Thyrotropin receptor autoantibody (TRAb) enhance the expression of thyrotropin receptor on mouse brain vascular endothelial cells: in vivo and in vitro.

The possibility that thyrotropin receptor (TSHR) expression in non-thyroid tissue is well-documented. However, there is insufficient data on the expression of TSHR in medulla oblongata regions, particularly when focusing on the background of encephalopathy associated with hyperthyroidism. In this study, we explored the expression of the functional TSHR in Graves' disease (GD) mouse cerebral vascular endothelial cells and the effects of thyrotropin receptor autoantibody (TRAb) on its expression. A mouse model of GD was constructed with an adenovirus overexpressing TSHR289. The location and expression of the TSHR gene and protein in vivo were determined via RT-qPCR, Western blot, and immunofluorescence techniques. The effect of TRAb on the expression of functional TSHR in vitro was investigated using bEnd.3 cells. Our results show that medulla oblongata vascular endothelial cells from GD mice expressed higher levels of TSHR compared to control mice. In an in vitro experiment, novel results demonstrated that after treatment with a monoclonal TSHR-specific agonistic antibody (M22), the expression of TSHR on the bEnd.3 cells increased at both the protein and mRNA levels. Furthermore, compared with bEnd.3 cells were treated with IBMX only, those treated with M22 showed increased cAMP production. This study suggested that TSHR is expressed and functionally active in the mouse medulla oblongata and in vitro-cultured bEnd.3 cells and TRAb (M22) increased the expression of TSHR on bEnd.3 cells.

Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated Lipid-Protein Interaction.

The Hippo pathway plays a crucial role in organ size control and tumor suppression, but its precise regulation is not fully understood. In this study, we discovered that phosphatidic acid (PA)-related lipid signaling is a key regulator of the Hippo pathway. Supplementing PA in various Hippo-activating conditions activates YAP. This PA-related lipid signaling is involved in Rho-mediated YAP activation. Mechanistically, PA directly interacts with Hippo components LATS and NF2 to disrupt LATS-MOB1 complex formation and NF2-mediated LATS membrane translocation and activation, respectively. Inhibition of phospholipase D (PLD)-dependent PA production suppresses YAP oncogenic activities. PLD1 is highly expressed in breast cancer and positively correlates with YAP activation, suggesting their pathological relevance in breast cancer development. Taken together, our study not only reveals a role of PLD-PA lipid signaling in regulating the Hippo pathway but also indicates that the PLD-PA-YAP axis is a potential therapeutic target for cancer treatment.

Expression of LATS family proteins in ovarian tumors and its significance.

Epithelial ovarian cancer is composed of a diverse group of tumors that can be derived from the fallopian tube, endometrium, or ovary. In this study, we explored the expression levels of LATS family members in ovarian tumors using normal ovaries, fallopian tubes, and endometrium as controls. Immunohistochemistry studies of LATS1, LATS2, Pax8, and calretinin were performed on normal ovary, fallopian tube, normal endometrium, and ovarian tumor sections. Statistical analyses were conducted using the χ(2) test, Fisher exact test, or Kruskal-Wallis H test. Patient survival was analyzed using the Kaplan-Meier method. LATS1 was expressed in normal ovarian epithelia, endometrium, and fallopian tubes, whereas LATS2 expression was observed in the normal fallopian tubes and endometrium. High expressions of LATS1 and LATS2 in serous cystadenomas gradually decreased in borderline cystadenomas and carcinomas, respectively. However, an opposite expression pattern was observed in mucinous tumors. Low expressions of LATS1 and LATS2 were also detected in clear cell carcinoma. Both LATS1 and LATS2 expression levels significantly correlated with recurrence and stage; LATS1 levels were also related with tumor grades in serous carcinoma. However, univariate and multivariate Cox regression analyses revealed that high expression of LATS1 was associated with better prognosis in patients with serous carcinoma. Both LATS1 and LATS2 were not related with the clinical variables in mucinous and clear cell carcinoma. LATS1 expression levels might be a valuable survival indicator in ovarian serous carcinoma.

Different binding of stimulatory-type and blocking-type TSH receptor antibody with guinea-pig testis membrane.

A receptor assay using [125I]bTSH-binding to guinea-pig testis membrane was developed. Unlabelled hCG and FSH inhibited [125I]bTSH binding. In patients with Graves' disease and in untreated hyperthyroid patients, almost all long-acting thyroid stimulators and thyroid-stimulating antibodies, respectively did not inhibit [125I]bTSH binding, which on the other hand was inhibited by thyroid stimulation blocking antibodies in patients with primary hypothyroidism. When the inhibitory effect on the binding of [125I]hCG and 125I-synthetic alpha-subunit peptide (alpha 26-46) of hCG to testis membrane was examined, bTSH resulted in a significant inhibition. However, all three kinds of TSH receptor antibodies had no inhibitory effect. This study demonstrated 1. interaction of alpha-subunit of TSH and hCG with the testicular receptor; 2. binding of thyroid stimulation-blocking antibody and lack of binding of thyroid-stimulating antibody to the testicular TSH receptor in spite of binding of these TSH receptor antibodies to the thyroidal TSH receptor, and 3. lack of binding of thyroid-stimulating antibody and thyroid stimulation-blocking antibody to the testicular gonadotropin receptor.

Relapse of Graves' disease 23 years after treatment with radioactive iodine (131I).

The use of radioactive iodine (131I) in the treatment of Graves' disease results frequently in hypothyroidism requiring thyroid hormone supplementation. Relapse of Graves' disease months after inadequate treatment with 131I is well-recognized. However, late relapse of Graves' disease in a patient rendered hypothyroid by 131I years after therapy has not been reported. The authors discuss a patient who had a relapse of his Graves' disease 23 yr after treatment with 131I. Over the interval the patient had been on 1-thyroxine replacement for hypothyroidism and had persistently high levels of long acting thyroid stimulator or thyroid stimulating antibody. The authors speculate that the immune nature of Graves' disease may play a role in the observed clinical response to 131I.

Interaction of the mouse thyrotrophin receptor with thyrotrophin binding inhibitor immunoglobulins.

The species-specificity of thyrotrophin binding inhibitor immunoglobulin (TBII) for the thyroid TSH receptor was investigated using a preparation of thyroid plasma membranes (TPM) from propylthiouracil-treated mice, as well as from human glands. The interest in the mouse arose from its use as the bioassay animal for the long-acting thyroid stimulator (LATS). A comparison was made of the response in the two radioreceptor assays of serum immunoglobulins from ten normal subjects and twenty patients with Graves's disease, who had also been selected to have positive TBII activity in the assay based on human TPM. All the specimens from the patients with Graves's disease had detectable TBII activity in the mouse radioreceptor assay, inhibiting the binding of 125I-labelled TSH to a greater extent than did any of the specimens from normal subjects. There was evidence for a minor degree of species-specificity, since at least one of the specimens from the Graves' disease group had unexpectedly high activity in the assay based on mouse TPM and another had unexpectedly weak activity in that assay. However, this specificity appeared to be unrelated to the presence or absence of LATS. The effect of LATS on the response of serum immunoglobulins in the mouse radioreceptor assay was tested using nine patients with Graves's disease who had undetectable serum LATS and another eight patients with Graves's disease whose serum gave a positive LATS response. These patients had also all been selected to have positive TBII activity in their serum, as determined with human TPM. All samples from each of the LATS-positive and LATS-negative subgroups gave a positive TBII response in the radioreceptor assay based on mouse TPM, and there was extensive overlap between the individual values for the two subgroups. It is concluded that the failure of some TBII-positive serum immunoglobulins to stimulate the mouse thyroid gland and produce a positive LATS response is not due to species-specificity at the level of receptor binding.

Comparison of human and porcine thyroid membranes for radioreceptor assay of bovine thyrotrophin and thyrotrophin-binding inhibiting immunoglobulins.

A detailed comparison between the use of human and porcine thyroid membranes for the radioreceptor assay (RRA) of bovine TSH (bTSH) and thyrotrophin-binding inhibiting immunoglobulins (TBIIg) is reported. Bovine thyroid membranes were also investigated but were found to be far less satisfactory than either human or porcine thyroid membranes. The affinity constant (ka) of the interaction of bTSH with porcine thyroid membranes (Ka = 3.3 X 10(9) l/mol) measured b Scatchard analysis was higher than with human thyroid membranes (Ka = 2.1 X 10(8) l/mol). Porcine thyroid membranes were more sensitive for the assay of bTSH (detection limit 30 microunits, half-maximal inhibition 0.3 microunit) than human thyroid membranes (detection limit 200 microunits, half-maximal inhibition 7.4 mu.). Preincubation of membranes from either species with immunoglobulin rich in long-acting thyroid stimulator (LATS) inhibited the saturable binding of 125I-labelled TSH to a greater extent than did normal immunoglobulin. The binding of 125I-labelled TSH to porcine membranes was more sensitive to the inhibitory effect of LATS-immunoglobulin and was also less affected by normal immunoglobulin than was binding to human thyroid membranes. When assayed with each type of membrane preparation there was good correlation between the RRA of immunoglobulins prepared from patients with Grave's disease and from normal subjects (n = 18) (r = 0.85, P less than 0.001, n = 73). The incidence of positive TBIIg in untreated Grave's disease was greater for porcine than for human thyroid membranes.

Graves' IgG stimulation of continuously cultured rat thyroid cells: a sensitive and potentially useful clinical assay.

A continuously cultured line of normal rat thyroid (FRTL) cells can be stimulated by immunoglobulin preparations from patients with Graves' disease as measured by increases in intracellular cAMP levels. Responsiveness is concentration-dependent but is delayed in time relative to thyrotropin. Additionally, the cells respond to Graves' immunoglobulins which have no long-acting thyroid stimulator (LATS) activity and are negative when adenylate cyclase stimulation in human thyroid membrane preparations is assayed. No correlation exists between the stimulation activity and the ability of a Graves' immunoglobulin preparation to inhibit thyrotropin binding; cells are responsive even in the presence of such inhibitor activity.

An assay for antibodies to thyroid plasma membrane using the isotope-labeled protein A.

An assay for detection of serum IgG binding to thyroid membrane using 125I-Protein A was examined. The test serum is incubated with purified thyroid membranes, and the IgG bound to the membrane is detected by its interaction with 125I-Protein A. Most of the bound IgG is not bound to the TSH receptor, because TSH does not induce any appreciable decrease in the binding of the IgG to thyroid membranes. Increased serum IgG binding to thyroid membrane is found in most patients with Graves' disease and Hashimoto's thyroiditis, but not in patients with thyroid cancer or simple goiter. Many sera with positive binding activity showed positive microsomal antibody. Serum IgG binding to thyroid membrane in Graves' disease correlates neither with LATS activity nor thyroglobulin antibody. This finding suggests that TSH receptor is not involved in the reaction. The assay method is useful for measuring the binding immunoglobulin for thyroid membrane that is frequently increased in autoimmune thyroid disease, and the present data provide further support to the concept that thyroid autoimmune disorders are associated with antibodies to thyroid cell surface components.

Discrepancy between thyroid-stimulating and thyrotropin-binding inhibitory activities of Graves' immunoglobulin Gs assessed in the mouse.

TSH receptor binding-inhibiting (TBI) and thyroid-stimulating (TSI) activities of Graves' immunoglobulin Gs (IgGs) were measured using the murine TSH receptor (mTBI) and the McKenzie LATS bioassay. In contrast to the expectation that only the IgGs positive for LATS showed mTBI activity, mTBI activity was closely correlated with TBI activity measured at the human TSH receptor regardless of LATS potency (r = 0.79; P less than 0.01), indicating a lack of species specificity of IgG in this particular aspect. In 7 to 15 IgGs positive for both mTBI and LATS, there was a significant correlation between the 2 parameters (r = -0.89; P less than 0.01). However, 4 IgGs among the 15 were positive for mTBI but negative for LATS even when tested at 3- to 7-fold concentrations of IgG.

TSH binding by globulins from patients with Graves' disease.

Forty one globulin preparations from patients with Graves' disease were tested for their specific TSH binding properties and compared with their TSI activity in: 1. human thyroid membrane competition assay; 2. adenylate cyclase stimulation; 3. colloid droplet formation. The preparations tested were divided into two well distinguishable groups according to their specific TSH binding: 25 samples out of 41 contained thyrotropin binding sites similar to the globulins from normal healthy persons (A-type binding, Kd: 1.2 pmol-1 g and Bmax: 0.8 pmol g-1) and 17 samples bound TSH with lower affinity but higher capacity (B-type binding mean Kd: 15.3 pmol-1 g, mean Bmax: 2.7 pmol g-1). both A- and B-type TSH binding globulins containing samples were active in binding to human thyroid membrane, in droplet formation and adenylate cyclase stimulation. However, there was a linear correlation between the potential THS binding capacity of a B-type sample and the percentage adenylate cyclase stimulation and droplet formation, whereas the reverse correlation was found with their thyroid membrane binding activity. It was concluded that B-type TSH binding globulins may play a role in the formation and/or modification of the pathogenetic TSH action.

Binding of thyrotrophin to low molecular weight fragments of human thyroid membranes.

The LATS absorbing activity from thyroid homogenates which is associated with the soluble thyroid fraction (4S-LAA) binds TSH in addition to LATS. This may indicate that 4S-LAA originates from the receptor sites for TSH at the thyroid cell surface. The affinity of 4S-LAA for TSH is much lower than that of thyroid membranes suggesting that 4S-LAA represents incomplete receptor sites or receptor sites which are altered in their structure.

On incubation of long acting thyroid stimulator with human thyroid and adrenal homogenates.

In view of the ability of long acting thyroid stimulator (LATS) containing sera and IgG samples to stimulate the adrenal cortex of the mouse, it was decided to study the effect of incubation of LATS containing IgG with human thyroid and adrenal homogenates. Whereas incubation with human thyroid gland abolished (loss greater than 80%) both the thyroid and adrenocortical stimulatory effects of LATS, incubation with human adrenal gland resulted in a minimal loss (20%) of both of these stimulatory activities; the magnitude of this loss was no greater than that attributable to non-specific absorption observed with liver and kidney homogenates by previous workers. It is suggested that a, human and mouse thyroid receptors for TSH/LATS are similar and that adrenocortical receptor for ACTH/LATS are different; and b, the inability of human adrenal homogenates to neutralize LATS explains the absence of hyperadrenocorticism in patients with Graves' disease.

[Cell receptor defects as the cause of endocrine and metabolic diseases (author's transl)]. / Defekte an Zellrezeptoren als Ursache endokriner und metabolischer Krankheiten.

The following pathogenetic mechanisms, exemplified by three diseases (diabetes mellitus, hyperthyroidism and familial hypercholesterolemia), are discussed: 1. The impaired interaction between a chemical signal and a specific receptor can be the cause of a disease. 2. The cause for an imparied interaction can be a defect of the receptor, i.e., a reduced number of receptors or an altered receptor affinity, or a wrong signal. 3. A defect of the receptor can be induced by exogenous influences or it can be determined genetically. 4. The receptor and the signal can be modified by their interaction: the number of receptors is reduced by high concentrations of the chemical signal or by increased degradation due to binding to the receptor. 5. The receptor concept opens new perspectives for the pathogenetic understanding, diagnosis and therapy of some diseases.

Changes in thyroid-stimulating immunoglobulins during antithyroid therapy.

Thyroid-stimulating immunoglobulin (TSI) activity was measured by radioreceptor assay in sera from patients with Graves' disease, Hashimoto's thyroiditis, and thyroid cancer. In untreated Graves' disease (47 cases), TSI index was significantly lower [76.7 +/- 1.4 (SE)] than the average of a normal control group (30 cases; 94.4 +/- 1.9). In untreated Hashimoto's thyroiditis (25 cases), it was also significantly lower (83.0 +/- 2.4). In patients with thyroid cancer (19 cases), there was no significant difference from normal controls. After 131I treatment, the TSI index in Graves' disease decreased during 2--4 months, then increased and reached normal levels in 1 yr. During propylthiouracil treatment, the TSI index increased and reached a normal level in 5--6 months without the decreasing phase seen after 131I treatment. Free T4 index values were gradually decreased by both treatments. There was no significant relationship between TSI index and thyroid antibodies (microsomal antibodies and thyroglobulin antibodies) in untreated Graves' disease or Hashimoto's thyroiditis. It is concluded that 1) in the sera of patients with Graves' disease and Hashimoto's thyroiditis, there are immunoglobulin Gs that can displace TSH binding to thyroid membranes; 2) these immunoglobulins Gs are different from the classic antithyroid antibodies; and 3) 131T treatment of Graves' disease may enhance TSI production during the first 1--2 months after therapy.

[Pathogenesis of Basedow's disease]. / Zur Pathogenese der Basedow' schen Erkrankung.

Thyroid antibodies and thyroid-stimulating factors (LATS and LATS-Protector) have been controlled 53 patients with Graves' disease during antithyroid drug treatment. It has been demonstrated that dosage is higher and duration of treatment has been more protracted in antibody-positive thyrotoxicosis than in patients without these antibodies. Thyroid antibodis have been found only in patients with detectable thyroid-stimulating factors, which are identified as immunglobulins. These results indicate the importance of immunologic processes. Suppressibility of thyroid function proved 2 months after stopping medical treatment, was negative in 19 and positive in 17 patients. Thyroid-stimulating factors remained positive up to this time in 20 patients and disappeared in 18. In 8 patients the suppression-test was positive indicating a normal function of the thyroid stimulating hormone (TSH), but thyroid-stimulating factors still have been detected in these patients. These results are controversial to the autoimmune-concept of the pathogenesis of Graves' disease. As this concept cannot be refused, changes in the effectivity of LATS or LATS protector by crude serum factors or by changes of the receptor structures are discussed.