Transfected Caco-2 Cells Research Articles

Colon-derived Caco-2 cells support replication of hepatitis E virus genotype 1 strain Sar55 generated by reverse genetics

The hepatitis E virus (HEV) is infecting over 20 million people annually with a high morbidity especially in pregnant women and immune-suppressed individuals. While HEV genotype 1 (HEV-1) infects only humans, genotype 3 (HEV-3) is zoonotic and commonly transmitted from infected animals to humans. Whereas a few reverse genetics systems enabling targeted genome manipulations exist for HEV-3, those for HEV-1 are still very limited, mainly because of inefficient cell culture replication. Here, the generation of HEV-1 strain Sar55 and HEV-3 strain 47832mc by transfecting in vitro-transcribed and capped virus genomes into different cell lines was attempted. Culture supernatants of colon-derived colorectal adenocarcinoma cell line Caco-2 contained HEV-1 and HEV-3 capable of infecting Caco-2 cells. Density gradient centrifugation analyses of culture supernatants confirmed that HEV-1 particles were quasi-enveloped in analogy to HEV-3 and that non-virion-associated capsid protein was secreted from cells. Following transfection or infection of Caco-2 cells, HEV-1 consistently reached higher titers than HEV-3 in culture supernatants, but HEV-1 generated by transfection of Caco-2 cells was unable to efficiently infect hepatoma cell lines PLC/PRF/5 or HuH7-Lunet BLR. Taken together, our results indicate that HEV-1 is able to exert a complete replication cycle in Caco-2 cells. An efficient cell culture system for this genotype will be useful for studying species tropism, but further research is required to determine the significance of HEV-1 replication in colon-derived cells.

HuNoV Non-Structural Protein P22 Induces Maturation of IL-1β and IL-18 and N-GSDMD-Dependent Pyroptosis through Activating NLRP3 Inflammasome.

Norovirus infection is the leading cause of foodborne gastroenteritis worldwide, causing more than 200,000 deaths each year. As a result of a lack of reproducible and robust in vitro culture systems and suitable animal models for human norovirus (HuNoV) infection, the pathogenesis of HuNoV is still poorly understood. In recent years, human intestinal enteroids (HIEs) have been successfully constructed and demonstrated to be able to support the replication of HuNoV. The NLRP3 inflammasome plays a key role in host innate immune responses by activating caspase1 to facilitate IL-1β and IL-18 secretion and N-GSDMD-driven apoptosis, while NLRP3 inflammasome overactivation plays an important role in the development of various inflammatory diseases. Here, we found that HuNoV activated enteric stem cell-derived human intestinal enteroids (HIEs) NLRP3 inflammasome, which was confirmed by transfection of Caco2 cells with full-length cDNA clones of HuNoV. Further, we found that HuNoV non-structural protein P22 activated the NLRP3 inflammasome and then matured IL-1β and IL-18 and processed the cleavage of gasdermin-D (GSDMD) to N-GSDMD, leading to pyroptosis. Besides, berberine (BBR) could ameliorate the pyroptosis caused by HuNoV and P22 by inhibiting NLRP3 inflammasome activation. Together, these results reveal new insights into the mechanisms of inflammation and cell death caused by HuNoV and provide potential treatments.

Deficiency of microRNA-10b promotes DSS-induced inflammatory response via impairing intestinal barrier function

Inflammatory bowel disease (IBD) is a non-specific inflammatory disease of the intestine with the pathogenesis to be largely unknown. We found that microRNA (miR)-10b knock-out mice displayed mild IBD symptoms, suggesting that miR-10b may be involved in the onset and development of IBD. This study focuses on elucidating the role of miR-10b in IBD.The colitis model was induced by feeding the mice with 2.5% dextran sodium sulfate (DSS), and the expression levels of miR-10b in colon tissue and blood samples were examined. The severity of colitis was assessed by disease activity index, colon length, histopathological damage, intestinal permeability and ELISA. Then, after transfection of Caco-2 cells with miR-10b mimic and inhibitor, qRT-PCR was used to detect the expression levels of intestinal barrier related genes in colon tissues and cells.miR-10b levels were significantly reduced in mice with DSS-induced acute colitis. Compared with wild-type (WT) mice, miR-10b knockout mice were more sensitive to DSS-induced colitis characterized by increased inflammatory cell infiltration and more severe disruption of colonic barrier function. In addition, by inhibiting miR-10b and thus increasing intestinal barrier gene expression in Caco-2 cells, we found that miR-10b suppressed inflammatory responses and enhanced intestinal barrier function both in vivo and in vitro.miR-10b inhibits the inflammatory response in DSS-induced acute colitis mice in vivo and enhances intestinal barrier function in vitro, suggesting that miR-10b plays a key role in the developmental process of IBD. Thus, miR-10b may be expected to be a new target for the treatment of IBD.

Effect of modification of MTDH gene expression on colorectal cancer aggressiveness

BackgroundMetadherin (MTDH) is an oncogene that has been overexpressed in numerous types of malignancies including colorectal cancer (CRC). However, few investigations associated with the biological behavior of MTDH in CRC have been performed. The aim of the present study was to investigate the effect of modification of MTDH gene expression (knockdown and overexpression) on the biological behavior of CRC in vitro. MethodsMTDH gene expression was analyzed in two CRC cell lines (Caco-2 and HCT116) by qPCR. MTDH was down-regulated via siRNA-mediated knockdown of human MTDH in HCT116 cells, which express high endogenous levels of MTDH gene. Also, MTDH gene was up-regulated via transfection of Caco-2 cells, which express low endogenous levels of MTDH gene, with a plasmid carrying human MTDH gene. ResultsKnockdown of MTDH gene expression significantly decreased the gene expression of multidrug resistance gene (MDR1), Snail and NF-κB p65, but increased the gene expression of E-cadherin. Furthermore, MTDH-knockdown significantly decreased anaerobic glycolysis (glucose consumption and lactate production), cell proliferation ability and transformation into cancer stem cell. Moreover, up-regulation of MTDH gene significantly increased the gene expression of MDR1, Snail and NF-κB p65, deceased the gene expression of E-cadherin, enhanced cell proliferation, and anaerobic glycolysis and activated transformation into cancer stem cells. ConclusionsMTDH has an important role in promoting CRC aggravation. Also, inhibition of MTDH expression may attenuate the carcinogenic behavior of CRC cells. Furthermore, MTDH-associated NF-κB p65 signaling pathways may be involved in mediating the biological behavior of CRC.

Cloning and characterization of the human lactoferrin receptor gene promoter.

Lactoferrin (Lf) is a major protein in human milk. Multiple biological functions of Lf are postulated to be mediated by a Lf receptor (LfR). The Lf receptor (LfR) plays an important role in absorption of Lf and Lf-bound iron by intestinal epithelial cells. Here, we cloned and characterized the promoter from a~3.1kb 5'-flanking region of the human LfR gene. Neither a TATA box nor a CCAAT box is found at the typical positions. The transcription start site was identified as 298bp upstream of the translation start codon (+1) by 5' RLM-RACE. A series of deletions of 5'-flanking sequences of the human LfR gene were cloned into a promoter-less pGL3 luciferase reporter and transiently transfected into an intestinal enterocyte model (Caco-2 cells). A fragment of -299/+63 elicited the maximal promoter activity in transfected Caco-2 cells, suggesting that functional transcription factor binding sites appear in the region of -299/+63. Bioinformatics analysis indicates that the -299/+63 fragment contains two putative Sp1 binding sites. The promoter activity was significantly decreased when the Sp1 binding sites were mutated by site-directed mutagenesis. Additionally, the promoter activity was dramatically inhibited by treating cells with an Sp1 inhibitor. Binding of Sp1 to the promoter was confirmed by EMSA. Moreover, after Sp1 expression was significantly suppressed by RNA interference, LfR was significantly decreased at both RNA and protein levels. In conclusion, the LfR gene promoter contains downstream core promoter elements, and the Sp1 binding sites play critical roles in transcriptional regulation of the LfR gene.

Phosphorylation of Ser-525 in βPix impairs Nox1-activating ability in Caco-2 cells

βPix activates Nox1, an O2−-generating NADPH oxidase, through Rac activation. In this study, we found that S525E mutation of βPix eliminated its Nox1-activating ability in transfected Caco-2 cells. Unexpectedly, affinity for Rac was not diminished but rather enhanced by S525E mutation, and guanine nucleotide exchange factor (GEF) activity was not altered. The N-terminal fragment (amino acids 1–400) showed similar Rac-binding and GEF activity to wild-type βPix. In contrast, the C-terminal fragment (amino acids 408–646) had higher Rac-binding activity, particularly for Rac-GTP, than wild-type βPix, and showed no GEF activity. These data suggest that a second Rac-binding site within the C-terminal region is opened by phosphorylation of Ser-525. The site may bind not only Rac-GDP but also Rac-GTP released from the N-terminal catalytic region, which interrupts Rac-GTP translocation to the membrane where Nox1 resides. If one considers that S340E mutation enhances Nox1 activation (Kaito et al., 2014), the present study suggests that βPix can also play an inhibitory role in O2− production, depending on the sites of phosphorylation.

AMPK improves gut epithelial differentiation and barrier function via regulating Cdx2 expression.

Impairment in gut epithelial integrity and barrier function is associated with many diseases. The homeostasis of intestinal barrier is based on a delicate regulation of epithelial proliferation and differentiation. AMP-activated protein kinase (AMPK) is a master regulator of energy metabolism, and cellular metabolites are intrinsically involved in epigenetic modifications governing cell differentiation. We aimed to evaluate the regulatory role of AMPK on intestinal epithelial development and barrier function. In this study, AMPK activator (AICAR) improved the barrier function of Caco-2 cells as indicated by increased transepithelial electrical resistance and reduced paracellular FITC-dextran permeability; consistently, AICAR enhanced epithelial differentiation and tight junction formation. Transfection of Caco-2 cells with AMPK WT plasmid, which enhances AMPK activity, improved epithelial barrier function and epithelial differentiation, while K45R (AMPK dominant negative mutant) impaired; these changes were correlated with the expression of caudal type homeobox 2 (CDX2), the key transcription factor committing cells to intestinal epithelial lineage. CDX2 deficiency abolished intestinal differentiation promoted by AMPK activation. Mechanistically, AMPK inactivation was associated with polycomb repressive complex 2 regulated enrichment of H3K27me3, the inhibitory histone modification, and lysine-specific histone demethylase-1-mediated reduction of H3K4me3, a permissive histone modification. Those histone modifications provide a mechanistic link between AMPK and CDX2 expression. Consistently, epithelial AMPK knockout in vivo reduced CDX2 expression, impaired intestinal barrier function, integrity and ultrastructure of tight junction, and epithelial cell migration, promoted intestinal proliferation and exaggerated dextran sulfate sodium-induced colitis. In summary, AMPK enhances intestinal barrier function and epithelial differentiation via promoting CDX2 expression, which is partially mediated by altered histone modifications in the Cdx2 promoter.

Heat shock factor 2 levels are associated with the severity of ulcerative colitis.

Background and AimsThe morbidity of ulcerative colitis (UC) is increasing in China every year. In addition, there is a lack of accurate diagnostic indices with which to evaluate the activity of the disease. The aim of this study was to identify UC-associated proteins as biomarkers for the diagnosis, and objective assessment of disease activity.MethodsDifferential expression of serum proteins from UC patients compared to normal controls was analyzed by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The expression of heat shock factor 2(HSF2)in colonic mucosa in Crohn's disease, Behcet's disease, ulcerative colitis, intestinal tuberculosis, infective enteritis, intestinal lymphoma, and normal controls was investigated by immunohistochemistry (IHC). The expression of the HSF2 in colonic mucosa of UC subjects with varying severity of disease was measured by real time-PCR and Western Blots. The expression of HSF2 was inhibited by HSF2 small interfering RNA (siRNA) transfection in Caco-2 cells. The concentrations of HSF2, IL-1β, and TNF-α in serum and IL-1β, and TNF-α in the supernatants of transfected Caco-2 cells were determined by ELISA.ResultsHSF2 was differentially expressed in UC patients compared to normal controls. HSF2 expression was significantly higher in the intestinal mucosa of UC patients compared to other six groups. The results of immunohistochemistry, real time-PCR, Western Blots, and ELISA showed that the expression of HSF2 increased in parallel with the severity of UC. The serum concentration of HSF2 also positively correlated with levels of IL-1β and TNF-α. After down-regulation expression of HSF2 in Caco-2 cells by RNA interference, the productions of IL-1β and TNF-α stimulated by lipopolysaccharide (LPS) increased dramatically.ConclusionsHSF2 appears to be a potential novel molecular marker for UC activity, and may provide a basis for studies on the pathogenesis and novel therapeutic targets for UC.

The expression and significance of pShuttle-CMV-heme oxygenase-1 in human caco-2 cell line

Objective To investigate the potential role and localization of heme oxygenase-1 (HO-1) gene in human colorectal cancer Caco-2 cell line by constructing the overexpression vector of HO-1 gene.Methods A overexpression vector of HO-1 gene was constructed and transfected into Caco-2 cells by lipofectin-mediated method after the identification of sequencing and enzyme digestion.Cells (Caco-2-pS/HO-1)stably overexpressing HO-1 gene were selected by using G418.Cells were divided into two groups with three repetitive holes in each group.Caco-2 cells untransfected were used as controls.The expression levels of HO-1 gene were detected by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR)and Western blotting,the cellular localization of HO-1 in Caco-2 cells was observed by cell immunofluorescence and the biological function of HO-1 gene was analyzed by cell counting kit-8 (CCK-8).Results The result of sequencing and enzyme digestion showed the construction of overexpression vector of HO-1 gene in line with the expected.As compared with Caco-2-pS cells,the expression of ZO-1 mRNA in Caco-2-pS/HO-1 cells was markedly increased (63.55 ± 0.86 vs.3.66 ± 0.36,P ＜ 0.05),and HO-1 protein had the same tendency.In transfected Caco-2 cells in immunofluorescence with C-Y3 labeled antibody under confocal laser microscope,it was found HO-1 molecules were mainly distributed in the cytoplasm and cell membrane.The cell growth activity showed same tendency in Caco-2-pS/HO-1 group in comparison to Cacl-2-pS group (5.4300 ±0.2497 vs.3.2500 ±0.2477,P＜0.05).Conclusion The overexpression vector of HO-1 gene was constructed successfully and the high expression of HO-1 located in cytomembrane and cytoplasm could promote the proliferation of Caco-2 cells,which could protect intestinal mucosa in thein vitro model. Key words: Heme oxygenase-1 gene; Caco-2 cells; Overexpression vector; Intestinal mueosa barrier

Dose-Dependent Targeted Suppression of P-glycoprotein Expression and Function in Caco-2 Cells

The efflux transporter P-glycoprotein (Pgp), encoded by the ABCB1 gene, decreases the bioavailability of a wide range of orally administered drugs. Drug permeability studies using the in vitro Caco-2 cell model commonly rely on small molecule modulators to estimate the contribution of Pgp to drug efflux. The use of such modulators may be limited by their interactions with other membrane transporters. RNA interference, a tool allowing for the specific degradation of a target gene’s mRNA, has emerged as a technique to study gene expression and function. This manuscript describes the use of chemically modified small interfering RNA (siRNA) for a dose-dependent suppression of ABCB1 in Caco-2 cells and the subsequent drug permeability assay. We transfected Caco-2 cells while in suspension with chemically modified synthetic siRNA–lipid complexes and then seeded the cells on polycarbonate semipermeable supports. Once the monolayer of Caco-2 cells formed tight junctions and expressed brush border enzymes, we determined the dose-dependent suppression of the ABCB1 gene using RT-qPCR. We measured the duration of silencing at the optimal siRNA dose by Western blot for Pgp protein. The utility of this in vitro model was determined by performing bidirectional transport studies using a well-established substrate for Pgp, rhodamine 123. A single 4 h transfection of the Caco-2 cells with ≥100 nM siRNA reduced the expression of ABCB1 mRNA by >85% at day five in culture. The time-course study revealed that the single transfection reduces Pgp protein levels for 9 days in culture. This magnitude of silencing was sufficient to reduce the efflux of rhodamine 123 as measured by the apparent permeability coefficient and intracellular accumulation. In this study, we demonstrate the dose-dependent, targeted degradation of Pgp in Caco-2 cells as a new model for assessing drug efflux from enterocytes. The dose-dependent nature of the Pgp silencing in this study offers significant improvements over other approaches to creating a Caco-2 model with suppressed ABCB1 expression. We envision that this technique, in conjunction with better small molecule inhibitors, will provide a useful tool for future drug permeability studies.

Casein phosphopeptides modulate calcium uptake and apoptosis in Caco2 cells through their interaction with the TRPV6 calcium channel

Dietary calcium intake is associated with colon cancer incidence due to its ability to modulate proliferation and apoptosis, cellular function directly linked to normal or tumour cell phenotype. In milk calcium is associated with casein whose hydrolysis produces the casein phosphopeptides (CPPs). CPPs induce calcium uptake in differentiated HT-29 and Caco2 cells. The aim of the present study was to explore: (i) the interaction between CPPs and the TRPV6 calcium channel in HT-29 and Caco2 cells; (ii) CPP effects on Caco2 cell functions. By reducing the expression of TRPV6 through small interfering RNA (siRNA), a decrease in cell response to CPPs was monitored in Caco2 cells (about 56%) but not in HT-29 cells. CPPs increased apoptosis both in undifferentiated and in transfected Caco2 cells. Based on the reported involvement of TRPV6 in cancer development, the results presented for CPPs may be helpful for their consideration as functional food ingredients.

Constitutive expression of an antioxidant enzyme, glutathione S-transferase P1, during differentiation of human intestinal Caco-2 cells

In this study, we investigated the molecular mechanism of the constitutive expression of an antioxidant enzyme, glutathione S-transferase (GST), during differentiation of human intestinal epithelial Caco-2 cells. We observed that the class π GST isozyme (GST P1) expression correlated with the increased expression of caudal-related homeodomain protein 2 (CDX2), a member of the mammalian homeobox family of transcription factors. In addition, transfection of Caco-2 cells with the human CDX2 cDNA resulted in enhanced expression of the GST P1 gene and protein. Chromatin immunoprecipitation assay demonstrated that CDX2 binds to the GST P1 promoter containing the putative consensus CDX-binding element, TTTAC, located at −247 upstream from the established site for transcription initiation. Using the dsDNA pull-down assay, it was revealed that CDX2 recognized and bound to the putative consensus CDX-binding element within the human GST P1 promoter region and that the amount of the CDX2 bound to the putative consensus CDX-binding element increased during Caco-2 cell differentiation. Furthermore, we demonstrated that CDX2 formed the transcriptional complex with Sp1 and bound to the putative consensus CDX-binding element within the human GST P1 promoter region. These data suggest that CDX2 binds to the human GST P1 promoter via complex formation with Sp1 and controls the constitutive expression of GST P1 during Caco-2 cell differentiation.

Prebiotic Oligosaccharides Reduce Proinflammatory Cytokines in Intestinal Caco-2 Cells via Activation of PPARγ and Peptidoglycan Recognition Protein 31–3

Prebiotic oligosaccharides modulate the intestinal microbiota and beneficially affect the human body by reducing intestinal inflammation. This immunomodulatory effect was assumed to be bacterial in origin. However, some observations suggest that oligosaccharides may exert an antiinflammatory effect per se. We hypothesized that oligosaccharides affect the intestinal immunity via activation of peptidoglycan recognition protein 3 (PGlyRP3), which reduces the expression of proinflammatory cytokines. Caco-2 cells were treated with the oligosaccharides, α3-sialyllactose, or fructooligosaccharides (Raftilose p95), and the effects of these treatments on PGlyRP3 and PPARγ expression, the release and expression of some proinflammatory cytokines, and NF-ĸB translocation were tested. Both oligosaccharides had antiinflammatory activity; they significantly reduced IL-12 secretion in Caco-2 cells and gene expression of IL-12p35, IL-8, and TNFα. They also reduced the gene expression and nuclear translocation of NF-ĸB. Both oligosaccharides dose and time dependently induced the production of PGlyRP3, the silencing of which by transfection of Caco-2 cells with specific small interfering RNA targeting PGlyRP3 abolished the antiinflammatory role of both oligosaccharides. Incubation of Caco-2 cells with both oligosaccharides induced PPARƳ. Antagonizing PPARγ by culturing the cells with GW9662 for 24 h inhibited the oligosaccharide-induced PGlyRP3 production and the antiinflammatory effect of the oligosaccharides. We conclude that oligosaccharides may exert an antiinflammatory effect by inducing the nuclear receptor PPARƳ, which regulates the antiinflammatory PGlyRP3.

A T/C polymorphism in the GPX4 3′UTR affects the selenoprotein expression pattern and cell viability in transfected Caco-2 cells

BackgroundSynthesis of selenoproteins such as glutathione peroxidases (GPx) requires a specific tRNA and a stem-loop structure in the 3′untranslated region (3′UTR) of the mRNA. A common single nucleotide polymorphism occurs in the GPX4 gene in a region corresponding to the 3′UTR. MethodsThe two variant 3′UTR sequences were linked to sequences from a selenoprotein reporter gene (iodothyronine deiodinase) and expressed in Caco-2 cells. Clones expressing comparable levels of deiodinase (assessed by real-time PCR) were selected and their response to tert-butyl hydroperoxide assessed by cell viability and measurement of reactive oxygen species. Selenoprotein expression was assessed by real-time PCR, enzyme activity and immunoassay. ResultsWhen selenium supply was low, cells overexpressing the C variant 3′UTR showed lower viability after oxidative challenge, increased levels of reactive oxygen species and lower GPx activity and SelH mRNA expression compared to cells overexpressing the T variant. After selenium supplementation, cell viability and GPx4 expression were higher in the cells overexpressing the C variant. Expression of transgenes incorporating the T/C variant GPX4 (rs713041) sequences in Caco-2 cells leads to alterations in both cell viability after an oxidative challenge and selenoprotein expression. This suggests that the two variants compete differently in the selenoprotein hierarchy. General SignificanceThe data provide evidence that the T/C variant GPX4 (rs713041) alters the pattern of selenoprotein synthesis if selenium intake is low. Further work is required to assess the impact on disease susceptibility.

Human intestinal acyl-CoA synthetase 5 is sensitive to the inhibitor triacsin C

To investigate whether human acyl-CoA synthetase 5 (ACSL5) is sensitive to the ACSL inhibitor triacsin C. The ACSL isoforms ACSL1 and ACSL5 from rat as well as human ACSL5 were cloned and recombinantly expressed as 6xHis-tagged enzymes. Ni(2+)-affinity purified recombinant enzymes were assayed at pH 7.5 or pH 9.5 in the presence or absence of triacsin C. In addition, ACSL5 transfected CaCo2 cells and intestinal human mucosa were monitored. ACSL5 expression in cellular systems was verified using Western blot and immunofluorescence. The ACSL assay mix included TrisHCl (pH 7.4), ATP, CoA, EDTA, DTT, MgCl(2), [9,10-(3)H] palmitic acid, and triton X-100. The 200 μL reaction was initiated with the addition of solubilized, purified recombinant proteins or cellular lysates. Reactions were terminated after 10, 30 or 60 min of incubation with Doles medium. Expression of soluble recombinant ACSL proteins was found after incubation with isopropyl beta-D-1-thiogalactopyranoside and after ultracentrifugation these were further purified to near homogeneity with Ni(2+)-affinity chromatography. Triacsin C selectively and strongly inhibited recombinant human ACSL5 protein at pH 7.5 and pH 9.5, as well as recombinant rat ACSL1 (sensitive control), but not recombinant rat ACSL5 (insensitive control). The IC50 for human ACSL5 was about 10 μmol/L. The inhibitory triacsin C effect was similar for different incubation times (10, 30 and 60 min) and was not modified by the N- or C-terminal location of the 6xHis-tag. In order to evaluate ACSL5 sensitivity to triacsin C in a cellular environment, stable human ACSL5 CaCo2 transfectants and mechanically dissected normal human intestinal mucosa with high physiological expression of ACSL5 were analyzed. In both models, ACSL5 peak activity was found at pH 7.5 and pH 9.5, corresponding to the properties of recombinant human ACSL5 protein. In the presence of triacsin C (25 μmol/L), total ACSL activity was dramatically diminished in human ACSL5 transfectants as well as in ACSL5-rich human intestinal mucosa. The data strongly indicate that human ACSL5 is sensitive to triacsin C and does not compensate for other triacsin C-sensitive ACSL isoforms.

Transcriptional inhibition of intestinal NHE8 expression by glucocorticoids involves Pax5

Sodium/hydrogen exchangers (NHEs) are a family of proteins that transport sodium ions into the cells by moving protons out of the cells. They play a major role in sodium absorption, cell volume regulation, and intracellular pH regulation. Three out of nine identified NHEs (NHE2, NHE3, and NHE8) are expressed on the apical membrane of intestinal epithelial cells. Glucocorticoids have been found to regulate NHE3 function in the intestine, but it is unknown if they have a similar function on NHE8 expression. Interestingly, high glucocorticoid levels in the intestine coincide chronologically with the change from high expression of NHE8 to high expression of NHE3. Studies were performed to explore the role of glucocorticoids on NHE8 expression during intestinal maturation. Brush-border membrane vesicles were isolated from intestinal epithelia, and Western blotting was performed to determine NHE8 protein expression of suckling male rats treated with methylpredisolone. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcription factors involved in glucocorticoid-mediated regulation. Our results showed that the expression of NHE8 mRNA and protein was decreased in glucocorticoid-treated rats and human intestinal epithelial cells (Caco-2). The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by glucocorticoid treatment. GMSAs suggested that the reduction in promoter activity in the presence of glucocorticoids was due to enhanced transcription factor Pax5 binding on the NHE8 proximal promoter region. In conclusion, this study showed that glucocorticoids inhibit NHE8 gene expression by increasing Pax5 binding on NHE8 gene promoter, suggesting an important role for Pax5 during intestinal maturation.

Epidermal growth factor inhibits intestinal NHE8 expression via reducing its basal transcription

Sodium/hydrogen exchangers (NHEs) play a major role in Na(+) absorption, cell volume regulation, and intracellular pH regulation. Of the nine identified mammalian NHEs, three (NHE2, NHE3, and NHE8) are localized on the apical membrane of epithelial cells in the small intestine and the kidney. Although the regulation of NHE2 and NHE3 expression has been extensively studied in the past decade, little is known about the regulation of NHE8 gene expression under physiological conditions. The current studies were performed to explore the role of epidermal growth factor (EGF) on NHE8 expression during intestinal maturation. Brush-border membrane vesicles (BBMV) were isolated from intestinal epithelia, and Western blot analysis was performed to determine NHE8 protein expression of sucking male rats treated with EGF. Real-time PCR was used to quantitate NHE8 mRNA expression in rats and Caco-2 cells. Human NHE8 promoter activity was characterized through transfection of Caco-2 cells. Gel mobility shift assays (GMSAs) were used to identify the promoter sequences and the transcriptional factors involved in EGF-mediated regulation. Our results showed that intestinal NHE8 mRNA expression was decreased in EGF-treated rats and Caco-2 cells, and NHE8 protein abundance was also decreased in EGF-treated rats. The activity of the human NHE8 gene promoter transfected in Caco-2 cells was also reduced by EGF treatment. This could be explained by reduced binding of transcription factor Sp3 on the NHE8 basal promoter region in the presence of EGF. Pretreatment with MEK1/2 inhibitor UO-126 could prevent EGF-mediated inhibition of NHE8 gene expression. In conclusion, this study showed that EGF inhibits NHE8 gene expression through reducing its basal transcription, suggesting an important role of EGF in regulating NHE expression during intestinal maturation.

Molecular cloning and characterization of the human lactoferrin receptor gene promoter

Lactoferrin (Lf) is an iron‐binding protein in human milk. Multiple functions of Lf are postulated to be mediated by a Lf receptor (LfR). We have demonstrated that the Lf receptor (LfR) plays an important role in Lf endocytosis by Caco‐2 cells via a clathrin‐mediated pathway, which focused our interest to investigate the transcriptional regulation of LfR. In the present study, we cloned and characterized the promoter from a 3100 bp 5′‐flanking region of the human LfR gene. The transcription start site was identified as 298 bp upstream of the translation start site (+1) by 5′ RLM‐RACE. The promoter region contains neither a TATA box nor a CCAAT box. A series of deletions of 5′‐flanking sequence were cloned into the promoter‐less luciferase reporter vector and transiently transfected into Caco‐2 cells. The transfection assays demonstrated a sequence of −419/+1 sufficient for eliciting maximal promoter activity and the region −507/−729 exhibited inhibitory effects on reporter gene expression in transiently transfected Caco‐2 cells. The −419/+1 fragment contains a Sp1 binding site, which was confirmed by electrophoretic mobility supershift assay. Mutations of this binding site decreased Sp1 binding affinity in gel shift assays. In conclusion, the isolated LfR gene promoter contains downstream core promoter elements and the Sp1 binding site plays a critical role in transcriptional regulation of the LfR gene.

Curcumin: a novel nutritionally derived ligand of the vitamin D receptor with implications for colon cancer chemoprevention

The nuclear vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25D) to regulate gene transcription. Recently, the secondary bile acid, lithocholate (LCA), was recognized as a novel VDR ligand. Using reporter gene and mammalian two-hybrid systems, immunoblotting, competitive ligand displacement and quantitative real-time PCR, we identified curcumin (CM), a turmeric-derived bioactive polyphenol, as a likely additional novel ligand for VDR. CM (10 −5 M) activated transcription of a luciferase plasmid containing the distal vitamin D responsive element (VDRE) from the human CYP3A4 gene at levels comparable to 1,25D (10 −8 M) in transfected human colon cancer cells (Caco-2). While CM also activated transcription via a retinoid X receptor (RXR) responsive element, activation of the glucocorticoid receptor (GR) by CM was negligible. Competition binding assays with radiolabeled 1,25D confirmed that CM binds directly to VDR. In mammalian two-hybrid assays employing transfected Caco-2 cells, CM (10 −5 M) increased the ability of VDR to recruit its heterodimeric partner, RXR, and steroid receptor coactivator-1 (SRC-1). Real-time PCR studies revealed that CM-bound VDR can activate VDR target genes CYP3A4, CYP24, p21 and TRPV6 in Caco-2 cells. Numerous studies have shown chemoprotection by CM against intestinal cancers via a variety of mechanisms. Small intestine and colon are important VDR-expressing tissues where 1,25D has known anticancer properties that may, in part, be elicited by activation of CYP-mediated xenobiotic detoxification and/or up-regulation of the tumor suppressor p21. Our results suggest the novel hypothesis that nutritionally-derived CM facilitates chemoprevention via direct binding to, and activation of, VDR.

ERK regulates strain‐induced migration and proliferation from different subcellular locations

Repetitive deformation like that engendered by peristalsis or villous motility stimulates intestinal epithelial proliferation on collagenous substrates and motility across fibronectin, each requiring ERK. We hypothesized that ERK acts differently at different intracellular sites. We stably transfected Caco-2 cells with ERK decoy expression vectors that permit ERK activation but interfere with its downstream signaling. Targeting sequences constrained the decoy inside or outside the nucleus. We assayed proliferation by cell counting and migration by circular wound closure with or without 10% repetitive deformation at 10 cycles/min. Confocal microscopy confirmed localization of the fusion proteins. Inhibition of phosphorylation of cytoplasmic RSK or nuclear Elk confirmed functionality. Both the nuclear-localized and cytosolic-localized ERK decoys prevented deformation-induced proliferation on collagen. Deformation-induced migration on fibronectin was prevented by constraining the decoy in the nucleus but not in the cytosol. Like the nuclear-localized ERK decoy, a Sef-overexpressing adenovirus that sequesters ERK in the cytoplasm also blocked the motogenic and mitogenic effects of strain. Inhibiting RSK or reducing Elk ablated both the mitogenic and motogenic effects of strain. RSK isoform reduction revealed isoform specificity. These results suggest that ERK must translocate to the nucleus to stimulate cell motility while ERK must act in both the cytosol and the nucleus to stimulate proliferation in response to strain. Selectively targeting ERK within different subcellular compartments may modulate or replace physical force effects on the intestinal mucosa to maintain the intestinal mucosal barrier in settings when peristalsis or villous motility are altered and fibronectin is deposited into injured tissue.