scholarly journals Characterization of apical and basal thiol-disulfide redox regulation in human colonic epithelial cells

2010 ◽  
Vol 299 (2) ◽  
pp. G523-G530 ◽  
Author(s):  
Yanci O. Mannery ◽  
Thomas R. Ziegler ◽  
Li Hao ◽  
Yvonne Shyntum ◽  
Dean P. Jones
Keyword(s):  
Redox Regulation ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Disulfonic Acid ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Synthesis Inhibitor ◽  
Basal Surface ◽  
Permeable Membrane

Control of extracellular thiol-disulfide redox potential (Eh) is necessary to protect cell surface proteins from external oxidative and reductive stresses. Previous studies show that human colonic epithelial Caco-2 cells, which grow in cell culture with the apical surface exposed to the medium, regulate extracellular cysteine/cystine Eh to physiological values (approximately −80 mV) observed in vivo. The present study tested whether extracellular Eh regulation occurs on the basal surface of Caco-2 cells and investigated relevant mechanisms. Experiments were performed with confluent, differentiated cells grown on a permeable membrane surface. Cells were exposed to an oxidizing potential (0 mV) using a fixed cysteine-to-cystine ratio, and culture medium was sampled over time for change in Eh. Regulation of extracellular thiol-disulfide Eh on the basal domain was faster, and the extent of change at 24 h was greater than on the apical surface. Mechanistic studies showed that redox regulation on the basal surface was partially sodium dependent and inhibited by extracellular lysine, a competitive inhibitor of cystine transport by the y+L system and by quisqualic acid, an inhibitor of the xc− system. Studies using the thiol-reactive alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid and the glutathione synthesis inhibitor buthionine sulfoximine showed that extracellular redox regulation was not attributable to plasma membrane cysteine/cystine interconversion or intracellular glutathione, respectively. Thus the data show that redox regulation occurs at different rates on the apical and basal surfaces of the polarized Caco-2 epithelial cell line and that the y+L and xc− systems function in extracellular cysteine/cystine redox regulation on the basal surface.

Polarized distribution of oxalate transport systems in LLC-PK1 cells, a line of renal epithelial cells

1994 ◽  
Vol 266 (2) ◽  
pp. F266-F274 ◽  
Author(s):  
H. Koul ◽  
S. Ebisuno ◽  
L. Renzulli ◽  
M. Yanagawa ◽  
M. Menon ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Factors Affecting ◽  
Membrane Surfaces ◽  
Transport Inhibitors ◽  
Porcine Origin ◽  
Oxalate Transport

Although oxalate is a major component of kidney stones, the factors affecting renal oxalate handling are poorly understood. This uncertainty stems in part from complexities inherent to available preparations; thus the present studies examined oxalate handling in a simpler model system, LLC-PK1 cells, an epithelial cell line of porcine origin. Initial studies on monolayers in dishes demonstrated that these cells accumulate oxalate via a process or processes sensitive to the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Subsequent studies using LLC-PK1 monolayers on membrane filters examined the characteristics and distribution of these transporter(s). At the apical surface, DIDS-sensitive uptake was sensitive to [Cl-] but not [SO4(2-)] or [HCO3-] and was unaffected by alterations in pH or membrane potential. At the basolateral surface, oxalate uptake was [Cl-] insensitive but markedly affected by variation in pH, [SO4(2-)], or [HCO3-]. Uptake at the two membrane surfaces was also differentially affected by transport inhibitors and organic acids. Thus LLC-PK1 cells appear to express unique transporters at each membrane surface: oxalate/Cl- exchange at the apical surface and oxalate/SO4(2-) (or HCO3-) exchange at the basolateral surface.

scholarly journals Brush border protocadherin CDHR2 promotes the elongation and maximized packing of microvilli in vivo

2019 ◽  
Vol 30 (1) ◽  
pp. 108-118 ◽  
Author(s):  
Julia A. Pinette ◽  
Suli Mao ◽  
Bryan A. Millis ◽  
Evan S. Krystofiak ◽  
James J. Faust ◽  
...  
Keyword(s):  
Functional Capacity ◽  
Brush Border ◽  
Intestinal Tract ◽  
Membrane Surface ◽  
Careful Examination ◽  
Tissue Cell ◽  
Apical Surface ◽  
Membrane Surface Area ◽  
Solute Uptake

Transporting epithelial cells optimize their morphology for solute uptake by building an apical specialization: a dense array of microvilli that serves to increase membrane surface area. In the intestinal tract, individual cells build thousands of microvilli, which pack tightly to form the brush border. Recent studies implicate adhesion molecule CDHR2 in the regulation of microvillar packing via the formation of adhesion complexes between the tips of adjacent protrusions. To gain insight on how CDHR2 contributes to brush border morphogenesis and enterocyte function under native in vivo conditions, we generated mice lacking CDHR2 expression in the intestinal tract. Although CDHR2 knockout (KO) mice are viable, body weight trends lower and careful examination of tissue, cell, and brush border morphology revealed several perturbations that likely contribute to reduced functional capacity of KO intestine. In the absence of CDHR2, microvilli are significantly shorter, and exhibit disordered packing and a 30% decrease in packing density. These structural perturbations are linked to decreased levels of key solute processing and transporting factors in the brush border. Thus, CDHR2 functions to elongate microvilli and maximize their numbers on the apical surface, which together serve to increase the functional capacity of enterocyte.

Role of the scaffold protein RACK1 in apical expression of CFTR

2007 ◽  
Vol 293 (1) ◽  
pp. C294-C304 ◽  
Author(s):  
Michael Auerbach ◽  
Carole M. Liedtke
Keyword(s):  
Surface Proteins ◽  
Apical Plasma Membrane ◽  
Epithelial Cell Line ◽  
Spectrometric Analysis ◽  
Apical Surface ◽  
Airway Epithelial ◽  
Binding Assays ◽  
Direct Binding

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC)ε in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKCε to RACK1, a receptor for activated C kinase, and of RACK1 to human Na+/H+ exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKCε-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.

Adhesion of calcium oxalate monohydrate crystals to anionic sites on the surface of renal epithelial cells

1996 ◽  
Vol 270 (1) ◽  
pp. F192-F199 ◽  
Author(s):  
J. C. Lieske ◽  
R. Leonard ◽  
H. Swift ◽  
F. G. Toback
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Calcium Oxalate ◽  
Kidney Stones ◽  
Membrane Surface ◽  
Calcium Oxalate Monohydrate ◽  
Apical Surface ◽  
Renal Epithelial Cells ◽  
Renal Tubular Cells

Adhesion of microcrystals to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones. The role of membrane surface charge as a determinant of the interaction between renal epithelial cells (BSC-1 line) and the most common crystal in kidney stones, calcium oxalate monohydrate (COM), was studied in a tissue culture model system. Adhesion of COM crystals to cells was blocked by cationized ferritin. Other cations that bind to cells including cetylpyridinium chloride and polylysine, as well as cationic dyes such as Alcian blue, also inhibited adhesion of COM crystals, but not all polycations shared this effect. Specific lectins including Triticum vulgaris (wheat germ agglutinin) blocked crystal binding to the cells. Furthermore, treatment of cells with neuraminidase inhibited binding of crystals. Therefore, anionic cell surface sialic acid residues appear to function as COM crystal receptors that can be blocked by specific cations or lectins. In vivo, alterations in the structure, function, quantity, or availability of these anionic cell surface molecules could lead to crystal retention and formation of renal calculi.

cAMP-dependent exocytosis and vesicle traffic regulate CFTR and fluid transport in rat jejunum in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C429-C438 ◽  
Author(s):  
Nadia A. Ameen ◽  
Christopher Marino ◽  
Pedro J. I. Salas
Keyword(s):  
Fluid Transport ◽  
Fluid Secretion ◽  
Cell Types ◽  
Surface Proteins ◽  
Apical Surface ◽  
Rat Jejunum ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Vesicle Traffic

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is regulated by cAMP-dependent vesicle traffic and exocytosis to the apical membrane in some cell types, but this has not been demonstrated in the intestinal crypt. The distribution of CFTR, lactase (control), and fluid secretion were determined in rat jejunum after cAMP activation in the presence of nocodazole and primaquine to disrupt vesicle traffic. CFTR and lactase were localized by immunofluorescence, and surface proteins were detected by biotinylation of enterocytes. Immunoprecipitates from biotinylated and nonbiotinylated cells were analyzed by streptavidin detection and immunoblots. Immunolocalization confirmed a cAMP-dependent shift of CFTR but not lactase from a subapical compartment to the apical surface associated with fluid secretion that was reduced in the presence of primaquine and nocodazole. Analysis of immunoblots from immunoprecipitates after biotinylation revealed a 3.8 ± 1.7-fold ( P < 0.005) increase of surface-exposed CFTR after vasoactive intestinal peptide (VIP). These measurements provide independent corroboration supporting a role for vesicle traffic in regulating CFTR and cAMP-induced fluid transport in the intestine.

scholarly journals MT1-MMP mediates MUC1 shedding independent of TACE/ADAM17

2004 ◽  
Vol 382 (1) ◽  
pp. 363-373 ◽  
Author(s):  
Amantha THATHIAH ◽  
Daniel D. CARSON
Keyword(s):  
Tumour Progression ◽  
Tyrosine Phosphatase ◽  
Embryo Implantation ◽  
Critical Role ◽  
Matrix Metalloproteases ◽  
Epithelial Cell Line ◽  
Apical Surface ◽  
Uterine Epithelial Cell ◽  
Hes Cells

MUC1, a transmembrane mucin, plays a critical role in embryo implantation, protection of mucosal epithelia from microbial and enzymic attack and various aspects of tumour progression. In some species, a decrease in uterine epithelial MUC1 protein and mRNA expression accompanies embryo implantation. In other species, such as rabbits and humans, MUC1 appears to be locally removed at blastocyst attachment sites, suggesting the action of a protease. We previously demonstrated that MUC1 is proteolytically released from the surface of a human uterine epithelial cell line, HES, and identified TACE/ADAM17 (where TACE stands for tumour necrosis factor-α converting enzyme and ADAM for ADisintegrin And Metalloprotease-like) as a constitutive and PMA-stimulated MUC1 sheddase [Thathiah, Blobel and Carson (2003) J. Biol. Chem. 274, 3386–3394]. Further characterization of the proteolytic activity(ies) mediating MUC1 release indicates that MUC1 shedding is also accelerated by the tyrosine phosphatase inhibitor pervanadate. Pervanadate, but not PMA, stimulates MUC1 shedding in TACE-deficient cells, indicating activation of a metalloproteolytic activity(ies) distinct from TACE. Pervanadate-stimulated MUC1 release is inhibited by the TIMP-2 (tissue inhibitor of metalloprotease-2) and TIMP-3, but is unaffected by TIMP-1, consistent with the MT-MMPs (membrane-type matrix metalloproteases). Pervanadate stimulation of MUC1 shedding is absent from MUC1-transfected MT1-MMP-deficient fibroblasts, but is restored after MUC1 and MT1-MMP co-transfection. Furthermore, overexpression of MT1-MMP in HES cells enhances pervanadate-stimulated MUC1 release, and MT1-MMP co-localizes with MUC1 in vivo at the apical surface of receptive-phase human uterine epithelia. Taken together, these studies characterize a MUC1 sheddase activity in addition to TACE and identify MT1-MMP as a pervanadate-stimulated MUC1 sheddase.

scholarly journals The Na+/I− symporter mediates active iodide uptake in the intestine

2009 ◽  
Vol 296 (4) ◽  
pp. C654-C662 ◽  
Author(s):  
Juan Pablo Nicola ◽  
Cécile Basquin ◽  
Carla Portulano ◽  
Andrea Reyna-Neyra ◽  
Monika Paroder ◽  
...  
Keyword(s):  
Small Intestine ◽  
Thyroid Hormone ◽  
Epithelial Cell Line ◽  
Central Component ◽  
Apical Surface ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Thyroid Hormone Biosynthesis ◽  
Hormone Biosynthesis

Absorption of dietary iodide, presumably in the small intestine, is the first step in iodide (I−) utilization. From the bloodstream, I− is actively taken up via the Na+/I− symporter (NIS) in the thyroid for thyroid hormone biosynthesis and in such other tissues as lactating breast, which supplies I− to the newborn in the milk. The molecular basis for intestinal I− absorption is unknown. We sought to determine whether I− is actively accumulated by enterocytes and, if so, whether this process is mediated by NIS and regulated by I− itself. NIS expression was localized exclusively at the apical surface of rat and mouse enterocytes. In vivo intestine-to-blood transport of pertechnetate, a NIS substrate, was sensitive to the NIS inhibitor perchlorate. Brush border membrane vesicles accumulated I− in a sodium-dependent, perchlorate-sensitive manner with kinetic parameters similar to those of thyroid cells. NIS was expressed in intestinal epithelial cell line 6, and I− uptake in these cells was also kinetically similar to that in thyrocytes. I− downregulated NIS protein expression and its own NIS-mediated transport both in vitro and in vivo. We conclude that NIS is functionally expressed on the apical surface of enterocytes, where it mediates active I− accumulation. Therefore, NIS is a significant and possibly central component of the I− absorption system in the small intestine, a system of key importance for thyroid hormone biosynthesis and thus systemic intermediary metabolism.

scholarly journals Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 110
Author(s):  
Dina Aweida ◽  
Shenhav Cohen
Keyword(s):  
Protein Quality ◽  
Protein Structures ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Catalytic Core ◽  
Complex Protein ◽  
Ubiquitin Proteasome ◽  
Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

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