© Borgis - Postępy Nauk Medycznych 11/2011, s. 924-928
*Urszula Piotrowska1, Grażyna Adler1, Urszula Mackiewicz2
TSH signal transduction in thyroid cells of Nthy-ori 3-1 line**
Przekazywanie sygnału TSH w komórkach tarczycy linii Nthy-ori 3-1
1Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education, Warsaw
Head of Department: prof. dr hab. Barbara Czarnocka
2Department of Clinical Physiology, Medical Center of Postgraduate Education, Warsaw
Head of Department: prof. dr hab. Andrzej Beręsewicz
W poniższej pracy badano białka błonowe oraz wewnątrzkomórkowe szlaki sygnalizacyjne uruchamiane pod wpływem TSH w komórkach tarczycy linii Nthy-ori 3-1. Białka w solubilizowanych błonach komórkowych charakteryzowano metodą immunoblotingu z użyciem przeciwciał przeciwko receptorowi TSH i podjednostkom α i β regulatorowego białka G. Sygnalizację wewnątrzkomórkową uruchamianą przez TSH badano przez pomiar aktywności cyklazy adenylanowej i pomiar stężenia wewnątrzkomórkowego Ca2+. Wykazano występowanie w błonach komórek linii Nthy-ori 3-1 receptora TSH oraz wszystkich czterech podstawowych klas białka G: Gs, Gq, Gi i G12/13 jak również występowanie podjednostek Gβ. Stwierdzono, że egzogenny TSH aktywuje procesy zachodzące z udziałem wykazanych białek, a więc stymuluje aktywność cyklazy adenylanowej i wzrost wewnątrzkomórkowego stężenia jonów Ca2+. Poziom tej stymulacji w komórkach Nthy-ori 3-1 jest jednak niższy niż w komórkach tarczycy z pierwotnej hodowli oraz transferowanych komórkach CHO prezentujących receptor TSH.
The paper characterizes proteins and intracellular signaling activating under TSH stimulation in thyroid cells of Nthy-ori 3-1 line. Solubilized cell membrane proteins from Nthy-ori 3-1 cells were characterized by immunoblotting with antibodies against TSH receptor and G-protein α and β subunits. Intracellular G-protein-dependent signal transmission was investigated by assaying adenylate cyclase activity and intracellular Ca2+ concentration under TSH stimulation. TSH receptor and all four classes of G protein: Gs, Gq, Gi, and G12/13 and the G protein subunits β were present in membrane fraction of Nthy-ori 3-1 cells. In accordance with the presence of these proteins known to participate in signal transduction in thyroid cells, TSH treatment led to adenylate cyclase stimulation and intracellular Ca2+ rise. The results show, however, that the response of Nthy-ori 3-1 thyroid cells to TSH stimulation as adenylate cyclase activation and intracellular Ca2+ mobilization is weaker than that of thyroid cells in primary culture or transfected CHO cells expressing TSH receptor.
In vitro models that preserve a functional characteristics of the normal thyroid gland have been a challenging objective of recent experimental thyroidology. The earliest experimental thyroid models were based on organ culture or tissue slice preparations or, alternatively, short-term cell suspensions (1).The first permanently growing normal cell line named FRTL originated from rat thyroid (2). Many authors have studied human thyroid cell growth and expression of differentiated functions in primary cultures and thyroid cancer cell lines, but for a long time, normal human cell lines were unavailable. The cell line named Nthy-ori 3-1 was derived from normal thyroid follicular epithelium of an human adult (3). The cells were transfected with a plasmid encoding the SV40 large T gene. The resultant immortalized cell line has retained an epithelial morphology. It was active in the iodide trapping assay, but this activity was much lower compared with that of human thyroid follicular cells after extended passage. Initial research on newly established thyroid cell lines focuses on thyroglobulin detection, quantification, and hormonal regulation of its production, because this protein is the marker of thyroid tissue unequivocally defining the thyroid character of the cells. Assay of thyroglobulin production by Nthy-ori 3-1 cell line has confirmed the specific function of this line. Its thyroglobulin production, however, was about 10-fold lower than that of primary normal thyroid cell culture (3). The transfected human thyroid follicular cell line has been an attractive model of tumorigenesis (4, 5). In other studies this cell line has been used as a control. For example, RNA from Nthy-ori 3-1 cells stably transfected with a plasmid carrying the Pax8/PPARG fusion gene was used as a positive control in a clinical assay for the detection of such rearrangements in patients (6).
Material and Methods
Nthy-ori 3-1, normal human differentiated thyroid cells (European Collection of Human Cell Cultures), were grown in 5% CO2 at 37°C in RPMI 1640 AQmedia with 10% FBS and 1% penicillin – streptomycin solution (all medium components from Sigma-Aldrich). The medium was changed every 3 days.
TSH receptor and G protein estimation
The cells were detached with trypsin/EDTA (GIBCO BRL), or with non-enzymatic Cell Dissociation Solution (Sigma), washed and frozen. The frozen sample of 1.2x108 cells were suspended in 3 ml of homogenization buffer, 20 mM Tris pH 7.4, 50 mM NaCl, 10 mM EDTA, 10 mM EGTA and protease inhibitor cocktail (Roche) and homogenized in glass/teflon homogenizer. The homogenates were centrifuged for 30 min at 800 x g. The supernatants were further centrifuged for 30 min at 25000 x g. After washing, the membrane pellets were resuspended in 90 μl of the above buffer with 1.5% Triton X 100, stirred for 3 h on ice, centrifuged for 1 h at 60 000 x g and supernatant adjusted to 100 μl. Proteins (20 μg corresponding to 1x106 cells per well) were separated in nonreducing or reducing conditions by electrophoresis on 10% polyacrylamide gel 0.1% SDS in a Mini Protean apparatus (Bio Rad, Richmond CA). Dual color protein standards (Bio-Rad) were included in each gel. After electrophoresis proteins were blotted onto nitrocellulose membranes (Whatman, Dassel, Germany), blots were rocked for 1 h with 5% defatted milk in PBS and incubated overnight in the cold room with a antibody suitably diluted in PBS, 0.2% BSA, 0.1% Tween20. The following antibodies were used for specific protein recognition: anti-TSH receptor subunit A (A9, Advanced Targeting System, dilution 1:1500), TSH receptor subunit B (peptide 398-415, dilution 1:2000), anti-Gαs (Abcam, dilution 1:2000), anti-Gαq/11 (Santa Cruz, dilution 1:100), anti-Gαi (Santa Cruz, dilution 1:100), anti-Gα13 (Santa Cruz, dilution 1:100) and anti-Gβ (Santa Cruz, dilution 1:500). The blots were then rinsed and incubated for 2 h at room temperature with the appropriate horsereadish peroxidase-conjugated second antibodies diluted 1:4000 (Dako). The signal was developed with Super Signal West-Pico chemiluminescent substrate (Pierce) and exposed against Kodak X-ray film.
Adenylate cyclase assay
For cAMP measurements 1 x 105 cells per well were seeded into 12-well plate and cultured overnight. Before the assay the medium was aspirated and 0.5 ml of fresh medium without atibiotic or FBS, containing 0.5 mM 3-isobutyl-1-methyl-xantine (IBMX) (Sigma-Aldrich) and the indicated in figure 2 concentration of bovine TSH (Sigma-Aldrich) was added. Control samples were without TSH. Samples were incubated for 0, 30 or 120 minutes at room temperature, then the medium was aspirated and the reaction was stopped with 250 μl 0.1M HCl. Concentration of cellular cAMP in non-stimulated and TSH stimulated samples was estimated with a cAMP enzyme immunoassay kit (Sigma Aldrich) according to the supplier’s instruction.
Measurements of intracellular Ca2+ concentration in single cells
The cells grown on the glass coverslips in flexi Perm (Greiner Bio-One GmbH) were rapidly washed with Tyrode solution containing (in mM): 144 NaCl, 5 KCl, 1.5 CaCl2 1 MgCl2, 0.43 NaH2PO4, 10 Hepes, 11 glucose, pH 7.4. For estimation of changes of intracellular Ca2+ concentration cells were incubated with 2.5 μM Indo-1 acetoxymethyl ester for 15 min at 37°C and after incubation rinsed in Tyrode solution. Then cells on the coverlips were placed on superfusion chamber mounted on the stage of inverted microscope (Nicon) equipped for epifluorescence and perfused with Tyrode solution at 37°C or 24°C. Changes in intracellular Ca2+ concentration were traced by monitoring the ratio of 405 nm to 495 nm Indo-1 fluorescence obtained from the output of Dual Channel Ratio Fluorometer (Biomedical Instrumentation Group, University of Pennsylvania). Once a stable baseline of fluorescence was obtained, the stimulator, TSH (Sigma-Aldrich) or ATP (Boehringer) were added to 10 mU/ml and 100 μM, respectively, from 100-fold concentrated stock solution. In some experiments two minutes before TSH application the purinergic receptor agonist N-(L-2-phenylisopropyl)-adenosine (PIA) (Sigma-Aldrich) was added.
Membrane protein characterization
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