Intestinal Epithelial Cells Cells Research Articles

Individual and Combined Cytotoxic Effects of Co-Occurring Fumonisin Family Mycotoxins on Porcine Intestinal Epithelial Cell.

Human health is seriously threatened by mycotoxin contamination, yet health risk assessments are typically based on just one mycotoxin, potentially excluding the additive or competitive interactions between co-occurring mycotoxins. In this investigation, we evaluated the individual or combined toxicological effects of three fumonisin-family B mycotoxins: fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3), by using porcine intestinal epithelial cells (IPEC). IPEC cells were exposed to various concentrations (2.5-40 μM) for 48 h, and a cell counting kit (CCK8) was used to determine cell vitality. Firstly, we discovered that they might inhibit cell viability. Additionally, the cytotoxicity of FB1 was significantly greater than that of FB2 and FB3. The results also indicated that the combinations of FB1-FB2, FB2-FB3, and FB1-FB2-FB3 showed synergistically toxicological effects at the ID10-ID50 levels and antagonistic effects at the ID75-ID90 levels. In addition, the FB1-FB3 exposure was also synergistic at the ID10-ID25 level. We also found that myriocin and resveratrol alleviated the cytotoxicity induced by fumonisin in IPEC cells. In all, this study may contribute to the determination of legal limits, the optimization of risk assessment for fumonisins in food and feed, and the development of new methods to alleviate fumonisin toxicity.

Butyrate ameliorates Paneth cell defects, total inflammation, and epithelial cell apoptosis during mitochondrial dysfunction driven by Prohibitin 1-deficiency

Introduction: Prohibitin 1 (PHB1) is a mitochondrial chaperone protein important for maximal activity of the electron transport chain. PHB1 deficiency in intestinal epithelial cells (IECs) or specifically in Paneth cells (PCs) induces epithelial mitochondrial dysfunction, PC defects, gut microbiota dysbiosis, and spontaneous ileitis in mice by 20 weeks-of-age. Dysbiotic gut microbiota has been implicated in inflammatory bowel diseases and is associated with the decreased abundance of short chain fatty acids (SCFAs; acetate, propionate, and butyrate)-producing bacteria. SCFAs play a significant role in reducing intestinal inflammation and enhancing epithelial barrier dysfunction. We tested the hypothesis that luminal SCFA levels were decreased in mice with PHB1 deficiency and that supplementation of SCFAs will ameliorate intestinal inflammation during mitochondrial dysfunction driven by loss of PHB1. Methods: Ileal luminal contents were collected from Phb1fl/fl (control) and Phb1iΔIEC (Phb1-deficient in IECs) mice at 20 weeks of age and SCFAs were measured by gas chromatography-mass spectrometry. Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 20 mM butyrate dissolved in their drinking water from 16 to 20 weeks-of-age and PC defects were measured by antimicrobial peptide expression (Lysozyme, Cryptdin 3, Cryptdin 5, and Ang4) and Lysozyme staining allocation into secretory granules. Ileitis was measured by histological scoring and pro-inflammatory cytokine expression. To test epithelial intrinsic responses, ileal crypt enteroids derived from Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 200 μg/ml healthy ileal stool lysates (from Phb1fl/fl mice) or dysbiotic stool lysates (from Phb1iΔIEC mice) for 16 hours with or without 0.05 mM butyrate, 0.5 mM propionate, 0.5 mM acetate, alone or in combination. Enteroid viability and apoptosis were measured by morphological change and cleaved Caspase 3 expression. Results: Butyrate was significantly decreased in ileal stool of Phb1iΔIEC mice. Butyrate supplementation in Phb1iΔIEC mice protected against ileitis and PC defects. Dysbiotic stool lysate decreased viability and increased apoptosis in Phb1-deficient enteroids that were prevented by butyrate supplementation. Conclusion: Mitochondrial health in IECs cells is crucial for healthy gut microbiota composition and for epithelial defense when the microbiome becomes dysbiotic. Butyrate rescues IECs from insults caused by dysbiotic gut microbiota during mitochondrial dysfunction driven by PHB1 deletion. Funding: APS Postdoctoral Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chitinase A, a tightly regulated virulence factor of Salmonella enterica serovar Typhimurium, is actively secreted by a Type 10 Secretion System.

As a facultative intracellular pathogen, Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne diseases in humans. With the ingestion of fecal contaminated food or water, S. Typhimurium reaches the intestine. Here, the pathogen efficiently invades intestinal epithelial cells of the mucosal epithelium by the use of multiple virulence factors. Recently, chitinases have been described as emerging virulence factors of S. Typhimurium that contribute to the attachment and invasion of the intestinal epithelium, prevent immune activation, and modulate the host glycome. Here we find that the deletion of chiA leads to diminished adhesion and invasion of polarized intestinal epithelial cells (IEC) compared to wild-type S. Typhimurium. Interestingly, no apparent impact on interaction was detected when using non-polarized IEC or HeLa epithelial cells. In concordance, we demonstrate that chiA gene and ChiA protein expression was solely induced when bacteria gain contact with polarized IEC. The induction of chiA transcripts needs the specific activity of transcriptional regulator ChiR, which is co-localized with chiA in the chitinase operon. Moreover, we established that after chiA is induced, a major portion of the bacterial population expresses chiA, analyzed by flow cytometry. Once expressed, we found ChiA in the bacterial supernatants using Western blot analyses. ChiA secretion was completely abolished when accessory genes within the chitinase operon encoding for a holin and a peptidoglycan hydrolase were deleted. Holins, peptidoglycan hydrolases, and large extracellular enzymes in close proximity have been described as components of the bacterial holin/peptidoglycan hydrolase-dependent protein secretion system or Type 10 Secretion System. Overall, our results confirm that chitinase A is an important virulence factor, tightly regulated by ChiR, that promotes adhesion and invasion upon contact with polarized IEC and is likely secreted by a Type 10 Secretion System (T10SS).

BUTYRATE AMELIORATES PANETH CELL DEFECTS, TOTAL INFLAMMATION, AND EPITHELIAL CELL APOPTOSIS DURING MITOCHONDRIAL DYSFUNCTION DRIVEN BY PROHIBITIN 1-DEFICIENCY

Abstract INTRODUCTION Prohibitin 1 (PHB1) is a mitochondrial chaperone protein important for maximal activity of the electron transport chain. PHB1 deficiency in intestinal epithelial cells (IECs) or specifically in Paneth cells (PCs) induces epithelial mitochondrial dysfunction, PC defects, gut microbiota dysbiosis, and spontaneous ileitis in mice by 20 weeks-of-age. Dysbiotic gut microbiota has been implicated in inflammatory bowel diseases and is associated with the decreased abundance of short chain fatty acids (SCFAs; acetate, propionate, and butyrate)-producing bacteria. SCFAs play a significant role in reducing intestinal inflammation and epithelial barrier dysfunction. We tested the hypothesis that luminal SCFA levels were decreased in mice with PHB1 deficiency and that supplementation of SCFAs will ameliorate intestinal inflammation during mitochondrial dysfunction driven by loss of PHB1. METHODS Ileal luminal contents were collected from Phb1fl/fl (control) and Phb1iΔIEC (Phb1-deficient in IECs) mice at 20 weeks of age and SCFAs were measured by gas chromatography-mass spectrometry. Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 20 mM butyrate dissolved in their drinking water from 16 to 20 weeks-of-age and PC defects were measured by antimicrobial peptide expression (Lysozyme, Cryptdin 3, Cryptdin 5, and Ang4) and Lysozyme staining allocation into secretory granules. Ileitis was measured by histological scoring and pro-inflammatory cytokine expression. To test epithelial intrinsic responses, ileal crypt enteroids derived from Phb1iΔIEC and Phb1fl/fl littermate mice were treated with 200 µg/ml healthy ileal stool lysates (from Phb1fl/fl mice) or dysbiotic stool lysates (from Phb1iΔIEC mice) for 16 hours with or without 0.05 mM butyrate, 0.5 mM propionate, 0.5 mM acetate, alone or in combination. Enteroid viability and apoptosis were measured by morphological change and cleaved Caspase 3 expression. RESULTS Butyrate was significantly decreased in ileal stool of Phb1iΔIEC mice. Butyrate supplementation in Phb1iΔIEC mice protected against ileitis and PC defects. Dysbiotic stool lysate decreased viability and increased apoptosis in Phb1-deficient enteroids that were prevented by butyrate supplementation. CONCLUSION Mitochondrial health in IECs cells is crucial for healthy gut microbiota composition and for epithelial defense when the microbiome becomes dysbiotic. Butyrate rescues IECs from insults caused by dysbiotic gut microbiota during mitochondrial dysfunction driven by PHB1 deletion.

Human Milk Oligosaccharide 2'-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells.

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2′-fucosyllactose (2′FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2′FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4+ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNβ, IFNλ1 and CCL5 secretion. Pre-incubation with 2′FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2′FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2′FL. These results provide insights regarding the protective capacity of human milk components in early life.

A1 USING HUMAN NEONATAL ORGANOIDS TO EXPLORE GUT-IMMUNE SYSTEM INTERACTIONS OF THE NEONATAL INTESTINE

BackgroundNecrotizing Enterocolitis (NEC) affects around 10% of preterm babies and is one of the leading causes of death for newborns. NEC is characterized by exaggerated inflammation of the intestinal mucosa, possibly triggered by aberrant exposure to gut microbes, leading to hypoxic conditions and the death of intestinal tissues. It has been hypothesized that NEC develops when the immature intestine (epithelium and immune system) is unable to properly balance these new microbial interactions. To date, NEC is poorly understood and due to the difficulty of modeling the human neonatal intestine, few therapeutic options are available.Intestinal epithelial cells (IEC) are important players in promoting beneficial host-microbe interactions in the gut, being the primary barrier that separates the host’s mucosal immune system from luminal microbiota, as well as key players in mediating signaling between microbes and the host. Based on their location, IEC are also subject to injury associated with maladaptive immune responses against gut microbes. Many studies have shown that immune cells (such as T helper 17 cells) interact with IEC to promote gut health and function. These interactions include educating IEC on how to respond to, and fight pathogenic microbes, yet also remain tolerant to commensal microbes.AimsThis project seeks to develop an in vitro human neonatal intestinal organoid model to study developmental changes in IEC and their functional interactions with neonatal Th17 cells.Methods3D organoids were established from human neonatal intestinal biopsies and then co-cultured with the supernatant of differentiated Th17 cells or with recombinant cytokines, IL-17 and IL-22. Changes in barrier function, cell proliferation, production of mucins and anti-microbial peptides (AMP) were analyzed by qPCR and immunostaining.ResultsUsing 3D neonatal organoids we observed that the supernatants from neonatal Th17 cells (containing IL-17, IL-22 etc.) promoted the proliferation, differentiation and barrier function of the neonatal epithelium. By using specific recombinant cytokine (IL22, IL17) and neutralizing IL-22 antibodies in parallel, we demonstrated that the high levels of IL-22 produced by neonatal Th17 cells specifically induced proliferation of IEC, AMP and mucus production as compared to control media treated organoids, as shown by increases in Ki67, Reg3γ and Muc2 markers.ConclusionsThis experimental model mimicking the neonatal intestinal environment can be used to study interactions between neonatal IEC and immune cells. Our findings can provide clinically relevant information and clues to how developmental changes in the newborn intestine can influence susceptibility to NEC while demonstrating our development of a simple, yet accurate and clinically applicable model of the neonatal gut.Funding AgenciesCAG, CIHRBCCHRI, CCC, C.H.I.L.D. Fdn

Inflammatory Responses of Porcine MoDC and Intestinal Epithelial Cells in a Direct-Contact Co-culture System Following a Bacterial Challenge.

Intestinal epithelial cells (IEC) and immune cells, such as dendritic cells (DC), jointly control the immune response towards luminal pathogens in the intestinal mucosa. Crosstalk between IEC and DC is crucial for coordinating immune responses and occurs via soluble factors and direct cell-cell contacts. The present study aimed at establishing a direct-contact co-culture model of porcine IEC and DC to mimic these interactions. The effects of (1) co-cultivation of the two cell types and (2) bacterial infection on the inflammatory response patterns of each of the cell types were determined with a special focus on the canonical and non-canonical inflammasome signaling pathways. In infection experiments, in vitro cultures were exposed to either the probiotic Enterococcus (E.) faecium NCIMB 10415 or enterotoxigenic Escherichia coli (ETEC). In porcine IEC (IPEC-J2), co-cultivation with porcine monocyte-derived DC (MoDC) resulted in reduced basal NLRP3 (nucleotide oligomerization domain [NOD]-like receptor [NLR] family, pyrin domain containing 3) inflammasome mRNA levels in unstimulated conditions. In porcine MoDC, the presence of IPEC-J2 cells evoked a noticeable decrease of interleukin (IL)-8 and transforming growth factor-β (TGF-β) mRNA and protein expression. ETEC, in contrast to E. faecium, modulated the inflammasome pathway in IPEC-J2 cells and porcine MoDC. Co-cultured IPEC-J2 cells showed an augmented inflammasome response to ETEC infection. By contrast, MoDC revealed a weakened ETEC response under such co-culture conditions as indicated by a reduction of inflammasome-related IL-1β protein release. Our data indicate that the close contact between IEC and resident immune cells has a major effect on their immunological behavior.

The influence of porcine epidemic diarrhea virus on pig small intestine mucosal epithelial cell function.

Porcine epidemic diarrhea (PED) is a highly contagious, acute enteric tract infectious disease of pigs (Sus domesticus) caused by porcine epidemic diarrhea virus (PEDV). PED is characterized by watery diarrhea, dehydration, weight loss, vomiting and death. PEDV damages pig intestinal epithelial tissue, causing intestinal hyperemia and atrophy of intestinal villi, with formation of intestinal epithelial cell cytoplasmic vacuoles. Since pig small intestinal epithelial cells (IECs) are target cells of PEDV infection, IEC cells were utilized as a model for studying changes in cellular activities post-PEDV infection. Monitoring of Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities demonstrated that PEDV infection decreased these activities. In addition, IECs proliferation was shown to decrease after PEDV infection using an MTT assay. Moreover, IECs apoptosis detected by flow cytometry with propidium iodide (PI) staining was clearly shown to increase relative to the control group. Meanwhile, animal experiments indicated that PEDV virulence for IEC cells was greater than viral virulence for Vero cells, although this may be due to viral attenuation after numerous passages in the latter cell line. Collectively, these studies revealed viral pathogenic mechanisms in PEDV-infected IECs and offer a theoretical basis for PEDV prevention and control.

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

Crohnʼs disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy

The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

Arsenic is a naturally occurring metalloid and the drinking water contamination by inorganic arsenic remains a major public health problem. The trivalent arsenic (arsenite) is more toxic than the pentavalent form (arsenate), and is known to cause gastrointestinal toxicity. Specific immortal cell lines are considered to be suitable for toxicity screening and testing of chemicals as they are easy to handle and possess most of the biochemical pathways present in the corresponding cells present in vivo. The present study was designed to evaluate and compare the in vitro toxicity of arsenite on rat intestinal epithelial cell line (IEC-6) and primary cultures of rat intestinal epithelial cells (IEC). To evaluate in vitro toxicity, cultures of IEC and IEC-6 cells were assessed for viability, morphometric analysis, membrane transport enzymes and structural constituents for membrane damage, dehydrogenase activity test for respiratory and energy producing processes and esterase activity test for intra and extra cellular degradation, following the post exposures to arsenite (0–20 ppm). Significantly similar concentration-dependent changes in these toxicity-screening parameters in IEC and IEC-6 were observed. Highest tested concentration of arsenite (20 ppm) was found to be detrimental in both IEC and IEC-6. Furthermore, to evaluate arsenite toxicity in epithelial cells of rat intestine, intestinal loops were filled with arsenite solutions and incubated for 30 min in situ. In situ studies also showed a significant arsenite concentration-dependent decline in epithelial cell membrane transport enzyme activities and total hexose and sialic acid contents. Concomitant release of membrane enzymes, hexose and sialic acid in the intestinal luminal fluid following higher arsenite exposures further indicated partial membrane damage. Similar morphological changes in IEC and IEC-6 were also evident. These findings also suggest that IEC-6 cell lines are suitable for initial screening of gastrointestinal cellular toxicity caused by arsenite.

Cell differentiation dependent expressed CCR6 mediates ERK‐1/2, SAPK/JNK, and Akt signaling resulting in proliferation and migration of colorectal cancer cells

The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Precursor peptide progastrin(1-80) reduces apoptosis of intestinal epithelial cells and upregulates cytochrome c oxidase Vb levels and synthesis of ATP.

We recently reported that downregulation of gastrin gene expression in colon cancer cells significantly suppresses relative levels of mitochondrial cytochrome c (cyt c) oxidase Vb (Cox Vb) RNA and protein. These unexpected findings suggested the possibility that gastrin gene products [mainly progastrin (PG)] may be directly or indirectly mediating the observed effects in colon cancer cells. Because colon cancer cells do not respond to exogenous PG, we examined the possibility of whether PG regulates Cox Vb expression in gastrin-responsive intestinal epithelial cells (IECs) in vitro. Levels of Cox Vb RNA and protein were significantly increased in a dose-dependent manner in response to PG. Mitochondrial synthesis of ATP was also increased by approximately three- to fivefold in response to optimal concentrations (0.1-1.0 nm) of PG. Possible antiapoptotic effects of PG were additionally examined, because activation of caspases 9 and 3 had been noted in colon cancer cells downregulated for gastrin gene expression. We measured a significant loss in the levels of cyt c in the cytosol of PG-treated vs. control IEC cells, which correlated with a significant loss in the activation of caspases 9 and 3, resulting in a significant loss in DNA fragmentation on PG treatment of the cells. Our results thus suggest the novel possibility that the precursor PG peptide exerts direct antiapoptotic effects on IECs, which may contribute to the observed growth effects of PG on these cells. Additionally, Cox Vb gene appears to be an important intracellular target of PG, resulting in an increase in ATP levels, which may also contribute to the observed increase in the growth of target cells in response to PG.

Transforming growth factor-beta1 enhances the secretion of monocyte chemoattractant protein-1 by the IEC-18 intestinal epithelial cell line.

Intestinal epithelial cells (IEC) are known to produce monocyte chemoattractant protein-1 (MCP-1). However, MCP-1 production, as with many other cytokines, can be regulated by a network of cytokines present in the environment of the IEC. Both IEC and inflammatory cells have been shown to produce transforming growth factor-beta (TGF-beta), and the regulatory effect of this cytokine on MCP-1 secretion by IEC has not been determined. Using the IEC-18 cell line, we have found that TGF-beta1 alone induced the secretion of high levels of MCP-1. Treatment with TGF-beta1 also enhanced the levels of MCP-1 messenger ribonucleic acid. However, costimulation of the cells with TGF-beta1 and interleukin-1beta (IL-1beta) resulted in significant, but less than additive, increases in MCP-1 secretion. Finally, the enhancing effect of TGF-beta1 on MCP-1 secretion was not due to IL-6. These results suggest that TGF-beta1 from IEC or inflammatory cells may significantly enhance the secretion of MCP-1 by IEC and play an important role in inflamed mucosal tissues.

Pp60c-Src Kinase mediates growth effects of the full-length precursor progastrin1-80 peptide on rat intestinal epithelial cells, in vitro.

Growth factor effects of precursor forms of gastrins have become evident in recent years. However, intracellular pathways that mediate growth effects of the precursor molecules are not known. In previous studies, we reported an increase in Tyr phosphorylation of pp60(c-Src) in intestinal epithelial cells (IEC) in response to the fully processed form of gastrin [gastrin(1-17) (G17)]. We have now examined whether c-Src kinase is similarly phosphorylated and activated in response to the full-length precursor molecule, progastrin (PG)(1-80), (recombinant human PG) in IEC cells. We found a significant increase in pp60(c-Src) kinase activity in response to both G17 and PG (0.1-1.0 nM), suggesting that growth effects of both the precursor and fully processed gastrin molecules may be mediated via similar pathways. On the other hand, pp62(c-Yes) was not phosphorylated or activated in response to either G17 or PG. To examine whether c-Src kinase mediates proliferative effects of PG, IEC cells were microinjected with anti-Src-IgG and (3)H-thymidine ((3)H-Tdr) uptake of the cells measured. Control cells received nonimmune IgG. The (3)H-Tdr uptake of cells stimulated with 1.0 nM PG was significantly reduced in cells microinjected with anti-c-Src-IgG; control IgG had no effect. In cells stimulated with 1.0% fetal calf serum, microinjection with c-Src-IgG had no effect on (3)H-Tdr uptake. The specificity of the effect was further confirmed by blocking the inhibitory effect of anti-c-Src-IgG with antigenic Src peptide. These results suggest that activation of c-Src kinase likely represents a critical step in mediating proliferative effects of both the precursor and fully processed forms of gastrins on IEC.

Progastrin1-80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites.

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG(1-80)) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK(1) and CCK(2) receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK(1)-R and CCK(2)-R on IEC cells. High-affinity (K(d) = 0.5-1.0 nM) binding sites for (125)I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG >or= COOH-terminally extended G17 >or= G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

Transmural pressure induces IL-6 secretion by intestinal epithelial cells

Although intestinal epithelial cells (IECs) are known as an important source for IL-6, it is not known whether mechanical forces affect IL-6 production. We investigated how transmural pressure modulates IL-6 synthesis and activation of transcription factors in IECs. Pressure was loaded onto IEC-18 cells by introducing compressed helium gas into the cell culture flask for 1-48 h. IL-6 release into the culture media was determined by cell proliferation bioassay using an IL-6-dependent mouse hybridoma cell line (7TD1). Exposure to pressure (80 mmHg) significantly enhanced IL-6 release into the culture media from IEC-18 cells at 12 h. Under control conditions, IL-6 secretion was directed to the basolateral side, but after exposure to pressure IL-6 secretion was increased in both the apical and basolateral sides. A nuclear factor kappa B (NF-kappaB) decoy reversed completely the pressure-induced increase of IL-6 secretion by IEC-18 cells. Pressure treatment enhanced IL-6 mRNA expression in IECs within 6 h. Pressure loading significantly enhanced the activation of both NF-kappaB and NF-IL-6 from 1h in the nuclear protein of IEC-18 cells as assessed by the electrophoretic mobility shift assay using FITC-conjugated specific primers. Increased phosphorylation of I-kappa B was also demonstrated in the cytosol of IEC cells within 1h by Western blot analysis. These results suggest a possible role for pressure loading in immune modulation of the intestinal mucosa by the stimulation of IL-6 release from intestinal epithelial cells.

Polarized secretion of IL-6 by IEC-6 intestinal epithelial cells: differential effects of IL-1 beta and TNF-alpha.

Intestinal epithelial cells (IEC) can exist as polarized cells and are capable of secreting interleukin-6 (IL-6), yet it has not been determined if this IL-6 is secreted in a polarized fashion. Using the non-transformed rat IEC-6 intestinal epithelial cell line grown on microporous membrane inserts, we have determined that these cells were capable of secreting IL-6 preferentially to the basal surface when stimulated basally with IL-1 beta. In contrast, stimulation of the cells with TNF-alpha resulted in an equal level of IL-6 secretion to the apical and basal surfaces, regardless of whether the cells were stimulated by the apical or basal route. Experiments designed to test the permeability of the IEC-6 cell layer to apically added sodium fluorescein confirmed that neither IL-1 beta nor TNF-alpha altered the integrity of the cell layer after three days. These results suggest that IEC may have the capacity to secret IL-6 in different patterns depending upon the stimulation received. This would allow communication between the IEC and lamina propria cells via basal secretion and rapid communication between IEC via apical secretion.