Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.     

Targeting thyroid diseases with TSH receptor analogs

The thyroid-stimulating hormone (TSH) receptor (TSHR) is a major regulator of thyroid function and growth, and is the key antigen in several pathological conditions including hyperthyroidism, hypothyroidism, and thyroid tumors. Various effective treatment strategies are currently available for many of these clinical conditions such as antithyroid drugs or radioiodine therapy, but they are not devoid of side effects. In addition, treatment of complications of Graves’ disease such as Graves’ ophthalmopathy is often difficult and unsatisfactory using current methods. Recent advances in basic research on both in vitro and in vivo models have suggested that TSH analogs could be used for diagnosis and treatment of some of the thyroid diseases. The advent of high-throughput screening methods has resulted in a group of TSH analogs called small molecules, which have the potential to be developed as promising drugs. Small molecules are low molecular weight compounds with agonist, antagonist and, in some cases, inverse agonist activity on TSHR. This short review will focus on current advances in development of TSH analogs and their potential clinical applications. Rapid advances in this field may lead to the conduct of clinical trials of small molecules related to TSHR for the management of Graves’ disease, thyroid cancer, and thyroid-related osteoporosis in the coming years.     

Increased susceptibility to apoptosis of human hepatocarcinoma cells transfected with antisense N-acetylglucosaminyltransferase V cDNA.

The antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V, EC 2. 4.1.155) was constructed as pcDNA3/GnT-V-AS plasmid and transfected into 7721 cells, a human hepatocarcinoma cell line. The transfection was confirmed with Northern blot. By using HPLC and HRP-lectin staining, it was found that the cells transfected with pcDNA3/GnT-V-AS (GnT-V-AS/7721) expressed less GnT-V activity and beta-1,6-GlcNAc branching in the cell glycoproteins compared with the cells mock-transfected with the vector pcDNA3 (pcDNA3/7721). The growth rate of GnT-V-AS/7721 was decreased in serum-containing medium, while the cell death was accelerated in serum-free medium. The GnT-V-AS/7721 cells were more susceptible to the apoptosis induced by ATRA than the mock-transfected cells. This was evidenced by the obvious appearance of a hypoploid sub-G(1) fraction in the DNA histogram using FCM analysis, the more condensed new moon-type nuclei under morphological observation, and the more intensive TUNEL reaction for assaying the fragmented DNA. At the same time as GnT-V down-regulation by GnT-V-AS, an increase of another N-aceylglusaminyltransferase, GnT-III (EC 2.4.1.144), was observed, and the biological significance of this finding was discussed.     

Cyclic AMP level of human thyroid cells in monolayer culture. TSH induced refractoriness to TSH action.

Thyroid cells from euthyroid patients with Graves' disease were cultured in a chemically defined medium. The cells preserved the ability to respond to TSH with 8-fold increase in cyclic AMP concentration. This cyclic AMP response to TSH was diminished by prior exposure of cells to TSH. The decrease in cyclic AMP response to TSH induced to TSH was reversible, was not associated with a similar decrease to cyclic AMP response to PGE1, and could not be attributed to increased phosphodiesterase activity or to decreased adenyl cyclase activity. The partial resistence to TSH stimulation of thyroid cells previously exposed to TSH may be due to changes in the TSH receptor, possibly caused by TSH itself.     

TSH receptor in adipose cells.

Although there have been reports supporting the presence of the TSH receptor (TSHR) in human adipose tissue, these findings are still not universally accepted. Contributing to the controversy is a paucity of data about the physiological role the TSH receptor might play in adipose cells. In addition to mature lipid-filled adipocytes, adipose tissue also harbors a pool of specialized, fibroblast-like preadipocytes within the stromal-vascular compartment. Upon appropriate induction, preadipocytes can either differentiate into adipocytes or undergo apoptosis. Since TSHR has been detected in preadipocytes and adipocytes, its potential impact on adipose tissue function may relate to differentiation stage-specific cellular properties.     

Deactivation of TSH receptor signaling in filter-cultured pig thyroid epithelial cells

Thyrotropin [thyroid-stimulating hormone (TSH)] receptor on-off signaling was studied in polarized monolayers of pig thyrocytes cultured on permeable support. Transepithelial resistance (R) and potential difference (PD) were used as parameters to monitor the effect of altered TSH concentrations on vectorial electrolyte transport. TSH induced rapid but long-lasting changes in R (decrease) and PD (increase) that were cAMP-dependent and related to enhanced transcellular conductance of sodium and chloride. Withdrawal of TSH from cultures prestimulated with TSH (0.1 mU/ml) for 48 h resulted in restitution of R to control level within 30 min. Such deactivation was markedly accelerated by mild trypsinization, which degraded receptor-bound ligand without affecting TSH receptor responsiveness or ion transporting capacity. Small alterations in the TSH concentration (0.01-0.1 mU/ml) were followed almost instantaneously by adjustments of R. In contrast, the reversal of R after acute TSH stimulation (30 min) and subsequent TSH washout was delayed for several hours independently of cell surface trypsinization. The observations indicate that, during continuous exposure to physiological concentrations, TSH exerts a close minute-to-minute surveillance of thyroid function and the rate-limiting step of deactivation is the dissociation of ligand from the TSH receptor at the cell surface. TSH-deprived cells briefly exposed to TSH are refractory to rapid deactivation, probably because of altered metabolism downstream of TSH receptor signal transduction.     

Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA.

Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment.     

Growth hormone (GH) induction of tyrosine phosphorylation and activation of mitogen-activated protein kinases in cells transfected with rat GH receptor cDNA.

The mechanism of growth hormone (GH) action was studied in Chinese hamster ovary (CHO) cells transfected with GH receptor cDNA. Cytosolic extracts from GH- or phorbol ester (12-O-tetradecanoyl 4 beta-phorbol 13-acetate)-treated cells, transfected with full-length GH receptor cDNA, had an enhanced ability to phosphorylate myelin basic protein. Myelin basic protein, a substrate for mitogen-activated protein (MAP) kinase, was maximally phosphorylated using extracts from cells treated with 50 nM bovine GH for 10 min. In addition, GH treatment resulted in an increased cell proliferation by 30-60%. GH and 12-O-tetradecanoyl 4 beta-phorbol 13-acetate cause tyrosine phosphorylation of two proteins with M(r) of 40,000 and 42,000 that are also recognized by MAP kinase antibodies. These proteins were identified as MAP kinases by analyzing phosphotyrosine immunoprecipitates on Western blots using MAP kinase antibodies. In addition, GH induces mitogenicity, as well as MAP kinase activation, in CHO cells expressing a receptor in which 184 amino acids had been deleted in the carboxyl-terminal part of the intracellular domain. No GH effects were seen in untransfected cells, in CHO cells expressing a truncated GH receptor containing only 5 of 349 amino acids in the intracellular domain, or in cells expressing the soluble GH-binding protein. In conclusion, our data show that GH treatment of CHO cells, reconstituted with GH receptors, initiates a phosphorylation cascade which includes MAP kinase.     

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.     

Thyroglobulin binding and TSH regulation of the RHL-1 subunit of the asialoglycoprotein receptor in rat thyroid.

The ability of asialo-thyroglobulin to bind the thyroid RHL-1 subunit of the asialoglycoprotein receptor has been investigated. Ligand blot assays show that the recombinant carbohydrate recognition domain of the thyroid RHL-1 subunit specifically interacts with rat desialated thyroglobulin. Moreover, RT-PCR and Western blot assays show that TSH deprivation decreases RHL-1 expression in PC C13 thyroid differentiated cells whereas insulin deprivation does not have any effect. The simultaneous absence of both TSH and insulin dramatically decreases the level of RHL-1 expression.     

High-throughput classification of images of cells transfected with cDNA clones

The sequence of the human genome has been determined. The next task is to determine the function of the genes. Classifying cellular forms of proteins encoded by human cDNA clones is a primary step toward understanding the biological role of proteins and their coding genes. We report here our ongoing work on an automatic system to facilitate this classification. Our system handles the transfection, incubation, acquisition of microscopic images of the cells, and the classification of forms there appearing in the images. Our system correctly classified proteins by their forms at a rate of 90% in feasibility studies.     

Effect of TSH and melatonin on thyroid activity in the rat.

Melatonin and TSH, injected separately, caused no change of the blood thyroxine level at 30 min after treatment. Simultaneous or subsequent administration of the two hormones induced an increase of the level. Thus, melatonin is capable of potentiating acute, thyroxine mobilizing effect of TSH.     

Amplified expression of tumor necrosis factor receptor in cells transfected with Epstein-Barr virus shuttle vector cDNA libraries

As an approach to isolate the cell-surface receptor for tumor necrosis factor (TNF), we have developed transfectants of human B-lymphoblastoid cells (UC cells) that overexpress the TNF receptor. These transfectants were isolated from UC cells transfected with cDNA libraries of HeLa or NG108 cells constructed in the mammalian expression vector EBO-pcD. This vector contains the Epstein-Barr virus origin of replication (ori-P) plus the EBNA-1 gene conferring replication function to ori-P and, therefore, the ability to replicate autonomously within the transfected cell (Margolskee, R.F., Kavathas, P., and Berg, P. (1988) Mol. Cell. Biol. 8, 2837-2947). Cells overexpressing the TNF receptor were identified and separated by the binding of fluoresceinated TNF and flow cytometric selection. Scatchard analysis of 125I-TNF binding data revealed a single class of high affinity receptors with a dissociation constant (Kd) of 0.2 to 2 nM and a receptor density of about 150,000 per cell, an increase of approximately 150-fold over UC cells. Cross-linking of receptor-ligand with bis-sulfosuccinimidyl suberate followed by polyacrylamide gel electrophoresis gave estimates of 87 and 104 kDa for the size of the complex. Based on its ability to bind TNF, a 68-kDa receptor protein was identified in cell extracts enriched for the receptor by using immobilized wheat germ agglutinin and TNF affinity chromatography. The difference in the estimated size of the receptor and the receptor-ligand complexes demonstrates that TNF binds to the receptor as a monomer or a dimer. Analysis of cDNA sequences conferring receptor amplification in transfectants revealed that plasmid DNA was present at 30 or more copies per cell, most likely integrated into the genomic DNA or organized into high molecular weight catenanes, and autonomously replicating units could not be recovered. Therefore, while this vector was useful in generating stable receptor-amplified cells, it was not maintained as a recoverable episome.     

Proalbumin is processed to serum albumin in COS-1 cells transfected with cDNA for rat albumin

Synthesis and processing of rat albumin were investigated in COS-1 cells transiently expressing rat albumin. Analysis using isoelectric focusing revealed that serum-type albumin, which is indistinguishable from the counterpart isolated from rat hepatocyte cuture medium, was secreted from the transfected COS-1 cells, indicating that proalbumin is effectively converted into serum albumin in the COS-1 cells, if not completely. Furthermore methylamine was found to cause the diminution of serum albumin released from the cells, substantiating that the proteolytical conversion of proalbumin occurs in the Golgi complex before discharge from the COS-1 cells.     

Cloning of the human glucocorticoid receptor cDNA.

We show that the human glucocorticoid receptor (GR), isolated from the breast cancer cell line MCF-7, has an apparent molecular weight identical to that of rat liver GR (94 kDa) and reacts with antibodies raised against the latter. These antibodies were used to clone cDNA sequences corresponding to the human GR from a lambda gt11 expression library constructed using MCF-7 poly(A)+ RNA. Three non-homologous cDNA clones with inserts of 125, 220 and 350 bp, which express epitopes recognised by the rat liver GR antibodies, were isolated. Rat liver GR antibodies, immunopurified using the immobilised purified beta-galactosidase fusion proteins, detect partially purified rat liver and human GRs on Western blots. In addition, these antibodies immuno-adsorb rat liver and human GRs affinity-labelled with [3H] triamcinolone acetonide. Northern blot analysis, using all three cDNA probes, reveals the presence of a major MCF-7 poly(A)+ RNA species of approximately 7 kb.     

Elimination of apolipoprotein B48 formation in rat hepatoma cell lines transfected with mutant human apolipoprotein B cDNA constructs.

Rat hepatoma McA-RH7777 cell lines transfected with full-length human apolipoprotein (apo) B constructs produce mostly human apoB48 and only small amounts of apoB100, as a result of mRNA editing at codon 2153 (C to U conversion at nucleotide 6666). To abolish the formation of apoB48 and increase the yield of apoB100 and other forms of apoB longer than apoB48, site-specific mutations were introduced at or near the site of apoB mRNA editing. Among four mutations examined, only that in which codon 2153 was converted from CAA (Gln) to CTA (Leu) effectively precluded the formation of apoB48. In this mutant, a stop codon would not be generated even if the C to U conversion occurred. The three other mutations were introduced to disrupt the proposed stem-loop structure encompassing the editing site. Changes made in the third positions of five codons on the 5' side of the edited base or of four codons 3' of the edited base failed to eliminate the production of a protein with the approximate size of apoB48. A construct in which codon 2153 was changed from CAA to GAT (Asp) also failed to eliminate the production of a protein the size of apoB48. Analysis of the region between nucleotides 6200 and 6900 of the cDNA did not detect any prevalent alternate editing sites. Immunoblot analysis using polyclonal antibodies raised against synthetic peptides of human apoB100 indicated that the carboxyl terminus of the apoB48-like proteins probably resides between amino acid residues 2068 and 2129 of apoB100. These results provide some insight into the mechanism of apoB mRNA editing and will facilitate further studies on apoB-containing lipoproteins.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)