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Molecular Endocrinology 14 (3): 440-448
Copyright © 2000 by The Endocrine Society

Thyrotropin Induces SOCS-1 (Suppressor of Cytokine Signaling-1) and SOCS-3 in FRTL-5 Thyroid Cells

Eun Shin Park, Ho Kim, Jae Mi Suh, Soo Jung Park, O-Yu Kwon, Young Kun Kim, Heung Kyu Ro, Bo Youn Cho, Jongkyeong Chung and Minho Shong

Department of Internal Medicine (E.S.P., H.K., J.M.S., S.J.P., Y.K.K., H.K.R., M.S.) Department of Anatomy (O.-Y.K.) Chungnam National University School of Medicine Taejon, 301–040, Korea
Department of Internal Medicine (B.Y.C.) Seoul National University School of Medicine Seoul, 110–744, Korea
Department of Biological Sciences (J.C.) Korea Advanced Institute of Science and Technology Taejon 305–701, Korea


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TSH has multiple physiological roles: it is required for growth, differentiation, and function of the thyroid gland, and it regulates transcription of interferon-{gamma} (IFN-{gamma})-responsive genes in thyrocytes, including genes for the major histocompatibility complex and intercellular adhesion molecule-1. This report demonstrates that TSH induces the expression of suppressor of cytokine signaling (SOCS)-1 and -3 proteins and alters the phosphorylation state of signal transducer and activator of transcription (STAT) proteins STAT1 and STAT3. The expression of SOCS-1 and SOCS-3 and the phosphorylation state of STAT1 and STAT3 were examined after treatment with TSH or IFN-{gamma} in either TSH-sensitive FRTL-5 thyroid cells or TSH-insensitive FRT and buffalo rat liver (BRL) cells, which lack functional TSH receptors. SOCS-1 and SOCS-3 are constitutively expressed in FRTL-5 cells, but not in FRT and BRL cells. IFN-{gamma} up-regulated SOCS-1 and SOCS-3 RNA and protein in FRTL-5 cells, as reported previously for nonthyroid cells. Interestingly, TSH also significantly induced SOCS-1 and SOCS-3 in FRTL-5 cells, but not in FRT and BRL cells. When SOCS-1 or SOCS-3 was overexpressed in FRTL-5 cells, STAT1 phosphorylation at Y701 and STAT1/DNA complex formation in response to IFN-{gamma} were reduced. Furthermore, overexpression of either SOCS-1 or SOCS-3 significantly inhibited the IFN-{gamma}-mediated transactivation of the rat ICAM-1 (intercellular adhesion molecule-1) promoter. TSH and IFN-{gamma} had different effects on STAT1 and STAT3 phosphorylation. The phosphorylation of Y701 in STAT1, which is responsible for homodimer formation, nuclear translocation, and DNA binding, was specifically stimulated by IFN-{gamma}, but not by TSH or forskolin. However, the phosphorylation of S727 in STAT1 was induced by IFN-{gamma}, TSH, and forskolin. TSH induced phosphorylation of both Y705 and S727 in STAT3, while IFN-{gamma} phosphorylated only the Y705. In addition, we found that SOCS-3 was associated with JAK1 and JAK2 and that these associations were stimulated by TSH. These findings demonstrate that TSH induces SOCS in thyroid cells and provides the evidence of signal cross-talk between TSH and cytokines in thyroid cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cytokines are elicited during the progression of autoimmune thyroid diseases, and they exert multiple biological effects such as abnormal regulation of immune response genes and altered growth and function of thyroid cells (1, 2). The interactions of type I and type II interferons and interleukin-6 type cytokines with their specific receptors activate receptor-associated Janus kinases (JAKs) (3, 4). The activation of the JAKs is crucial for propagation of the signal cascade via proteins such as STAT (signal transducer and activator of transcription) factors (3, 5, 6, 7, 8, 9). The activated JAKs phosphorylate receptors and STATs, leading to altered gene expression, cellular differentiation, and cell growth properties (5, 6, 7, 8, 9).

The proinflammatory cytokine interferon-{gamma} (IFN-{gamma}) has pleiotropic effects on cells and is involved in a variety of pathophysiological conditions associated with autoimmune thyroid disease (10). The thyrocyte response to IFN-{gamma} is mediated by two distinct classes of proteins, Janus kinases (JAK1 and JAK2) and STAT1 (11). The activation of STAT1 participates in the regulation of many genes by binding to an IFN-{gamma}-activated site (GAS) in the promoter region (12, 13, 14, 15, 16). IFN-{gamma}-activated genes include interferon regulatory factor-1 (IRF-1) (12), IRF-2 (13), intercellular adhesion molecule-1 (ICAM-1) (14), and MHC class II transactivator (CIITA) (15, 16). Recently, four proteins were identified that are members of the suppressors of cytokine signaling family, SOCS-1, SOCS-2, SOCS-3, and cytokine-inducible SH2 domain-containing protein (CIS)(17, 18, 19, 20, 21, 22). These proteins appear to provide negative regulation of cytokine signal transduction pathways (17, 18, 19, 20, 21, 22).

TSH has long been regarded as an important physiological regulator of the growth and function of the thyroid gland (2). The function of TSH depends on the receptor-mediated activation of adenylate cyclase leading to cAMP synthesis and protein kinase A (PKA)-mediated phosphorylation of transcription factors that regulate thyroid gene expression (23, 24). Despite the pivotal roles of cAMP and PKA in TSH signaling, it is likely that additional mechanisms are involved in the regulation of thyroid-specific gene expression and cellular function in the thyroid. It was recently observed that TSH also negatively regulates the expression of MHC class I (25, 26, 27) and ICAM-1 (28, 29) by inhibiting STAT1 (30). These findings suggest that TSH down-regulates the IFN-{gamma}-activated JAK-STAT signaling pathway.

This report demonstrates that TSH induces SOCS (suppressor of cytokine signaling) proteins, which are negative regulators of cytokine signaling, and alters the phosphorylation state of STAT factors. These observations indicate that TSH signaling interacts with the JAK-STAT pathway; thus, there is signaling cross-talk between TSH and cytokines, and this functional interaction contributes to maintaining thyrocyte function during normal and pathological conditions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TSH-Mediated Expression of SOCSs and CIS RNA in FRTL-5, FRT, and BRL Cells
The expression of SOCS-1, SOCS-2, SOCS-3, and CIS was examined by Northern blot using total RNA from FRTL-5 cells maintained in 5% calf serum and 3H medium lacking hydrocortisone, insulin, and TSH. IFN-{gamma} increases SOCS-1, SOCS-3, and CIS RNA (Fig. 1AGo, lanes 2 and 7). After addition of IFN-{gamma}, CIS RNA expression was greater at 2 h than at 4 h (Fig. 1AGo, lane 2), but SOCS-1 and SOCS-3 RNA expression was greater at 4 h than at 2 h (Fig. 1AGo, lane 7). SOCS-2 RNA was not detected in FRTL-5 cells even after stimulation with IFN-{gamma} (data not shown). TSH, which is important for the growth and function of FRTL-5 cells (24, 25, 26), significantly augmented the SOCS-1 and SOCS-3 RNA levels (Fig. 1AGo, lane 3 and lane 8) as compared with the control (Fig. 1AGo, lane 1 and lane 6). Forskolin (100 µM) mimicked the action of TSH on SOCS-1 and SOCS-3 RNA. In contrast to SOCS-1 and SOCS-3, CIS RNA was not induced by TSH or forskolin at 2 h or 4 h (Fig. 1AGo, lanes 4 and 9). The induction of SOCS-1 and SOCS-3 expression by TSH and forskolin was transient; maximal induction was within 2 h of treatment, and within 4 h of treatment near-basal expression was restored. The antithyroid drug, methimazole (MMI), had no effect on SOCS-1, -3, or CIS RNA expression.



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  		Figure 1. Effects of IFN-{gamma}, TSH, Forskolin (FSK), and Methimazole (MMI) on SOCS-1, SOCS-3, and CIS RNA Levels in Rat FRTL-5, FRT, and BRL Cells

A, After FRTL-5 cells were grown to near confluency in complete 6H medium with 5% serum, cells were maintained for 6 days with 3H medium that does not contain hydrocortisone, insulin, or TSH. The medium was replaced with fresh medium including the following additions as indicated: 100 U/ml IFN-{gamma}, 1 x 10-9 M TSH, 100 µM FSK (forskolin), and 0.5 mM MMI. B, FRT and BRL cells were grown to near confluency in their respective media. The cells were then treated with or without 100 U/ml IFN-{gamma} or 1 x 10-9 M TSH. RNA was isolated 2 h after the final treatment and subjected to Northern analysis (20 µg/lane) using probes for ICAM-1, MHC class I, and rat ß-actin. Representative Northern analyses are shown.

 
Expression of SOCS-1, -2, -3, and CIS was also examined in FRT thyroid cells and BRL cells, which lack thyroid transcription factor-1 (TTF-1) (31, 32) and thyroid-specific gene expression (TSH receptor, thyroglobulin, and thyroperoxidase). In these cells, IFN-{gamma} induced SOCS-1 RNA (Fig. 1BGo, lanes 2 and 5), but TSH did not (Fig. 1BGo, lanes 3 and 6). SOCS-2, SOCS-3, and CIS RNA were not detectable in FRT or BRL cells with or without IFN-{gamma} stimulation (data not shown).

Western blot analysis (Fig. 2Go) demonstrated the TSH-mediated induction of SOCS-1 and SOCS-3 proteins in FRTL-5 cells (Fig. 1Go). The SOCS-1 and -3 proteins were readily detectable in FRTL-5 cells maintained in 3H 5% medium (Fig. 2Go, lane 1), and the level increased and was maintained at a high level after the addition of TSH (Fig. 2Go, lanes 2–4). As expected, IFN-{gamma} augmented the expression of SOCS-1 and SOCS-3 protein; the level of expression at 6 h after treatment of IFN-{gamma} was higher than with treatment of TSH (Fig. 2Go, lane 2 vs. lane 4). SOCS-2 protein was not detected by Western blot, which is consistent with its Northern blot analyses (data not shown).



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  		Figure 2. Effects of TSH and IFN-{gamma} on the Level of SOCS-1 and SOCS-3 Proteins

FRTL-5 cells were treated with TSH and IFN-{gamma} as indicated. Untreated cells are shown in lane 1. Total cell lysates were resolved by SDS/PAGE and analyzed using anti-SOCS-1 and anti-SOCS-3 antibodies.

 
Overexpression of SOCS-1 and SOCS-3 Protein in FRTL-5 Cells Inhibits Tyrosine Phosphorylation, Nuclear Translocation, and Transactivation of STAT1 in Response to IFN-{gamma}
STATs participate in cytokine-stimulated signaling pathways in the thyroid. The following experiments were designed to test whether SOCS-1 and SOCS-3 may be involved in signaling in FRTL-5 cells stimulated by TSH and IFN-{gamma}. In particular, the effect of SOCS RNA and protein induction on tyrosine 701 phosphorylation of STAT1 was directly examined using a transient transfection experiment. FRTL-5 cells were transfected with expression vectors for SOCS-1 (pEF-SOCS-1) or SOCS-3 (pEF-SOCS-3) (17) and treated with IFN-{gamma} (100 U/ml) for 30 min. Western blot and electrophoretic mobility shift assays (EMSA) were then carried out with whole-cell lysates and nuclear extracts, respectively. The level of phosphorylation of Y701 in STAT1 was evaluated using an antibody that specifically recognizes the Y701 phosphotyrosine in STAT1. IFN-{gamma} stimulated the phosphorylation of Y701 on STAT1 in FRTL-5 cells transfected with the empty expression vector, pEF (Fig. 3AGo, lane 3). However, FRTL-5 cells overexpressing SOCS-1 or SOCS-3 had a significantly lower level of Y701 phosphorylation after IFN-{gamma} stimulation (Fig. 3AGo, lanes 4 and 5).



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  		Figure 3. Effects of SOCS-1 and SOCS-3 Overexpression on IFN-{gamma}-Mediated Activation of STAT1

A, Effects of SOCS-1 and SOCS-3 overexpression on IFN-{gamma}-mediated phosphorylation of Y701 in STAT1. FRTL-5 cells transfected with pEF-SOCS-1 and pEF-SOCS-3 were treated with IFN-{gamma} (30 min) and used to prepare whole-cell lysates. Total cell lysates were resolved by SDS/PAGE, and phosphorylated forms of STAT1 were detected by antibodies specific for Y701-phosphorylated STAT1. B, IFN-{gamma}-mediated formation of the STAT1/DNA complex inhibited by overexpression of SOCS-1 and SOCS-3 in FRTL-5 cells. FRTL-5 cells transfected with pEF-SOCS-1 and pEF-SOCS-3 expression vectors were treated with 100 U/ml IFN-{gamma} for 30 min, and nuclear extracts were prepared. The nuclear extracts were incubated with radiolabeled oligonucleotide with the GAS sequence of the rat ICAM-1 promoter (-154 to -120 bp). EMSA was carried out as described in Materials and Methods.

 
EMSA was carried out with nuclear extracts from these cells and a radiolabeled probe including the GAS from the 5'-flanking region of the rat ICAM-1 gene. Overexpression of SOCS-1 or SOCS-3 significantly decreased the amount of STAT1/DNA complex detected in extracts from IFN-{gamma}-treated FRTL-5 cells (Fig. 3BGo, lane 3 vs. lanes 4 and 5).

Reporter constructs were prepared with promoters containing or lacking a functional GAS element and the luciferase reporter gene. The function of these constructs was tested in FRTL-5 cells after cotransfection in the presence or absence of SOCS-1 and SOCS-3 expression vectors. The promoter variants included 5'-deleted fragments of the rat ICAM-1 promoter, pCAM-431, pCAM-175, pCAM-175 GAS mut, and pCAM-97 (Fig. 4Go). pCAM-431 includes Sp1, nuclear factor-{kappa}B (NF-{kappa}B), GAS, and TATA regulatory elements, but pCAM-175 includes only the GAS element (11-bp core element, 5'-tttccgggaaa-3') and the TATA element. pCAM-175 GAS mut has a mutated nonpalindromic GAS element. Figure 5AGo shows that the promoter activities of pCAM-431 and pCAM-175, but not pCAM-175 GAS mut, were strongly stimulated by IFN-{gamma}, suggesting that the GAS is required for IFN-{gamma} inducibility of this promoter. Addition of TSH dramatically inhibited the promoter activities of pCAM-431 and pCAM-175 in the presence or absence of IFN-{gamma} (Fig. 5AGo). Thus, TSH inhibits both the induced expression level and the basal expression level of these constructs. These results strongly suggest that the promoter function of the GAS element is positively regulated by IFN-{gamma} and negatively regulated by TSH.



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  		Figure 4. Schematic Representation of the 5'-Region of the Rat ICAM-1 Promoter-Luciferase Reporter Constructs

The fragments of the ICAM-1 promoter region cloned upstream of a promoterless firefly luciferase cDNA in the pGL2-basic vector is shown (see Materials and Methods).

 


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  		Figure 5. Effects of TSH and SOCS on ICAM-1 Promoter Activity

A, Effects of TSH and forskolin on ICAM-1 promoter activities with and without IFN-{gamma} treatment. FRTL-5 cells stably transfected with the indicated ICAM-1-luciferase chimera were maintained with 5H 5% medium that does not contain TSH for 6 days before addition of 1 x 10-9 M TSH, 100 U/ml IFN-{gamma}, or both as noted. B, The effects of SOCS-1 and SOCS-3 overexpression on IFN-{gamma}-mediated transactivation of ICAM-1 promoter. FRTL-5 cells were transiently transfected with 20 µg of the indicated luciferase chimera. Luciferase assays were performed as described above (see Materials and Methods). The luciferase activity from untreated cells was used as the control. All experiments were repeated at least three times. Data are normalized for transfection efficiency and are presented as the mean ± SE of these experiments; significance, P < 0.005, was determined by two-way ANOVA.

 
The promoter activity and IFN-{gamma}-responsiveness of pCAM-431 and pCAM-175 were also tested in the absence or presence of SOCS-1 or SOCS-3 overexpression. The activities of pCAM-431 and pCAM-175 were increased about 8- and 7-fold in the presence of IFN-{gamma}, respectively. However, cotransfection of pEF-SOCS-1 or pEF-SOCS-3 with the reporter plasmids strongly decreased not only the basal activities of pCAM-431 and pCAM-175 (Fig. 5BGo), but also their induced activities by IFN-{gamma} (Fig. 5BGo). The inhibitory abilities of SOCS-1 and SOCS-3 to the IFN-{gamma}-mediated stimulation of the promoter activities of pCAM-431 and pCAM-175 were similar (Fig. 5BGo).

TSH Induces Phosphorylation of Tyrosine 705 and Serine 727 Residues of STAT3, but Only Serine 727 of STAT1
The results presented above show that TSH stimulates the expression of SOCS-1 and SOCS-3 in FRTL-5 cells (Fig. 1Go) and that SOCS overexpression inhibits IFN-{gamma}-mediated tyrosine phosphorylation, DNA binding, and transactivation activities of STAT1 (Figs. 3Go and 5Go). As previously described by others (17, 18, 19), the expression of SOCS is dependent on the activation of STAT1 and STAT3 by various cytokines. To understand whether the TSH-mediated induction of SOCS-1 and SOCS-3 was also regulated by STAT(s), we examined phosphorylation of tyrosine 701 (Y701) and serine 727 (S727) residues of STAT1 and phosphorylation of tyrosine 705 (Y705) and S727 of STAT3 in FRTL-5 cells. Figure 6AGo shows that IFN-{gamma}, but not TSH or forskolin, increased phosphorylation of tyrosine 701 in STAT1. In contrast, IFN-{gamma}, TSH, and forskolin all stimulated phosphorylation of serine 727 in STAT1 (Fig. 6AGo). Different results were observed for STAT3 (Fig. 6BGo); TSH induced the phosphorylation of both Y705 and S727 in STAT3 (Fig. 6BGo, lane 3), and IFN-{gamma} induced phosphorylation of Y705 without affecting S727 phosphorylation (Fig. 6BGo, lane 2). To address whether stimulation of FRTL-5 cells with TSH interferes with the phosphorylation of STAT1, proteins from lysates were immunoblotted with a specific antibody against tyrosine-phosphorylated (Y701) STAT1 (Fig. 6CGo). Stimulation of FRTL-5 cells with IFN-{gamma} (100 U/ml) resulted in a dramatic increase of phosphorylation in tyrosine 701 of STAT1 (Fig. 6CGo, lane 2), as expected. However, pretreatment with TSH significantly inhibited the IFN-{gamma}-induced phosphorylation of Y701 (Fig. 6CGo, lane 4).



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  		Figure 6. TSH Modulates the Phosphorylation of STAT1 and STAT3

After FRTL-5 cells were grown to near confluency in complete 6H medium with 5% serum, cells were maintained for 6 days with 5H medium that did not contain TSH. The medium was replaced with fresh medium including the following additions as indicated: 100 U/ml IFN-{gamma}, 1 x 10-9 M TSH, and 100 µM FSK. Total cell lysates were prepared 30 min after treatment and analyzed by SDS/PAGE. Phosphorylated forms of STAT1 were detected by phosphospecific antibodies. In panel C, the FRTL-5 cells were pretreated with TSH for 4 h, and medium was replaced with fresh medium including the following additions as indicated: 100 U/ml IFN-{gamma}, 1 x 10-9 M TSH, for 30 min.

 
For many cytokines, the signaling pathway involves the following steps: binding to their receptors, activation of receptor-associated cytoplasmic kinases (JAKs), and JAK-dependent phosphorylation of the receptor cytoplasmic domain and its associated STATs. SOCS proteins recognize and inhibit activated signaling molecules, including JAKs and cytokine receptors, through their SH2 and N-terminal domains. Thus, the effect of TSH on the association between SOCS-3 and JAK1/JAK2 was investigated in FRTL-5 cells. This experiment demonstrated that SOCS-3 was associated with JAK1 and JAK2 and that these associations increased in cells treated with TSH (Fig. 7Go).



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  		Figure 7. Association of JAK1 with SOCS-1 and SOCS-3 in Vivo

After FRTL-5 cells were grown to near confluency in complete 6H medium with 5% serum, cells were maintained for 6 days with 5H medium that did not contain TSH. Medium was replaced with fresh medium including 1 x 10-9 M TSH as indicated. Cell extracts were immunoprecipitated (IP) with antibodies specific for JAK1 and immunoblotted with anti-SOCS-1 (panel A) and anti-SOCS-3 (panel B). The filter was washed and reprobed with anti-JAK-1 and anti-IgG.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present report shows that TSH treatment of FRTL-5 cells induces SOCS proteins and alters the phosphorylation state of STATs and suggests that TSH-induced SOCS-1 and -3 are negative regulators of IFN-{gamma} signaling in these cells. Somatic cell genetics coupled with expression cloning has identified proteins that mediate interferon signal transduction (3, 4, 5, 6, 7, 8). Several groups have described a new family of cytokine-inducible proteins that inhibit the Jak-STAT signaling cascade. These proteins have been termed suppressors of cytokine signaling (SOCS) (17, 20, 21), STAT-induced STAT inhibitors (SSI) (19), CIS (18), and Jak binding protein (JAB) (18). The basal expressions of SOCS-1, SOCS-2, SOCS-3, and CIS RNA were observed in limited organs; SOCS-1 in thymus, spleen, and lung, SOCS-2 in testis, liver, and lung, SOCS-3 in the lung, spleen, and thymus, whereas CIS expression was more widespread, including testis, heart, lung, and liver. In this study, IFN-{gamma} increased the expression of SOCS-1, SOCS-3 and CIS, but not SOCS-2, in FRTL-5 thyroid cells. All of these expressions are related to the negative regulation of the IFN-{gamma} signaling cascade, but the relative potencies and time-dependent mechanisms for inhibiting JAK-STAT signaling cascade have not yet been determined. TSH increases the expression of SOCS-1 and SOCS-3, but not CIS, through the activation of TSH signaling in thyroid cells. These findings suggest that SOCS-1 and SOCS-3 are more important than CIS in TSH-mediated negative regulation of IFN-{gamma} signaling.

Recent studies show that TSH has multiple effects on the regulation of immune response genes in thyroid cells, including MHC class I (25, 26, 27), MHC class II (33, 34), and ICAM-1 genes (28). TSH suppresses MHC class I and ICAM-1 gene expression by modulating the action of specific transcription factors for these genes (25, 26, 27). These studies also showed that TSH can interfere with the induction of these genes by IFN-{gamma} (28). TSH typically triggers the cAMP signaling pathway, which can interrupt JAK-STAT, and NF-{kappa}B activation (35, 36). This study shows that induction of SOCS-1 and SOCS-3 is another mechanism contributing to signal interference and cross-talk between TSH and IFN-{gamma} in thyroid cells.

The SOCS proteins are relatively small molecules comprised mainly of an SH2 domain and a C-terminal homology domain termed SOCS box (20, 37). SOCS proteins recognize activated signaling molecules including JAK kinases and cytokine receptors through their SH2 and N-terminal domains and inhibit their activity (18, 19, 37). Zhang et al. (39) reported that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates, JAK kinases or cytokine receptors, to the proteosomal degradation pathway (37, 38). SOCSs expression is induced in response to a range of cytokines (17, 18, 19, 20, 21, 22) or hormones (22), and these proteins are therefore thought to form part of a negative feedback loop. In addition to their roles as negative regulators of cytokine signal transduction, SOCSs may have multiple biological functions mediated through their SOCS box, SH2 domain, and GTPase domains.

Although the regulation of SOCS-1 and -3 in specific tissues is not yet fully identified, their expression is dependent on STAT1 or STAT3 activation (19). With respect to the current study, it appears that TSH-mediated induction of SOCS-1 and -3 may be related to TSH-dependent phosphorylation of S727 in STAT1 and Y705 and S727 in STAT3. The phosphorylation of S727 of STAT1 and STAT3 is required for maximal transcriptional activation (5, 7) and cell survival (39, 40). It has been demonstrated that the MEK inhibitor PD98059-sensitive kinase can phosphorylate S727 in STAT1 and STAT3 (7), but it is not clear which kinase(s) mediates the TSH-induced phosphorylation of S727 in FRTL-5 thyroid cells. TSH strongly induced the phosphorylation of Y705 in STAT3 but not the phosphorylation of Y701 in STAT1 (Fig. 6AGo, lane 3, vs. Fig. 6BGo, lane 3). The molecular mechanism of the phosphorylation of Y705 tyrosine in STAT3 by TSH is not currently known, but it may involve the activation of the TSH receptor and JAKs, especially JAK2 (data not shown). IFN-{gamma} is more potent than TSH in the induction of SOCS-1 and SOCS-3 in FRTL-5 cells (Fig. 1AGo). When we treated the cells with IFN-{gamma} and TSH in FRTL-5 cells, they did not show synergistic effects on SOCS RNA induction (data not shown) because TSH inhibits IFN-{gamma}-mediated STAT1 tyrosine phosphorylation (Fig. 3CGo).

If TSH acts as a general negative regulator of cytokine action in thyroid tissue, then this study may be relevant to the pathogenesis of autoimmune thyroid diseases such as Graves’ disease and Hashimoto’s thyroiditis. Many cytokines are involved in the amplification or maintenance of the autoimmune process in the thyroid gland. Without doubt, TSH is one of the most important physiological factors required to maintain cellular survival and differentiated functions in thyroid gland. STAT1 activation (both constitutive and cytokine-stimulated) is involved in growth inhibition or apoptosis in other cell types, and IFN-{gamma} induced STAT1 activation is also involved in growth inhibition in FRTL-5 thyroid cells (our unpublished data). IFN-{gamma} is involved in many immunological perturbations during autoimmune thyroid diseases. Many cytokines, including type 1 interferons and interleukin-6 type cytokines, act through the same JAK-mediated signaling pathway as IFN-{gamma}. It is possible that TSH-mediated negative regulation via SOCS-1 and SOCS-3 may apply to other cytokine signals beside IFN-{gamma}. Thus, TSH could be a major rescue factor for maintaining thyrocyte survival and function during autoimmune thyroid disease.

One of the major factors in the pathogenesis of Graves’ hyperthyroidism is thyroid- stimulating autoantibodies. The effect of these autoantibodies on thyroid signal transduction is not known, but it is possible that they could induce SOCSs in thyroid cells. Continued studies of TSH regulation of cytokine signaling may elucidate the pathogenesis of such autoimmune thyroid diseases.

In summary, this study demonstrates the stimulatory effects of TSH on SOCS-1 and SOCS-3 expression in FRTL-5 cells and its effect on the TSH-induced phosphorylation/activation state of STAT1 and STAT3. Further studies of these cytokines and their effects on thyroid tissue will improve understanding of the mechanisms and physiological importance of cross-talk between the hormone and cytokine signaling pathways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Highly purified bovine TSH was obtained either from the hormone distribution program of the National Institute of Diabetes and Digestive and Kidney Diseases, NIH (NIDDK-bTSH I-1; 30 U/mg) or was a previously described preparation, 26 ± 3 U/mg, homogeneous in the ultracentrifuge, about 27,500 in mol wt, with the amino acid and carbohydrate composition of TSH. Rat recombinant IFN-{gamma} was from Life Technologies (Gaithersburg, MD). [{alpha}-32P] dCTP (3000 Ci/mmol) and [{alpha}-32P]ATP were from DuPont-Merck Pharmaceutical Co. (Wilmington, DE). The source of all other materials was Sigma (St. Louis, MO) unless otherwise noted.

Cell Culture
Buffalo rat liver cells (BRL 3A, ATCC No. CRL 1442) were grown in Coon’s modified Ham’s F-12 (Sigma, St. Louis, MO) supplemented with 5% FCS (Biofluids, Rockville, MD). FRT rat thyroid cells were grown in Coon’s modified F12 medium supplemented with 5% calf serum and 1 mM nonessential amino acids (Life Technologies, Inc.). FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) that had all properties previously detailed (42). Their doubling time with TSH was 36 ± 6 h; without TSH, they did not proliferate. After 6 days in medium with no TSH, addition of 1 x10-10 M TSH stimulated thymidine incorporation into DNA by at least 10-fold. Cells were diploid and between their 5th and 20th passage. Cells were grown in 6H medium consisting of Coon’s modified F12 supplemented with 5% calf serum, 1 mM nonessential amino acids, and a mixture of six hormones: bovine TSH (1 x10-10 M, insulin (10 µg/ml), cortisol (0.4 ng/ml), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all cells every 2 or 3 days, and cells were passed every 7–10 days. In individual experiments, cells were shifted to 3H (which is devoid of hydrocortisone, insulin, and TSH) or 5H medium (which is devoid of TSH) with 5% calf serum during or after TSH, IFN-{gamma}, and forskolin, or other agents were added.

RNA Isolation and Northern Analysis
Total cellular RNA was isolated by standard procedures and Northern analysis was performed as described (25, 26, 27). Final washes were carried out at 65 C in 1x SSPE (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4). The SOCS-1, -2, -3, and CIS probes were the purified insert fragments of the expression vectors pEF-SOCS-1, -2, -3, and CIS, respectively [provided by Dr. Starr, The Walter Eliza Hall Institute for Medical Research, Victoria, Australia (17)]. All probes were radiolabeled by a random priming protocol (Amersham Pharmacia Biotech, Arlington Heights, IL).

Construction of Promoter/Luciferase Chimeric Plasmids
Chimeric expression constructs using fragments of pCAM-1822, containing 1822 bp of the 5'-flanking region of the rat ICAM-1 gene, were made using high-fidelity PCR (28). Promoter segments were amplified with pfu polymerase using appropriate forward and reverse primers including a BglII site (5'-end) and HindIII site (3'-end). Mutations of promoter sequences were generated by PCR using primers incorporating the mutated sequence. Amplified fragments were ligated into the pGL2-basic vector containing a luciferase reporter gene, and the correct DNA sequence was confirmed by DNA sequencing analysis. The 5'-deletion mutants included pCAM-431, pCAM-175, pCAM-175 GAS mut, and pCAM-95 (see Fig. 4Go), containing the indicated fragment of the ICAM-1 promoter starting from the numbered nucleotide at the 5'-end and extending to +1 bp, the start of protein translation. All plasmid preparations were purified twice by CsCl gradient centrifugation.

Transfection
Transient transfections were performed in FRTL-5 or FRT cells at 80% confluency. For transfection, 20 µg of pCAM-431 or equivalent molar amounts of the deletion mutants or pGL2-basic (negative control) were cotransfected with 5 µg pSVGH, and then cultured for 48 h. Medium was taken for RIA of hGH (Nichols Institute Diagnostics, San Juan Capistrano, CA), and cells were harvested for luciferase assay. Luciferase assays were performed as described previously (25, 26, 27).

Stably transfected FRTL-5 cells were constructed with pGL2-basic, pCAM-431, pCAM-175, or pCAM-95. Near-confluent FRTL-5 cells in 6H medium were cotransfected with 20 µg of plasmid DNA and 10 µg of pRcNeo. PRcNeo contains a portion of human early cytomegalovirus promoter from pRc/CMV vector. After 2 days, 400 µg/ml of G418 (Life Technologies) was added to the medium, and after 3 weeks the G418-resistant colonies were pooled and used for experiments. To test the effect of cytokines and hormones, cells were grown to 70–80% confluency in 6H medium and then maintained without TSH (5H medium) for 5 days, at which time they were exposed to various concentrations of the noted agents (IFN-{gamma}, TSH) for 24 h before luciferase activity was measured.

Nuclear Extracts
FRTL-5 cells were grown in the presence of complete 6H medium to approximately 80% confluency and then maintained in 5H medium (lacking TSH) for the time periods as noted. After cells were exposed to IFN-{gamma}, nuclear extracts were prepared from the FRTL-5 cells as described (25, 26, 27) with the exception that they were washed with Dulbecco’s modified PBS without Mg2+ and Ca2+. After centrifugation at 500 x g, the cells were resuspended in 5 pellet volumes of 0.3 M sucrose and 2% Tween-40 in Buffer A [10 mM HEPES-potassium hydroxide (KOH); pH 7.9, containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml leupeptin, and 2 µg/ml pepstatin A]. After freezing, thawing, and gently homogenizing, nuclei were isolated by centrifugation at 25,000 x g in Buffer A with a 1.5 M sucrose cushion. Isolated nuclei were lysed in Buffer B [10 mM HEPES-KOH; pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A], and centrifuged at 100,000 x g for 1 h. The supernatant was dialyzed against a buffer containing 10 mM Tris (pH 7.9), 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol, and aliquoted. Samples were stored at -70 C.

EMSA
Oligonucleotides derived from the rat ICAM-1 gene, 5'-CGAGGTTTCCGGGAAAGT GGCCCC-3' (a putative GAS is underlined), were used as the DNA binding substrate for EMSA. Gel-purified oligonucleotides were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase and purified on an 8% native polyacrylamide gel. EMSA was performed as previously described (25, 26, 27). Binding reactions in low salt without detergent were carried out in a volume of 20 µl for 30 min at room temperature. The reaction mixtures contained 1.5 fmol of [32P] DNA, 4 µg cell or nuclear extract, and 0.5 µg poly (dI-dC) in 10 mM Tris-Cl (pH 7.9), 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 5% glycerol. Binding reactions in high salt with detergent were carried out in a solution of 1.5 fmol of [32P]DNA, 2 µg nuclear extract, and 0.5 µg poly(dI-dC) in 10 mM Tris-Cl (pH 7.9), 5 mM MgCl2, 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol. Where indicated, unlabeled double-stranded oligonucleotides were also added as competitor to the binding reaction and incubated for 20 min before the addition of labeled DNA. After incubation, reaction mixtures were analyzed by electrophoresis on 4 or 4.5% native polyacrylamide gels at 160 V in 0.5x TBE buffer. Gels were dried and autoradiographed. For the supershift assay, extracts were incubated in the same buffer containing specific antibodies or a control antibody at 20 C for 1 h before being processed as indicated above.

Immunoprecipitation and Western Blot Analysis
Cells were washed with ice-cold STE [NaCl 150 mM, 50 mM Tris (pH 7.4), 1 mM EDTA] and were lysed in lysis buffer [20 mM Tris (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerophosphate, 2 mM DTT, 1 mM Na3VO4, 1 mM PMSF, 4 µg/ml aprotinin]. Cell lysates were incubated with antibodies against JAK1 (Transduction Laboratories, Inc., Lexington, KY) at 4 C for 2 h, and immune complexes were precipitated by protein A or G. They were washed twice in lysis buffer with 500 mM NaCl and once with ST (50 mM Tris, pH 7.4, 150 mM NaCl). Precipitated proteins were released from protein A beads by boiling in the presence of 1x SDS sample buffer and separated by SDS-PAGE. Immunoblot analyses were performed using anti-SOCS-1 or anti-SOCS-3 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For Western blot, adherent FRTL-5 cells were stimulated in the presence or absence of TSH or IFN-{gamma} for the indicated period of time at 37 C. The treated cells were scraped, lysed by addition of SDS sample buffer [62.5 mM Tris-HCl (pH 6.8), 6% (wt/vol) SDS, 30% glycerol, 125 mM DTT, 0.03% (wt/vol) bromophenol blue] and separated by 10% SDS-PAGE along with biotinylated mol wt standards. The proteins were transferred to a nitrocellulose membrane by electrotransfer for 2 h. After soaking the membrane in blocking buffer (1x TBS, 0.1% Tween-20 with blocking reagent 5% milk) the membrane was incubated with the primary antibodies overnight at 4 C. Primary antibodies were rabbit polyclonal IgG, affinity purified (New England Biolabs, Inc., Beverly, MA; and Upstate Biotechnology, Inc., Lake Placid, NY) specific for STAT1 or STAT3 or phosphorylated forms of STAT1 (Y701, S727) or STAT3 (Y705, S727). Blots were developed using horseradish peroxidase-linked antirabbit secondary antibody and chemiluminescent detection system (Phototope-HRP Western Blot Detection Kit, New England Biolabs).

Other Assays
Protein concentration was determined by Bradford’s method (Bio-Rad Laboratories, Inc. Hercules, CA) and used recrystallized BSA as the standard.

Statistical Significance
All experiments were repeated at least three times with different batches of cells. Values are the mean ± SE of these experiments. Significance between experimental values was determined by two-way ANOVA.


   ACKNOWLEDGMENTS
 
Acknowledgements

We thank Drs. Starr and Hilton for the gift of expression vectors pEF-mSOCS-1, pEF-mSOCS-2, pEF-mSOCS-3, and pEF-mCIS, and helpful suggestions. We are also grateful to Dr. Kohn for helpful discussion and critical reading of this manuscript.


   FOOTNOTES
 
Address requests for reprints to: Minho Shong, M.D. Associate Professor, Department of Internal Medicine, School of Medicine, Chungnam National University, 640 Daesadong Chungku, Taejon 301–040, Korea.

This work was supported by the Biotech 2000 project, Grant 98-N1–02-04-A-01, and Molecular Medicine Research Group Program, Ministry of Science and Technology, Korea.

Received for publication June 23, 1999. Revision received December 6, 1999. Accepted for publication December 13, 1999.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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