EXPRESSION OF MENIN IN THE HUMAN THYROID GLAND.

INTRODUCTION: The expression of menin in the thyroid gland has long been debated. Animal models with targeted inactivation of menin in the thyroid gland have shown that its inactivation might play a role in the progression to a more aggressive type of cancer. Human studies are conflicting, some have identified mutations in the MEN1 gene in a sub-type of oncocytic thyroid carcinomas, while others have not identified a higher prevalence of thyroid cancer in MEN1 patients. OBJECTIVE: To analyze the immunohistochemical expression of menin in different types of thyroid carcinomas. MATERIALS AND METHODS: 48 thyroid tumours (12 papillary thyroid carcinomas (PTC), 6 anaplastic thyroid carcinomas (ATC), 12 poorly differentiated thyroid carcinomas (PDTC), 5 medullary thyroid carcinomas (MTC), 5 oncocytic follicular carcinomas (OC), 3 oncocytic adenomas (OA) and 5 goiters (G)) were tested for nuclear expression of menin using an anti-menin antibody. The expression was considered positive, negative or decreased. RESULTS: The expression of menin was positive, identical to normal tissue, in 39 cases (81.25%). The expression was decreased (n=8) or absent (n=1) in 9 tumours (18.75% - 2 PTC, 5 PDTC, 2 OC) accounting for 42% (5/12) of the PDTC and 40% (2/5) of the OC. CONCLUSIONS: Our results show that the expression of menin is generally preserved in human thyroid carcinomas, but it can be decreased or absent in certain types of thyroid cancer. Further molecular studies are needed to evaluate to potential of menin protein in tumorigenesis.

Connexin 32 fused to the green fluorescent protein retains its ability to control the proliferation of thyroid cells.

Cell-to-cell exchanges of signaling molecules are thought to be involved in the control of cell proliferation. Connexins, which are encoded by a family of genes expressed in a cell type-specific manner, are considered as tumor suppressors. Thyroid epithelial cells co-express connexin 32 (Cx32) and connexin 43 (Cx43) that form distinct and delocalized gap junctions in vivo. The communication-deficient rat thyroid-derived cell lines, FRTL-5 and FRT, stably transfected with the Cx32 cDNA, have a reduced proliferation rate related to a prolonged G1 cell cycle phase. To determine whether Cx32-gap junctions exert the same regulatory role in vivo, we have undertaken a program of production of transgenic mice over-expressing Cx32 specifically in thyrocytes. To this purpose, we designed a vector in which the Cx32 cDNA was fused to the gene encoding the enhanced green fluorescent protein (EGFP) and placed under the control of a strong and thyroid-specific promoter, the thyroglobulin gene promoter (pTg). In stably transfected FRTL-5 cells, the Cx32/EGFP chimeric protein forms functional gap junction channels and induces the same proliferation slowdown as native Cx32. The pTg-Cx32/EGFP construct should thus allow us to obtain the thyroid-targeted over-expression of Cx32 in the mouse to investigate the involvement of Cx32-gap junctions in thyroid growth, functional activity and propensity to form tumors.

Role of the conserved C-terminal region of thyroid hormone receptor-alpha in ligand-dependent transcriptional activation.

The ligand binding domain (LBD) of thyroid hormone (T3) receptors contains subdomains that participate in transcriptional activation, hormone-relieved repression and dimerization. A sequence conserved within the nuclear receptor superfamily is found at positions 397-405 of the 408-amino acid chicken T3 receptor-alpha (cTR alpha) and is deleted in the related avian v-erbA. Since v-erbA exhibits compromised ligand binding and transcriptional activation, this conserved region may play a role in ligand-dependent transcriptional activation. Transfections reveal that cTR alpha(1-392) and site-directed mutants cTR alpha(L398R) and cTR alpha(F399E) are inactive, while cTR alpha(1-403) displays reduced ligand-dependent transcriptional activity. The loss of transcriptional activity in cTR alpha(1-392) is not caused by impaired DNA binding or receptor dimer formation. Proteolytic protection assays reveal that both transcriptionally active and inactive cTR alpha derivatives undergo T3-mediated conformational changes. Gal4 chimeras containing the final 16, 35 or 44 amino acids of cTR alpha indicate that the conserved C-terminal region does not function as an independent transactivation domain. Our results are consistent with a model in which ligand plays a structural role to position the conserved C-terminal regions of cTR alpha and related receptors in a transcriptionally active conformation.

Expression of alpha- and beta-subunits and activity of Na+K+ ATPase in pig thyroid cells in primary culture: modulation by thyrotropin and thyroid hormones.

Na+ K+ ATPase located at the basolateral pole of thyroid epithelial cells, contributes to thyroid hormone synthesis by generating the driving force for the uptake of the substrate, iodide. We have investigated whether the expression of the alpha- and beta-subunits and activity of Na+ K+ ATPase were subjected to variations in response, (a) to TSH, that controls the expression of differentiation in thyroid cells and (b) to thyroid hormones as potential autocrine factors. Studies were carried out on pig thyroid cells cultured (a) without TSH to obtain thyroid cell monolayers (TCM) in basal state or (b) with TSH in the form of cell monolayers (TCM-T) or as reconstituted thyroid follicles (RTF). Iodide uptake activity, thyroperoxidase protein and thyroglobulin mRNA taken as parameters of thyroid cell differentiation were 6 to 25-fold higher in RTF and TCM-T than in TCM. Western blot analyses of Na+ K+ ATPase subunits revealed that the alpha-subunit (105 kDa) content of TCM-T and RTF was similar but 8-fold higher than that of TCM. In contrast, the beta-subunit (50 kDa) content of TCM-T and RTF was only about twice that of TCM. Similar relative variations were observed at the mRNA level for both alpha- and beta-subunits. Na+ K+ ATPase activity was only 40% higher in RTF and TCM-T than in TCM. A 48 h treatment of RTF by either T4 or T3 (1-100 nM) induced a 3-fold increase of the alpha-subunit but did neither alter the beta-subunit nor the Na+ K+ ATPase activity. In conclusion, Na+ K+ ATPase activity and the level of expression of its beta-subunit, known to control the assembly and targetting of alpha-beta oligomers and thus the amount of functional sodium pump at the plasma membrane, are only moderately altered when thyroid cells undergo major changes in their differentiation status. Our data show that the expression of the alpha-subunit of Na+ K+ ATPase by thyroid cells is up-regulated by TSH and thyroid hormones.

Restoration of cell-to-cell communication in thyroid cell lines by transfection with and stable expression of the connexin-32 gene. Impact on cell proliferation and tissue-specific gene expression.

Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.

Characterization of the rat thyroid iodide transporter using anti-peptide antibodies. Relationship between its expression and activity.

Anti-peptide antibodies directed against the C-terminal portion (amino acids 603-618) of the rat thyroid iodide transporter (rTIT) have been produced to characterize the molecular forms of rTIT in the rat thyroid and in the functional rat thyroid cell line, FRTL-5. rTIT is located on the basolateral membrane of rat thyroid follicular cells and randomly distributed on the plasma membrane of FRTL-5 cells that do not exhibit cell polarity. The major rTIT component corresponds to an 80-90-kDa glycosylated protein. After treatment of cell membrane fractions with N-glycosidase F or incubation of FRTL-5 cells with tunicamycin, rTIT has an apparent molecular mass of about 55 kDa. FRTL-5 cells cultured in the presence of TSH exhibit a high rTIT content and a high iodide uptake activity (IUA). Upon either removal of TSH or addition of cycloheximide, IUA declines more rapidly than rTIT. The half-life of rTIT was about 4 days. Re-exposure of 7-day TSH-deprived FRTL-5 cells to TSH causes a rapid synthesis of the glycosylated rTIT but a delayed re-induction of IUA. Tunicamycin totally prevents the TSH-dependent re-expression and activity of rTIT. Our data bring basic information on the location, structure, and turnover of rTIT and suggest that its activity is subjected to diverse control mechanisms including regulatory proteins.

Analysis of the functional state of T3 nuclear receptors expressed in thyroid cells.

T3 nuclear receptors (TR) are present in thyroid cells. We have analyzed the ability of thyroid TR to function as transcriptional regulators. Studies were performed on pig thyrocytes in primary culture. Messenger RNA corresponding to TR alpha 1, alpha 2 and beta were detected in pig thyrocytes by RT-PCR and Northern blot; the alpha 2 mRNA was more abundant than the alpha 1 mRNA. Thyrocytes were transiently transfected with different plasmids containing the CAT (chloramphenicol acetyl transferase) gene placed under the control of different promoters (delta MTV, TK or delta SV40) and bearing a thyroid hormone response element, TREp or TRE DR + 4. It was found that TSH induced a concentration-dependent increase of the transfection efficiency, an effect reproduced by (Bu)2cAMP and Forskolin. Cells transfected with either delta MTV-, TK- or delta SV40-TREp-CAT expressed similar basal CAT activities. Addition of T3 produced a 3-fold increase of CAT activity expressed from each of these vectors. In contrast, CAT activity expressed from a vector containing the TRE DR + 4 was decreased by about 50% by T3. Thus, TREp and TRE DR + 4 gave distinct responses. These data demonstrate that TR physiologically expressed in thyroid cells can act as transcriptional regulators in a T3-dependent manner. This finding directly substantiates the concept of autocrine regulatory actions of thyroid hormones.

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.