Biology subjects

Donowitz, M.

Publications and source records attributed to Donowitz, M..

14 recordsLinked to original sources

bioRxiv2025

Differential Regulation of NHE3 Expression in Type 1 and Type 2 Diabetic Intestine: Impaired Endosomal Regulation of NHE3 Expression in Type 1 Diabetes

Chronic diarrhea is a frequent gastrointestinal complication in both type 1 (T1D) and type 2 diabetes (T2D), though the underlying mechanisms differ: T1D is linked to autonomic neuropathy and disrupted transporter regulation, while T2D is often linked to medications and intestinal inflammation. Using streptozotocin-induced mouse models of T1D and T2D, we observed increased luminal fluid in the small intestine of both. Given the role of Na+/H+ exchanger 3 (NHE3) in fluid absorption and its loss in most diarrheal diseases, we examined NHE3 expression across intestinal segments. In T1D, NHE3 protein was significantly reduced in the duodenum and jejunum without changes in mRNA, suggesting post-transcriptional regulation. In contrast, T2D mice exhibited reduced NHE3 protein and mRNA, restricted to the proximal colon. To investigate mechanisms underlying NHE3 loss in T1D, we evaluated endosomal scaffolding proteins involved in NHE3 trafficking. While our previous work showed that the Sorting Nexin-27 (SNX27)-retromer complex does not regulate NHE3 protein stability, we found that SNX17 was significantly decreased in the small intestine of T1D mice but unchanged in T2D. SNX17 knockdown in SK-CO15 cells reduced NHE3 activity and stability. A GST pull-down assay showed that SNX17 interacts with the C-terminus of NHE3. Mutation of the NHE3 distal NPxY motif disrupted this interaction, leading to reduced NHE3 expression and increased degradation. These findings reveal segment-specific and mechanistically distinct causes of diabetic diarrhea in T1D versus T2D, and identify SNX17 loss as a contributor to reduced NHE3 stability and activity in T1D, likely promoting diabetic diarrhea. New and NoteworthyThis study identifies distinct mechanisms of impaired sodium absorption contributing to diabetic diarrhea in type-1 and type-2 diabetes. We identify SNX17 as a novel regulator of NHE3 in the small intestine, showing that SNX17 loss in T1D contributes to post-translational NHE3 destabilization. In contrast, T2D-associated NHE3 downregulation is transcriptional and confined to the colon. These findings reveal disease- and region-specific regulatory pathways that drive impaired fluid absorption in diabetes, with direct implications for the development of targeted therapies. Graphical abstract Schematic overview of differential NHE3 regulation in diabetic diarrhea.In Type 1 Diabetes (T1D), reduced SNX17 expression in the small intestine causes increased degradation of NHE3 and impaired sodium absorption. In Type 2 Diabetes (T2D), SNX17 levels remain unchanged in the small intestine, but NHE3 expression is transcriptionally downregulated in the proximal colon. These distinct mechanisms contribute to segment-specific differential regulation of NHE3 in T1 and T2D diabetes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/675364v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@eabf0aorg.highwire.dtl.DTLVardef@1a46891org.highwire.dtl.DTLVardef@cd2d5corg.highwire.dtl.DTLVardef@5d185b_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Host-derived CEACAM-laden vesicles engage enterotoxigenic E. coli for elimination and toxin neutralization.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of diarrheal illnesses annually ranging from mildly symptomatic cases to severe, life-threatening cholera-like diarrhea. Although ETEC are associated with long-term sequelae including malnutrition, the acute diarrheal illness is largely self-limited. Recent studies indicate that in addition to causing diarrhea, the ETEC heat-labile toxin (LT) modulates the expression of many genes in intestinal epithelia, including carcinoembryonic cell adhesion molecules (CEACAMs) which ETEC exploit as receptors, enabling toxin delivery. Here however, we demonstrate that LT also enhances the expression of CEACAMs on extracellular vesicles (EV) shed by intestinal epithelia and that CEACAM-laden EV increase in abundance during human infections, mitigate pathogen-host interactions, scavenge free ETEC toxins, and accelerate ETEC clearance from the gastrointestinal tract. Collectively, these findings indicate that CEACAMs play a multifaceted role in ETEC pathogen-host interactions, transiently favoring the pathogen, but ultimately contributing to innate responses that extinguish these common infections. Significance statementEnterotoxigenic E. coli, characterized by the production of heat-labile (LT) and heat-stable (ST) toxins, are a very common cause of diarrhea in low-income regions responsible for hundreds of millions of infections each year, and the major cause of diarrhea in travelers to endemic areas. Although these infections may be severe and cholera-like, they are typically self-limited. These studies demonstrate that extracellular vesicles produced by host intestinal cells can capture the bacteria and its secreted toxins at a distance from the cell surface, potentially acting as molecular decoys to neutralize the enterotoxins and extinguish the infection.

microbiology↗

bioRxiv2023

Disruption of Epithelium Integrity by Inflammation-Associated Fibroblasts through Prostaglandin Signaling

Inflammation-associated fibroblasts (IAFs) are associated with the progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial function and architecture is unknown. In this study, we developed an in vitro model whereby human colon fibroblasts are induced to become IAFs by specific cytokines and recapitulate key features of IAFs in vivo. When co-cultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid swelling and barrier disruption due to swelling and rupture of individual epithelial cells. Epithelial cells co-cultured with IAFs also exhibit increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated through a paracrine pathway involving prostaglandin E2 (PGE2) and the PGE2 receptor EP4, leading to PKA-dependent activation of the CFTR chloride channel. Importantly, EP4-specific chemical inhibitors effectively prevented colonoid swelling and restored normal proliferation and genome stability of IAF-exposed epithelial cells. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a potential treatment to mitigate inflammation-associated epithelial injury. TeaserInflammation-associated fibroblasts compromise colon epithelial barrier integrity and genome stability via PGE2-EP4 signaling.

cell biology↗

bioRxiv2022

Air-Liquid-Interface Reorganizes Membrane Lipid and Enhance Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colon

Background and AimsCholesterol-rich membrane domains, also called lipid rafts (LR), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LR that further aggregate into larger platform-like structures that are enriched in ceramide and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down Regulated in Adenoma (DRA) is an intestinal Cl-/HCO3- antiporter which is enriched in LR. However, little is known regarding the mechanisms involved in the regulation of DRA activity. MethodsAir liquid interface (ALI) was created by removing apical media for a specified number of days from 12-14 days post confluency Caco-2/BBe cells or confluent colonoid monolayer grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contain DRA. ResultsDRA expression and activity were enhanced by culturing Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 m compared to cells grown as submerged cultures. ASMase inhibitor, desipramine disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced differentiation of colonoids along with enhancing basal and forskolin-stimulated DRA activity. ConclusionsALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of normal human colonoids. SynopsisAir-liquid interface (ALI) enhances total and brush border DRA expression and activity in Caco-2/BBe cells and human colonoids by causing differentiation of enterocytes and acid sphingomyelinase-dependent enhanced retention of DRA in ceramide-rich platform-like structures at the plasma membrane.

cell biology↗

bioRxiv2022

SLC26A3 (DRA) is stimulated in a synergistic, intracellular Ca2+ dependent manner by cAMP AND ATP in intestinal epithelial cells

In polarized intestinal epithelial cells, DRA is a brush border (BB) Cl-/HCO3- exchanger that is part of neutral NaCl absorption under baseline conditions but in cAMP driven diarrheas it is stimulated and contributes to increased anion secretion. To further understand regulation of DRA in conditions mimicking some diarrheal diseases, differentiated Caco-2/BBE cells were exposed to forskolin and ATP. Forskolin and ATP both acutely stimulated DRA in a concentration dependent manner. Forskolin at 1{micro}M and ATP at 0.25 {micro}M had minimal to no effect on DRA activity given individually; however, together, they stimulated DRA activity to levels seen with maximum concentrations of forskolin and ATP alone. In Caco-2/BBE cells expressing the Ca2+ indicator GCaMP6s, ATP alone increased Ca2+ in a concentration dependent manner, while forskolin (1 {micro}M), that by itself did not significantly alter Ca2+, followed by 0.25 {micro}M ATP produced a large increase in Ca2+ that was [~]equal to the elevation caused by 1 {micro}M ATP. BAPTA-AM pretreatment prevented the ATP and forskolin/ATP synergistic increased DRA activity and the increase in intracellular Ca2+ caused by ATP/forskolin. Conclusion: In Caco-2/BBE cells subthreshold concentrations of forskolin (cAMP) and ATP (Ca2+) synergistically increased intracellular Ca2+ and stimulated DRA activity with both being blocked by BAPTA-AM pretreatment. Diarrheal diseases such as bile acid diarrhea, in which both cAMP and Ca2+ are elevated, are likely to be associated with stimulated DRA activity contributing to increased anion secretion, while separation of DRA from NHE3 contributes to reduced NaCl absorption.

physiology↗

bioRxiv2022

Adipokine C1q/Tumor necrosis factor-related protein 3 (CTRP3) Attenuates Intestinal Inflammation via Histone Deacetylase Sirtuin 1 (SIRT1)/NF-κB Signaling

BACKGROUND & AIMSThe adipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) has anti-inflammatory effects in several non-intestinal disorders. Although CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD), its function in IBD has not been established. We aimed to elucidate the function of CTRP3 and related molecular mechanisms in intestinal inflammation using a colitis model of genetically-modified CTRP3 mice and intestinal epithelial tissue from patients with Crohns disease (CD), one of the two main forms of IBD. METHODSCTRP3 knockout (KO) and overexpressing transgenic (Tg) mice along with their corresponding wild-type (WT) littermates were subjected to drinking water containing dextran sulfate sodium (DSS) for 6-10 days to induce acute colitis. Mouse colitis symptoms and histological data were analyzed. CTRP3-mediated signaling was examined in the intestinal tissue of mice and patients with CD. RESULTSCTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. In DSS-induced acute colitis models, CTRP3 KO mice developed more severe colitis than their WT littermates, while CTRP3 overexpressing Tg mice developed less severe colitis than their WT littermates. In both water- and DSS-treated CTRP3 KO mice, reduced CTRP3 levels correlated with decreased levels of Sirtuin 1 (SIRT1), a histone deacetylase, increased levels of phosphorylated nuclear factor kappa B (NF-{kappa}B) subunit p65, resulting in increased expression of pro-inflammatory cytokines tumor necrosis factor- (TNF-) and interleukin 6 (IL-6). The results from CTRP3 Tg mice mirrored those from CTRP3 KO mice in most respects. This CTRP3/SIRT1/NF-{kappa}B relationship was also observed in the intestinal epithelial tissue of patients with active and inactive CD. CONCLUSIONSCTRP3 expression levels correlate negatively with intestinal inflammation in mouse colitis models and CD patients. CTRP3 attenuates intestinal inflammation via SIRT1/NF-{kappa}B signaling to suppress pro-inflammatory cytokines in mouse colitis models and patients with IBD. The manipulation of CTRP3 signaling, including through the use of SIRT1 agonists, may offer translational potential in the treatment of IBD. WHAT YOU NEED TO KNOWO_ST_ABSBACKGROUND AND CONTEXTC_ST_ABSAdipokine C1q/tumor necrosis factor-related protein 3 (CTRP3) is a novel adipokine with known non-intestinal anti-inflammatory effects. CTRP3 is reduced in the serum of patients with inflammatory bowel disease (IBD). However, little is known about whether and how CTRP3 influences intestinal inflammation in IBD. NEW FINDINGSCTRP3 mRNA and protein were detected in murine and human intestinal epithelial cells, as well as in murine intestinal smooth muscle cells and mesenteric fat. CTRP3 deletion was associated with more severe acute dextran sulfate sodium (DSS)-induced colitis, while CTRP3 overexpression was associated with less severe colitis. In both mice and humans, reduced CTRP3 levels correlated with reduced levels of the histone deacetylase Sirtuin 1 (SIRT1), resulting in the up-regulation of phosphorylated nuclear factor-kappa B (NF-{kappa}B) p65 and pro-inflammatory cytokine synthesis. LIMITATIONSThis study was performed using genetically modified mice and human tissue samples. An acute DSS-induced colitis model was used; additional mouse colitis models designed to mimic other aspects of IBD will be examined in future studies. The specific source of the secreted CTRP3 protein which influences intestinal inflammation is yet to be identified. The use of recombinant CTRP3 protein supplementation and SIRT1 agonists to mitigate intestinal inflammation also requires further study. IMPACTCTRP3 is a novel anti-inflammatory adipokine that attenuates intestinal inflammation in colitis mouse models and intestinal epithelial tissue of patients with IBD. CTRP3 attenuates intestinal inflammation by activating SIRT1, which suppresses the pro-inflammatory transcriptional activity of phosphorylated NF-{kappa}B p65. CTRP3 and SIRT1 agonists have potential as novel IBD drug targets.

pathology↗

bioRxiv2021

SARS-COV-2 induced Diarrhea is inflammatory, Ca2+ Dependent and involves activation of calcium activated Cl channels

Diarrhea occurs in 2-50% of cases of COVID-19 ([~]8% is average across series). The diarrhea does not appear to account for the disease mortality and its contribution to the morbidity has not been defined, even though it is a component of Long Covid or post-infectious aspects of the disease. Even less is known about the pathophysiologic mechanism of the diarrhea. To begin to understand the pathophysiology of COVID-19 diarrhea, we exposed human enteroid monolayers obtained from five healthy subjects and made from duodenum, jejunum, and proximal colon to live SARS-CoV-2 and virus like particles (VLPs) made from exosomes expressing SARS-CoV-2 structural proteins (Spike, Nucleocapsid, Membrane and Envelope). Results: 1) Live virus was exposed apically for 90 min, then washed out and studied 2 and 5 days later. SARS-Cov-2 was taken up by enteroids and live virus was present in lysates and in the apical>>basolateral media of polarized enteroids 48 h after exposure. This is the first demonstration of basolateral appearance of live virus after apical exposure. High vRNA concentration was detected in cell lysates and in the apical and basolateral media up to 5 days after exposure. 2) Two days after viral exposure, cytokine measurements of media showed significantly increased levels of IL-6, IL-8 and MCP-1. 3) Two days after viral exposure, mRNA levels of ACE2, NHE3 and DRA were reduced but there was no change in mRNA of CFTR. NHE3 protein was also decreased. 4) Live viral studies were mimicked by some studies with VLP exposure for 48 h. VLPs with Spike-D614G bound to the enteroid apical surface and was taken up; this resulted in decreased mRNA levels of ACE2, NHE3, DRA and CFTR. 4) VLP effects were determined on active anion secretion measured with the Ussing chamber/voltage clamp technique. S-D614G acutely exposed to apical surface of human ileal enteroids did not alter the short-circuit current (Isc). However, VLPS-D614G exposure to enteroids that were pretreated for [~]24 h with IL-6 plus IL-8 induced a concentration dependent increase in Isc indicating stimulated anion secretion, that was delayed in onset by [~]8 min. The anion secretion was inhibited by apical exposure to a specific calcium activated Cl channel (CaCC) inhibitor (AO1) but not by a specific CFTR inhibitor (BP027); was inhibited by basolateral exposure to the K channel inhibit clortimazole; and was prevented by pretreatment with the calcium buffer BAPTA-AM. 5) The calcium dependence of the VLP-induced increase in Isc was studied in Caco-2/BBe cells stably expressing the genetically encoded Ca2+ sensor GCaMP6s. 24 h pretreatment with IL-6/IL-8 did not alter intracellular Ca2+. However, in IL-6/IL-8 pretreated cells, VLP S-D614G caused appearance of Ca2+waves and an overall increase in intracellular Ca2+ with a delay of [~]10 min after VLP addition. We conclude that the diarrhea of COVID-19 appears to an example of a calcium dependent inflammatory diarrhea that involves both acutely stimulated Ca2+ dependent anion secretion (stimulated Isc) that involves CaCC and likely inhibition of neutral NaCl absorption (decreased NHE3 protein and mRNA and decreased DRA mRNA).

physiology↗

bioRxiv2020

Loss of PI3-kinase activity of inositol polyphosphate multikinase impairs PDK1-mediated AKT activation, cell migration, and intestinal homeostasis

Inositol polyphosphate multikinase (IPMK) is a rate-limiting enzyme in the inositol phosphate (IP) pathway which converts IP3 to IP4 and IP5. In mammalian cells, IPMK can also act as a phosphoinositol-3-kinase (PI3-kinase). We previously found that IPMK is a critical PI3-kinase activator of AKT. Here, we show that IPMK mediates AKT activation by promoting membrane localization and activation of PDK1. The PI3-kinase activity of IPMK is dispensable for membrane localization of AKT, which is entirely controlled by classical PI3-kinase (p110,{beta},{gamma} , {delta}). By contrast, we found that PDK1 membrane localization was largely independent of classical PI3-kinase. Membrane localization of PDK1 stimulates cell migration by dissociating ROCK1 from inhibitory binding to RhoE and promoting ROCK1-mediated myosin light chain (MLC) phosphorylation. Deletion of IPMK impairs cell migration associated with the abolition of PDK1-mediated ROCK1 disinhibition and subsequent MLC phosphorylation. To investigate the physiological relevance of IPMK-mediated AKT activation, we generated mice selectively lacking IPMK in epithelial cells of the intestine, where IPMK is highly expressed. Deletion of IPMK in intestinal epithelial cells markedly reduced AKT phosphorylation and diminished numbers of Paneth cells - a crypt-resident epithelial cell type that generates the physiological niche for intestinal stem cells. Ablation of IPMK impaired intestinal epithelial cell regeneration basally and after; chemotherapy-induced damage, suggesting a broad role for IPMK in the activation of AKT and intestinal tissue regeneration. In summary, the PI3-kinase activity of IPMK promotes membrane localization of PDK1, a critical kinase whereby AKT maintains intestinal homeostasis. One Sentence SummaryPI3-kinase activity of IPMK is essential for activation of AKT.

cell biology↗

bioRxiv2020

Chronic exposure of ErbB TKIs inhibits DRA expression and activity through an ERK/Elk-1/CREB/AP-1 dependent pathway

Diarrhea is the major side effect of first- and second-generation ErbB tyrosine kinase inhibitors (TKI), the mechanism of which remains incompletely understood. The current studies were carried out over the time frame that ErbB TKIs usually initiate diarrhea. We report in Caco-2/bbe cells that exposure of ErbB TKIs, but not non-ErbB TKIs for six days at clinically-relevant concentrations significantly reduced the expression of DRA and inhibited apical Cl-/HCO3-exchange activity. The ErbB TKIs decreased DRA expression through an ERK/Elk-1/CREB/AP-1 dependent pathway. The blockade of ERK phosphorylation by ErbB TKIs decreased the phosphorylation of Elk-1 and the amount of total and p-CREB, and reduced the expression of C-Fos, which is part of the AP-1 complex that maintain DRA expression. Altogether, our studies demonstrate that ErbB TKIs decrease expression and activity of DRA, which occurs over the time frame that these drugs clinically cause diarrhea, and since DRA is part of the intestinal neutral NaCl absorptive process, the reduced absorption is likely to represent a major contributor to the ErbB TKI-associated diarrhea.

pharmacology and toxicology↗

bioRxiv2019

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

BackgroundRotavirus (RV) is the 5th leading cause of death in children <5 years old but the leading cause of diarrhea related deaths in this age group. The mechanism of RV diarrhea involves decreased activity of Na+-dependent solute transporters with increased luminal secretion of Cl- in the absence of significant histologic damage. While our understanding of RV diarrhea has come from studies in animal models and cancer cell lines, the mechanism of the diarrhea and the transport proteins affected in human RV disease remains only partially understood. This understanding is likely to impact drug development therapy for RV diarrhea.\n\nMethodsFormalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibodies) were analyzed by immunofluorescence for changes in apical/basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins. Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated to resemble villus epithelial cells, and infected with human RV.\n\nResultsRV diarrhea was associated with reduced expression and intracellular localization of transport proteins normally found in the brush border membranes, including SGLT1, NHE3, NHE2, the Na+-dependent amino acid transporter SLC6A19, and CFTR. In contrast, basolateral proteins, including Na+/K+-ATPase, NKCC1, and {beta}-catenin, the brush border marker ezrin, as well as the tight junction protein, ZO-1, were expressed and localized normally. RV-induced mislocalization of NHE3, SGLT1, SLC6A19 and CFTR was also seen when human small intestinal enteroids were infected with RV.\n\nConclusionsThese data demonstrate a new pathophysiologic mechanism of acute diarrhea in which expression of multiple apical transport proteins are reduced. This acute diarrhea is likely to be caused by an effect on a common apical trafficking pathway, as exemplified by RV diarrhea, and its contribution to other enteric pathogen-induced diarrheal diseases should be determined.

cell biology↗

bioRxiv2019

Mechanical Stimuli Affect E. coli Heat Stable Enterotoxin-cyclic GMP Signaling in a Human Enteroid Intestine-Chip Diarrhea Model

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a pathogen virulence factor, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips), exposed to enterotoxigenic E. coli heat-stable enterotoxin A (ST), and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height, transcription of the cyclic nucleotide transporting protein MRP4, and apical and basolateral secretion of cGMP under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to static conditions, but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to static culture conditions under both baseline conditions and with exposure to ST enterotoxin, and suggests that further investigations of application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resembles intact intestine than study under static conditions.

microbiology↗

bioRxiv2019

Organoid-derived Duodenum Intestine-Chip for preclinical drug assessment in a human relevant system

Induction of intestinal drug metabolizing enzymes can complicate the development of new drugs, owing to potential to cause drug-drug interactions (DDIs) leading to changes in pharmacokinetics, safety and efficacy. The development of a human relevant model of the adult intestine that accurately predicts CYP450 induction could help address this challenge as species differences preclude extrapolation from animals. Here, we combined organoids and Organ-Chip technology to create a human Duodenum Intestine-Chip that emulates intestinal tissue architecture and functions, that are relevant for the study of drug transport, metabolism, and DDI. Duodenum Intestine-Chip demonstrates the polarized cell architecture, intestinal barrier function, presence of specialized cell subpopulations, and in vivo-relevant expression, localization, and function of major intestinal drug transporters. Notably, in comparison to Caco-2, it displays improved CYP3A4 expression and induction capability. This model could enable improved in vitro to in vivo extrapolation for better predictions of human pharmacokinetics and risk of DDIs.

cell biology↗

bioRxiv2018

Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli secreted cytotoxin, EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

physiology↗

bioRxiv2018

cAMP stimulates SLC26A3 activity in human colon by a CFTR-dependent mechanism that does not require CFTR activity

Background & AimsDRA (SLC26A3) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute cAMP-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates or does not affect its activity.\n\nMethodsThis study reevaluated cAMP regulation of DRA using new \"tools\" including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on non-specific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2.\n\nResultsDRA is an apical protein in human proximal colon, differentiated colonoid monolayers and Caco-2 cells. It is glycosylated and appears as two bands. cAMp(forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with IC50 of 0.5 mol/L and 0.2 {micro}mol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked CFTR. When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172.\n\nConclusionsDRA is acutely stimulated by cAMP by a process that is CFTR-dependent but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

physiology↗