Urinary excretion of LATS in a patient with Graves' disease complicated by nephrotic syndrome

A case report of a female patient with Graves' disease complicated by nephrotic syndrome with high LATS activity in urinary gamma-globulin is presented. When in the hyperthyroid state with high LATS activity in the serum, she was treated with antithyroid drugs, excess iodine, and finally radioisotopes. Mild hypothyroidism occurred transiently without any significant change in serum LATS activity. Nephrotic syndrome suddenly appeared. Urinary IgG was purified by salting out with ammonium sulfate, DEAE and protein A-Sepharose, and LATS activity in the purified urinary IgG fraction was demonstrated. The specific activity of LATS activity in urinary IgG protein was slightly lower than that of the serum. This case is the first demonstration of LATS activity in urine from a patient with hyperthyroidism and nephrotic syndrome.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Effects of Long-acting Thyroid Stimulator (LATS) and LATS Protector on Human Thyroid Adenyl Cyclase Activity

The long-acting thyroid stimulator (LATS) has been thought to be responsible for the hyperthyroidism of Graves''s disease. It is detected by its effect on the mouse thyroid gland but cannot be found in all patients with hyperthyroidism. In an attempt to clarify the problem of LATS-negative hyperthyroidism, serum was obtained from untreated patients and its effect in vitro on human thyroid tissue examined, using the activation of adenyl cyclase as a measure of stimulation. Human thyroid adenyl cyclase was activated by both thyroid-stimulating hormone (TSH) and LATS. Thyroid tissue obtained from patients with Graves''s disease was relatively less responsive to LATS than was non-toxic thyroid tissue. Of the 24 samples studied five contained LATS and all of these activated adenyl cyclase. The presence of LATS protector in LATS-negative hyperthyroid patients was confirmed but LATS-negative sera had no effect on human thyroid adenyl cyclase activity.     

Serum long acting thyroid stimulator protector in pregnancy complicated by Graves' disease.

Activities of serum long acting thyroid stimulator protector were measured in a series of nine pregnancies in eight mothers who had Graves'' disease, one of whom had been successfully treated by surgery. In all but two instances the activities tended to decline as pregnancy progressed. After delivery activities rose in three out of five patients in whom these had disappeared in pregnancy and, as this occurred, the patients relapsed. In the two patients whose activities did not decline thyrotoxicosis persisted throughout pregnancy and after delivery. None of the nine babies in this study suffered from neonatal thyrotoxicosis because maternal activities of the thyroid stimulator protector, though high enough to induce Graves'' disease in adults, were not above the threshold for the induction of thyroid overactivity in neonates.     

LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase-targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.     

LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1

LATS (large tumour suppressor) is a family of conserved tumour suppressors identified in Drosophila and mammals. Here we show that human LATS1 binds to LIMK1 in vitro and in vivo and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. LATS1 inhibits both the phosphorylation of cofilin by LIMK1 and LIMK1-induced cytokinesis defects. Inactivation of LATS1 by antibody microinjection or RNA-mediated interference in cells, or gene knockout in mice, abrogates cytokinesis and increases the percentage of multinucleate cells. Our findings indicate that LATS1 is a novel cytoskeleton regulator that affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1.     

Post-partum transient thyrotoxicosis: report of two cases

Two patients with post-partum transient thyrotoxicosis associated with painless thyroiditis and low radioactive iodine uptake were described. The surreptitious use of thyroid hormones or iodine was excluded. Although the clinical course was compatible with that of subacute thyroiditis, passing through the hyperthyroid, euthyroid, hypothyroid and recovery phase, the patients showed a sustained elevation of serum anti-thyroglobulin antibody titer during the entire phase of the disease. Moreover, the histological findings obtained by the thyroid biopsy performed in a case were characteristic of chronic lymphocytic thyroiditis. Furthermore, it was of interest that the disease recurred following every delivery in 2 cases, suggesting the possible role of immunological changes induced by pregnancy and delivery in the etiology of this disease.     

Correlation between Long-acting Thyroid Stimulator Protector Level and Thyroid 131I Uptake in Thyrotoxicosis

Out of 50 consecutive untreated patients with diffuse toxic goitre 15 showed long-acting thyroid stimulator (LATS), 30 showed LATS protector only, and five showed neither. LATS protector was present in all the patients with LATS. Infiltrative ophthalmopathy was less common in patients with LATS protector only (40%) than in patients with LATS also (67%), but the difference was not significant. There was a correlation between LATS protector level and thyroid ¹³¹I uptake rate factor (k₁), the coefficient (r) being 0·68 (P < 0·001). LATS level showed no such correlation. The results support the hypothesis that LATS protector is a pathogenic thyroid stimulator in patients with diffuse toxic goitre.     

Comparison of LATS activity and TSH receptor antibody in Graves' disease

TSH-receptor antibody (TRAb) activity and LATS activity of Graves' sera were compared. All of 50 LATS-positive cases were TRAb positive, although only 63% of LATS-negative cases were TRAb positive. Binding of 125I-TSH to the TSH receptors was inhibited dose-dependently by LATS-immunoglobulin. However, no correlation between TRAb activity and LATS activity was observed. TRAb was positive in 2 LATS-positive cases even when the symptoms of hyperthyroidism were controlled by treatment (antithyroid or radioisotope). The positive TRAb was not changed in 4 Graves' disease patients whose LATS activity had disappeared following antithyroid treatment. These clinical studies show that TRAb is more sensitive than LATS and suggest that LATS may be one of a heterogenous population of antibodies to the TSH receptor in Graves' disease.     

Clinical applications of assays for thyrotropin-receptor antibodies in Graves' disease.

Graves'' disease is characterized by hyperthyroidism, diffuse goitre, infiltrative ophthalmopathy and, rarely, pretibial myxedema. In 1956 a substance capable of prolonged thyroid stimulation was discovered in the serum of some patients with Graves'' disease and termed long-acting thyroid stimulator (LATS). It was shown to be an antibody that could interact with the receptor for thyroid-stimulating hormone (TSH). The term LATS is usually reserved for the activity measured in a laborious in-vivo bioassay in mice. Today the activity of TSH-receptor antibodies (TSH-R Ab) can be measured by in-vitro bioassays or by radioreceptor assays. These assays are now becoming commercially available. TSH-R Ab assays may be useful in predicting the response to therapy for Graves'' disease, investigating euthyroid ophthalmopathy and predicting the likelihood of neonatal hyperthyroidism.     

Management of Thyrotoxicosis: Preconception,Pregnancy, and the Postpartum Period

Objective: To review the diagnosis and management of thyrotoxicosis in women who are preconception, pregnant, and in the postpartum period.Methods: Literature review of English-language papers published between 1980 and 2018.Results: Overt thyrotoxicosis occurs in 0.2% of pregnancies and subclinical thyrotoxicosis in 2.5%. Hyperthyroidism in women of childbearing age most frequently is caused by Graves disease (GD). Gestational thyrotoxicosis, transient human chorionic gonadotropin (hCG)-mediated hyperthyroidism, may develop in the first trimester. In the first year following delivery, postpartum thyroiditis, which frequently includes a thyrotoxic phase, occurs in 5% of women. Hyperthyroidism from nodular autonomy is uncommon in women of childbearing age. It is essential to understand the underlying etiology for thyrotoxicosis in order to recommend appropriate treatment. Gestational thyrotoxicosis requires supportive care, without antithyroid drug therapy. GD may be treated with antithyroid drugs, radioactive iodine, or thyroidectomy. Pregnancy, plans for pregnancy, and lactation have important implications for the choice of GD treatment. When thyrotoxicosis presents following delivery, postpartum thyroiditis must be differentiated from GD.Conclusion: The diagnosis and management of thyrotoxicosis in the peripregnancy period present specific challenges. In making management decisions, it is essential to weigh the risks and benefits of treatments not just for the mother but also for the fetus and for breastfed infants. A team approach to management is critical, with close collaboration among endocrinologists, maternal-fetal medicine specialists, and neonatologists.Abbreviations: GD = Graves disease; hCG = human chorionic gonadotropin; MMI = methimazole; PPT = postpartum thyroiditis; PTU = propylthiouracil; T3 = triiodothyronine; T4 = thyroxine; TBG = thyroxine-binding globulin; TRAb = TSH receptor antibody; TSH = thyroid-stimulating hormone     

Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator)

TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species.     

WWP1 E3 Ligase Targets LATS1 for Ubiquitin-Mediated Degradation in Breast Cancer Cells

The Large Tumor Suppressor 1 (LATS1) is a serine/threonine kinase and tumor suppressor found down-regulated in various human cancers. LATS1 has recently been identified as a central player of the emerging Hippo signaling pathway, which plays important roles in organ size control, tumorigenesis, and stem cell differentiation and renewal, etc. Although mounting evidence supports a role of LATS1 in tumor suppression and tumorigenesis, how LATS1 is regulated at the molecular level is not fully understood. Recently several positive regulators of LATS1 (Mst1/2, MOB1, Kibra, etc) have been identified but how LATS1 is negatively regulated is still largely unknown. We have recently identified Itch, a member of the NEDD4-like family E3 ubiquitin ligases, as a novel negative regulator of LATS1. However, whether other ubiquitin ligases modulate LATS1 stability and function is unclear. By screening many E3 ligases of the NEDD4-like family using over-expression and short-interference RNA knockdown approaches, we have identified WWP1 E3 ligase as another novel negative regulator of LATS1. We have provided in vitro and in vivo evidence that WWP1 is essential for LATS1 stability and negatively regulate LATS1 by promoting LATS1 degradation through polyubiquitination and the 26S proteasome pathway. Importantly, we also showed that degradation of LATS1 is critical in mediating WWP1-induced increased cell proliferation in breast cancer cells. Since WWP1 is an oncogene and LATS1 is a tumor suppressor gene in breast cancer, our studies provide a promising therapeutic strategy in which developed drugs targeting WWP1 cause activation of LATS1 in suppressing breast cancer cell growth.     

The human tumour suppressor LATS1 is activated by human MOB1 at the membrane

Downregulation of the LATS1 tumour suppressor protein kinase contributes to tumour formation in mammals and flies. Strikingly, the tumour suppressor activity depends on the interaction with Dmob (Drosphila Mps1-One binder) in Drosophila melanogaster. Recently, human LATS1 was reported to interact with human MOB1 (hMOB1), but the activation of LATS1 was not addressed. Here, we identified a highly conserved hMOB1-binding motif within LATS1's primary structure. While co-expression of LATS1 with hMOB1 did not elevate LATS1 kinase activity in mammalian cells, membrane-targeting of hMOB1 resulted in a significant increase of LATS1 activity. This stimulation was dependent on intact activation segment and hydrophobic motif phosphorylation sites, and was further found to occur a few minutes after membrane association. Therefore, we suggest a potential in vivo mechanism of LATS1 activation through rapid recruitment to the plasma membrane by hMOB1 followed by multi-site phosphorylation, thereby providing insight into the molecular regulation of the LATS tumour suppressor.     

Thyrotoxicosis with low serum total T3 level in patients with destructive thyroiditis and non-thyroidal illness.

The serum total T3 level, evaluated in 687 patients with thyrotoxicosis diagnosed by an elevated serum free T4 level and suppressed serum TSH level, was found to be high in 98.1% and normal in 1.9% of 592 patients with Graves' hyperthyroidism, and high in 75.8%, normal in 21.1% and low in 3.2% of 95 patients with destructive thyroiditis. Non-thyroidal illness was found in about a third of the patients with thyrotoxicosis and a normal serum total T3 level. The serum total T3 level was low with elevated serum thyroglobulin and reverse T3 levels in three patients with severe non-thyroidal illness, in whom the thyroidal radioactive iodine uptake was suppressed and the thyrotoxicosis resolved spontaneously with a normalization of the serum total T3 level after recovery from the destructive thyroiditis and non-thyroidal illness. It is therefore concluded that thyrotoxicosis with a low serum total T3 level, partially due to associated non-thyroidal illness, is more frequently found in patients with destructive thyroiditis than in those with Graves' hyperthyroidism.     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

LATS2-Ajuba complex regulates gamma-tubulin recruitment to centrosomes and spindle organization during mitosis

LATS2 is a human homolog of Drosophila tumor suppressor lats/warts, and encodes a mitotic kinase whose physiological roles remain to be elucidated. We performed yeast two-hybrid screening and identified a LIM protein Ajuba, as a binding partner of LATS2. LATS2 was localized to the centrosomes throughout the cell cycle and was associated with Ajuba during mitosis, contributing to latter's mitotic phosphorylation. Depletion of LATS2 or Ajuba impaired centrosomal accumulation of gamma-tubulin and spindle formation at the onset of mitosis, suggesting that the LATS2-Ajuba complex regulates organization of the spindle apparatus through recruitment of gamma-tubulin to the centrosome.