A mutation in Tpst2 encoding tyrosylprotein sulfotransferase causes dwarfism associated with hypothyroidism

The growth-retarded (grt) mouse has an autosomal recessive, fetal-onset, severe thyroid hypoplasia related to TSH hyporesponsiveness. Through genetic mapping and complementation experiments, we show that grt is a missense mutation of a highly conserved region of the tyrosylprotein sulfotransferase 2 (Tpst2) gene, encoding one of the two Tpst genes implicated in posttranslational tyrosine O-sulfation. We present evidence that the grt mutation leads to a loss of TPST2 activity, and TPST2 isoform has a high degree of substrate preference for TSH receptor (TSHR). The expression of TPST2 can restore TSH-TSHR-mediated cAMP production in fibroblasts derived from grt mice. Therefore, we propose that the tyrosine sulfation of TSHR by TPST2 is crucial for TSH signaling and resultant thyroid gland function.     

Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptor

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.     

Functional TSH receptor in human abdominal preadipocytes and orbital fibroblasts

Controversy continues about whether, and to whatlevels of abundance, thyroid-stimulating hormone receptors (TSHR) arefound in human tissues other than the thyroid gland. Restrictedexpression to the thyroid and orbit would suggest that TSHR representsthe target autoantigen in thyroid-associated ophthalmopathy. A more generalized pattern of tissue expression would be inconsistent withTSHR acting as the autoantigen that is solely responsible forselectively targeting the immune system to the orbit. We have detectedTSHR mRNA in human abdominal adipose tissue by Northern blot analysis.TSHR protein was also detected, by immunoblotting with two differentantibodies, in preadipocytes isolated from human abdominal subcutaneousand omental adipose tissue and in derivative adipocytes differentiatedin primary culture. Preadipocytes treated with thyroid-stimulatinghormone (TSH) exhibited a sevenfold increase in the activity of p70 S6kinase, a serine/threonine kinase recently recognized as a downstreamtarget of TSHR in thyroid cells. Activation of p70 S6 kinase by TSH wasalso observed in orbital fibroblasts. Thus TSHR protein expression isfound in fibroblasts from several anatomic locations, suggesting thatfactors other than site-limited TSHR expression must be involved inrestricting the distribution of Graves' disease manifestations.Furthermore, the presence of functional TSHR in preadipocytes raisesthe possibility of a novel role for TSHR signaling in adipose tissue development.

     

Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes

To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.     

A lack of thyroid hormones rather than excess thyrotropin causes abnormal skeletal development in hypothyroidism

By proposing TSH as a key negative regulator of bone turnover, recent studies in TSH receptor (TSHR) null mice challenged the established view that skeletal responses to disruption of the hypothalamic-pituitary-thyroid axis result from altered thyroid hormone (T(3)) action in bone. Importantly, this hypothesis does not explain the increased risk of osteoporosis in Graves' disease patients, in which circulating TSHR-stimulating antibodies are pathognomonic. To determine the relative importance of T(3) and TSH in bone, we compared the skeletal phenotypes of two mouse models of congenital hypothyroidism in which the normal reciprocal relationship between thyroid hormones and TSH was intact or disrupted. Pax8 null (Pax8(-/-)) mice have a 1900-fold increase in TSH and a normal TSHR, whereas hyt/hyt mice have a 2300-fold elevation of TSH but a nonfunctional TSHR. We reasoned these mice must display opposing skeletal phenotypes if TSH has a major role in bone, whereas they would be similar if thyroid hormone actions predominate. Pax8(-/-) and hyt/hyt mice both displayed delayed ossification, reduced cortical bone, a trabecular bone remodeling defect, and reduced bone mineralization, thus indicating that the skeletal abnormalities of congenital hypothyroidism are independent of TSH. Treatment of primary osteoblasts and osteoclasts with TSH or a TSHR-stimulating antibody failed to induce a cAMP response. Furthermore, TSH did not affect the differentiation or function of osteoblasts or osteoclasts in vitro. These data indicate the hypothalamic-pituitary-thyroid axis regulates skeletal development via the actions of T(3).     

TSH induces co‐localization of TSH receptor and Na/K‐ATPase in human erythrocytes

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra‐thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K‐ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K‐pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K‐pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co‐immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K‐ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co‐localization of TSHR with Na/K‐ATPase and co‐immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K‐ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control. Copyright © 2009 John Wiley & Sons, Ltd.     

Relation of structural polarity to responses of cyclic 3', 5'-adenosine monophosphate accumulation and thyroid hormone release in cultured human thyroid follicles

The relationship of structural polarity to functional activities was examined in cultured human thyroid follicles, which were isolated from the thyroid gland of patients with Graves' disease by collagenase treatment. Structural polarity was examined morphologically by electron microscopy, while the functional response to bovine TSH was examined by measuring intracellular cAMP accumulation and T3 release. In freshly isolated thyroid follicles, structural polarity was normal and TSH induced significant cAMP accumulation but no significant release of T3. After culture for 5 days the structural polarity of thyroid follicles became inverted in the absence of thyroid stimulators, but normal polarity was retained in the presence of TSH or dibutyryl cAMP [Bu)2 cAMP). The response to TSH of cAMP accumulation increased markedly after culture in either the presence or absence of TSH, suggesting that cAMP accumulation in response to TSH is not related to structural polarity. In contrast, thyroid follicles cultured without thyroid stimulators showed no significant T3 release in response to TSH, whereas those cultured with TSH or (Bu)2 cAMP showed significant T3 release in response to TSH. These data indicate that in cultured human thyroid follicles, the responses to TSH of cAMP accumulation and T3 release are not always correlated. Among many other explanations, the results were at least compatible with the idea that normal structural polarity is necessary for thyroid hormone release in response to TSH.     

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.     

Vitamin D: a hormonal regulator of the cAMP signaling pathway

In the rat thyroid FRTL-5 cell line calcitriol, the biologically most active of the naturally occurring vitamin D metabolites, attenuates both TSH-stimulated cAMP production and the effects of cAMP. Calcitriol treatment abolishes the upregulation of the TSHR number occurring in cells cultivated in the absence of TSH. In addition, the level of G(i-2)alpha increases, which may further attenuate the transmembrane signaling of TSH and facilitate the effects of IGFs. The effect of cAMP on PKAI stimulation is inhibited by increasing the level of the PKA subunit RIIbeta. Regulation of TSHR, G(i-2)alpha and RIIbeta is associated with altered cell proliferation and differentiation in several cells and tissues. Effects of calcitriol on these proteins indicate how the vitamin D endocrine system may regulate cAMP signaling in both classical and nonclassical target tissues.     

Dominant Negative Effect of Mutated Thyroid Stimulating Hormone Receptor (P556L) Causes Hypothyroidism in C.RF-Tshr(hyt/wild) Mice

C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.     

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.