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Abstract Transferrin receptor (TFRC) is the major mediator for iron entry into a cell. Under excessive iron conditions, TFRC is expected to be reduced to lower iron uptake and toxicity. However, the mechanism whereby TFRC expression is maintained at high levels in iron‐enriched cancer cells and the contribution of TFRC to cancer development are enigmatic. Here the work shows TFRC is induced by adenomatous polyposis coli (APC) gene loss‐driven β‐catenin activation in colorectal cancer, whereas TFRC‐mediated intratumoral iron accumulation potentiates β‐catenin signaling by directly enhancing the activity of tankyrase. Disruption of TFRC leads to a reduction of colonic iron levels and iron‐dependent tankyrase activity, which caused stabilization of axis inhibition protein 2 (AXIN2) and subsequent repression of the β‐catenin/c‐Myc/E2F Transcription Factor 1/DNA polymerase delta1 (POLD1) axis. POLD1 knockdown, iron chelation, and TFRC disruption increase DNA replication stress, DNA damage response, apoptosis, and reduce colon tumor growth. Importantly, a combination of iron chelators and DNA damaging agents increases DNA damage response and reduces colon tumor cell growth. TFRC‐mediated iron import is at the center of a novel feed‐forward loop that facilitates colonic epithelial cell survival. This discovery may provide novel strategies for colorectal cancer therapy.

Ulcerative Colitis (UC) is a chronic inflammatory disease of the intestinal tract for which a definitive etiology is yet unknown. Both genetic and environmental factors have been implicated in the development of UC. Recently, single cell RNA sequencing (scRNA-seq) technology revealed cell subpopulations contributing to the pathogenesis of UC and brought new insight into the pathways that connect genome to pathology. This review describes key scRNA-seq findings in two major studies by Broad Institute and University of Oxford, investigating the transcriptomic landscape of epithelial cells in UC. We focus on five major findings: (1) the identification of BEST4 + cells, (2) colonic microfold (M) cells, (3) detailed comparison of the transcriptomes of goblet cells, and (4) colonocytes and (5) stem cells in health and disease. In analyzing the two studies, we identify the commonalities and differences in methodologies, results, and conclusions, offering possible explanations, and validated several cell cluster markers. In systematizing the results, we hope to offer a framework that the broad scientific GI community and GI clinicians can use to replicate or corroborate the extensive new findings that RNA-seq offers.

Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.

Intestinal enteroids are ex vivo primary cultured single-layer epithelial cell spheroids of average diameter ∼150 μm with luminal surface facing inward. Measurement of enteroid swelling in response to secretagogues has been applied to genetic testing in cystic fibrosis and evaluation of drug candidates for cystic fibrosis and secretory diarrheas. The current measurement method involves manual addition of drugs and solutions to enteroids embedded in a Matrigel matrix and estimation of volume changes from confocal images of fluorescently stained enteroids. We developed a microfluidics platform for efficient trapping and immobilization of enteroids for quantitative measurement of volume changes. Multiple enteroids are trapped in a "pinball machine-like" array of polydimethylsiloxane posts for measurement of volume changes in unlabeled enteroids by imaging of an extracellular, high-molecular weight fluorescent dye. Measurement accuracy was validated using slowly expanding air bubbles. The method was applied to measure swelling of mouse jejunal enteroids in response to an osmotic challenge and cholera toxin-induced chloride secretion. The microfluidics platform allows for parallel measurement of volume changes on multiple enteroids during continuous superfusion, without an immobilizing matrix, and for quantitative volume determination without chemical labeling or assumptions about enteroid shape changes during swelling.

Breastfeeding has been associated with long lasting health benefits. Nutrients and bioactive components of human breast milk promote cell growth, immune development, and shield the infant gut from insults and microbial threats. The molecular and cellular events involved in these processes are ill defined. We have established human pediatric enteroids and interrogated maternal milk’s impact on epithelial cell maturation and function in comparison with commercial infant formula. Colostrum applied apically to pediatric enteroid monolayers reduced ion permeability, stimulated epithelial cell differentiation, and enhanced tight junction function by upregulating occludin. Breast milk heightened the production of antimicrobial peptide α-defensin 5 by goblet and Paneth cells, and modulated cytokine production, which abolished apical release of pro-inflammatory GM-CSF. These attributes were not found in commercial infant formula. Epithelial cells exposed to breast milk elevated apical and intracellular pIgR and enabled maternal IgA translocation. Proteomic data revealed a breast milk-induced molecular pattern associated with tissue remodeling and homeostasis. Using a novel ex vivo pediatric enteroid model, we have identified distinct cellular and molecular events involved in human milk-mediated improvement of human intestinal physiology and immunity.

Diarrheal diseases remain a leading cause of morbidity and mortality for children in developing countries, while representing an important cause of morbidity worldwide. The World Health Organization recommended that low osmolarity oral rehydration solutions plus zinc save lives in patients with acute diarrhea, but there are no approved, safe drugs that have been shown to be effective against most causes of acute diarrhea. Identification of abnormalities in electrolyte handling by the intestine in diarrhea, including increased intestinal anion secretion and reduced Na(+) absorption, suggest a number of potential drug targets. This is based on the view that successful drug therapy for diarrhea will result from correcting the abnormalities in electrolyte transport that are pathophysiologic for diarrhea. We review the molecular mechanisms of physiologic regulation of intestinal ion transport and changes that occur in diarrhea and the status of drugs being developed to correct the transport abnormalities in Na(+) absorption that occur in diarrhea. Mechanisms of Cl(-) secretion and approaches to anti-Cl(-) secretory therapies of diarrhea are discussed in a companion review.

Colonic epithelium–commensal interactions play a very important role in human health and disease development. Colonic mucus serves as an ecologic niche for a myriad of commensals and provides a physical barrier between the epithelium and luminal content, suggesting that communication between the host and microbes occurs mainly by soluble factors. However, the composition of epithelia-derived metabolites and how the commensal flora influences them is less characterized. Here, we used mucus-producing human adult stem cell-derived colonoid monolayers exposed apically to probiotic E. coli strain Nissle 1917 to characterize the host–microbial communication via small molecules. We measured the metabolites in the media from host and bacterial monocultures and from bacteria-colonoid co-cultures. We found that colonoids secrete amino acids, organic acids, nucleosides, and polyamines, apically and basolaterally. The metabolites from host-bacteria co-cultures markedly differ from those of host cells grown alone or bacteria grown alone. Nissle 1917 affects the composition of apical and basolateral metabolites. Importantly, spermine, secreted apically by colonoids, shows antibacterial properties, and inhibits the growth of several bacterial strains. Our data demonstrate the existence of a cross-talk between luminal bacteria and human intestinal epithelium via metabolites, which might affect the numbers of physiologic processes including the composition of commensal flora via bactericidal effects.

Background: In intestinal epithelial cells, acute regulation of the brush border Na+/H+ exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca2+ ([Ca2+]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. Methods: The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches. Results: Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K+ depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers. Conclusion: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42.

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Shiga toxin (Stx) produced by the invasive Shigella dysenteriae serotype 1 (S. dysenteriae1) causes gastrointestinal and kidney complications. It has been assumed that Stx is released intracellularly after enterocyte invasion by S. dysenteriae1. However, there is little information about Stx distribution inside S. dysenteriae1-infected enterocytes. Here, we use intestinal epithelial T84 cells to characterize the trafficking of Stx delivered into the cytosol, in ways that mimic aspects of S. dysenteriae1 infection. We find that cytoplasmic Stx is transported into nucleoli. Stx nucleolar movement is carrier- and energy-dependent. Stx binding to the nucleoli of normal human enterocytes in vitro supports possible roles for nucleolar trafficking in toxin-induced intestinal pathology.

Wehunan E J, Steplock D, Cha B, Kovbasnjuk O, Frost NA, Cunningham R, Shenolikar S, Blanpied TA, Donowitz M. PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP. Am J Physiol Renal Physiol 297: F1560-F1565, 2009. First published September 30, 2009; doi: 10.1152/ajprenal.90657.2008.--Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ~17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-I. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis. sodium-dependent phosphate transporter type 2a; fluorescence recovery after photobleaching; parathyroid hormone; OK cells; PDZ domains doi: 10.1152/ajprenal.90657.2008

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of nonreceptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases. O157:H7 colonization; toxin endocytosis; transport across the monolayer

Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.