Dog Thyrocytes In Primary Culture Research Articles

Identification of differentially expressed genes in thyrotropin stimulated dog thyroid cells by the cDNA-AFLP technique

In dog thyrocytes in primary culture, thyrotropin (TSH), through cAMP, positively controls proliferation and differentiation. As until now, the key events and the genes involved in the action of TSH remain largely uncharacterized, our goal was to identify new differentially expressed genes in TSH-induced thyroid proliferation. Using cDNA-AFLP, we vizualized 105 different transcripts showing significant differential expression during the stimulation of dog thyrocytes with TSH for different times, in the presence of insulin. Northern blot and RT-PCR analyses confirmed the pattern expression of 5 clones encoding known proteins: thrombospondine 1, TNFr1, RhoE, RalB, and annexin A2. These regulations provide molecular counterparts of in vivo physiological effects of TSH: angiogenesis (decreased thrombospondin 1), decreased apoptosis (decreased TNFR1) and actin filament disruption, macropinocytosis and thyroid hormone secretion (decreased RhoE).

Study of Gene Expression in Thyrotropin-Stimulated Thyroid Cells by cDNA Expression Array: ID3 Transcription Modulating Factor as an Early Response Protein and Tumor Marker in Thyroid Carcinomas

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with 32P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

TTF-2 Does Not Appear to Be a Key Mediator of the Effect of Cyclic AMP on Thyroglobulin Gene Transcription in Primary Cultured Dog Thyrocytes

TTF-2 is a thyroid-specific winged-helix transcription factor which has been proposed to play a key role in the hormonal control of thyroglobulin and thyroperoxidase genes transcription in FRTL-5 cells. We have analyzed TTF-2 DNA-binding activity in primary cultures of dog thyrocytes maintained in control condition or in the presence of the cAMP agonist forskolin. Binding of35S-labelled nuclear proteins to the TTF-2 recognition sequence identified the presence of two molecular species of 41.5 and 42.5 kDa. TTF-2 DNA-binding activity was clearly detectable in nuclear extracts from unstimulated cells and appeared increased in forskolin-treated cells. Thus, the presence of TTF-2 DNA-binding activity does not correlate with the cAMP-dependent activity of thyroglobulin and thyroperoxidase genes in this cell system. In addition, the mutation of the TTF-2 binding site in the thyroglobulin promoter resulted in a very reduced but still clearly cAMP-dependent promoter activity when assayed by transient expression in the same cells. These results do not support a dominant role for TTF-2 in the cAMP-dependent control of thyroglobulin gene transcription in primary cultured thyrocytes.

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.

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

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

Identification of a thyroid-specific and cAMP-responsive enhancer in the upstream sequences of the human thyroglobulin promoter

Functional analysis of remote 5′-flanking sequences from the human thyroglobulin gene in primary cultured dog thyrocytes led to the identification of a partly cAMP-responsive enhancer, located between −3.6 to −2.2 kb from the transcriptional start site. Deletion analysis of the 1.4 kb-long region localised the enhancer activity in a 0.5 kb-long fragment (located between −3.2 and −2.7 kb relative to transcription start), which could be divided into two functional sub-fragments of 0.2 and 0.3 kb. A potential binding site for the CREB/ATF transcription factors was found in the 0.3 kb element. The complete enhancer region had no detectable activity when assayed in Hela cells, suggesting that it constituted a thyroid-specific regulatory element. Accordingly, footprinting experiments revealed the presence of several binding sites for Thyroid Transcription Factor-1 (TTF-1) in both the 0.2 and 0.3 kb elements.

Study of TTF-1 gene expression in dog thyrocytes in primary culture

TTF-1 is a homeodomain-containing transcription factor mainly expressed in the thyroid where it controls the tissue-specific expression of the thyrogobulin, thyroperoxidase and TSH receptor genes. It is therefore potentially implicated in the hormonal control exerted by thyrotropin via the second messenger cyclic AMP on the transcription of these genes in thyrocytes. In order to investigate whether there exists a relationship between the stimulation of the cAMP pathway and TTF-1 gene expression in these cells, we have compared the amounts of TTF-1 protein, its state of phosphorylation and its subcellular distribution in control and cAMP-stimulated dog thyrocytes in primary culture. Dog TTF-1 was expressed in bacteria as a fusion protein and antibodies were raised against the dog TTF-1 moiety. Stimulation of the thyrocytes by cyclic AMP agonist only marginally increased TTF-1 gene expression as shown for the mRNA by RNase protection assay and for the protein by immunoblotting and immunoprecipitation of extracts from 35S-methionine labelled cells. The phosphorylation state of TTF-1 was investigated by immunoprecipitation of extracts from 32P-labelled thyrocytes. Phosphorylation level appeared to be essentially unaffected by forskolin treatment of the cells. We also looked for differences in the use of phosphorylation sites by partial proteolytic digestion of immunoprecipitated 32P-labelled TTF-1 with Glu-C and Asp-N endoproteases. Comparison of radioactivity distribution amongst the generated fragements did not reveal any difference in the pattern of TTF-1 phosphorylation in control and forskolin conditions. Lastly, in situ detection of TTF-1 by immunofluorescence demonstrated that the protein was localized in the nucleus of the cells, irrespective of the culture conditions. No major change in TTF-1 gene expression upon stimulation of the thyrocyte with a cAMP agonist could thus be detected in this study. The absence of an obvious modification of the TTF-1 protein itself in response to cAMP stimulation may indicate that other transcription factor(s) or co-factor(s) are involved in the control exerted by cAMP on the expression of thyroid-specific genes.

Jun B Expression Is Regulated Differently by Three Mitogenic Pathways in Thyrocytes

In dog thyrocytes in primary culture, thyrotropin (TSH), acting through cyclic AMP, induces proliferation and differentiation expression, while tetradecanoylphorbol acetate (TPA) or epidermal growth factor (EGF) induces proliferation and dedifferentiation. In this work, we have investigated the regulation of mRNA expression of the protooncogene jun B in these cells. TSH stimulated jun B expression very transiently with biphasic kinetics similar to those obtained for c- myc mRNA accumulation. Forskolin reproduced these effects, suggesting that they are, as other effects of TSH in this system, mediated by cyclic AMP. As shown by nuclear run-on experiments, jun B is regulated by the cAMP pathway at the transcriptional level. As in other cell types, EGF or TPA caused a more sustained increase in mRNA levels. In thyroid slices, in which DNA synthesis appears to be induced by the wounding process, jun B is also induced, suggesting a correlation with the proliferative status of the cell. Interestingly, two jun B mRNAs of 2.1 and 2.3 kb were induced by all the mitogenic pathways. The kinetics of their accumulation were different; i.e., TPA induced the smaller transcript with some delay after the longer one and cycloheximide induced the progressive shortening of the first appearing heavier mRNA. The 2.3-kb messenger has a longer poly(A) tail, and kinetics in the presence of actinomycin D suggested it could represent a precursor form of the 2.l-kb messenger. It is suggested that the specific kinetics of cyclic-AMP-induced accumulation of jun B mRNA could be related to the dual stimulation of differentiation and proliferation by TSH in dog thyrocytes.

Regulation of the Max Gene Expression by Different Mitogenic Pathways in Dog Primary Thyrocytes

The family of Myc proteins appears to function through heterodimerization with the bHLH-Zip protein Max. We have investigated the regulation of Max mRNA in primary cultured dog thyrocytes whose proliferation is stimulated by three distinct mitogenic pathways: (1) the thyrotropin (TSH) cascade mediated by cyclic AMP, (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA), (3) a protein tyrosine kinase system activated by epidermal growth factor (EGF). Among these cascades only the first is compatible with differentiation. We have observed that the mRNA of Max is stable and regulated differentially by the three pathways in our system. TSH decreases the level of the messenger while EGF or TPA increases it with early delayed kinetics. The effect of cyclic AMP on the accumulation of Max is observed even in the presence of cycloheximide, while the EGF- or TPA-induced increase is cycloheximide sensitive, suggesting differences in regulation pathways. Max mRNA is regulated with the same intensity, but in a more delayed fashion than the messenger of c-myc. As Max protein mediates the transcriptional effects of c-Myc our results are compatible with a mediator role of Max in modulating the cell sensitivity to the proliferative signal c-Myc.

Human, bovine, canine and rat thyroglobulin promoter sequences display species-specific differences in an in vitro study

The proximal promoter regions of the thyroglobulin gene from man, beef, dog and rat were compared by transient expression in primary cultured dog thyrocytes. All four promoter regions were able to control properly the expression of a reporter gene in response to cyclic AMP stimulation. Surprisingly, despite extensive sequence conservation, the transcriptional activities of these four mammalian thyroglobulin promoters were differently affected by equivalent mutations. Homologous sequence elements from these promoter regions also exhibited distinct binding characteristics in mobility-shift experiments conducted in the presence of nuclear proteins from bovine thyroids. Our observations show that the highly conserved thyroglobulin promoters may exhibit unexpected functional differences in a specific assay and indicate that some of the molecular mechanisms involved in the control of thyroglobulin gene expression have evolved differently within mammals.

Identification of a transcriptional enhancer upstream from the bovine thyroglobulin gene

The DNA sequences corresponding to a DNasel-hypersensitive region identified previously in bovine thyroglobulin gene chromatin (Hansen et al. (1988) Eur. J. Biochem. 178, 387–393) exhibited the properties of a transcriptional enhancer in a transient assay in primary cultured dog thyrocytes, but did not so in transfected HeLa cells. By contrast to the thyroglobulin proximal promoter, the enhancer element did not require cyclic AMP stimulation of the thyrocytes to be active. Using a bi-directional deletion approach, the minimal region displaying enhancer activity has been localized between positions − 1906 and − 1744 relative to the thyroglobulin gene transcription start. DNA-footprinting experiments revealed the presence of several binding sites for the thyroid-specific transcription factor TTF-1 within the enhancer sequence.

Functional role of TTF-1 binding sites in bovine thyroglobulin promoter

We have studied the binding of purified TTF-1 on the bovine thyroglobulin gene promoter. DNase I footprinting experiments revealed three binding sites which corresponded in location to the A, B and C sites found in the rat thyroglobulin promoter. Mutants in the A and C regions showing reduced binding of TTF-1, also exhibited largely decreased promoter activity in transient expression experiments in primary-cultured dog thyrocytes. Two mutants in the B site that exhibited a reduced capacity to bind TTF-1 also displayed a drastically affected transcriptional activity in transient assays. As in the rat, sites A and C only are critical for promoter activity, these results suggest that full occupancy of the B site is required for thyroglobulin promoter activity in the cow only.

Isolation and characterization of the canine thyroglobulin gene promoter region

The 5' flanking sequences from the canine thyroglobulin gene were isolated by homology screening with the evolutionary conserved sequence from the bovine thyroglobulin promoter and sequenced. Transient expression in primary cultured dog thyrocytes demonstrated that the canine clone contains a functional promoter inducible by cAMP. DNAse I footprinting assays showed that the thyroid-specific transcription factor TTF-1, purified from bovine thyroid, also recognizes the canine thyroglobulin promoter. Similar footprints were obtained with crude nuclear extracts from primary cultured dog thyrocytes.

Control of the intracellular Ca2+‐concentration and the inositol phosphate accumulation in dog thyrocyte primary culture: Evidence for different kinetics of Ca2+‐phosphatidylinositol cascade activation and for involvement in the regulation of H2O2 production

Carbachol, through a muscarinic receptor, thyrotropin-releasing hormone (TRH), prostaglandin F2 alpha (PGF2 alpha), bradykinin, and adenosine triphosphate (ATP) increased the apparent [Ca2+]i (intracellular free Ca2(+)-concentration) of dog thyrocytes in primary culture. The [Ca2+]i measured by the Quin-2 technique rose immediately after the addition of the agonists and reached a maximal value after less than 30 seconds. Afterwards, the [Ca2+]i declined to a plateau higher than the basal level when the cells were triggered with carbachol. By contrast, in most experiments with PGF2 alpha and in the case of bradykinin, TRH, and ATP, the [Ca2+]i returned to the basal value. If the extracellular Ca2+ was chelated by excess of EGTA, the addition of all agents caused a sharp reduced transient rise in the [Ca2+]i followed by a decline of the [Ca2+]i often below the basal level (especially in the case of carbachol). It is suggested that the first transient phase of these responses is due at least in part to the mobilisation of Ca2+ from intracellular stores whereas the second sustained phase of the response to carbachol mainly originates from an increased Ca2+ influx into the thyrocytes. Carbachol, bradykinin, TRH, PGF2 alpha, and ATP also increased generation of inositol phosphates in dog thyrocytes. This effect was sustained when the cells were triggered with carbachol and was more transient with bradykinin, TRH, PGF2 alpha, or ATP. All these agents and the phorbdester TPA as well as forskolin enhanced to various extent the thyrocyte H2O2 generation. This enhancement was severely reduced in the absence of extracellular Ca2+ and was mimicked by Ca2+ ionophores in the presence of extracellular Ca2+ especially in synergy with protein kinase C activators. These data suggest that the dog thyrocyte H2O2 generation, the limiting step of the thyroid hormone synthesis, is modulated by carbachol, TRH, PGF2 alpha, bradykinin, and ATP through their action on the Ca2(+)-phosphatidylinositol cascade.

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.

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 protoon-congenes 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 neosynthetized 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.

Identification of a cAMP-responsive region in thyroglobulin gene promoter

The DNA sequences involved in transcription control by a cAMP-dependent mechanism have been localized in the thyroglobulin gene promoter region by a functional assay. The proximal 5'-flanking sequences from the bovine thyroglobulin gene were linked to the bacterial chloramphenicol acetyl-transferase gene. Transient expression of this reporter gene was studied in dog thyrocytes in primary culture in the presence, or absence, of cAMP stimulation. Deletion analysis showed that the cAMP-responsive region is contained within the first 250 base-pairs of the promoter, and suggests that it could correspond to a sequence conserved between species. These DNA sequences do not bear significant homology with cAMP-responsive elements (CRE) described previously. By contrast, some similarities were found with the fat-specific element (FSE2) of genes under cAMP control in adipocytes and with DNA elements mediating cAMP-dependent regulation of expression of two different genes in the lower eukaryote Dictyostelium discoideum. This suggests that control of Tg gene transcription by cAMP could involve a mechanism different from the one mediated by a classical CRE.

Induction of DNA synthesis in dog thyrocytes in primary culture: synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin.

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.