OmpV is an important adhesin of S. typhimurium. Further, our study showed that OmpV binds to the extracellular matrix component fibronectin and signals through α1β1 integrin receptor on the IECs and OmpV‐mediated activation of α1β1, resulting in the activation of focal adhesion kinase and F‐actin modulation. Actin modulation is crucial for bacterial invasion. To the best of our knowledge, this is the first report of an adhesin mediated its effect through integrin in S. typhimurium. Further, we have observed a decrease in pathogenicity in terms of increased LD₅₀ dose, lesser bacterial numbers in stool, and less colonisation of bacteria in different organs of mice infected with Δompv mutant compared with the wild‐type bacteria, thus confirming the crucial role of OmpV in the pathogenesis of S. typhimurium.     

Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems

Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.     

Alpha-tocopherylquinone differentially modulates claudins to enhance intestinal epithelial tight junction barrier via AhR and Nrf2 pathways

1. Download : Download high-res image (187KB)
2. Download : Download full-size image

     

乳杆菌微小膜蛋白对肠上皮细胞Caco-2的细胞毒性研究

目的 利用脂多糖（lipopolysaccharide,LPS）刺激模拟体外炎症环境,观察不同浓度下乳杆菌微小膜蛋白（micro integral membrane protein,MIMP）对肠上皮细胞Caco-2的生物学影响,评估其细胞毒性作用。方法 首先通过CCK-8实验检测在LPS刺激后不同浓度MIMP（0.01、0.1和1 ng/mL）对Caco-2细胞增殖活性的影响,并利用Toll样受体4（Toll-like receptor 4,TLR4）抑制剂作为阳性对照。其次利用流式细胞术检测不同浓度下MIMP对Caco-2细胞凋亡及细胞周期的影响。结果 在12 h的特定孵育时间内,单独应用不同浓度MIMP及TLR4抑制剂对Caco-2细胞增殖活性无显著影响（P＞0.05）,但是MIMP可以拮抗LPS对Caco-2细胞的促增殖作用（P<0.05）。不同浓度的MIMP对Caco-2细胞的周期和凋亡无明显影响（P＞0.05）。结论 不同浓度MIMP对Caco-2细胞的增殖、凋亡及细胞周期无明显影响,并可以拮抗LPS的促细胞增殖作用,因此具有较高的安全性,有望用于临床对炎症性肠病的治疗。     

Anti-inflammatory properties of intestinal Bifidobacterium strains isolated from healthy infants

Certain Bifidobacterium strains have been shown to inhibit inflammatory responses in intestinal epithelial cells. However, the precise mechanisms of these effects, including the chemical nature of the active compounds, remain to be elucidated. Here partial characterization of the anti-inflammatory properties of Bifidobacterium strains isolated from feces of healthy infants is reported. It was found that conditioned media (CM) of all strains studied are capable of attenuating tumor necrosis factor-α (TNF-α) and lipopolysaccharide- (LPS) induced inflammatory responses in the HT-29 cell line. In contrast, neither killed bifidobacterial cells, nor cell-free extracts showed such activities. Further investigations resulted in attribution of this activity to heat-stable, non-lipophilic compound(s) resistant to protease and nuclease treatments and of molecular weight less than 3 kDa. The anti-inflammatory effects were dose- and time-dependent and associated with inhibition of IκB phosphorylation and nuclear factor-κ light chain enhancer of activated B cells (NF-κB)-dependent promoter activation. The combined treatments of cells with CMs and either LPS or TNF-α, but not with CMs alone, resulted in upregulation of transforming growth factor-β1, IκBζ, and p21(CIP) mRNAs. Our data suggest certain species-specificities of the anti-inflammatory properties of bifidobacteria. This observation should prompt additional validation studies using larger set of strains and employing the tools of comparative genomics.     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Enhanced osteoinduction by mesenchymal stem cells transfected with a fiber-mutant adenoviral BMP2 gene

BACKGROUND: Bone regeneration therapy using mesenchymal stem cells (MSCs) is beginning to come into clinical use. To overcome the difficulty of healing large bone defects, we previously reported the efficacy of using rat mesenchymal stem cells (rMSCs) carrying a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). METHODS: Firstly, we evaluated the transduction efficiency of Adv-F/RGD into bone-marrow-derived human MSCs (hMSCs) using a beta-galactosidase chemiluminescent assay. Next, we evaluated whether the vector AxCAhBMP2-F/RGD carrying the human bone morphogenetic protein 2 (BMP2) gene could enhance the osteogenic activity of hMSCs in vitro and in vivo (in an ectopic model). In the ectopic model, transduced hMSCs, hMSCs in the presence of recombinant human BMP2 (rhBMP2) or hMSCs alone were implanted into a subcutaneous site of nude mice. We also applied this vector system to an orthotopic model (large bone defect model) using rMSCs. RESULTS: The transduction efficiency of Adv-F/RGD into hMSCs was increased 10-fold over the vector containing the wild-type fiber (Adv-F/wt), as assessed by a beta-galactosidase chemiluminescent assay. AxCAhBMP2-F/RGD increased the osteogenic activity of hMSCs in vitro. In the ectopic model, AxCAhBMP2-F/RGD-transduced hMSCs were found to induce new bone at 1 week after transplantation, and a greater quantity of new bone was formed than in other groups. Similarly, AxCAhBMP2-F/RGD-transduced rMSCs induced a greater quantity of new bone than other groups (AxCAhBMP2-F/wt-transduced rMSCs, rMSCs in the presence of rhBMP2, rMSCs alone, or scaffolds alone) in the orthotopic model. CONCLUSIONS: These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue-engineering applications.