0013-7227/92/1305-2769$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 5 Printed
Society
Regulation of Thyroid Hormone Ovine Thyroid Follicles* GREGORY P. BECKS, AND DAVID J. HILL
KEVIN
D. BUCKINGHAM,
Synthesis
JIA-F.
WANG,
in U.S.A.
in Cultured
IAN D. PHILLIPSt,
The Lawson Research Institute, St. Joseph’s Health Centre and Departments of Medicine (G. P. B., D. J.H.) and Physiology (J-F. W., D.J.H.), The University of Western Ontario, London, Ontario, Canada, N6A 4V2
ABSTRACT. Primary cultures of sheep thyroid follicles were used to study the regulatory control mechanisms of thyroid hormone production. When maintained under serum-free conditions in vitro these follicles exhibit hormone-dependent iodide transport, iodine organification, and physiological de nouo thyroid hormone formation. In previous studies we have also shown that thyroid follicles condition their culture media with insulinlike growth factors (IGFs) and IGF-binding proteins which are of potential autocrine or paracrine significance in thyroid hormonogenesis. TSH (100 rU/ml) alone modestly stimulated iodine uptake and organification, which was further potentiated by pharmacological or physiological concentrations of insulin and by physiological concentrations of IGF-I or IGF-II. A combination of TSH and cortisol (10 nM) optimally stimulated iodine uptake and organification without additive or synergistic effects among combinations of cortisol with insulin or IGFs. Insulin, IGF-I, IGF-II, or cortisol alone were without effect on iodine uptake and organification. The effect of TSH was mimicked by forskolin or (BuhcAMP, and the synergistic effect of
cortisol with TSH was duplicated in incubations of dexamethasone with TSH. In long term studies of the same experimental conditions, with lo-G111 NaI added to the culture medium, an increase in radioimmunoassayable T, and T, in conditioned cell culture media and cell layer extracts was confirmed for all conditions, with the exception of physiological concentrations of insulin. IGF-I and IGF-II were equipotent in their stimulation of thyroid hormonogenesis in the presence of TSH. The effect of high concentrations of insulin may be explained by a combined action through insulin and type I IGF receptors. We have previously reported that the stimulation of iodine uptake and organification (de nouo thyroid hormone formation here) by TSH and cortisol is inversely correlated with their inhibition of IGF-binding proteins released by the cells while IGF release is unchanged. Overall, these data suggest that the regulation of thyroid hormonogenesis involves the endocrine hormones TSH and cortisol, acting in synergy with locally produced IGFs. (Endocrinology 130: 2789-2794,1992)
T
binding proteins (IGFBPs) by cultured sheep thyroid epithelial cells (9-11). A recent report (12) in porcine thyroid follicles suggested that CAMP was sufficient to stimulate physiological de nova thyroid hormone formation but failed to account for the possible interactions of TSH or CAMP with the other hormones present within this widely used defined culture media, which contains transferrin, somatostatin, glycyl-histidyl-lysine, insulin, and cortisol, hormone supplements originally described for the growth and maintenance of rat thyroid cells (13). Among other reports in primary bovine thyroid cell cultures (14), an ovine thyroid cell strain (15) and the FRTL-5 rat thyroid cell line (16, 17), cortisol, insulin, and IGF-I are variably reported to enhance or inhibit TSH- and CAMP-stimulated iodide transport and thyroglobulin expression. Whether these effects necessarily result in corresponding changes in the synthesis and secretion of thyroid hormones is unknown and which factor(s) are necessary, sufficient, or optimal for thyroid hormonogenesis is unclear. We have now studied in detail the hormonal regulation of differentiated thyroid function, including de novo syn-
HYROTROPIN (TSH) and iodide are traditionally regarded as the major factors involved in the regulation of thyroid function and growth (1,2). Many effects of TSH are mediated through intracellular CAMP generation. However, previous in vivo and in vitro studies with thyroid tissues, and thyroid epithelial cells employing low- or serum-free culture media, indicate that there are important interactions between TSH or CAMP and other hormones such as insulin and cortisol in controlling thyroid cell function and growth (3-7). Such regulatory controls may ultimately involve local autocrine or paracrine mechanisms within the thyroid microenvironment. For example, insulin is known to cross-react with type I insulin-like growth factor (IGF) receptors (8). We and others have reported the secretion of IGFs and IGFReceived November 18, 1991. Address correspondence and reprint requests to: Dr. G. P. Becks, Room 4-738, St. Joseph’s Health Centre, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * Supported by grants from the Cancer Research Society Inc. and the Medical Research Council of Canada. Presented in part at the 10th International Thyroid Conference, The Hague, The Netherlands, February 1991 (Abstract 126). t Research Fellow of the Thyroid Foundation of Canada. 2789
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thesis of T4 and TB, in primary cultures of sheep thyroid follicles, which is the subject of this report. The data indicate that TSH or CAMP are necessary, but not wholly sufficient, for thyroid hormone formation, their effects being significantly enhanced by insulin or physiological concentrations of IGF-I or IGF-II. Moreover, a combination of TSH and cortisol is optimal for thyroid hormone formation while insulin or IGF combinations with TSH/cAMP and cortisol show no additive effect.
Materials
and Methods
Materials
Chemicals, hormones, and enzymes of reagent grade or higher were purchasedfrom Sigma Chemical Co. (St. Louis, MO) unlessotherwiseindicated. Isolation
and culture of thyroid follicles
Primary cultures of sheepthyroid follicles were preparedby digestionwith collagenase(type IV) and neutral protease(Dispase;Boehringer Mannheim, Dorval, Quebec, Canada) using procedurespreviously described(18). Follicles were grown to confluenceover 1 weekon 24-well culture plates (Costar; Johns Scientific Inc., Toronto, Ontario, Canada)in a 37 C humidified incubator in 0.5 ml Coon’s modified Ham’s F-12 M medium (Gibco, Burlington, Ontario, Canada) containing 0.5% calf serum, 100 IU/ml penicillin, and 100 pg/ml streptomycin (Gibco); supplementedwith 5 pg/ml transferrin, 10 rig/ml glycyl-histidyl-lysine acetate, and 10 rig/ml somatostatin (this combination designatedas 3H); 10 pg/ml bovine insulin and 4 rig/ml (10 nM) cortisol. The mediumwaschangedevery second or third day. Confluent cultures were switched to serum-free basalor 3H mediumfor 2 days before experimental manipulations in studiesof differentiated thyroid function. Stimulation
of differentiated
thyroid
function
After preincubation, triplicate wells on a single plate were incubated with bovine TSH, alone or in combination with cortisol, insulin, recombinant human IGF-I (IMCERA Bioproducts Inc., Terre Haute, IN), or recombinant IGF-II (Bachem Inc., Torrance, CA) for 48 or 96 h. Preliminary experimentsrevealedthat TSH alone stimulated iodine uptake and organification with an optimal dose-response between lOO500 pU/ml. A standard TSH concentration of 100 pU/ml was usedin the experimentspresentedherein. Where indicated, the CAMP agonist forskolin (Sigma) or CAMP analog (Bu& (Sigma)wassubstituted for TSH, and dexamethasonewasused in place of cortisol. Assay of iodine uptake and organification
Iodine uptake and organification was determined on each well after experimental manipulation after a 3 h incubation with lo6 cpm carrier-free Na”“I (Amersham,Oakville, Ontario, Canada) in 1 ml 37 C basal medium in an incubator (10, 18). The incubation wasterminated by aspirating the mediumafter which the cell layer wasrinsed in 1 ml ice cold Hank’s balanced salt solution (HBSS), solubilized in 1 ml 0.1 M NaOH, and counted in a y-counter. The per cent of cell-associatediodine organifiedwasdeterminedafter protein precipitation with 1 ml
IN THYROID
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Endo. 1992 Vol 130. No 5
ice-cold 10% trichloroacetic acid (5% final concentration) at 4 C for 10 min. After centrifugation at 1,000X g for 10 min, the supernatant was aspirated followed by washingof the precipitate with a further 1 ml 10% trichloroacetic acid and a final aspiration of the supernatant before repeat y-spectrometry. Assay of thyroid
hormone
synthesis and secretion
To determine whether stimulation of iodine uptake and organification under in vitro conditions was indicative of thyroid hormonogenesis,additional experimental incubations included 10m6M NaI in the culture media, a concentration of iodide we have previously shown to be optimal in these cells for de novo T, and TB synthesisin fully supplementeddefined culture media with TSH (18). The conditioned medium was aspirated after 48 or 96 h and centrifuged at 1000X g for 10 min, and the supernatant was stored at -20 C for later assay. The cell layer was solubilizedin 0.5 ml 0.05% Triton X-100 in HBSS and the nuclei, which remained intact as assessed by light microscopy, were gently scraped from the well with a rubber spatula. Nuclei were pelleted by centrifugation at 1000 x g for 10 min, and the supernatant, representing solubilized cytosol and follicle contents, was aspirated and stored at -20 C aswasthe nuclear pellet. Aliquots of conditioned mediumor cytosol extract wereassayedin duplicate for their concentration of T, and T3 (nanogramsper ml) by double antibody RIA as previously described(18) using tracer and antibody reagents obtained from Joldon Diagnostics(Scarborough,Ontario, Canada). Aqueous standardsfor T, and T3 were prepared in basal mediumor 0.05% Triton X-100 in HBSS from stock hormones in 70% ethanol-O.1M NaOH. Intra- and interassayvariability was 10% or lessfor either assay.Cross-reactivity of Tq on the TSassaywas lessthan 1% and observedonly at T, concentrations above 80 rig/ml. Cross-reactivity of T, on the T, assay was 1%. None of the usualculture mediasupplementsor IGFs exhibited detectable displacementof radiotracer in the assay incubations. The absoluteamounts of Tq and T3 in the media and cell fractions were added to derive the total T, and T3 content of each well. DNA assay and cell counts
The DNA content of eachwell wasdeterminedon the protein precipitate used for iodine organification, or on the nuclear pellet, by the diphenylaminereaction (18,19) and demonstrated lessthan 10% variability within and between replicate conditions on individual plates. Cell number was determined on someplates on an aliquot of trypsinized cell suspensionfrom confluent wells using a Coulter particle counter (Coulter Electronics, Hialeah, FL), demonstrating approximately 5-8 X lo” cellsper well, which was uniform between wells on individual plates under the various experimental conditions and stable over the secondweek of culture. Statistics
and data analysis
All experimentswere performedusing triplicate wells on 24well plates for each experimental condition and were repeated at least three times. Results are the mean f SE of pooled or singlerepresentative experimentsas indicated. Differences betweenmeanswereassessed by analysisof variance and unpaired t tests. Resultswere significant at the P C 0.05 level.
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THYROID
HORMONOGENESIS
Results
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iodine
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1. Effect of cortisol and dexamethasone uptake and organification
on TSH-stimulated
Regulation of iodine uptake and organification
Exposure to TSH alone for 48 h significantly (P < 0.05) enhanced total iodine uptake in cells maintained in 3H medium as shown in Fig. 1, the organified iodine also increasing from 14 f 0.9% (mean f SE) to 25 f 0.5%; P < 0.005 us. 3H. This effect of TSH was qualitatively and quantitatively similar when cells were maintained in basal serum-free culture media (data not shown). Coincubation with the usual insulin concentration (10 pg/ml) potentiated the TSH effect on total iodine uptake nearly 4-fold (P < 0.005) as shown in Fig. 1, with a further increase in organified iodine to 35 f 5.2%; P < 0.05 vs. TSH alone. A high physiological concentration of insulin (100 rig/ml) also stimulated iodine uptake and organification in coincubation experiments with TSH, similar to the 10 pg/ml insulin dose (data not shown). Optimal stimulation of total iodine uptake was seen with a combination of TSH and cortisol; up to 6-fold elevation compared to TSH alone (P < 0.005) as shown in Fig. 1, and the organified fraction rose to 74 + 2.5%; P < 0.005 us. TSH alone. There was no evidence of an additive or synergistic effect on iodine uptake and organification with a combination of TSH, insulin, and cortisol (Fig. 1). Neither insulin nor cortisol had any effect in the absence of TSH (data not shown). The effect of cortisol with TSH was reproduced by incubation of dexamethasone with TSH. Results of a representative experiment are shown in Table 1. Physiological concentrations of IGF-I and IGF-II (l30 rig/ml) produced a significant (P < 0.05) 2- to 3-fold potentiation of TSH-stimulated iodine uptake as shown in Fig. 2, and Fig. 3, respectively. The iodine organified also increased from 24 f 1.2% (mean f SE) with TSH alone to 31 + 0.9% with addition of 30 rig/ml IGF-I (Fig. **
0 ezd
Iodine uptake (cpm X 10m3)
Addition 3H TSH (100 rU/ml) +Cortisol (4 rig/ml) +Dexamethasone (1 rig/ml)
% organified
3.2 + 0.6 6.9 + 0.4 29.2 37.3
29 + 2 33 + 2 67 + 3* 75 f 3d
+ 2.0” + 11.4
Confluent wells on a 24-well plate were incubated with lo6 cpm Na? after exposure to the various additives and iodine uptake and organification determined as described in Materials and Methods. Values are mean + SE (n = 3). a.*. ‘P < 0.005 us. TSH. ‘P c 0.025 us. TSH. 0 E?iI
tot01 organified
** 1
**
TSH (1 OOuU/ml) IGF I (rig/ml)
-
+
+ -
1
2. Effect of IGF-I on TSH-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean + SE of triplicates of three experiments. *, P < 0.05 us. 3H; **, P < 0.005 us. TSH. FIG.
**
0 eZa
total organified
total orgonified
800 C e z 8 z E 2 0 4 p ‘5
600
400 TSH (lOOuU/ml) IGF II (rig/ml)
200
TSH (1 OOuU/ml) Insulin (lOug/ml) Cortisol (1 OnM)
+ -
+ +
+ +
+ + +
regulation of iodine uptake and organification. Effect of TSH, insulin, and cortisol in 3H culture medium. Components of 3H medium are as defined in Materials and Methods. Open bars indicate total iodine uptake, and hatched areas indicate the proportion organified. TSH value for total iodine uptake is arbitrarily assigned a value of 100%. Results are the mean +SE of triplicates of three experiments. *, P c 0.01 us. 3H; **, P < 0.005 us. TSH. FIG.
-
+ -
+ 1
+ 10
+ 30
FIG. 3. Effect of IGF-II on TSH-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean f SE of triplicates of three experiments. *, P < 0.005 us. 3H; **, P c 0.005 us. TSH.
1. Hormonal
2), P < f 4.2% 0.05. Either < 0.01)
0.05; and from 25 + 1.4% with TSH alone to 35 with addition of 30 rig/ml IGF-II (Fig. 3), P < forskolin or (Bu)~cAMP alone significantly stimulated iodine uptake and organification
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(P
in
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3H media, similar to TSH, and in all cases the effects were also potentiated by cortisol (Fig. 4). Regulation of thyroid hormone synthesis
During the first 48 h following addition of TSH and low6 M NaI to the cultures, with or without insulin or cortisol, there was no measurable increase in T4 or T3 content of the conditioned media or cell layer extract, levels being at or below the limits of detection. In the interval from 48 to 96 h there was a marked increase in T4 and T3 content of the cultures incubated with TSH and iodide (P < 0.05) and a further 2- to 3-fold increase (P < 0.005) in the presence of 10 pg/ml insulin or 10 nM cortisol as shown in Fig. 5. In long term studies these effects and differences persisted for up to 1 week after the various hormone additions (data not shown). In contrast to the similar effects of physiological and pharmacological concentrations of insulin on TSH-stimu0 EZ
total orgonified
”
200 e z 8 150 6
E 2
100
P .p -0 _o
50
n I
FOLLICLES
I (10nM)
(lOOuU/ml)
-
Fonkolin
+
-
(lOhI)
Bu.+AW
+
-
(1mM)
1992 No 5
lated iodine uptake and organification, there was no measurable increase in the Tq and T3 content of TSHtreated cultures incubated with 100 rig/ml insulin (Table 2), whereas the potentiation of de novo Tq and T3 production by 10 pg/ml insulin was readily evident, also shown in Table 2. The effect of cortisol on TSH-stimulated T, and T3 production was duplicated by dexamethasone (Table 3). Optimal concentrations of IGF-I or IGFII (30 rig/ml) produced a slight but significant (P < 0.05) 1.5-fold increase in thyroid hormone content of the cultures over that of TSH alone (Fig. 6). Discussion Our results have elucidated the role in thyroid hormonogenesis of the various hormones originally utilized to maintain rat FRTL thyroid cell function (13) and additionally may reveal something of the endocrine/ autocrine/paracrine interactions likely to regulate thyroid function in vivo. TSH or CAMP are necessary, and indeed sufficient, for de novo thyroid hormone formation as suggested (12) but clearly are not optimal in this regard. The effect of TSH reported here is not dependent on the 3H medium components, transferrin, somatostatin, and glycl-histidyl-lysine. In order of increasing apparent efficacy, the effect of TSH was potentiated by IGFs, insulin, and cortisol.
T, content T, content (na/well) (ndwell) 3H 14 * 3.3 ND TSH (100 rU/ml) 46 f 11.4 1.5 + 0.3 +Insulin (100 rig/ml) 36 + 17.1" 1.3 + o.3b +Insulin (10 &ml) 77 f 8.0 5.1 + 0.4d Confluent wells on a 24-well plate were incubated with lOWeM NaI and the additions shown and de nouo T, and T, production determined as described in Materi& and Methods. Values are the mean + SE (n = 3). ND, Not detected. ‘.‘P > 0.05 us. TSH. ‘P < 0.025 us. TSH. dP < 0.005 us. TSH. Addition
+
FIG. 4. Effect of cortisol on TSH-, forskolin-, and (B&CAMP-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean + SE of triplicates of two experiments. *, P < 0.01 us. 3H; **, P < 0.01 us. TSH, forskolin, or (Bu),cAMP alone. l *
10 CB < r
“i
TABLE 3. Effect of cortisol and dexamethasone on TSH-stimulated and T3 production
0 TSH (1 OOuU/ml) lnrulln (lOug/ml) (1 Onhl)
+ -
+ +
+
+
FIG. 5. Hormonal regulation of thyroid hormone formation. Effect of TSH, insulin, and cortisol in 3H culture medium. The total Tq (open bars) and T, (hatched bars) content of each culture well was calculated after measurement by RIA as described in Materials and Methods. Results are the mean + SE of triplicates of a representative experiment. *, P < 0.05 us. 3H; **, P < 0.05 vs. TSH.
T,
T, content Ta content (&well) (r&well) 3H 13 + 1.1 ND TSH (100 rU/ml) 35 + 4.7 1.8 * 0.1 +Cortisol (4 rig/ml) 53 + 3.2" 3.8 + 0.2* +Dexamethasone (1 nalml) 64 + 6.0 7.0 + 0.5d Confluent wells on a 24-well plate were incubated with 10e6 M NaI and the additions shown and de MUO T, and T3 production determined as described in Materials and Methods. Values are mean + SE (n = 3). ND, Not detected. ‘1 bP < 0.025 us. TSH. c.dP < 0.005 us. TSH. Addition
COrtlSOl
Endo. Voll30.
TABLE 2. Effect of pharmacological and physiological concentrations of insulin on TSH-stimulated T, and Ta production 7%
cortisol
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6
BBSH
TSH (100uU/ml) ICF I (30ng/ml) ICF II (3Ong/ml)
+ +
!
” $ 4' P 2;
0
+
+
FIG. 6. Effect of IGF-I and IGF-II on TSH-stimulated T, and T, production in 3H medium. Results are the mean f SE of triplicates of a representative experiment. *,P< 0.05 us. 3H; **,P< 0.05 us. TSH.
Our data are in accord with previous reports in cultured porcine, ovine, and rat thyroid cells on the potentiation of TSH-stimulated iodide transport, iodine organification, and thyroglobulin expression by insulin or IGFI (3, 16, 20) but additionally now confirm an actual increase in Tq and T3 formation. Still, the precise mechanisms by which insulin or IGF-I potentiate thyroidal iodine metabolism are unclear. A minimal effect of insulin/IGF-I on thyroid peroxidase messenger RNA has been shown in comparison to that of TSH alone (21). Previous studies of thyroid cell growth and thyroglobulin expression suggest that IGF-I and high concentrations of insulin, as used in this study, interact with TSH via the type-1 IGF receptor upon events distal to generation of CAMP (16, 22). Insulin may also act through its own receptor. Physiological concentrations of insulin were recently reported to enhance pertechnetate uptake, iodine organification, and thyroglobulin expression, in addition to DNA synthesis, in sheep thyroid cells (20). In our studies, similar concentrations of insulin also enhanced iodine uptake and organification, but we were unable to demonstrate any increase in T4 and TB production during prolonged culture with NaI. Thus, physiological concentrations of insulin acting via the insulin receptor may not have a major impact on resultant thyroid hormonogenesis, in contrast to the significant effects of physiological concentrations of IGFs, which are presumably acting through type-1 IGF receptors. An alternative explanation for the negative results at low insulin concentrations would be its degredation during prolonged cultured (23). This also raises an other important issue with respect to in vitro assessments of differentiated thyroid function; that iodine uptake and organification may not always reflect thyroid hormone formation. The superior effect of high-dose insulin on thyroid hormone synthesis compared to IGF-I and IGF-II in our studies may reflect a combined action of pharmacological concentrations of insulin through type-1 IGF receptors and insulin receptors. Recently, inhibitory effects of insulin
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and IGF-I upon TSH- and CAMP-stimulated iodide transport have been reported in FRTL-5 rat thyroid cells maintained in serum-depleted medium, an effect opposite to that seen when cells are incubated in 5% serum (17, 24). The significance of these findings, the former which contrast the present study is unclear but may relate to the differences between primary cultures as opposed to a passaged cell line. To our knowledge, this is the first report of significant bioactivity of IGF-II on thyroid hormone synthesis although IGF-II is an important autocrine factor in the growth of FRTL-5 rat thyroid cells (25). We find IGF-II to be equipotent to IGF-I in stimulating thyroid hormone formation even though IGF-II exhibits reduced affinity for the type-1 IGF receptor in sheep thyroid cells (11). This raises the possibility that IGF-II may also act in the thyroid through the typeII IGF/mannose-6-phosphate receptor which is supported by in vivo studies in hyperplastic rat goiters demonstrating increased IGF-II, but not IGF-I, binding (26).
Moreover, we find that cortisol optimally stimulates iodine uptake and organification in the presence of TSH or CAMP, in agreement with earlier reports that cortisol, along with TSH, stimulated thyroglobulin expression or iodide uptake in bovine and canine thyroid cells (6, 14). We extend these results in demonstrating increased T4 and T3 production. Cortisol requires the presence of IGFI or insulin to enhance TSH-dependent iodide efflux in FRTL-5 cells (27) and in an ovine thyroid cell strain thyroglobulin production was potentiated by cortisol only in the presence of insulin (15). The effect of cortisol on thyroid hormonogenesis reported in this study is notable in that there is no requirement for exogenous insulin or IGFs. On the other hand, inhibition of iodide transport by cortisol has been reported recently in FRTL-5 cells (4), and previous in vivo studies in humans subjects suggest that cortisol inhibits thyroid function overall by decreasing TSH secretion and increasing urinary iodide clearance (5, 28, 29). The precise mechanism(s) of glucocorticoid action, whether stimulatory or inhibitory, upon thyroidal iodine metabolism remains unclear. Posttranscriptional regulation of iodide transporter activity has been suggested (4). The present studies indicate a complex hormonal regulation of thyroid hormone biosynthesis by cortisol, insulin, or IGFs, dependent upon prior TSH or CAMP stimulation. The effect of insulin at high concentrations is consistent with an action, in part, through type-1 IGF receptors. This action may be met in situ by an autocrine or paracrine production of IGFs as reported by ourselves and others in sheep thyroid cells (9-11). These same studies have also demonstrated the release of IGFBPs in vitro. While IGFs appear to be constituitively expressed, we have shown the biosynthesis and release of IGFBPs to be hormonally regulated (10,30) and recently reported
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an inverse correlation between inhibition of IGFBP presence in conditioned culture media by TSH and cortisol and the stimulation of iodine organification (10). The precise function(s) of IGFBPs are unclear but may include modulation of tissue IGF availability to IGF receptors and their resultant biological activity (31,32). Thus, the possibility exists that in sheep thyroid cells, TSH and cortisol control thyroid hormone formation, in part, through interactions with locally produced IGFs and their associated IGFBPs. Acknowledgement The authors wish to thank Ms. Michele assistance in preparation of the manuscript.
IN THYROID
14. 15. 16.
17.
18. Falconer for excellent
References 1. Bidey SP, Tomlinson S 1988 The regulation and integration of thvroid follicular differentiation and function. Clin Endocrinol (dxf) 28:434-444 2. Bidev SP. Lambert A. Robertson WR 1988 Thvroid cell erowth. differentiation and function in the FRTL-5 celi line: a s&ey. J Endocrinol119:365-376 3. Sho K, Kondo Y 1984 Insulin modulates thyrotropin-induced follicle reconstruction and iodine metabolism in hog thyroid cells in chemically defined medium. Biochem Biophys Res Commun 118385-391 4. Saji M, Kohn LD 1990 Effect of hydrocortisone on the ability of thyrotropin to increase deoxyribonucleic acid synthesis and iodide uptake in FRTL-5 rat thyroid cells: opposite regulation of adenosine 3’,5’-monophosphate signal action. Endocrinology 127:18671876 5. Berson SA, Yalow RS 1952 The effect of cortisone on the iodine accumulating function of the thyroid gland in euthyroid subjects. J Clin Endocrinol Metab 12:407-422 6. Roger PP, Dumont JE 1983 Thyrotropin and the differential expression of proliferation and differentiation in dog thyroid cells in primary culture. J Endocrinol96:241-249 7. Nataf BM, Chaikoff IL 1965 The effect of insulin on iodine metabolism of fetal thyroid glands in organ culture. Biochim Biophys Acta 3:422-428 8. Jolly JMP, Wass JAH 1989 Insulin-like growth factors; autocrine, paracrine or endocrine? New perspectives of the somatomedin hypothesis in the light of recent developments. J Endocrinol 122:611-618 9. Wang J-F, Becks GP, Buckingham KD, Hill DJ 1990 Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells. J Endocrinol 125:439-448 10. Wane J-F. Becks GP. Hanada E. Buckineham KD. Phillins ID. Hill “DJ 1991 Hormonal regulation of insulin-like growth-factor binding proteins secreted by sheep thyroid epithelial cells: relationship with iodine organification. J Endocrinol 130:129-140 11. Bachrach LK, Eggo MC, Hintz RL, Burrow GN 1988 Insulin-like growth factors in sheep thyroid cells: actions, receptors and production. Biochem Biophys Res Commun 154:861-867 12. Murakami S, Summer CN, Iida-Klein A, Anderson DG, Sugawara M 1990 Physiological de nouo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3’,5’-monophosphate alone is sufficient for thyroid hormone formation. Endocrinology 1261692-1698 13. Ambesi-Impiombato FS, Parks LAM, Coon HG 1980 Culture of
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hormone-dependent functional epithelial cells from rat thyroids. Proc Nat1 Acad Sci USA 77:3455-3459 Gerard CM, Roger PP, Dumont JE 1989 Thyroglobulin gene expression as a differentiation marker in primary cultures of calf thvroid cells. Mol Cell Endocrinol 61:23-35 Aouani A, Hovsepian S, Fayet G 1988 Multihormonal regulation of thyroglobulin production by the OVNIS 6H thyroid cell strain. Horm Metab Res 20:91-95 Santisteban P, Kohn LD, DiLauro R 1987 Thyroglobulin gene expression is regulated by insulin and insulin-like growth factor I, as well as thvrotronin. in FRTL-5 thvroid cells. J Biol Chem 262:4048-4052 Saji M, Kohn LD 1991 Insulin and insulin-like growth factor-I inhibit thyrotropin-increased iodide transport in serum-depleted FRTL-5 rat thyroid cells: modulation of adenosine 3’,5’-monophosphate signal action. Endocrinology 1281136-1143 Becks GP. Eeeo MC. Burrow GN. 1987 Iodide reeulation of differentiated function: preferential inhibitory effect of excess iodide on thyroid hormone secretion in sheep thyroid cell culture. Endocrinology 120:2569-2576 Burton K 1956 A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem J 62:315-323 Eggo MC, Bachrach LK, Burrow GN 1990 Interaction of TSH, insulin and insulin-like growth factors in regulating thyroid growth and function. Growth Factors 2:99-109 Isozaki 0, Kohn LD, Kozak CA, Kimura S 1989 Thyroid peroxidase: rat cDNA sequence, chromosomal localization in mouse and regulation of gene expression by comparison to thyroglobulin in rat FRTL-5 cells. Mol Endocrinol 3:1681-1692 Ingbar SH, Tramontano D, Ambsei-Impiombato S 1987 Observations on the regulation of thyroid cell growth. Horm Metab Res [sUDP11 17:43-51 Trischitta V, Damante G, Foti D, Filetti S 1987 Insulin binding and biological activities in the FRTLB rat thyroid cell line. Metabolism 36379-383 Zakariia M. McKenzie JM 1989 Variation in the culture medium for FRTLB’cells: effects on growth and iodide uptake. Endocrinology 125:1253-1259 Maciel RMB. Moses AC. Villone G. Tramontano D. Ingbar SH 1988 Demonstration of the production and physiological role of insulin-like growth factor II in rat thyroid follicular cells in culture. J Clin Invest 82:546-553 Polychronakos C, Guyda HJ, Pate1 B, Posner B 1986 Increase in the number of type II insulin-like growth factor receptors during propythiouracil-induced hyperplasia in the rat thyroid. Endocrinology 119:1204-1209 Sho K, Okajima F, Akiyama H, Shoka Y, Kobayashi I, Kondo Y 1989 Requirement of insulin growth factor I plus hydrocortisone for the regeneration of thyrotropin (TSH)-dependent mechanisms of I-efflux and Ca*+ mobilization in FRTL-5 cells during TSHdepletion. Endocrinology 124:598-604 Ingbar SH 1953 The effect of cortisone on the thyroidal and renal metabolism of iodine. Endocrinology 53:171-181 Hill Jr RS, Reiss RS, Forsham PH, Thorn GW 1950 The effect of adrenocorticotropin and cortisone on thyroid function: thyroidadrenocortical interrelationships. J Clin Endocrinol Metab l&1375-1400 Buckingham KD, Phillips ID, Delhanty PJD, Wang J-F, Becks GP, Han VKM, Hill DJ 1991 Hormonal regulation of insulin-like growth factor binding protein mRNA abundance in cultured ovine thyroid follicles. J Endocrinol [Suppl] 129:270 (Abstract) Baxter RC, Martin JL 1989 Binding proteins for the insulin-like growth factors: structure, regulation and function. Prog Growth Factor Res 1:49-68 Clemmons DR 1990 Insulin-like growth factor binding proteins. Trends Endocrinol Metab 1:412-417 I--
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Vol. 130, No. 5 Printed
Society
Regulation of Thyroid Hormone Ovine Thyroid Follicles* GREGORY P. BECKS, AND DAVID J. HILL
KEVIN
D. BUCKINGHAM,
Synthesis
JIA-F.
WANG,
in U.S.A.
in Cultured
IAN D. PHILLIPSt,
The Lawson Research Institute, St. Joseph’s Health Centre and Departments of Medicine (G. P. B., D. J.H.) and Physiology (J-F. W., D.J.H.), The University of Western Ontario, London, Ontario, Canada, N6A 4V2
ABSTRACT. Primary cultures of sheep thyroid follicles were used to study the regulatory control mechanisms of thyroid hormone production. When maintained under serum-free conditions in vitro these follicles exhibit hormone-dependent iodide transport, iodine organification, and physiological de nouo thyroid hormone formation. In previous studies we have also shown that thyroid follicles condition their culture media with insulinlike growth factors (IGFs) and IGF-binding proteins which are of potential autocrine or paracrine significance in thyroid hormonogenesis. TSH (100 rU/ml) alone modestly stimulated iodine uptake and organification, which was further potentiated by pharmacological or physiological concentrations of insulin and by physiological concentrations of IGF-I or IGF-II. A combination of TSH and cortisol (10 nM) optimally stimulated iodine uptake and organification without additive or synergistic effects among combinations of cortisol with insulin or IGFs. Insulin, IGF-I, IGF-II, or cortisol alone were without effect on iodine uptake and organification. The effect of TSH was mimicked by forskolin or (BuhcAMP, and the synergistic effect of
cortisol with TSH was duplicated in incubations of dexamethasone with TSH. In long term studies of the same experimental conditions, with lo-G111 NaI added to the culture medium, an increase in radioimmunoassayable T, and T, in conditioned cell culture media and cell layer extracts was confirmed for all conditions, with the exception of physiological concentrations of insulin. IGF-I and IGF-II were equipotent in their stimulation of thyroid hormonogenesis in the presence of TSH. The effect of high concentrations of insulin may be explained by a combined action through insulin and type I IGF receptors. We have previously reported that the stimulation of iodine uptake and organification (de nouo thyroid hormone formation here) by TSH and cortisol is inversely correlated with their inhibition of IGF-binding proteins released by the cells while IGF release is unchanged. Overall, these data suggest that the regulation of thyroid hormonogenesis involves the endocrine hormones TSH and cortisol, acting in synergy with locally produced IGFs. (Endocrinology 130: 2789-2794,1992)
T
binding proteins (IGFBPs) by cultured sheep thyroid epithelial cells (9-11). A recent report (12) in porcine thyroid follicles suggested that CAMP was sufficient to stimulate physiological de nova thyroid hormone formation but failed to account for the possible interactions of TSH or CAMP with the other hormones present within this widely used defined culture media, which contains transferrin, somatostatin, glycyl-histidyl-lysine, insulin, and cortisol, hormone supplements originally described for the growth and maintenance of rat thyroid cells (13). Among other reports in primary bovine thyroid cell cultures (14), an ovine thyroid cell strain (15) and the FRTL-5 rat thyroid cell line (16, 17), cortisol, insulin, and IGF-I are variably reported to enhance or inhibit TSH- and CAMP-stimulated iodide transport and thyroglobulin expression. Whether these effects necessarily result in corresponding changes in the synthesis and secretion of thyroid hormones is unknown and which factor(s) are necessary, sufficient, or optimal for thyroid hormonogenesis is unclear. We have now studied in detail the hormonal regulation of differentiated thyroid function, including de novo syn-
HYROTROPIN (TSH) and iodide are traditionally regarded as the major factors involved in the regulation of thyroid function and growth (1,2). Many effects of TSH are mediated through intracellular CAMP generation. However, previous in vivo and in vitro studies with thyroid tissues, and thyroid epithelial cells employing low- or serum-free culture media, indicate that there are important interactions between TSH or CAMP and other hormones such as insulin and cortisol in controlling thyroid cell function and growth (3-7). Such regulatory controls may ultimately involve local autocrine or paracrine mechanisms within the thyroid microenvironment. For example, insulin is known to cross-react with type I insulin-like growth factor (IGF) receptors (8). We and others have reported the secretion of IGFs and IGFReceived November 18, 1991. Address correspondence and reprint requests to: Dr. G. P. Becks, Room 4-738, St. Joseph’s Health Centre, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * Supported by grants from the Cancer Research Society Inc. and the Medical Research Council of Canada. Presented in part at the 10th International Thyroid Conference, The Hague, The Netherlands, February 1991 (Abstract 126). t Research Fellow of the Thyroid Foundation of Canada. 2789
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THYROID
2790
HORMONOGENESIS
thesis of T4 and TB, in primary cultures of sheep thyroid follicles, which is the subject of this report. The data indicate that TSH or CAMP are necessary, but not wholly sufficient, for thyroid hormone formation, their effects being significantly enhanced by insulin or physiological concentrations of IGF-I or IGF-II. Moreover, a combination of TSH and cortisol is optimal for thyroid hormone formation while insulin or IGF combinations with TSH/cAMP and cortisol show no additive effect.
Materials
and Methods
Materials
Chemicals, hormones, and enzymes of reagent grade or higher were purchasedfrom Sigma Chemical Co. (St. Louis, MO) unlessotherwiseindicated. Isolation
and culture of thyroid follicles
Primary cultures of sheepthyroid follicles were preparedby digestionwith collagenase(type IV) and neutral protease(Dispase;Boehringer Mannheim, Dorval, Quebec, Canada) using procedurespreviously described(18). Follicles were grown to confluenceover 1 weekon 24-well culture plates (Costar; Johns Scientific Inc., Toronto, Ontario, Canada)in a 37 C humidified incubator in 0.5 ml Coon’s modified Ham’s F-12 M medium (Gibco, Burlington, Ontario, Canada) containing 0.5% calf serum, 100 IU/ml penicillin, and 100 pg/ml streptomycin (Gibco); supplementedwith 5 pg/ml transferrin, 10 rig/ml glycyl-histidyl-lysine acetate, and 10 rig/ml somatostatin (this combination designatedas 3H); 10 pg/ml bovine insulin and 4 rig/ml (10 nM) cortisol. The mediumwaschangedevery second or third day. Confluent cultures were switched to serum-free basalor 3H mediumfor 2 days before experimental manipulations in studiesof differentiated thyroid function. Stimulation
of differentiated
thyroid
function
After preincubation, triplicate wells on a single plate were incubated with bovine TSH, alone or in combination with cortisol, insulin, recombinant human IGF-I (IMCERA Bioproducts Inc., Terre Haute, IN), or recombinant IGF-II (Bachem Inc., Torrance, CA) for 48 or 96 h. Preliminary experimentsrevealedthat TSH alone stimulated iodine uptake and organification with an optimal dose-response between lOO500 pU/ml. A standard TSH concentration of 100 pU/ml was usedin the experimentspresentedherein. Where indicated, the CAMP agonist forskolin (Sigma) or CAMP analog (Bu& (Sigma)wassubstituted for TSH, and dexamethasonewasused in place of cortisol. Assay of iodine uptake and organification
Iodine uptake and organification was determined on each well after experimental manipulation after a 3 h incubation with lo6 cpm carrier-free Na”“I (Amersham,Oakville, Ontario, Canada) in 1 ml 37 C basal medium in an incubator (10, 18). The incubation wasterminated by aspirating the mediumafter which the cell layer wasrinsed in 1 ml ice cold Hank’s balanced salt solution (HBSS), solubilized in 1 ml 0.1 M NaOH, and counted in a y-counter. The per cent of cell-associatediodine organifiedwasdeterminedafter protein precipitation with 1 ml
IN THYROID
FOLLICLES
Endo. 1992 Vol 130. No 5
ice-cold 10% trichloroacetic acid (5% final concentration) at 4 C for 10 min. After centrifugation at 1,000X g for 10 min, the supernatant was aspirated followed by washingof the precipitate with a further 1 ml 10% trichloroacetic acid and a final aspiration of the supernatant before repeat y-spectrometry. Assay of thyroid
hormone
synthesis and secretion
To determine whether stimulation of iodine uptake and organification under in vitro conditions was indicative of thyroid hormonogenesis,additional experimental incubations included 10m6M NaI in the culture media, a concentration of iodide we have previously shown to be optimal in these cells for de novo T, and TB synthesisin fully supplementeddefined culture media with TSH (18). The conditioned medium was aspirated after 48 or 96 h and centrifuged at 1000X g for 10 min, and the supernatant was stored at -20 C for later assay. The cell layer was solubilizedin 0.5 ml 0.05% Triton X-100 in HBSS and the nuclei, which remained intact as assessed by light microscopy, were gently scraped from the well with a rubber spatula. Nuclei were pelleted by centrifugation at 1000 x g for 10 min, and the supernatant, representing solubilized cytosol and follicle contents, was aspirated and stored at -20 C aswasthe nuclear pellet. Aliquots of conditioned mediumor cytosol extract wereassayedin duplicate for their concentration of T, and T3 (nanogramsper ml) by double antibody RIA as previously described(18) using tracer and antibody reagents obtained from Joldon Diagnostics(Scarborough,Ontario, Canada). Aqueous standardsfor T, and T3 were prepared in basal mediumor 0.05% Triton X-100 in HBSS from stock hormones in 70% ethanol-O.1M NaOH. Intra- and interassayvariability was 10% or lessfor either assay.Cross-reactivity of Tq on the TSassaywas lessthan 1% and observedonly at T, concentrations above 80 rig/ml. Cross-reactivity of T, on the T, assay was 1%. None of the usualculture mediasupplementsor IGFs exhibited detectable displacementof radiotracer in the assay incubations. The absoluteamounts of Tq and T3 in the media and cell fractions were added to derive the total T, and T3 content of each well. DNA assay and cell counts
The DNA content of eachwell wasdeterminedon the protein precipitate used for iodine organification, or on the nuclear pellet, by the diphenylaminereaction (18,19) and demonstrated lessthan 10% variability within and between replicate conditions on individual plates. Cell number was determined on someplates on an aliquot of trypsinized cell suspensionfrom confluent wells using a Coulter particle counter (Coulter Electronics, Hialeah, FL), demonstrating approximately 5-8 X lo” cellsper well, which was uniform between wells on individual plates under the various experimental conditions and stable over the secondweek of culture. Statistics
and data analysis
All experimentswere performedusing triplicate wells on 24well plates for each experimental condition and were repeated at least three times. Results are the mean f SE of pooled or singlerepresentative experimentsas indicated. Differences betweenmeanswereassessed by analysisof variance and unpaired t tests. Resultswere significant at the P C 0.05 level.
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THYROID
HORMONOGENESIS
Results
IN THYROID TABLE
iodine
FOLLICLES
2791
1. Effect of cortisol and dexamethasone uptake and organification
on TSH-stimulated
Regulation of iodine uptake and organification
Exposure to TSH alone for 48 h significantly (P < 0.05) enhanced total iodine uptake in cells maintained in 3H medium as shown in Fig. 1, the organified iodine also increasing from 14 f 0.9% (mean f SE) to 25 f 0.5%; P < 0.005 us. 3H. This effect of TSH was qualitatively and quantitatively similar when cells were maintained in basal serum-free culture media (data not shown). Coincubation with the usual insulin concentration (10 pg/ml) potentiated the TSH effect on total iodine uptake nearly 4-fold (P < 0.005) as shown in Fig. 1, with a further increase in organified iodine to 35 f 5.2%; P < 0.05 vs. TSH alone. A high physiological concentration of insulin (100 rig/ml) also stimulated iodine uptake and organification in coincubation experiments with TSH, similar to the 10 pg/ml insulin dose (data not shown). Optimal stimulation of total iodine uptake was seen with a combination of TSH and cortisol; up to 6-fold elevation compared to TSH alone (P < 0.005) as shown in Fig. 1, and the organified fraction rose to 74 + 2.5%; P < 0.005 us. TSH alone. There was no evidence of an additive or synergistic effect on iodine uptake and organification with a combination of TSH, insulin, and cortisol (Fig. 1). Neither insulin nor cortisol had any effect in the absence of TSH (data not shown). The effect of cortisol with TSH was reproduced by incubation of dexamethasone with TSH. Results of a representative experiment are shown in Table 1. Physiological concentrations of IGF-I and IGF-II (l30 rig/ml) produced a significant (P < 0.05) 2- to 3-fold potentiation of TSH-stimulated iodine uptake as shown in Fig. 2, and Fig. 3, respectively. The iodine organified also increased from 24 f 1.2% (mean f SE) with TSH alone to 31 + 0.9% with addition of 30 rig/ml IGF-I (Fig. **
0 ezd
Iodine uptake (cpm X 10m3)
Addition 3H TSH (100 rU/ml) +Cortisol (4 rig/ml) +Dexamethasone (1 rig/ml)
% organified
3.2 + 0.6 6.9 + 0.4 29.2 37.3
29 + 2 33 + 2 67 + 3* 75 f 3d
+ 2.0” + 11.4
Confluent wells on a 24-well plate were incubated with lo6 cpm Na? after exposure to the various additives and iodine uptake and organification determined as described in Materials and Methods. Values are mean + SE (n = 3). a.*. ‘P < 0.005 us. TSH. ‘P c 0.025 us. TSH. 0 E?iI
tot01 organified
** 1
**
TSH (1 OOuU/ml) IGF I (rig/ml)
-
+
+ -
1
2. Effect of IGF-I on TSH-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean + SE of triplicates of three experiments. *, P < 0.05 us. 3H; **, P < 0.005 us. TSH. FIG.
**
0 eZa
total organified
total orgonified
800 C e z 8 z E 2 0 4 p ‘5
600
400 TSH (lOOuU/ml) IGF II (rig/ml)
200
TSH (1 OOuU/ml) Insulin (lOug/ml) Cortisol (1 OnM)
+ -
+ +
+ +
+ + +
regulation of iodine uptake and organification. Effect of TSH, insulin, and cortisol in 3H culture medium. Components of 3H medium are as defined in Materials and Methods. Open bars indicate total iodine uptake, and hatched areas indicate the proportion organified. TSH value for total iodine uptake is arbitrarily assigned a value of 100%. Results are the mean +SE of triplicates of three experiments. *, P c 0.01 us. 3H; **, P < 0.005 us. TSH. FIG.
-
+ -
+ 1
+ 10
+ 30
FIG. 3. Effect of IGF-II on TSH-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean f SE of triplicates of three experiments. *, P < 0.005 us. 3H; **, P c 0.005 us. TSH.
1. Hormonal
2), P < f 4.2% 0.05. Either < 0.01)
0.05; and from 25 + 1.4% with TSH alone to 35 with addition of 30 rig/ml IGF-II (Fig. 3), P < forskolin or (Bu)~cAMP alone significantly stimulated iodine uptake and organification
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(P
in
THYROID
2792
HORMONOGENESIS
3H media, similar to TSH, and in all cases the effects were also potentiated by cortisol (Fig. 4). Regulation of thyroid hormone synthesis
During the first 48 h following addition of TSH and low6 M NaI to the cultures, with or without insulin or cortisol, there was no measurable increase in T4 or T3 content of the conditioned media or cell layer extract, levels being at or below the limits of detection. In the interval from 48 to 96 h there was a marked increase in T4 and T3 content of the cultures incubated with TSH and iodide (P < 0.05) and a further 2- to 3-fold increase (P < 0.005) in the presence of 10 pg/ml insulin or 10 nM cortisol as shown in Fig. 5. In long term studies these effects and differences persisted for up to 1 week after the various hormone additions (data not shown). In contrast to the similar effects of physiological and pharmacological concentrations of insulin on TSH-stimu0 EZ
total orgonified
”
200 e z 8 150 6
E 2
100
P .p -0 _o
50
n I
FOLLICLES
I (10nM)
(lOOuU/ml)
-
Fonkolin
+
-
(lOhI)
Bu.+AW
+
-
(1mM)
1992 No 5
lated iodine uptake and organification, there was no measurable increase in the Tq and T3 content of TSHtreated cultures incubated with 100 rig/ml insulin (Table 2), whereas the potentiation of de novo Tq and T3 production by 10 pg/ml insulin was readily evident, also shown in Table 2. The effect of cortisol on TSH-stimulated T, and T3 production was duplicated by dexamethasone (Table 3). Optimal concentrations of IGF-I or IGFII (30 rig/ml) produced a slight but significant (P < 0.05) 1.5-fold increase in thyroid hormone content of the cultures over that of TSH alone (Fig. 6). Discussion Our results have elucidated the role in thyroid hormonogenesis of the various hormones originally utilized to maintain rat FRTL thyroid cell function (13) and additionally may reveal something of the endocrine/ autocrine/paracrine interactions likely to regulate thyroid function in vivo. TSH or CAMP are necessary, and indeed sufficient, for de novo thyroid hormone formation as suggested (12) but clearly are not optimal in this regard. The effect of TSH reported here is not dependent on the 3H medium components, transferrin, somatostatin, and glycl-histidyl-lysine. In order of increasing apparent efficacy, the effect of TSH was potentiated by IGFs, insulin, and cortisol.
T, content T, content (na/well) (ndwell) 3H 14 * 3.3 ND TSH (100 rU/ml) 46 f 11.4 1.5 + 0.3 +Insulin (100 rig/ml) 36 + 17.1" 1.3 + o.3b +Insulin (10 &ml) 77 f 8.0 5.1 + 0.4d Confluent wells on a 24-well plate were incubated with lOWeM NaI and the additions shown and de nouo T, and T, production determined as described in Materi& and Methods. Values are the mean + SE (n = 3). ND, Not detected. ‘.‘P > 0.05 us. TSH. ‘P < 0.025 us. TSH. dP < 0.005 us. TSH. Addition
+
FIG. 4. Effect of cortisol on TSH-, forskolin-, and (B&CAMP-stimulated iodine uptake and organification in 3H culture medium. TSH is assigned an arbitrary value of 100%. Results are the mean + SE of triplicates of two experiments. *, P < 0.01 us. 3H; **, P < 0.01 us. TSH, forskolin, or (Bu),cAMP alone. l *
10 CB < r
“i
TABLE 3. Effect of cortisol and dexamethasone on TSH-stimulated and T3 production
0 TSH (1 OOuU/ml) lnrulln (lOug/ml) (1 Onhl)
+ -
+ +
+
+
FIG. 5. Hormonal regulation of thyroid hormone formation. Effect of TSH, insulin, and cortisol in 3H culture medium. The total Tq (open bars) and T, (hatched bars) content of each culture well was calculated after measurement by RIA as described in Materials and Methods. Results are the mean + SE of triplicates of a representative experiment. *, P < 0.05 us. 3H; **, P < 0.05 vs. TSH.
T,
T, content Ta content (&well) (r&well) 3H 13 + 1.1 ND TSH (100 rU/ml) 35 + 4.7 1.8 * 0.1 +Cortisol (4 rig/ml) 53 + 3.2" 3.8 + 0.2* +Dexamethasone (1 nalml) 64 + 6.0 7.0 + 0.5d Confluent wells on a 24-well plate were incubated with 10e6 M NaI and the additions shown and de MUO T, and T3 production determined as described in Materials and Methods. Values are mean + SE (n = 3). ND, Not detected. ‘1 bP < 0.025 us. TSH. c.dP < 0.005 us. TSH. Addition
COrtlSOl
Endo. Voll30.
TABLE 2. Effect of pharmacological and physiological concentrations of insulin on TSH-stimulated T, and Ta production 7%
cortisol
IN THYROID
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THYROID
HORMONOGENESIS
6
BBSH
TSH (100uU/ml) ICF I (30ng/ml) ICF II (3Ong/ml)
+ +
!
” $ 4' P 2;
0
+
+
FIG. 6. Effect of IGF-I and IGF-II on TSH-stimulated T, and T, production in 3H medium. Results are the mean f SE of triplicates of a representative experiment. *,P< 0.05 us. 3H; **,P< 0.05 us. TSH.
Our data are in accord with previous reports in cultured porcine, ovine, and rat thyroid cells on the potentiation of TSH-stimulated iodide transport, iodine organification, and thyroglobulin expression by insulin or IGFI (3, 16, 20) but additionally now confirm an actual increase in Tq and T3 formation. Still, the precise mechanisms by which insulin or IGF-I potentiate thyroidal iodine metabolism are unclear. A minimal effect of insulin/IGF-I on thyroid peroxidase messenger RNA has been shown in comparison to that of TSH alone (21). Previous studies of thyroid cell growth and thyroglobulin expression suggest that IGF-I and high concentrations of insulin, as used in this study, interact with TSH via the type-1 IGF receptor upon events distal to generation of CAMP (16, 22). Insulin may also act through its own receptor. Physiological concentrations of insulin were recently reported to enhance pertechnetate uptake, iodine organification, and thyroglobulin expression, in addition to DNA synthesis, in sheep thyroid cells (20). In our studies, similar concentrations of insulin also enhanced iodine uptake and organification, but we were unable to demonstrate any increase in T4 and TB production during prolonged culture with NaI. Thus, physiological concentrations of insulin acting via the insulin receptor may not have a major impact on resultant thyroid hormonogenesis, in contrast to the significant effects of physiological concentrations of IGFs, which are presumably acting through type-1 IGF receptors. An alternative explanation for the negative results at low insulin concentrations would be its degredation during prolonged cultured (23). This also raises an other important issue with respect to in vitro assessments of differentiated thyroid function; that iodine uptake and organification may not always reflect thyroid hormone formation. The superior effect of high-dose insulin on thyroid hormone synthesis compared to IGF-I and IGF-II in our studies may reflect a combined action of pharmacological concentrations of insulin through type-1 IGF receptors and insulin receptors. Recently, inhibitory effects of insulin
IN THYROID
FOLLICLES
2193
and IGF-I upon TSH- and CAMP-stimulated iodide transport have been reported in FRTL-5 rat thyroid cells maintained in serum-depleted medium, an effect opposite to that seen when cells are incubated in 5% serum (17, 24). The significance of these findings, the former which contrast the present study is unclear but may relate to the differences between primary cultures as opposed to a passaged cell line. To our knowledge, this is the first report of significant bioactivity of IGF-II on thyroid hormone synthesis although IGF-II is an important autocrine factor in the growth of FRTL-5 rat thyroid cells (25). We find IGF-II to be equipotent to IGF-I in stimulating thyroid hormone formation even though IGF-II exhibits reduced affinity for the type-1 IGF receptor in sheep thyroid cells (11). This raises the possibility that IGF-II may also act in the thyroid through the typeII IGF/mannose-6-phosphate receptor which is supported by in vivo studies in hyperplastic rat goiters demonstrating increased IGF-II, but not IGF-I, binding (26).
Moreover, we find that cortisol optimally stimulates iodine uptake and organification in the presence of TSH or CAMP, in agreement with earlier reports that cortisol, along with TSH, stimulated thyroglobulin expression or iodide uptake in bovine and canine thyroid cells (6, 14). We extend these results in demonstrating increased T4 and T3 production. Cortisol requires the presence of IGFI or insulin to enhance TSH-dependent iodide efflux in FRTL-5 cells (27) and in an ovine thyroid cell strain thyroglobulin production was potentiated by cortisol only in the presence of insulin (15). The effect of cortisol on thyroid hormonogenesis reported in this study is notable in that there is no requirement for exogenous insulin or IGFs. On the other hand, inhibition of iodide transport by cortisol has been reported recently in FRTL-5 cells (4), and previous in vivo studies in humans subjects suggest that cortisol inhibits thyroid function overall by decreasing TSH secretion and increasing urinary iodide clearance (5, 28, 29). The precise mechanism(s) of glucocorticoid action, whether stimulatory or inhibitory, upon thyroidal iodine metabolism remains unclear. Posttranscriptional regulation of iodide transporter activity has been suggested (4). The present studies indicate a complex hormonal regulation of thyroid hormone biosynthesis by cortisol, insulin, or IGFs, dependent upon prior TSH or CAMP stimulation. The effect of insulin at high concentrations is consistent with an action, in part, through type-1 IGF receptors. This action may be met in situ by an autocrine or paracrine production of IGFs as reported by ourselves and others in sheep thyroid cells (9-11). These same studies have also demonstrated the release of IGFBPs in vitro. While IGFs appear to be constituitively expressed, we have shown the biosynthesis and release of IGFBPs to be hormonally regulated (10,30) and recently reported
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2794
THYROID
HORMONOGENESIS
an inverse correlation between inhibition of IGFBP presence in conditioned culture media by TSH and cortisol and the stimulation of iodine organification (10). The precise function(s) of IGFBPs are unclear but may include modulation of tissue IGF availability to IGF receptors and their resultant biological activity (31,32). Thus, the possibility exists that in sheep thyroid cells, TSH and cortisol control thyroid hormone formation, in part, through interactions with locally produced IGFs and their associated IGFBPs. Acknowledgement The authors wish to thank Ms. Michele assistance in preparation of the manuscript.
IN THYROID
14. 15. 16.
17.
18. Falconer for excellent
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