Biomedical subjects

L D Kohn

Publications and source records attributed to L D Kohn.

At least 37 records · Page 2Linked to original sources

PubMed2000

Increased major histocompatibility complex (MHC) expression in nontoxic goiters is associated with iodide depletion, enhanced ability of the follicular thyroglobulin to increase MHC gene expression, and thyroid autoantibodies.

Recent studies suggest that thyroglobulin (TG) accumulated in the follicular lumen of colloid nodular goiters can increase major histocompatibility complex (MHC) class I gene expression in FRTL-5 thyrocytes. Iodide deficiency, also present in these patients, was separately suggested to enhance thyroidal MHC class I and class II gene expression in vivo and in vitro. To test the clinical relevance of these observations, we examined 41 nontoxic goiters surgically removed from patients who had compression problems. Northern analysis revealed that there was a mean 3.9-fold increase in MHC class I expression and a 8.3-fold increase in class II expression by comparison to 9 normal glands. In situ hybridization showed that thyrocytes were the main source of class I and class II transcripts; histological examination revealed that lymphocytic infiltration was minimal to non-existent. The iodine content of the 41 nontoxic goiters was significantly lower than in normal glands, consistent with increased MHC class I and class II. There is also a profound accumulation of TG in the follicles of the nontoxic goiters, and TG purified from the follicles of these glands increased MHC class I gene expression in FRTL-5 thyroid cells significantly more than TG from normal glands per mg protein. Nearly all patients with nontoxic goiter had low, but significantly elevated, levels of antibodies against thyroid peroxidase and/or against TG in their sera compared with those in normal individuals. Moreover, there was a positive correlation between the titer of the serum antibodies against thyroid peroxidase and against TG and MHC class I and class II expression in the thyroid. The data support the possibility that the TG accumulated in the follicular lumen of nontoxic goiters together with relative iodine deficiency contributes to increased MHC expression in thyroid cells in vivo and that increased MHC gene expression contributes to the ability of thyroid antigens to trigger an autoimmune reaction.

Adult↗

PubMed2000

Hashimoto's thyroiditis with heterogeneous antithyrotropin receptor antibodies: unique epitopes may contribute to the regulation of thyroid function by the antibodies.

Blocking-type TSH-binding inhibitor Igs (TBIIs) are known to cause hypothyroidism and an atrophic thyroid gland in patients with primary myxedema. They can block the activity of thyroid-stimulating antibodies (TSAbs) in Graves' patients as well as the activity of TSH. The majority of the epitopes for these blocking-type TBIIs have been, and are shown herein, to be present on the C-terminal region of the extracellular domain of the human TSH receptor (TSHR), whereas those for Graves' TSAbs are on the N-terminus. We report on a patient with Hashimoto's thyroiditis who suffered from mild hypothyroidism and a moderately sized goiter. Her serum had a potent blocking-type TBII and a weak TSAb in human and porcine TSHR systems. Using human TSHR/lutropin-CG receptor chimeras, we determined that the functional epitope of her blocking-type TBII was uniquely present on the N-terminal, rather than the C-terminal, region of the extracellular domain of the TSHR, unlike the case for blocking-type TBIIs in primary myxedema patients. The epitope of her TSAb was also unusual. Although the functional epitopes of most TSAbs are known to involve the N-terminal region of the receptor, her TSAb epitope did not seem to be present solely on the N- or C-terminus of the extracellular domain of the receptor. Blocking-type TBIIs from patients with primary myxedema blocked her TSAb activity as well as stimulation by TSH; her blocking-type TBII was able to only partially block her TSAb. In contrast, her blocking-type TBII almost completely blocked TSAbs from Graves' patients. Thus, we suggest that the unique epitopes of this patient's heterogeneous population of TSH receptor antibodies, at least in part, contribute to regulation of her thyroid function.

Animals↗

PubMed2000

Major histocompatibility class I gene transcription in thyrocytes: a series of interacting regulatory DNA sequence elements mediate thyrotropin/cyclic adenosine 3',5'-monophosphate repression.

In response to TSH, thyroid cells decrease major histocompatibility (MHC) class I expression and transcription, providing an excellent model for studying the dynamic modulation of transcription of MHC class I genes. Here we show that protein kinase A (PKA), a downstream effector of the TSH/cAMP pathway, reproduces the effects of TSH in repressing class I transcription. PKA/cAMP-mediated repression of transcription involves multiple interacting upstream response elements in the class I promoter: an element extending from -127 to -90 bp containing a CRE-like core, and at least two elements within an upstream 30-bp segment (-160 to -130 bp), which overlaps with the interferon regulatory element. ICER (inducible cAMP early response), a transcriptional repressor induced by TSH/cAMP can decrease class I promoter activity when introduced into FRTL-5 thyroid cells in the absence of TSH/cAMP. ICER binds to both the CRE-like element and the upstream 30-bp segment, generating a novel TSH-induced ternary complex. The present studies led to the proposal that TSH-mediated repression of class I transcription is the result of integrating signals from transcription factors through the higher order interactions of multiple regulatory elements.

Animals↗

PubMed2000

Transforming growth factor-beta1 down-regulation of major histocompatibility complex class I in thyrocytes: coordinate regulation of two separate elements by thyroid-specific as well as ubiquitous transcription factors.

Transforming growth factor (TGF)-beta1-decreased major histocompatibility complex (MHC) class I gene expression in thyrocytes is transcriptional; it involves trans factors and cis elements important for hormone- as well as iodide-regulated thyroid growth and function. Thus, in rat FRTL-5 thyrocytes, TGF-beta1 regulates two elements within -203 bp of the transcription start site of the MHC class I 5'-flanking region: Enhancer A, -180 to -170 bp, and a downstream regulatory element (DRE), -127 to -90 bp, that contains a cAMP response element (CRE)-like sequence. TGF-beta1 reduces the interaction of a NF-kappaB p50/fra-2 heterodimer (MOD-1) with Enhancer A while increasing its interaction with a NF-kappaB p50/p65 heterodimer. Both reduced MOD-1 and increased p50/p65 suppresses class I expression. Decreased MOD-1 and increased p50/p65 have been separately associated with the ability of autoregulatory (high) concentrations of iodide to suppress thyrocyte growth and function, as well as MHC class I expression. TGF-beta1 has two effects on the downstream regulatory element (DRE). It increases DRE binding of a ubiquitously expressed Y-box protein, termed TSEP-1 (TSHR suppressor element binding protein-1) in rat thyroid cells; TSEP-1 has been shown separately to be an important suppressor of the TSH receptor (TSHR) in addition to MHC class I and class II expression. It also decreases the binding of a thyroid-specific trans factor, thyroid transcription factor-1 (TTF-1), to the DRE, reflecting the ability of TGF-beta1 to decrease TTF-1 RNA levels. TGF-beta1-decreased TTF-1 expression accounts in part for TGF-beta1-decreased thyroid growth and function, since decreased TTF-1 has been shown to decrease thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR gene expression, coincident with decreased MHC class I. Finally, we show that TGF-beta1 increases c-jun RNA levels and induces the formation of new complexes involving c-jun, fra-2, ATF-1, and c-fos, which react with Enhancer A and the DRE. TGF-beta1 effects on c-jun may be a pivotal fulcrum in the hitherto unrecognized coordinate regulation of Enhancer A and the DRE.

Animals↗

PubMed2000

A novel mouse model of Graves' disease: implications for a role of aberrant MHC class II expression in its pathogenesis.

Mice immunized with fibroblasts expressing an MHC class II molecule and human thyrotropin receptor (TSHR), but not either alone, develop major features characteristic of Graves' disease (GD), such as thyroid-stimulating autoantibodies directed against TSHR, increased serum thyroid hormone levels, and enlarged thyroid glands. The results indicate the need for the simultaneous expression of a class II molecule and the TSHR on the surface of the fibroblasts to develop stimulating anti-TSHR antibodies and full-blown GD in our model. A T cell line established from a mouse with hyperthyroidism proliferates in response to fibroblasts expressing a class II molecule and TSHR, but not to the fibroblasts expressing only TSHR, indicating that the class II molecules on the fibroblasts present TSHR-derived peptide(s) to T cells. These results strongly suggest that the acquisition of antigen-presenting ability by thyrocytes can lead to the induction or progression of GD. We identified a T cell epitope of TSHR by the proliferative response of spleen cells from mice immunized with fibroblasts expressing a class II molecule and TSHR to 80 overlapping peptides spanning the extracellular domain of human TSHR. The identification of a major T cell epitope provides an important clue to a novel therapy of GD.

Animals↗

PubMed2000

Graves' disease: a host defense mechanism gone awry.

In this report we summarize evidence to support a model for the development of Graves' disease. The model suggests that Graves' disease is initiated by an insult to the thyrocyte in an individual with a normal immune system. The insult, infectious or otherwise, causes double strand DNA or RNA to enter the cytoplasm of the cell. This causes abnormal expression of major histocompatibility (MHC) class I as a dominant feature, but also aberrant expression of MHC class II, as well as changes in genes or gene products needed for the thyrocyte to become an antigen presenting cell (APC). These include increased expression of proteasome processing proteins (LMP2), transporters of antigen peptides (TAP), invariant chain (Ii), HLA-DM, and the co-stimulatory molecule, B7, as well as STAT and NF-kappaB activation. A critical factor in these changes is the loss of normal negative regulation of MHC class I, class II, and thyrotropin receptor (TSHR) gene expression, which is necessary to maintain self-tolerance during the normal changes in gene expression involved in hormonally-increased growth and function of the cell. Self-tolerance to the TSHR is maintained in normals because there is a population of CD8- cells which normally suppresses a population of CD4+ cells that can interact with the TSHR if thyrocytes become APCs. This is a host self-defense mechanism that we hypothesize leads to autoimmune disease in persons, for example, with a specific viral infection, a genetic predisposition, or even, possibly, a TSHR polymorphism. The model is suggested to be important to explain the development of other autoimmune diseases including systemic lupus or diabetes.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

73-kDa heat shock cognate protein interacts directly with P27Kip1, a cyclin-dependent kinase inhibitor, during G1/S transition.

Although heat shock proteins (HSPs) were discovered as inducible proteins by the physical stress to protect cells, recent evidence has suggested that HSPs are likely involved in cell cycle control under normal conditions without stress. In the present study, we demonstrated that 73hsc (heat shock cognate protein), which belongs to the HSP70 family of molecular chaperones, interacts with P27Kip1, an inhibitor of cyclin-dependent kinase, during G1/S transition. 73hsc was detected in the immunoprecipitates with anti-P27Kip1 antibody and, vice versa, P27Kip1 was present in the immunoprecipitates with anti-73hsc antibody by Western blotting using growth-stimulated rat thyroid FRTL-5 cells. This complex formation of 73hsc and P27Kip1 was cell cycle dependent and its maximum formation was observed at G1/S transition where the level of P27Kip1 dramatically decreased. ATP dissociated this complex formation in a dose-dependent manner. These data indicated that 73hsc might be involved in the cell cycle progression through the regulation of cell cycle regulators such as P27Kip1.

Adenosine Diphosphate↗

PubMed1999

Nicotinamide potentiates TSHR and MHC class II promoter activity in FRTL-5 cells.

Here we show that nicotinamide modulates the promoter activity of rat thyrotropin (TSHR) and major histocompatibility complex (MHC) class II genes in rat FRTL-5 thyroid cells, and have identified a novel mechanism for its action. TSHR and MHC class II, are potentiated through reduced expression of a common repressor of these two genes, TSEP-1 (TSHR suppressor element binding protein-1)/YB-1. Thus we show that TSHR mRNA is increased and TSHR promoter activity was concentration-dependently activated from 0 to 40 mM nicotinamide. The promoter lengths of TSHR and MHC class II containing TSEP/YB-1 binding sites were enhanced by 40 mM nicotinamide, but not the ones deleted of these binding sites. TSEP-1/YB-1 binding to the recognition sites in both TSHR and MHC class II promoters was reduced in nicotinamide-treated FRTL-5 nuclear extracts. Nicotinamide reduced the expression of TSEP-1/YB-1 mRNA and TSEP-1/YB-1 protein in the nucleus.

Animals↗

PubMed1999

Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides.

Abnormal expression of major histocompatibility complex (MHC) class I and class II in various tissues is associated with autoimmune disease. Autoimmune responses can be triggered by viral infections or tissue injuries. We show that the ability of a virus or a tissue injury to increase MHC gene expression is duplicated by any fragment of double-stranded (ds) DNA or dsRNA introduced into the cytoplasm of nonimmune cells. Activation is sequence-independent, is induced by ds polynucleotides as small as 25 bp in length, and is not duplicated by single-stranded polynucleotides. In addition to causing abnormal MHC expression, the ds nucleic acids increase the expression of genes necessary for antigen processing and presentation: proteasome proteins (e.g., LMP2), transporters of antigen peptides; invariant chain, HLA-DM, and the costimulatory molecule B7.1. The mechanism is different from and additive to that of gamma-interferon (gammaIFN), i.e., ds polynucleotides increase class I much more than class II, whereas gammaIFN increases class II more than class I. The ds nucleic acids also induce or activate Stat1, Stat3, mitogen-activated protein kinase, NF-kappaB, the class II transactivator, RFX5, and the IFN regulatory factor 1 differently from gammaIFN. CpG residues are not responsible for this effect, and the action of the ds polynucleotides could be shown in a variety of cell types in addition to thyrocytes. We suggest that this phenomenon is a plausible mechanism that might explain how viral infection of tissues or tissue injury triggers autoimmune disease; it is potentially relevant to host immune responses induced during gene therapy.

3T3 Cells↗

PubMed1999

Differing MHC class I requirements for induction and propagation of experimental systemic lupus erythematosus.

Mice deficient in beta2-microglobulin expression are resistant to the induction of experimental systemic lupus erythematosus (SLE). The present studies were designed to identify the beta2-microglobulin-dependent cell surface molecule(s) that confers sensitivity to experimental SLE, and to determine its role in disease development. We report hat mice lacking the transporter associated with antigen presentation (TAP-/-) were also resistant to disease, whereas CD1-/- and CD8-/- mice were susceptible; susceptibility also did not correlate with neonatal Fc receptor or HEPH expression. These data indicate that disease susceptibility is determined by expression of MHC class I. Furthermore, by analyzing both adoptive transfer and radiation bone marrow chimeras, we demonstrate that MHC class I expression is necessary for propagation of disease, but not for induction of pathogenic cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

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.

Animals↗

PubMed1999

In vivo expression of thyroid transcription factor-1 RNA and its relation to thyroid function and follicular heterogeneity: identification of follicular thyroglobulin as a feedback suppressor of thyroid transcription factor-1 RNA levels and thyroglobulin synthesis.

We used in situ hybridization to evaluate thyroid transcription factor-1 (TTF-1) RNA expression in individual follicles and related this to thyroglobulin (Tg) synthesis in vivo, as estimated by immunohistochemical analysis. We studied the thyroids of Wistar rats treated with thyroxine (T4) or propylthiouracil (PTU), each of which modulates TSH levels, but affects follicular function and Tg accumulation in the follicular lumen very differently. We show that TTF-1 RNA levels in vivo correlate directly with an increase in the cytoplasmic accumulation of Tg within the cells of individual follicles. Because TTF-1 increases Tg gene expression, RNA levels, and protein synthesis in thyroid cell cultures and because there is no correlation with TSH-increased Tg degradation within the follicular lumen, the increased cytoplasmic accumulation of Tg in vivo is interpreted to reflect TTF-1-increased Tg synthesis. Increases in serum TSH levels in the PTU or T4 treated animals did not always correlate with increases in this measure of increased Tg synthesis; and TSH levels did not always correlate with changes in TTF-1 RNA levels that would be expected to accompany increased Tg synthesis. As one possibility, this suggested there might be a hitherto unrecognized suppressor of TTF-1 RNA levels and TSH-induced Tg synthesis in individual follicles. The immunohistochemical data suggested that this suppressor might be follicular Tg itself. Supporting this possibility, we show that physiological concentrations of highly purified 19S follicular Tg decrease TTF-1 RNA levels in rat FRTL-5 thyroid cells and inhibit the action of TSH to increase Tg synthesis. We therefore suggest that follicular Tg is a feedback autoregulator of thyroid function that can counterregulate TSH actions on thyroid function in vivo and in thyroid cells in culture. We suggest this phenomenon contributes to follicular heterogeneity in vivo.

Animals↗

PubMed1999

Two Graves' disease patients who spontaneously developed hypothyroidism after antithyroid drug treatment: characteristics of epitopes for thyrotropin receptor antibodies.

Few reports have identified blocking thyrotropin receptor antibodies (TSHRAbs) as a pathogenic mechanism explaining spontaneous hypothyroidism after antithyroid drug (ATD) treatment of Graves' disease. Here we report 2 Graves' patients who showed different courses of hypothyroidism after ATD treatment. The first patient had Graves' hyperthyroidism and was treated with ATD for 1 year. After a short period of euthyroidism, she developed permanent hypothyroidism with blocking TSHRAb. The second patient became euthyroid after 1 year of ATD treatment. After 3 years, however, she presented with hypothyroidism with blocking TSHRAb activity. Her hypothyroidism was transient, and restoration of euthyroidism was followed by disappearance of blocking TSHRAb. Blocking and stimulating TSHRAbs activities of these 2 patients were serially measured using Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR (CHO-hTSHR) and 2 TSHR chimeras with residues 8-165 (Mc1+2) or 90-165 (Mc2) substituted by equivalent residues of the luteinizing hormone/chorionic gonadotropin receptor (LH/CGR). During their hypothyroid phases, blocking TSHRAbs activities were positive in all 3 kinds of assays and stimulating TSHRAbs activities were negative in CHO-hTSHR or in Mc 1+2 assay. Mc2 stimulating TSHRAb activity was detected in sera of hypothyroid phase of the second patient who had transient hypothyroidism but not in the first whose hypothyroidism was permanent. In these 2 cases, we demonstrate the causative role of blocking TSHRAb in the development of hypothyroidism after ATD treatment in Graves' patients. Interestingly, the difference in the course of blocking TSHRAb-induced hypothyroidism was associated with the difference in epitope reactivities of TRAb during hypothyroid phase that developed after ATD treatment of Graves' disease.

Adult↗

PubMed1999

Epitope heterogeneity of thyrotropin receptor-blocking antibodies in Graves' patients as detected with wild-type versus chimeric thyrotropin receptors.

The stable transfectants of wild-type (W25) and mutant thyrotropin-receptor (TSH-R) allow detection of the bioactivities of TSH-R antibodies in Graves' patients. A mutant Chinese hamster ovary (CHO) cell line (Mc1+2) transfected with a chimeric construct, where residues 8 to 165 of the TSH-R are replaced with residues 10 to 166 of the lutropin/choriogonadotropin (LH/CGR) receptor, lacks the cyclic adenosine monophosphate (cAMP) response to most thyrotropin stimulating antibodies (TSAb), yet retains the response to TSH and acquires the response to LH/CG. We compared Mc1+2 cells with wild-type W25 cells for their ability to detect TSAb as well as thyrotropin-blocking antibodies (TBAb) in Graves' sera. Eighteen normal and 39 Graves' sera were tested for TSAb and TBAb levels by in vitro bioassays using W25 and Mc1+2 cells. In addition, these sera were also tested for thyrotropin-binding inhibitory activity (TBII) by a radioreceptor assay. Eighteen (47%) Graves' sera had TBAb activity measured with W25 cells but not with Mc1+2 cells. These TBAbs were, therefore, a population of antibodies with functional epitopes on the N-terminus of the extracellular domain. This TBAb activity by W25 cells exhibited a high degree of correlation with TBII levels by a radioreceptor assay (r = 0.70, p = 0.001). Ten (25.6%) Graves' sera had positive TBAb activity in both W25 and Mc1+2 cells; moreover, their activity in both assays was similar (r = 0.83, p < 0.001). The TBAb activity in these sera, however, did not correlate with TBII activity. Eleven (28%) Graves' sera had no TBAb activity. Overall, thyroid-stimulating antibodies were detected in 87% and 28% of the 39 Graves' sera by W25 and Mc1 +2 cells, respectively. Thus, using the 2 cell lines, at least 2 distinct populations of TBAbs were detected. One is detected in a similar fashion by both W25 and Mc1+2 cell lines and likely interacts with the epitopes residing in the unaltered C-terminus of the TSH-R. The other is reactive in W25 cells only, indicating the loss of TBAb epitope in the chimeric receptor located in the N-terminus of the TSH-R. Furthermore, our results indicate that the TBAb binding epitope in 8-165 residues of the native TSH-R is highly associated with TBII activity in Graves' disease. These results indicate that patients with Graves' disease harbor TBAbs with epitope heterogeneity and favor the notion that there are different sites and mechanisms by which TBAbs act in Graves' patients. It remains to be determined whether or not TBAb subtyping will have a useful predictive role in the management of patients with Graves' disease.

Adult↗

PubMed1999

Hormone-dependent regulation of intercellular adhesion molecule-1 gene expression: cloning and analysis of 5'-regulatory region of rat intercellular adhesion molecule-1 gene in FRTL-5 rat thyroid cells.

Intercellular adhesion molecule-1 (ICAM-1) has been suggested to play an important role in the perpetuation of autoimmune thyroid disease. To clarify the regulation of ICAM-1 gene in thyroid cells, we investigated ICAM-1 expression in the FRTL-5 thyroid cell model and defined several elements in the 5'-regulatory region that are important for transcriptional regulation of the rat ICAM-1 gene. Cells maintained in medium with 5% serum but without hydrocortisone, insulin, and thyrotropin (TSH) express the highest levels of ICAM-1 RNA. TSH/forskolin downregulate ICAM-1 RNA levels independent of the presence or absence of hydrocortisone or insulin. Moreover, TSH/forskolin decrease ICAM-1 RNA levels that are maximally induced by two cytokines: 100 ng/mL tumor necrosis factor-alpha (TNF-alpha) or 100 U/ml interferon-gamma (IFN-gamma). The effect of TSH/forskolin, as well as TNF-alpha and IFN-gamma, on ICAM-1 RNA levels is transcriptional. Thus, we cloned a 1.8-kb fragment of the 5'-flanking region of the rat ICAM-1 gene, upstream of the translational start site, and showed that TNF-alpha or IFN-gamma caused a 3.5- and greater than 12-fold increase respectively, in its promoter activity, when linked to a luciferase reporter gene and stably transfected into FRTL-5 cells. TSH or forskolin, in contrast, halved the activity of the full length chimera within 24 hours and significantly suppressed the TNF-alpha and IFN-gamma-induced increase (>50%; p < 0.02). Using 5'-deletion mutants, we located the element important for the TNF-alpha effect between -431 and -175 bp; we additionally show that deletion of a NF-kappaB core element within this region, TTGGAAATTC (-240 to -230 bp), causes the loss of TNF-alpha inducibility. The effect of IFN-gamma could be localized between -175 bp and -97 bp from the start of translation. This region contains 2 regulatory elements known to be involved in IFN-gamma action in other eukaryotic cells, an IFN-gamma activated site (GAS), -138 to -128 bp, and Spl site, -112 to -108 bp. Deletion of the 10 bp GAS sequence resulted in the complete loss of IFN-gamma induction of pCAM-175 promoter activity. TSH and forskolin action was also mapped between -175 bp and -97 bp from the start of translation. The mutant construct, pCAM-175delGAS mutl, which has no GAS sequence, exhibited no TSH-mediated suppression of promoter activity. We thus show that TSH/cAMP can downregulate ICAM-1 gene expression and inhibit the activity of cytokines (TNF-alpha and IFN-gamma) to increase ICAM-1 gene expression in FRTL-5 thyroid cells. We also localized elements on the 5'-flanking region of ICAM-1 important for these actions. We propose that this TSH/cyclic adenosine monophosphate (cAMP) action is a component of the mechanism to preserve self-tolerance of the thyroid during hormone-induced growth and function of the gland, and it may attenuate cytokine action during inflammatory reactions.

Animals↗

PubMed1999

Selective binding of thyrotropin receptor autoantibodies to recombinant extracellular domain of thyrotropin/lutropin-chorionic gonadotropin receptor chimeric proteins.

The extracellular domain of the glycosylated human thyrotropin receptor (ET-gp) contains epitopes that can adsorb pathogenic antibodies from sera of patients with Graves' disease (GD). In an attempt to define the regions within the ETSHR with which autoantibodies interact, we expressed extracellular domains of eight thyrotropin receptor/chorionic gonadotropin receptor (TSHR/LH-CGR) chimeric proteins in insect cells. The levels of expression were high and chimeric proteins were glycosylated. Chimeric proteins designated as EMc2+4 and EMc2+3+4, in which amino acids (aa) 90-165 and 261-370, and aa 90-370, respectively, of TSHR were replaced with corresponding aa of LH-CGR, partially reversed the thyrotropin binding inhibitory immunoglobulin (TBII) activity of experimental anti-TSHR antisera (anti-ET-gp). The other six chimeras almost completely reversed the TBII activity of these anti-ET-GP antisera. Next, we tested the ability of these chimeric proteins to reverse the TBII activity of GD patients' sera. Similar to our earlier study, ET-gp protein reversed the TBII activity of all eight GD patients' sera tested. Chimera EMc2, in which aa 90-165 of TSHR has been replaced with corresponding aa of LH-CGR, and EMc2+4 partially reversed the TBII activity of only three of the eight GD patients' sera. However, the other six chimeric proteins failed to neutralize the TBII activity of any of GD patients' sera. These data showed the following: (1) There is considerable heterogeneity amongst autoantibodies in GD patients' sera, (2) The TBII activity of some, but not others, is dependent on aa 90-165 and 261-370, and (3) Most Graves' sera, with TBII activity, failed to react with chimeric proteins in which either N-terminal or C-terminal regions of the extra cellular domain of the TSHR were replaced with corresponding regions of LH-CGR. These results suggest that the TBII activity of GD patients' sera is dependent on conformational epitopes and replacement of certain regions of TSHR with homologous regions of LH-CGR results in sufficient alteration in the conformation of the protein leading to loss of reactivity.

Animals↗

PubMed1999

Follicular thyroglobulin suppresses iodide uptake by suppressing expression of the sodium/iodide symporter gene.

A major function of the thyrocyte is to take up and concentrate iodide. This is needed for thyroid hormone synthesis and is accomplished by the sodium iodide symporter (NIS), whose expression and activity are up-regulated by TSH. Recently, we reported that follicular thyroglobulin (TG) is a potent suppressor ofthyroid-specific gene expression and can overcome TSH-increased gene expression. We suggested this might be a negative feedback, autoregulatory mechanism that counterbalanced TSH stimulation of follicular function. In this report, we support this hypothesis by coordinately evaluating TG regulation of NIS gene expression and iodide transport. We show that physiological concentrations of TG similarly and significantly suppress TSH-increased NIS promoter activity, NIS protein, and NIS-dependent iodide uptake as well as RNA levels. We show, in vivo, that TG accumulation at the apical membrane of a thyrocyte facing the follicular lumen is associated with decreased uptake ofradioiodide. It is likely, therefore, that TG suppresses NIS-dependent iodide uptake and NIS gene expression in vivo, as is the case in vitro. RNA levels of NIS and vascular endothelial growth factor/vascular permeability factor, which has been reported to be TSH regulated and possibly associated with TSH-increased iodide uptake, are coordinately decreased by follicular TG as a function of concentration and time. Also, removal of follicular TG from the medium, but not TSH, coordinately returns NIS and vascular endothelial growth factor/vascular permeability factor RNA levels to their TSH-stimulated state. TG accumulated in the follicular lumen appears, therefore, to be a negative feedback regulator of critical TSH-increased follicular functions, iodide uptake, and vascular permeability.

Animals↗