Paraben transport and metabolism in the biomimetic artificial membrane permeability assay (BAMPA) and 3-day and 21-day Caco-2 cell systems

Noncellular and cellular in vitro models for predicting intestinal absorption were used to investigate the transport and metabolism of parabens. The biomimetic artificial membrane permeability assay (BAMPA) membrane was constructed by impregnating a lipid solution on a hydrophobic filter. Caco-2 cells at passage numbers 65 to 80 were cultured in either the accelerated 3-day Biocoat system or the standard 21-day Transwell cell culture system. Paraben transport across the BAMPA system showed a parabolic relationship. The lowest log P (p-hydroxybenzoic acid) and highest log P compounds (heptyl and octyl parabens) had apparent permeabilities (Papp) less than 1.0 x 10(-6) cm/s and Papp was maximal at approximately 8.5 x 10(-6)cm/s for the intermediate log P (ethylparaben) compound. With the Biocoat, a similar parabolic relationship was found. In the 21-day Caco-2 cells, the parabens were metabolized by esterases at to p-hydroxybenzoic acid. In conclusion, the in vitro models added complementary insight into the absorption process, such as the transport route, intrinsic permeability, and extent of metabolism of the parabens. This study indicated that presystemic metabolism of orally ingested parabens to the p-hydroxybenzoic acid in the intestine may limit systemic exposure to alkyl-paraben esters in vivo.     

Differentiation-dependent expression and localization of the class B type I scavenger receptor in intestine

The current study used the human Caco-2 cell line and mouse intestine to explore the topology of expression of the class B type I scavenger receptor (SR-BI) in intestinal cells. Results showed that intestinal cells expressed only the SR-BI isoform with little or no expression of the SR-BII variant. The expression of SR-BI in Caco-2 cells is differentiation dependent, with little or no expression in preconfluent undifferentiated cells. Analysis of Caco-2 cells cultured in Transwell porous membranes revealed the presence of SR-BI on both the apical and basolateral cell surface. Immunoblot analysis of mouse intestinal cell extracts demonstrated a gradation of SR-BI expression along the gastrocolic axis of the intestine, with the highest level of expression in the proximal intestine and decreasing to minimal expression levels in the distal intestine. Immunofluorescence studies with SR-BI-specific antibodies also confirmed this expression pattern. Importantly, the immunofluorescence studies also revealed that SR-BI immunoreactivity was most intense in the apical membrane of the brush border in the duodenum. The crypt cells did not show any reactivity with SR-BI antibodies. The localization of SR-BI in the jejunum was found to be different from that observed in the duodenum. SR-BI was present on both apical and basolateral surfaces of the jejunum villus. Localization of SR-BI in the ileum was also different, with little SR-BI detectable on either apical or basolateral membranes.Taken together, these results suggest that SR-BI has the potential to serve several functions in the intestine. The localization of SR-BI on the apical surface of the proximal intestine is consistent with the hypothesis of its possible role in dietary cholesterol absorption, whereas SR-BI present on the basolateral surface of the distal intestine suggests its possible involvement in intestinal lipoprotein uptake.     

α-Amylase expressed in human small intestinal epithelial cells is essential for cell proliferation and differentiation

α-Amylase, which plays an essential role in starch degradation, is expressed mainly in the pancreas and salivary glands. Human α-amylase is also detected in other tissues, but it is unclear whether the α-amylase is endogenously expressed in each tissue or mixed exogenously with one expressed by the pancreas or salivary glands. Furthermore, the biological significance of these α-amylases detected in tissues other than the pancreas and salivary glands has not been elucidated. We discovered that human α-amylase is expressed in intestinal epithelial cells and analyzed the effects of suppressing α-amylase expression. α-Amylase was found to be expressed at the second-highest messenger RNA level in the duodenum in human normal tissues after the pancreas. α-Amylase was detected in the cell extract of Caco-2 intestinal epithelial cells but not secreted into the culture medium. The amount of α-amylase expressed increased depending on the length of the culture of Caco-2 cells, suggesting that α-amylase is expressed in small intestine epithelial cells rather than the colon because the cells differentiate spontaneously upon reaching confluence in culture to exhibit the characteristics of small intestinal epithelial cells rather than colon cells. The α-amylase expressed in Caco-2 cells had enzymatic activity and was identified as AMY2B, one of the two isoforms of pancreatic α-amylase. The suppression of α-amylase expression by small interfering RNA inhibited cell differentiation and proliferation. These results demonstrate for the first time that α-amylase is expressed in human intestinal epithelial cells and affects cell proliferation and differentiation. This α-amylase may induce the proliferation and differentiation of small intestine epithelial cells, supporting a rapid turnover of cells to maintain a healthy intestinal lumen.     

Increased curvature of hollow fiber membranes could up‐regulate differential functions of renal tubular cell layers

Tissue engineering devices as in vitro cell culture systems in scaffolds has encountered the bottleneck due to their much lower cell functions than real tissues/organs in vivo. Such situation has been improved in some extent by mimicking the cell microenvironments in vivo from either chemical or physical ways. However, microenvironmental curvature, commonly seen in real tissues/organs, has never been manipulated to regulate the cell performance in vitro. In this regard, this paper fabricated polysulfone membranes with or without polyethylene glycol modification to investigate the impact of curvature on two renal tubular cells. Regardless the varying membrane curvatures among hollow fiber membranes of different diameters and flat membrane of zero curvature, both renal cells could well attach at 4 h of seeding and form similar confluent layers at 6 days on each membrane. Nevertheless, the renal cells on hollow fibers, though showing confluent morphology as those on flat membranes, expressed higher renal functions and, moreover, the renal functions significantly increased with the membrane curvature among hollow fibers. Such upregulation on functions was unassociated with mass transport barrier of hollow fibers, because the cultures on lengthwise cut hollow fibers without mass transfer barrier showed same curvature effect on renal functions as whole hollow fibers. It could be proposed that the curvature of hollow fiber membrane approaching to the large curvature in kidney tubules increased the mechanical stress in the renal cells and thus might up‐regulate the renal cell functions. In conclusion, the increase of substrate curvature could up‐regulate the cell functions without altering the confluent cell morphology and this finding will facilitate the design of functional tissue engineering devices. Biotechnol. Bioeng. 2013; 110: 2173–2183. © 2013 Wiley Periodicals, Inc.     

Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: selective secretion of PON1 and PON2

The paraoxonase (PON) family contains three genes (PON1/2/3) that are believed to be involved in the protection against oxidative stress. PON1 and PON3 are circulating in serum attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/3 mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON3 were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased paraoxonase and lactonase activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON3 to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

A radial flow hollow fiber bioreactor for the large-scale culture of mammalian cells

A radial flow hollow fiber bioreactor has been developed that maximizes the utilization of fiber surface for cell growth while eliminating nutrient and metabolic gradients inherent in conventional hollow fiber cartridges. The reactor consists of a central flow distributor tube surrounded by an annular bed of hollow fibers. The central flow distributor tube ensures an axially uniform radial convective flow of nutrients across the fiber bed. Cells attach and proliferate on the outer surface of the fibers. The fibers are pretreated with polylysine to facilitate cell attachment and long-term maintenance of tissuelike densities of cell mass. A mixture of air and CO(2) is fed through the tube side of the hollow fibers, ensuring direct oxygenation of the cells and maintenance of pH. Spent medium diffuses across the cell layer into the tube side of the fibers and is convected away along with the spent gas stream. The bioreactor was run as a recycle reactor to permit maximum utilization of nutrient medium. A bioreactor with a membrane surface area of 1150 cm(2) was developed and H1 cells were grown to a density of 7.3 x 10(6) cells/cm(2).     

Enteric glia inhibit intestinal epithelial cell proliferation partly through a TGF-beta1-dependent pathway

Although recent studies have shown that enteric neurons control intestinal barrier function, the role of enteric glial cells (EGCs) in this control remains unknown. Therefore, our goal was to characterize the role of EGCs in the control of intestinal epithelial cell proliferation using an in vivo transgenic and an in vitro coculture model. Assessment of intestinal epithelial cell proliferation after ablation of EGCs in transgenic mice demonstrated a significant increase in crypt cell hyperplasia. Furthermore, mucosal glial network (assessed by immunohistochemical detection of S-100beta) is altered in colon adenocarcinoma compared with control tissue. In an in vitro coculture model of subconfluent Caco-2 cells seeded onto Transwell filters with EGCs, Caco-2 cell density and [3H]thymidine incorporation were significantly lower than in control (Caco-2 cultured alone). Flow cytometry analysis showed that EGCs had no effect on Caco-2 cell viability. EGCs induced a significant increase in Caco-2 cell surface area without any sign of cellular hypertrophy. These effects by EGCs were also seen in various transformed or nontransformed intestinal epithelial cell lines. Furthermore, TGF-beta1 mRNA was expressed, and TGF-beta1 was secreted by EGCs. Exogenously added TGF-beta1 reproduced partly the EGC-mediated effects on cell density and surface area. In addition, EGC effects on Caco-2 cell density were significantly reduced by a neutralizing TGF-beta antibody. In conclusion, EGCs have profound antiproliferative effects on intestinal epithelial cells. Functional alterations in EGCs may therefore modify intestinal barrier functions and be involved in pathologies such as cancer or inflammatory bowel diseases.     

The Transition from Proliferation to Differentiation in Colorectal Cancer Is Regulated by the Calcium Activated Chloride Channel A1

Breaking the balance between proliferation and differentiation in animal cells can lead to cancer, but the mechanisms maintaining this balance remain largely undefined. The calcium activated chloride channel A1 (CLCA1) is a member of the calcium sensitive chloride conductance family of proteins and is expressed mainly in the colon, small intestine and appendix. We show that CLCA1 plays a functional role in differentiation and proliferation of Caco-2 cells and of intestinal tissue. Caco-2 cells spontaneously differentiate either in confluent culture or when treated with butyrate, a molecule present naturally in the diet. Here, we compared CLCA1 expressional levels between patients with and without colorectal cancer (CRC) and determined the functional role of CLCA1 in differentiation and proliferation of Caco-2 cells. We showed that: 1) CLCA1 and CLCA4 expression were down-regulated significantly in CRC patients; 2) CLCA1 expression was up-regulated in Caco-2 cells induced to differentiate by confluent culture or by treatment with sodium butyrate (NaBT); 3) Knockdown of CLCA1 with siRNA significantly inhibited cell differentiation and promoted cell proliferation in Caco-2 confluent cultures, and 4) In Caco-2 3D culture, suppression of CLCA1 significantly increased cell proliferation and compromised NaBT-induced inhibition of proliferation. In conclusion, CLCA1 may contribute to promoting spontaneous differentiation and reducing proliferation of Caco-2 cells and may be a target of NaBT-induced inhibition of proliferation and therefore a potential diagnostic marker for CRC prognosis.     

中空纤维灌注培养模拟骨髓造血研究

采用细胞工程技术探索造血细胞体外扩增技术以维持其自我更新潜能,抑制过度分化。方法：首先建立微载体 基质细胞体外造血模型（G1组,即瓶培养模式）,设置单纯微载体 基质细胞培养（G2组）和单纯骨髓细胞液体悬浮培养（G3组）作对照。检测各组粒系 巨噬系造血祖细胞集落产率（CFU GM/105）。自行设计1对引物,以检测Balb/c小鼠原始造血细胞c kit基因mRNA表达水平。试用中空纤维模拟血管灌注功能（Gh组,即中空纤维灌注模式）,以G1、G2、G3作对照,并对各组培养效果进行评价。结果：微载体 基质细胞体外造血模型实验结果显示：小鼠骨髓细胞培养2周后,CFU GM/105检测G1组比G3组高7.7倍（P<0.05）,是2个对照组（G2+G3）集落产率总和的1.9倍。原始造血细胞c kit mRNA表达水平：模型G1组比G2组高3.7倍,比G3组高62.3倍,且差异均显著。在成功建立微载体 基质细胞体外造血模型基础上进行中空纤维灌注培养实验,CFU GM/105检测显示：Gh组比G3组高4.6倍,并且略高于G2组;Gh组与G1组集落产率差别不明显。在原始造血细胞c kit mRNA表达水平上Gh组最高,从Gh、G1、G2到G3依次呈下降趋势。结论：在没有外加细胞因子的条件下,微载体 基质细胞和中空纤维灌注造血模型可抑制造血干、祖细胞过度分化与耗竭,维持其c kit较高的表达水平。     

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

The lipocalin 2//NGAL/24p3 receptor (NGAL-R/24p3-R) is expressed in rodent distal nephron where it mediates protein endocytosis. The mechanisms of apical endocytosis and transcytosis of proteins and peptides in the intestine are poorly understood. In the present study, the expression and localization of rodent 24p3-R (r24p3-R) and human NGAL-R (hNGAL-R) was investigated in intestinal segments by immunofluorescence and confocal laser scanning microscopy, immunohistochemistry and immunoblotting. r24p3-R/hNGAL-R was also studied in human Caco-2 BBE cells and CHO cells transiently transfected with r24p3-R by immunofluorescence microscopy, RT-PCR and immunoblotting of plasma membrane enriched vesicles (PM). To assay function, endocytosis/transcytosis of putative ligands phytochelatin (PC₃), metallothionein (MT) and transferrin (Tf) was assayed by measuring internalization of fluorescence-labelled ligands in Caco-2 BBE cells grown on plastic or as monolayers on Transwell inserts. The binding affinity of Alexa 488-PC₃ to colon-like Caco-2 BBE PM was quantified by microscale thermophoresis (MST). r24p3-R/hNGAL-R expression was detected apically in all intestinal segments but showed the highest expression in ileum and colon. Colon-like, but not duodenum-like, Caco-2 BBE cells expressed hNGAL-R on their surface. Colon-like Caco-2 BBE cells or r24p3-R transfected CHO cells internalized fluorescence-labelled PC₃ or MT with half-maximal saturation at submicromolar concentrations. Uptake of PC₃ and MT (0.7 µM) by Caco-2 BBE cells was partially blocked by hNGAL (500 pM) and an EC ₅₀ of 18.6 ± 12.2 nM was determined for binding of Alexa 488-PC₃ to PM vesicles by MST. Transwell experiments showed rapid (0.5-2 h) apical uptake and basolateral delivery of fluorescent PC₃/MT/Tf (0.7 µM). Apical uptake of ligands was significantly blocked by 500 pM hNGAL. hNGAL-R dependent uptake was more prominent with MT but transcytosis efficiency was reduced compared to PC₃ and Tf. Hence, r24p3-R/hNGAL-R may represent a high-affinity multi-ligand receptor for apical internalization and transcytosis of intact proteins/peptides by the lower intestine.     

Human cytomegalovirus infects Caco-2 intestinal epithelial cells basolaterally regardless of the differentiation state

Human cytomegalovirus (CMV) causes severe disease in immunosuppressed patients and notably infects the gastrointestinal tract. To understand the interaction of CMV with intestinal epithelial cells, which are highly susceptible to CMV infection in vivo, we used the intestinal epithelial cell line Caco-2 and demonstrated that CMV enters predominantly through the basolateral surface of polarized Caco-2 cells. As shown by expression of all three classes of CMV proteins and by visualization of nucleocapsids by transmission electron microscopy, both poorly and fully differentiated Caco-2 cells were permissive to CMV replication. However, infection failed to produce infectious particles in Caco-2 cells, irrespective of the state of differentiation.     

Permeation of Insulin,Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

Objectives

Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs.

Methods

The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer.

Results

Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures.

Conclusions

Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.     

Rbm19 is a nucleolar protein expressed in crypt/progenitor cells of the intestinal epithelium

Intestinal development and homeostasis rely on the coordination of proliferation and differentiation of the epithelium. To better understand this process, we are studying Rbm19, a gene expressed in the gut epithelium that is essential for intestinal morphogenesis and differentiation in the zebrafish (Development 130, 3917). Here we analyzed the expression of Rbm19 in several biological contexts that feature proliferation/differentiation cell fate decisions. In the undifferentiated embryonic gut tube, Rbm19 is expressed throughout the epithelium, but then becomes localized to the crypts of Lieberkühn of the adult intestine. Consistent with its expression in adult crypt/progenitor cells, expression is widespread in human colorectal carcinomas and dividing Caco-2 cells. Its expression in Caco-2 cells recapitulates the in vivo pattern, declining when the cells undergo confluence-induced arrest and differentiation. Rbm19 protein localizes to the nucleolus during interphase and to the perichromosomal sheath during mitosis, in accordance with the pattern described for other nucleolar proteins implicated in ribosome biogenesis. Interestingly, the loss of nucleolar rbm19, nucleolin/C23, and nucleophosmin/B23 in confluent Caco-2 cells did not signify loss of nucleoli as detected by electron microscopy. Taken together, these data point to the nucleolus as a possible locus for regulating the proliferation/differentiation cell fate decision in the intestinal epithelium.     

Suppressor of cytokine signaling-2 limits intestinal growth and enterotrophic actions of IGF-I in vivo

Suppressors of cytokine signaling (SOCS) typically limit cytokine receptor signaling via the JAK-STAT pathway. Considerable evidence demonstrates that SOCS2 limits growth hormone (GH) action on body and organ growth. Biochemical evidence that SOCS2 binds to the IGF-I receptor (IGF-IR) supports the novel possibility that SOCS2 limits IGF-I action. The current study tested the hypothesis that SOCS2 normally limits basal or IGF-I-induced intestinal growth and limits IGF-IR signaling in intestinal epithelial cells. Intestinal growth was assessed in mice homozygous for SOCS2 gene deletion (SOCS2 null) and wild-type (WT) littermates at different ages and in response to infused IGF-I or vehicle or EGF and vehicle. The effects of SOCS2 on IGF-IR signaling were examined in ex vivo cultures of SOCS2 null and WT intestine and Caco-2 cells. Compared with WT, SOCS2 null mice showed significantly enhanced small intestine and colon growth, mucosal mass, and crypt cell proliferation and decreases in radiation-induced crypt apoptosis in jejunum. SOCS2 null mice showed significantly greater growth responses to IGF-I in small intestine and colon. IGF-I-stimulated activation of IGF-IR and downstream signaling intermediates were enhanced in the intestine of SOCS2 null mice and were decreased by SOCS2 overexpression in Caco-2 cells. SOCS2 bound directly to the endogenous IGF-IR in Caco-2 cells. The intestine of SOCS2 null mice also showed enhanced growth responses to infused EGF. We conclude that SOCS2 normally limits basal and IGF-I- and EGF-induced intestinal growth in vivo and has novel inhibitory effects on the IGF-IR tyrosine kinase pathway in intestinal epithelial cells.     

Differential Regulation of Intestinal and Liver Fatty Acid-Binding Proteins in Human Intestinal Cell line (Caco-2): Role of Collagen

Fatty acid-binding proteins (FABP) are small cytosolic proteins which are thought to play a key role in fatty acid metabolism. The intestine contains the intestinal (I-FABP) and the liver (L-FABP) isoforms, but their regulation is still poorly documented. In order to find suitable conditions for studying the regulation of the two FABP isoforms in Caco-2 cells, we investigated the effects of the presence of collagen during cell proliferation or differentiation. When collagen was present only during cell proliferation on culture dishes, I-FABP expression was enhanced, whereas sucrase-isomaltase was unaffected and L-FABP expression was merely accelerated. In contrast, when collagen was present during cell differentiation on filter inserts, both I-FABP and sucrase-isomaltase were strongly reduced, but L-FABP was not affected. Under the former conditions (the more suitable for studying FABP regulation), the peroxysome proliferator-activated receptor (PPAR) activators, clofibrate and α-bromopalmitate, enhanced the two isoforms. This study, which is the first one providing a quantitative protein analysis of I-FABP and L-FABP in Caco-2 cells, demonstrates different time courses of expression of these proteins during cell differentiation. It also shows that I-FABP is specifically regulated by collagen and that, under conditions optimal for their expression, both isoforms are modulated by metabolic factors.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.