Human Intestinal Caco2-BBE Cells Research Articles

Identification and Molecular Mechanism of Anti-inflammatory Peptides Isolated from Jack Bean Protein Hydrolysates: in vitro Studies with Human Intestinal Caco-2BBe Cells

Jack bean (JB), Canavalia ensiformis (L.) DC, is a commonly cultivated legume in Indonesia. It is rich in protein, which can be hydrolyzed, making it potentially a good source of bioactive peptides. Intestinal inflammation is associated with several diseases, and the production of interleukin-8 (IL-8) in intestinal epithelial cells induced by tumor necrosis factor (TNF)-α has an important role in inflammatory reaction. The present study investigated the anti-inflammatory effects of peptides generated from enzymatic hydrolysis of JB protein on human intestinal Caco-2BBe cells. Additionally, in silico approaches were used to identify potential bioactive peptides. JB protein hydrolysate (JBPH) prepared using pepsin and pancreatin reduced the IL-8 expression at protein and mRNA levels in Caco-2BBe cells stimulated with TNF-α. Immunoblot analysis showed that the JBPH reduced the TNF-α-induced phosphorylation of c-Jun-NH(2)-terminal kinase, nuclear factor kappa B (NF-κB), and p38 proteins. Anti-inflammatory activity was observed in the 30% acetonitrile fraction of JBPH separated on a Sep-Pak C18 column. An ultrafiltration method revealed that relatively small peptides (< 3 kDa) had a potent inhibitory effect on the IL-8 production. Purification of the peptides by reversed-phase and anion-exchange high performance chromatography produced three peptide fractions with anti-inflammatory activities. A combination of mass spectrometry analysis and in silico approaches identified the potential anti-inflammatory peptides. Peptides derived from JB protein reduces the TNF-α-induced inflammatory response in Caco-2BBe cells via NF-κB and mitogen-activated protein kinase signaling pathways. Our results may lead to a novel therapeutic approach to promote intestinal health.

Re: Activation of the PKA Signaling Pathway Stimulates Oxalate Transport by Human Intestinal Caco2-BBE Cells.

Re: Activation of the PKA Signaling Pathway Stimulates Oxalate Transport by Human Intestinal Caco2-BBE Cells.

Activation of the PKA signaling pathway stimulates oxalate transport by human intestinal Caco2-BBE cells.

Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [14C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [14C]oxalate transport as a function of increasing [14C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in Vmax and reduction (2-fold) in Km. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced Km. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.

Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor.

Most kidney stones (KS) are composed of calcium oxalate, and small increases in urine oxalate affect the stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 (PAT1) plays a crucial role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and related KS, reflecting the importance of understanding regulation of intestinal oxalate transport. We previously showed that ATP and UTP inhibit oxalate transport by human intestinal Caco2-BBE cells (C2). Since ATP is rapidly degraded to adenosine (ADO), we examined whether intestinal oxalate transport is regulated by ADO. We measured [14C]oxalate uptake in the presence of an outward Cl gradient as an assay of Cl-oxalate exchange activity, ≥49% of which is PAT1-mediated in C2 cells. We found that ADO significantly inhibited oxalate transport by C2 cells, an effect completely blocked by the nonselective ADO receptor antagonist 8- p-sulfophenyltheophylline. ADO also significantly inhibited oxalate efflux by C2 cells, which is important since PAT1 mediates oxalate efflux in vivo. Using pharmacological antagonists and A2B adenosine receptor (A2B AR) siRNA knockdown studies, we observed that ADO inhibits oxalate transport through the A2B AR, phospholipase C, and PKC. ADO inhibits oxalate transport by reducing PAT1 surface expression as shown by biotinylation studies. We conclude that ADO inhibits oxalate transport by lowering PAT1 surface expression in C2 cells through signaling pathways including the A2B AR, PKC, and phospholipase C. Given higher ADO levels and overexpression of the A2B AR in inflammatory bowel disease (IBD), our findings have potential relevance to pathophysiology of IBD-associated hyperoxaluria and related KS.

Competitive regulation of human intestinal β-carotene 15,15′-monooxygenase 1 (BCMO1) gene expression by hepatocyte nuclear factor (HNF)-1α and HNF-4α

AimAmong the pro-vitamin A carotenoids, β-carotene is an excellent source of vitamin A. β-Carotene 15,15′-monooxygenase 1 (BCMO1) is a critical enzyme involved in the conversion of β-carotene into vitamin A (retinal) in the small intestine of many vertebrates. In the present study, we investigated the regulation of human BCMO1 gene expression using human intestinal Caco-2 BBe cells. Main methodsWe performed electrophoretic mobility shift assays and chromatin immunoprecipitation assays to investigate the binding properties of hepatocyte nuclear factor (HNF)-1α and HNF-4α to the proximal promoter of the human BCMO1 gene. Caco-2 BBe cells were also transfected with HNF-1α and HNF-4α siRNAs, and BCMO1 gene expression levels and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and luciferase reporter assays, respectively. Key findingsWe identified overlapping binding sites for HNF-1α and HNF-4α in the human BCMO1 gene proximal promoter. Endogenous nuclear HNF-1α and HNF-4α proteins competitively bound these sites in Caco-2 BBe cells. BCMO1 gene expression levels and promoter activity were significantly decreased in HNF-1α siRNA-transfected Caco-2 BBe cells. In contrast, HNF-4α siRNA-transfected cells exhibited a significant increase in BCMO1 gene expression and promoter activity. Mutation of these overlapping binding sites dramatically decreased BCMO1 promoter activity. SignificanceOur study indicates that the competitive actions of HNF-1α and HNF-4α on their overlapping binding sites in the human BCMO1 gene promoter oppositely regulate BCMO1 gene expression in the human small intestine.

Hepatocyte nuclear factor-4α regulates human cellular retinol-binding protein type II gene expression in intestinal cells

Cellular retinol-binding protein type II (CRBPII) is abundantly expressed in the small intestinal enterocytes of many vertebrates and plays important physiological roles in intestinal absorption, transport, and metabolism of vitamin A. In the present study, we investigated regulation of human CRBPII gene expression using human intestinal Caco-2 BBe cells. We found that the human CRBPII gene contained a direct repeat 1 (DR-1)-like nuclear receptor response element in the proximal promoter region and that endogenous hepatocyte nuclear factor-4alpha (HNF-4alpha) was a major transcription factor binding to the DR-1-like element. Cotransfection of HNF-4alpha expression vector transactivated the human CRBPII gene promoter activity, whereas mutation of the DR-1-like element abolished the promoter activity. Stably transfected Caco-2 BBe cells overexpressing HNF-4alpha significantly increased endogenous CRBPII gene expression and retinyl ester synthesis. Reduction of HNF-4alpha protein levels by HNF-4alpha small interference RNA decreased CRBPII gene expression. Caco-2 BBe cells treated with phorbol 12-myristate 13-acetate, a protein kinase C activator, decreased nuclear HNF-4alpha protein level and binding activity to the human CRBPII gene DR-1-like element, as well as CRBPII gene expression. Moreover, nuclear HNF-4alpha protein levels, HNF-4alpha protein binding to human CRBPII DR-1-like elements, and CRBPII gene expression level were coordinately increased during Caco-2 BBe cell differentiation. These results suggest that HNF-4alpha is an important transcriptional factor that regulates human CRBPII gene expression and provide the possibility for a novel function of HNF-4alpha in the regulation of human intestinal vitamin A absorption and metabolism.

Aldosterone regulation of intestinal Na absorption involves SGK-mediated changes in NHE3 and Na+pump activity

Aldosterone-induced intestinal Na(+) absorption is mediated by increased activities of apical membrane Na(+)/H(+) exchange (aNHE3) and basolateral membrane Na(+)-K(+)-ATPase (BLM-Na(+)-K(+)-ATPase) activities. Because the processes coordinating these events were not well understood, we investigated human intestinal Caco-2BBE cells where aldosterone increases within 2-4 h of aNHE3 and alpha-subunit of BLM-Na(+)-K(+)-ATPase, but not total abundance of these proteins. Although aldosterone activated Akt2 and serum glucorticoid kinase-1 (SGK-1), the latter through stimulation of phosphatidylinositol 3-kinase (PI3K), only the SGK-1 pathway mediated its effects on Na(+)-K(+)-ATPase. Ouabain inhibition of the early increase in aldosterone-induced Na(+)-K(+)-ATPase activation blocked most of the apical NHE3 insertion, possibly by inhibiting Na(+)-K(+)-ATPase-induced changes in intracellular sodium concentration ([Na](i)). Over the next 6-48 h, further increases in aNHE3 and BLM-Na(+)-K(+)-ATPase activity and total protein expression were observed to be largely mediated by aldosterone-activated SGK-1 pathway. Aldosterone-induced increases in NHE3 mRNA, for instance, could be inhibited by RNA silencing of SGK-1, but not Akt2. Additionally, aldosterone-induced increases in NHE3 promoter activity were blocked by silencing SGK-1 as well as pharmacological inhibition of PI3K. In conclusion, aldosterone-stimulated intestinal Na(+) absorption involves two phases. The first phase involves stimulation of PI3K, which increases SGK-dependent insertion and function of BLM-Na(+)-K(+)-ATPase and subsequent increased membrane insertion of aNHE3. The latter may be caused by Na(+)-K(+)-ATPase-induced changes in [Na] or transcellular Na flux. The second phase involves SGK-dependent increases in total NHE3 and Na(+)-K(+)-ATPase protein expression and activities. The coordination of apical and BLM transporters after aldosterone stimulation is therefore a complex process that requires multiple time- and interdependent cellular processes.

Triiodothyronine stimulates CMO1 gene expression in human intestinal Caco-2 BBe cells

Vitamin A is derived from provitamin A carotenoids, mainly β-carotene, by β-carotene 15,15′-monooxygenase (CMO1; EC 1.13.11.21). We previously found that enhancement of CMO1 mRNA expression was related to the levels of hormones, such as thyroid hormones, in chick duodenum. We investigated whether CMO1 expression was increased by triiodothyronine (T 3), a thyroid hormone, using human intestinal Caco-2 BBe cells. Treatment of 7 days post-confluent Caco-2 BBe cells with T 3 significantly enhanced CMO1 mRNA levels in both dose- and time-dependent manners. This T 3-inducing effect on CMO1 mRNA level was blocked by actinomycin D. The levels of mRNAs for the thyroid hormone receptors TRα1 and TRβ1 were significantly increased in 7 days post-confluent Caco-2 BBe cells. CMO1 enzyme activity was also significantly increased by T 3 treatment in medium supplemented with fetal bovine serum. Furthermore, T 3 treatment also increased the level of mRNA for lecithin:retinol acyltransferase (LRAT), but not those for cellular retinol-binding protein, type II (CRBPII) and retinal dehydrogenase 1 (RALDH1), in Caco-2 BBe cells. These results indicate that T 3 is an important hormone for the regulation of vitamin A and β-carotene metabolism-related gene expression in human small intestinal cells.

Directionality of transepithelial Na gradients affects NHE2 and NHE3 activity and expression in human intestinal Caco2bbe cells

Directionality of transepithelial Na gradients affects NHE2 and NHE3 activity and expression in human intestinal Caco2bbe cells

Directionality of transepithelial Na gradients affects NHE2 and NHE3 activity and expression in human intestinal Caco2bbe cells

Directionality of transepithelial Na gradients affects NHE2 and NHE3 activity and expression in human intestinal Caco2bbe cells