Subepithelial Serotonin Reduces Small Intestinal Epithelial Cell Tightness via Reduction of Occluding Expression.

BACKGROUND: The precise pathogenesis of irritable bowel syndrome (IBS) remains unresolved; however, recent studies have reported that patients with diarrhea-predominant IBS exhibit an increased small intestinal permeability and increased number of enterochromaffin cells containing high 5-hydroxytryptamine (5HT; serotonin) levels. In this study, we investigated whether 5HT has the potential to modulate small intestinal epithelial cell permeability, focusing on tight junction-associated proteins. METHODS: The differentiated Caco-2 cell monolayer on porous filters (Millicell) was used. Then, 5HT was added to the lower Millicell compartment for 7 days. Intestinal epithelial cell permeability was assessed by measuring the flux of paracellular permeability markers. We further assessed the expression of occludin in the 5HT-stimulated Caco-2 monolayer. RESULTS: We found that 5HT did not affect the viability of Caco-2 cells at concentrations up to 100 µM during the experimental period. Administration of 5HT to the basal side of Caco-2 cells increased the flux of 3H-labeled mannitol (182 Da) but did not increase that of FITC-dextran (4000 Da). Among the tight junction proteins, the expression of occludin was specifically decreased by stimulation with 5HT at a concentration of 100 µM. CONCLUSION: In conclusion, excessive 5HT in the basal side increased the permeability of intestinal epithelial cells via reduction of occludin expression.

Diurnal Transcriptome and Gene Network Represented through Sparse Modeling in Brachypodium distachyon.

We report the comprehensive identification of periodic genes and their network inference, based on a gene co-expression analysis and an Auto-Regressive eXogenous (ARX) model with a group smoothly clipped absolute deviation (SCAD) method using a time-series transcriptome dataset in a model grass, Brachypodium distachyon. To reveal the diurnal changes in the transcriptome in B. distachyon, we performed RNA-seq analysis of its leaves sampled through a diurnal cycle of over 48 h at 4 h intervals using three biological replications, and identified 3,621 periodic genes through our wavelet analysis. The expression data are feasible to infer network sparsity based on ARX models. We found that genes involved in biological processes such as transcriptional regulation, protein degradation, and post-transcriptional modification and photosynthesis are significantly enriched in the periodic genes, suggesting that these processes might be regulated by circadian rhythm in B. distachyon. On the basis of the time-series expression patterns of the periodic genes, we constructed a chronological gene co-expression network and identified putative transcription factors encoding genes that might be involved in the time-specific regulatory transcriptional network. Moreover, we inferred a transcriptional network composed of the periodic genes in B. distachyon, aiming to identify genes associated with other genes through variable selection by grouping time points for each gene. Based on the ARX model with the group SCAD regularization using our time-series expression datasets of the periodic genes, we constructed gene networks and found that the networks represent typical scale-free structure. Our findings demonstrate that the diurnal changes in the transcriptome in B. distachyon leaves have a sparse network structure, demonstrating the spatiotemporal gene regulatory network over the cyclic phase transitions in B. distachyon diurnal growth.

Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation.

Mucin is produced and secreted by epithelial goblet cells and is a key component of the innate immune system, acting as a barrier in the intestinal tract. However, no studies have been conducted investigating the increase in mucin secretion to enhance the intestinal barrier function. The present study investigated whether rebamipide (Reb) acts as a secretagogue of intestinal mucin and the underlying mechanisms involved, thereby focusing on the effect on goblet cells. The LS174T cell line was used as goblet cell­like cells. Using Reb­treated LS174T cells, the level of mucin content was assessed by periodic acid­Schiff (PAS) staining, and mucin 2, oligomeric mucus/gel­forming (MUC2) mRNA expression was assessed using quantitative polymerase chain reaction (PCR). Furthermore, MUC2 secretion in the supernatant was quantified by the dot blot method. The present study additionally investigated the involvement of the epidermal growth factor receptor/Akt serine/threonine kinase 1 (Akt) pathway in mucin secretion by western blotting. The results suggested that Reb strongly enhanced the positivity of PAS staining in LS174T cells, thereby suggesting increased intracellular mucin production. The PCR results indicated that Reb significantly increased MUC2 mRNA in whole cell lysate of LS174T cells. In order to assess the subsequent secretion of mucin by LS174T, MUC2 protein expression in the supernatant was assessed using the dot blot method and it was demonstrated that Reb significantly increased the secretion of MUC2 in a concentration­dependent manner. The p­Akt was significantly increased by Reb treatment, and an Akt inhibitor specifically suppressed MUC2 secretion. Overall, Reb increased mucin secretion directly via p­Akt. Reb­increased mucin may act as a strong non­specific barrier against pathogenic stimulants in various intestinal diseases.

Real-time monitoring of trans-epithelial electrical resistance in cultured intestinal epithelial cells: the barrier protection of water-soluble dietary fiber.

OBJECTIVES: In this study we aimed to verify a real-time trans-epithelial electrical resistance (TEER) monitoring system in a Caco-2 monolayer and to investigate the therapeutic effect of partially hydrolyzed guar gum (PHGG), a dietary fiber, against interferon (IFN)-γ-induced intestinal barrier dysfunction using this monitoring system. METHODS: We measured TEER using a real-time monitoring system and evaluated epithelial paracellular permeability using fluorescein isothiocyanate-conjugated dextran (4 kDa; FD4) in Caco-2 monolayers treated with IFN-γ for 48 h. The expression and distribution of tight junction (TJ)-associated proteins, ZO-1 and occludin, were analyzed by Western blot and immunocytochemistry, respectively. In some experiments PHGG was added prior to IFN-γ treatment in order to investigate its protective effect on barrier function. RESULTS: IFN-γ treatment significantly decreased TEER and increased FD4 flux across Caco-2 monolayers, indicating a great influence of IFN-γ on the intestinal epithelial paracellular permeability. In contrast, the pretreatment of PHGG significantly reduced the IFN-γ-induced increment of FD4 flux without affecting TEER. Neither IFN-γ nor PHGG treatment affected the expressions of TJ-associated proteins, while immunocytochemistry showed that IFN-γ-induced redistribution of occludin was clearly restored by PHGG. CONCLUSIONS: Real-time TEER monitoring enabled us to evaluate the dynamic changes of intestinal epithelial barrier function. PHGG may have a protective effect against IFN-γ-induced barrier dysfunction by attenuating the paracellular hyperpermeability; thus, its promotion as a functional food is anticipated.

The wavy growth 3 E3 ligase family controls the gravitropic response in Arabidopsis roots.

Regulation of the root growth pattern is an important control mechanism during plant growth and propagation. To better understand alterations in root growth direction in response to environmental stimuli, we have characterized an Arabidopsis thaliana mutant, wavy growth 3 (wav3), whose roots show a short-pitch pattern of wavy growth on inclined agar medium. The wav3 mutant shows a greater curvature of root bending in response to gravity, but a smaller curvature in response to light, suggesting that it is a root gravitropism-enhancing mutation. This wav3 phenotype also suggests that enhancement of the gravitropic response in roots strengthens root tip impedance after contact with the agar surface and/or causes an increase in subsequent root bending in response to obstacle-touching stimulus in these mutants. WAV3 encodes a protein with a RING finger domain, and is mainly expressed in root tips. RING-containing proteins often function as an E3 ubiquitin ligase, and the WAV3 protein shows such activity in vitro. There are three genes homologous to WAV3 in the Arabidopsis genome [EMBRYO SAC DEVELOPMENT ARREST 40 (EDA40), WAVH1 and WAVH2 ], and wav3 wavh1 wavh2 triple mutants show marked root gravitropism abnormalities. This genetic study indicates that WAV3 functions positively rather than negatively in root gravitropism, and that enhancement of the gravitropic response in wav3 roots is dependent upon the function of WAVH2 in the absence of WAV3. Hence, our results demonstrate that the WAV3 family of proteins are E3 ligases that are required for root gravitropism in Arabidopsis.

Alkoxy-auxins are selective inhibitors of auxin transport mediated by PIN, ABCB, and AUX1 transporters.

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

Role of the phytochrome and cryptochrome signaling pathways in hypocotyl phototropism.

Unilateral blue-light irradiation activates phototropin (phot) photoreceptors, resulting in asymmetric distribution of the phytohormone auxin and induction of a phototropic response in higher plants. Other photoreceptors, including phytochrome (phy) and cryptochrome (cry), have been proposed as modulators of phototropic responses. We show here that either phy or cry is required for hypocotyl phototropism in Arabidopsis thaliana under high fluence rates of blue light, and that constitutive expression of ROOT PHOTOTROPISM 2 (RPT2) and treatment with the phytohormone gibberellin (GA) biosynthesis inhibitor paclobutrazol partially and independently complement the non-phototropic hypocotyl phenotype of the phyA cry1 cry2 mutant under high fluence rates of blue light. Our results indicate that induction of RPT2 and reduction in the GA are crucial for hypocotyl phototropic regulation by phy and cry. We also show that GA suppresses hypocotyl bending via destabilization of DELLA transcriptional regulators under darkness, but does not suppress the phototropic response in the presence of either phyA or cryptochromes, suggesting that these photoreceptors control not only the GA content but also the GA sensing and/or signaling that affects hypocotyl phototropism. The metabolic and signaling regulation of not only auxin but also GA by photoreceptors therefore appears to determine the hypocotyl growth pattern, including phototropic and gravitropic responses and inhibition of hypocotyl elongation, for adaptation to various light environments.

The ABC subfamily B auxin transporter AtABCB19 is involved in the inhibitory effects of N-1-naphthyphthalamic acid on the phototropic and gravitropic responses of Arabidopsis hypocotyls.

N-1-Naphthylphthalamic acid (NPA) causes the abnormal growth and development of plants by suppressing polar auxin transport. The mechanisms underlying this inhibition, however, have remained elusive. In Arabidopsis, we show that a defect in the ABC subfamily B auxin transporter AtABCB19 suppresses the inhibitory effects of NPA on hypocotyl phototropism and gravitropism, but not on hypocotyl elongation. Expression analysis using the auxin reporter gene DR5:GUS further suggests that NPA partially inhibits the asymmetric distribution of auxin in an AtABCB19-dependent manner. These data thus suggest that AtABCB19 plays an important role in the inhibitory effects of NPA on hypocotyl tropism induced by auxin.

Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19-dependent and a PGP19-independent manner.

Photoreceptors, phytochromes and cryptochromes regulate hypocotyl growth under specific conditions, by suppressing negative gravitropism, modulating phototropism and inhibiting elongation. Although these effects seem to be partially caused via the regulation of the phytohormone auxin, the molecular mechanisms underlying this process are still poorly understood. In our present study, we demonstrate that the flabby mutation enhances both phytochrome- and cryptochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes and cryptochromes. Our current results therefore indicate that the phytochromes and cryptochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis: the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the using an auxin polar transport assay and DR5:GUS expression analysis, we further find that the phytochromes inhibit basipetal auxin transport, and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes and cryptochromes is a critical regulatory component of hypocotyl growth in response to light.

Armadillo repeat-containing kinesins and a NIMA-related kinase are required for epidermal-cell morphogenesis in Arabidopsis.

The involvement of kinesin motor proteins in both cell-tip growth and cell-shape determination has been well characterized in various organisms. However, the functions of kinesins during cell morphogenesis in higher plants remain largely unknown. In the current study, we demonstrate that an armadillo repeat-containing kinesin-related protein, ARMADILLO REPEAT KINESIN1 (ARK1), is involved in root-hair morphogenesis. Microtubule polymers are more abundant in ark1 null allele root hairs, but analysis shows that these extra microtubules are concentrated in the endoplasm, and not in the cortical array, suggesting that ARK1 regulates tip growth by limiting the assembly and distribution of endoplasmic microtubules. The ARK1 gene has two homologues in the Arabidopsis genome, ARK2 and ARK3, and our results show that ARK2 is involved in root-cell morphogenesis. We further reveal that a NIMA-related protein kinase, NEK6, binds to the ARK family proteins and has pleiotropic effects on epidermal-cell morphogenesis, suggesting that NEK6 is involved in cell morphogenesis in Arabidopsis via microtubule functions associated with these armadillo repeat-containing kinesins. We discuss the function of NIMA-related protein kinases and armadillo repeat-containing kinesins in the cell morphogenesis of eukaryotes.

Identification of a novel Cys2/His2-type zinc-finger protein as a component of a spermine-signaling pathway in tobacco.

In a previous work, we identified a Cys(2)/His(2)-type zinc-finger transcription repressor, (ZFT1), that functions in a spermine-mediated signal transduction pathway in tobacco plants. Database search disclosed the presence of another Cys(2)/His(2)-type zinc-finger protein ZFP1 (accession number AAC06243) in tobacco plants. In this work, we characterized ZFP1 and investigated whether this protein is also involved in a Spm-signaling pathway. This factor showed the highest identity to petunia ZPT2-2 and higher similarity to petunia ZPT2-3, Arabidopsis STZ/ZAT10, soybean SCOF-1, red pepper CAZFP1/CaPIF1 as well as to tobacco ZFT1. ZFP1 localized to the nucleus and had a specific DNA-binding activity, supportive to be a transcription factor. Furthermore, the protein had a mild repression activity on transcription in plant cells. The expression of ZFP1, encoding ZFP1, was upregulated during tobacco mosaic virus-induced hypersensitive response. ZFP1 expression was also induced by exogenously applied spermine and its induction was repressed by inhibitors of amine oxidase/polyamine oxidase. Collectively, our data indicate that ZFP1 is a new transcription factor which functions in a spermine-signaling pathway in tobacco.

Tobacco ZFT1, a transcriptional repressor with a Cys2/His2 type zinc finger motif that functions in spermine-signaling pathway.

We previously proposed that a spermine (Spm)-mediated signal transduction pathway is involved in the hypersensitive response induced by Tobacco mosaic virus (TMV) in tobacco plants. To identify regulatory component(s) of this pathway, we surveyed a tobacco cDNA library and found that the ZFT1 gene, which encodes a Cys2/His2 type zinc-finger protein, is Spm-responsive. ZFT1 was not induced by two other polyamines, putrescine and spermidine, or by salicylic acid (SA), jasmonic acid or ethylene. Furthermore, ZFT1 was upregulated in TMV- inoculated tobacco plants in an N gene-dependent manner. Notably, induction of ZFT1 by Spm and by TMV infection was unimpaired in NahG-transgenic tobacco plants, indicating that cross-talk with an SA signaling pathway is not involved in this response. Within the Spm-signaling pathway, we found that ZFT1 functioned downstream of both mitochondrial dysfunction and mitogen-activated protein kinase activation. The ZFT1 protein has two zinc finger motifs and shows a high degree of similarity to ZPT2-3 in petunia and SCOF1 in soybean. However, unlike the latter two proteins, ZFT1 binds to the EP1S sequence and functions as a transcription repressor. Moreover, interestingly, ZFT1 overexpression rendered tobacco plants more tolerant to TMV. Based on the results presented here, we propose that ZFT1 functions as a transcription repressor in a Spm signaling pathway, thereby accelerating necrotic local region formation in tobacco leaves.

A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco.

A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin-induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm-responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre-treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm-induced activation of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain-of-function and loss-of-function studies on MAPK cascade members reveal that the expression of Spm-induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.

Identification of tobacco HIN1 and two closely related genes as spermine-responsive genes and their differential expression during the Tobacco mosaic virus -induced hypersensitive response and during leaf- and flower-senescence.

Previously we showed that the polyamine spermine (Spm) specifically leads to mitochondrial dysfunction in tobacco that is followed by the activation of salicylic acid-induced protein kinase and wound-induced protein kinase. To identify the possible downstream components of the Spm signalling pathway, we isolated Spm-responsive genes by a differential hybridization approach. This showed that the harpin-induced 1 (HIN1) gene is responsive to Spm. Genomic Southern analysis showed that HIN1 constitutes a multi-gene family and this led to the isolation of two novel HIN1 -like tobacco cDNAs that we designated as HIN9 and HIN18. Both genes are also responsive to Spm, albeit HIN18 is induced weakly compared to HIN1 and HIN9. As HIN1 is up-regulated both during the hypersensitive response (HR) generated by an incompatible plant-pathogen interaction and during senescence, we compared the expression of the three HIN1 family genes in these situations. All three were responsive to HR due to Tobacco mosaic virus infection, although HIN18 was less efficiently induced, and HIN1 and HIN18 were both strongly up-regulated during leaf- and flower-senescence. This suggests that the signalling pathways in the HR and senescence overlap somehow but are distinct. That HIN1 and its closely related genes are Spm-responsive genes also supports the idea that Spm plays a role as a signal transmitter in the HR process.