Class II transactivator suppresses transcription of thyroid-specific genes.

Class II transactivator (CIITA) is the master regulator of MHC class II genes, and mediates their induction by interferon gamma (IFN gamma). To study the role of CIITA in modulating the expression of thyroid-specific genes, we cloned the full-length rat CIITA and use it to transfect a rat thyroid cell line. We found that only one type of CIITA, type IV, is induced in thyroid cells upon IFN gamma stimulation, and that CIITA is capable not only of inducing the expression of MHC genes in the thyroid, but also of differentially suppressing the expression of thyroid-specific genes. These findings suggest new avenues for the development of thyroid autoimmune diseases.

Role of insulin and serum on thyrotropin regulation of thyroid transcription factor-1 and pax-8 genes expression in FRTL-5 thyroid cells.

Thyrotropin (TSH), via its cyclic adenosine monophosphate (cAMP) signal, decreases thyrotropin receptor (TSHR) gene expression in FRTL-5 thyroid cells, whereas it increases expression of the thyroglobulin (Tg) gene. Despite the opposite effects of TSH on TSHR and Tg expression, both genes are positively controlled by thyroid transcription factor-1 (TTF-1) and evidence has accumulated that TSH can decrease TTF-1 mRNA levels. In this report, we further characterize the action of TSH on TTF-1 in order to understand its different activities on the TSHR and Tg genes better. The effect of TSH on the TSHR requires the presence of insulin and serum and we show here that also both factors are necessary for the TSH effect to decrease TTF-1 mRNA levels. The decrease is paralleled by a downregulation of TTF-1 protein levels as well as by a decrease in TTF-1/DNA complex when the TTF-1 site of the TSHR promoter was used as probe. Again, the decrease requires insulin and serum. The TSH downregulation of TTF-1 mRNA levels is due to a decrease in its transcription rate. Using a luciferase-linked chimera construct spanning 5.18 kb of the TTF-1 5'-flanking region, we show that TSH decreases TTF-1 promoter activity and that this effect depends on insulin and serum. These data contrast with the action of TSH on Tg and Pax-8 gene expression. TSH increases Pax-8 mRNA levels and the increase is evident whether insulin and serum are present or not. Moreover, this increase is paralleled by an increase in Pax-8 protein binding to an oligonucleotide derived from the C site of the Tg promoter, which can bind both TTF-1 and Pax-8. The present data thus show that TTF-1 gene expression is interdependently regulated by TSH and serum growth factors including insulin. They also show this interdependent-regulation is not duplicated in the case of Pax-8. We suggest that these differences may contribute to the distinct ability of TSH to regulate TSHR versus Tg gene expression in FRLT-5 thyroid cells.

Follicular thyroglobulin (TG) suppression of thyroid-restricted genes involves the apical membrane asialoglycoprotein receptor and TG phosphorylation.

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A. , and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.

Thyroglobulin regulates follicular function and heterogeneity by suppressing thyroid-specific gene expression.

Thyroglobulin (TG) is the primary synthetic product of the thyroid and the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and lysosomal degradation can each modulate thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (the TSHR), increases thyroid hormone levels by upregulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of the thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex (MHC) class I gene expression, which is also regulated by TTF-1 and Pax-8 in the thyroid, is simultaneously decreased; this maintains self tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. We now show that follicular TG, 27S > 19S > 12S, counter-regulates TSH-increased thyroid-specific gene transcription by suppressing the expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS and TSHR genes, but increases class I expression. TG action involves an apical membrane TG-binding protein; however, it acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the start of TTF-1 transcription. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS and/or TSHR gene expression. TG activity is not duplicated by thyroid hormones or iodide. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism which regulates follicular function, regulates thyroid hormone secretion, and contributes to follicular heterogeneity.

Efficient syntheses of (E)-5-(2-bromovinyl)-2'-deoxy-4'-thiouridine; a nucleoside analogue with potent biological activity.

(E)-5-(2-Bromovinyl)-2'-deoxy-4'-thiouridine (S-BVDU) is a potent antiherpesvirus agent and its use in gene therapy as an anticancer agent has recently been described. We here outline 2 efficient methods for the synthesis of S-BVDU. The decision as to which method is to be used depends upon the starting materials available but starting from BVU, an overall yield of beta-nucleoside of 35% can be expected. From 5-ethyl-2'-deoxy-4'-thiouridine, radical bromination using bromine will give a quantitative conversion to S-BVDU if unreacted starting material is recycled (50%) or using N-bromosuccinimide, a one step yield in excess of 80% can be obtained.

Autoregulation of thyroid-specific gene transcription by thyroglobulin.

Thyroglobulin (TG), the primary synthetic product of the thyroid, is the macromolecular precursor of thyroid hormones. TG synthesis, iodination, storage in follicles, and degradation control thyroid hormone formation and secretion into the circulation. Thyrotropin (TSH), via its receptor (TSHR), increases thyroid hormone levels by up-regulating expression of the sodium iodide symporter (NIS), thyroid peroxidase (TPO), and TG genes. TSH does this by modulating the expression and activity of several thyroid-specific transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, which coordinately regulate NIS, TPO, TG, and the TSHR. Major histocompatibility complex class I gene expression, which also is regulated by TTF-1 and Pax-8 in the thyroid, is decreased simultaneously. This helps maintain self-tolerance in the face of TSH-increased gene products necessary for thyroid hormone formation. In this report we show that follicular TG counter-regulates TSH-increased, thyroid-specific gene transcription by suppressing expression of the TTF-1, TTF-2, and Pax-8 genes. This decreases expression of the TG, TPO, NIS, and TSHR genes, but increases class I expression. TG acts transcriptionally, targeting, for example, a sequence within 1.15 kb of the 5' flanking region of TTF-1. TG does not affect ubiquitous transcription factors regulating TG, TPO, NIS, and/or TSHR gene expression. The inhibitory effect of TG on gene expression is not duplicated by thyroid hormones or iodide and may be mediated by a TG-binding protein on the apical membrane. We hypothesize that TG-initiated, transcriptional regulation of thyroid-restricted genes is a normal, feedback, compensatory mechanism that limits follicular function and contributes to follicular heterogeneity.

Regulation of major histocompatibility (MHC) class II human leukocyte antigen-DR alpha gene expression in thyrocytes by single strand binding protein-1, a transcription factor that also regulates thyrotropin receptor and MHC class I gene expression.

The single strand binding protein (SSBP-1) is a positive regulator of TSH receptor gene expression and binds to an element with a GXXXXG motif. The S box of the mouse major histocompatibility class II gene has multiple GXXXXG motifs and can also bind SSBP-1. The S box is one of four highly conserved elements on the 5'-flanking region of class II genes that are necessary for interferon-gamma (IFNgamma) to overcome the normally suppressed state of the gene and induce aberrant class II expression. In this report we show that SSBP-1, when overexpressed in FRTL-5 thyroid cells, is a positive regulator of human leukocyte antigen (HLA)-DR alpha class II gene expression, as is IFNgamma or the class II trans-activator (CIITA). This is evidenced by increased exogenous promoter activity, increased endogenous RNA levels, and increased endogenous antigen expression after transfecting full-length SSBP-1 complementary DNA together with a HLA-DR alpha promoter-reporter gene chimera into TSH-treated FRTL-5 thyroid cells whose endogenous SSBP-1 levels are low. IFNgamma reverses the ability of TSH to decrease endogenous SSBP-1 RNA levels. Also, whereas SSBP-1 transfection does not cause any increase in IFNgamma-induced exogenous promoter activity, transfection of SSBP-1 and CIITA additively increases endogenous class II RNA levels to levels measured in cells treated with IFNgamma. Further, competition studies show that SSBP-1 binding is necessary for formation of the double strand protein/DNA complexes that are seen in electrophoretic mobility shift assays when the class II 5'-flanking region is incubated with extracts from IFNgamma-treated FRTL-5 cells and that have been previously associated with IFNgamma-induced aberrant class II expression. These data suggest that SSBP-1 is involved in the action of IFNgamma to overcome the normally suppressed state of the class II gene; it functions together with CIITA, whose expression is independently increased by IFNgamma. The effect of SSBP-1 as a positive regulator of class II promoter activity is lost in cells maintained without TSH, in which endogenous SSBP-1 RNA levels are already high in the absence of aberrant class II gene expression. These data suggest that high levels of endogenous SSBP-1 are insufficient to cause aberrant class II expression, but, rather, TSH or IFNgamma treatment additionally modulates the cell, albeit differently, such that transfected or endogenous SSBP-1, respectively, can express its positive regulatory activity. The effect of TSH is consistent with reports indicating that TSH enhances the ability of IFNgamma to increase class II gene expression despite the fact IFNgamma increases endogenous SSBP-1 to only the same levels as in cells untreated with TSH. Finally, the effect of SSBP-1 as a positive regulator is lost when GXXXXG motifs, which exist on both the coding and noncoding strands of the S box, are mutated. Consistent with this, mutation and oligonucleotide competition studies show that GXXXXG motifs are necessary for either strand of the S box to bind protein/DNA complexes containing SSBP-1 in FRTL-5 cell extracts or to bind to recombinant SSBP-1. They also suggest that the SSBP-1-binding sites on either strand of the HLA-DR alpha S box are functionally distinct. We conclude from these data that the positive regulatory action of SSBP-1 on class II gene expression involves GXXXXG motifs on each strand of the highly conserved S box of the class II 5'-flanking region. As SSBP-1 is modulated by IFNgamma and is involved in class I and TSH receptor as well as class II gene expression in FRTL-5 cells, the sum of the data supports the hypotheses that common transcription factors regulate all three genes, and their altered activities may contribute to the development of autoimmunity.