Molecular and Cdlular Endocrb~k~~~. X6 (1992) I I-20

% 1992 Elsevier Scientific Publishers Ireland, Ltd. 030.1-7207/92/$05.00

II

MOLCEL 02772

Transfected insulin-like growth factor II modulates the mitogenic response of rat thyrocytes in culture

Bianca Maria Veneziani, Cinzia Di Marino, Paola Salvatore, Giovanni Villone, Nicola Perrotti, Rodolfo Frunzio and Donatella Tramontano Dpt. di Medicinu Spr~rimcvtale c C‘liniccr, Uni/wxitic tii Keg,+ C‘aluhriu, 88100 Catunzaro, Iialy,

and CEOS / CNR and Dpt. di Biologia L’ Patologiu Cellulrrrc~ e Molecolore, Unrr~cvzsitri di Nupoli, X01.31 Nuples, II&

(Received 16 October 1991: accepted 2X February 1902)

KcJy ~rrls: Somatomedin; Cell growth; FRTLS cell: Thyroid

Summary

Rat thyroid cells (FRTLSI, transfected with the sequence coding for rat insulin-like growth factor 11 (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [“Hlthymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTLS cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.

Introduction

Although thyrotropin is a potent mitogen for thyroid follicular cells (Roger et al., 1983, 1988; Dere and Rapoport, 1986; Jin et al., 1986; Tra- montano et al., 1986a, 1988; Ingbar et al., 1987), studies on the regulation of thyrocite prolifera- tion in vitro have indicated that growth factors

like insulin, insulin-like growth factor I (IGF-I) and IGF-II, or epidermal growth factor (EGF),

play a key role in this complex phenomenon. In several thyroid systems in vitro, growth factors induce cell proliferation per se and, more impor- tantly, modulate the mitogenic effect of thy- rotropin (TSH) (Dumont and Roger, 1982; West- ermark and Westermark, 1982; Westermark et al., 1983; Smith et al., 1986; Tramontano et al., 1986b; Takasu et al., 1989; Takahashi et al., 1990).

Correspondence to: Prof. Donatella Tramontano, Dpt. di

Medicina Sperimentale e Clinica, Policlinico Mater Domini, via T. Campanella, 88100 Catanzaro. Italy. Tel. 961.770278;

Fax 961.775373.

Recently, data from several laboratories sug- gest that thyroid cells produce growth factors, mainly of the family of IGFs (Clemmons et al., 1985; Mak et al., 1985; Maciel et al., 1988; Min-

uto et al., 1989; Villone et al., 1989) and may respond to them in an autocrine fashion. In this respect, FRTLS cells, rat thyroid cells in culture, seem to represent a useful model since they re- spond to the mitogenic stimuli of insulin and

IGFs and they possess on their membrane insulin receptors and both type I and type II IGF recep-

tors (Tramontano et al., 1987). In addition they have been shown to produce in the culture medium small amounts of a peptide immunologi- cally related to IGF-II, whose presence influ- ences the proliferative response of FRTLS cells to TSH (Maciel et al., 1988; Villone et al., 1989). In the present study we have investigated whether an increased production of endogenous IGFs would influence FRTLS cell proliferation stimu- lated via TSH and via insulin-like peptides. We have addressed this question transfecting into FRTLS cells a rat IGF-II expression vector to- gether with a vector expressing a selectable marker. Several subclones were obtained and an- alyzed for their proliferative parameters.

Materials and methods

Materials Materials were purchased from the following

sources: Coon’s modified Ham F12 tissue culture medium from Hazleton Research Products (Den- ver, CO, USA); calf serum and t_-glutamine from

Gibco (Grand Island, NY, USA); Dulbecco modi- fied Eagle’s medium (DMEM) from Flow (Irvine, Scotland, UK); bovine serum albumin (BSA) from Rehesis Chemical Co. (Phoenix, AR, USA); biosynthetic IGF-I (ThrSy-IGF-I) from AMgen Biologicals (Thousand Oaks, CA, USA); tissue culture dishes (24-well, 16 mm2 or 72 mm2) from Costar (Cambridge, MA, USA); tissue culture dishes (100 x 20 mm and 60 X 15 mm) and cen- trifugation tubes (15 ml) from Falcon, Becton and Dickinson Co. (Oxnard, CA, USA); bovine TSH (bTSH) from Sigma Chemical Co. (St. Louis, MO, USA); [“HImethyl-thymidine (1.0 mCi/ml) and [ ““I]IGF-I from Amersham. Highly purified bovine TSH AFP-5555B was kindly provided by the National Hormone and Pituitary Program, NIDDK, NIH, University of Maryland, School of Medicine; sm1.2 (antibody against IGF-II) was a

gift of Dr. L.E. Underwood and Dr. J.J. Van Wyk, Chapel Hill, NC, USA).

Culture techniques

All the cell strains were routinely cultured in Coon’s modified Ham F12 medium supplemented with 5% calf serum and with three ‘hormones’:

bTSH (1 mU/ml>, insulin (1 pg/ml) and trans- ferrin (5 pg/ml) (3H medium). Cells were cul- tured in 100 X 20 mm dishes or 24-well Costar plates at 37°C in an atmosphere of 95% air-5% CO, in a humidified incubator. Techniques for the subculture of cells have been described in detail previously (Tramontano et al., 1986a). In some studies, as indicated, cells were maintained in the absence of bTSH, insulin or transferrin

(H-free medium).

Construction of an expression elector for the rat

IGF-II A genomic DNA fragment of rat IGF-II gene,

from the KpnI site at the beginning of exon 1 to the first Bgf II site of exon 3 which comprises the first two coding exons of 143 and 169 bp, inter- vening introns and a portion of exon 3 which includes the remaining of the coding region and approximately 600 bp of 3’ non-coding regions (Frunzio et al., 19861, was transferred from a pUC vector into an RSV expression vector. The

expression vector RSV-globin (Gorman et al., 1983) was cut with a restriction enzyme in the Hind111 and BglII sites and the globin sequences

were thus deleted and replaced with the IGF-II fragment described above. The obtained plasmid pRSV-IGF-II was then used in the cotransfection experiments, along with pRSVneo (Gorman et al., 1983). The plasmids pRSV-globin and pRSV- neo were a gift of Dr. B. Howard.

Transfection techniques FRTLS cells were seeded in 100 mm dishes in

3H medium (lo6 cells per dish). After 24 h medium was replaced with fresh medium and 3 h later Ca,PO, precipitated DNA was added and cells were incubated for 3 h at 37°C. Cells were then washed 3 times with serum-free medium, incubated with 3H medium for 48 h, trypsinized and seeded (approximately 5 x lo4 cells per dish) in 3H medium supplemented with geneticin (G418, 400 pug/ml).

RNA purification Total RNA was purified from FRTLS and Tr

cells as previously described (Chirgwin et al., 1979). Briefly, FRTLS and Tr cells were grown to confluence into 100 mm culture dishes; then monolayers were solubilized with guanidine iso- thiocyanate 4 M (2.5 ml/dish). The cell lysates

were applied to a cushion of 5.7 M CsCl, pH 5.2 and centrifuged 18 h at 42,000 rpm in an SW55 rotor. Supernatants were discarded and the pellet precipitated. 10 ng of total RNA were separated

on agarose gel and transferred to nitrocellulose and, then, hybridized with a “‘P-labeled IGF-II probe (Thomas, 1980).

/ -7H]Thymidine incorporation For studies of [ “Hlthymidine incorporation into

DNA, cells were sparsely seeded in H-free medium in 24-well Costar plates. Five days later, medium was removed, cells were washed 2 times and then incubated for 24 h with Coon’s modified Ham F12 medium devoid of serum. This medium was replaced by 500 ~1 of fresh medium contain- ing 0.1% BSA and the appropriate concentra- tions of the agents to be tested. After the incuba- tion time, cells were incubated for an additional 2 h with 250 ~1 of DMEM containing [‘Hlmethyl- thymidine (5 pCi/ml). The labeling was stopped by aspirating the medium and washing the cells

twice with phosphate-buffered saline (PBS) and 3 times with 10% ice-cold trichloroacetic acid (TCA). TCA precipitable material was solubilized with 500 ~1 of 2% sodium dodecyl sulfate (SDS). Cell-associated radioactivity was then counted in a scintillation spectrometer.

Growth curues For long-term experiments, cells were plated

in 60 mm dishes at a concentration of 1.5 x lO’/dish, in medium supplemented with serum and hormones as indicated in the legends to the figures. Replicate dishes were periodically trypsinized and counted in a Neubauer chamber.

Radioreceptor assay Binding studies were performed as previously

described (Tramontano et al., 1987). Final spe- cific activity of [‘251]IGF-I was 280 pCi/Fg. Briefly, for studies of the binding of IGF-I, cells

were grown to confluence in 24-well Costar plates in 3H medium replaced by H-free medium for an additional 5 days. Immediately before the binding studies, media were aspirated and monolayers were washed 3 times with buffer. [‘*“I]IGF-I and

decreasing concentrations of unlabeled peptide were then added to each well in 250 ~1 of modi- fied Krebs-Ringer buffer (KRB) containing 0.1%

BSA (mKRB). Steady-state binding experiments were carried out overnight at 4°C. At the end of

the incubation, cells were washed 3 times with mKRB. Cells were then solubilized in 1 N NaOH and “‘1 content of the cell lysates was deter-

mined. Protein concentration was measured in

aliquots of cell lysate by the method of Lowry (Lowry et al., 1951). Non-specific binding was

determined in the presence of 2.6 x lo-” M IGF- I. Scatchard plot analysis was performed as al- ready described (Scatchard, 1949).

Statistical analyses In all studies at least three separate samples

were studied for each experimental point. Analy- sis of difference in the values obtained in the various experimental groups was conducted by analysis of variance followed by Neuman-Keuls test for the significance of difference among mul- tiple experimental groups (Zar, 1974). All the

experiments were performed at least twice, and usually more often, with good agreement between the results obtained.

Results

Isolation of rIGF-II expressing clones The sequence coding for rat IGF-II was cloned

under the control of retroviral long terminal re- peat (pRSVIGF-II). FRTLS cells were cotrans- fected with pRSVIGF-II along with the pRSV- neo plasmid that confers the G418 resistance. Drug-resistant colonies were expanded and ana- lyzed for the presence of a messenger RNA for the transfected rat IGF-II. Three clones have been chosen and subjected to extensive studies. Clones Tr7 and Tr12 show high to intermediate levels of mRNA for the transfected rat IGF-II by Northern blot analysis, while clone Trll, like FRTLS cells, was negative under this experimen- tal condition (Fig. 1). Furthermore, the isolation

-4Kb

- 1.7Kb

FRTL5 7Tr 11Tr 12Tr Fig. 1. Northern blot analysis of the total RNA derived from

FRTLS cells and from Tr7, Trll and Tr12 clones, FRTLS, as

Trl I cells, does not show a signal corresponding to the

molecular weight of the mRNA for IGF-II. A clear band was

observed in the case of Tr7 and Trl2. Each lane contained 20

pg of total RNA. Ethidium bromide staining of the agarose

gel revealed that the amount of RNA analyzed was equal in

all samples.

of genomic DNA showed the presence of unrear- ranged plasmid of the IGF-II vector integrated in the Tr7 and the Tr12 genome in low copy num- ber. The Southern blot of the clone Trll genomic DNA showed the integration of the near plasmid but failed to reveal the integration of IGF-II plasmid (data not shown).

The effect of exogenous mitogens on the prolifera- tion of Tr cells

In order to investigate whether the presence of transfected IGFII RNA would influence the growth parameters of Tr cells we have analyzed [“Hlthymidine incorporation of Tr cells and FRTLS cells in the absence of added mitogens. Increasing numbers of the cells to be tested were

seeded and maintained for 4 days in H-free

medium, then t3H]thymidine incorporation was measured. In clones Tr7 and Tr12 the basal level of [ ‘Hlthymidine incorporation was significantly higher than that observed in Trl 1 or FRTLS cells at all cell numbers analyzed (Table 1).

FRTLS cells and Tr7 and Tr12 cells have been

cultured over 30 days in medium supplemented with 0.1% BSA, in the presence or in the absence

of TSH and IGF-I respectively or in combination. In the absence of mitogens, FRTLS cells remain quiescent; TSH and IGF-I, independently added, induced cell proliferation, and the effect was

enhanced when the two peptides were added together, as expected (Tramontano et al., 1986b) (Fig. 2, panel a). As for transfected cells, in the absence of added mitogens the number of Tr12 increased 50% over a 30-day period while Tr7 were more than doubled over the same length of time. The IGF-I effect on Tr cell proliferation was greatly reduced when compared to the IGF-I effect on parental cells (Fig. 2, panels b and c). On the other hand, Tr cells showed a normal proliferative response to TSH. The effect was not modified by the concomitant addition of IGF-I

(Fig. 2, panels b and c). The effect of IGF-I, IGF-II or insulin on DNA

synthesis in the Tr clones was studied (Fig. 3). IGF-I and IGF-II had no effect on DNA synthe- sis in Tr7 and Tr12 cells at concentrations able to induce dose-dependent [ -?H]thymidine incorpora- tion into DNA of quiescent FRTLS and Trll

TABLE I

EFFECT OF INCREASING CELL NUMBER ON TRITI-

ATED THYMIDINE INCORPORATION INTO DNA

(cpm/WELL)

Increasing numbers of FRTLS and Tr cells were seeded in

H-free medium; 5 days later cells were incubated for 36 h with

fresh H-free medium devoid of serum containing 0.1%~ BSA. Values represent the mean + SD of triplicate samples for each

experimental point.

Cell FRTLS Trll Tr7 Tr12 number

xx 10” 122k74 125k 13 4.172+ 431 2,192+ 549 2x 10’ 221+4Y 209k 22 16,352+4,188 8,37Y+ 4x0

4x IO’ 420 f 28 471 f 102 27,200 f 2,620 22,399 k 1,555 8X 10s 662k44 702+ S8 29,464k 8X0 17.612k 2,783

0-o conlrol *-AT!% 1nM D0IGF-I UnM l -•TSH+IGF-I

td 1 10 20 30 10 30 30

TIME (days)

L

10 20 30

Fig. 2. The effect of TSH and IGF-I on the proliferation of FRTLS cells and Tr7 and Tr12 clones. Cells were trypsinized and

counted at various time intervals; values represent the mean of triplicate samples.

cells (Fig. 3). Insulin retained a week stimulatory effect only at high doses.

The effect of sm1.2 on the proliferation of Tr cells sm1.2, a monoclonal antibody against IGF-II

with a sligth cross-reactivity with IGF-I, inhibits basal [‘Hlthymidine incorporation into DNA of FRTLS cell. We have investigated the effect of sm1.2 on the proliferation of the transfected clones. sm1.2 induced a dose-dependent reduc- tion of basal [ “Hlthymidine incorporation already significant at a concentration of 1 ng/ml in all cells tested. As shown in Table 2, [3H]thymidine incorporation in Tr7 and Tr12 clones was re- duced up to 82-85% at concentration of sm1.2 of 50 ng/ml.

Binding of [‘2-iI]IGF-I to Tr cells It is generally accepted that IGF-II binds the

type I receptor with affinity almost equal to IGF-I and that the type I IGF receptor mediates the biological effect of IGF-II (Furlanetto et al., 1987; Steele-Perkins et al., 1988; Adashy et al., 1989;

Rosenfeld et al., 1991). Then, to determine whether the rIGF-II peptide secreted into the medium of the Tr7 and Tr12 cells interacted with a surface receptor for IGFs in those cells, the affinity and the total binding capacity of type I receptor was measured by steady-state binding of [1251]IGF-I. Scatchard plot analysis (Fig. 4) of

TABLE 2

EFFECT OF sm1.2 ON BASAL [‘HITHYMIDINE INCOR-

PORATION (PERCENT OF INHIBITION)

Effect of sm1.2 on the autonomous proliferation of FRTLS,

Tr7, Trll and Tr12. Increasing concentrations of sm1.2 were

added to cells to be tested for 36 h, then [‘Hlthymidine

incorporation was measured. Values are the mean of tripli-

cate samples (SD < 5%).

Cell smI.2 concentration

1 ng/ml 10 ng/,ml 50 ng/ml

FRTLS _ 46 _

Tr7 24 59 82 Trll 31 43 46

Tr12 55 79 85

lh

competition studies showed that Tr7 and Tr12 possessed a type I IGF receptor with the same affinity of that present in the FRTLS cells, but a reduced binding capacity of almost 70%.

Biological effects of media conditioned by trans- fected cells

The ability of the media conditioned by Tr clones to induce L3Hlthymidine incorporation into DNA of FRTLS cells was studied. Conditioned media undiluted or diluted 1: 5 or 1: 2 with Coon’s

FRTL 5 / 0

. ?l,, / / .

0

-10 -e -8 -7 -6

7 Tr

l o/O .-:A0 /

-10 r I I I 1 4 -8 -7 -6

modified Ham F12 medium containing 0.1% BSA were examined. Added alone, media conditioned by Tr7 and Tr12 cells slightly increased [“Hlthymidine incorporation into DNA of quies- cent FRTLS cells. As for medium conditioned by FRTLS cells, it consistently induced only a small increase of [“Hlthymidine incorporation into DNA of FRTLS cells, as already reported else- where (Maciel et al., 1988). More importantly, when FRTLS cells were incubated with the media conditioned by the transfected clones or FRTLS

2(

l!

1C

s

0

10

5

0

11 Tr

/ .

#

0

.

I -To -2

I I 1 -8 -7 -0

12 Tr

Fig. 3. The effect of varying concentrations of insulin, IGF-I and IGF-II on [“Hlthymidine incorporation into DNA of parental and transfected cells. The results are the mean of triplicate experimental points. The SD was less than 5%.

o-o ML5 m-9 7 Tr

a--allTr

B/F l -.12Tr

I 0 25 50

BOUND (fmoles)

Fig, 4. Scatchard plot analysis of competition studies revealed

that the Tr7 and Tr12 cells possess receptors for IGF-I with

the same affinity as those present in FRTLS and Trll cells

but a reduced maximum binding capacity.

FRTLs medium

--- blSH OlnM

o-0 CM 1lons

l -• CM+TSn

7Tr medium

17

cells (Fig. 5) in the presence of TSH, values of [ 3H]thymidine incorporation were significantly in- creased in respect to those observed in FRTLS cells treated with TSH alone.

Discussion

Growth of rat thyroid cells in culture, FRTLS, is regulated by the complex interactions of the post-receptoral pathways triggered by TSH and IGFs via specific receptors present on the mem- brane of FRTLS cells (Jin et al., 1986; Tramon- tano et al., 1987, 1988). TSH has a potent and independent stimulating effect on FRTLS cell proliferation; however, its mitogenic activity is greatly amplified by the addition of appropriate concentrations of IGF-I, IGF-II or insulin (Tramontano et al., 1986b). In addition, Maciel and coworkers (1988) have reported that FRTLS cells produce and secrete in their culture medium a peptide immunologically related to IGF-II, which influences their proliferative response to

12Tr mrd iua

CONDITIONED MEDIA CONCENTRATION (%I

11Tr medium

.

-\

.

Fig. 5. Effect of media conditioned by FRTLS and transfected cells on i3H]thymidine incorporation into DNA of quiescent FRTLS

cells. Values represent the mean of triplicate samples (SD < 5%).

IX

TSH, as demonstrated by treatment of FRTLS

cells with sm1.2, an antibody against IGF-II,

which strongly inhibited cell proliferation induced

by TSH. sm1.2 binds with high affinity the IGF-II secreted by the cells and in consequence IGF-II

is unable to bind the type I IGF receptor which has been demonstrated to mediate the mitogenic effect of IGF-II. Although IGF-II also binds with

high affinity to the type II IGF receptor, which has proven to be identical to the mannose-6- phosphate receptor, the ability of the IGF-II/M- 6-P receptor to mediate classical ‘somatomedin- like’ actions of IGF-II remains in doubt (Fur- lanetto et al., 1987; Steele-Perkins et al., 1988; Adashy et al., 1989; Rosenfeld et al., 1991).

The results reported above supported the hy- pothesis that IGFs produced by thyroid cells act

as autocrine factor required for the full expres- sion of the mitogenic effect of thyrotropin (Vil- lone et al., 1989). Following this line of reasoning, we have investigated in which way, if any, in-

creased production of transfected IGFs would influence the proliferative response of rat thyroid cells in culture. To do so, FRTLS cells have been cotransfected with a vector coding for rIGF-II along with a vector coding for the neomycin resis- tance gene (German et al., 1983; Frunzio et al., 1986) Clones Tr7, Trl 1 and Tr12 have been in- tensively investigated. Clone Trll, although neomycin resistant, does not contain the trans- fected rIGF-II. Clones Tr7 and Tr12 presented in their genome the transfected insert, and a mRNA which hybridizes with an IGF-II probe.

The analysis of the proliferative properties of the transfected clones showed that clones Tr7 and Tr12 have a basal [‘Hlthymidine incorpora- tion significantly higher than the parental FRTLS cells and the Trll clone. Moreover, contrary to parental FRTLS cells, clones Tr7 and Tr12 are able to proliferate in the absence of exogenous stimuli. This result supports the hypothesis that the Tr clones expressing the IGF-II insert behave in an autocrine fashion and respond to trans- fected mitogens. Several lines of evidence indi- cate that the mitogenic activity present in the culture media of Tr7 and Tr12 clones is related to the IGF family insofar as sm1.2, an antibody raised against IGF-11, reduces the basal [“HIthy- midine incorporation in the Tr7 and Tr12 cells.

IGFs failed to stimulate Tr7 and Tr12 DNA

synthesis at concentrations that vigorously induce

[3H]thymidine incorporation in the FRTLS and Trll cells. On the other hand, a week-long in-

sulin-dependent proliferative response is shown by Tr7 and Tr12 cells; this effect may be medi- ated primarily by the insulin receptor.

Similarly to FRTLS parental cells, Tr clones

are responsive to the mitogenic action of thy- rotropin alone, even in the absence of serum. However, the presence of a mitogenic activity in the culture media of Tr7 and Tr12 ceils influ- ences their response to TSH, as demonstrated by the ability of TSH to maximally stimulate Tr cell growth even in the absence of exogenous IGF.

IGF-I, IGF-II and insulin, at a concentration which induced an abundant increase in [‘HIthy- midine incorporation into DNA of FRTLS and Trll cells, failed to stimulate DNA synthesis in

clones Tr7 and Tr12. This result could be ex- plained by a reduced capacity of the type I IGF receptors present on the membrane of Tr7 and Tr12 cells. In that, type I IGF receptors might be continuously occupied by the peptide coded by the transfected gene and in consequence might not be available for exogenous peptide. It could be speculated that the number of available recep- tors is a limiting factor on the biological activity of the growth factor. Release of insulin-like growth factors has been demonstrated in the cul- ture medium of a variety of cells in culture and, more recently, evidence has been provided for their synthesis in human and rat tissues (D’Ercole et al., 1980, 1984; Clemmons and Van Wyk, 1985; Stiles et al., 1985; Brown et al., 1986; Han et al.,

1987). The media conditioned by Tr7 and Tr12 cells

stimulated both basal and TSH-stimulated [ “Hlthymidine incorporation in quiescent FRTLS cells, suggesting that the peptide produced by Tr7 and Tr12 cells is biologically active. In addition, TSH amplifies the effect of mitogenic activity present in the media conditioned by Tr clones as it does that of exogenous IGFs. The data pre- sented here support once more the key role of the mitogenic pathway induced by TSH which cannot be substituted by increasing concentra- tions of endogenous IGFs. However, the ability of high concentrations of IGFs produced and se-

19

creted by thyroid cells to influence their prolifer-

ative response in culture is, in our view, a clear indication that thyroid cell proliferation is regu- lated by more than one pathway and, moreover,

that the various mitogens cannot be considered as interchangeable with thyroid cells. On the con- trary, we believe that only the concomitant activa- tion of the various pathways, and their coordinate action, allow the full expression of the prolifera- tive response of the thyrocytes.

Production of IGFs has been demonstrated in ovine thyroid in primary culture, in the FRTLS cells and also in human tissues (Clemmons et al., 1985; Mak et al., 1985; Maciel et al., 1988; Min- uto et al., 1989; Villone et al., 1989) and it is likely that IGFs, produced locally within the thy-

roid, alone or in concert with TSH, play a role both in the physiological and pathological state of the thyroid gland. It is known that IGF-II is mainly produced during fetal life (Brown et al., 1986). since in the human fetus TSH is produced only after 14 weeks of gestation (Toran-Allerand, 1986), when thyroid cells are already present and thyroglobulin is being produced, and conse- quently it is possible that IGFs are involved in regulating the early stages of development of the gland. Finally, disorders in the production of en- dogenous IGFs could be responsible for goiter of unknown origin in the adult.

Acknowledgments

This work was partially supported by a grant from AIRC Italy and from Progetto Finalizzato

BTBS, CNR Italy.

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