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Biomedical subjects

C Maenhaut

Publications and source records attributed to C Maenhaut.

At least 55 records · Page 3Linked to original sources
PubMed1992

Differential regulation of thyrotropin receptor and thyroglobulin mRNA accumulation at the cellular level: an in situ hybridization study.

Regulation of TSH receptor (TSHr) mRNA accumulation has been investigated in canine thyrocytes in primary culture by in situ hybridization experiments; the effects of the mitogenic thyrotropin (TSH), epidermal growth factor (EGF), and phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) have been compared. Apart from their mitogenic action, TSH enhances, while EGF and phorbol ester inhibit, the expression of differentiation. The TSHr gene was transcribed in almost all the cells cultured in control conditions (serum free medium supplemented with insulin). Addition of TSH slightly upregulated (twofold) the expression (mRNA) of the TSHr gene. This positive effect was maintained for 20 and 44 h of treatment. EGF and TPA reduced transiently the TSHr mRNA accumulation but did not suppress it. In these different conditions, the TSHr mRNA was homogeneously distributed within the cell population. This contrasted strongly with the effects of TSH, EGF, and TPA on the expression of the thyroglobulin gene, a prominent marker of thyroid cell differentiation: in this case, the regulation was much tighter (high range of stimulation by TSH, strong inhibition by EGF, and suppression of Tg gene expression by TPA) and displayed a great variability of the level of individual cellular response. The fact that the TSHr gene was little modulated and remained expressed regardless of the treatment may reflect the physiological role of the receptor which is the main connection of the thyrocyte to the regulation network.

Animals↗
PubMed1992

Gene expression of differentiation- and dedifferentiation markers in normal and malignant human thyroid tissues.

Steady state mRNA transcript levels of thyroid differentiation markers such as TSH receptor (TSHR), thyroglobulin (Tg) and thyroid peroxidase (TPO) as well as a potential marker of dedifferentiation, c-myc, marker were investigated in patients with thyroid tumors and in normal controls using Northern blot analysis. Blots were normalized by acridine orange staining whereas analysis of beta-actin mRNA levels revealed highly variable levels already in normal tissue suggesting regulation of this "constitutively" expressed gene. Determination of c-myc mRNA revealed increased steady state mRNA levels in anaplastic carcinomas (ATC) as compared to normal tissues. However, in some patients c-myc transcript levels were lower in the tumor than in the adjacent normal tissue reducing the significance of c-myc as a marker of dedifferentiation. High levels of TSH mRNA were found in control thyroids, whereas in ATC no normal TSHR mRNA was detected. In PTC and follicular thyroid carcinomas (FTC) the transcripts varied from increased to markedly reduced levels. In one patient with FTC 2 independent preparations of the tumor revealed different results, undetectable and clearly detectable TSHR mRNA levels. Xenotransplantation of this tissue on nude rats showed a variable expression pattern in the individual xenotransplantations suggesting heterogeneity of the tumor tissue. Tg and TPO mRNA were strongly expressed in normal tissues and completely lost in all ATC. In differentiated thyroid tumors the transcript levels of Tg and TPO varied from normal to complete loss of expression of either Tg or TPO, or both.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridine Orange↗
PubMed1991

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor.

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1991

Activation of the cyclic AMP cascade as an oncogenic mechanism: the thyroid example.

Three cascades activate thyroid cell proliferation: the EGF-protein tyrosine kinase pathway, the phorbol ester-protein kinase C pathway and the thyrotropin-cyclic AMP pathway. While the first 2 cascades converge early, they remain distinct from the cyclic AMP cascade until very late in G1. The cyclic AMP cascade is characterized by an early and transient expression of c-myc, which may explain why it induces proliferation and differentiation expression. Constitutive activation of this cascade causes growth and hyperfunction, ie, hyperfunctioning adenomas. The various possible defects that could lead to such a constitutive activation are discussed.

Adenoma↗
PubMed1991

Human thyrotropin receptor gene: expression in thyroid tumors and correlation to markers of thyroid differentiation and dedifferentiation.

Human thyrotropin (TSH) receptor steady-state transcript levels were analyzed by Northern blot analysis in thyroids of patients with thyroid carcinoma, with hyperfunctioning adenoma and in normal controls. In control tissue and benign tumors expression levels of TSH receptor mRNA were high whereas in anaplastic carcinomas no normal TSH receptor mRNA was detected. In papillary and follicular tumors it varied from normal to markedly reduced levels. Thyroid peroxidase (TPO) and thyroglobulin (Tg) mRNA were strongly expressed in normal tissue and in hyperfunctioning adenomas but were completely lost in all anaplastic tumors. In papillary tumors expression of TPO and Tg mRNA varied from normal to a complete loss of expression of either TPO, Tg or both. Tg and TPO steady-state expression did not correlate to TSH receptor transcript levels. C-myc mRNA was highly expressed in anaplastic carcinomas, very variable in normal controls and in differentiated thyroid tumors and low in hyperfunctioning adenomas. In summary, TSH receptor mRNA is persistently expressed in all differentiated thyroid tissues and tumors but lost in undifferentiated carcinomas. Its persistence far along the transformation pathway further supports the concept that this gene which inserts the thyrocytes in the physiological regulatory network is almost constitutively expressed in this cell.

Adolescent↗
PubMed1991

The TSH cyclic AMP cascade in the control of thyroid cell proliferation: the story of a concept.

Thyrotropin stimulates the growth and proliferation of thyroid cells in vivo. Starting in the 1970, we have progressively shown that these effects could be reproduced in vitro in dog thyroid cells in primary culture. They are accompanied by the expression of differentiation. All the effects of thyrotropin are mediated by the cyclic AMP cascade. The best argument that this concept applies in vivo is the generation of hyperfunctioning adenoma involving the whole gland in transgenic mice expressing the constitutively active adenosine A2 receptor in the thyroid.

Animals↗
PubMed1990

RDC8 codes for an adenosine A2 receptor with physiological constitutive activity.

The cDNA of an unidentified recently cloned G protein-coupled receptor, RDC8, has been expressed in Y1 adrenal cells, in dog thyrocytes in primary culture and in Xenopus oocytes. In all these systems this resulted in the activation of adenylyl cyclase and of the cyclic AMP cascade in the absence of any added external signal. However, this physiologically constitutive activator was inhibited by adenosine deaminase and by inhibitors of the adenosine A2 receptor. Cos 7 cells transfected with RDC8 cDNA constructs acquired binding characteristics of an adenosine A2 receptor. Moreover, RDC8 mRNA and adenosine A2 receptors display a very similar distribution in the brain. RDC8 therefore codes for an A2 adenosine receptor. Whether the physiologically constitutive activation of this receptor is entirely explained by endogeneously produced adenosine is as yet unknown.

Adenosine↗
PubMed1990

Thyrotropin activates both the cyclic AMP and the PIP2 cascades in CHO cells expressing the human cDNA of TSH receptor.

The effect of thyrotropin (TSH) on cyclic AMP accumulation, phosphatidylinositol bisphosphate (PIP2) hydrolysis and [Ca2+]i rise has been studied in CHO cells stably transfected with human TSH receptor (hTSHR) cDNA. In human thyroid slices, TSH activates these two intracellular cascades with a higher affinity for the adenylate cyclase activation (from 0.1 to 1 mU/ml TSH) than for phospholipase C activation (from 1 to 10 mU/ml TSH). The CHO cells transfected with the recently cloned cDNA of human TSH receptor respond in the same way to TSH. They respond between 0.1 and 1 mU/ml TSH for cyclic AMP accumulation and between 1 and 10 mU/ml TSH for inositol monophosphate (IP1) increase. In these same cells, TSH 10 mU/ml, but not forskolin (10 microM), or dibutyryl cyclic AMP (100 microM), clearly enhances intracellular calcium concentration [( Ca2+]i). Our results demonstrate unequivocally that a single transcription unit has the potential to encode receptor molecules coupled to both cascades.

Adenylyl Cyclases↗
PubMed1990

Molecular cloning of a dog thyrotropin (TSH) receptor variant.

A clone coding for a variant form of thyrotropin receptor was isolated from a dog thyroid cDNA library. It was characterized by a 75 bp deletion in the coding region, additionally to minor modifications in the 3' untranslated region. The corresponding 25 amino acids deletion is located in the long NH2 terminal extracellular domain which is characteristic of the glycoprotein hormone receptors. This region of the protein is composed of imperfect repeats and the deletion corresponds exactly to one of the repeat units. This suggests that the repeats correspond to individual exons in the thyrotropin receptor chromosomal gene. It is not known whether the deletion of the repeat and the concomitant suppression of one of the N-glycosylation sites of the molecule do alter the receptor function.

Amino Acid Sequence↗
PubMed1990

An efficient screening morphological test for the identification and characterization of cyclic AMP-coupled hormone receptors.

We describe here a new method for the identification of adenylate cyclase-regulating receptors using the morphological response of Y1 cells to cAMP. The efficiency of this method was demonstrated with a cloned beta 2-adrenergic receptor: the coding region of a genomic beta 2-adrenergic receptor clone was amplified using the polymerase chain reaction, so that in vitro RNA transcripts of this DNA could be obtained. Y1 cells were microinjected with this RNA and assayed for sensitivity to isoproterenol. In the presence of this agonist, the microinjected cells rapidly showed morphological changes identical to those observed in the presence of ACTH, forskolin, or cholera toxin, agents that enhance cAMP accumulation in the control uninjected cells. This suggests the integration, within the injected cell membrane, of a functional beta 2-adrenergic receptor, whose activation by isoproterenol resulted in the intracellular formation of cAMP. This new qualitative screening method for the identification of adenylate cyclase-regulating receptors can be extended to any unknown receptor coupled to adenylate cyclase and absent in these cells. It has been applied to the identification of the dog thyrotropin receptor.

Adrenal Gland Neoplasms↗
PubMed1990

Regulation of protooncogenes c-fos and c-myc expressions by protein tyrosine kinase, protein kinase C, and cyclic AMP mitogenic pathways in dog primary thyrocytes: a positive and negative control by cyclic AMP on c-myc expression.

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes.

Animals↗
PubMed1989

Molecular cloning of the thyrotropin receptor.

The pituitary hormone thyrotropin, or thyroid-stimulating hormone (TSH), is the main physiological agent that regulates the thyroid gland. The thyrotropin receptor (TSHR) was cloned by selective amplification with the polymerase chain reaction of DNA segments presenting sequence similarity with genes for G protein-coupled receptors. Out of 11 new putative receptor clones obtained from genomic DNA, one had sequence characteristics different from all the others. Although this clone did not hybridize to thyroid transcripts, screening of a dog thyroid complementary DNA (cDNA) library at moderate stringency identified a cDNA encoding a 4.9-kilobase thyroid-specific transcript. The polypeptide encoded by this thyroid-specific transcript consisted of a 398-amino acid residue amino-terminal segment, constituting a putative extracellular domain, connected to a 346-residue carboxyl-terminal domain that contained seven putative transmembrane segments. Expression of the cDNA conferred TSH responsiveness to Xenopus oocytes and Y1 cells and a TSH binding phenotype to COS cells. The TSHR and the receptor for luteinizing hormone-choriogonadotropin constitute a subfamily of G protein-coupled receptors with distinct sequence characteristics.

Amino Acid Sequence↗