Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2)

Lysosomes of intestinal epithelial cells in vivo and in culture display strong immunoreactivity with monoclonal antibodies against various brush border enzymes as visualized by immunoelectron microscopy. Novel subcellular fractionation procedures were developed to study, by the pulse-chase technique and by internalization assays, the pathway along which two microvillar hydrolases, sucrase-isomaltase and dipeptidylpeptidase IV, are transported to lysosomes in the differentiated colon adenocarcinoma cell line Caco-2. 7-9% of metabolically labeled sucrase-isomaltase of dipeptidylpeptidase IV were present in lysosomes after 7-8 h of chase as intact complex-glycosylated molecules. Appearance of these enzymes in lysosomes was biphasic. Endocytosis studies with radioiodinated antienzyme monoclonal antibodies (monovalent antigen-binding fragments) and by means of cell surface iodination revealed only slow transport of the enzymes to lysosomes at a low level. However, both enzymes were internalized with different efficiencies and recycled to the cell surface via endosomes. These results suggest that in Caco-2 cells a significant amount of newly synthesized sucrase-isomaltase and dipeptidylpeptidase IV is directly imported into lysosomes bypassing the brush border membrane.     

乳杆菌微小膜蛋白对肠上皮细胞Caco-2的细胞毒性研究

目的 利用脂多糖（lipopolysaccharide,LPS）刺激模拟体外炎症环境,观察不同浓度下乳杆菌微小膜蛋白（micro integral membrane protein,MIMP）对肠上皮细胞Caco-2的生物学影响,评估其细胞毒性作用。方法 首先通过CCK-8实验检测在LPS刺激后不同浓度MIMP（0.01、0.1和1 ng/mL）对Caco-2细胞增殖活性的影响,并利用Toll样受体4（Toll-like receptor 4,TLR4）抑制剂作为阳性对照。其次利用流式细胞术检测不同浓度下MIMP对Caco-2细胞凋亡及细胞周期的影响。结果 在12 h的特定孵育时间内,单独应用不同浓度MIMP及TLR4抑制剂对Caco-2细胞增殖活性无显著影响（P＞0.05）,但是MIMP可以拮抗LPS对Caco-2细胞的促增殖作用（P<0.05）。不同浓度的MIMP对Caco-2细胞的周期和凋亡无明显影响（P＞0.05）。结论 不同浓度MIMP对Caco-2细胞的增殖、凋亡及细胞周期无明显影响,并可以拮抗LPS的促细胞增殖作用,因此具有较高的安全性,有望用于临床对炎症性肠病的治疗。     

Iron-induced oxidative stress up-regulates calreticulin levels in intestinal epithelial (Caco-2) cells

Calreticulin, a molecular chaperone involved in the folding of endoplasmic reticulum synthesized proteins, is also a shock protein induced by heat, food deprivation, and chemical stress. Mobilferrin, a cytosolic isoform of calreticulin, has been proposed to be an iron carrier for iron recently incoming into intestinal cells. To test the hypothesis that iron could affect calreticulin expression, we investigated the possible associations of calreticulin with iron metabolism. To that end, using Caco-2 cells as a model of intestinal epithelium, the mass and mRNA levels of calreticulin were evaluated as a function of the iron concentration in the culture media. Increasing the iron content in the culture from 1 to 20 microM produced an increase in calreticulin mRNA and a two-fold increase in calreticulin. Increasing iron also induced oxidative damage to proteins, as assessed by the formation of 4-hydroxy-2-nonenal adducts. Co-culture of cells with the antioxidants quercetin, dimethyltiourea and N-acetyl cysteine abolished both the iron-induced oxidative damage and the iron-induced increase in calreticulin. We postulate that the iron-induced expression of calreticulin is part of the cellular response to oxidative stress generated by iron.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells.

When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.     

Epithelial properties of human intestinal Caco-2 cells cultured in a serum-free medium

Human intestinal Caco-2 cells were cultured under serum-free conditions on an insoluble collagen and FCS matrix (Caco-2-SF), and a comparison was made between several characteristics of Caco-2 and Caco-2-SF cells. Their morphological appearance was identical. Slight differences were found in cell growth and expression of brush border enzymes between Caco-2 and Caco-2-SF cells. Similar levels of activity of Gly-Gly transport were expressed in both types of cell. Caco-2 cells cultured on permeable filters showed high transepithelial electrical resistance (TEER), indicating the high monolayer integrity. The transepithelial transport activity for glucose, alanine and Gly-Gly was detected by measuring the change in short-circuit current (Isc) after adding each of these nutrients to the apical chamber. In Caco-2-SF cells, such parameters as TEER and Isc were reduced drastically, suggesting that the monolayer integrity and cell polarity that are important for transepithelial transport were not attained. These parameters, however, could be restored by adding FCS or by milk whey. The result suggested that FCS and milk whey contain factors which regulate the formation of the tight junctions and, consequently, the development of cell polarity. Thus the Caco-2-SF cell-culture system will provide a useful model for studying factors which regulate the intestinal transepithelial transport functions.Abbreviations BCECF 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein - TEER transepithelial electrical resistance - LY lucifer yellow CH lithium salt     

Effects of valerate on intestinal barrier function in cultured Caco-2 epithelial cell monolayers

Molecular Biology Reports - Short-chain fatty acids (SCFAs) are a group of microbial metabolites of undigested dietary fiber, protein and unabsorbed amino acids in the colon, well-known for their...     

Relationship between structure and permeability of tryptophan derivatives across human intestinal epithelial (Caco-2) cells

L-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mM, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-to-basolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco-2 cells was proposed.     

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.     

Transport of bile acids in a human intestinal epithelial cell line, Caco-2

The transport of taurocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (BL) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [14C]TA to the AP or BL side of the monolayers (30 min) were approximately equal (54.4 +/- 2.7 and 64.6 +/- 2.8 fmol/mg protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vmax and Km for transport were 13.7 pmol/mg protein per min and 49.7 microM, respectively. The transport of TA had an activation energy of 13.2 kcal.mol-1, required Na+ and glucose. AP-to-BL transport of [14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.     

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.     

Sodium-dependent, concentrative nucleoside transport in cultured intestinal epithelial cells

In a simple salts medium, monolayers of IEC-6 intestinal cells achieved concentrations of unmetabolized formycin B (an analog of inosine) about 6-fold higher than in the medium. Rates of formycin B influx were a saturable function of Na+ concentrations in the medium. Although IEC-6 cells possess sites with high affinity for nitrobenzylthioinosine, a potent inhibitor of equilibrative (facilitated diffusion) nucleoside transport systems in certain cell types, the inhibitor had only minor effects on formycin B uptake in IEC-6 cells, but reduced efflux of the analog from these cells. These findings indicate the joint presence in IEC-6 cells of nucleoside transporters of two types, one that is concentrative and Na+-dependent, and another that is sensitive to nitrobenzylthioinosine and apparently equilibrative.     

Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells

The absorption of dietary non-heme iron by intestinal enterocytes is crucial to the maintenance of body iron homeostasis. This process must be tightly regulated since there are no distinct mechanisms for the excretion of excess iron from the body. An insight into the cellular mechanisms has recently been provided by expression cloning of a divalent cation transporter (DCT1) from rat duodenum and positional cloning of its human homologue, Nramp2. Here we demonstrate that Nramp2 is expressed in the apical membrane of the human intestinal epithelial cell line, Caco 2 TC7, and is associated with functional iron transport in these cells with a substrate preference for iron over other divalent cations. Iron transport occurs by a proton-dependent mechanism, exhibiting a concurrent intracellular acidification. Taken together, these data suggest that the expression of the Nramp2 transporter in human enterocytes may play an important role in intestinal iron absorption.     

Modulation of human Caco-2 intestinal epithelial cell phenotype by protein kinase C inhibitors

Protein kinase C (PKC) isoforms are altered in colon tumors and upon exposure of intestinal mucosa to nutrients. We evaluated the effects of the PKC inhibitors staurosporine and calphostin C on human Caco-2 intestinal epithelial proliferation, motility, and differentiation. Motility was quantitated by monolayer expansion and the brush border enzymes dipeptidyl dipeptidase (DPDD) and alkaline phosphatase (AP) by synthetic substrate digestion. Staurosporine (0.03-1.0 ng/ml) and calphostin C (10^-12M-10^-4 M) dose-dependently inhibited monolayer expansion and AP but stimulated DPDD. Proliferation was also inhibited but the effects of each inhibitor on motility, AP, and DPDD were preserved after mitomycin C proliferative blockade, suggesting that these effects were proliferation-independent. PKC inhibitors independently inhibit motility, AP and proliferation in human intestinal Caco-2 epithelial cells, but selectively stimulate the small intestinal differentiation marker DPDD. PKC may regulate small intestinal epithelial differentiation.     

Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.