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1.
Appl Environ Microbiol ; 82(24): 7227-7235, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27742679

ABSTRACT

Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCE: Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.


Subject(s)
Bacterial Proteins/metabolism , Cyanothece/enzymology , Hydrogen/metabolism , Nitrogenase/metabolism , Oxidative Stress , Bacterial Proteins/genetics , Cyanothece/genetics , Cyanothece/metabolism , Cyanothece/radiation effects , Light , Nitrogenase/genetics , Oxidation-Reduction , Oxygen/metabolism , Photosynthesis/radiation effects
2.
Sci Rep ; 5: 16004, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26525576

ABSTRACT

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.


Subject(s)
Cyanothece/metabolism , Hydrogen/metabolism , Nitrogenase/metabolism , Oxygen/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cluster Analysis , Cyanothece/enzymology , Cyanothece/genetics , Energy Metabolism , Gene Expression Profiling , Glycogen/chemistry , Glycogen/metabolism , Hydrogen/chemistry , Hydrogenase/genetics , Hydrogenase/metabolism , Kinetics , Nitrogenase/genetics , Oxidation-Reduction , Photosynthesis , Photosystem II Protein Complex/genetics , Photosystem II Protein Complex/metabolism , Proteomics , RNA, Messenger/metabolism , Water/chemistry
3.
PLoS One ; 9(2): e87614, 2014.
Article in English | MEDLINE | ID: mdl-24586284

ABSTRACT

PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs), a small noncoding RNAs (21-23 nucleotides), post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs) of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA) expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis). The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.


Subject(s)
Cell Communication , Colitis/metabolism , Colon/metabolism , Intestinal Mucosa/metabolism , MicroRNAs/genetics , Symporters/metabolism , Up-Regulation , Animals , Biological Transport , Colitis/genetics , Colitis/pathology , Colon/pathology , Humans , Intestinal Mucosa/pathology , Male , Mice , MicroRNAs/metabolism , Peptide Transporter 1 , Symporters/genetics
4.
Gastroenterology ; 146(5): 1289-300.e1-19, 2014 May.
Article in English | MEDLINE | ID: mdl-24503126

ABSTRACT

BACKGROUND & AIMS: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. METHODS: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. RESULTS: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. CONCLUSIONS: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.


Subject(s)
Colitis/prevention & control , Colon/metabolism , Fusion Regulatory Protein-1/genetics , Fusion Regulatory Protein-1/immunology , Genetic Therapy/methods , Nanomedicine/methods , Nanoparticles , RNA Interference , RNA, Small Interfering/administration & dosage , Single-Chain Antibodies/administration & dosage , Administration, Oral , Alginates/chemistry , Animals , Cell Line , Chitosan/chemistry , Colitis/chemically induced , Colitis/genetics , Colitis/immunology , Colitis/metabolism , Colitis/pathology , Colon/immunology , Colon/pathology , Dextran Sulfate , Disease Models, Animal , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Hydrogels , Interleukin-12/metabolism , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Small Interfering/metabolism , Severity of Illness Index , Time Factors , Tumor Necrosis Factor-alpha/metabolism
5.
Biomaterials ; 34(30): 7471-82, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23820013

ABSTRACT

The application of RNA interference (RNAi) for inflammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-α is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-α siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-α (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-α level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.


Subject(s)
Inflammatory Bowel Diseases/therapy , Macrophages/metabolism , Mannose/metabolism , Nanoparticles/chemistry , RNA Interference , Tumor Necrosis Factor-alpha/metabolism , Animals , Caco-2 Cells , Cell Death/drug effects , Electrophoresis, Agar Gel , Endocytosis/drug effects , Humans , Macrophages/cytology , Macrophages/drug effects , Magnetic Resonance Spectroscopy , Male , Mice , Nanoparticles/ultrastructure , Particle Size , Polymers/chemical synthesis , Polymers/chemistry , Polymers/pharmacology , Polyphosphates/chemistry , Polyphosphates/pharmacology , RNA Interference/drug effects , RNA, Small Interfering/metabolism , Titrimetry
6.
Lab Invest ; 93(8): 888-99, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23797361

ABSTRACT

We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-γ were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site-leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.


Subject(s)
Colitis/immunology , Gene Expression , Immunity, Cellular/immunology , Macrophages/metabolism , Symporters/genetics , Animals , Anti-Bacterial Agents/pharmacology , Colitis/chemically induced , Colitis/pathology , Colon/drug effects , Colon/enzymology , Colon/microbiology , Cytokines/genetics , Cytokines/metabolism , Dextran Sulfate/toxicity , Disease Models, Animal , Female , Gene Knockdown Techniques , Homeostasis , Immunity, Cellular/drug effects , Macrophages/drug effects , Macrophages/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptide Transporter 1 , Peroxidase/metabolism , RNA, Messenger/metabolism , Symporters/metabolism
7.
Infect Immun ; 81(3): 923-34, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23297381

ABSTRACT

CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.


Subject(s)
Enterobacteriaceae Infections/immunology , Fusion Regulatory Protein-1/metabolism , Immunity, Innate , Intestinal Mucosa/metabolism , Animals , Caco-2 Cells , Citrobacter rodentium , Colon , Enterobacteriaceae Infections/microbiology , Enteropathogenic Escherichia coli , Epithelial Cells/immunology , Epithelial Cells/metabolism , Female , Fusion Regulatory Protein-1/genetics , Gene Expression Regulation/immunology , Humans , Male , Mice , Mice, Transgenic , Peroxidase , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods
8.
Am J Physiol Gastrointest Liver Physiol ; 302(11): G1282-91, 2012 Jun 01.
Article in English | MEDLINE | ID: mdl-22499850

ABSTRACT

The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.


Subject(s)
Colitis/metabolism , Colon/metabolism , Fusion Regulatory Protein-1/biosynthesis , Intestinal Mucosa/metabolism , MicroRNAs/metabolism , Animals , Colitis/genetics , Epithelial Cells/metabolism , Inflammation , Mice , MicroRNAs/genetics
9.
PLoS One ; 7(3): e32084, 2012.
Article in English | MEDLINE | ID: mdl-22427817

ABSTRACT

Inflammatory bowel diseases (IBDs), primarily ulcerative colitis and Crohn's disease, are inflammatory disorders caused by multiple factors. Research on IBD has often used the dextran sodium sulfate (DSS)-induced colitis mouse model. DSS induces in vivo but not in vitro intestinal inflammation. In addition, no DSS-associated molecule (free glucose, sodium sulfate solution, free dextran) induces in vitro or in vivo intestinal inflammation. We find that DSS but not dextran associated molecules established linkages with medium-chain-length fatty acids (MCFAs), such as dodecanoate, that are present in the colonic lumen. DSS complexed to MCFAs forms nanometer-sized vesicles ~200 nm in diameter that can fuse with colonocyte membranes. The arrival of nanometer-sized DSS/MCFA vesicles in the cytoplasm may activate intestinal inflammatory signaling pathways. We also show that the inflammatory activity of DSS is mediated by the dextran moieties. The deleterious effect of DSS is localized principally in the distal colon, therefore it will be important to chemically modify DSS to develop materials beneficial to the colon without affecting colon-targeting specificity.


Subject(s)
Colitis/chemically induced , Dextran Sulfate/adverse effects , Dextran Sulfate/metabolism , Fatty Acids/metabolism , Macromolecular Substances/metabolism , Nanostructures/chemistry , Analysis of Variance , Animals , Colitis/metabolism , Colitis/pathology , Cytokines/blood , DNA Primers/genetics , Diet, High-Fat , Electric Impedance , Endoscopy, Gastrointestinal , Female , Histological Techniques , Mice , Mice, Inbred C57BL , Particle Size , Peroxidase/metabolism , Transport Vesicles/metabolism
10.
Am J Physiol Gastrointest Liver Physiol ; 302(5): G484-92, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22194420

ABSTRACT

Intestinal inflammation is characterized by epithelial disruption, leading to loss of barrier function and the recruitment of immune cells, including neutrophils. Although the mechanisms are not yet completely understood, interactions between environmental and immunological factors are thought to be critical in the initiation and progression of intestinal inflammation. In recent years, it has become apparent that the di/tripeptide transporter PepT1 may play an important role in the pathogenesis of such inflammation. In healthy individuals, PepT1 is primarily expressed in the small intestine and transports di/tripeptides for metabolic purposes. However, during chronic inflammation such as that associated with inflammatory bowel disease, PepT1 expression is upregulated in the colon, wherein the protein is normally expressed either minimally or not at all. Several recent studies have shown that PepT1 binds to and transports various bacterial di/tripeptides into colon cells, leading to activation of downstream proinflammatory responses via peptide interactions with innate immune receptors. In the present review, we examine the relationship between colonic PepT1-mediated peptide transport in the colon and activation of innate immune responses during disease. It is important to understand the mechanisms of PepT1 action during chronic intestinal inflammation to develop future therapies addressing inappropriate immune activation in the colon.


Subject(s)
Gastroenteritis/etiology , Inflammatory Bowel Diseases/etiology , Symporters/physiology , Animals , Colorectal Neoplasms/physiopathology , Gastroenteritis/drug therapy , Humans , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/physiopathology , Nod Signaling Adaptor Proteins/physiology , Oligopeptides/metabolism , Peptide Transporter 1 , Symporters/genetics
11.
J Biol Chem ; 286(35): 31003-31013, 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21757725

ABSTRACT

The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 µM. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 µM. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 µM) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.


Subject(s)
Diaminopimelic Acid/analogs & derivatives , Leucine/chemistry , Nod1 Signaling Adaptor Protein/chemistry , Oligopeptides/chemistry , Biophysics/methods , Caco-2 Cells , Diaminopimelic Acid/chemistry , Diaminopimelic Acid/metabolism , Humans , Immunity, Innate , Inflammation , Microscopy, Atomic Force/methods , Nucleotides/chemistry , Oligopeptides/metabolism , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction
12.
Gastroenterology ; 141(4): 1334-45, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21762661

ABSTRACT

BACKGROUND & AIMS: The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1. METHODS: Transgenic mice were generated in which hPepT1 expression was regulated by the ß-actin or villin promoters; colitis was induced using 2,4,6-trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulfate (DSS) and the inflammatory responses were assessed. The effects of NOD2 deletion in the hPepT1 transgenic mice also was studied to determine the involvement of the PepT1-NOD2 signaling pathway. RESULTS: TNBS and DSS induced more severe levels of inflammation in ß-actin-hPepT1 transgenic mice than wild-type littermates. Intestinal epithelial cell-specific hPepT1 overexpression in villin-hPepT1 transgenic mice increased the severity of inflammation induced by DSS, but not TNBS. Bone marrow transplantation studies showed that hPepT1 expression in intestinal epithelial cells and immune cells has an important role in the proinflammatory response. Antibiotics abolished the effect of hPepT1 overexpression on the inflammatory response in DSS-induced colitis in ß-actin-hPepT1 and villin-hPepT1 transgenic mice, indicating that commensal bacteria are required to aggravate intestinal inflammation. Nod2-/-, ß-actin-hPepT1 transgenic/Nod2-/-, and villin-hPepT1 transgenic/Nod2-/- littermates had similar levels of susceptibility to DSS-induced colitis, indicating that hPepT1 overexpression increased intestinal inflammation in a NOD2-dependent manner. CONCLUSIONS: The PepT1-NOD2 signaling pathway is involved in aggravation of DSS-induced colitis in mice.


Subject(s)
Colitis/metabolism , Colon/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Signal Transduction , Symporters/metabolism , Actins/genetics , Animals , Anti-Bacterial Agents/pharmacology , Bone Marrow Transplantation , Colitis/chemically induced , Colitis/genetics , Colitis/immunology , Colitis/microbiology , Colitis/pathology , Colitis/prevention & control , Colon/drug effects , Colon/immunology , Colon/microbiology , Colon/pathology , Dextran Sulfate , Disease Models, Animal , Humans , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/genetics , Nod2 Signaling Adaptor Protein/deficiency , Nod2 Signaling Adaptor Protein/genetics , Peptide Transporter 1 , Promoter Regions, Genetic , Severity of Illness Index , Signal Transduction/drug effects , Symporters/genetics , Time Factors , Trinitrobenzenesulfonic Acid
13.
PLoS One ; 6(4): e19293, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21559394

ABSTRACT

Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3'-untranslated regions (3'-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3'-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.


Subject(s)
Metagenome/genetics , MicroRNAs/genetics , 3' Untranslated Regions , Animals , Cell Line , Computational Biology/methods , DNA, Complementary/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Mice , Multidrug Resistance-Associated Proteins/metabolism , Oligonucleotide Array Sequence Analysis
14.
Article in English | MEDLINE | ID: mdl-21030610

ABSTRACT

MicroRNAs (miRNAs), which are noncoding RNAs that posttranscriptionally inhibit expression of target genes, have recently emerged as important regulators of many cellular functions such as cell differentiation. The epithelial di/tripeptide membrane transporter PepT1 is expressed in highly differentiated cells (the villous tip) but not in undifferentiated cells (the crypt) of the small intestine. Here, we investigated the regulation of PepT1 expression by miRNAs and its functional consequences. We observed a reverse correlation between the expression levels of PepT1 and mature miRNA-92b (miR-92b) during the differentiation of intestinal epithelial Caco2-BBE cells, suggesting a miR-92b-mediated regulation of PepT1 expression. We demonstrate that miR-92b suppressed PepT1 expression at both mRNA and protein levels, with subsequent reduced PepT1 transport activity, in Caco2-BBE cells by directly targeting the PepT1 3'-untranslated region. In addition, miR-92b suppresses bacterial peptide-induced proinflammatory responses in intestinal epithelial cells by inhibiting PepT1 expression. Altogether, our study provides for the first time evidence for the regulation of PepT1 expression at a posttranscriptional level by miRNAs in intestinal epithelial cells during pathophysiological states.


Subject(s)
Intestinal Mucosa/metabolism , MicroRNAs/physiology , Symporters/biosynthesis , Caco-2 Cells , Cell Differentiation , Diaminopimelic Acid/analogs & derivatives , Diaminopimelic Acid/pharmacology , Down-Regulation , Humans , Inflammation/chemically induced , Inflammation/prevention & control , Intestinal Mucosa/cytology , Oligopeptides/pharmacology , Peptide Transporter 1
15.
J Bacteriol ; 192(12): 3227-30, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20400540

ABSTRACT

Shewanella oneidensis is a metal reducer that uses the cyclic AMP receptor protein, CRP, to regulate anaerobic respiration. In addition, ArcA(So) is required for anaerobic growth with dimethyl sulfoxide (DMSO) and plays a role in aerobic respiration. The sensor kinase that activates ArcA(So) in S. oneidensis is not known. ArcB1(So), a homolog of the Escherichia coli sensor kinase ArcB(Ec), was identified and found to be required for DMSO reductase gene expression. In combination with HptA, ArcB1(So) complemented an E. coli arcB(Ec) mutant. ArcA(So), ArcB1(So), and HptA appear to constitute a two-component signal transduction system that regulates DMSO reduction in S. oneidensis.


Subject(s)
Dimethyl Sulfoxide/metabolism , Escherichia coli Proteins/chemistry , Membrane Proteins/chemistry , Protein Kinases/chemistry , Shewanella/physiology , Aerobiosis , Anaerobiosis , Gene Expression Regulation, Bacterial/physiology , Oxygen Consumption , Receptors, Cyclic AMP/metabolism , Shewanella/classification
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