Enterocyte Differentiation Marker Research Articles

P035 3D bioprinting of gelatin derivatives: towards novel small intestinal in vitro models

Abstract Background Despite significant progress achieved in the field of inflammatory bowel disease research, the precise cause of the disease has yet to be elucidated. Intestinal in vitro models can provide a fast and inexpensive alternative for in vivo studies. Nonetheless, many in vitro models lack the crypt or crypt-villus architecture as seen in the large and small intestine respectively and therefore fail to mimic the complexity of the gut. In this study, we aim to improve the physiology of small intestinal in vitro models by the development of hydrogels that mirror the villi and crypts of the digestive tract. Methods This study focused on the development of gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-, and gelatin-methacryloyl-norbornene (gel-MA-NB)-based biomaterial inks to fabricate 3D constructs, mimicking villi or a combination of villi and crypts, with digital light processing. The constructs were mechanically and morphologically evaluated with parallel plate rheology and cryo-scanning electron microscopy respectively. To assess the biocompatibility of the 3D constructs, a Caco-2/HT29-MTX co-culture in a 9:1 ratio was maintained for 21 days and confluency was confirmed with immunofluorescence. Gene expression, transepithelial electrical resistance (TEER) and paracellular permeability of the cells cultured on the constructs were compared to cells cultured on flat gel-MA-AEMA and gel-MA-NB hydrogels, a collagen type I coating or uncoated tissue culture plastic. Results Both gel-MA-AEMA and gel-MA-NB hydrogels exhibited physiologically relevant stiffness (1.9 ± 0.63 kPa and 1.64 ± 0.63 kPa respectively), but only the gel-MA-AEMA based biomaterial ink could be successfully utilized for printing constructs with villi and crypts. On all construct designs, confluency was reached and paracellular permeability of small sized marker molecules in combination with TEER measurements suggested the formation of a functional barrier over time, which was further confirmed by immunofluorescence and increased gene expression of tight junction proteins, occludin and ZO-1. The gene expression of enterocyte differentiation markers, villin-1, sucrase isomaltase and alkaline phosphatase, suggested the superior differentiation of Caco-2 cells on the ‘only villi’ and ‘villi and crypts’ constructs compared to flat hydrogels, collagen type I coating or uncoated tissue culture plastic. Conclusion Although both hydrogels promoted functional barrier formation and enterocyte differentiation, gel-MA-AEMA was more suited for DLP than gel-MA-NB. In addition, culturing intestinal epithelial cells on the 3D constructs ameliorated cell differentiation compared to conventional 2D setups.

Altered colonocyte differentiation may lead to decreased electrolyte and fluid absorption in the ileocolon of IBD patients by affecting the expression pattern of the responsible ion transporters

Crohn's disease and ulcerative colitis are chronic inflammatory bowel diseases (IBD) characterized by disturbed homeostasis of the immune system, the intestinal mucosa and the microbiome, causing ‘leaky’ intestinal epithelial barrier with altered fluid and electrolyte absorption leading to diarrhea. Fluid absorption in the ileocolonic region is predominantly mediated by the Cl-/HCO3 - exchanger SLC26A3 (DRA) and the Na+/H+ exchanger SLC9A3 (NHE3), while in the distal colon, the epithelial Na+ channel (ENaC) also plays a role. The objective of the current study is to evaluate if the downregulation of specific absorptive ion transporters or general defect of the enterocyte differentiation correlate with the degree of diarrhea and inflammation. For this reason, we have analyzed the mRNA expression and/or protein abundance of DRA, NHE3 and ENaC, as well as a panel of genes that correlate with the differentiation state of the colonic epithelium, in different segments of the ileocolon of healthy individuals and IBD patients with an acute flare or in remission. Endoscopy and histopathologic scoring and TNFα mRNA levels were used to grade inflammation, clinical scores for disease activity and diarrhea. mRNA expression and immunohistochemical staining of DRA and NHE3 in healthy colonic tissue revealed differences in the expression pattern of DRA and NHE3 along the human colon, with DRA being most abundant in the transverse and descending colon and NHE3 in the ascending colon (n=10). The reduction of DRA and NHE3 mRNA expression/abundance in the membrane correlated with the degree of inflammation as well as the diarrhea score (n=42), while in inactive disease the expression and abundance of DRA and NHE3 did not differ from healthy controls (n=13). Furthermore, significant decrease of the ENaC subunit ENaCγ expression was detected in inflamed colon sigmoideum (n=30). Since the addressed ion transporters are expressed on differentiated intestinal epithelial cells, we further investigated if their altered expression is linked to a possible defect of the epithelial differentiation program. It was found that the mRNA expression of the absorptive enterocyte differentiation markers intestinal alkaline phosphatase (iALP), villin and HES1 was significantly decreased in inflamed colon, while the expression of the proliferative marker Ki67, HOPX, BM1 and Claudin2 was significantly increased. However, the expression of the stem cell marker LGR5 was not altered (n=30). The results showed a disrupted transcriptional regulation of the epithelial ion transporters DRA, NHE3 and ENaCγ in the inflamed epithelium of IBD patients, which will lead to their reduced function and cause electrolyte/water accumulation leading to diarrhea. The decreased expression of the epithelial ion transporters could be a consequence of the altered enterocyte differentiation of the inflamed epithelium, most likely due to signaling pathways triggered by the proinflamatory cytokines. Grant Support: DFG SE 460/21-1 and 22-1, and VW Z1953. 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.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

The therapeutic efficacy of mesenchymal stromal cells on experimental colitis was improved by the IFN-\u03b3 and poly(I:C) priming through promoting the expression of indoleamine 2,3-dioxygenase

BackgroundInflammatory bowel disease is a chronic and excessive inflammation of the colon and small intestine. We previously reported that priming of mesenchymal stromal cells (MSCs) with poly(I:C) induced them to express indoleamine 2,3-dioxygenase (IDO). We tried to find out whether the IFN-γ and poly(I:C)-primed MSCs have better therapeutic efficacy on the experimental colitis in the IDO1-dependent manner.MethodsTo compare the therapeutic effects between the unstimulated MSCs and primed MSCs on murine colitis, mice (C57BL6) were administered with 2.5% dextran sodium sulfate (DSS) in drinking water for 5 days and injected with MSCs intraperitoneally on days 1 and 3 following DSS ingestion. The disease activity index score and body weight loss were assessed daily until day 9.ResultsMice receiving the IFN-γ and poly(I:C)-primed MSCs showed a reduced disease activity index and less weight loss. Colon tissue from the same mice presented attenuated pathological damage, increased Paneth cells, increased IDO1-expressing cells, and better proliferation of enterocytes. The primed MSC treatment upregulated the mRNA expression of intestinal stem cell markers (Lgr5, Olfm4, and Bmi1), enterocyte differentiation markers (Muc2, Alpi, Chga, and occludin), and regulatory T (Treg) cells (Foxp3). The same treatment decreased inflammatory cell infiltration to lymphoid organs and the level of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, and MCP-1) in colon tissue. Notably, in vivo pharmacologic inhibition of the IDO1 activity blocked the Foxp3 upregulation in colon tissue and diminished the protective effects of the primed MSC.ConclusionsThe priming of MSCs with the IFN-γ and poly(I:C) is a promising new strategy to improve the therapeutic efficacy of MSC and is worth further research.

Loss of Slfn3 induces a sex-dependent repair vulnerability after 50% bowel resection.

Bowel resection accelerates enterocyte proliferation in the remaining gut with suboptimal absorptive and digestive capacity because of a proliferation-associated decrease in functional differentiation markers. We hypothesized that although schlafen 3 (Slfn3) is an important regulator of enterocytic differentiation, Slfn3 would have less impact on bowel resection adaptation, where accelerated proliferation takes priority over differentiation. We assessed proliferation, cell shedding, and enterocyte differentiation markers from resected and postoperative bowel of wild-type (WT) and Slfn3-knockout (Slfn3KO) mice. Villus length and crypt depth were increased in WT mice and were even longer in Slfn3KO mice. Mitotic marker, Phh3+, and the proliferation markers Lgr5, FoxL1, and platelet-derived growth factor-α (PDGFRα) were increased after resection in male WT, but this was blunted in male Slfn3KO mice. Cell-shedding regulators Villin1 and TNFα were downregulated in female mice and male WT mice only, whereas Gelsolin and EGFR increased expression in all mice. Slfn3 expression increased after resection in WT mice, whereas other Slfn family members 1, 2, 5, 8, and 9 had varied expressions that were affected also by sex difference and loss of Slfn3. Differentiation markers sucrase isomaltase, Dpp4, Glut2, and SGLT1 were all decreased, suggesting that enterocytic differentiation effort is incompatible with rapid proliferation shift in intestinal adaptation. Slfn3 absence potentiates villus length and crypt depth, suggesting that the differentiating stimulus of Slfn3 signaling may restrain mucosal mass increase through regulating Villin1, Gelsolin, EGFR, TNFα, and proliferation markers. Therefore, Slfn3 may be an important regulator not only of "normal" enterocytic differentiation but also in response to bowel resection.NEW & NOTEWORTHY The differentiating stimulus of Slfn3 signaling restrains an increase in mucosal mass after bowel resection, and there is a Slfn3-sex interaction regulating differentiation gene expression and intestinal adaptation. This current study highlights the combinatory effects of gender and Slfn3 genotype on the gene expression changes that contribute to the adaptation in intestinal cellular milleu (i.e. villus and crypt structure) which are utilized to compensate for the stress-healing response that the animals display in intestinal adaptation.

The Sodium/Hydrogen Exchanger 2 (Slc9a2/NHE2) is Involved in the Differentiation of Colonic Intestinal Epithelial Cells

BackgroundBoth sodium/hydrogen exchanger 2 and 3 (NHE2 and NHE3) isoforms of the Slc9 family Na+/H+ exchangers are expressed in the luminal membrane of the intestinal enterocytes, but in contrast to the deletion of NHE3, the deletion of NHE2 does not result in diarrhea, hyperaldosteronism, or reduced survival in mice.Aim and methodsTo further analyze the physiological role of NHE2 in the intestinal epithelial cells, we fluorometrically measured the intracellular pH (pHi) along the colonic cryptal axis of NHE2−/− mice, and in the intestinal epithelial Caco 2Bbe (C2Bbe) after stable lentiviral sh‐mediated NHE2 silencing. RNA and protein expression were assessed by qPCR, Western analysis and immunohistochemistry, and proliferative, migratory, and functional features of colonic cells in vivo and in vitro were assessed by enzymatic and functional assays.ResultsSteady‐state pHI was significantly decreased in C2Bbe/shNHE2 compared to empty‐vector transfected C2Bbe cells. In the colonic crypts, the pHi in the cryptal base was significantly lower than in the surface region, and the acidic zone along the crypt axis was significantly longer in colonic crypts from NHE2−/− mice. NHE2−/− colonic crypts were elongated with a much longer proliferation zone but with less proliferating cells per crypt area. Cell proliferation in C2Bbe/shNHE2 cells was reduced compared to empty‐vector transfected C2Bbe. The expression and activity of alkaline phosphatase (an enterocyte differentiation marker), was significantly reduced in NHE2−/− colonic mucosa and in C2Bbe/shNHE2 cells. However, the number of goblet cells and mucin 2 (Muc2) expression was increased in the NHE2−/− compared to the WT colon, accompanied by formation of a thicker mucus layer. The shift from absorptive to secretory differentiation program was accompanied by a decrease in Hes1 expression, as a downstream transcription factor of Notch signaling that supports the development of absorptive enterocytes.ConclusionsThe results suggest that NHE2 expression and/or function is activated when the intestinal cells emerge from the stem cell niche, and that this is essential for the establishment of the pHi gradient along the colonic crypt axis. Its activity facilitates enterocyte proliferation and differentiation along the crypt axis.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Use of hydrogel scaffolds to develop an in vitro 3D culture model of human intestinal epithelium.

Forty percent of hospital referrals are linked to disorders of the digestive tract. Current studies have utilised animal models or simple cultures of isolated cells which do not behave in the same manner as human intestine. Thus new models are required which more closely mimic the behaviour of intestinal cells. Here, we tested a number of scaffolds and conditions to develop a cell culture model which closely represents the 3D environment seen within the human small intestine. We successfully created structures seen within the intestine which have not previously been possible with other culture models. These models could be used to investigate tissue engineering, drug discovery, and used asan alternative to in vivo animal models in drug toxicity studies.

Spontaneous maturation of human colonic organoids (1119.2)

The intestinal epithelium is a complex structure consisting of distinct cell types derived from stem cells residing in the crypt base. Here we investigated the spontaneous maturation of organoids grown from human colonic crypts. Crypts were isolated by Ca2+ chelation from proximal colonic biopsies and transferred to Matrigel and growth media. The organoids were grown for 15 days and the gross appearance, ultrastructure, histology of the organoids and gene expression of proliferation, differentiation, maturation and stem cell activity markers analysed at day 4 and 15 by qPCR. At day 4, 71±7% of the organoids were thin walled spheroids, while at day 15 there was a mixture of large convoluted organoids (51±10%) and spheroids. At day 4 the organoids consisted of thin undifferentiated cells, whereas at day 15 the convoluted organoids consisted of differentiated columnar epithelial cells. Associated with this there was an increase (p < 0.0001) in expression of mature enterocyte and goblet cell differentiation marker, KRT20, a decrease (p<0.001) in CAECAM7, an indicator of undifferentiated cells, and an increase (p <0.01) in expression of CFTR, an anion channel expressed in colonic crypt epithelial cells. Although the cells appear to have differentiated they were still capable of proliferation, as indicated by the lack of a change in expression of MKi67, a marker of proliferation, and increased levels of the intestinal stem cell markers LGR5 (p< 0.0001) and OLFM4 (p<0.05). These findings suggest that over 15 days, cultured human intestinal colonic organoids form a secretory epithelium resembling that found in the colonic crypts.Grant Funding Source: Supported by University of Otago Research Grant

Sa1731 Neurotensin-HIF-1α-MicroRNA 210 Axis Orchestrates Hypoxia, Colonic Inflammation and Intestinal Angiogenesis

G A A b st ra ct s or constructs overexpressing HA-tagged 14-3-3σ plasmids. 14-3-3σ, Rictor (an mTORC2 protein), MUC2, cleaved Notch1, p-Akt(S473), Akt1 and Akt2 protein expression was determined by Western blotting. Enterocyte differentiation was induced by treatment of HT29 cells with sodium butyrate (NaBT) and assayed by measuring intestinal alkaline phosphatase (IAP) activity, an enterocyte differentiation marker. The expression of the goblet cell differentiation marker MUC2 and the Notch1 target gene HES1 was also determined by RT-PCR. The interaction between Akt andmTORC2was determined by immunoprecipitation followed by Western blot analysis. RESULTS. Knockdown of 14-3-3 σ did not alter basal or NaBT-induced of IAP activity. However, knockdown of 14-3-3 σ significantly increased MUC2 expression associated with the inhibition of Notch1 signaling as shown by decreased cleaved Notch1 protein and decreased HES1 mRNA expression. These results suggested that 14-3-3σ plays an important role in the regulation of goblet cell differentiation without impacting enterocyte differentiation. In addition, overexpression of 14-3-3σ decreased Akt (Ser473) phosphorylation and knockdown of either Akt1 or Akt2 decreased MUC2 expression. Moreover, overexpression of 14-3-3σ decreased the interaction between Akt and mTORC2. These results suggest that 14-3-3σmay regulate Akt activity through the inhibition of mTORC2-mediated Akt (Ser473) phosphorylation. CONCLUSIONS. Our results suggest a role for 14-3-3σ in goblet cell differentiation through the alteration of TSC2, Notch and Akt signaling pathways. Importantly, our data demonstrate that 14-3-3 σ regulation of Akt phosphorylation is mediated through the altered interaction between mTORC2 and Akt.

Hypoxia and lineage specification of cell line-derived colorectal cancer stem cells

Hypoxia is an important regulator of normal and cancer stem cell (CSC) differentiation. Colorectal CSCs from SW1222, LS180, and CCK81 colorectal cancer-derived cell lines are able to differentiate into complex 3D lumen-containing structures in normoxia, whereas in hypoxia, they form undifferentiated dense colonies that have reduced expression of the enterocyte differentiation marker CDX1, lack goblet cell formation, and have increased expression of BMI1 and activated Notch1. Hypoxia increases the clonogenicity of CSCs, which is cumulative as each round of hypoxia enriches for more CSCs. The hypoxic phenotype is reversible, because cells from hypoxic-dense colonies are able to reform differentiated structures when regrown in normoxia. We show that CDX1 is able to stimulate the generation of lumens even in hypoxia and has a negative feedback on BMI1 expression. Knockdown of CDX1 reduces lumen formation but does not affect goblet cell formation, suggesting that enterocytes and goblet cells form from different progenitor cells. Notch inhibition by dibenzazepine (DBZ) allowed CSCs to form goblet cells in both normoxia and hypoxia. Finally, we show that Hif1α, but not CA9, is an important mediator of the effects of hypoxia on the clonogenicity and differentiation of CSCs. In summary, hypoxia maintains the stem-like phenotype of colorectal cell line-derived CSCs and prevents differentiation of enterocytes and goblet cells by regulating CDX1 and Notch1, suggesting that this regulation is an important component of how hypoxia controls the switch between stemness and differentiation in CSCs.

RNA-Binding Protein Quaking, a Critical Regulator of Colon Epithelial Differentiation and a Suppressor of Colon Cancer

Colon cancer is one of the best understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death. Post-transcriptional regulation mediated by RNA-binding proteins or microRNAs coordinately targets multiple genes, holding promise involved in colon cancer initiation and development. Here we studied the role of RNA-binding protein quaking (QKI) in colon cancer. We observed the expression pattern of QKI in normal colon and colon cancers through reverse-transcription polymerase chain reaction and Western blot. Bisulfite sequencing and methylation-specific PCR were applied for QKI promoter methylation analysis. We used enterocyte differentiation markers and soft agar assay to test the role of QKI in colon differentiation and colon cancer development. 3' Untranslated region (UTR) reporter assay and RNA-immunoprecipitation were used to confirm the interaction between QKI and beta-catenin or p27. QKI is significantly down-regulated and even absent in some colon cancers, which is at least partially because of the promoter hypermethylation. Forced expression of QKI in the colon cancer cells increased the expression of enterocyte differentiation marker intestinal alkaline phosphatase and lactase, together with the enhancement of p27Kip1 protein level, and membrane localized beta-catenin. Finally, QKI overexpression reduced the proliferation and tumorigenesis ability. Our study establishes that QKI functions as a principal regulator in the differentiation of colon epithelium and a suppressor of carcinogenesis through coordinately targeting multiple genes associated with cell growth and differentiation, whose deregulation by methylation is involved in colon cancer onset and progress.

The Pro-inflammatory Cytokines, IL-1βand TNF-α, Inhibit Intestinal Alkaline Phosphatase Gene Expression

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Intestinal alkaline phosphatase gene expression is activated by ZBP-89

Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (ChIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5'-CCTCCTCCC-3') located between -1018 and -1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption.

Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2

We have examined the role that the caudal-related homeobox transcription factors Cdx1 and Cdx2 play in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). Human colon cancer Caco-2 cells were transiently transfected with Cdx1 and/or Cdx2, and semiquantitative RT-PCR was used to study the effects on IAP mRNA expression. Transfections with a variety of IAP-luciferase reporter constructs were used to identify a Cdx response element located within the human IAP gene promoter. Protein-DNA interactions were examined by EMSA. Results showed that Cdx1 markedly induced IAP mRNA expression, whereas Cdx2 did not, and, in fact, inhibited the Cdx1 effects. Functional analysis revealed that Cdx1 transactivates (fourfold, P < 0.05) the IAP promoter through a novel Cdx response element (GTTTAGA) located between -2369 and -2375 upstream of the translational start site. EMSA showed that both Cdx1 and Cdx2 could bind to the cis element, but in cotransfection experiments, Cdx2 inhibited the Cdx1 effects by approximately 50%. Thus we have identified a previously unrecognized interaction between two important gut transcription factors, Cdx1 and Cdx2, in the context of IAP gene regulation. Cdx1 activates the IAP gene via a novel cis element, whereas Cdx2 inhibits the Cdx1 effects.

Thyroid hormone positively regulates the enterocyte differentiation marker intestinal alkaline phosphatase gene via an atypical response element.

Thyroid hormone (T3) is a critical regulator of intestinal epithelial development and homeostasis, but its mechanism of action within the gut is not well understood. We have examined the molecular mechanisms underlying the T3 activation of the enterocyte differentiation marker intestinal alkaline phosphatase (IAP) gene. RT-PCR and Western blotting showed that thyroid hormone receptors TRalpha1 and TRbeta1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T3. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5' deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (-620) of the ATG start codon. EMSAs using TRalpha1-retinoid X receptor alpha (RXRalpha) on sequential 5' and 3' single nucleotide deletions defined the TRE between -632 and -612 (5'-TTGAACTCAgccTGAGGTTAC-3'). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TRalpha1 nor RXRalpha binds to the IAP-TRE; however, TRbeta1 binds to this TRE with minimal affinity. In the presence of TR and RXRalpha, only the TR-RXRalpha heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T3-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase.

Convergence of the thyroid hormone and gut-enriched Krüppel-like factor pathways in the context of enterocyte differentiation.

The gut-enriched Krüppel-like factor (KLF4) and the ligand-bound thyroid hormone receptor (TR) have each been shown to play a critical role in mammalian gut development and differentiation. We investigated an interrelationship between these two presumably independent pathways using the differentiation marker gene, intestinal alkaline phosphatase (IAP). Transient transfections were performed in Cos-7 cells using luciferase reporter plasmids containing a 2.5 kb segment of the proximal human IAP 5' regulatory region, as well as multiple deletions. Cells were cotransfected with TR and/or KLF4 expression vectors and treated+/-100 nmol/L thyroid hormone (T3). IAP reporter gene transactivation was increased independently by KLF4 (ninefold) and ligand-bound TR beta 1 (sevenfold). Cells cotransfected with KLF4 and TR beta 1 in the presence of T3 showed synergistic activation (70-fold). A similar pattern was seen with the other T3 receptor isoform, TR alpha 1. The synergistic effect was lost with deletions of the T3 and KLF4 response elements in the IAP promoter and was completely or partially abolished in the case of mutant KLF4 expression vectors. The thyroid hormone receptor complex and KLF4 synergistically activate the enterocyte differentiation marker gene IAP, suggesting a previously unrecognized interrelationship between these two transcription factor pathways.

Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Kruppel-like factor.

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

Thyroid hormone positively regulates an enterocyte differentiation marker gene through an atypical response element

the physiologic relevance of our previous investigations. Methods: Transgenic mice were denved to express a human growth hormone (HGH) reporter under control of nt -218 to + 118 of the rat ASBT promoter, c-los antisense oligonucleotides were used to generate clos negative Caco-2 cells, c-los knock out (ko) mice were bred from commercially available heterozygotes. Results: Nucleotides -218 to + 118 of the rat ASBT promoter directed HGH expression ubiquitously throughout multiple tissues from five different transgenic mouse lines, lndomethacin Induced.acute ileitis in these transgenic mice resulted in repression of both ASBT (-49%) and HGH (-52%). ASBT promoter activity In the c-los negative Caco-2 cells was enhanced (+ 51%) compared to standard Caco-2 cells. 1L-1B treatment of Caco2 cells resulted in reduced ASBT promoter activity (-80%), while c-los antisense treatment abrogated the cytokine mediated response, c-los expression was absent from c-los ko mice Indomethacin induced ileitis was associated with up-regulation (700%) of c-jun in both ~ld type and ko mice. Steady-state ASBT mRNA levels were repressed (-56%) in indomethatin treated wild type mice, while ASBT was induced (+ 80%) in ko mice. Conclusion: Basal activity of the ASBT promoter is dependent upon the balance of negative (c-los) and positive (ojun) AP-1 elements. Cis elements located between -218 and + 118 are sufficient to mediate the inflammatory response of ASBT. ASBT transcription is enhanced when negative tone is removed (i.e. c-los antisense or knock-out). Anomalous inflammatory mediated induction at ASBT expression is observed in o-los knock-out mice, since up-regulated c-jun is unopposed by c-los.

Transcriptional Activation of the Enterocyte Differentiation Marker Intestinal Alkaline Phosphatase Is Associated With Changes in the Acetylation State of Histone H3 at a Specific Site Within Its Promoter Region In Vitro

Enterocyte differentiation is thought to occur through the transcriptional regulation of a small subset of specific genes. A recent growing body of evidence indicates that post-translational modifications of chromatin proteins (histones) play an important role in the control of gene transcription. Previous work has demonstrated that one such modification, histone acetylation, occurs in an in vitro model of enterocyte differentiation, butyrate-treated HT-29 cells. In the present work, we sought to determine if the epigenetic signal of histone acetylation occurs in an identifiable pattern in association with the transcriptional activation of the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). HT-29 cells were maintained under standard culture conditions and differentiated with sodium butyrate. The chromatin immunoprecipitation (ChIP) assay was used to compare the acetylation state of histones associated with specific regions of the IAP promoter in the two cell populations (undifferentiated vs. differentiated). Chromatin was extracted from cells and cleaved by sonication or enzymatic digestion to obtain fragments of approximately 200 to 600 base-pairs, as confirmed by polymerase chain reaction using primers designed to amplify the IAP segments of interest. The ChIP assay selects DNA sequences that are associated with acetylated histones by immunoprecipitation. Unbound segments represent DNA sequences whose histones are not acetylated. After immunoprecipitation, sequences were detected by radiolabeled polymerase chain reaction, and the relative intensity of the bands was quantified by densitometry. The relative acetylation state of histones at specific sites was determined by comparing the ratios of bound/unbound segments. We determined that in a segment of the IAP promoter between −378 and −303 base-pairs upstream from the transcriptional start site, the acetylation state of histone H3 increased twofold in the differentiated, IAP expressing cells, whereas that of histone H4 remained essentially constant. Additionally, at a distant site, between −1378 and −1303 base-pairs, the acetylation state of H3 and H4 did not change appreciably between the undifferentiated and differentiated cells. We conclude that butyrate-induced differentiation is associated with specific and localized changes in the histone acetylation state within the IAP promoter. These changes within the endogenous IAP gene may underlie its transcriptional activation in the context of the enterocyte differentiation program. ( J Gastrointest Surg 2003;7:237–245.)

Enterocyte Response to Ischemia Is Dependent on Differentiation State

Enterocytes at the tips of microvilli are more sensitive to an ischemic insult than those cells residing in the crypts, an effect thought to be due to a relative lack of collateral flow. We speculated that this increased cellular sensitivity to ischemia might be an intrinsic feature of the cells related to their differentiated phenotype. To test this hypothesis, enterocyte response to ischemia was determined using both in vivo and in vitro models. For the in vivo studies, male Sprague-Dawley rats underwent laparotomy, and small intestinal ischemia was induced by clamping the superior mesenteric artery for 30 or 60 minutes, after which reperfusion was allowed for various time points up to 4 days. Injury was assessed histologically, as well as with Northern blots, probing for the enterocyte differentiation markers intestinal alkaline phosphatase and lactase, as well as the gut-epithelial marker villin. Mucosal changes consistent with ischemia/reperfusion injury were evident—that is, a rapid inflammatory response followed by progressive villus cell loss beginning at the tips and progressing to the crypts, depending on the degree of insult, with an eventual return to normal microanatomy. Intestinal alkaline phosphatase and lactase were lost immediately after ischemia and returned with reperfusion, confirming that the differentiated cells are particularly sensitive to ischemic injury. The in vitro studies employed two separate models of enterocyte differentiation: sodium butyrate–treated HT-29 cells and Caco-2 cells maintained for 7 days after confluence. In both models, undifferentiated and differentiated cells were subjected to treatment with 2-deoxyglucose and oligomycin-A (in vitro model of ischemia) and apoptosis was assessed by fluorescence-activated cell sorting analysis. Differentiation of both cell lines resulted in a significantly greater apoptotic response to ischemia compared to undifferentiated cells exposed to an identical insult. We conclude that differentiated enterocytes may be inherently more sensitive to ischemia-induced injury than their undifferentiated counterparts. These findings call into question the popularly held belief that villus tip cells are more susceptible to ischemia because of their location relative to the microvascular anatomy. ( J Gastrointest Surg 2002;6:403–409.)