Circadian regulation of uroguanylin and guanylin in the rat intestine

Uroguanylin (UGN) and guanylin (GN) are theendogenous intestinal ligands for guanylyl cyclase C (GC-C). Weexamined the circadian expression of UGN, GN, and GC-C in the jejunum,ileum, and proximal colon of young adult rats by Northern blotanalyses. These assays revealed that UGN is more abundant in theproximal small intestine, whereas GN and GC-C are more abundant in theproximal colon. mRNA levels showed significant circadian variation forUGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-foldpeak/trough difference), and GC-C (3- to 5-fold peak/troughdifference). The maximal abundance occurred in the dark period for allthree mRNAs, although peak UGN and GN expression occurred later in thedark period in the jejunum relative to the ileum and colon. Immunoblotanalyses using monospecific polyclonal antibodies against UGN and GNprohormones confirmed the regional and circadian variation detected byNorthern assays. Thus the expression of these genes is regulated notonly by histological position but also by circadian time.

     

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.     

Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.     

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C₁ cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C₁ was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10) (m) increased furosemideinsensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanideinsensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloridedepleted cells. When cells with normal chloride content were depolarized (135 mm medium potassium + 10 m valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl^–Br^–>NO ₃ ^– >I^–. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.     

Altered expression of sialylated carbohydrate antigens in HT29 colonic carcinoma cells

To determine whether cell growth conditions impacted carbohydrate expression, HT29 cells were gradually transferred from a conventional glucose-containing media to a glucose-free galactose containing media. Indirect immunofluorescence on acetone fixed cells showed increased expression of sialyl Lewis A antigen (CA19-9), sialyl Lewis C (DUPAN2) and Tn/sialyl-Tn on the surface of HT29 cells grown in the glucose-free galactose containing media compared to those grown in the glucose containing media. Sialyltransferases responsible for the synthesis for these sialylated epitopes were increased in the galactose-fed HT29 cells. Media overlying the cells was subjected to isopycnic ultracentrifugation in cesium chloride and the fractions derived from both glucose and galactose media with equivalent buoyant densities of 1.56 g/L, which are predicted to contain mucin glycoforms, were further separated by HPLC using a Mono-Q anion exchange column. The chromatograph of eluent from the sample derived from the cells growing in the galactose containing media showed an increased peak that reacted with the anti-sialyl Lewis A antibody, CA19-9. These results show that alteration of in vitro culture conditions may cause HT29 colonic carcinoma cells to alter the expression of sialylated carbohydrates.     

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 microM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide.     

Decreased phospholipase A2 activity in butyrate differentiated HT29 cells.

Our earlier work has shown that in butyrate differentiated colonic HT29 cells, there is an alteration in phospholipid composition as compared to control. To know more about these changes, butyrate treated and control cell homogenates were incubated in presence of calcium and phospholipids were analyzed. It was observed that incubation with calcium was associated with increase in lysophosphatidylcholine (lysoPC) and free fatty acids and the increase was much higher in control as compared to butyrate treated cells. There was no alteration in lysoPC content. These products are formed by the action of phospholipase A2 (PLA2) which is activated by calcium and suggests that butyrate-induced differentiation is associated with decrease in PLA2 activity.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

Interaction of atrial natriuretic peptide, urodilatin, guanylin and uroguanylin in the isolated perfused rat kidney

Escherichia coli heat-stable enterotoxin (STa), guanylin and uroguanylin are novel natriuretic and kaliuretic peptides that bind to and activate membrane guanylate cyclase (GC) receptors such as GC-C and OK-GC that are expressed in the kidney and intestine. Atrial natriuretic peptide (ANP) and its renal form (urodilatin, UROD) elicit natriuretic effects by activation of a different membrane guanylate cyclase, GC-A. Experiments were done in perfused rat kidneys to search for possible synergistic interactions between ANP, UROD, guanylin and uroguanylin on renal function. Pretreatment with ANP (0.03 nM) enhanced guanylin (0.19 microM) natriuretic activity (%ENa(+); from 18.5+/-4.25 to 31.5+/-1.69, P<0.05, 120 min) and its kaliuretic activity (%EK(+); from 24.5+/-4.43 to 50.6+/-3.84, P<0.05, 120 min). Furthermore, ANP increased the natriuretic (29.05+/-3.00 to 37.8+/-2.95, P<0.05, 120 min) and kaliuretic (from 33.2+/-3.52 to 42.83+/-2.45, P<0.05, 120 min) responses of perfused kidneys treated with low-dose (0.06 microM) uroguanylin. In contrast, ANP clearly inhibited the uroguanylin-induced (0.31 microM) increase in %ENa(+) (from 35.9+/-2.37 to 14.8+/-1.93, P<0.05, 120 min), and in %EK(+) (from 51.0+/-4.43 to 38.8+/-3.61, P<0.05, 120 min). UROD (0.03 nM) also enhanced the guanylin-induced natriuresis (to %ENa(+)=31.0+/-1.93, P<0.05, 120 min) and kaliuresis (to %EK(+)=54.2+/-3.61, P<0.05, 120 min), and inhibited the %ENa(+) of uroguanylin (0.31 microM) to 17.9+/-1.67 as well as its %EK(+) to 24.3+/-3.13 (both at 120 min, P<0.05). The synergism between ANP and UROD with either guanylin or uroguanylin at sub-threshold doses and the unexpected antagonism between ANP and UROD with uroguanylin at a pharmacological dose point to possible interactions between natriuretic peptide receptor (NPR) and uroguanylin/guanylin receptor signaling pathways. The interactions herein described may play a contributory role in the regulation of kidney function in many pathophysiological states, such as in the saliuresis following ingestion of salty meals.     

The binding of lactoferrin to glycosaminoglycans on enterocyte-like HT29-18-C1 cells is mediated through basic residues located in the N-terminus

Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K_d: 63±22 nM; 0.7±0.2 μM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G¹RRRR⁵ is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A¹PRK⁴ in bLf and K¹ATT⁴ in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.     

Sorting of sphingolipids in the endocytic pathway of HT29 cells

The intracellular flow and fate of two fluorescently labeled sphingolipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoyl glucosyl sphingosine (C6-NBD-glucosylceramide) and C6-NBD-sphingomyelin, was examined in the human colon adenocarcinoma cell line HT29. After their insertion into the plasma membrane at low temperature and subsequent warming of the cells to 37 degrees C, both sphingolipid analogues were internalized by endocytosis, but their intracellular site of destination differed. After 30 min of internalization, C6-NBD-glucosylceramide was localized in the Golgi apparatus, as demonstrated by colocalization with fluorescently labeled ceramide, a Golgi complex marker, and by showing that monensin-induced disruption of the Golgi structure was paralleled by a similar perturbation of the fluorescence distribution. By contrast, C6-NBD-sphingomyelin does not colocalize with the tagged ceramide. Rather, a colocalization with ricin, which is internalized by endocytosis and predominantly reaches the lysosomes, was observed, indicating that the site of delivery of this lipid is restricted to endosomal/lysosomal compartments. Also, in monensin-treated cells no change in the distribution of fluorescence was observed. Thus, these results demonstrate that (sphingo)lipid sorting can occur in the endocytic pathway. Interestingly, the observed sorting phenomenon was specific for glucosylceramide, when compared to other glycolipids, while only undifferentiated HT29 cells displayed the different routing of the two lipids. In differentiated HT29 cells the internalization pathway of sphingomyelin and glucosylceramide was indistinguishable from that of transferrin.     

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.     

The modulation of growth by HMBA in PKC overproducing HT29 colon cancer cells.

To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses high levels of PKC beta 1. We found that although HMBA markedly inhibited the growth of the control cells, no inhibition was seen with the HT29-PKC7 cells. On the other hand the tumor promoter 12-0-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and TPA. These results may be relevant to the use of HMBA in combination with other agents in the therapy of specific cancers.     

Rapid formation of tight junctions in HT 29 human adenocarcinoma cells by hypertonic salt solutions

The human colon adenocarcinoma cell line HT 29 grows virtually without tight junctions (TJ) under standard culture conditions. Earlier studies have shown that focal TJ (fasciae occludentes) can be rapidly assembled in this cell line under the influence of various proteases. Here we show that focal TJ can be induced in this cell line by a brief treatment with appropriate salt solutions. Induction by ammonium sulfate in Hanks' buffer reached a maximum value after 15 to 30 min. The amount of TJ increased with the salt concentration and reached a plateau value at a concentration of 160 mM ammonium sulfate. The amount and complexity of TJ induced by ammonium sulfate were similar to those in experiments using trypsin as inducing agent as shown by morphometric analysis. At 0 degrees C, no TJ were formed under the influence of the salt. A comparative study of TJ induction using a variety of inorganic and organic salts gave the following results. All alkali sulfates induced TJ, although with different yield. Both calcium and magnesium chloride were potent inducers. Ammonium and sodium salts encompassing a variety of anions covered a wide range from maximum induction (sulfate, citrate) to almost complete absence of induction (nitrate). Sodium chloride did not induce any TJ. It follows that the induction of TJ is a specific effect of individual ionic components of the solution as opposed to a general effect of osmolarity and ionic strength. The data suggest tentatively that antichaotropic but not chaotropic ions have the potential to trigger the formation of TJ in this experimental system.