Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD

1995 ◽  
Vol 269 (2) ◽  
pp. C487-C495 ◽  
Author(s):  
J. Du ◽  
P. D. Wilson
Keyword(s):  
Epithelial Cell ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Scatchard Analysis ◽  
Egf Receptors ◽  
Renal Epithelial Cell ◽  
Western Blots ◽  
High Affinity ◽  
Cyst Fluids

The underlying mechanism of the hyperproliferative response of human autosomal dominant polycystic kidney disease (ADPKD) epithelia was studied. Epidermal growth factor (EGF) protein is highly expressed in ADPKD cyst epithelia in vivo, and primary cultures are hyperesponsive to mitogenic stimulation by EGF in vitro. Doses of > 1 ng/ml EGF were highly mitogenic to ADPKD epithelia. 3H-labeled thymidine proliferation assays showed that cyst fluids and ADPKD epithelial cell-conditioned media also stimulated renal epithelial cell proliferation and contained EGF immunoreactivity (6, 30, and 37 kDa) as detected by Western blots. Radioimmunoassays detected mean levels of 2.87 and 1.4 ng/ml EGF in cyst fluids from early (proliferative) and end-stage ADPKD cysts, respectively. Scatchard analysis of 125I-labeled EGF binding to apical and basolateral membrane showed high-affinity binding to basolateral membranes of normal and ADPKD kidneys but additional unique high-affinity receptor binding to apical membranes of ADPKD but not normal kidneys. Cross-linking analysis and antiphosphotyrosine Western analysis demonstrated functionally active apical EGF receptors at 150-170 kDa. These results suggest mediation of cyst expansion via an autocrine loop involving EGF synthesis and processing by cyst epithelial cells, apical secretion into cyst lumens, and subsequent binding to and phosphorylation of apical membrane EGF receptors. These findings are consistent with a membrane protein polarization defect in ADPKD cyst epithelia.

Axonal targeting of agrin in cultured rat dorsal horn neurons

1996 ◽  
Vol 109 (13) ◽  
pp. 2959-2966
Author(s):  
G. Escher ◽  
C. Bechade ◽  
S. Levi ◽  
A. Triller
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Neuromuscular Junction ◽  
Dorsal Horn ◽  
Nicotinic Acetylcholine Receptors ◽  
Primary Cultures ◽  
Western Blots ◽  
Dorsal Horn Neurons ◽  
The Central Nervous System

Agrin, a synaptic basal lamina protein synthesized by motoneurons is involved in the aggregation of nicotinic acetylcholine receptors (nAchRs) at the neuromuscular junction. Agrin transcripts are broadly expressed in the central nervous system (CNS) including non-cholinergic regions. This wide distribution of agrin mRNAs raises the question of its function in these areas. To approach this question, we analysed the expression and cellular distribution of agrin in primary cultures of rat embryonic dorsal horn neurons. Polymerase chain reaction analysis demonstrated that the four agrin isoform (B0, B8, B11, B19) mRNAs are expressed as early as 4 days in vitro, before the formation of functional synaptic contacts. Western blots also showed that agrin-like proteins are secreted in conditioned medium from 7 days cultures. We analysed the subcellular distribution of agrin by double immunolabeling and fluorescence microscopy. We found that agrin is synthesized by almost all neurons and was present in the somata and in the axons but not in dendrites within the sensitivity of the detection. This intra-axonal localisation of agrin could only be seen after permeabilization. Furthermore, agrin immunoreactive axons were found adjacent to gephyrin, the postsynaptic glycine receptor-associated protein. Altogether, our results suggest that, as established at the neuromuscular junction, agrin may be involved in pre- to postsynaptic interactions in the central nervous system.

Role of IFN-γ and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury

2004 ◽  
Vol 286 (1) ◽  
pp. L4-L14 ◽  
Author(s):  
Olivier Lesur ◽  
Marcel Brisebois ◽  
Alexandre Thibodeau ◽  
Frédéric Chagnon ◽  
Denis Lane ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Primary Cultures ◽  
Fold Increase ◽  
Protective Role ◽  
Scatchard Analysis ◽  
Membrane Expression ◽  
Extracellular Signal ◽  
Ifn Γ

In the present study, IFN-γ exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common γ-chain of the IL-2 receptor (∼2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-γ exposure: 1) IFN-γ priming promoted a fivefold increase of IL-2-driven TIIP locomotion ( P < 0.05 vs. control at 100 U/ml) and 2) IFN-γ coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by ∼70% (TdT-mediated dUTP nick end labeling/4′,6′-diamidino-2-phenylindole assay) as well as in vivo by ∼90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 μM) significantly reversed bleomycin-induced TIIP apoptosis ( P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-γ and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.

Estrogen-dependent expression of the cystic fibrosis transmembrane regulator gene in a novel uterine epithelial cell line

1994 ◽  
Vol 107 (9) ◽  
pp. 2439-2448
Author(s):  
L. Rochwerger ◽  
S. Dho ◽  
L. Parker ◽  
J.K. Foskett ◽  
M. Buchwald
Keyword(s):  
Epithelial Cells ◽  
Cell Line ◽  
Steroid Hormones ◽  
Primary Cultures ◽  
T Antigen ◽  
Epithelial Cell Line ◽  
Normal Female ◽  
Western Blots ◽  
Uterine Epithelial Cell

We have demonstrated previously the modulation of CFTR expression by estrogen in vivo in the rat uterine epithelium. The purpose of this study was to establish a suitable in vitro system to investigate the regulation of CFTR by steroid hormones. Primary cultures of rat uterine epithelial cells, which showed high levels of CFTR expression in vitro, were infected with an adeno/SV40 virus. One clone, UIT 1.16, which retained the morphology of the primary epithelial cells yet proliferated beyond the life span of the primary culture, was isolated and characterized. Successful immortalization of UIT 1.16 cells was verified by the presence of a band corresponding to the SV40 large T-antigen in western blots, as well as by their ability to proliferate continuously. Transmission electron microscopy studies revealed that these cells maintained the characteristics of a polarized epithelium with well-established membrane domains and specialized intercellular junctions. A high transepithelial electrical resistance was also observed when cells were assayed in modified Ussing chambers. When the basolateral cellular membrane of cells grown in vitrogen-coated filters was permeabilized with nystatin, a forskolin-stimulated Cl- permeability was observed in the apical membrane, similar to that present in other CFTR-expressing epithelial cells. UIT 1.16 cells showed high levels of CFTR expression on northern blots. The expression of CFTR was dependent on the presence of estrogen in the culture medium, since almost undetectable levels of CFTR mRNA were observed when the cells were cultured in medium containing serum depleted of steroid hormones. However, addition of estrogen to this medium prevented the disappearance of CFTR mRNA, confirming estrogen-regulated expression of CFTR in the UIT 1.16 cell line. The newly developed UIT 1.16 cell line provides a valuable model to analyze the regulation of CFTR expression by steroid hormones. Moreover, the cell line could also be used to investigate the role of CFTR in the uterus during the normal female cycle as well as for the study of other uterine epithelial functions and the agents that regulate them.

scholarly journals A novel renal epithelial cell in vitro assay to assessCandida albicansvirulence

Virulence ◽  
2013 ◽  
Vol 5 (2) ◽  
pp. 286-296 ◽  
Author(s):  
Edina K Szabo ◽  
Donna M MacCallum
Keyword(s):  
Epithelial Cell ◽  
In Vitro Assay ◽  
Renal Epithelial Cell ◽  
Vitro Assay

Epidermal growth factor receptor status in four carcinoma of the cervix cell lines

1991 ◽  
Vol 1 (4) ◽  
pp. 185-192 ◽  
Author(s):  
K. S. Tonkin ◽  
M. Berger ◽  
M. Ormerod
Keyword(s):  
Cell Lines ◽  
Egf Receptor ◽  
Receptor Gene ◽  
Flow Cytometric Analysis ◽  
Staining Intensity ◽  
Immunocytochemical Staining ◽  
Scatchard Analysis ◽  
Cervix Carcinoma ◽  
Egf Receptors ◽  
High Affinity

We have evaluated aspects of the EGF receptor content of four human cell lines derived from patients with previously untreated carcinoma of the cervix. Scatchard analysis revealed that three of the lines possessed approximately 2×105 low-affinity and 2×104 high-affinity receptors, whereas the fourth line had approximately 9×104 low-affinity receptors and 9×103 high-affinity receptors. Immunocytochemical staining using the monoclonal antibody EGFR1 showed wide intra- and inter-line variation in staining intensity. Flow cytometric analysis of EGFR1 demonstrated a fivefold difference in staining intensity between lines. Thirteen cloned derivatives of one of the lines exhibited a 200% variation in EGFR1 staining intensity. There were no differences in radiosensitivity in four of the cloned lines with different EGF receptor levels. Southern blotting analysis did not reveal any rearrangement or amplication of the EGF receptor gene. These three different methods for determining receptor content produced variations in the ranking of receptor number across the four cell lines. These studies with cervix carcinoma cell lines demonstrate the presence of varied levels of EGF receptors according to the methodology used. This may reflect differences in biological characteristics of the cell lines evaluated.

scholarly journals Acceptor sites for the oestrogen receptor in hen oviduct chromatin

1983 ◽  
Vol 210 (3) ◽  
pp. 905-912 ◽  
Author(s):  
T S Ruh ◽  
T C Spelsberg
Keyword(s):  
Receptor Binding ◽  
Oestrogen Receptor ◽  
Guanidine Hydrochloride ◽  
Scatchard Analysis ◽  
Oestrogen Receptors ◽  
Receptor Complexes ◽  
High Affinity ◽  
Acceptor Activity ◽  
Specificity Of Binding

Partially purified hen oviduct oestrogen receptors, charged with [3H]oestradiol, were shown to specifically bind in vitro to purified hen oviduct chromatin. Maximal binding occurred within 60min at 0 degrees C in a Tris buffer containing 0.1 M-KCl and 0.5 mM-phenylmethanesulphonyl fluoride. The binding of the [3H]oestradiol-receptor complexes to intact purified chromatin was saturable, whereas the receptor binding to hen DNA remained linear. Saturation was further demonstrated by the minimal acceptor binding of receptor charged with [3H]oestradiol plus 200-fold oestradiol compared with [3H]oestradiol receptors at equal [3H]oestradiol concentrations. Scatchard analysis of [3H]oestradiol-receptor binding to chromatin above DNA levels gave indications of high-affinity binding with a low capacity. Further, the nuclear binding was tissue-specific since the binding to hen spleen chromatin was negligible. To further uncover the specific acceptor sites, proteins were removed from hen oviduct chromatin by increasing concentrations of guanidine hydrochloride (1-7M). Those residual fractions extracted with 3-7 M-guanidine hydrochloride had the highest acceptor activity (above DNA levels) with the peak activity uncovered by 5 M-guanidine hydrochloride. To further characterize the oestrogen-receptor acceptor sites, oviduct chromatin was bound to hydroxyapatite in the presence of 3 M-NaCl and then protein fractions were extracted sequentially with 1-7 M-guanidine hydrochloride. Each fraction was then reconstituted to pure hen DNA by reverse gradient dialysis. [3H]Oestradiol receptors were found to bind to the greatest degree to the fraction reconstituted from the 5 M-guanidine hydrochloride protein extract. Reconstituted nucleoacidic proteins (NAP) from combined 4-7 M-guanidine hydrochloride protein extracts showed saturable binding by [3H]-oestradiol receptors, whereas binding to hen DNA did not saturate. The high affinity, low capacity, and specificity of binding of oestrogen receptors to NAP was similar to that found in intact chromatin. Thus, chromatin acceptor proteins for the oestrogen receptor have been partially isolated and characterized in the hen oviduct and display properties similar to that reported for the acceptor proteins of the progesterone receptor.

scholarly journals In Vitro Cell Culture Models of Hyperoxaluric States: Calcium Oxalate and Renal Epithelial Cell Interactions

Crystals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 735
Author(s):  
Ana Petrović ◽  
Tomislav Kizivat ◽  
Ines Bilić Ćurčić ◽  
Robert Smolić ◽  
Martina Smolić
Keyword(s):  
Epithelial Cell ◽  
Calcium Oxalate ◽  
Stone Formation ◽  
In Vitro Models ◽  
High Recurrence Rate ◽  
Published Data ◽  
Calcium Oxalate Crystals ◽  
Renal Epithelial Cell ◽  
Oxalate Crystals

Urolithiasis is a multifactorial disease with a high incidence and high recurrence rate, characterized by formation of solid deposits in the urinary tract. The most common type of these stones are calcium oxalate stones. Calcium oxalate crystals can, in hyperoxaluric states, interact with renal epithelial cells, causing injury to the renal epithelia. Pathogenesis of urolithiasis is widely investigated, but underlying mechanisms are still not completely clarified. In vitro models offer insight into molecular processes which lead to renal stone formation and are significant for evaluation of prophylactic and therapeutic management of patients with urolithiasis. In this review, we summarize recently published data from in vitro studies investigating interactions of calcium oxalate crystals with renal epithelial cell lines, anti-urolithiatic mechanisms, and the results from studies exploring possible therapeutic and prophylactic options for calcium oxalate urolithiasis in cell cultures.

scholarly journals Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Michelle Reed ◽  
Anny-Claude Luissint ◽  
Veronica Azcutia ◽  
Shuling Fan ◽  
Monique N. O’Leary ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Wound Repair ◽  
Inflammatory Responses ◽  
Critical Role ◽  
Primary Cultures ◽  
Intestinal Barrier ◽  
Mucosal Repair

Abstract CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial β1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-β1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.

scholarly journals Primary Cultures of Female Swine Genital Epithelial Cells In Vitro: a New Approach for the Study of Hormonal Modulation of Chlamydia Infection

2003 ◽  
Vol 71 (8) ◽  
pp. 4700-4710 ◽  
Author(s):  
Natalia V. Guseva ◽  
Stephen T. Knight ◽  
Judy D. Whittimore ◽  
Priscilla B. Wyrick
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Chlamydial Infection ◽  
Primary Cultures ◽  
Transmitted Disease ◽  
Reproductive Hormones ◽  
Chlamydia Infection ◽  
Cell Physiology ◽  
Hormonal Modulation

ABSTRACT Previous studies have demonstrated that female reproductive hormones influence chlamydial infection both in vivo and in vitro. Due to the reduced availability of human genital tissues for research purposes, an alternative hormone-responsive model system was sought to study chlamydial pathogenesis. Mature female swine eliminated from breeding programs were selected as the animals of choice because of the similarity of a sexually transmitted disease syndrome and sequelae in swine to a disease syndrome and sequelae found in humans, because of the near identity of a natural infectious chlamydial isolate from swine to Chlamydia trachomatis serovar D from humans, and because a pig's epithelial cell physiology and the mean length of its estrous cycle are similar to those in humans. Epithelial cells from the cervix, uterus, and horns of the uterus were isolated, cultivated in vitro in Dulbecco's minimum essential medium-Hanks' F-12 (DMEM-F-12) medium with and without exogenous hormone supplementation, and analyzed for Chlamydia suis S-45 infectivity. The distribution of chlamydial inclusions in swine epithelial cells was uneven and was influenced by the genital tract site and hormone status. This study confirmed that, like primary human endometrial epithelial cells, estrogen-dominant swine epithelial cells are more susceptible to chlamydial infection than are progesterone-dominant cells. Further, the more differentiated luminal epithelial cells were more susceptible to infection than were glandular epithelial cells. Interestingly, chlamydial growth in mature luminal epithelia was morphologically more active than in glandular epithelia, where persistent chlamydial forms predominated. Attempts to reprogram epithelial cell physiology and thereby susceptibility to chlamydial infection by reverse-stage, exogenous hormonal supplementation were unsuccessful. Freshly isolated primary pig epithelial cells frozen at −80°C in DMEM-F-12 medium with 10% dimethyl sulfoxide for several weeks can, after thawing, reform characteristic polarized monolayers in 3 to 5 days. Thus, primary swine genital epithelia cultured ex vivo appear to be an excellent cell model for dissecting the hormonal modulation of several aspects of chlamydial pathogenesis and infection.

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