Radiation-induced oral mucositis hamster model using a linear accelerator enhances clinical relevance of preclinical studies for treatment strategy investigation.

Translational animal models for oral mucositis (OM) are necessary to simulate and assess the bioclinical effects and response in humans. These models should simulate high levels of radiation exposure that leads to oxidative stress and inflammatory-initiated tissue changes. Hamster models have been extensively studied to observe pathological effects of radiation exposure and help in the development of effective treatments. To successfully evaluate the potential for treatment regimens with consistency and relevance, a radiation-induced OM hamster model was developed using a clinical linear accelerator utilized by cancer patients daily. The dose exposure to the isolated, everted cheek pouch of a hamster, as well as the progression of injury, pro-inflammatory marker, histological, and elasticity analyses of the buccal pouch were conducted to verify replicability and reproducibility of the injury model. The findings from this model demonstrated its ability to consistently induce injury and resolution over 28 days using an acute dose of 60 Gy. This model was developed to enhance clinical relevance when evaluating potential efficacious treatments and can now be utilized in efficacy studies to better evaluate developed therapeutics in a preclinical model that is easy to translate to clinical studies..

The mitochondrial retrograde signaling regulates Wnt signaling to promote tumorigenesis in colon cancer.

Cancer cells are known to upregulate aerobic glycolysis to promote growth, proliferation, and survival. However, the role of mitochondrial respiration in tumorigenesis remains elusive. Here we report that inhibition of mitochondrial function by silencing TFAM, a key transcription factor essential for mitochondrial DNA (mtDNA) replication and the transcription of mtDNA-encoded genes, markedly reduced tumor-initiating potential of colon cancer cells. Knockdown of TFAM significantly decreased mitochondrial respiration in colon cancer cells; however, the cellular levels of ATP remained largely unchanged as a result of increased glycolysis. This metabolic alteration rendered cancer cells highly susceptible to glucose deprivation. Interestingly, upregulation of glycolysis was independent of hypoxia-inducible factor-1 (HIF1) as TFAM knockdown cells fail to stabilize HIF1α under hypoxic conditions. Moreover, knockdown of TFAM results in decreased expression of genes-associated cancer stem cells downstream of Wnt/ß-catenin signaling. Metabolic analysis reveals that the level of α-ketoglutarate (α-KG) was significantly upregulated in TFAM knockout cells. Silencing of prolyl hydroxylase domain-containing protein 2 (PHD2), a α-KG-dependent dioxyenase, rescued the expression of target genes of both HIF1α and Wnt/ß-catenin. Furthermore, intestinal-specific knockout of TFAM prevents tumor formation in Apc-mutant mouse models of colon cancer. Taken together, our findings identify a novel role of mitochondria-mediated retrograde signaling in regulating Wnt signaling and tumor initiation in colon cancer.

Beta-catenin cleavage enhances transcriptional activation.

Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

Myo-inositol reduces ß-catenin activation in colitis.

AIM: To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate pß-cateninS552 as a biomarker of recurrent dysplasia. METHODS: We examined the effects of dietary myo-inositol treatment on inflammation, pß-cateninS552 and pAkt levels by histology and western blot in IL-10-/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear pß-cateninS552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia. RESULTS: In mice, pß-cateninS552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, pß-cateninS552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease. CONCLUSION: Enumerating crypts with increased numbers of pß-cateninS552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/ß-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/ß-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/ß-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/ß-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ß-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/ß-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Epithelial PIK3R1 (p85) and TP53 Regulate Survivin Expression during Adaptation to Ileocecal Resection.

Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of ß-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome.

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

AIM: To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

A Cytosolic Multiprotein Complex Containing p85α Is Required for ß-Catenin Activation in Colitis and Colitis-associated Cancer.

Wnt/ß-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated ß-catenin (pß-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in ß-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with ß-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial ß-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces ß-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the ß-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.

The combination of ezetimibe and ursodiol promotes fecal sterol excretion and reveals a G5G8-independent pathway for cholesterol elimination.

Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination from the body. We hypothesized that a combination of ursodiol (Urso) and ezetimibe (EZ) could increase biliary secretion and reduce cholesterol reabsorption, respectively, to promote cholesterol excretion. Treatment with Urso increased hepatic ABCG5 ABCG8 (G5G8) protein and both biliary and fecal sterols in a dose-dependent manner. To determine whether the drug combination (Urso-EZ) further increased cholesterol excretion, mice were treated with Urso alone or in combination with two doses of EZ. EZ produced an additive and dose-dependent increase in fecal neutral sterol (FNS) elimination in the presence of Urso. Finally, we sequentially treated wide-type and G5G8-deficient mice with Urso and Urso-EZ to determine the extent to which these effects were G5G8 dependent. Although biliary and FNS were invariably lower in G5G8 KO mice, the relative increase in FNS following treatment with Urso alone or the Urso-EZ combination was not affected by genotype. In conclusion, Urso increases G5G8, biliary cholesterol secretion, and FNS and acts additively with EZ to promote fecal sterol excretion. However, the stimulatory effect of these agents was not G5G8 dependent.

Placental syncytium forms a biophysical barrier against pathogen invasion.

Fetal syncytiotrophoblasts form a unique fused multinuclear surface that is bathed in maternal blood, and constitutes the main interface between fetus and mother. Syncytiotrophoblasts are exposed to pathogens circulating in maternal blood, and appear to have unique resistance mechanisms against microbial invasion. These are due in part to the lack of intercellular junctions and their receptors, the Achilles heel of polarized mononuclear epithelia. However, the syncytium is immune to receptor-independent invasion as well, suggesting additional general defense mechanisms against infection. The difficulty of maintaining and manipulating primary human syncytiotrophoblasts in culture makes it challenging to investigate the cellular and molecular basis of host defenses in this unique tissue. Here we present a novel system to study placental pathogenesis using murine trophoblast stem cells (mTSC) that can be differentiated into syncytiotrophoblasts and recapitulate human placental syncytium. Consistent with previous results in primary human organ cultures, murine syncytiotrophoblasts were found to be resistant to infection with Listeria monocytogenes via direct invasion and cell-to-cell spread. Atomic force microscopy of murine syncytiotrophoblasts demonstrated that these cells have a greater elastic modulus than mononuclear trophoblasts. Disruption of the unusually dense actin structure--a diffuse meshwork of microfilaments--with Cytochalasin D led to a decrease in its elastic modulus by 25%. This correlated with a small but significant increase in invasion of L. monocytogenes into murine and human syncytium. These results suggest that the syncytial actin cytoskeleton may form a general barrier against pathogen entry in humans and mice. Moreover, murine TSCs are a genetically tractable model system for the investigation of specific pathways in syncytial host defenses.

Occludin S408 phosphorylation regulates tight junction protein interactions and barrier function.

Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13-induced, claudin-2-dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.

Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 ß1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Redistribution of the tight junction protein ZO-1 during physiological shedding of mouse intestinal epithelial cells.

We questioned how tight junctions contribute to intestinal barrier function during the cell shedding that is part of physiological cell renewal. Intravital confocal microscopy studied the jejunal villus epithelium of mice expressing a fluorescent zonula occludens 1 (ZO-1) fusion protein. Vital staining also visualized the cell nucleus (Hoechst staining) or local permeability to luminal constituents (Lucifer Yellow; LY). In a cell fated to be shed, ZO-1 redistributes from the tight junction toward the apical and then basolateral cell region. ZO-1 rearrangement occurs 15 ± 6 min (n = 28) before movement of the cell nucleus from the epithelial layer. During cell extrusion, permeation of luminal LY extends along the lateral intercellular spaces of the shedding cell only as far as the location of ZO-1. Within 3 min after detachment from the epithelial layer, nuclear chromatin condenses. After cell loss, a residual patch of ZO-1 remains in the space previously occupied by the departed cell, and the size of the patch shrinks to 14 ± 2% (n = 15) of the original cell space over 20 min. The duration of cell shedding measured by nucleus movement (14 ± 1 min) is much less than the total duration of ZO-1 redistribution at the same sites (45 ± 2 min). In about 15% of cell shedding cases, neighboring epithelial cells also undergo extrusion with a delay of 5-10 min. With the use of normal mice, ZO-1 immunofluorescent staining of fixed tissue confirmed ZO-1 redistribution and the presence of ZO-1 patches beneath shedding cells. Immunostaining also showed that redistribution of ZO-1 occurred without corresponding mixing of apical and basolateral membrane domains as marked by ezrin or E-cadherin. ZO-1 redistribution is the earliest cellular event yet identified as a herald of physiological cell shedding, and redistribution of tight junction function along the lateral plasma membrane sustains epithelial barrier during cell shedding.

CLIC5 mutant mice are resistant to diet-induced obesity and exhibit gastric hemorrhaging and increased susceptibility to torpor.

Chloride intracellular channel 5 (CLIC5) and other CLIC isoforms have been implicated in a number of biological processes, but their specific functions are poorly understood. The association of CLIC5 with ezrin and the actin cytoskeleton led us to test its possible involvement in gastric acid secretion. Clic5 mutant mice exhibited only a minor reduction in acid secretion, Clic5 mRNA was expressed at only low levels in stomach, and Clic5 mutant parietal cells were ultrastructurally normal, negating the hypothesis that CLIC5 plays a major role in acid secretion. However, the mutants exhibited gastric hemorrhaging in response to fasting, reduced monocytes and granulocytes suggestive of immune dysfunction, behavioral and social disorders suggestive of neurological dysfunction, and evidence of a previously unidentified metabolic defect. Wild-type and mutant mice were maintained on normal and high-fat diets; plasma levels of various hormones, glucose, and lipids were determined; and body composition was studied by quantitative magnetic resonance imaging. Clic5 mutants were lean, hyperphagic, and highly resistant to diet-induced obesity. Plasma insulin and glucose levels were reduced, and leptin levels were very low; however, plasma triglycerides, cholesterol, phospholipids, and fatty acids were normal. Indirect calorimetry revealed increased peripheral metabolism and greater reliance on carbohydrate metabolism. Because Clic5 mutants were unable to maintain energy reserves, they also exhibited increased susceptibility to fasting-induced torpor, as indicated by telemetric measurements showing episodes of reduced body temperature and heart rate. These data reveal a requirement for CLIC5 in the maintenance of normal systemic energy metabolism.

Volume density, distribution, and ultrastructure of secretory and basolateral membranes and mitochondria predict parietal cell secretory (dys)function.

Acid secretion in gastric parietal cells requires highly coordinated membrane transport and vesicle trafficking. Histologically, consensus defines acid secretion as the ratio of the volume density (Vd) of canalicular and apical membranes (CAMs) to tubulovesicular (TV) membranes, a value which varies widely under normal conditions. Examination of numerous achlorhydric mice made it clear that this paradigm is discrepant when used to assess most mice with genetic mutations affecting acid secretion. Vd of organelles in parietal cells of 6 genetically engineered mouse strains was obtained to identify a stable histological phenotype of acid secretion. We confirmed that CAM to TV ratio fairly represented secretory activity in untreated and secretion-inhibited wild-type (WT) mice and in NHE2-/- mice as well, though the response was significantly attenuated in the latter. However, high CAM to TV ratios wrongly posed as active acid secretion in AE2-/-, GHKAalpha-/-, and NHE4-/- mice. Achlorhydric genotypes also had a significantly higher Vd of basolateral membrane than WT mice, and reduced Vd of mitochondria and canaliculi. The Vd of mitochondria, and ratio of the Vd of basolateral membranes/Vd of mitochondria were preferred predictors of the level of acid secretion. Alterations in acid secretion, then, cause significant changes not only in the Vd of secretory membranes but also in mitochondria and basolateral membranes.

AE2 Cl-/HCO3- exchanger is required for normal cAMP-stimulated anion secretion in murine proximal colon.

Anion secretion by colonic epithelium is dependent on apical CFTR-mediated anion conductance and basolateral ion transport. In many tissues, the NKCC1 Na(+)-K(+)-2Cl(-) cotransporter mediates basolateral Cl(-) uptake. However, additional evidence suggests that the AE2 Cl(-)/HCO(3)(-) exchanger, when coupled with the NHE1 Na(+)/H(+) exchanger or a Na(+)-HCO(3)(-) cotransporter (NBC), contributes to HCO(3)(-) and/or Cl(-) uptake. To analyze the secretory functions of AE2 in proximal colon, short-circuit current (I(sc)) responses to cAMP and inhibitors of basolateral anion transporters were measured in muscle-stripped wild-type (WT) and AE2-null (AE2(-/-)) proximal colon. In physiological Ringer, the magnitude of cAMP-stimulated I(sc) was the same in WT and AE2(-/-) colon. However, the I(sc) response in AE2(-/-) colon exhibited increased sensitivity to the NKCC1 inhibitor bumetanide and decreased sensitivity to the distilbene derivative SITS (which inhibits AE2 and some NBCs), indicating that loss of AE2 results in a switch to increased NKCC1-supported anion secretion. Removal of HCO(3)(-) resulted in robust cAMP-stimulated I(sc) in both AE2(-/-) and WT colon that was largely mediated by NKCC1, whereas removal of Cl(-) resulted in sharply decreased cAMP-stimulated I(sc) in AE2(-/-) colon relative to WT controls. Inhibition of NHE1 had no effect on cAMP-stimulated I(sc) in AE2(-/-) colon but caused a switch to NKCC1-supported secretion in WT colon. Thus, in AE2(-/-) colon, Cl(-) secretion supported by basolateral NKCC1 is enhanced, whereas HCO(3)(-) secretion is diminished. These results show that AE2 is a component of the basolateral ion transport mechanisms that support anion secretion in the proximal colon.

Reduced NHE3-mediated Na+ absorption increases survival and decreases the incidence of intestinal obstructions in cystic fibrosis mice.

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 Na+/H+ exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target.

Colonic anion secretory defects and metabolic acidosis in mice lacking the NBC1 Na+/HCO3- cotransporter.

The NBC1 Na+/HCO3- cotransporter is expressed in many tissues, including kidney and intestinal epithelia. NBC1 mutations cause proximal renal tubular acidosis in humans, consistent with its role in HCO3- absorption in the kidney. In intestinal and colonic epithelia, NBC1 localizes to basolateral membranes and is thought to function in anion secretion. To test the hypothesis that NBC1 plays a role in transepithelial HCO3- secretion in the intestinal tract, null mutant (NBC1-/-) mice were prepared by targeted disruption of its gene (Slc4a4). NBC1-/- mice exhibited severe metabolic acidosis, growth retardation, reduced plasma Na+, hyperal-dosteronism, splenomegaly, abnormal dentition, intestinal obstructions, and death before weaning. Intracellular pH (pH(i)) was not altered in cAMP-stimulated epithelial cells of NBC1-/- cecum, but pH(i) regulation during sodium removal and readdition was impaired. Bioelectric measurements of NBC1-/- colons revealed increased amiloride-sensitive Na+ absorption. In Ringer solution containing both Cl- and HCO3-, the magnitude of cAMP-stimulated anion secretion was normal in NBC1-/- distal colon but increased in proximal colon, with the increase largely supported by enhanced activity of the basolateral NKCC1 Na+-K+-2Cl- cotransporter. Anion substitution studies in which carbonic anhydrase was inhibited and transepithelial anion conductance was limited to HCO3- revealed a sharp decrease in both cAMP-stimulated HCO3- secretion and SITS-sensitive current in NBC1-/- proximal colon. These results are consistent with the known function of NBC1 in HCO3- absorption in the kidney and demonstrate that NBC1 activity is a component of the basolateral mechanisms for HCO3- uptake during cAMP-stimulated anion secretion in the proximal colon.

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice.

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.