Among sex steroids, especially estrogen metabolism has been considered to play a role in the function and pathology of human veins. We investigated the expression and activity of the estrogen-producing enzyme aromatase and estrogen receptor (ER) in human vena cava to assess possible in situ biosynthesis of estrogens and their modes of action. We first examined aromatase expression by immunohistochemistry in human inferior vena cava obtained from 29 autopsy cases (11 males, 18 females, 63.6±3.0 years old). We then semiquantitated the level of aromatase mRNA by reverse transcriptase-polymerase chain reaction in 24 cases and aromatase activity by 3H-water assay in 15 cases to examine whether or not and in which cell types aromatase was expressed. We also studied alternative use of multiple exon is of its gene and immunolocalization of 17β-hydroxysteroid dehydrogenase type I (17β-HSD I), which converts estrone produced by aromatase to estradiol, a biologically active estrogen and ER. Aromatase and 17β-HSD I immunoreactivity were both detected in smooth muscle cells (SMC) of the media in all the cases and in endothelial cells (EC) in 20 and 22 cases, respectively. ER immunoreactivity was detected in SMC of vena cava in 21 cases. The amount of aromatase mRNA was significantly greater in the cases utilizing 1c (I.3) or id (P.II) of exon 1 (9 cases, 191.1±26.3 attomol/ng total RNA) than those utilizing 1b (I.4) as the promoter (14 cases, 50.6±13.0 attomol/ng total RNA) (p<0.01). Significant correlation (p<0.05) was observed between the amount of aromatase mRNA and aromatase activity in 15 cases examined. No significant correlation was detected between the amount of aromatase mRNA or aromatase labeling index and the ER status. These results suggest that estrone and estradiol are produced in the human vena cava and that their production is mediated by aromatase and 17β-HSD I, respectively but not all of these locally synthesized estrogens may not work directly in situ.
To examine whether synthetic vitamin D3 analog, 22-oxa-1, 25(OH)2D3 (OCT) has an inhibitory effect on the growth of thyroid carcinoma, we tested the in vitro and in vivo effects of OCT on the growth of a well- differentiated thyroid cancer cell line, NPA. OCT bound to its receptor at the same rate as 1, 25(OH)2D3, and inhibited the proliferation of NPA cells in vitro in a dose-dependent manner, similar to that observed with 1, 25 (OH)2D3. Northern blot analysis showed that steady-state and fetal bovine serum-stimulated levels of c-myc mRNA were suppressed after 0.5-4 hour treatment with OCT. Transfection studies with the deletion mutants of the 5''- up-stream flanking region of c-myc/chroramphenicol acetyltransferase chimera genes indicated the presence of an OCT responsive element between -410 and -106. Next, we examined OCT effects in implanted NPA tumor cells in nude mice. OCT showed no remarkable hypercalcemic effect compared to 1, 25(OH2)D3, but OCT and 1, 25 (OH2)D3, had no significant inhibitory effect in vivo after either intra-tumor or intra-peritoneum injection. Our results demonstrate that OCT inhibits the proliferation of well-differentiated thyroid cancer in an in vitro system associated with the suppression of c-myc mRNA, but this inhibitory effect was not reproducible in in vivo model.
We have previously reported that type I transforming growth factor beta (TGF-β1) is a potent stimulator of cell growth in articular chondrocytes. In this study, we examined the mechanism of TGF-β1 induced cellular proliferation by using cultured rat articular chondrocytes (CRAC). A time-course study of [3H]thymidine incorporation upon TGF-β1 (1ng/mL) or 10% fetal bovine serum stimulation revealed that TGF-β1 directly stimulates DNA synthesis in CRAG. Pretreatment with H7, an inhibitor for protein kinase C (PKC), completely blocks TGF-β1-induced proliferation. Since TGF-β1 has been shown to transduce signals through MAP kinase cascades, we investigated the induction of several protooncogenes by Northern blotting. TGF-β1 addition causes an immediate and transient induction of c-fos but not myc or jun mRNA. Furthermore, this c-fos expression is not inhibited by cycloheximide, but is completely abolished by pretreatment with TPA, so that the c-fos gene is a direct target of TGF-β1 signalling and PKC is involved in this c-fos induction. To refine our understanding of TGF-β1 regulation of the c-fos promoter region, we performed chloramphenicol acetyltransferase (CAT) assays. A serial deletion analysis of the c-fos promoter region reveals a TGF-β1 responsive element in a region between -403 and - 329bp upstream of the transcription initiation site. We attempted gel shift assays on this response element with CRAC nuclear extracts. Although this region contains a sis-inducible binding element, we fail to detect specific DNA- protein complexes. Our results, however, suggest that TGF-β1 acts as a primary mitogen in CRAC and this mitogenic activity requires PKC activation. Finally, the subsequent induction of c-fos expression occurs through an as yet unidentified transactivation mechanism.
This is a case report of an 18-year-old man with central diabetes incipidus (DI). An MRI done three months after the onset of the DI did not disclose a responsible lesion. Four months later, a second MRI showed the location of the tumor origin at the upper pituitary stalk and median eminence. Eight months later, the tumor occupied the hypothalamic area. The tumor became large and contrast-making enough to be visible on MRI between 3 and 4 months after the onst of DI. Besides the suprasellar tumor, another mass was noted in the pineal region. The growth pattern of the latter mass corresponded well to that of the former. Although the MRI is a sensitive diagnostic tool for the detection of intracranial tumors, no adequate rationale has been given as to how the MRI might be repeated for children and adolescents who have been diagnosed as having the central DI, when their initial MRIs may have been normal. In our patient, the superconductive thin slice MRI revealed the suprasellar germinoma 4 months after the onset. The suprasellar and pineal tumors in this report originated and developed simultaneously. This may indicate a multi-center origin of the tumor. Another possibility is a very early dissemination from the onset of the tumor development.
To clarify the characteristics of vasopressin (AVP) secretion in patients with the syndrome of inappropriate antidiuresis (SIAD) related to central nervous system disorders, we examined the response of AVP secretion to osmotic stimulus by hypertonic saline infusion and analyzed the possible causative factors in six patients with SIAD associated with head trauma or cerebral infarction. Hyponatremia developed after head trauma in four patients and cerebral infarction in two patients. In all patients the clinical state and laboratory findings fulfilled the criteria for SIAD, which was supported by either nonsuppressible plasma AVP levels or effectiveness of treatments with water restriction, demeclocycline, nonpeptide V2 AVP antagonist or diphenylhydantoin. Although patterns of plasma AVP response to the osmotic stimulus varied, plasma AVP concentrations neither increased nor decreased to undetectable levels with a rise in plasma osmolality. In one patient, plasma AVP levels responded to increasing plasma osmolality when plasma osmolality normalized; in which the threshold and the sensitivity of osmostat were normal. In two other patients, AVP secretion responded to plasma osmolality after the treatment. The changes in AVP secretion were not due to nonosmotic stimuli for AVP release. In conclusion, this study shows that patients with SIAD and central nervous system disorders may have persistent AVP secretion with a loss of hypotonic suppression such as found in patients with adrenal insufficiency or depletional hyponatremia in central nervous system disorders, indicating that careful evaluation is necessary to determine the relationship between persistent AVP secretion and the pathogenesis of hyponatremic disorders.
Some of the recently identified coactivators which interact with members of nuclear hormone receptors contain a stretch of homopolymeric glutamines (poly-Q). Length of poly-Q in several genes are known to be polymorphic in healthy subjects, and extraordinary expansion of poly-Q in specific genes is known to cause neurodegenerative disorders. In the present study, we investigated whether such polymorphism can be observed in two Coactivators, CBP (CREB [cyclic AMP responsive element binding protein]-binding protein) and AIB1/ACTR (amplified in breast cancer-1/ACTR, also called RAC3/TRAM-1). The genomic regions encoding the poly-Q were amplified by means of PCR using fluorescence labeled primer and analyzed by an automatic sequencer. While contiguous glutamine residues inAIB1/ACTR ranged from 26 to 32 with a heterozygosity of 54%, no polymorphism could be observed in poly-Q of CBP among 54 unrelated subjects. These results suggest that the residue in CBP may play a critical role in the function so that individuals with CBP containing different sizes of poly-Q might have been eliminated. It has been reported that AIB1/ACTR is overexpressed in some of the cell lines derived from breast cancer. If the length of poly-Q alters the stability of AIB1/ACTR and/or potency to enhance hormone action through nuclear receptors, the length of poly-Q is likely to be one of the genetic factors affecting not only susceptibility to breast cancers but also the sensitivity to hormones. This polymorphism should also be tested in patients with neurodegenerative disorders of unknown cause.
We report the case of a 17-year-old boy with delayed puberty, who presented a complexity of clinical problems. An analysis of steroid hormones led to a diagnosis of 17α-hydroxylase/17, 20-lyase deficiency (17OHD) .Unlike typical cases of 17OHD, however, the patient had pubertal development without medical intervention. I naddition, he never exhibited the symptoms of mineralocorticoid excess, showing instead the symptoms of glucocorticoiddeficiency, including fatigability, emaciation, and weight-loss induced by minor infection. He also had dysmorphic features, which comprised marfanoid habitus, arachnodactyly and putative craniosynostosis. The combination of these malformations substantially resembled that of Shprintzen-Goldberg syndrome. Direct sequencing o fthe CYP17 gene did not reveal any significant aberrations in the exons or exon-intron boundaries. We speculate tha tthe association of partial combined 17OHD with the Shprintzen-Goldberg phenotype in the present patient may resultfrom an aberration of a hitherto unknown gene that controls both steroid hormone synthesis and skeletal development.
The changes in interleukin-1(IL-1)β mRNA expression and the number of macrophages were studied in the ovary during the estrous cycle in rats and after intraperitoneal injection of lipopolysaccharide (LPS, 2mg/body )2 hours before autopsy. IL-1β mRNA expression was very low in the ovary, and there was no statistically significan tchange during the estrous cycle. Hybridization signals of IL-1β mRNA were localized intensely in the thecal layer, moderately in the corpora lutea, and slightly in granulosa cells of the ovary during the cycle. The number o fmacrophages seen mainly in the hilum and interstitium significantly increased on proestrus compared with otherestrous days. LPS significantly increased IL-1β mRNA expression on each day with the highest response to LPS at 1500h on proestrus, and caused an increase in the number of macrophages in the ovary within 2 hours. These results indicate that IL-1β mRNA expressions are low during the estrous cycle in rats, and proestrus is the day of maximal IL-1β synthesis in response to LPS. The increase in IL-1β synthesis caused by LPS might be due to at least the influx of macrophages into the ovary.
We encountered a young man treated with anticonvulsant therapy who had greatly reduced bone mineral density. An 18-year-old man was admitted to our hospital for shoulder pain and further evaluation of decrease dbone mineral density. He had been treated with anticonvulsants, including phenytoin, phenobarbital, valproic acidand zonisamide for seizures. Although testosterone was found within the normal range for adult men, the serum estrogen concentration was below the detection limit (<10pg/ml) and his wrist epiphyses were not yet closed. After 10 months of treatment with the conjugated estrogen, both his height and weight showed improvement, while his bone mineral density and bone age were increased. These findings suggested that estrogen therapy had a significant effect on his skeletal growth and bone maturation in man. This is the first report showing the beneficial effect of estrogen supplementation in an epileptic man receiving treatment with anticonvulsants.
Apoptosis appears to play important roles in physiological and pathological processes in the endocrine system including the thyroid, but little is known about the regulation of apoptosis in the thyroid. The functioningrat thyroid cell line (FRTL-5), a cloned cell line of differentiated thyroid cells, hardly undergoes apoptosis. In this study we examined the factors which prevent FRTL-5 cells from undergoing apoptosis. After culturing FRTL-5 cells in medium with and without TSH, actinomycin D (AMD) or cycloheximide (CHX) was added. CHX did not induce apoptosis. AMD induced apoptosis in FRTL-5 cells cultured in medium lacking TSH, as confirmed by the presence of DNA fragmentation, together with nuclear fragmentation and condensation, but AMD did not induce apoptosis in FRTL-5 cells cultured in the presence of TSH. Furthermore, the fact that AMD did not induce apoptosis in FRTL-5 cells cultured with dibutyryl cyclic AMP (Bt2cAMP), or forskolin suggests that TSH has an inhibitory effect on apoptosis in FRTL-5 cells via the TSH-CAMP pathway.
We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1α, 25-dihydroxyvitamin D3(1, 25-(OH)2D3)-induced 24-hydroxylation activity inHL-60 cells by increasing 1, 25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol- 13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1, 25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1, 25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1, 25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1, 25-(OH)2D3, besides up-regulation of VDR.
Familial multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited disorder characterized by tumors of the parathyroid, anterior pituitary and gastro-entero-pancreatic endocrine tissues. The MEN1 gene has recently been cloned and its germline mutations have been considered to play an important role in the tumorigenesis of MEN1. We analyzed a Japanese MEN1 patient and her daughter for germline mutations of the MEN1 gene. The proband (60 y.o.) had primary hyperparathyroidism (PHP) and gastrinoma, and her daughter (30 y.o.) had prolactinoma. Clinical examinations revealed no evidence of PHP in the daughter. We identified a novel heterozygous germline mutation (712 A del) at codon 201 in exon 3 of the MEN1 gene in the proband. Restriction digestion analysis revealed the same mutation pattern in her daughter. These findings suggest that this family has familial MEN1 including a rare case of MEN1 with a single lesion of the pituitary. Genetic examinations are useful as diagnostic tools for any rare or variant case of familial MEN1.
We report on a case of rapid and marked hormone release as a result of rapid tumor reduction due to chemotherapy in a 36-year-old woman with ectopic ACTH syndrome due to small cell lung cancer. Treatment of thecancer with cisplatin and etoposide resulted in an 80% reduction in tumor size on computed tomographic scan within two weeks. Concurrently, plasma ACTH exhibited an unexpected and astonishing increase from 373pg/ml before treatment to more than 1200pg/ml. There were no biochemical characteristics observed in tumor lysis syndrome of solid tumors such as azotemia, increased LDH and hyperkalemia. The present case indicates that anticancer chemotherapy instituted in patients with ectopic ACTH syndrome could result in an acute increase of plasma ACTH and exacerbation of hypercortisolism, similar to tumor lysis syndrome, which is a potentially fatal complication following anti-cancer chemotherapy.
The purpose of this prospective study was to characterize the changes in serum levels of two proteins produced during the synthesis and degradation of type I collagen, i.e., the carboxyterminal propeptide of type Iprocollagen (PICP) and the pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), respectively, after oophorectomy, and to assess the degree of correlation between changes in the serum values of these proteins and changes in bone mineral density (BMD) of the lumbar spine. Serum levels of PICP, ICTP and bone gla protein (BGP) were determined in 18 women before oophorectomy (baseline) and at 7 days, and 1, 2, 3, 6, 9 and 12 months post-oophorectomy (PO). The BMD of the lumbar spine was measured at baseline, and at 6 months and 12 months PO. ICTP had increased significantly at 7 days PO and peaked between 1 and 3 months PO. PICP and BGP had increased significantly at 2 months PO and remained at high levels thereafter. The percent changes in lumbar BMD from baseline values (% CFB) at 6 months and at 12 months PO were significantly correlated with % CFB in ICTP, but not with % CFB in PICP or BGP. Accordingly, bone resorption is a main determinant of bone mineral loss after oophorectomy and the change in recently-developed bone resorption markers, such as ICTP, is of clinical utility in predicting a degree of subsequent bone loss after surgical menopause.