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The Expression of the Thyroid-stimulating Hormone (TSH) Receptor and the cAMP-dependent Protein Kinase RII β Regulatory Subunit Confers TSH-cAMP-dependent Growth to Mouse Fibroblasts

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m307501200

1083-351X

Antonio Porcellini, Samantha Messina, G. de Gregorio, Antonio Feliciello, Annalisa Carlucci, Maria Vittoria Barone, Antonietta Picascia, Antonio De Blasi, Enrico V. Avvedimento,

Animal Genetics and Reproduction

TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIβ) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIβ-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIβ-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments. TSH activates its specific receptor in thyroid cells and induces cAMP, a robust stimulator of thyroid cell proliferation. Conversely, cAMP is a potent inhibitor of growth in mouse fibroblasts. To dissect the signals mediating cAMP-dependent growth, we have expressed in mouse fibroblasts the human thyrotropin receptor (TSHR) or a constitutively active mutant, under the control of the tetracyclin promoter. Both TSHR and cAMP levels were modulated by tetracyclin. In the presence of serum, activation of TSHR by TSH induced growth arrest. In the absence of serum, cells expressing TSHR stimulated with TSH, replicated their DNA, but underwent apoptosis. Co-expression of cAMP-dependent protein kinase (PKA) regulatory subunit type II (RIIβ) inhibited apoptosis and stimulated the growth of cells only in the presence of TSH. Expression of RIIβ-PKA, in the absence of TSHR, induced apoptosis, which was reversed by cAMP. Growth, stimulated by TSHR-RIIβ-PKA in mouse fibroblasts, was also dependent on Rap1 activity, indicating cAMP-dependent growth in thyroid cells. As for the molecular mechanism underlying these effects, we found that in normal fibroblasts, TSH induced AKT and ERK1/2 only in cells expressing TSHR and RII. Similarly, activation of TSHR increased cAMP levels greatly, but was unable to stimulate CREB phosphorylation and transcription of cAMP-induced genes in the absence of RII. These data provide a simple explanation for the anti-proliferative and proliferative effects of cAMP in different cell types and indicate that RII-PKAII complements TSHR action by stably propagating robust cAMP signals in cell compartments. cAMP stimulates or inhibits the growth of many cell types (1Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (848) Google Scholar, 2Dumont J.E. Jauniaux J.C. Roger P.P. Trends Biochem. Sci. 1989; 14: 67-71Abstract Full Text PDF PubMed Scopus (451) Google Scholar, 3Heldin N.E. Paulsson Y. Forsberg K. Heldin C.H. Westermark B. J. Cell. Physiol. 1989; 138: 17-23Crossref PubMed Scopus (68) Google Scholar, 4Magnaldo I. Pouyssegur J. Paris S. FEBS Lett. 1989; 245: 65-69Crossref PubMed Scopus (64) Google Scholar) Thyroid cells are exquisitely dependent on TSH 1The abbreviations used are: TSH, thyroid-stimulating hormone; PKA, cAMP-dependent protein kinase; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunoadsorbent assay; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; FACS, fluorescent-activated cell sorting; ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.1The abbreviations used are: TSH, thyroid-stimulating hormone; PKA, cAMP-dependent protein kinase; CAT, chloramphenicol acetyltransferase; ELISA, enzyme-linked immunoadsorbent assay; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; FACS, fluorescent-activated cell sorting; ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. for growth and differentiation (5Vassart G. Dumont J.E. Endocr. Rev. 1992; 13: 596-611PubMed Google Scholar, 6Avvedimento V.E. Tramontano D. Ursini M.V. Monticelli A. Di Lauro R. Biochem. Biophys. Res. Commun. 1984; 122: 472-477Crossref PubMed Scopus (62) Google Scholar). cAMP mediates most TSH effects (7Weiss S.J. Philp N.J. Ambesi-Impiombato F.S. Grollman E.F. Endocrinology. 1984; 114: 1099-1107Crossref PubMed Scopus (184) Google Scholar). Conversely, cAMP arrests mouse 3T3 fibroblasts in G1 (8Chen J. Iyengar R. Science. 1994; 263: 1278-1281Crossref PubMed Scopus (107) Google Scholar). In both cases, the downstream effects of cAMP are mediated by PKA (9Taylor S.S. J. Biol. Chem. 1989; 264: 8443-8446Abstract Full Text PDF PubMed Google Scholar) and by cAMP-binding proteins, GEF, that stimulate GTP binding effectors, such as Rap1 (10Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1167) Google Scholar). Rap1 has been shown to mediate both positive and negative effects of cAMP on cell proliferation (11Schmitt J.M. Stork P.J. Mol. Cell. Biol. 2001; 21: 3671-3683Crossref PubMed Scopus (126) Google Scholar, 12Schmitt J.M. Stork P.J. Mol Cell. 2002; 9: 85-94Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Iacovelli L. Capobianco L. Salvatore L. Sallese M. D'Ancona G.M. De Blasi A. Mol Pharmacol. 2001; 60: 924-933Crossref PubMed Scopus (82) Google Scholar). Paradoxically, the same downstream targets appear to mediate negative or positive cAMP effects. The cdk2 inhibitor p27 has been shown to be induced by cAMP-PKA both in thyroid cells (14Feliciello A. Gallo A. Mele E. Porcellini A. Troncone G. Garbi C. Gottesman M.E. Avvedimento E.V. J. Biol. Chem. 2000; 275: 303-311Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and in other cell lines arrested in G1 by cAMP (15van Oirschot B.A. Stahl M. Lens S.M. Medema R.H. J. Biol. Chem. 2001; 276: 33854-33860Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 16Miskimins W.K. Wang G. Hawkinson M. Miskimins R. Mol. Cell. Biol. 2001; 21: 4960-4967Crossref PubMed Scopus (84) Google Scholar). To dissect the signals involved in cAMP stimulation of growth, we have expressed the TSH receptor (TSHR) in mouse fibroblasts under the control of an inducible promoter. Note that a previous study reported the permanent expression of the receptor in mouse fibroblasts, stably transfected with wild-type TSHR and constitutively active mutants. Under these conditions, a strong selection was applied to the cells, and the phenotypes of the clones were not easily interpretable in terms of cAMP-dependent growth (17Du Villard J.A. Wicker R. Crespo P. Russo D. Filetti S. Gutkind J.S. Sarasin A. Suarez H.G. Oncogene. 2000; 19: 4896-4905Crossref PubMed Scopus (16) Google Scholar). We report here that under conditional expression of TSHR, only the co-expression of RIIβ subunit of PKA and TSHR resulted in TSH-dependent growth. Both TSHR or RIIβ PKA expressed alone induced apoptosis or growth arrest. Their growth-stimulating activity was dependent on the ability not only to generate but to propagate cAMP signals to downstream nuclear substrates. Materials and Reagents—Unless otherwise specified, drugs and chemicals were obtained from Sigma, and cell culture supplies were purchased from standard suppliers, e.g. Falcon, Invitrogen, Hyclone. Cell Lines—Mouse fibroblasts NIH 3T3 were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mm glutamine (standard medium). The tTA-repressor-expressing clones were grown in standard medium containing puromycin (2.5 μg/ml), tetracyclin (1.0 μg/ml), and geneticin (G418) (200 μg/ml). The selection and tetracyclin were removed 48 h before starting the experimental procedures described below. Plasmids and Transfections—We obtained stable cell lines using the Tet-off repressor system consisting of the t-TA gene expression vector (a pUHD15.1-modified vector conferring puromycin resistance) and the CMV-tet-operator cloning vector pUHD10.3 (18Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4232) Google Scholar). Several NIH3T3 clones, expressing the t-TA gene, were isolated by Luc reporter activity under Tet-repressor control (NTC or control). The NTC clone was used to generate: 1) hTSHR-WT and 2) T632I-espressing cells (TSHR, T632I). TSHR constructs were obtained by subcloning the 2300-bp EcoRI fragment (19Porcellini A. Ciullo I. Pannain S. Fenzi G. Avvedimento E. Oncogene. 1995; 11: 1089-1093PubMed Google Scholar) into the pUHD10.3 vector. For each NTC line, we have generated permanent clones, which expressed the regulative subunit of the PKA type II β (RIIβ) by transfecting a plasmid encoding the human RIIβ cDNA (3.6-kb EcoRI fragment) under the control of the CMV promoter, carrying the geneticin resistance gene (G-418) (20Tortora G. Budillon A. Yokozaki H. Clair T. Pepe S. Merlo G. Rohlff C. Cho-Chung Y.S. Cell Growth Differ. 1994; 5: 753-759PubMed Google Scholar). For cAMP-dependent transcription assay each clone was transiently transfected in triplicte samples by the calcium phosphate procedure (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.30-16.62Google Scholar) with 5 μg of pRSV-LacZ and 5 μg of pCRE-CAT (22Harada H. Becknell B. Wilm M. Mann M. Huang L.J. Taylor S.S. Scott J.D. Korsmeyer S.J. Mol. Cell. 1999; 3: 413-422Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar). 24 h after transfection, cells were serum-starved for 48 h (0.5% fetal bovine serum). Control cells were stimulated with forskolin (25 μm) for 4 h. The cells were harvested and total protein extracted (see below). CAT enzymatic assay was performed as described (21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 16.30-16.62Google Scholar). Transfection efficiency was normalized for β-galactosidase assay. Total RNA Extraction and Northen Blot Analysis—Total RNA was extracted from 107 cells grown in a 150-mm dish by the guanidium isothiocyanate procedure; 20 μg of each RNA sample was electrophoresed on a 1% agarose gel containing formaldehyde and transferred to nylon membranes (Amersham Biosciences) using standard capillary techniques. The filter was then hybridized with specific radioactive probes overnight at 65 °C in Church Buffer (0.5 m Na2PO4, pH 7.2, 7% SDS, 1 mm EDTA). Radioactivity was revealed by autoradiography using preflashed films. The following probes were used: a 0.9-kb DNA fragment corresponding to hTSHR cDNA region coding for codons 58–381; and the fulllength rat glyceraldehyde-3-phosphate dehydrogenase cDNA. All probes were labeled with [32P]dGTP by nick translation. Quantitative analysis of the hTSHR mRNA amount was performed by densitometry using the NIH Image software for Apple Macintosh. cAMP Assays—For each experimental point 2 × 105 cells of each clone were seeded in a 6-well tissue culture tray. After 24 h the cells were serum-starved for 18 h; then serum-free medium with 0.5 mm IBMX and different TSH concentrations (0, 0.1, 1, and 10 mU/ml) were added for 40 min. Cells were lysed with cold ethanol containing 1% 1 m HCl and incubated overnight at +4 °C. The medium was collected, neutralized, lyophilized, and resuspended in 0.2 ml of 10 mm Tris-HCl pH 7.8, 1 mM EDTA and used for cAMP assay (Amersham Biosciences, cyclic AMP 3H assay system). Each experiment was repeated three times in triplicate samples. Growth Analysis—Proliferation was analyzed under different conditions: normal medium or low serum (0.5%) with and without TSH 10 mU/ml. For each experiment 104 cells were seeded in a 12-well tissue culture tray; cells were collected and counted after 24 h or 3, 6, 9, or 15 days. The MTT colorimetric assay for proliferation was performed in a 96-well flat-bottomed tissue culture tray for each time point: 2500 cells for each clone were seeded in eight replicas for each experimental point; 6 h after plating the standard medium was removed, and 100 μl of culture medium without serum were applied. The determinations were carried out every 24 h as follow: 0.01 ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) stock (5 mg/ml) was added to each well. After 4 h of incubation at 37 °C the medium was removed and 0.1 ml of isopropyl alcohol/0.04 n HCl was added. The absorbance was measured after 1 h on an ELISA plate reader. We have used a Dynatech MR580 reader with a test wavelength of 570 nm and a reference wavelength of 630 nm. [3H]thymidine incorporation assays were carried out as follows. 5000 cells were seeded in a 96-well flat-bottomed tissue culture tray. After 72 h of growth in the absence of tetracyclin serum was removed for 18 h (0.5% fetal bovine serum). The following day the low serum medium was replaced by standard medium, and [3H]thymidine (0.5 μCi) and TSH 10 mU/ml (as required) were added. Cells were collected 6, 12, and 18 h after stimulation. DNA was extracted with the Cell Harvester; radioactivity was determined by liquid scintillation. All experiments were repeated three times in triplicate. Cell cycle analysis was carried out by FACS. 106 cells were plated in 100-mm dishes; 8 h after seeding, cells were subject to different treatments. After 48 h, 2.5–3 × 106 cells were collected, washed, and resuspended in 1 ml of PBS. Ethanol fixation was carried out by adding 10 ml of ice-cold 70% ethanol. After 3 h, fixed cells were washed and stained for 30 min at room temperature in 0.1% Triton X-100, 0.2 mg/ml DNase-free RNaseA, 20 μg/ml propidium iodide. Fluorescence was determined by FACS and analyzed by Cell Fit Cell-Cycle Analysis Version 2. Protein Extraction and Western Blot Analysis—107 cells were lysed in TBS, 1% Triton X-100 lysis buffer (150 mm NaCl, 50 mm Tris-HCl, pH 8, 5 mm EDTA, 1 mm NaF, 1 mm Na4P2O7, 1.5 mm KH2PO4, 0.4 mm Na3VO4) and incubated on ice for 20 min. After centrifugation at 12,000 × g, the protein concentrations were determined using the BioRad protein assay reagent (Bio-Rad, Hercules, CA). 100 μg of protein were added to an equal volume of 2× sample buffer (125 mm Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, 2% mercaptoethanol), boiled for 5 min, separated on 7% SDS-PAGE, and electrophoretically transferred onto nitrocellulose membranes (Schleicher & Schuell, Germany). After transferring, Ponceau staining was used to confirm equal protein loading. The membranes were incubated for 1 h at room temperature with PBS containing 5% nonfat dry milk (Bio-Rad) and then incubated overnight at 4 °C in PBS/2.5% nonfat dry milk containing 1 μg/ml of specific antibody. The membranes were washed twice with PBS and reincubated for 1 h with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (Amersham Biosciences). Signals were detected using enhanced chemiluminescence according to the manufacturer's instruction (ECL detection system; Amersham Biosciences). Anti-rat PCREB antibodies (23Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (742) Google Scholar) were purchased from Upstate Biotechnology, Lake Placid, NY. Specific anti-RIIβ antibodies were generated by immunizing rabbits with a synthetic RIIβ peptide (peptide 31–57 from the AUG of the rat sequence), cross-linked to soybean trypsin inhibitor (24Paolillo M. Feliciello A. Porcellini A. Garbi C. Bifulco M. Schinelli S. Ventra C. Stabile E. Ricciardelli G. Schettini G. Avvedimento E.V. J. Biol. Chem. 1999; 274: 6546-6552Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Anti-Rap-1b was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. TUNEL Assay—104 cells were grown on slides (ordinary glass slides washed in ethanol and soaked in 0.01% poly-l-lysine). After treatment, slides were fixed in 4% paraformaldehyde/1× PBS, 10 min, at room temperature and washed one time in PBS + 50 mm glycine, 10 min at room temperature, and three times for 5 min in PBS. Cells were permeabilized with 0.5% Triton X-100, 1× PBS for 10 min, washed 3 × 5 min in PBS, and equilibrated with 200 μl of 1× TdT buffer + 1 mm cobalt chloride (under a 60 × 24 mm coverslip), for 5 min. The TUNEL reaction was carried out using the In Situ Cell Death Detection Kit (Roche Applied Science). Apoptotic rate was determined by FACS analysis on propidium iodide-stained cells. For FACS analysis, after 48 of tet starvation, 106 cells for each clone were plated in 100-mm dishes and starved from serum for 18 h. Terminal transferase reaction (Roche Applied Science) and staining with antiBRDU-FITC (BD Pharmingen) was performed on paraformaldheyde/ethanol-fixed cells, according to the manufacturer's instructions. After TUNEL reaction, cells were washed and stained for 30 min at room temperature in 0.1% Triton X-100, 0.2 mg/ml DNase-free RNaseA, 20 μg/ml propidium iodide. Fluorescence was determined by FACS and analyzed by Cell Quest software. Recombinant Adenovirus Expressing the Dominant-negative RAP-1b Variant—RAP-1b dominant-negative mutant plasmid was previously described (13Iacovelli L. Capobianco L. Salvatore L. Sallese M. D'Ancona G.M. De Blasi A. Mol Pharmacol. 2001; 60: 924-933Crossref PubMed Scopus (82) Google Scholar). The cDNA insert was subcloned into the multiple cloning site of the shuttle plasmid (pAd-CMV-TRK) by standard cloning procedures. The purified shuttle plasmid was digested with the restriction enzymes PmeI to obtain the "rescue" fragment. The fragment was then purified on agarose gel, and 2 μg of purified rescue fragment was used for homologous recombination. The adenoviral plasmid pAdEasy-1 (25He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3237) Google Scholar) was then mixed with the rescue fragment, and the DNA mixture was transformed into the BJ5183 bacterial strain and incubated overnight. Colonies were screened by digesting the DNA with BglII and performing a Southern blot to confirm the presence of the cDNA insert. The DNA with the proper orientation was transformed into the DH5α bacterial strain. The recombinant construct, purified using Qiagen Maxi preparation kit, was digested overnight with PacI and transfected into 293 cells using LipofectAMINE (Invitrogen). Adenoviral plaques were twice purified by infecting 293 cells in agar. Virus was purified by CsCl gradient centrifugation, dialyzed and titrated by plaque assay. Conditional Expression of Wild-type and Mutated TSH Receptor in Mouse Fibroblasts—TSH receptor was cloned under the control of the inducible tetracyclin promoter. In the configuration we have used, Tet-off (see "Experimental Procedures"), the tetracyclin repressor (tTA), in the presence of the tetracyclin, inhibits the transcription of the downstream gene. Removal of the drug from the medium results in the activation of transcription. We transfected NIH 3T3 fibroblasts with the tTA repressor, isolated several clones, and re-transfected them with the Tet-TSHReceptor (TSHR) construct. Several clones were isolated and screened for inhibition of expression of TSHR in the presence of tetracyclin (tet). Fig. 1 shows the levels of TSHR mRNA in two clones, transfected with the wild-type TSHR and the constitutively active mutant T632I, which contains a mutated residue in position 632 (isoleucine mutated to threonine). TSHR mRNA levels were critically dependent on the time and the concentration of tet added to the medium. Maximal expression was observed 12–24 h after removal of the drug and it remained constant for 15 days of continuous culture in the absence of the drug (Fig. 1). Down-regulation of TSHR mRNA by tet was maximal after 48–72 h of continuous drug exposure. To test if TSHR was functionally coupled to Gs, we determined the levels of intracellular cAMP. In cells, expressing the TSHR (i.e. in the absence of tet), basal cAMP levels were unchanged, while TSH induced a rapid (30Feliciello A. Giuliano P. Porcellini A. Garbi C. Obici S. Mele E. Angotti E. Grieco D. Amabile G. Cassano S. Li Y. Musti A.M. Rubin C.S. Gottersman M.E. Avvedimento V.E. J. Biol. Chem. 1996; 271: 25350-25359Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar min) accumulation of cAMP. We screened for clones displaying the same range of cAMP response found in thyroid cells (FRTL-5, see Ref. 26Porcellini A. Ruggiano G. Pannain S. Ciullo I. Amabile G. Fenzi G. Avvedimento E.V. Oncogene. 1997; 15: 781-789Crossref PubMed Scopus (20) Google Scholar). TSH induction of cAMP was abolished by tet treatment (Fig. 2, panel c). In cells transfected with the constitutively active receptor T632I, basal cAMP levels were increased when the receptor was expressed (i.e. when tet was removed from the medium) (Fig. 2, panel d). In these cells, TSH further increased cAMP levels (Fig. 2, panels c and d) (19Porcellini A. Ciullo I. Pannain S. Fenzi G. Avvedimento E. Oncogene. 1995; 11: 1089-1093PubMed Google Scholar). These data validate our experimental model, since cAMP levels mirror the expression of both the TSHR and T632I receptors. Thus, cAMP basal levels were higher in cells expressing T632I than in cells expressing TSHR in the absence of TSH. This indicates that the wild-type receptor was not expressed at levels that induced its activity in the absence of TSH. Expression of TSH Receptor Inhibits DNA Synthesis and Growth of Mouse Fibroblasts—It is known that cAMP inhibits the growth of mouse fibroblasts (27Stork P.J. Schmitt J.M. Trends Cell Biol. 2002; 12: 258-266Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, 28Dumaz N. Light Y. Marais R. Mol. Cell. Biol. 2002; 22: 3717-3728Crossref PubMed Scopus (68) Google Scholar, 29Dhillon A.S. Pollock C. Steen H. Shaw P.E. Mischak H. Kolch W. Mol. Cell. Biol. 2002; 22: 3237-3246Crossref PubMed Scopus (189) Google Scholar). We asked whether the regulated production of cAMP by Gs-coupled TSHR could modulate the growth of NIH 3T3 fibroblasts. To this end, we have used the wild-type receptor, which activates cAMP production only when exogenous TSH is present, or a mutant version of the receptor, T632I, which constitutively activates adenylyl cyclase in the absence TSH (26Porcellini A. Ruggiano G. Pannain S. Ciullo I. Amabile G. Fenzi G. Avvedimento E.V. Oncogene. 1997; 15: 781-789Crossref PubMed Scopus (20) Google Scholar, 19Porcellini A. Ciullo I. Pannain S. Fenzi G. Avvedimento E. Oncogene. 1995; 11: 1089-1093PubMed Google Scholar). Growth of fibroblasts was measured under different conditions (in the absence or presence of tet, serum, or TSH). A general scheme illustrating the temporal order of the various assays is shown in Fig. 3. Under each experimental condition indicated in Fig. 3, we have monitored the expression of TSHR and cAMP levels, following the treatment of the cell with 1 mU TSH for 40 min, to ascertain that the observed effects were not caused by variations in TSHR levels or activity (data not shown). Tetracyclin, tet, at the concentrations used (1.0 μg/ml) did not alter the growth of control (cells expressing the tTA-repressor only) and TSHR-expressing fibroblasts (Fig. 4, panels a and b). Control cells displayed a doubling time of ∼28.4 h. When tet was removed from the medium, the growth of TSHR expressing cells was slightly reduced (doubling time = 29.3 h). Continuous exposure of these cells to TSH (10 mU/ml) inhibited robustly cell growth (Fig. 4, panel b). The doubling time was 42 h, 45% longer than the control. This inhibition was dependent on the expression of TSHR, because it was abolished by tet and was strictly dependent on TSH in cell lines expressing TSHR (Fig. 4, panels a and b). In cells transfected with T632I, the expression of the active receptor, following removal of tet from the medium, inhibited cell growth in the absence of TSH (Fig. 4, panel c). These data were confirmed by thymidine incorporation curves (Fig. 4, panels d–f) and by MTT assay (data not shown). FACS analysis confirmed that inhibition by TSH was caused by delayed S phase entry (Fig. 5C). The data shown in Figs. 2 and 4 indicate that in our experimental settings, the biological effects on cAMP and DNA synthesis were dependent on TSH. Note that in the absence of TSH, the growth and cAMP levels were similar between TSHR transfected and control cell.Fig. 4TSH receptor expression inhibits the growth of mouse fibroblasts. The upper panel shows the growth profile of control fibroblasts (tTA-repressor-expressing clone) and cells expressing wild-type receptor or T632I, respectively. The plots indicate the mean ± S.E. of three independent experiments in triplicate samples. The lower panel shows thymidine incorporation curves of the same cell lines indicated in A. Briefly, after 72 h of growth in the absence of tetracyclin, serum was removed (0.5%) for 18 h. At time 0, serum and [3H]thymidine were added. DNA was extracted by Cell Harvester, and radioactivity was determined. All these experiments were repeated three times in triplicate samples. At the concentrations used (1.0 μg/ml), tetracyclin did not affect thymidine incorporation in control cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Co-expression of TSHR and PKA, regulatory subunit RIIβ, stimulates the growth of mouse fibroblasts. A, expression of exogenous RIIβ PKA subunit in NIH 3T3 fibroblasts. Western blot analysis for PKA-regulatory subunits type II-β in control and transfected clones. Control cells (ctrl), TSHR- and T632I-expressing clones contain low levels of RIIβ PKA subunit. Stable clones, transfected with the RIIβ expression vector contain significantly higher levels of the protein. All samples were normalized for transfection and for β-actin expression (lower panel). The expression of the RIIβ subunit does not affect the cAMP basal levels: 1.81 ± 0.13 pmol/2 × 105 cells in NTC versus 1.68 ± 0.19 in NTC/RIIβ) B, co-expression of TSHR and PKA, regulatory subunit RIIβ, stimulates the growth of mouse fibroblasts. The upper panel shows the growth curve of control fibroblasts (tTA-repressor-expressing clone) and cells expressing the TSHR-WT or T632I and regulatory subunit RIIβ. The plots are the mean ± S.E. of three independent experiments in triplicate samples. The lower panel shows the thymidine incorporation profile of the same cell lines indicated above. The cells were processed as described in Fig. 4. C, cell cycle progression of cells expressing TSHR and RIIβ. The cell lines indicated were analyzed by FACS. 1 × 106 cells were plated in 100-mm dishes; tetracyclin was removed 48 h before seeding, if required, and growth was monitored for 48 h in standard medium in the presence 10 mU/ml of TSH, as indicated. For FACS analysis, cells were harvested at 60% of confluence, ethanol-fixed, and stained with propidium iodide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Co-expression of PKA Regulatory Subunit, RIIβ, Reverses Growth Inhibition by TSHR—A common feature of thyroid cells and other cell types, stimulated by cAMP, is the robust expression of type II regulatory PKA subunits, in particular, the RIIβ subtype. RIIβ binds to A kinase anchor proteins (AKAPs) with high affinity, is associated to membranes and its affinity for cAMP is lower than that of the RI subunits (14Feliciello A. Gallo A. Mele E. Porcellini A. Troncone G. Garbi C. Gottesman M.E. Avvedimento E.V. J. Biol. Chem. 2000; 275: 303-311Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 24Paolillo M. Feliciello A. Porcellini A. Garbi C. Bifulco M. Schinelli S. Ventra C. Stabile E. Ricciardelli G. Schettini G. Avvedimento E.V. J. Biol. Chem. 1999; 274: 6546-6552Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 30Feliciello A. Giuliano P. Porcellini A. Garbi C. Obici S. Mele E. Angotti E. Grieco D. Amabile G. Cassano S. Li Y. Musti A.M. Rubin C.S. Gottersman M.E. Avvedimento V.E. J. Biol. Chem. 1996; 271: 25350-25359Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mouse 3T3 fibroblasts contain mostly RI subunits, which form cytosolic PKAI holoenzyme. These cells express low levels of RIIβ subunit (data not shown and Ref. 31Amieux P.S. Cummings D.E. Motamed K. Brandon E.P. Wailes L.A. Le K. Idzerda R.L. McKnight G.S. J. Biol. Chem. 1997; 272: 3993-3998Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). To define the role of RIIβ subunit in TSHR signaling, we have inves