Jacky M. Burrin

Clinical manifestations of familial paraganglioma and phaeochromocytomas in succinate dehydrogenase B (SDH-B) gene mutation carriers

Objectiveu2002 Phaeochromocytomas and paragangliomas are familial in up to 25% of cases and can result from succinate dehydrogenase (SDH) gene mutations. The aim of this study was to describe the clinical manifestations of subjects with SDH‐B gene mutations.

The release of leptin and its effect on hormone release from human pituitary adenomas

BACKGROUND Leptin is the protein product of the obese gene, known to play an important role in body energy balance. The leptin receptor exists in numerous isoforms, the long isoform being the major form involved in signal transduction. Leptin expression has recently been demonstrated in the human pituitary, both in normal tissue and in pituitary adenomas. The long isoform of the leptin receptor has also been shown to be present in pituitary adenomas; however, contrasting results have been obtained regarding its expression in the normal human pituitary.

Regulation of Human Glycoprotein Hormone α-Subunit Gene Transcription in LβT2 Gonadotropes by Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2

Abstract Transcriptional activation of the human glycoprotein hormone α-subunit (αGSU) promoter in response to GnRH and phorbol-12-myristate-13-acetate (PMA) has been well characterized in αT3-1 gonadotropes but not investigated in the more differentiated LβT2 clonal gonadotrope. We have evaluated αGSU transcription in the more mature LβT2 cell line, using deletion and heterologous constructs of the αGSU promoter linked to a luciferase reporter gene. Basal αGSU-promoter activity was significantly less in LβT2 cells than in αT3-1 cells, but stimulation of transfected cells with GnRH and PMA resulted in similar increases in αGSU-promoter activity. Deletional analysis of the human αGSU promoter in LβT2 cells indicated that sequences between −398 and −244 and between −244 and −195 base pairs (bp) were involved in regulating basal αGSU-promoter transcription, whereas the region between −244 and −195 bp regulated PMA-stimulated promoter activity. Deletion of this promoter region containing a steroidogenic factor-1 (SF-1) binding site abolished basal and PMA-stimulated transcription. Site-directed mutagenesis of the SF-1 binding site resulted in a significant attenuation of basal and PMA-stimulated αGSU transcription. Pretreatment of LβT2 cells with a mitogen-activated protein kinase kinase-specific inhibitor, U0126, abolished the PMA-stimulated increase in MAPK activity and significantly reduced basal and PMA-stimulated promoter activity. Electrophoretic mobility shift assays for SF-1 and GATA revealed that PMA failed to affect SF-1 binding but enhanced GATA binding to a consensus GATA oligonucleotide, an effect that was blocked with U0126 pretreatment, suggesting that GATA may mediate ERK activation of αGSU transcription. Our data suggests that, in the mature LβT2 gonadotrope cell line, two regions of the human αGSU promoter regulate basal transcription and that SF-1 is involved in mediating basal and PMA-stimulated promoter activity. Furthermore, PKC-stimulated transcription partially relies on ERK acting on elements downstream of −244 bp of the human αGSU promoter.

Contrasting clinical manifestations of SDHB and VHL associated chromaffin tumours

Mutations in succinate dehydrogense-B (SDHB) and the von Hippel-Lindau (VHL) genes result in an increased risk of developing chromaffin tumours via a common aetiological pathway. The aim of the present retrospective study was to compare the clinical phenotypes of disease in subjects developing chromaffin tumours as a result of SDHB mutations or VHL disease. Thirty-one subjects with chromaffin tumours were assessed; 16 subjects had SDHB gene mutations and 15 subjects had a diagnosis of VHL. VHL-related tumours were predominantly adrenal phaeochromocytomas (22/26; 84.6%), while SDHB-related tumours were predominantly extra-adrenal paragangliomas (19/25; 76%). Median age at onset of the first chromaffin tumour was similar in the two cohorts. Tumour size was significantly larger in the SDHB cohort in comparison with the VHL cohort (P=0.002). Multifocal disease was present in 9/15 (60%) of the VHL cohort (bilateral phaeochromocytomas) and only 3/16 (19%) of the SDHB cohort, while metastatic disease was found in 5/16 (31%) of the SDHB cohort but not in the VHL cohort to date. The frequency of symptoms, hypertension and the magnitude of catecholamine secretion appeared to be greater in the SDHB cohort. Renal cell carcinomas were a feature in 5/15 (33%) of the VHL cohort and 1/16 (6%) of the SDHB cohort. These data indicate that SDHB-related tumours are predominantly extra-adrenal in location and associated with higher catecholamine secretion and more malignant disease, in subjects who appear more symptomatic. VHL-related tumours tend to be adrenal phaeochromocytomas, frequently bilateral and associated with a milder phenotype.

Effects of ‘Non-Calcaemic’ Vitamin D Analogues on 24-Hydroxylase Expression in MG-63 Osteoblast-Like Cells

Background: New ‘non-calcaemic’ analogues of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are entering the clinical arena and some of them have been shown to have differential effects in bone. This may have a bearing on the evolution of bone lesions in uraemic patients receiving vitamin D therapies. A potential mechanism for differential effects of analogues lies in their target cell inactivation. Methods: Using a human osteoblastic cell line, MG-63, three analogues, 22-oxacalcitriol (OCT), 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol) and 1α,25-dihydroxydihydrotachysterol₂ (1,25(OH)₂DHT₂), were compared with 1,25(OH)₂D₃ for (1) their affinity for the vitamin D receptor (VDR) by competitive displacement of tritiated 1,25(OH)₂D₃ from calf thymus VDR; (2) effects on 24-hydroxylase mRNA expression using comparative RT-PCR, and (3) rates of metabolism, using high performance liquid chromatography, over a 24-hour time course. Results: Relative VDR-binding affinities (IC₅₀) were 1,25(OH)₂D₃ (100%), OCT (25%), paricalcitol (14%) and 1,25(OH)₂DHT₂ (0.3%). A ≧3-fold increase in 24-hydroxylase mRNA expression was observed for all compounds at 2 h peaking at 7- to 8-fold above control levels by 12 h, with no significant difference between the analogues and 1,25(OH)₂D₃. Differences in their rates of metabolism were observed [calculated t½ values = OCT (1.2 h) > paricalcitol (2.3 h) > 1,25(OH)₂D₃ (2.6 h) > 1,25(OH)₂DHT₂ (3.4 h)], with OCT having a significantly shorter half-life. Conclusion: In MG-63 cells these analogues up-regulate 24-hydroxylase mRNA expression with similar potency, in each case accelerating ligand inactivation, despite significant differences in VDR affinity. VDR affinity did not correspond to either 24-hydroxylase mRNA expression or the rates of ligand disappearance, suggesting cellular metabolism is one of several factors that determine the analogue specificity of these agents in bone.

Leptin in Pituitary Adenomas–A Novel Paracrine

A growing number of physiological and pathophysiological processes have been shown to be influenced by leptin apart from its first recognised role as a modulator of hypothalamic appetite and weight control centers. We investigated the presence and pattern of distribution of leptin mRNA and the mRNA of the long isoform of the leptin receptor in the normal pituitary and in different types of pituitary adenomas. We also studied leptin secretion from human pituitary tumors in culture, and the in vitro pituitary hormone release following stimulation with human leptin. Leptin mRNA expression was detected at a low level of expression in 50% of tumors but in none of the normal pituitaries. By immunohistochemistry, leptin was present in occasional scattered cells in the normal pituitary and in pituitary tumors. The leptin receptor long isoform was detected in the majority (65%) of pituitary tumors and in all normal pituitaries. It did not segregate with any particular tumor type, and varying levels of expression were detected between the tissues studied. 34% of pituitary adenomas showed leptin release into the incubation media during in vitro culture. Leptin mRNA, the mRNA of the long isoform of the receptor, or in vitro leptin release, did not correlate with tumor type or with any of the other pituitary hormones released. In vitro leptin stimulation of pituitary tumors caused stimulation of FSH and α-subunit secretion from a non-functioning adenoma and TSH secretion from a somatotroph adenoma. As the co-localisation of ACTH and leptin in corticotroph cells was previously suggested, we investigated whether in vivo ACTH release is accompanied by a simultaneous plasma leptin level rise (i) in peripheral plasma samples after food intake-induced ACTH rise in healthy obese and nonobese individuals and (ii) in petrosal sinus samples after CRH injection in Cushings disease patients. Our data suggest that a rise in ACTH levels is not accompanied by detectable rise in leptin levels in peripheral and in petrosal sinus blood samples. In summary, leptin is synthesized and stored within the pituitary and may modulate other pituitary hormone secretion, although probably do not contribute to plasma leptin level changes. Pituitary leptin may therefore be a novel paracrine regulator of pituitary function.