scholarly journals Alternative splicing of the Menkes copper Atpase (Atp7a) transcript in the rat intestinal epithelium

2009 ◽  
Vol 297 (4) ◽  
pp. G695-G707 ◽  
Author(s):  
James F. Collins ◽  
Ping Hua ◽  
Yan Lu ◽  
P. N. Ranganathan
Keyword(s):  
Splice Variants ◽  
Copper Binding ◽  
Intestinal Epithelial ◽  
Rat Intestine ◽  
Protein Variant ◽  
Iron Deprivation ◽  
Rapid Amplification ◽  
Vitro Model ◽  
64 Kda Protein

The intestinal Menkes copper Atpase ( Atp7a) gene is strongly induced by iron deficiency in the rat intestine. We sought to develop an in vitro model to understand the mechanism of this induction by performing molecular studies in native rat intestine and in intestinal epithelial (IEC-6) cells. IEC-6 cells express Atp7a, and induction was noted with iron deprivation. 5′ Rapid amplification of cDNA ends and PCR experiments revealed three splice variants in rat intestine and IEC-6 cells; all variants were strongly induced during iron deprivation (five- to sevenfold). The splice variants presumably encode proteins that would either contain the extreme NH2 terminus of the protein (containing copper binding domain 1) or not. We thus hypothesized that more than one version of Atp7a protein exists. Antibodies against this NH2-terminal region of the protein were developed (named N-term) and used along with previously reported antibodies (against more COOH-terminal regions, termed 54–10) to perform immunoblotting and immunolocalization studies. Results with the 54–10 antiserum revealed an Atp7a protein variant of ∼190 kDa that localized to the trans-Golgi network of IEC-6 cells and trafficked to the plasma membrane with copper loading. Using the N-term antiserum, however, we noted protein of ∼97 and 64 kDa. The 97-kDa protein was cytosolic and nuclear, whereas the 64-kDa protein was nuclear specific. Immunolocalization analyses with the N-term antiserum showed strong staining of nuclei in IEC-6 and Caco-2 cells and in rat intestine. We conclude that novel Atp7a protein variants may exist in rat and human intestinal epithelial cells, with different intracellular locations and potentially distinct physiological functions.

scholarly journals Copper stabilizes the Menkes copper-transporting ATPase (Atp7a) protein expressed in rat intestinal epithelial cells

2013 ◽  
Vol 304 (3) ◽  
pp. C257-C262 ◽  
Author(s):  
Liwei Xie ◽  
James F. Collins
Keyword(s):  
Iron Deficiency ◽  
Copper Accumulation ◽  
Copper Binding ◽  
Intestinal Epithelial ◽  
Divalent Metal Transporter ◽  
Protein Levels ◽  
Iron Deprivation ◽  
Intracellular Copper

Iron deficiency decreases oxygen tension in the intestinal mucosa, leading to stabilization of hypoxia-inducible transcription factor 2α (Hif2α) and subsequent upregulation of genes involved in iron transport [e.g., divalent metal transporter (Dmt1) and ferroportin 1 (Fpn1)]. Iron deprivation also alters copper homeostasis, reflected by copper accumulation in the intestinal epithelium and induction of an intracellular copper-binding protein [metallothionein (Mt)] and a copper exporter [Menkes copper ATPase (Atp7a)]. Importantly, Atp7a is also a Hif2α target. It was, however, previously noted that Atp7a protein expression was induced more strongly than mRNA in the duodenum of iron-deprived rats, suggesting additional regulatory mechanisms. The current study was thus designed to decipher mechanistic aspects of Atp7a regulation during iron deprivation using an established in vitro model of the mammalian intestine, rat intestinal epithelial (IEC-6) cells. Cells were treated with an iron chelator and/or copper loaded to mimic the in vivo situation. IEC-6 cells exposed to copper showed a dose-dependent increase in Mt expression, confirming intracellular copper accumulation. Iron chelation with copper loading increased Atp7a mRNA and protein levels; however, contrary to our expectation, copper alone increased only protein levels. This suggested that copper increased Atp7a protein levels by a posttranscriptional regulatory mechanism. Therefore, to determine if Atp7a protein stability was affected, the translation inhibitor cycloheximide was utilized. Experiments in IEC-6 cells revealed that the half-life of the Atp7a protein was ∼41 h and, furthermore, that intracellular copper accumulation increased steady-state Atp7a protein levels. This investigation thus reveals a novel mechanism of Atp7a regulation in which copper stabilizes the protein, possibly complementing Hif2α-mediated transcriptional induction during iron deficiency.

scholarly journals Copper Transport and Disease: What Can We Learn from Organoids?

2019 ◽  
Vol 39 (1) ◽  
pp. 75-94 ◽  
Author(s):  
Hannah Pierson ◽  
Haojun Yang ◽  
Svetlana Lutsenko
Keyword(s):  
Three Dimensional ◽  
Transport Processes ◽  
Intestinal Epithelial ◽  
Metabolic Coupling ◽  
Significant Research ◽  
Cu Homeostasis ◽  
Vitro Model ◽  
Cu Uptake ◽  
Fat Processing

Many metals have biological functions and play important roles in human health. Copper (Cu) is an essential metal that supports normal cellular physiology. Significant research efforts have focused on identifying the molecules and pathways involved in dietary Cu uptake in the digestive tract. The lack of an adequate in vitro model for assessing Cu transport processes in the gut has led to contradictory data and gaps in our understanding of the mechanisms involved in dietary Cu acquisition. The recent development of organoid technology has provided a tractable model system for assessing the detailed mechanistic processes involved in Cu utilization and transport in the context of nutrition. Enteroid (intestinal epithelial organoid)-based studies have identified new links between intestinal Cu metabolism and dietary fat processing. Evidence for a metabolic coupling between the dietary uptake of Cu and uptake of fat (which were previously thought to be independent) is a new and exciting finding that highlights the utility of these three-dimensional primary culture systems. This review has three goals: ( a) to critically discuss the roles of key Cu transport enzymes in dietary Cu uptake; ( b) to assess the use, utility, and limitations of organoid technology in research into nutritional Cu transport and Cu-based diseases; and ( c) to highlight emerging connections between nutritional Cu homeostasis and fat metabolism.

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia

2009 ◽  
Vol 296 (6) ◽  
pp. G1332-G1343 ◽  
Author(s):  
Annabelle Cesaro ◽  
Abakar Abakar-Mahamat ◽  
Patrick Brest ◽  
Sandra Lassalle ◽  
Eric Selva ◽  
...  
Keyword(s):  
High Activity ◽  
Early Phase ◽  
Active Form ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Intestinal Epithelia ◽  
Tnf Α ◽  
Vitro Model

The acute phase of Crohn's disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-α. Conversion of inactive TNF-α into an active form requires the cleavage of a transmembrane TNF-α precursor by the TNF-α-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-α induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H2O2, or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.

scholarly journals Heat-Labile Enterotoxin Promotes Escherichia coli Adherence to Intestinal Epithelial Cells

2008 ◽  
Vol 191 (1) ◽  
pp. 178-186 ◽  
Author(s):  
Amber M. Johnson ◽  
Radhey S. Kaushik ◽  
David H. Francis ◽  
James M. Fleckenstein ◽  
Philip R. Hardwidge
Keyword(s):  
Escherichia Coli ◽  
Surface Expression ◽  
Host Cells ◽  
Intestinal Epithelial ◽  
Host Interactions ◽  
Heat Labile ◽  
Vitro Model ◽  
Bacterial Sensing

ABSTRACT Given recent evidence suggesting that the heat-labile enterotoxin (LT) provides a colonization advantage for enterotoxigenic Escherichia coli (ETEC) in vivo, we hypothesized that LT preconditions the host intestinal epithelium for ETEC adherence. To test this hypothesis, we used an in vitro model of ETEC adherence to examine the role of LT in promoting bacterium-host interactions. We present data demonstrating that elaboration of LT promotes a significant increase in E. coli adherence. This phenotype is primarily dependent on the inherent ADP-ribosylation activity of this toxin, with a secondary role observed for the receptor-binding LT-B subunit. Rp-3′,5′-cyclic AMP (cAMP), an inhibitor of protein kinase A, was sufficient to abrogate LT's ability to promote subsequent bacterial adherence. Increased adherence was not due to changes in the surface expression of the host receptor for the K88ac adhesin. Evidence is also presented for a role for bacterial sensing of host-derived cAMP in promoting adherence to host cells.

Villus contraction aids repair of intestinal epithelium after injury

1989 ◽  
Vol 257 (2) ◽  
pp. G274-G283 ◽  
Author(s):  
R. Moore ◽  
S. Carlson ◽  
J. L. Madara
Keyword(s):  
In Vitro Model ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Villus Height ◽  
Epithelial Barrier Function ◽  
Epithelial Repair ◽  
Vitro Model ◽  
Energy Dependent ◽  
Cytoplasmic Processes

A highly reproducible in vitro model of intestinal epithelial injury in guinea pig ileum was used to study the structural and functional events that accompany rapid epithelial repair. This model is characterized by denudation of the villus tip followed by rapid restitution of the epithelial barrier. Using standard electrophysiological and quantitative morphometric techniques, we found that immediately after injury the number of cells lost exceeded the number of empty cell positions on the denuded basement membrane by 100%. Concurrently, cytoplasmic processes of subepithelial myofibroblasts contained condensations of microfilaments that were not apparent in controls. Additionally, villus height was diminished immediately after injury, and progressively decreased during the restitution period. In tissues that were depleted of ATP using the uncoupler dinitrophenol and in tissues functionally denervated by tetrodotoxin, villus shortening after injury was significantly reduced. Denervation also retarded functionally and structurally defined reestablishment of epithelial barrier function. These data suggest that intestinal epithelial repair is aided by energy-dependent, neurally mediated villus contraction. We speculate that the subepithelial network of myofibroblasts is responsible for this process, which effectively minimizes the denuded surface area to be reepithelialized.

Effect of amyloid curli fibrils and curli CsgA monomers from Escherichia coli on in vitro model of intestinal epithelial barrier stimulated with cytokines

2019 ◽  
Vol 309 (5) ◽  
pp. 274-282
Author(s):  
Beata Sobieszczańska ◽  
Barbara Pawłowska ◽  
Anna Duda-Madej ◽  
Krzysztof Pawlik ◽  
Jerzy Wiśniewski ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
In Vitro Model ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Vitro Model
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