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C Maenhaut

Publications and source records attributed to C Maenhaut.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation by thyroid stimulating hormone of nerve growth factor-induced gene-B expression in thyrocytes in culture: relation with proliferation and specific gene expression.

Nerve growth factor-induced gene-B (NGFI-B) is an immediate early gene first found as a part of the PC12 cell response to NGF (Milbrandt, J., Science 238 (1987) 797-799). We have previously reported that NGFI-B mRNA is strongly upregulated by thyroid-stimulating hormone (TSH) in dog thyrocytes in culture (Pichon et al., Endocrinology 137 (1996) 4691-4698). In this study, we have analyzed the regulation of NGFI-B mRNA expression by a variety of agents acting on thyrocytes proliferation and/or differentiation. We show that: (1) the induction of NGFI-B mRNA is stronger after stimulation of the cAMP cascade, but it is not restricted to this signaling pathway; (2) the powerful mitogens for thyroid cells EGF and HGF have little or no effect on NGFI-B mRNA induction; (3) NGFI-B mRNA is induced by anisomycin at a subinhibitory concentration for protein synthesis, and is superinduced by the combination of TSH and anisomycin; this treatment decreases the TSH-induced proliferation levels, but does not inhibit the induction of some differentiation markers; and (4) both in dog and in pig thyrocytes, NGFI-B mRNA induction is observed after a variety of treatments stimulating differentiation, but without proliferative effects. Our results therefore suggest that NGFI-B mRNA induction might not be related to TSH-induced thyrocyte proliferation, but could participate in the differentiation program triggered by TSH.

Animals↗
PubMed1997

Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells.

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues. several potential phosphorylation sites. and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epidermal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.

Adenoviridae↗
PubMed1997

Moderate doses of iodide in vivo inhibit cell proliferation and the expression of thyroperoxidase and Na+/I- symporter mRNAs in dog thyroid.

The function and the growth of adult thyroid gland is controlled by the opposite actions of thyrotropin (TSH) and iodide, the main substrate of the gland. Iodide deprivation leads to stimulation of the thyroid, improving the efficiency of iodide transport for hormone biosynthesis. We have investigated cell proliferation and thyroid specific gene expression 24 and 48 h after administering KI to dogs previously treated with goitrogens and perchlorate. In the hypothyroid dogs T3 and T4 serum levels decreased from 53 +/- 4 to < 30 ng/dl and from 1.6 +/- 0.6 to < 1 microg/dl respectively; TSH concentration increased from 0.16 +/- 0.02 to 2.7 +/- 0.4 ng/ml. After a 24 h moderate KI treatment (300 microg KI/dog of +/- 10 kg) serum T3 concentrations rose higher than the initial normal values, while T4 concentrations increased to reach values equivalent to the normal level. The high TSH concentration did not change significantly. The hyperplasia of the chronically stimulated thyroid resulting from goitrogens/NaClO4 treatment was not modified by this short term treatment with KI. In contrast, KI decreased the weight of the total gland and the level of cell proliferation, as determined by the fraction of cells incorporating BrdU. The effect of acute administration of KI on the expression of four major thyroid genes, the TSH receptor (TSHr), thyroglobulin (Tg), thyroperoxidase (TPO), and Na+/I- symporter (NIS) was analyzed by Northern blot. Tg, TPO and NIS mRNA expressions were up-regulated by chronic stimulation. The expression of the mRNAs of TSHr and Tg did not significantly differ between hyperstimulated and KI-treated dogs while TPO and NIS mRNA expression decreased after a 48 h KI treatment. TPO and NIS are therefore the only of these four genes whose expression is acutely modulated by iodide in vivo. Under TSH stimulation low doses of iodide resulted in: (1) decreased cell proliferation, (2) reestablished synthesis and secretion of thyroid hormones, (3) diminished TPO and NIS mRNA expression. Notably low doses of iodide under the same conditions had no effect on Tg and TSHr mRNA expression.

Animals↗
PubMed1997

Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin.

Thyrotropin (TSH) through the cAMP cascade and in the presence of insulin induces the proliferation of dog thyroid cells. In this work, it is shown that TSH via cAMP causes the phosphorylation of the three members of the pRb family, pRb, p107, and p130, with the same kinetics as those observed when these cells are stimulated by mitogens acting through a tyrosine kinase receptor or through activation of kinase C. It is the first described point of convergence of cAMP-dependent and -independent mitogenic pathways in dog thyrocytes and suggests that the phosphorylation of the three proteins may be involved in the initiation of DNA synthesis in these cells. We also show that insulin, which induces hypertrophy and is permissive for the TSH mitogenic action, does not provoke the phosphorylation of any pRb family member, suggesting that none of these phosphorylations is required for this effect.

Animals↗
PubMed1997

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

Animals↗
PubMed1996

Identification and characterization of novel genes modulated in the thyroid of dogs treated with methimazole and propylthiouracil.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific stimulation or repression of a large number of genes. To better understand differentiated epithelial cell growth regulation, we have initiated a study to identify genes which are regulated by the thyrotropin-dependent mitogenic pathway in dog thyroid cells. A thyroid cDNA library was prepared from a methimazole and propylthiouracil-treated dog and differentially screened with probes derived from control or stimulated thyroids. Among 19 clones isolated, 6 encode known proteins (inwardly rectifying potassium channel, nucleosome assembly protein, ribosomal protein L7, elongation factor 1alpha, non-muscle myosin light chain, and heat shock protein 90beta). The 13 others correspond to proteins whose function is unknown. Among them, 5 correspond to mRNAs whose expression was modulated by mitogenic stimulation of thyrocytes in primary culture. A preliminary characterization of two of these cDNAs is reported: clone 5, which might represent a novel, atypical protein kinase, and clone 3, which contains ankyrin-like repeats, suggesting that it might interact with other proteins.

Animals↗
PubMed1996

Induction of nerve growth factor-induced gene-B (NGFI-B) as an early event in the cyclic adenosine monophosphate response of dog thyrocytes in primary culture.

We investigated the induction of nerve growth factor-induced gene-B (NGFI-B) in dog thyrocytes in primary culture stimulated by different agents. The dog NGFI-B complementary DNA (cDNA) was cloned from a cDNA library of dog thyrocytes and used to study, by Northern blotting, the level of NGFI-B messenger RNA (mRNA) in those cells. We have shown that TSH and forskolin, which both induce proliferation and differentiation of the thyroid cells by activation of the protein kinase A pathway, lead to a strong and transient expression of two NGFI-B mRNA species, which differ in the length of the poly(A) tail. In contrast, 12-O-tetradecanoyl-13-phorbol-acetate (TPA) and epidermal growth factor, which induce proliferation and dedifferentiation of those cells by activation of the protein kinase C and the protein tyrosine kinase cascade, respectively, lead to a weaker expression of NGFI-B mRNA. In parallel, we studied the transactivation capacity of NGFI-B in the same cell system by transient transfection of a chloramphenicol acetyl transferase reporter construction containing a NGFI-B-dependent synthetic promoter. The highest transactivation was observed after forskolin stimulation, whereas transactivation after TPA stimulation was weak and no significant transactivation was observed after epidermal growth factor stimulation. Taken together, these results show that NGFI-B is an immediate early gene product that is mainly induced by the cAMP-dependent pathway in dog thyrocytes. Moreover they suggest that NGFI-B expression could be one of the early transcriptional changes induced specifically by this cascade and leading to differentiation and/or proliferation of these cells.

Amino Acid Sequence↗
PubMed1995

Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro.

E-cadherin, a cell-cell adhesion protein specifically expressed at the basolateral membrane of thyrocytes, is variably dysregulated in thyroid carcinomas in parallel to the dedifferentiation of the tumors. No data are currently available on the regulation of E-cadherin in messenger RNA (mRNA) expression by physiological stimulators of thyroid proliferation and differentiation. The present study investigated the control of E-cadherin steady state mRNA levels and protein expression in primary cultures of dog and human thyrocytes under the influence of physiological regulators of thyroid differentiation and dedifferentiation using Northern blot analysis and immunohistochemistry. Following dedifferentiation by epidermal growth factor and fetal calf serum in primary cultures of dog and human thyrocytes, E-cadherin steady state mRNA expression was low but easily detectable. Stimulation of the cells by TSH (1 mU/ml) or forskolin (10 microM) induced an increase in E-cadherin mRNA levels with a maximal effect after 20 h. An up-regulation of E-cadherin protein levels are also observed by immunostaining with anti-E-cadherin antibodies. A concentration-response relation determined for TSH stimulation (10 microU/ml to 10 mU/ml) led to a concentration-dependent stimulation of E-cadherin mRNA levels and a parallel increase in protein expression with a minimal effective concentration of 10-30 microU/ml. These effects depend on protein synthesis as they are completely blocked by the presence of 10 micrograms/ml cycloheximide. Treatment with EGF did not markedly alter E-cadherin mRNA expression, whereas removal of insulin from the medium slightly decreased E-cadherin mRNA and protein levels. There is, therefore, a qualitative parallelism between the effect of the various factors on E-cadherin protein and mRNA levels. These results suggest that the cell-cell adhesion protein E-cadherin is under the control of the TSH-cAMP-dependent pathway and may play an important physiological role on the action of this pathway in proliferation and differentiation.

Animals↗
PubMed1994

Cloning of cDNA specifically involved in the thyroid cAMP mitogenic pathway.

The activation of the cyclic AMP cascade in dog and human thyroid cells in primary culture induces the expression of differentiated gene expression, hyperfunction and proliferation. These programs are developed simultaneously in quiescent dedifferentiated cells. In this paper the strategy followed by our group to define the genes involved in the cAMP mitogenic cascade is outlined.

Animals↗
PubMed1994

Expression of thyrotropin receptor (TSH-R), thyroglobulin, thyroperoxidase, and calcitonin messenger ribonucleic acids in thyroid carcinomas: evidence of TSH-R gene transcript in medullary histotype.

We studied the expression of the TSH receptor (TSH-R), thyroglobulin (Tg), thyroperoxidase (TPO), and calcitonin (CT) genes in a total of 53 tissues from 30 patients with thyroid carcinoma and from 9 patients with benign thyroid diseases. By Northern blot analysis of total RNA preparations, CT mRNA was expressed in all cases (n = 6) of medullary thyroid carcinoma (MTC). Surprisingly, 3 of them expressed the TSH-R mRNA, in association with the Tg and TPO mRNAs in 1. The presence of the TSH-R transcript in the neoplastic C-cells was confirmed in 1 MTC by in situ hybridization using a mixture of 3 oligonucleotide probes derived from dog TSH-R cDNA. With various degrees of expression, all differentiated thyroid carcinomas (20 papillary and 2 follicular) expressed TSH-R, Tg, and TPO, but not CT mRNAs. On the contrary, samples from 2 patients with anaplastic carcinoma did not express TSH-R, Tg, or TPO mRNA, but 1 of them expressed CT mRNA. All of the transcripts obtained from thyroid carcinomas (both primary and metastatic) were of the same size as the transcripts from normal or benign thyroid tissues, with the exception of 2 cases of differentiated thyroid cancer, in which TSH-R mRNA of lower mol wt (approximately 4.0 kilobases) was found in the absence of alteration in cDNA size and restriction map. The main conclusions of our study are that 1) the TSH-R gene is expressed in some MTC, which supports, at molecular level, the hypothesis of the existence of mixed follicular-medullary thyroid tumors; and 2) the expression of TSH-R, Tg, and TPO in undifferentiated thyroid cancer is lost.

Blotting, Northern↗
PubMed1993

The human and dog 5-HT1D receptors can both activate and inhibit adenylate cyclase in transfected cells.

The cloned human serotonin 1D (5-HT1D) receptor has been shown to inhibit adenylate cyclase while the corresponding cloned dog receptor has been characterized by its enhancement of cAMP accumulation. To resolve this apparent discrepancy, the human 5-HT1D receptor has been cloned and expressed in Chinese hamster ovary (CHO) cells and the corresponding dog receptor expressed in mutant Y1 adrenal (Y1 Kin-8) cells. It is shown that both receptors when activated by sumatriptan depress forskolin induced adenosine 3'5'-cyclic monophosphate (cAMP) accumulation by a pertussis toxin sensitive mechanism, presumably involving Gi (the adenylate cyclase inhibitory GTP transducing protein). In the absence of forskolin, the dog receptor enhances cAMP accumulation, thus activating Gs (the adenylate cyclase stimulatory GTP transducing protein). When its overriding action on Gi is blocked by pertussis toxin pretreatment, the human receptor also enhances cAMP accumulation. Thus both 5-HT1D receptors activate markedly Gi and to a lesser extent Gs and can exert opposite effects on the same effector system, adenylate cyclase.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

In vitro and in vivo regulation of thyrotropin receptor mRNA levels in dog and human thyroid cells.

Regulation of thyrotropin (TSH) receptor (TSHr) mRNA accumulation as compared with two other thyroid differentiation markers (thyroglobulin and thyroperoxidase (TPO] has been investigated by Northern blot. In dogs in vivo, chronic stimulation of the thyroid TSHr mRNA although it increased the levels of thyroglobulin and TPO mRNA. In dogs treated with thyroxin, the quiescent thyroids expressed normal levels of TSHr and TPO mRNA but depressed levels of thyroglobulin mRNA. In primary cultures of dog thyrocytes, dedifferentiation of the cells by treatment with epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate led to decreased TSHr mRNA levels and nearly abolished thyroglobulin and TPO gene expression. However, TSHr mRNA was always present, compatible with the fact that these cells, when treated by TSH, reexpress differentiation. Treatment of the cells with TSH or forskolin transiently increased the TSHr mRNA level after 20 h, an effect inhibited by cycloheximide. This up-regulation was confirmed at the protein level: forskolin-treated cells showed an enhanced cAMP response to TSH and an increased binding of labeled TSH to their membranes. Long term TSH treatment led to a slight down-regulation of TSHr mRNA in dog thyrocytes, but in human thyroid cells no marked down-regulation was observed.

Animals↗