0021-972x/92/7406-1477$03.00/0 Journal of Clinical Endocrinology and Metabolism CopyrightO1992byThe EndocrineSociety
Vol. 74, No. 6 Printed
HUMAN CHORIONIC GONADOTROPIN (hCG) INTERAC TS DIRECTLY WITH RECOMBINANT HUMAN TSH RECEPTORS Yaron
Tomer,
Gerold
Division of Endocrinology Mount Sinai School
K. Iiuber,
and Terry
F. Davies
and Metabolism, Department of Medicine, of Medicine, New York, New York 10029.
m: We have previously shown that highly purified urinary hCG has the potential to both stimulate the intracellular accumulation of cyclic AMP and induce growth of immortalized rat thyroid cells. We have now compared the ability of recombinant human TSH and purified urinary hCG preparations to stimulate Chinese hamster ovary (CHO) cells which have been transfected with the human TSH receptor. Only transfected CHO cells expressing recombinant TSH receptors, but not control CHO cells, were stimulated by hCG to release cyclic AMP in a dose-related manner and the effect of 100 IU of HCG was equivalent to approximately 9.2 UU of ret-hTSH. These data demonstrate that hCG interacts directly with the human TSH receptor. Several lines of evidence point to intrinsic thyroid the expression of stimulating activity by human chorionic normal early gonadotropin (hCG). During pregnancy some reports have shown elevated free T4 and free T3 levels, and decreased circulating TSH when measured by sensitive immunoassay. Both biochemical and clinical hyperthyroidism have been observed in trophoblastic tumors patients with [3[reviewed in 11. We [2] and others 4] have demonstrated that such purified hCG stimulates growth, iodide uptake, and cyclic AMP generation by immortalized rat thyroid cells. However, some investigators have claimed that while hCG may be a potent stimulator of the rat thyroid it fails to stimulate human thyroid tissue [5] even though it is able to inhibit TSH to human binding of radiolabeled thyroid membranes. Others have, therefore, non-hCG thyroid suggested that a stimulating molecule may also be secreted by trophoblast cells [6]. To complicate matters even further, it was reported that human thyroid tissue may contain specific mRNA for the LH/HCG receptor itself [7]. We have been trying to clarify some of these issues and in our laboratory we were
unable to demonstrate LH/hCG receptor mRNA in human thyroid cells [S]. We have now reexamined the interaction of hCG with human rather than rat TSH receptors Using a recombinant receptor approach. BY stimulating Chinese hamster ovary (CHO) cells transfected with human TSH receptor cDNA and using purified hCG we were able to show that the thyroid stimulating activity of hCG is secondary to its direct interaction with the human TSH receptor itself. MTERIALS AND MBTBODB The continuously proliferating TSHdependent lB-6 subclone of FRTL-5 cells described were cultured as previously supplement and a 6 hormone [9lP using Ham's modified F-12 medium designated 6H. The medium contained 5% newborn calf serum, and antibiotic supplement. CHO cells stably transfected with the plasmid kindly pSV2-NEO-ECE-hTSH were [lOI, provided by Dr. Basil Rapoport (UCSF) (controls and (called CHO-TSHR). CHO cells transfected) were grown in Ham's F-12 medium supplemented with 10% bovine fetal serum, and an antibiotic-antimycotic preparation containing penicillin, streptomycin and amphotericin B (Gibco laboratories, Grand Island, N.Y.). All cells were grown in 5% COz, 95% air humidity, fed twice weekly, and passaged
1477
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in U.S.A.
1478
RAPID every
COMMUNICATIONS
2-3 weeks. Highly purified hCG (hCG-CR 127, 14,900 U/mg (95% confidence limits lO,lOO21,700 U/mg) was supplied by the National Pituitary Agency (Baltimore, MD). The hCG tested for TSH preparation was contamination by a supersensitive third TSH chemiluminescent assay generation (Cirrus Immulite System, NJ), and showed no detectable TSH in the hCG preparation. (ret-hTSH, Recombinant human TSH bioactivity 7.0 IU/mg) was kindly supplied by Genzyme Inc. MA [ll]. For hTSH and hCG stimulation experiments 3 x lo4 cells/well (CHO or lB-6) were transferred to 96-well plates and stimulated with graded concentrations of hTSH and hCG as Prior to previously described 191’ bioassay, lB-6 cells were grown in medium devoid of TSH for 5 days to allow for recovery from TSH desensitization. For hormone stimulation, medium was replaced by increasing concentrations of hTSH or hCG diluted 1:l in modified hypotonic Hank's balanced salt solution [I21 containing 40 ml4 Hepes (Sigma) and 4mM 3iso-butyl-1-methyl-xanthine (Sigma) at Ph 7.4; the cells were incubated with the hormones for 60 min. at 37' C. Incubations were terminated by medium aspiration and the supernatants acetylated and subjected to cyclic AMP (CAMP) radioimmunoassay [2]. RESULTS TSH stimulation - As expected [2], recextracellular CAMP hTSH stimulated accumulation in hTSH receptor expressing CHO cells and lB-6 rat thyroid cells, but not in control CHO cells (Figures 1A & 1B). However, the responsiveness of CHOTSHR cells to hTSH was markedly lower than that of the lB-6 cells. At maximum hTSH concentration the CHO-TSHR cells exhibited a 276% increase in CAMP production while the lB-6 cells showed a 1747% increase. J'ICG stimulation - The TSH receptor expressing CHO cells and the rat lB-6 cells demonstrated significant increases upon in extracellular CAMP production
JCE%M.1662 Vo174*No6
stimulation CHO cells 1B). In purified response 100 IU bioactivity
with hCG, while the control were unresponsive (Figures 1A & the CHO-TSHR cells the highly hCG (CR 127) gave a similar dose curve to that of hTSH, such that of hCG was equivalent to the of 9.2 UU of human TSH.
250 -
200 -
T-
!1 ;::l’/, , , , , _i/, 0 10’
lo*
lo=
lo4
0
[TSH] mU/L
rig.
1A.
10’
lo*
1 lo6
10’
The extracellular CAMP response of CRO-TSHR cells to increasina concentrations of ret-hTSH and hCG-CR-127. Data expressed as mean + SEM. The data are representative of 3 experiments.
T
0
pig.
1B.
lo6
[hCC] q/ml or x14.9 U/L
TSH
d 0
hCC
B
1
1 i
0 TSH
0 hCG
lB-6 CHO-control CAMP production by lB-6 cells and untransfected CHO cells: without hormone stimulation (0 and after stimulation with 101 mu/L of ret-hTSH (TSH), or 10' nG/ml (14.9x105 U/L) of hCGCR127 (hCG). Data were obtained from the same experiments as in figure 1A. Results expressed as mean -+ SD.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 April 2015. at 16:53 For personal use only. No other uses without permission. . All rights reserved.
RAPID
COMMUNICATIONS
DISCUSSION data demonstrate that highly purified hCG-CR127 stimulates CHO cells which have been stably transfected with human TSH receptor cDNA and expressing the human TSH receptor. Such observations confirm the intrinsic human TSH receptor stimulating capacity of hCG, as suggested by earlier studies [Z-4]. Using the CHOTSHR cells the thyroid-stimulating potency of 100 IU of hCG was equivalent to 9.2 UU of recombinant human TSH. This potency is different to that not significantly previously calculated using a thyroid cell bioassay [Z]. This implies that thyroid cells and CHO-TSHR cells bind human TSH and hCG in a similar manner. show that the hCG These results molecule is capable of stimulating the TSH thereby inducing cyclic receptor itself, AMP generation and release by CHO cells expressing the human TSH receptor. This phenomenon probably results from cross reaction secondary to the known homologies between molecules and their these respective These receptors [1,71observations are also consistent with our previous results demonstrating that hCG stimulates thyroid cells [Z], and that such thyroid cells do not transcribe LH Our results continue receptor mRNA [8]. to be consistent with the central role of hCG influencing thyroid function during early normal pregnancy and in patients with hydatidiform moles and other trophoblastic tumors. These
RBFBRBNCES 1. Kennedy RL, and Darne J. The role of hCG in regulation of the thyroid gland in normal and abnormal pregnancy. 1991;78:298-307. Obstet Gynecol. 2. Davies TF, and Platzer M. hCGinduced receptor activation and growth acceleration in FRTL-5 thyroid cells. Endocrinology 1986:118:2149-2151. 3. Kennedy RL, Darne J, Griffiths H, Price A, Davies R, and Cohn M. Thyroidstimulatory effects of human chorionic gonadotrophin in early pregnancy. In vivo and in vitro studies. Horm Res. 1990;33:177-183.
Her&man RM, Lee HY, Sugawara M, Kirell CJ, Pang XP, Yanagisawa M, and Pekary AE. Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxribonucleic acid synthesis in cultured rat thyroid cells. J Clin Endocrinol Metab. 1988;67:74-79. 5. Amir SM, Endo K, Osathanondh R, and Ingbar SH. Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro. Mol Cell Endocrinol. 1985;39:31-37. 6. Avivi A, Schreiber AB, and Shemesh M. Isolation and characterization of a 94,000-dalton protein with thyrotropic activity from early bovine placenta. J Bio Chem. 1982;257:1138411389. 7. Frazier AL, Robbins LS, stork PJ, Sprengel R, Segaloff DL, and Cone RD. Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing. Mol Endocrinol. 1990;4:1264-1267. 8. Graves PN, and Davies TF. Absence of lutropin (LH) receptor mRNA in the rat thyroid: further evidence for specificity cross-over at the thyroidstimulating hormone receptor level. Mol Cell Endocrinol. 1991;79:21-28. 9. Davies, T.F., Yang, C., and Platzer, M. Cloning the Fisher rat thyroid cell (FRTL-5): variability in clonal growth and 3,5-cyclic adenosine monophosphate response to thyrotropin. Endocrinology 1987;121:78-83. lO.Chazenbalk GD. Nagayama Y, Kaufman KD, and Rapoport B. The functional expression of recombinant human thyrotropin receptors in nonthyroidal eukaryotic cells provides evidence that homologous desensitization to thyrotropin stimulation requires a cell-specific factor. Endocrinology 1990;127:1240-1244. ll.Huber GK, Fong P, Conception ES, and Davies TF. Recombinant human thyroid stimulating hormone: Initial bioactivity assessment using human fetal thyroid cells. J Clin Endocrinol Metab. 1991;72:1328-1331. lZ.Kasagi Km Konishi J, Iida Y, Ikebuko K, Mori T, Kuma K, and Torizuka K. A new in-vitro assay for human thyroid stimulators using cultured thyroid cells: effect of sodium chloride on adenosine 3,.5 - monophosphate increase. J Clin Endocrinol Metab. 1982;54:108-114. 4.
Acknowledgments: Supported in part by grants DK28243 and DK35764 from NIDDK, and Al-Zohar Foundation, Sheba Medical Center, Israel. Address for correspoadeace: Y. Tomer MD, Box 1055, Mount Sinai School of Medicine, One Gustave Levy Place, N.Y., N.Y., 10029.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 April 2015. at 16:53 For personal use only. No other uses without permission. . All rights reserved.
1479
Vol. 74, No. 6 Printed
HUMAN CHORIONIC GONADOTROPIN (hCG) INTERAC TS DIRECTLY WITH RECOMBINANT HUMAN TSH RECEPTORS Yaron
Tomer,
Gerold
Division of Endocrinology Mount Sinai School
K. Iiuber,
and Terry
F. Davies
and Metabolism, Department of Medicine, of Medicine, New York, New York 10029.
m: We have previously shown that highly purified urinary hCG has the potential to both stimulate the intracellular accumulation of cyclic AMP and induce growth of immortalized rat thyroid cells. We have now compared the ability of recombinant human TSH and purified urinary hCG preparations to stimulate Chinese hamster ovary (CHO) cells which have been transfected with the human TSH receptor. Only transfected CHO cells expressing recombinant TSH receptors, but not control CHO cells, were stimulated by hCG to release cyclic AMP in a dose-related manner and the effect of 100 IU of HCG was equivalent to approximately 9.2 UU of ret-hTSH. These data demonstrate that hCG interacts directly with the human TSH receptor. Several lines of evidence point to intrinsic thyroid the expression of stimulating activity by human chorionic normal early gonadotropin (hCG). During pregnancy some reports have shown elevated free T4 and free T3 levels, and decreased circulating TSH when measured by sensitive immunoassay. Both biochemical and clinical hyperthyroidism have been observed in trophoblastic tumors patients with [3[reviewed in 11. We [2] and others 4] have demonstrated that such purified hCG stimulates growth, iodide uptake, and cyclic AMP generation by immortalized rat thyroid cells. However, some investigators have claimed that while hCG may be a potent stimulator of the rat thyroid it fails to stimulate human thyroid tissue [5] even though it is able to inhibit TSH to human binding of radiolabeled thyroid membranes. Others have, therefore, non-hCG thyroid suggested that a stimulating molecule may also be secreted by trophoblast cells [6]. To complicate matters even further, it was reported that human thyroid tissue may contain specific mRNA for the LH/HCG receptor itself [7]. We have been trying to clarify some of these issues and in our laboratory we were
unable to demonstrate LH/hCG receptor mRNA in human thyroid cells [S]. We have now reexamined the interaction of hCG with human rather than rat TSH receptors Using a recombinant receptor approach. BY stimulating Chinese hamster ovary (CHO) cells transfected with human TSH receptor cDNA and using purified hCG we were able to show that the thyroid stimulating activity of hCG is secondary to its direct interaction with the human TSH receptor itself. MTERIALS AND MBTBODB The continuously proliferating TSHdependent lB-6 subclone of FRTL-5 cells described were cultured as previously supplement and a 6 hormone [9lP using Ham's modified F-12 medium designated 6H. The medium contained 5% newborn calf serum, and antibiotic supplement. CHO cells stably transfected with the plasmid kindly pSV2-NEO-ECE-hTSH were [lOI, provided by Dr. Basil Rapoport (UCSF) (controls and (called CHO-TSHR). CHO cells transfected) were grown in Ham's F-12 medium supplemented with 10% bovine fetal serum, and an antibiotic-antimycotic preparation containing penicillin, streptomycin and amphotericin B (Gibco laboratories, Grand Island, N.Y.). All cells were grown in 5% COz, 95% air humidity, fed twice weekly, and passaged
1477
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 April 2015. at 16:53 For personal use only. No other uses without permission. . All rights reserved.
in U.S.A.
1478
RAPID every
COMMUNICATIONS
2-3 weeks. Highly purified hCG (hCG-CR 127, 14,900 U/mg (95% confidence limits lO,lOO21,700 U/mg) was supplied by the National Pituitary Agency (Baltimore, MD). The hCG tested for TSH preparation was contamination by a supersensitive third TSH chemiluminescent assay generation (Cirrus Immulite System, NJ), and showed no detectable TSH in the hCG preparation. (ret-hTSH, Recombinant human TSH bioactivity 7.0 IU/mg) was kindly supplied by Genzyme Inc. MA [ll]. For hTSH and hCG stimulation experiments 3 x lo4 cells/well (CHO or lB-6) were transferred to 96-well plates and stimulated with graded concentrations of hTSH and hCG as Prior to previously described 191’ bioassay, lB-6 cells were grown in medium devoid of TSH for 5 days to allow for recovery from TSH desensitization. For hormone stimulation, medium was replaced by increasing concentrations of hTSH or hCG diluted 1:l in modified hypotonic Hank's balanced salt solution [I21 containing 40 ml4 Hepes (Sigma) and 4mM 3iso-butyl-1-methyl-xanthine (Sigma) at Ph 7.4; the cells were incubated with the hormones for 60 min. at 37' C. Incubations were terminated by medium aspiration and the supernatants acetylated and subjected to cyclic AMP (CAMP) radioimmunoassay [2]. RESULTS TSH stimulation - As expected [2], recextracellular CAMP hTSH stimulated accumulation in hTSH receptor expressing CHO cells and lB-6 rat thyroid cells, but not in control CHO cells (Figures 1A & 1B). However, the responsiveness of CHOTSHR cells to hTSH was markedly lower than that of the lB-6 cells. At maximum hTSH concentration the CHO-TSHR cells exhibited a 276% increase in CAMP production while the lB-6 cells showed a 1747% increase. J'ICG stimulation - The TSH receptor expressing CHO cells and the rat lB-6 cells demonstrated significant increases upon in extracellular CAMP production
JCE%M.1662 Vo174*No6
stimulation CHO cells 1B). In purified response 100 IU bioactivity
with hCG, while the control were unresponsive (Figures 1A & the CHO-TSHR cells the highly hCG (CR 127) gave a similar dose curve to that of hTSH, such that of hCG was equivalent to the of 9.2 UU of human TSH.
250 -
200 -
T-
!1 ;::l’/, , , , , _i/, 0 10’
lo*
lo=
lo4
0
[TSH] mU/L
rig.
1A.
10’
lo*
1 lo6
10’
The extracellular CAMP response of CRO-TSHR cells to increasina concentrations of ret-hTSH and hCG-CR-127. Data expressed as mean + SEM. The data are representative of 3 experiments.
T
0
pig.
1B.
lo6
[hCC] q/ml or x14.9 U/L
TSH
d 0
hCC
B
1
1 i
0 TSH
0 hCG
lB-6 CHO-control CAMP production by lB-6 cells and untransfected CHO cells: without hormone stimulation (0 and after stimulation with 101 mu/L of ret-hTSH (TSH), or 10' nG/ml (14.9x105 U/L) of hCGCR127 (hCG). Data were obtained from the same experiments as in figure 1A. Results expressed as mean -+ SD.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 April 2015. at 16:53 For personal use only. No other uses without permission. . All rights reserved.
RAPID
COMMUNICATIONS
DISCUSSION data demonstrate that highly purified hCG-CR127 stimulates CHO cells which have been stably transfected with human TSH receptor cDNA and expressing the human TSH receptor. Such observations confirm the intrinsic human TSH receptor stimulating capacity of hCG, as suggested by earlier studies [Z-4]. Using the CHOTSHR cells the thyroid-stimulating potency of 100 IU of hCG was equivalent to 9.2 UU of recombinant human TSH. This potency is different to that not significantly previously calculated using a thyroid cell bioassay [Z]. This implies that thyroid cells and CHO-TSHR cells bind human TSH and hCG in a similar manner. show that the hCG These results molecule is capable of stimulating the TSH thereby inducing cyclic receptor itself, AMP generation and release by CHO cells expressing the human TSH receptor. This phenomenon probably results from cross reaction secondary to the known homologies between molecules and their these respective These receptors [1,71observations are also consistent with our previous results demonstrating that hCG stimulates thyroid cells [Z], and that such thyroid cells do not transcribe LH Our results continue receptor mRNA [8]. to be consistent with the central role of hCG influencing thyroid function during early normal pregnancy and in patients with hydatidiform moles and other trophoblastic tumors. These
RBFBRBNCES 1. Kennedy RL, and Darne J. The role of hCG in regulation of the thyroid gland in normal and abnormal pregnancy. 1991;78:298-307. Obstet Gynecol. 2. Davies TF, and Platzer M. hCGinduced receptor activation and growth acceleration in FRTL-5 thyroid cells. Endocrinology 1986:118:2149-2151. 3. Kennedy RL, Darne J, Griffiths H, Price A, Davies R, and Cohn M. Thyroidstimulatory effects of human chorionic gonadotrophin in early pregnancy. In vivo and in vitro studies. Horm Res. 1990;33:177-183.
Her&man RM, Lee HY, Sugawara M, Kirell CJ, Pang XP, Yanagisawa M, and Pekary AE. Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxribonucleic acid synthesis in cultured rat thyroid cells. J Clin Endocrinol Metab. 1988;67:74-79. 5. Amir SM, Endo K, Osathanondh R, and Ingbar SH. Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro. Mol Cell Endocrinol. 1985;39:31-37. 6. Avivi A, Schreiber AB, and Shemesh M. Isolation and characterization of a 94,000-dalton protein with thyrotropic activity from early bovine placenta. J Bio Chem. 1982;257:1138411389. 7. Frazier AL, Robbins LS, stork PJ, Sprengel R, Segaloff DL, and Cone RD. Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing. Mol Endocrinol. 1990;4:1264-1267. 8. Graves PN, and Davies TF. Absence of lutropin (LH) receptor mRNA in the rat thyroid: further evidence for specificity cross-over at the thyroidstimulating hormone receptor level. Mol Cell Endocrinol. 1991;79:21-28. 9. Davies, T.F., Yang, C., and Platzer, M. Cloning the Fisher rat thyroid cell (FRTL-5): variability in clonal growth and 3,5-cyclic adenosine monophosphate response to thyrotropin. Endocrinology 1987;121:78-83. lO.Chazenbalk GD. Nagayama Y, Kaufman KD, and Rapoport B. The functional expression of recombinant human thyrotropin receptors in nonthyroidal eukaryotic cells provides evidence that homologous desensitization to thyrotropin stimulation requires a cell-specific factor. Endocrinology 1990;127:1240-1244. ll.Huber GK, Fong P, Conception ES, and Davies TF. Recombinant human thyroid stimulating hormone: Initial bioactivity assessment using human fetal thyroid cells. J Clin Endocrinol Metab. 1991;72:1328-1331. lZ.Kasagi Km Konishi J, Iida Y, Ikebuko K, Mori T, Kuma K, and Torizuka K. A new in-vitro assay for human thyroid stimulators using cultured thyroid cells: effect of sodium chloride on adenosine 3,.5 - monophosphate increase. J Clin Endocrinol Metab. 1982;54:108-114. 4.
Acknowledgments: Supported in part by grants DK28243 and DK35764 from NIDDK, and Al-Zohar Foundation, Sheba Medical Center, Israel. Address for correspoadeace: Y. Tomer MD, Box 1055, Mount Sinai School of Medicine, One Gustave Levy Place, N.Y., N.Y., 10029.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 02 April 2015. at 16:53 For personal use only. No other uses without permission. . All rights reserved.
1479
