Positional cloning of the APECED gene.

Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.

Mutations in PROP1 cause familial combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1, which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report, we have identified four CPHD families with homozygosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

Do centimetres matter? Self-reported versus estimated height measurements in parents.

AIM: An impressive discrepancy between reported and measured parental height is often observed. The aims of this study were: (a) to assess whether there is a significant difference between the reported and measured parental height; (b) to focus on the reported and, thereafter, measured height of the partner; (c) to analyse its impact on the calculated target height range. METHODS/RESULTS: A total of 1542 individual parents were enrolled. The parents were subdivided into three groups: normal height (3-97th Centile), short (<3%) and tall (>97%) stature. Overall, compared with men, women were far better in estimating their own height (p < 0.001). Where both partners were of normal, short or tall stature, the estimated heights of their partner were quite accurate. Women of normal stature underestimated the short partner and overestimated the tall partner, whereas male partners of normal stature overestimated both their short as well as tall partners. Women of tall stature estimated the heights of their short partners correctly, whereas heights of normal statured men were underestimated. On the other hand, tall men overestimated the heights of their female partners who are of normal and short stature. Furthermore, women of short stature estimated the partners of normal stature adequately, and the heights of their tall partners were overestimated. Interestingly, the short men significantly underestimated the normal, but overestimated tall female partners. CONCLUSION: Only measured heights should be used to perform accurate evaluations of height, particularly when diagnostic tests or treatment interventions are contemplated. For clinical trails, we suggest that only quality measured parental heights are acceptable, as the errors incurred in estimates may enhance/conceal true treatment effects.

Catch-up growth in autosomal dominant isolated growth hormone deficiency (IGHD type II).

OBJECTIVE: Data on the GH-induced catch-up growth of severely GH-deficient children affected by monogenetic defects are missing. PATIENTS: Catch-up growth of 21 prepubertal children (6 females, 15 males) affected with IGHD type II was analyzed in a retrospective chart review. At start of therapy, mean age was 6.2 years (range, 1.6-15.0), mean height SDS was -4.7 (-7.6 to -2.2), mean IGF-I SDS was -6.2 (-10.1 to -2.2). GH was substituted using a mean dose of 30.5microg/kg*d. RESULTS: Catch-up growth was characterized by a mean height gain of +0.92, +0.82, and +0.61 SDS after 1, 2, and 3 years of GH therapy, respectively. Mean height velocities were 10.7, 9.2 and 7.7cm/year during the first three years. Mean duration of complete catch-up growth was 6 years (3-9). Mean height SDS reached was -0.97 (-2.3 to +1.1), which was within the range of the estimated target height of -0.60 SDS (-1.20 to -0.15). The younger and shorter the children were at start of therapy the better they grew during the first year independent of the dose. Mean bone age was delayed at start by 2.1 years and progressed by 2.5 years during the first two years of therapy. Incomplete catch-up growth was caused by late initiation or irregular administration of GH in four cases. CONCLUSIONS: Our data suggest that GH-treated children with severe IGHD show a sustained catch-up growth over 6 years (mean) and reach their target height range. This response to GH is considered to be characteristic for young children with severe growth retardation due to IGHD.

[Impaired calcium homeostasis, clinical impact]. / Störungen der Kalziumhomöostase im Kindesalter--wann muss man daran denken?

Abnormalities of the calcium homeostasis are, with exception of the neonatal period, not often to diagnose in childhood. However, as the clinical features may not only be quite heterogeneous but also present with a very changing pattern, abnormalities of calcium homeostasis have to be considered in many differential diagnoses. Extracellular fluid calcium or plasma calcium is very carefully controlled by fluxes of calcium, which occur between the extracellular fluid and the skeleton, as well as between gut and the kidneys. Therefore, in this review, first, the factors physiologically regulating calcium homeostasis and bone formation are summarized; and then, the situations in which the plasma calcium level should be measured in daily clinical practices are discussed.

[Doping in adolescence]. / Doping im Jugendalter.

Doping in sport is often very present in the media. Doping does not only concern top level sports but it also has an impact on sport as a whole. Young athletes may be influenced by its role models. In Switzerland there are no exact data on this influence or on the use of doping by adolescents. In this article, the effects and side-effects especially on adolescents of some current doping substances are discussed. As well, the regulations of the therapeutic use exemptions for doping substances are explained.

[Hypoglycaemia--diagnosis and therapy in emergencies]. / Hypoglykämien--Diagnostik und Therapie im Notfall.

Hypoglycaemia defined as blood sugar below 2.5 mmol/L, is always an important medical emergency. The primary therapy is simple and consists of iv glucose. The occurrence of hypoglycaemia is directly connected to fuel balance, determined by the availability of glucose, free fatty acids and ketone bodies. An intact fuel balance and maintenance of normal blood sugar concentration is dependent upon: (1) an adequate caloric and qualitative dietary intake; (2) afunctionally intact hepatic glucogenolytic and gluconeogenic enzyme system; (3) an adequate supply of endogenous gluconeogenic substrates (lactate, amino acids and glycerol) (4) an adequate energy supply provided by the beta-oxidation of fatty acids to synthesize glucose and ketone bodies and (5) a normal endocrine system (insulin, glucagon, catecholamines and growth hormone) for integrating and modulating these processes. Disturbances in each of these factors may lead to hypoglycaemia. Glucose, like oxygen, is of essential and fundamental importance for the brain metabolism. The major contribution of the brain to the basal metabolic rate (70% in neonates versus 20% in adults) is an important factor contributing to the frequency and severity of a hypoglycaemic syndrome in the paediatric age. If hypoglycaemia is suspected, blood should be obtained prior to treatment for serum glucose determination and an extra tube (5 ml) serum should be obtained and refrigerated for further investigations. The first voided urine has to be tested for ketones using a dipstick and also refrigerated for further investigations. Basic laboratory investigations, anamnestic informations and clinical findings allow rapid tentative diagnosis and determine the specialized investigations out of the refrigerated material. A rapid definitive diagnosis is important for the specific treatment and to avoid recurrent and prolonged hypoglycaemia.

[The diabetic child in the emergency room]. / Das diabetische Kind auf der Notfallstation.

The WHO announced diabetes mellitus as one of the main threats to human health in the 21st century. In children and adolescents the prevalence of both the autoimmune type 1 and the obesity-related type 2 diabetes is increasing. Common to all types of diabetes is an absolute or relative lack of insulin to keep glucose homeostasis under control. Thus children and adolescents with newly diagnosed diabetes present with hyperglycemia which is often accompanied by ketoacidosis bearing the risk of cerebral edema. Children and adolescents with known diabetes treated with insulin or orale antidiabetic agents may also suffer from hyperglycemia or even ketoacidosis during times of non-compliance with diet and drugs or during concomitant illnesses. Hyperglycemia with ketoacidosis is an emergency situation for which patients need to be admitted to the next hospital for administration of insulin, fluids and potassium. In contrast, insulin treatment in diabetic patients may also lead to a hypoglycemia, the sudden drop in blood glucose, at any moment. Thus recognition and correction of mild hypoglycemia should be familiar to every diabetic child and their caretaker. Severe hypoglycemia with or without seizures may bring the diabetic child in a sudden emergency situation for which the administration of glucagon intramuscularly or glucose intravenously is mandatory. After every severe hypoglycemia the insulin and diet regimen of the diabetic child or adolescent must be reviewed with the diabetes specialist. For unexplained hypoglycemia or major treatment adjustments the diabetic child or adolescent may need to be readmitted to the diabetic ward of a hospital to avoid repeat, potentially life-threatening hypoglycemia.

Autosomal dominant GH deficiency due to an Arg183His GH-1 gene mutation: clinical and molecular evidence of impaired regulated GH secretion.

G to A transition at position 6664 of the GH-1 gene results in the substitution of Arg183 by His (R183H) in human GH protein and causes a new form of autosomal dominant isolated GH deficiency (type II). Although a weak GH release after standard pharmacological provocation tests is observed in these affected individuals, the dominant inheritance pattern is postulated to be caused by a blockade of the GH-regulated secretion in the somatotrophs. The aim of this study was to analyze the impact of this autosomal dominant mutation not only at a clinical, but also at a cellular, level. The results of the different stimulation tests showed first that the patient possesses a severely impaired, but releasable, GH store, and second that the GH secretion is blocked in a time-dependent and reversible way. To confirm these clinical data, cell culture studies were performed looking at the regulated secretory pathway of GH using AtT-20 cells. Importantly, we were able to show that when the R183H mutant GH was expressed in AtT-20 cells, secretagogue (forskolin) induced a normal R183H GH-regulated secretion, but in AtT-20 cells coexpressing both the R183H mutant GH and the normal GH, forskolin-induced GH secretion was markedly reduced. Together, the experiments seem to support the hypothesis that R183H mutant GH severely impaired the GH-regulated secretion and may, therefore, be the cause of this specific form of isolated GH deficiency type II.

Isolated growth hormone (GH) deficiency type IA associated with a 45-kilobase gene deletion within the human GH gene cluster.

A Turkish family of seven individuals (two parents and five offspring) is described in which three children presented with isolated GH deficiency type IA, as defined by Illig et al. The gene deletion responsible for the isolated GH deficiency was characterized by Southern blotting and hybridization analysis of genomic DNA using a 32P-labeled hGH cDNA clone as a probe. In the affected patients, a total of approximately 45 kilobases of DNA, encompassing the human (h) GH-1, human chorionic somatomammotropin-L (hCS-L), hCS-A, and hGH-2 genes, were deleted. The end points of the deletion lay within two regions of highly homologous DNA sequence situated 5' to the hGH-1 gene and 5' to the hCS-B gene. The retention of only the hCS-B gene was associated with normal weight and length at birth and normal postpartum lactation in the mother heterozygous for the deletion. The parents, who are consanguineous, both presented with a DNA restriction pattern consistent with heterozygosity for this deletion.

Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood.

We report on a female who is compound heterozygote for two new point mutations in the CYP19 gene. The allele inherited from her mother presented a base pair deletion (C) occurring at P408 (CCC, exon 9), causing a frameshift that results in a nonsense codon 111 bp (37 aa) further down in the CYP19 gene. The allele inherited from her father showed a point mutation from G-->A at the splicing point (canonical GT to mutational AT) between exon and intron 3. This mutation ignores the splice site and a stop codon 3 bp downstream occurs. Aromatase deficiency was already suspected because of the marked virilization occurring prepartum in the mother, and the diagnosis was confirmed shortly after birth. Extremely low levels of serum estrogens were found in contrast to high levels of androgens. Ultrasonographic follow-up studies revealed persistently enlarged ovaries (19.5-22 mL) during early childhood (2 to 4 yr) which contained numerous large cysts up to 4.8 x 3.7 cm and normal-appearing large tertiary follicles already at the age of 2 yr. In addition, both basal and GnRH-induced FSH levels remained consistently strikingly elevated. Low-dose estradiol (E2) (0.4 mg/day) given for 50 days at the age of 3 6/12 yr resulted in normalization of serum gonadotropin levels, regression of ovarian size, and increase of whole body and lumbar spine (L1-L4) bone mineral density. The FSH concentration and ovarian size returned to pretreatment levels shortly (150 days) after cessation of E2 therapy. Therefore, we recommend that affected females be treated with low-dose E2 in amounts sufficient to result in physiological prepubertal E2 concentrations using an ultrasensitive estrogen assay. However, E2 replacement needs to be adjusted throughout childhood and puberty to ensure normal skeletal maturation and adequate adolescent growth spurt, normal accretion of bone mineral density, and, at the appropriate age, female secondary sex maturation.

Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP1 gene mutation resulting in the substitution of Arg-->Cys at codon 120 (R120C).

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). A mutation in a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (PROP1), has been identified as causing the Ames dwarf (df) mouse phenotype, and thereafter, different PROP1 gene alterations have been found in humans with CPHD. We report on the follow-up of two consanguineous families (n = 12), with five subjects affected with CPHD (three males and two females) caused by the same nucleotide C to T transition, resulting in the substitution of Arg-->Cys in PROP1 at codon 120. Importantly, there is a variability of phenotype, even among patients with the same mutation. The age at diagnosis was dependent on the severity of symptoms, ranging from 9 months to 8 yr. Although in one patient TSH deficiency was the first symptom of the disorder, all patients became symptomatic by exhibiting severe growth retardation and failure to thrive, which was mainly caused by GH deficiency (n = 4). The secretion of the pituitary-derived hormones (GH, PRL, TSH, LH, and FSH) declined gradually with age, following a different pattern in each individual; therefore, the deficiencies developed over a variable period of time. All of the subjects entered puberty spontaneously, and the two females also experienced menarche and periods before a replacement therapy was necessary.

Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy.

We identified a new point mutation in the CYP19 gene responsible for aromatase (P450arom) deficiency in a 46,XY male infant with unremarkable clinical findings at birth. This boy is homozygote for a 1-bp (C) deletion in exon 5 of the aromatase gene causing a frame-shift mutation. The frame-shift results in a prematurely terminated protein that is inactive due to the absence of the functional regions of the enzyme. Aromatase deficiency was suspected prenatally because of the severe virilization of the mother during the early pregnancy, and the diagnosis was confirmed shortly after birth. Four weeks after birth, the baby boy showed extremely low levels of serum estrogens, but had a normal level of serum free testosterone; in comparison with the high serum concentration of androstenedione at birth, a striking decrease occurred by 4 weeks postnatally. We previously reported elevated basal and stimulated FSH levels in a female infant with aromatase deficiency in the first year of life. In contrast, in the male infant, basal FSH and peak FSH levels after standard GnRH stimulation tests were normal. This finding suggests that the contribution of estrogen to the hypothalamic-pituitary gonadotropin-gonadal feedback mechanism is different in boys and girls during infancy and early childhood. In normal girls, serum estradiol concentrations strongly correlate with circulating inhibin levels, and thus, low inhibin levels may contribute to the striking elevation of FSH in young girls with aromatase deficiency. In contrast, estradiol levels are physiologically about a 7-fold lower in boys than in girls, and serum inhibin levels remain elevated even though levels of FSH, LH, and testosterone are decreased.

New autosomal recessive mutation of the TSH-beta subunit gene causing central isolated hypothyroidism.

We identified a new nonsense mutation of the TSH-beta subunit gene responsible for a severe isolated TSH deficiency in two children from the same consanguineous kindred. These affected children are homozygous for a C-to-T transition at nucleotide 654 of the TSH-beta subunit gene, leading to the conversion of a glutamine (CAG) to a premature stop codon (TAG) in the codon 49 (Q49X). The resulting nascent peptide does not contain the seat belt region (amino acid residues 88-105), a TSH-beta subunit region crucial for the dimerization with the alpha-subunit, and, hence, the correct secretion of the mature TSH heterodimer is hampered. Free T(3), free T(4) as well as basal TSH levels were extremely low in both affected individuals and, importantly, TRH stimulations failed to increase serum TSH, but not PRL, confirming isolated TSH deficiency. Using the new StyI endonuclease restriction site generated by the mutation, we confirmed that the affected children were homozygous for the Q49X TSH-beta mutation whereas their unaffected parents as well as their unaffected brother were heterozygous. Consequently, this isolated TSH deficiency follows an autosomal recessive mode of inheritance.

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.

Regulation of human GH receptor gene transcription by 20 and 22 kDa GH in a human hepatoma cell line.

The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.

Transcription factors in pituitary development.

Many homeodomain transcription factors having a distinct temporal and spatial expression pattern have been described in the developing anterior pituitary gland. By interacting with each other, as well as with other extrinsic and intrinsic signals, they control cell determination, cell differentiation and eventually maintenance of cell function which is most important for the life long secretion of the pituitary derived hormones in an appropriate manner. The different phenotypes, as mainly studied in the mouse, may help to analyse the consequences of disruption of a known or yet unknown individual transcription factor in humans. Therefore, to study the different steps in morphogenesis will shed light onto developmental processes which will open a most fascinating time not only for basic scientists, biologist but also for clinicians.

Regulation of human growth hormone-binding protein production by human growth hormone in a hepatoma cell line.

The mechanism by which growth hormone-binding protein (GH-BP) is generated in humans remains unclear. To address this question, we analysed human GH-receptor/GH-BP gene expression in a human hepatoma cell line (HuH7). Northern hybridisation showed that HuH7 cells contain a single mRNA species hybridising with a probe for the sequences encoding the extracellular domain of the hGH-receptor/GH-BP. These data were confirmed by solution hybridisation methods. Thereafter, the cells were treated with r-hGH at physiological (12.5, 25, 50 ng/ml) and supra-physiological (150, 500 ng/ml) concentrations over the period of 48 h. At intervals, RNase protection assays were performed to determine GH-receptor/GH-BP mRNA levels, nuclear run-on assays were carried out to determine whether changes in mRNA levels represented changes in transcription rate, and a radio-ligand binding assay was performed to measure levels of GH-BP in the medium. We found that the r-hGH-regulated changes in GH-receptor/GH-BP mRNA levels detected with the probe for sequences encoding the extracellular domain of human GH-receptor/GH-BP were identical to those previously detected using a probe for the sequences encoding the transmembrane/intracellular domain of the human GH-receptor. In addition, we found that r-hGH had a rapid effect on the levels of GH-BP in the culture medium, which differed from its effect on the GH-receptor/GH-BP mRNA levels. Furthermore, lowering of temperature resulted in a decrease of GH-BP released into the medium implying that enzymes may be involved in the releasing mechanism. These data support the idea that GH-receptor and GH-BP are encoded by a single mRNA species in humans. In addition, they suggest that GH-BP levels are not an accurate reflection of GH-receptor/GH-BP mRNA levels, but that GH-BP production is subject to r-hGH-dependent post-transcriptional regulation, perhaps at the level of post-translational cleavage of the full-length GH-receptor protein. The notion that GH-BP measurements might represent GH-receptor status at the functional level must therefore be taken with caution.

Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein.

The hypothesis that growth hormone binding protein (GHBP) has an effect on its own on the regulation of the GH-receptor/GHBP transcription was tested. Three different forms of human GHBP (recombinant non-glycosylated GHBP, recombinant glycosylated GHBP and GHBP purified and extracted from serum) were added in different concentrations determined by LIFA [0 pmol/l; 50 pmol/l (low level), 200 pmol/l (average level) and 500 pmol/l (high level in circulation)] to a human hepatoma cell line (HuH7 cells) cultured in a serum free hormonally-defined medium. Following the incubation with GHBP for 0, 1 and 2 h, GH-receptor expression was quantitatively assessed by using polymerase chain reaction amplification. Treatment with a GHBP concentration of 50 pmol/l resulted in a significant increase of GH-receptor mRNA molecules given as number of molecules x 10(6)/microg total RNA. In contrast, the concentration of 500 pmol/l presented a significant decrease of GH-receptor mRNA molecules, whereas 200 pmol/l GHBP produced a GH-receptor gene expression which was in between the values of the experiments with 50 and 500 pmol/l of GHBP added. Furthermore, the three different forms of human GHBP used provided similar data and, therefore, did not effect in any variation of GH-receptor expression. In addition, nuclear run-on experiments confirmed the changes in GH-receptor expression; and cycloheximide (10 microg/ml) did not alter the transcription indicating that the up and down regulating effects of GHBP on the GH-receptor/GHBP gene transcription was dependent, at least partly, on pre-existing factors and does not require protein synthesis. In conclusion, we present data showing that GHBP on its own has an effect on GH-receptor gene expression.

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3).

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.