Role In Intestinal Development Research Articles

Improvement in growth performance and digestive function from amniotic injections of N-acetylglutamate in broiler chickens.

N-acetylglutamate (NAG) is the initial and essectial substrate in the process of de novo arginine synthesis, plays an important role in intestinal development. The aim of this study was to determine the effects of in ovo feeding of NAG, 1.5 mg/egg at 17.5 days of incubation (DOI) via amnion, on hatching performance, early intestinal histomorphometry, jejunal barrier, digestive function, and growth performance of broiler chickens between 1 and 14 days of age. Amniotic injection of NAG had no significant effect on hatching characteristics compared with the non-injected control group (NC group). Birds in the NAG solution-injected group (NAG group) exhibited lower average daily feed intake and better feed efficiency during a period of 1-14 days. In comparison with the NC group, the NAG group had decreased crypt depth (CD) in the ileum and increased villus height (VH) / CD in the jejunum at 7 days, and decreased CD in duodenum and significantly increased VH in the jejunum at 14 days. However, the effects of in ovo supplementation with NAG on the density of goblet cells, and gene expression of mucin 2 and alkaline phosphatase were not significant. Chicks in the NAG group had a significantly higher mRNA expression level of trypsin and maltase in jejunum at 7 days than the NC group but not at 14 days. Amniotic injections of NAG (1.5 mg/egg) at 17.5 DOI could improve early growth performance of broilers during 1-14 days after hatching by accelerating the development of the intestine and enhancing jejunal digestive function. © 2023 Society of Chemical Industry.

The regulatory relationship between RagA and Nprl2 in Drosophila gut development

The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.

S-adenosyl-L-methionine supplementation alleviates damaged intestinal epithelium and inflammatory infiltration caused by Mat2a deficiency.

Methionine is important for intestinal development and homeostasis in various organisms. However, the underlying mechanisms are poorly understood. Here, we demonstrate that the methionine adenosyltransferase gene Mat2a is essential for intestinal development and that the metabolite S-adenosyl-L-methionine (SAM) plays an important role in intestinal homeostasis. Intestinal epithelial cell (IEC)-specific knockout of Mat2a exhibits impaired intestinal development and neonatal lethality. Mat2a deletion in the adult intestine reduces cell proliferation and triggers IEC apoptosis, leading to severe intestinal epithelial atrophy and intestinal inflammation. Mechanistically, we reveal that SAM maintains the integrity of differentiated epithelium and protects IECs from apoptosis by suppressing the expression of caspases 3 and 8 and their activation. SAM supplementation improves the defective intestinal epithelium and reduces inflammatory infiltration sequentially. In conclusion, our study demonstrates that methionine metabolism and its intermediate metabolite SAM play essential roles in intestinal development and homeostasis in mice.

Effects of Oral Glutamine Supplementation, Birthweight and Age on Colonic Morphology and Microbiome Development in Male Suckling Piglets.

Mortality, impaired development and metabolic dysfunctions of suckling low-birthweight piglets may be influenced by modulating the intestinal microbiome through glutamine supplementation. Therefore, this study examined whether glutamine supplementation may affect the colonic development and microbiome composition of male low- and normal-birthweight piglets at 5 and 12 days of age. Suckling piglets were supplemented orally with glutamine or alanine. Colonic digesta samples were obtained for 16S rDNA sequencing, determination of bacterial metabolites and histomorphological tissue analyses. Glutamine-supplemented piglets had lower concentrations of cadaverine and spermidine in the colonic digesta (p < 0.05) and a higher number of CD3+ colonic intraepithelial lymphocytes compared to alanine-supplemented piglets (p < 0.05). Low-birthweight piglets were characterised by a lower relative abundance of Firmicutes, the genera Negativibacillus and Faecalibacterium and a higher abundance of Alistipes (p < 0.05). Concentrations of cadaverine and total biogenic amines (p < 0.05) and CD3+ intraepithelial lymphocytes (p < 0.05) were lower in low- compared with normal-birthweight piglets. In comparison to the factor age, glutamine supplementation and birthweight were associated with minor changes in microbial and histological characteristics of the colon, indicating that ontogenetic factors play a more important role in intestinal development.

The role of orally ingested milk fat globule membrane on intestinal barrier functions evaluated with a suckling rat pup supplementation model and a human enterocyte model

Milk fat globule membrane (MFGM), the membrane surrounding secreted fat droplets in milk, contains components involved in a wide range of bioprocesses including cell proliferation and differentiation. The intestine is relatively immature and permeable at birth. Since MFGM is partly resistant to digestion in infancy, we hypothesized that orally ingested MFGM promotes intestinal development by enhancing intestinal barrier functions in early life. An established suckling rat model was used; Sprague–Dawley rats were bred, and litters were culled to 10 pups/dam. Pups were supplemented orally with MFGM (0, 100, or 300 mg/kg/d) from postnatal day 1–20. Intestine samples were collected for histology, real-time quantitative PCR, immunoblotting, and immunohistochemistry analysis. Additionally, differentiated Caco-2 cells were used to assess effects of MFGM on the human intestinal barrier. Control and MFGM-supplemented rat pups showed similar growth. Intestinal differentiation and expression of tight junction proteins in jejunum and colon were significantly increased by orally ingested MFGM, and MFGM supplementation significantly activated PI3K/Akt/mTOR, mitogen-activated protein kinases, and myosin light chain kinase signaling pathways, suggesting that MFGM promotes intestinal development by triggering various signaling pathways. In human enterocytes (polarized Caco-2 cells), MFGM (400 µg/mL for 72 h) decreased permeability, as revealed by increased transepithelial electrical resistance. In Caco-2 cells, MFGM also enhanced expression of tight junction proteins, including claudin-4 and ZO-2. In conclusion, orally ingested MFGM may exert beneficial roles in intestinal development by activating various cell signaling pathways to upregulate tight junction proteins and thereby increasing intestinal barrier functions.

Supplemental L-Arginine Improves the Embryonic Intestine Development and Microbial Succession in a Chick Embryo Model.

Early colonization of intestinal microbiota plays an important role in intestinal development. However, the microbial succession at an embryonic stage and its assembly patterns induced by prenatal nutrition are unknown. In the present study, we used a chick embryo model to investigate the effects of in ovo feeding (IOF) of L-arginine (Arg) on the intestinal development and microbial succession of embryos. A total of 216 fertile eggs were randomly distributed into 2 groups including the non-injected control group and IOF of Arg group with 7 mg/egg. The results showed that IOF Arg increased the intestinal index, absolute weight of jejunum, and improved jejunal morphology in terms of villus width and surface area (p < 0.05). The relative mRNA expressions of mTOR and 4E-BP1 were up-regulated and accompanied by higher contents of Mucin-2 in the Arg group (p < 0.05). There was a significant elevation in contents of serum glucose and high-density lipoprotein cholesterol, whereas there was a decreased low-density lipoprotein cholesterol in the Arg group (p < 0.05). Additionally, Proteobacteria and Firmicutes were major intestinal bacteria species at the embryonic stage. However, Arg supplementation targeted to shape assembly patterns of microbial succession and then changed microbial composition (p = 0.05). Meanwhile, several short-chain fatty acids (SCFAs)-producing bacteria, such as Roseburia, Blautia, and Ruminococcus were identified as biomarkers in the Arg group (LDA > 3, p < 0.05). Accordingly, significant elevated concentrations of SCFAs, including lactic acid and formic acid, were observed in the Arg group (p < 0.05), accompanied by the higher concentration of butyric acid (0.05 < p < 0.10). In conclusion, prenatal Arg supplementation improved embryonic intestine development by regulating glucose and lipid homeostasis to supply more energy for chick embryos. The possible mechanism could be the roles of Arg in shaping the microbial assembly pattern and succession of the embryonic intestine, particularly the enrichment of potential probiotics. These findings may contribute to exploring nutritional strategies to establish health-promoting microbiota by manipulating prenatal host-microbe interactions for the healthy development of neonates.

Cdk5rap3 is essential for intestinal Paneth cell development and maintenance

Intestinal Paneth cells are professional exocrine cells that play crucial roles in maintenance of homeostatic microbiome, modulation of mucosal immunity, and support for stem cell self-renewal. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Cdk5 activator binding protein Cdk5rap3 (also known as C53 and LZAP) was originally identified as a binding protein of Cdk5 activator p35. Although previous studies have indicated its involvement in a wide range of signaling pathways, the physiological function of Cdk5rap3 remains largely undefined. In this study, we found that Cdk5rap3 deficiency resulted in very early embryonic lethality, indicating its indispensable role in embryogenesis. To further investigate its function in the adult tissues and organs, we generated intestinal epithelial cell (IEC)-specific knockout mouse model to examine its role in intestinal development and tissue homeostasis. IEC-specific deletion of Cdk5rap3 led to nearly complete loss of Paneth cells and increased susceptibility to experimentally induced colitis. Interestingly, Cdk5rap3 deficiency resulted in downregulation of key transcription factors Gfi1 and Sox9, indicating its crucial role in Paneth cell fate specification. Furthermore, Cdk5rap3 is highly expressed in mature Paneth cells. Paneth cell-specific knockout of Cdk5rap3 caused partial loss of Paneth cells, while inducible acute deletion of Cdk5rap3 resulted in disassembly of the rough endoplasmic reticulum (RER) and abnormal zymogen granules in the mature Paneth cells, as well as loss of Paneth cells. Together, our results provide definitive evidence for the essential role of Cdk5rap3 in Paneth cell development and maintenance.

Gut Microbiota-Derived Metabolite Signature in Suckling and Weaned Piglets.

The gut microbiota plays a key role in intestinal development at the suckling-to-weaning transition. The objective of this study was to analyze the production of metabolites by the gut microbiota in suckling and weaned piglets. We studied piglets raised in two separate maternity farms and weaned at postnatal day 21 in the same farm. The fecal metabolome (1H nuclear magnetic resonance) and the microbiota composition (16S rRNA gene amplicon sequencing) and its predicted functions (PICRUSt2) were analyzed in the same piglets during the suckling period (postnatal day 13) and 2 days after weaning (postnatal day 23). The relative concentrations of the bacterial metabolites methylamine, dimethylamine, cadaverine, tyramine, putrescine, 5-aminovalerate, succinate, and 3-(4-hydroxyphenylpropionate) were higher during the suckling period than after weaning. In contrast, the relative concentrations of the short-chain fatty acids acetate and propionate were higher after weaning than during the suckling period. The maternity of origin of piglets also influenced the level of some bacterial metabolites (propionate and isobutyrate). The fecal metabolome signatures observed in suckling and weaned piglets were associated with specific microbiota-predicted functionalities, structure, and diversity. Gut microbiota-derived metabolites, which are differentially abundant between suckling and weaned piglets (e.g., short-chain fatty acids and biogenic amines), are known to regulate gut health. Thus, identification of metabolome signatures in suckling and weaned piglets paves the way for the development of health-promoting nutritional strategies, targeting the production of bacterial metabolites in early life.

Comparisons between Animal Models and Intestinal Crypt Cultures when Investigating the Impact of Fatty Acids on Gene Expression in Intestinal Epithelial Cells

Developmental delay of UDP‐glucuronosyltransferase 1A1 (UGT1A1) expression is central toward the development of neonatal hyperbilirubinemia, observed clinically as increased total serum bilirubin (TSB) in newborns. UGT1A1 is the sole enzyme responsible for the glucuronidation and detoxification of bilirubin. We have developed a murine model that resembles neonatal hyperbilirubinemia. In humanized UGT1 (hUGT1) mice, the expression of intestinal but not hepatic UGT1A1 is inversely associated with TSB levels. In addition, the expression of intestinal UGT1A1 is closely associated with proliferation and maturation of intestinal epithelial cells (IECs). It is known that fatty acids (FAs), the major component of breast milk, plays an important role in intestinal development. We have treated hUGT1 neonatal mice with oleic acid (OA, 16:1), a monounsaturated fatty acid. Following OA exposure, TSB levels in hUGT1 neonatal mice were reduced significantly, and RT‐qPCR analysis demonstrated that intestinal human UGT1A1 was induced, along with increased expression of Cyp4a10, a target gene of peroxisome proliferator‐activated receptor alpha (PPARα). In addition, IEC maturation markers including sucrase‐isomaltase (Sis) and alkaline phosphatase 3 (Akp3) are also induced. To examine the mechanism of OA induction, intestinal crypt cells were isolated from hUGT1 mice. The organoids were exposed to different fatty acids, including OA, palmitoleic acid (PA), arachidonic acid (AA), linoleic acid (LA), and docosahexaenoic acid (DHA). Gene expression parameters were monitored 24 hours after exposure. We found that OA‐ and PA‐treated organoids have similar survival rates in comparison to controls, but at similar concentrations of LA, DHA or AA cell death and decreased organoid viability was observed. At lower FA concentrations, RT‐qPCR analysis demonstrated that the UGT1A1 gene was not induced in any of the samples, including the IEC maturation markers Sis and Akp3. However, increased expression of the Cyp4a10 gene resulted in DHA, AA, and LA‐treated organoids. By comparing the in vivo and in vitro experiments, it suggests that unlike Cyp4a10, the regulation of UGT1A1 by fatty acids is associated with the development of the intestinal tract. Under current culture conditions, the application of intestinal organoid cultures in the study of developmental‐associated events is limited.Support or Funding InformationSupported in part by NIH grants GM126074 and ES010337

Mechanically induced development and maturation of human intestinal organoids in vivo.

The natural ability of stem cells to self-organize into functional tissue has been harnessed for the production of functional human intestinal organoids. Although dynamic mechanical forces play a central role in intestinal development and morphogenesis, conventional methods for the generation of intestinal organoids have relied solely on biological factors. Here, we show that the incorporation of uniaxial strain, by using compressed nitinol springs, in human intestinal organoids transplanted into the mesentery of mice induces growth and maturation of the organoids. Assessment of morphometric parameters, transcriptome profiling, and functional assays of the strain-exposed tissue revealed higher similarities to native human intestine, with regards to tissue size and complexity, and muscle tone. Our findings suggest that the incorporation of physiologically relevant mechanical cues during the development of human intestinal tissue enhances its maturation and enterogenesis.

Exposure to the gut microbiota drives distinct methylome and transcriptome changes in intestinal epithelial cells during postnatal development

BackgroundThe interplay of epigenetic processes and the intestinal microbiota may play an important role in intestinal development and homeostasis. Previous studies have established that the microbiota regulates a large proportion of the intestinal epithelial transcriptome in the adult host, but microbial effects on DNA methylation and gene expression during early postnatal development are still poorly understood. Here, we sought to investigate the microbial effects on DNA methylation and the transcriptome of intestinal epithelial cells (IECs) during postnatal development.MethodsWe collected IECs from the small intestine of each of five 1-, 4- and 12 to 16-week-old mice representing the infant, juvenile, and adult states, raised either in the presence or absence of a microbiota. The DNA methylation profile was determined using reduced representation bisulfite sequencing (RRBS) and the epithelial transcriptome by RNA sequencing using paired samples from each individual mouse to analyze the link between microbiota, gene expression, and DNA methylation.ResultsWe found that microbiota-dependent and -independent processes act together to shape the postnatal development of the transcriptome and DNA methylation signatures of IECs. The bacterial effect on the transcriptome increased over time, whereas most microbiota-dependent DNA methylation differences were detected already early after birth. Microbiota-responsive transcripts could be attributed to stage-specific cellular programs during postnatal development and regulated gene sets involved primarily immune pathways and metabolic processes. Integrated analysis of the methylome and transcriptome data identified 126 genomic loci at which coupled differential DNA methylation and RNA transcription were associated with the presence of intestinal microbiota. We validated a subset of differentially expressed and methylated genes in an independent mouse cohort, indicating the existence of microbiota-dependent “functional” methylation sites which may impact on long-term gene expression signatures in IECs.ConclusionsOur study represents the first genome-wide analysis of microbiota-mediated effects on maturation of DNA methylation signatures and the transcriptional program of IECs after birth. It indicates that the gut microbiota dynamically modulates large portions of the epithelial transcriptome during postnatal development, but targets only a subset of microbially responsive genes through their DNA methylation status.

L-Glutamine Attenuates Apoptosis in Porcine Enterocytes by Regulating Glutathione-Related Redox Homeostasis

BackgroundProgrammed cell death plays a fundamental role in intestinal development and mucosal homeostasis. Dysregulation of these processes is associated with an impaired intestinal-mucosal barrier, reduced nutrient absorption, and initiation and progression of intestinal diseases. 4-Hydroxy-2-nonenal (4-HNE), a product of lipid peroxidation, is commonly used to induce oxidative stress in cells. l-Glutamine is known to protect cells from apoptosis. However, the underlying mechanisms are largely unknown. ObjectiveThis study was conducted to test the hypothesis that l-glutamine attenuates 4-HNE–induced apoptosis by modulating glutathione (GSH) and thioredoxin (TXN) antioxidant systems and the expression of genes involved in 4-HNE metabolism in enterocytes. MethodsIntestinal porcine epithelial cell line 1 (IPEC-1) cells were cultured with or without 4-HNE (30 μmol/L) in the presence of 0.05 or 0.25 mmol l-glutamine/L (a physiological concentration in the lumen of the small intestine) for indicated time periods. Cell viability, abundances of apoptotic proteins, mitochondrial membrane depolarization, production of reactive oxygen species (ROS) and GSH, and expression of genes involved in the biosynthesis of GSH, thioredoxin, and 4-HNE metabolism were determined. ResultsCompared with basal medium containing 0.05 mmol l-glutamine/L, 4-HNE enhanced apoptosis by 19.6% (P < 0.05) in a caspase-3-dependent manner. This effect was accompanied by elevated intracellular ROS production (39.5% and 85.3% for 2- and 4-h treatment, respectively), increased mitochondrial depolarization by 80%, and decreased intracellular GSH concentrations by 17.7%. These effects of 4-HNE were reduced by 0.25 mmol l-glutamine/L. Further study showed that the protective effect of l-glutamine was associated with the enhanced expression of genes involved in GSH production (including GCLC, GCLM, GSR, CBS, and CTH) by 3.9–14-fold, as well as genes involved in 4-HNE metabolism [e.g., glutathione S-transferase A (GSTA)1 and GSTA4] by 1.9–7.2-fold. The mRNA levels for ADH5, AKR1C1, AKR1A1, and TXNRD1 were enhanced 1.4–8.8-fold by 4-HNE but were not changed in cells co-treated with 4-HNE and l-glutamine. ConclusionThese findings indicate that l-glutamine attenuates 4-HNE–induced apoptosis by regulating GSH-related redox homeostasis and enhancing GSTA-mediated metabolism in enterocytes.

Maternal exposure to a Western-style diet causes differences in intestinal microbiota composition and gene expression of suckling mouse pups.

ScopeThe long‐lasting consequences of nutritional programming during the early phase of life have become increasingly evident. The effects of maternal nutrition on the developing intestine are still underexplored.Methods and resultsIn this study, we observed (1) altered microbiota composition of the colonic luminal content, and (2) differential gene expression in the intestinal wall in 2‐week‐old mouse pups born from dams exposed to a Western‐style (WS) diet during the perinatal period. A sexually dimorphic effect was found for the differentially expressed genes in the offspring of WS diet‐exposed dams but no differences between male and female pups were found for the microbiota composition.Integrative analysis of the microbiota and gene expression data revealed that the maternal WS diet independently affected gene expression and microbiota composition. However, the abundance of bacterial families not affected by the WS diet (Bacteroidaceae, Porphyromonadaceae, and Lachnospiraceae) correlated with the expression of genes playing a key role in intestinal development and functioning (e.g. Pitx2 and Ace2).ConclusionOur data reveal that maternal consumption of a WS diet during the perinatal period alters both gene expression and microbiota composition in the intestinal tract of 2‐week‐old offspring.

669 The CDC42/RAC Nucleotide Exchange Factor βPix Plays a Key Role in Intestinal Development and Neoplasia

669 The CDC42/RAC Nucleotide Exchange Factor βPix Plays a Key Role in Intestinal Development and Neoplasia

Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome.

BackgroundDNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited.ResultsWe use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3′ CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3′ CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3′ CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3′ CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions.ConclusionsOur results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0763-5) contains supplementary material, which is available to authorized users.

Bovine lactoferrin is a transcription factor stimulating IL‐1 beta gene expression in the small intestine

Lactoferrin (Lf) is an iron‐binding glycoprotein present in high concentration in human milk. It is a pleiotropic protein and involved in diverse bioactivities. Since Lf is partly resistant to proteolysis in the gastrointestinal tract, Lf may play important roles in intestinal development. Human Lf (hLf) is internalized by human intestinal epithelial cells (Caco‐2) and translocates to the nucleus where it functions as a transcription factor to up‐regulate expression of genes, such as IL‐1 beta. Bovine Lf (bLf) shares 69% amino acid identity with hLf and has been shown to have similar physiological functions as hLf. Therefore, we hypothesized that bLf is able to enter the nucleus of Caco‐2 and transactivate gene transcription. Confocal microscopy and immunoblotting using nuclear fractions showed that bLf was internalized and localized to the nucleus of Caco‐2. After Caco‐2 cells were treated with bLf, IL‐1 beta transcription was two fold up‐regulated as revealed by RT‐PCR. To determine if bLf binds to the promoter of IL‐1 gene, an IL‐1 beta promoter fragment containing a hLf potential DNA binding site were then inserted to a luciferase promoter‐less vector and luciferase assays were conducted. The results show that bLf binds to the IL‐1 beta promoter and activates IL‐1 beta transcription. Results from chromatin immunoprecipitation (ChIp) assays also demonstrated that bLf interacts with the potential hLf DNA binding site in the promoter of the IL‐1 beta gene in vivo. In conclusion, bLf is able to enter the nucleus of human intestinal epithelial cells, bind to the promoter of IL‐1 gene and functionally regulate gene transcription. It is therefore possible that bLf may insert some of the biological activities of Lf in human milk. Supported by Mead Johnson Nutrition.

Colocalization of β-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation.

The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

Transcriptomic profiling of intestinal epithelial cells in response to human, bovine and commercial bovine lactoferrins.

Lactoferrin (Lf) is an iron-binding glycoprotein present in high concentration in human milk. It is a pleiotropic protein and involved in diverse bioactivities, such as stimulation of cell proliferation and immunomodulatory activities. Lf is partly resistant to proteolysis in the gastrointestinal tract. Thus, Lf may play important roles in intestinal development. Due to differences in amino acid sequences and isolation methods, Lfs from human and bovine milk as well as commercially available bovine Lf (CbLf) may differ functionally or exert their functions via various mechanisms. To provide a potential basis for further applications of CbLf, we compared effects of Lfs on intestinal transcriptomic profiling using an intestinal epithelial cell model, human intestinal epithelial crypt-like cells (HIEC). All Lfs significantly stimulated proliferation of HIEC and no significant differences were found among these three proteins. Microarray assays were used to investigate transcriptomic profiling of intestinal epithelial cells in response to Lfs. Selected genes were verified by RT-PCR with a high validation rate. Genes significantly regulated by hLf, bLf, and CbLf were 150, 395 and 453, respectively. Fifty-four genes were significantly regulated by both hLf and CbLf, whereas 129 genes were significantly modulated by bLf and CbLf. Although only a limited number of genes were regulated by all Lfs, the three Lfs positively influenced cellular development and immune functions based on pathway analysis using IPA (Ingenuity). Lfs stimulate cellular and intestinal development and immune functions via various signaling pathways, such as Wnt/β-catenin signaling, interferon signaling and IL-8 signaling.

Transcriptomic profiling of intestinal epithelial cells in response to human, bovine and commercial bovine lactoferrins (623.2)

Lactoferrin (Lf) is an iron‐binding protein abundantly present in human milk. It is multifunctional and comparatively resistant to gastrointestinal proteolysis. Thus, Lf may play critical roles in intestinal development. Due to differences in sequences and isolation methods, Lfs from human and bovine milk as well as commercial available bovine Lf (CbLf) may differ functionally or exert functions diversely. The objective of the present study was to examine and compare effects of CbLf and Lfs isolated from human and bovine milk on intestinal epithelial cells and thus to provide a basis for applications of CbLf. Normal crypt‐like human intestinal epithelial crypt cells (HIEC) were used to evaluate effects of Lfs on intestinal cell proliferation and transcriptomic profiling. All Lfs (4‐50 µg/mL) significantly stimulated proliferation of HIEC and no differences were found among Lfs. Microarray assays (Illumina) were then conducted to investigate transcriptomic profiling of HIEC in response to Lfs. Genes markedly (&gt;1.5 or &lt; 0.5 fold) regulated by hLf, bLf, and CbLf were 152, 367, and 455, respectively. Although only few genes (16) were regulated by all Lfs, they remarkably stimulate signaling pathways tightly related to cellular development based on pathway analysis by IPA (Ingenuity). All Lfs triggered various signaling pathways, such as growth hormone signaling, cell cycle: G1/S checkpoint regulation, and IGF‐1 signaling. In summary, all Lfs may play important roles in intestinal development but through various mechanisms.

Columnar Cell Variant of Papillary Thyroid Carcinoma: A Study of 10 Cases with Emphasis on CDX2 Expression

Columnar cell variant is a recognized rare variant of papillary thyroid carcinoma (PTC) that has an uncertain clinical course. This variant has two subvariants, and one is regarded as a more aggressive form in comparison to the more common classical and follicular variants. These tumors have morphological resemblance with endometrial or colonic adenocarcinoma. CDX2, a transcription factor of the caudal homeobox family, plays a key role in intestinal development and differentiation, and it is widely used as a marker to detect adenocarcinomas of intestinal and colonic origin. CDX2 has been rarely reported in PTC. We studied 10 cases of columnar cell variant of PTC (CCV-PTC). The histological, architectural, and cytological features fulfilled the diagnostic criteria of the CCV-PTC, as defined by the current World Health Organization classification. Ten patients (six men and four women) ranging in age from 32 to 90 years (mean, 58.3 years) presented with tumors classified as indolent (four cases) or aggressive (six cases); three harbored a BRAF(V600E) mutation. All cases were β-catenin negative. The Ki-67 proliferative index was up to 50%. All cases were thyroid transcription factor-1-positive. Using paraffin-embedded blocks, immunohistochemistry for CDX2 was performed to evaluate the reactivity of this antibody to this variant of PTC. Nuclear positivity for CDX2 was detected in one out of the 10 cases studied (10%); the other nine cases did not express CDX2. Only one of our cases showed nuclear positivity for CDX2. Therefore, our study failed to confirm it as a marker for CCV-PTC. The absence of CDX2 in the majority of the cases does not support the theory of CDX2 playing a role in the intestinal phenotype of these tumors.