Activity Of Intestinal Cells Research Articles

Statins inhibit protein kinase D (PKD) activation in intestinal cells and prevent PKD1-induced growth of murine enteroids.

We examined the impact of statins on protein kinase D (PKD) activation by G protein-coupled receptor (GPCR) agonists. Treatment of intestinal IEC-18 cells with cerivastatin inhibited PKD autophosphorylation at Ser916 induced by angiotensin II (ANG II) or vasopressin in a dose-dependent manner with half-maximal inhibition at 0.2 µM. Cerivastatin treatment inhibited PKD activation stimulated by these agonists for different times (5-60 min) and blunted HDAC5 phosphorylation, a substrate of PKD. Other lipophilic statins, including simvastatin, atorvastatin, and fluvastatin also prevented PKD activation in a dose-dependent manner. Using IEC-18 cell lines expressing PKD1 tagged with EGFP (enhanced green fluorescent protein), cerivastatin or simvastatin blocked GPCR-mediated PKD1-EGFP translocation to the plasma membrane and its subsequent nuclear accumulation. Similar results were obtained in IEC-18 cells expressing PKD3-EGFP. Mechanistically, statins inhibited agonist-dependent PKD activation rather than acting directly on PKD catalytic activity since exposure to cerivastatin or simvastatin did not impair PKD autophosphorylation or PKD1-EGFP membrane translocation in response to phorbol dibutyrate, which bypasses GPCRs and directly stimulates PKC and PKD. Furthermore, cerivastatin did not inhibit recombinant PKD activity determined via an in vitro kinase assay. Using enteroids generated from intestinal crypt-derived epithelial cells from PKD1 transgenic mice as a model of intestinal regeneration, we show that statins oppose PKD1-mediated increase in enteroid area, complexity (number of crypt-like buds), and DNA synthesis. Our results revealed a previously unappreciated inhibitory effect of statins on receptor-mediated PKD activation and in opposing the growth-promoting effects of PKD1 on intestinal epithelial cells.

Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans.

The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.

Acetone effects on Buddleja scordioides polyphenol extraction process and assessment of their cellular antioxidant capacity and anti-inflammatory activity

The use of water, acetone, and hydroacetonic solutions as an extraction solvent for polyphenol compounds from Buddleja scordioides has been investigated. The phenolic profiles determined by liquid chromatography—electrospray ionization—mass spectrometry/mass spectrometry (LC–ESI–MS/MS) were compared with those obtained with the traditional aqueous infusion used for the treatment of gastrointestinal disorders. It was evaluated the effects of polyphenol extract at physiologically relevant conditions against H2O2-induced oxidative stress via attenuation of reactive oxygen species (ROS) levels and lipopolysaccharide (LPS)-induced inflammation in human HT-29 cells. The use of hydroacetonic solutions allows obtaining phytochemical extracts enriched with constituents that have antioxidant and anti-inflammatory activity in intestinal cells. This suggests that polyphenols of low and middle polarity from Buddleja scordioides may be better extracted with hydroacetonic solutions, and showing high nutraceutical potential to reduce oxidative stress associated with the onset and progress of inflammatory diseases.

Comparable Immune Function Inhibition by the Infliximab Biosimilar CT-P13: Implications for Treatment of Inflammatory Bowel Disease.

CT-P13 is the first biosimilar monoclonal antibody to infliximab, and was recently approved in the European Union, Japan, Korea, and USA for all six indications of infliximab. However, studies directly assessing the biologic activity of CT-P13 versus inflximab in the context of inflammatory bowel disease [IBD] are still scanty. In the present study, we aimed to compare the biological activities of CT-P13 and infliximab with specific focus on intestinal cells so as to gain insight into the potential biosimilarity of these two agents for treatment of IBD. CT-P13 and infliximab were investigated and compared by in vitro experiments for their neutralisation ability of soluble tumour necrosis factor alpha [sTNFα] and membrane-bound tumour necrosis factor alpha [mTNFα], suppression of cytokine release by reverse signalling, induction of regulatory macrophages and wound healing, and antibody-dependent cell cytotoxicity [ADCC]. CT-P13 showed similar biological activities to infliximab as gauged by neutralisation of soluble TNFα, as well as blockade of apoptosis and suppression of pro-inflammatory cytokines in intestinal Caco-2 cells. Infliximab and CT-P13 equally induced apoptosis and outside-to-inside signals through transmembrane TNFα [tmTNFα]. Moreover, regulatory macrophage induction and ensuing wound healing were similarly exerted by CT-P13 and infliximab. However, neither CT-P13 nor infliximab exerted any significant ADCC of ex vivo-stimulated peripheral blood monocytes or lamina propria mononuclear cells from IBD patients. These findings indicate that CT-P13 and infliximab exert highly similar biological activities in intestinal cells, and further support a mechanistic comparability of these two drugs in the treatment of IBD.

Quantifying Phenotypic Variation in Isogenic Caenorhabditis elegans Expressing Phsp-16.2::gfp by Clustering 2D Expression Patterns

Isogenic populations of animals still show a surprisingly large amount of phenotypic variation between individuals. Using a GFP reporter that has been shown to predict longevity and resistance to stress in isogenic populations of the nematode Caenorhabditis elegans, we examined residual variation in expression of this GFP reporter. We found that when we separated the populations into brightest 3% and dimmest 3% we also saw variation in relative expression patterns that distinguished the bright and dim worms. Using a novel image processing method which is capable of directly analyzing worm images, we found that bright worms (after normalization to remove variation between bright and dim worms) had expression patterns that correlated with other bright worms but that dim worms fell into two distinct expression patterns. We have analysed a small set of worms with confocal microscopy to validate these findings, and found that the activity loci in these clusters are caused by extremely bright intestine cells. We also found that the vast majority of the fluorescent signal for all worms came from intestinal cells as well, which may indicate that the activity of intestinal cells is responsible for the observed patterns. Phenotypic variation in C. elegans is still not well understood but our proposed novel method to analyze complex expression patterns offers a way to enable a better understanding.

Gliadins induce TNFα production through cAMP-dependent protein kinase A activation in intestinal cells (Caco-2)

Celiac disease is an autoimmune enteropathy caused by a permanent intolerance to gliadins. In this study the effects of two gliadin-derived peptides (PA2, PQPQLPYPQPQLP and PA9, QLQPFPQPQLPY) on TNFalpha production by intestinal epithelial cells (Caco-2) and whether these effects were related to protein kinase A (PKA) and/or -C (PKC) activities have been evaluated. Caco-2 cell cultures were challenged with several sets of gliadin peptides solutions (0.25 mg/mL), with/without different activators of PKA or PKC, bradykinin (Brdkn) and pyrrolidine dithiocarbamate (PDTC). The gliadin-derived peptides assayed represent the two major immunodominant epitopes of the peptide 33-mer of alpha-gliadin (56-88) (LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF). Both peptides induced the TNFalpha production triggering the inflammatory cell responses, the PA2 being more effective. The addition of the peptides in the presence of dibutyril cyclic AMP (cAMP), Brdkn or PDTC, inhibited the TNFalpha production. The PKC-activator phorbol 12-myristate 13-diacetate additionally increased the PA2- and PA9-induced TNFalpha production. These results link the gliadin-derived peptides induced TNFalpha production through cAMP-dependent PKA activation, where ion channels controlling calcium influx into cells could play a protective role, and requires NF-kappaB activation.

Modulation of cytochrome P450 1A1 expression and activity in intestinal Caco-2 cells by components of Ginkgo biloba-based dietary supplements

Ginkgo biloba is largely consumed as food supplement, because it could contribute to memory improvement by acting on cerebrovascular insufficiency. Among the large variety of phytochemicals contained in Ginkgo biloba leaves and extracts, flavonols (mainly kaempferol, quercetin and isorhamnetin) represent the largest part. Several unique terpenoids (ginkgolides A, B, C, J and bilobalide) are also present. In the present study, we investigated the modulation of cytochrome P450 (CYP) 1A1 enzymatic activity and expression in the intestinal Caco-2 cell line by either Ginkgo biloba extracts, by major individual secondary metabolites or by mixtures of these constituents in realistic proportions. Plausible used intestinal concentrations were calculated on the basis of recommended daily intake and quantitative analysis of major constituents in dietary supplements. Preliminary results suggest that (−)-bilobalide, but not the other terpenoids, induces a dose-dependent increase of CYP1A1 activity (measured through 7-ethoxyresorufin O-deethylation, EROD) after 6 h treatment of the Caco-2 cells. RT-PCR highlighted an increase of mRNA after 3 h treatment with (−)-bilobalide, suggesting the involvement of a transcriptional mechanism. Furthermore, a strong dose-dependent inhibition of benzo[a]pyrene (B[a]P)-induced EROD activity was shown after 6 h co-treatment with kaempferol, quercetin or isorhamnetin. This antagonistic effect was conserved with mixtures of the three flavonols and of the eight pre-cited major secondary metabolites. These preliminary results suggest that frequent consumption of Ginkgo biloba-based dietary supplements may influence CYP1A1 expression and activity in intestinal cells, leading to possible toxicological interactions.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-δ pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H2O2, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H2O2 in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC-delta inhibitor, and by small interfering RNA (siRNA) directed against PKC-delta, suggesting the regulation of PKD activity by upstream PKC-delta. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC-delta phosphorylation was inhibited by C3 treatment, further suggesting that PKC-delta is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC-delta and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).

Omega-3 fatty acids in colorectal cancer prevention.

Omega-3 fatty acids in colorectal cancer prevention.

Expression of the reduced folate carrier SLC19A1 in IEC-6 cells results in two distinct transport activities.

Intestinal absorption of folates has been characterized as a facilitative process with a low pH optimum. Studies with intestinal epithelial cells have suggested that this activity is mediated by the reduced folate carrier (RFC1). In this paper, we report on folate transport characteristics in an immortalized rat IEC-6 cell line that was found to exhibit the predominant influx activity for methotrexate (MTX) at pH 5.5 with a low level of activity at pH 7.4. Transfection of this cell line with an RFC1 construct resulted in clones exhibiting increased MTX uptake at both the pHs and high folic acid uptake only at the low pH. For the two clones with the highest level of transport activity, relative MTX influx at the two pHs was reversed. Moreover, the low pH MTX influx activity ([MTX](e) = 0.5 microM) was markedly inhibited by 20 microM folic acid while influx at neutral pH was not. Furthermore, in the presence and absence of glucose at low pH, MTX and folic acid influx activity was inhibited by azide, while MTX influx at pH 7.4 was stimulated by azide in the absence of glucose but was unchanged in the presence of glucose and azide. This was contrasted with the results of transfection of the same RFC1 construct into an L1210 murine leukemia cell line bearing a nonfunctional endogenous carrier. In this case, the activity expressed was only at pH 7.4. These data indicate that RFC1 can exhibit two distinct types of folate transport activities in intestinal cells that must depend on tissue-specific modulators.

Modulation of inducible nitric oxide synthase expression in rat intestinal cells by colon tumor promoters.

Metabolic epidemiological studies in humans and laboratory animal models demonstrate that consumption of diets high in fat and low in fiber excrete increased levels of 1,2-diacyl-sn-glycerol (DAG) and secondary bile acids such as deoxycholic acid (DA) and lithocholic acid that have been shown to promote colon carcinogenesis. The secondary bile acids and DAG have been shown to activate protein kinase C (PKC) and induce colonic cell proliferation. A large body of evidence indicates that iNOS, an inducible isoform of nitric oxide synthase, is over-expressed in human colon adenomas and in chemically-induced colon tumors of laboratory animals. However, the precise cascade of intracellular events that leads to the iNOS over-expression remains to be fully explored. In this study, we investigated the relationship between induction of iNOS and activation of the PKC pathway by DAG and DC, in an in vitro system using the rat intestinal cell line, RIE-1. As an initial step, we determined whether lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) modulate iNOS protein expression in RIE-1 cells. Treatment of RIE-1 cells with LPS and PMA for 4 h significantly elevated the iNOS protein expression. The induction of iNOS by the treatment with LPS/PMA was concentration- and time-dependent. Treatment with LPS/DAG or LPS/DC also caused iNOS over-expression in a concentration- and time-dependent fashion suggesting that DAG and DC induce iNOS activity in intestinal cells. Pretreatment with specific PKC inhibitors, bisindolylmaleimide I or Gö 6976, inhibited LPS/PMA, LPS/DAG, or LPS/DC-induced iNOS expression and activity. Extracts of the cells treated with LPS/PMA, LPS/DAG or LPS/DC had a high iNOS activity compared to that of control (p<0.04 to p<0.0001). Taken together, our data suggest a possible role of colon tumor promoters, DAG and DC, for iNOS over-expression through activation of the PKC pathway.

Linoleic acid and tumor necrosis factor- α increase manganese superoxide dismutase activity in intestinal cells

Manganese superoxide dismutase (MnSOD) protects mitochondria from oxidative damage. Alterations in the regulation of MnSOD plays an important role in the development of many types of cancer. Activity of this enzyme is induced by inflammatory cytokines and other conditions that increase oxygen radical production. High levels of dietary lipid have been shown to decrease MnSOD activity. This study was designed to define the effect of various type of fatty acids on MnSOD activity and MnSOD induction. IEC-6 cells were treated with 40 μmol/l of either linoleic acid (LA), eicosapentaenoic acid (EPA), or oleic acid (OA) in the presence or absence of 10 ng/ml tumor necrosis factor- α (TNF- α). Fatty acid supplementation increased MnSOD activity. MnSOD activity was greater in the LA group than in the EPA or OA groups. TNF- α induced MnSOD activity equally in all fatty acid-supplemented groups. High levels of MnSOD activity may be an indicator of chronic inflammation resulting from fatty acid, particularly LA, supplementation.

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

Studies of Cytotoxicity and Metabolic Competence with the Rat Intestinal Cell Line Iec-17

The characterization of metabolic activity of intestinal cells in culture may be of particular interest for the study of the effects of food additives and contaminants which, ingested through the diet, are absorbed primarily by the gut mucosa. IEC-17 cell line is derived from the intestine of newborn rat and has been shown to be competent for the metabolism of xenobiotics (Quaroni and Isselbacher, 1981), and to keep some degree of differentiation also in in vitro conditions. Our studies indicate that the xenobiotic metabolic activity of this cell line, as shown by measurement of 7-ethoxycoumarin (7-EC), can be induced by beta-naphthoflavone (NF) and not by phenobarbital (PB) and only at late subculturing stages, suggesting these cells undergo some kind of maturation in vitro. Induction of xenobiotic metabolism by beta-naphthoflavone seems also to elicit the toxicity of 9,10-dimethyl-1,2-benzathracene (DBA) but not that one of cyclophosphamide (CPA).

Vasoactive Intestinal Peptide Receptor Activity in Intestinal Cells Isolated from Human Fetuses with Cystic Fibrosis. Comparison with the Livera

Vasoactive Intestinal Peptide Receptor Activity in Intestinal Cells Isolated from Human Fetuses with Cystic Fibrosis. Comparison with the Liver^a

Release of phospholipase A and triglyceride lipase from rat liver

A new, rapid, and sensitive assay for phospholipase A, utilizing commercially available [14C]phosphatidylethanolamine with 14C label in both palmitic acid moieties, was used to study phospholipase A release from perfused liver, hepatocytes, and intestinal cells from rats. Heparin triggered a prompt release of phospholipase A from perfused liver. Phospholipase A and triglyceride lipase were released from hepatocytes at a linear rate for 1 h and 30 min, respectively. Heparin (20 u/ml) doubled the release of phospholipase A and triglyceride lipase from hepatocytes. Colchicine (0.1 mM), but not puromycin (0.2 mM), inhibited basal and heparin-stimulated phospholipase A release by 40%. Since the amount of phospholipase A and triglyceride lipase released into the medium greatly exceeded intracellular activities, it is possible that secretion is coupled with intracellular conversion from inactive to active forms of the enzymes. Dibutyryl cyclic AMP (1 mM) inhibited phospholipase A (48%) and triglyceride lipase (82%) release from hepatocytes. Epinephrine, dexamethasone, and clofibrate inhibited release of triglyceride lipase but not phospholipase A. Phospholipase A activity of intestinal cells was greater than in hepatocytes, but neither heparin nor dibutyryl cyclic AMP affected phospholipase A release from intestinal cells. These results suggest that the liver is a major source of phospholipase A of postheparin plasma. The fact that dibutyryl cyclic AMP affects the release of these enzymes suggests an additional mechanism for hormonal regulation of lipid and lipoprotein metabolism.

Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.

The Secretory Activity of Ascaris suum Intestine

Large amounts of granules, interpreted as secretory in nature, are found in the terminal web region of intestinal cells of freshly collected Ascaris suum. Morphologically identical granules are scattered throughout the apical half of the cell cytoplasm and appear to be formed at the lateral ends of the Golgi saccules. Silver-proteinate-stained sections indicate similar staining properties of glycoprotein or polysaccharide nature for the Golgi saccules and granules, the apical granules, and the cell coat. Unlike the microvillous border, lysosomes, multivesicular bodies, and some Golgi saccules, the secretory granules do not stain for acid phosphatase. Differences in secretory activity between intestinal cells from A. suum immediately fixed after collection and from parasites maintained in vitro for several hours are reported. The functional significance of this activity in digestive and absorption processes is discussed. Contrary to the large amount of work devoted to the secretory processes of intestinal cells in mammals and some parasites only scanty morphologic data are available on this subject for Ascaris suum (Kessel et al., 1961; Sheffield, 1964). The origin and the nature of secretory substances in intestinal cells of mammals have been the subjects of many investigations. The involvement of the Golgi apparatus in the production of cell coat material and the role of Golgi vesicles and lysosomes in the transport to the cell surface has been emphasized (Ito, 1969; Bennett, 1970; Bennett and Leblond, 1970, 1971; Rambourg et al., 1971; Whaley et al., 1972). Several hydrolytic and proteolytic enzymes have been located on or near the apical surface of A. suum intestinal cells (Lee, 1962a, b; Borgers et al., 1970; Gentner et al., 1972; Van den Bossche and Borgers, 1973; Beames et al., 1974). Recent observations in our laboratory indicated that the secretory status of A. suum intestine varied with the time of fixation after collection from the host and stressed the importance of working with parasites fixed immediately after removal from the host. The aim of this morphologic investigation is to describe the secretory activity of intestinal cells of freshly collected worms and to determine the nature and origin of the secretion products with the aid of ultrastructural cyto-