Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in ...mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.
ZIP5 localizes to the baso-lateral membranes of intestinal enterocytes and pancreatic acinar cells and is internalized and degraded coordinately in these cell-types during periods of dietary zinc ...deficiency. These cell-types are thought to control zinc excretion from the body. The baso-lateral localization and zinc-regulation of ZIP5 in these cells are unique among the 14 members of the Slc39a family and suggest that ZIP5 plays a role in zinc excretion.
We created mice with floxed Zip5 genes and deleted this gene in the entire mouse or specifically in enterocytes or acinar cells and then examined the effects on zinc homeostasis. We found that ZIP5 is not essential for growth and viability but total knockout of ZIP5 led to increased zinc in the liver in mice fed a zinc-adequate (ZnA) diet but impaired accumulation of pancreatic zinc in mice fed a zinc-excess (ZnE) diet. Loss-of-function of enterocyte ZIP5, in contrast, led to increased pancreatic zinc in mice fed a ZnA diet and increased abundance of intestinal Zip4 mRNA. Finally, loss-of-function of acinar cell ZIP5 modestly reduced pancreatic zinc in mice fed a ZnA diet but did not impair zinc uptake as measured by the rapid accumulation of (67)zinc. Retention of pancreatic (67)zinc was impaired in these mice but the absence of pancreatic ZIP5 sensitized them to zinc-induced pancreatitis and exacerbated the formation of large cytoplasmic vacuoles containing secretory protein in acinar cells.
These studies demonstrate that ZIP5 participates in the control of zinc excretion in mice. Specifically, they reveal a paramount function of intestinal ZIP5 in zinc excretion but suggest a role for pancreatic ZIP5 in zinc accumulation/retention in acinar cells. ZIP5 functions in acinar cells to protect against zinc-induced acute pancreatitis and attenuate the process of zymophagy. This suggests that it may play a role in autophagy.
Alcoholic liver disease (ALD) is a major health problem in the United States and worldwide without successful treatments. Chronic alcohol consumption can lead to ALD, which is characterized by ...steatosis, inflammation, fibrosis, cirrhosis, and even liver cancer. Recent studies suggest that alcohol induces both cell death and adaptive cell survival pathways in the liver, and the balance of cell death and cell survival ultimately decides the pathogenesis of ALD. This review summarizes the recent progress on the role and mechanisms of apoptosis, necroptosis, and autophagy in the pathogenesis of ALD. Understanding the complex regulation of apoptosis, necrosis, and autophagy may help to develop novel therapeutic strategies by targeting all 3 pathways simultaneously.
Cell death and cell survival are closely associated with alcoholic liver disease.
Ethanol metabolism leads to increased oxidative stress resulting in mitochondrial damage and apoptosis in hepatocytes.
Chronic alcohol consumption increases gut permeability and elevated systemic levels of gut‐derived endotoxins resulting in increased production of TNF‐α from activated Kupffer cells.
Depending on the cIAP levels and caspase‐8 activation in hepatocytes, TNF‐α either induces apoptosis or necroptosis by activating Bid‐mediated mitochondrial apoptotic pathway or ripoptosome or necrosome.
The cystic fibrosis intestine De Lisle, Robert C; Borowitz, Drucy
Cold Spring Harbor perspectives in medicine,
09/2013, Letnik:
3, Številka:
9
Journal Article
Recenzirano
Odprti dostop
The clinical manifestations of cystic fibrosis (CF) result from dysfunction of the cystic fibrosis transmembrane regulator protein (CFTR). The majority of people with CF have a limited life span as a ...consequence of CFTR dysfunction in the respiratory tract. However, CFTR dysfunction in the gastrointestinal (GI) tract occurs earlier in ontogeny and is present in all patients, regardless of genotype. The same pathophysiologic triad of obstruction, infection, and inflammation that causes disease in the airways also causes disease in the intestines. This article describes the effects of CFTR dysfunction on the intestinal tissues and the intraluminal environment. Mouse models of CF have greatly advanced our understanding of the GI manifestations of CF, which can be directly applied to understanding CF disease in humans.
Impaired macroautophagy/autophagy has been implicated in experimental and human pancreatitis. However, the transcriptional control governing the autophagy-lysosomal process in pancreatitis is largely ...unknown. We investigated the role and mechanisms of TFEB (transcription factor EB), a master regulator of lysosomal biogenesis, in the pathogenesis of experimental pancreatitis. We analyzed autophagic flux, TFEB nuclear translocation, lysosomal biogenesis, inflammation and fibrosis in GFP-LC3 transgenic mice, acinar cell-specific tfeb knockout (KO) and tfeb and tfe3 double-knockout (DKO) mice as well as human pancreatitis samples. We found that cerulein activated MTOR (mechanistic target of rapamycin kinase) and increased the levels of phosphorylated TFEB as well as pancreatic proteasome activities that led to rapid TFEB degradation. As a result, cerulein decreased the number of lysosomes resulting in insufficient autophagy in mouse pancreas. Pharmacological inhibition of MTOR or proteasome partially rescued cerulein-induced TFEB degradation and pancreatic damage. Furthermore, genetic deletion of tfeb specifically in mouse pancreatic acinar cells increased pancreatic edema, necrotic cell death, infiltration of inflammatory cells and fibrosis in pancreas after cerulein treatment. tfeb and tfe3 DKO mice also developed spontaneous pancreatitis with increased pancreatic trypsin activities, edema and infiltration of inflammatory cells. Finally, decreased TFEB nuclear staining was associated with human pancreatitis. In conclusion, our results indicate a critical role of impaired TFEB-mediated lysosomal biogenesis in promoting the pathogenesis of pancreatitis.
Abbreviations: AC: acinar cell; AMY: amylase; ATP6V1A: ATPase, H+ transporting, lysosomal V1 subunit A; ATP6V1B2: ATPase, H+ transporting, lysosomal V1 subunit B2; ATP6V1D: ATPase, H+ transporting, lysosomal V1 subunit D; ATP6V1H: ATPase, H+ transporting, lysosomal V1 subunit H; AV: autophagic vacuole; CDE: choline-deficient, ethionine-supplemented; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; EM: electron microscopy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; H & E: hematoxylin and eosin; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: normal donor; NEU: neutrophil; PPARGC1A/PGC1α: peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha; RIPA: radio-immunoprecipitation; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TM: tamoxifen; WT: wild-type; ZG: zymogen granule
The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis ...(CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160 % of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.
Cystic fibrosis (CF) has major effects on the intestinal tract with potential consequences on nutrition, but these are not fully understood. I investigated the possibility of altered enterocyte ...maturation in CF, as suggested by decreased enterocyte nutrient assimilation gene expression in published transcriptome analysis of the small intestine of CF mouse.
In CF and wild-type (WT) mice, enterocyte gene/protein expression was analyzed by quantitative realtime polymerase chain reaction (qRT-PCR), enzyme histochemistry, immunohistochemistry, and Western blot. One group of mice was maintained on a control liquid diet; to manipulate the gut microbiota, a second group was treated with oral antibiotics; to improve hydration of the gut lumen, a third group was given a laxative drinking solution.
On the control diet in the CF intestine, there were decreased levels (67%-85% reduction of WT levels) of enterocyte genes/proteins. Antibiotics did not normalize the expression of enterocyte markers in the CF mouse. In contrast, the laxative treatment of CF mice significantly increased expression to near WT levels.
These studies suggest that the environment of the CF intestinal lumen plays a role in reduced maturation of enterocytes. Because changing the gut lumen environment can affect enterocyte maturation, this is not a cell-autonomous effect of loss of CF transmembrane conductance regulator.
Zinc deficiency due to poor nutrition or genetic mutations in zinc transporters is a global health problem and approaches to providing effective dietary zinc supplementation while avoiding potential ...toxic side effects are needed.
Conditional knockout of the intestinal zinc transporter Zip4 (Slc39a4) in mice creates a model of the lethal human genetic disease acrodermatitis enteropathica (AE). This knockout leads to acute zinc deficiency resulting in rapid weight loss, disrupted intestine integrity and eventually lethality, and therefore provides a model system in which to examine novel approaches to zinc supplementation. We examined the efficacy of dietary clioquinol (CQ), a well characterized zinc chelator/ionophore, in rescuing the Zip4 (intest KO) phenotype. By 8 days after initiation of the knockout neither dietary CQ nor zinc supplementation in the drinking water was found to be effective at improving this phenotype. In contrast, dietary CQ in conjunction with zinc supplementation was highly effective. Dietary CQ with zinc supplementation rapidly restored intestine stem cell division and differentiation of secretory and the absorptive cells. These changes were accompanied by rapid growth and dramatically increased longevity in the majority of mice, as well as the apparent restoration of the homeostasis of several essential metals in the liver.
These studies suggest that oral CQ (or other 8-hydroxyquinolines) coupled with zinc supplementation could provide a facile approach toward treating zinc deficiency in humans by stimulating stem cell proliferation and differentiation of intestinal epithelial cells.
Lubiprostone is a synthetic bicyclic fatty acid derivative of prostaglandin E1 (PGE1) used for chronic constipation. The best known action of lubiprostone is simulation of Cl- dependent fluid ...secretion. In a mouse model of the genetic disease cystic fibrosis, we previously showed that in vivo administration of lubiprostone resulted in greater mucus accumulation in the small intestine. The aim of this study was to directly test whether lubiprostone stimulates intestinal mucin release.
Mucin release was measured by mounting segments (4-5 cm) of mouse proximal-mid small intestine in an organ bath, allowing access to the perfusate (luminal) and the bath (serosal) solutions. Nifedipine (10-6 M) and indomethacin (10-5 M) were included in all solutions to inhibit smooth muscle activity and endogenous prostaglandin production, respectively. The tissue was equilibrated under flow for 30 min, using the perfusate collected during the final 10 min of the equilibration period to measure unstimulated release rate. Stimulus was then added to either the perfusate or the bath and the perfusate was collected for another 30 min to measure the stimulated mucin release rate. Mucin in perfusates was quantified by periodic acid-Schiff's base dot-blot assay, using purified pig gastric mucin as a standard.
When applied luminally at 1 μM lubiprostone was ineffective at stimulating mucin release. When added to the serosal solution, 1 μM lubiprostone stimulated mucin release to ~300% of the unstimulated rate. As a positive control, serosal 1 μM prostaglandin E2 increased mucin release to ~400% of the unstimulated rate.
These results support the idea that lubiprostone has prostaglandin-like actions on the intestine, which includes stimulation of mucin release. Stimulation of mucin release by lubiprostone may be protective in gastrointestinal conditions where loss of mucus is believed to contribute to pathogenesis. Thus, in addition to chronic constipation, there is greater potential for the therapeutic applications of lubiprostone.
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