Biomedical subjects

S Ikuyama

Publications and source records attributed to S Ikuyama.

At least 19 recordsLinked to original sources

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

Novel germline mutations of the MEN1 gene in Japanese patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, the pancreatic islet cells, and the anterior pituitary. Germline mutations of the MEN1 gene in three independent Japanese cases with MEN1 were analyzed. Case 1 has revealed a 2-bp (TA) insertion at nucleotide position 341 (341insTA) in exon 2, which shifts the reading frame such that the mutant protein has a completely different amino acid sequence from codon 78 to the premature stop codon at 119. In case 2, a nucleotide substitution, i.e., TAG in place of TGG, which encodes tryptophan at codon 198 was identified (nonsense mutation). These mutations were heterozygously present and have not been reported previously. Case 3 showed no mutations in the protein-coding exons and exon-intron junctions of the MEN1 gene by single-strand conformation polymorphism or direct sequencing of the polymerase chain reaction (PCR) fragments. We confirmed the finding that patients with MEN1 carry heterozygous germline mutations in the MEN1 gene, which is compatible with the idea that the MEN1 gene is a tumor suppressor gene. The reason why mutations in the coding region of the MEN1 gene could not be detected by PCR-based analysis in some of the MEN1 patients, e.g. case 3, needs to be clarified further.

Adult↗

PubMed1998

Expression of an orphan nuclear receptor DAX-1 in human pituitary adenomas.

OBJECTIVES: An orphan nuclear receptor, DAX-1, is known to be involved in the development and differentiation of anterior pituitary cells. The present study aimed to examine 1) whether DAX-1 is expressed in human pituitary adenomas, and 2) if it is expressed, what types of adenoma express the factor. MATERIALS AND METHODS: Adenoma tissues examined included 18 clinically non-functioning adenomas, 14 GH-secreting adenomas and 7 PRL-secreting adenomas. The expression of the following genes were tested by reverse transcription-polymerase chain reaction (RT-PCR): DAX-1, Adrenal-4-binding protein/steroidogenic factor-1 (Ad4BP/SF-1), Pit-1, LH beta, FSH beta, gonadotrophin-releasing hormone receptor (GnRH-R), GH, PRL, and TSH beta, as well as beta-actin as a control. RESULTS: Eleven clinically non-functioning adenomas expressed DAX-1, 10 of which also expressed Ad4BP/SF-1. Nine out of the 11 DAX-1 expressing adenomas also expressed LH beta, FSH beta and GnRH-R as well, indicating that these adenomas possessed gonadotrophic properties. Nine clinically non-functioning adenomas expressed Pit-1 as well as GH, PRL and/or TSH beta, thus having somatomammotrophic or thyrotrophic properties, 3 of which overlapped with the above DAX-1-expressing adenomas. One non-functioning adenoma expressed Ad4BP/SF1 and FSH beta but not DAX-1, and another one expressed DAX-1 and Ad4BP/SF-1 with PRL. On the other hand, all GH-secreting and PRL-secreting adenomas expressed Pit-1 and GH and/or PRL, but neither DAX-1 nor Ad4BP/SF-1. CONCLUSIONS: The results shown here indicate that DAX-1 is expressed in the majority of human pituitary adenomas of gonadotrophic origin in parallel with Adrenal-4-binding protein/steroidogenic factor-1.

Adenoma↗

PubMed1998

Low level of glucocorticoid receptor messenger ribonucleic acid in pituitary adenomas manifesting Cushing's disease with resistance to a high dose-dexamethasone suppression test.

OBJECTIVES: The overnight 8-mg dexamethasone suppression test is often used to differentiate Cushing's disease, due to an oversecretion of ACTH from the pituitary gland, from other kinds of Cushing's syndrome. However, a few patients with ACTH-producing pituitary adenoma show no suppression of plasma cortisol after the administration of 8 mg of dexamethasone. To clarify the relationship between the level of glucocorticoid receptor (GR) in the pituitary adenoma and the sensitivity to dexamethasone in Cushing's disease, we thus examined the levels of GR alpha and GR beta mRNAs in the pituitary adenomas in six patients who were proven at surgery to have pituitary ACTH-producing adenomas. MATERIALS: Total RNA was extracted from six pituitary adenomas and pituitary tissue adjacent to one of the adenomas, and the mRNA levels of GR alpha, GR beta, pro-opiomelanocortin (POMC) and beta-actin in these samples were sampled by quantitative RT-PCR. RESULTS: The GR alpha mRNA levels in the adenomas from the two patients who showed no response to the 8-mg dexamethasone suppression test were significantly lower than those in the adenomas of four patients who showed suppression. The GR beta mRNA level was much lower than that of GR alpha mRNA but not significantly different among the six adenomas. CONCLUSIONS: These results suggest strongly that decreased expression of GR alpha in pituitary adenomas may be the major reason for the marked insensitivity to the 8-mg dexamethasone suppression test observed in two patients with Cushing's disease.

Actins↗

PubMed1998

A rare case of 46,XX true hermaphroditism with hidden mosaicism with sex-determining region Y chromosome-bearing cells in the gonads.

The sex-determining region Y chromosome (SRY) triggers testis determination. We report a 46,XX true hermaphrodite who had ambiguous genitalia at birth. A laparotomy at one year of age revealed this patient to have a testis on the right side and an ovotestis on the left side. By polymerase chain reaction analysis no SRY was detected in the DNA from the leukocytes but it was found in the DNA from the ovotestis. The hidden mosaicism with the Y-bearing cells in the gonads is likely the cause of the dual gonads in this patient.

Adult↗

PubMed1997

Cloning and characterization of the 4.2 kb region of the rat thyrotropin receptor promoter.

We previously identified an approximately 200 bp "minimal promoter" of the rat TSH receptor (TSHR) gene which is essential for the promoter activity. In the present study, we have cloned and characterized an upstream region of the TSHR promoter to disclose additional functional element(s). We screened a rat genomic library and obtained a DNA fragment which contained a 4.2 kb 5'-flanking region. This fragment was 2.5 kb longer than that we previously studied (1.7 kb). To assess the promoter activity, chimeric plasmids containing the 4.2 kb promoter and its 5'-deletions ligated to a chloramphenicol acetyltransferase gene were transfected into thyroid and non-thyroid cells. These plasmids expressed significant promoter activity in FRTL-5 and FRT thyroid cells, but not in BRL liver cells. The strongest promoter activity was expressed by the -199 bp promoter, and the longer promoter expressed rather decreased activity. Co-expression of thyroid transcription factor-1 (TTF-1) increased the activity of the promoter region from -3187 to -199 bp, which encompassed one or two TTF-1 binding sites we previously identified, but not the -4206 bp promoter. In addition, FRTL-5 stable transfectants each having a chimeric construct were cultured in the presence or absence of TSH. All transfectants expressed higher promoter activity in the absence of TSH than in the presence of TSH, in particular, the -3187 bp plasmid expressed significantly higher activity by comparison to the -2617 and -4206 bp constructs. This result indicates that the region between -3187 and -2617 bp may contribute to TSH/cAMP-induced suppression and also suggests that the region between -4206 and -3187 bp involves the element(s) for constitutive suppression of the promoter activity. These results not only suggest that the 4.2 kb upstream region of the TSHR gene possibly contains some elements for the regulation of the gene expression, but also emphasize the importance of the minimal promoter region which we previously identified for the efficient expression of the gene.

Animals↗

PubMed1996

Follicle stimulating hormone-beta subunit gene is expressed in parallel with a transcription factor Ad4BP/SF-1 in human pituitary adenomas.

OBJECTIVES: A transcription factor Ad4BP/SF-1 is implicated in the differentiation of gonadotrophs in the pituitary gland, but it is not known whether human pituitary cells express this factor. The present study aimed to disclose (1) whether human pituitary adenomas express Ad4BP/SF-1, and (2) if this is the case, what kinds of adenoma express the factor. MATERIAL: Total RNA was extracted from 23 pituitary adenomas obtained by transsphenoidal surgery, and subjected to Northern blot analyses using cDNAs of bovine Ad4BP/SF-1, porcine FSH-beta, LH-beta and glycoprotein hormone-alpha (GPH-alpha) subunts as radiolabelled probes. These adenomas included 13 clinically non-functioning adenomas, 1 GH/PRL-producing adenoma, 6 GH-producing adenomas, 2 PRL-producing adenomas and 1 ACTH-producing adenoma. RESULTS: The expression of Ad4BP/SF-1 exactly coincided with the expression of FSH-beta. Thus 5 out of 13 clinically non-functioning adenomas expressed Ad4BP/SF-1 and only these 5 adenomas expressed FSH-beta. Interestingly, only one of the GH-producing adenomas also expressed Ad4BP/SF-1 as well as FSH-beta. GPH-alpha was expressed in 4 non-functioning adenomas and 2 hormonally functioning adenomas, but did not necessarily coincide with Ad4BP/SF-1 expression. None of the 23 adenomas we tested expressed LH-beta, probably because LH-beta-producing adenomas are rather rare. CONCLUSIONS: The expression of FSH-beta was parallel with Ad4BP/SF-1 expression, indicating that the expression of Ad4BP/SF-1 is restricted to cells derived from gonadotroph lineages in human pituitary adenomas.

Adenoma↗

PubMed1996

Abnormalities of sex differentiation.

Sex differentiation is determined by a cascade of events proceeding from chromosomal sex to the completion of sexual maturation at puberty. Many factors involved in this cascade have been identified. Here we focus on DAX-1, androgen receptor and cytochrome P450c17, and discuss their functions in sex differentiation. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism using PCR amplification of genomic DNA and complete exonic sequencing, and established that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. Androgen insensitivity syndrome (AIS) is a good model to clarify the relationship between the structure and function of androgen receptor, the androgen receptor gene mutation and clinical phenotype. We analyzed 15 cases of AIS and demonstrate the structural and functional relationships of the androgen receptor. We have sequenced the CYP17 (P450c17) gene in DNA from several patients with 17 alpha-hydroxylase deficiency, reconstructed the mutations in a human P450c17 cDNA and expressed the mutant P450c17 in COSl cells to characterize the kinetic properties of 17 alpha-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17 alpha-hydroxylase deficiency have turned out to be complete or partial combined deficiencies of 17 alpha-hydroxylase/17,20-lyase.

Adolescent↗

PubMed1996

Interferon-gamma suppresses thyrotropin receptor promoter activity by reducing thyroid transcription factor-1 (TTF-1) binding to its recognition site.

Interferon-gamma (IFN gamma) is known to suppress the expression of thyroid-specific genes, such as thyroglobulin, thyroid peroxidase, and the TSH receptor (TSHR). In the present study, we show that this reflects, in part, a transcriptional action mediated by thyroid transcription factor-1 (TTF-1). Thus, transfected into rat FRTL-5 cells, the activity of reporter plasmids, containing rat TSHR promoter ligated to a chloramphenicol acetyltransferase gene, was significantly suppressed in the presence of rat IFN gamma. A -199-bp promoter construct showed the greatest suppression by IFN gamma whereas a -177-bp construct, in which the TTF-1 binding site was deleted, showed less suppressibility. The suppressive effect was rat IFN gamma-specific, since human IFN alpha, -beta, and -gamma exhibited no significant effects. The effect was concentration-dependent from 3-50 U/ml. In FRT rat thyroid cells that do not express TTF-1, IFN gamma-induced suppression on the promoter activity was not observed. In addition, when the TTF-1 binding site was mutated so that TTF-1 can not bind, IFN gamma-induced suppression was significantly reduced. In gel mobility shift analyses, a protein-DNA complex formed by TTF-1 was reduced when the nuclear extract prepared from IFN gamma-treated FRTL-5 cells was used; however, expression of TTF-1 mRNA and TTF-1 protein, which were assessed by Northern blot analysis and Western blot analysis, respectively, were not affected by IFN gamma treatment of FRTL-5 cells. Instead, reduction of DNA-binding affinity of TTF-1 was evident when competition analysis was performed in gel mobility shift analysis. From these results, we conclude that IFN gamma suppresses TSHR promoter activity, in part, by reducing TTF-1 binding to its recognition site. We also raise the possibility that the suppressive effect of IFN gamma on promoter activity is mediated by additional element(s) and factor(s) downstream of the TTF-1 site.

Animals↗

PubMed1996

Cyclic Cushing's disease in long-term remission with a daily low dose of bromocriptine.

A 56-year-old male patient with cyclic Cushing's disease remained in a state of remission for more than one year with a relatively low dose of bromocriptine (2.5-3.75 mg/day). It has been reported that bromocriptine treatment for cyclic Cushing's disease induces only a transient remission; in the most effective cases, a relatively high dose (40 mg/day) was necessary. In the hypercortisolemic state, plasma adrenocorticotropic hormone (ACTH) and serum cortisol were not suppressed by dexamethasone and did not respond to corticotropin-releasing factor (CRF). An antehypophysectomy was not effective, even though the resected tissue contained ACTH-positive microadenomas. The present observations thus indicate the effectiveness of bromocriptine for some patients with this rare disorder.

Adenoma↗

PubMed1995

Type 1 angiotensin II receptors of adrenal tumors.

The present study was designed to clarify the transcriptional regulation of the human type 1 angiotensin II receptor (AT1) gene and its pathophysiological roles in steroidogenesis by adrenal tumors. A cDNA encoding type 1 angiotensin II receptor (AT1) was isolated from a human liver cDNA library encoding a protein of 359 amino acids with seven transmembrane segments. It is very likely that human has only one type of AT1 receptor, in contrast with rodents. A genomic clone containing 217 bp of exon 1 and 2558 bp of the 5'-flanking region of human AT1 receptor gene was isolated. Its proximal promoter region contained putative TATA and GC boxes, CRE and AP1 sites. Aldosterone-producing adenoma (APA) contained significantly higher levels of mRNA for AT1 and ACTH receptors than normal tissues adjacent to APA. There were no mutations within the cytoplasmic third loops of AT1 and ACTH receptors in APAs examined. APA showed increased expression of the mRNA for P450c11 and decreased expression of the mRNA for P450c17. These results suggest that renin-independent overproduction and clinically observed ACTH-dependent production of aldosterone in APAs may results from the enhanced transcription of P450c11 and ACTH receptor genes. The mechanism of the discordantly increased expression of AT1 receptor in APA remains to be clarified.

Adenoma↗

PubMed1995

Aromatase in bone cell: association with osteoporosis in postmenopausal women.

To clarify the possible action of adrenal androgen on bone cell, the existence, characteristics and regulation of aromatase in human osteoblast-like osteosarcoma cells (HOS) and primary cultured osteoblast-like cells from normal human bones (HO) were examined in this study. Significant positive correlation between bone mineral density (BMD) and serum dehydroepiandrosterone sulfate (DHEA-S) was found in 120 postmenopausal women (51-99 years old) but no correlation was seen between BMD and serum estradiol (E2). In subset analysis, strongly positive correlation of serum DHEA-S and estrone (E1) with BMD was observed in postmenopausal women aged less than 69 years old. Administration of DHEA to ovariectomized rat significantly increased BMD and decreased relative osteoid volume in femur. These in vivo findings strongly suggested that serum adrenal androgen may be converted to estrogen in peripheral organ, especially, osteoblast and be important steroids to maintain BMD. [3H]DHEA was converted to [3H]androstenedione and [3H]androstenedione to [3H]estrone in primary cultured human osteoblast. Osteoblast-like cells showed aromatase activity, and an apparent Km and the Vmax were 4.74 +/- 0.78 nM (mean +/- SD, n = 3) and 0.83 +/- 0.79 fmol/mg protein/h for HOS, and 4.6 +/- 2.9 nM and 279 +/- 299 fmol/mg protein/h (mean +/- SD, n = 19) for HO, respectively. The aromatase activity was significantly increased by dexamethasone in a dose-dependent manner. Reverse transcription-polymerase chain reaction analysis revealed that dexamethasone increased the transcript of P450AROM gene. Osteoblast-specific promoters were also determined. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 synergistically enhanced aromatase activity and P450AROM mRNA expression. These results demonstrate that adrenal androgen, DHEA, is converted to E1 in osteoblast by P450AROM which is positively regulated by glucocorticoid and 1 alpha,25-dihydroxyvitamin D3 and important to maintain BMD in the 6 to 7th decade, after menopause.

Aged↗

PubMed1995

Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter.

A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression.

An element, -186 to -176 base pairs (bp), in the minimal TSH receptor (TSHR) promoter binds thyroid transcription factor-1 (TTF-1) and is important for both constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells. An element on the noncoding strand of the TSHR, contiguous with the 5'-end of the TTF-1 element, has single strand binding activity. It is distinct from the TTF-1 site, as evidenced by competition experiments using gel shift assays; but the association of the two elements is not random. Thus, the single strand binding protein (SSBP) element also exists contiguous to the 5'-end of an upstream TTF-1 site, -881 to -866 bp; mutation of two conserved nucleotides in each SSBP element results in the loss of SSBP binding and cross-competition. Transfection experiments indicate that full, constitutive TSHR gene expression in FRTL-5 thyroid cells requires the binding of both SSBPs and TTF-1, since mutation of either element halves thyroid-specific promoter activity, whereas mutation of both decreases promoter activity to values near those of a control vector. Transfection experiments with rat liver cells support their independent activities and show that the SSBP site contributes to TSHR gene expression in non-thyroid tissue. The SSBPs function conjointly with TTF-1 in thyroid-specific, TSH/cAMP-induced negative autoregulation of the TSHR. Thus, TSH or forskolin-treated FRTL-5 cells coordinately decrease TSHR RNA levels and TSHR DNA binding to both the SSBPs and TTF-1; also the maximal TSH/cAMP-induced decrease in gene expression requires both elements. The TSH-induced effect in each case is inhibited by cycloheximide; the TSH-induced decrease in SSBP/DNA complex formation requires the presence of insulin or calf serum, exactly as does TSH-induced down-regulation of TSHR RNA levels. In sum, full, constitutive expression of the TSHR in thyroid cells requires TTF-1 and the SSBPs to bind separate, contiguous elements on the TSHR promoter. TSH/cAMP decreases the binding of each factor to its respective site, thereby decreasing TSHR gene expression. The role of the SSBP and TTF-1 sites in constitutive TSHR expression and in TSH/cAMP-induced negative regulation of the TSHR is, therefore, additive and independent.

Animals↗

PubMed1995

In vivo and in vitro evidence for the production of inhibin-like immunoreactivity in human adrenocortical adenomas and normal adrenal glands: relatively high secretion from adenomas manifesting Cushing's syndrome.

To clarify whether adrenal gland secretes inhibin in vivo in physiological or pathological conditions, we measured the levels of inhibin-like immunoreactivity (inhibin-LI) in adrenal veins (A-vein) and compared them with those in inferior vena cava (IVC) using blood samples obtained at catheterization of adrenal vein in the patients with adrenal adenoma manifesting Cushing's syndrome (Cs), aldosterone-producing adenoma, clinically non-functioning adenoma and normal adrenal gland. The tumor sides of A-veins in the patients with adenomas and also both sides of A-veins in subjects with normal adrenal glands showed significantly higher contents of inhibin-LI than their IVC. When the inhibin-LI secretion rate from adrenal gland was estimated by the difference between the levels of A-vein (tumor side) and IVC, Cs adenomas showed the highest secretion rate. Similarly, the tissue inhibin-LI content and the basal secretion rate of inhibit-LI from primary cultured cells were the highest in Cs adenomas. These findings indicated that normal adrenal glands and adrenocortical adenomas produced and secreted inhibin-LI into the general circulation in vivo and Cs adenomas have relatively high capacity for secreting inhibin-LI, and the present study provided the first in vivo evidence for adrenal inhibin-LI production in pathological conditions.

Adrenal Cortex Neoplasms↗

PubMed1995

Cushing's disease preceding sarcoidosis.

We present a 32-year-old-woman with Cushing's disease and sarcoidosis. She had been diagnosed as having Cushing's disease in 1985. She had transsphenoidal surgery followed by pituitary radiation. Subsequently her plasma cortisol level gradually decreased to normal range. In 1991, she manifested bilateral hilar lymphadenopathy on chest X-ray, erythema nodosum, subcutaneous nodules and granulomatous uveitis. From the histological findings of the skin lesions, the patient was diagnosed as having sarcoidosis. Interestingly, an inverse relationship between the disease activity of sarcoidosis and the level of serum cortisol in Cushing's disease was observed in the clinical course of this patient. Co-existence of Cushing's disease and sarcoidosis is very rare. Since the therapeutic significance of steroids in sarcoidosis has been well established, this case provides insight to the relationship between sarcoidosis and steroids.

Adrenocorticotropic Hormone↗

PubMed1995

[Melatonin].

Explore the source record for details and available documents.

Adult↗

PubMed1994

Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter.

By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.

Animals↗