Takeo Iwata

The Role of the Aryl Hydrocarbon Receptor-Interacting Protein Gene in Familial and Sporadic Pituitary Adenomas

CONTEXT Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

Silefrin, a sodefrin-like pheromone in the abdominal gland of the sword-tailed newt, Cynops ensicauda

Sodefrin‐like female‐attracting pheromone was purified from the abdominal glands of male sword‐tailed newts, Cynops ensicauda, by gel‐filtration chromatography and reversed‐phase high‐performance liquid chromatography. The final product comprises 10 amino acid residues with the sequence SILSKDAQLK which coincided with the sequence deduced from its precursor cDNA. This peptide was designated silefrin. The sequence of silefrin was different from that of sodefrin by two amino acid residues, with substitutions Leu for Pro and Gln for Leu at positions 3 and 8, respectively. Both native and synthetic silefrin exerted an equipotent activity in attracting conspecific females.

The aryl hydrocarbon receptor-interacting protein gene is rarely mutated in sporadic GH-secreting adenomas

Background  Recently, germline mutations of aryl hydrocarbon receptor‐interacting protein (AIP) gene located on 11q13 were identified in patients with pituitary adenoma predisposition.

Hormonal control of urodele reproductive behavior

Hormonal control of expression of courtship behavior and of development of structures related to the reproductive behavior in two species of Japanese newts, Cynops pyrrhogaster and Cynops ensicauda, was described. Prolactin (PRL) and androgen were essential factors for eliciting courtship behavior. In addition, arginine vasotocin markedly enhanced the expression of courtship behavior. PRL induced migration to water, in which courtship and oviposition take place, and converted the integument from the terrestrial type to the aquatic one. PRL also stimulated the growth of the tail fin, which was blocked by estrogen. Cellular and nuclear size and number of synapses on the somata of Mauthner cells, which are involved in tail movement, were also increased by PRL and androgen. Synthesis of sodefrin, a female-attracting pheromone, in the abdominal gland as well as that of mucopolysaccharides constituting the sac of sperm in the lateral gland was enhanced by PRL and androgen. Structural development of oviducts was elicited by estrogen or PRL to a certain extent, and full oviducal development by the combination of these two hormones, PRL being indispensable for the oviducal jelly secretion.

Genetic analyses in patients with familial isolated hyperparathyroidism and hyperparathyroidism–jaw tumour syndrome

Background  A subset of familial isolated primary hyperparathyroidism (FIHP) is a variant of hyperparathyroidism–jaw tumour syndrome (HPT‐JT).

Peptide and protein pheromones in amphibians

Purification, characterization and biological activity of urodele and anuran sex-pheromones were reviewed. Female-attracting pheromones obtained from the abdominal gland of Cynops pyrrhogaster and C. ensicauda males are peptides consisting of 10 amino acid residues being designated sodefrin and silefrin, respectively. Each pheromone attracted only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed that both are generated from precursor proteins. Synthesis of these pheromones is regulated by prolactin (PRL) and androgen. Responsiveness of the female vomeronasal epithelium to sodefrin is enhanced by PRL and estrogen. The submandibular gland of the male terrestrial salamander, Plethodon jardani secretes a 22-kD proteinaceous pheromone that enhances female receptivity. It was revealed that every salamander synthesizes multiple isoforms of this pheromone, Plethodontid receptivity factor. The magnificent tree frog, Litoria splendida breed in an aquatic environment. The skin glands of the male secrete a female-attracting peptide pheromone, splendipherin, comprising 25 amino acid residues. The significance of the structure of the amphibian sex-pheromone as peptide and protein is discussed in terms of their species specificity.

The Action of D-Dopachrome Tautomerase as an Adipokine in Adipocyte Lipid Metabolism

Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.

Compressive force inhibits adipogenesis through COX-2-mediated down-regulation of PPARγ2 and C/EBPα

Various mechanical stimuli affect differentiation of mesoderm-derived cells such as osteoblasts or myoblasts, suggesting that adipogenesis may also be influenced by mechanical stimulation. However, effects of mechanical stimuli on adipogenesis are scarcely known. Compressive force was applied to a human preadipocyte cell line, SGBS. Levels of gene expression were estimated by real-time reverse transcription-polymerase chain reaction. The accumulation of lipids was evaluated by Sudan III or Oil Red O staining. In SGBS cells subjected to a compressive force of 226 Pa for 12 h before adipogenic induction, adipogenesis was inhibited. Compressive force immediately after adipogenic induction did not affect the adipogenesis. The expression of peroxisome proliferator-activated receptor (PPAR) gamma2 and CCAAT/enhancer binding protein (C/EBP) alpha mRNA during adipogenesis was inhibited by compressive force, whereas C/EBPbeta and C/EBPdelta mRNA levels were unaffected. In preadipocytes, compressive force increased mRNA levels of Krüppel-like factor 2, preadipocyte factor 1, WNT10b, and cyclooxygenase-2 (COX-2) which are known as negative regulators for the PPARgamma2 and C/EBPalpha genes. Furthermore, a COX-2 inhibitor completely reversed the inhibition of adipogenesis by compressive force. In conclusion, compressive force inhibited adipogenesis by suppressing expression of PPARgamma2 and C/EBPalpha in a COX-2-dependent manner.

Molecular cloning of newt sex pheromone precursor cDNAs: evidence for the existence of species‐specific forms of pheromones

Cloning of cDNA encoding a decapeptide pheromone (sodefrin) that attracts conspecific female newts was attempted. A cDNA clone encoding a protein consisting of 189 amino acid residues including a sodefrin sequence was isolated from a Cynops pyrrhogaster abdominal gland cDNA library. Likewise, a cDNA clone encoding a molecule comparable to the sodefrin precursor was obtained from a Cynops ensicauda abdominal gland cDNA library. This clone encoded a precursor protein of 192 amino acid residues, including a sodefrin‐like peptide sequence with substitutions of two amino acid residues. This is the first report of a peptide pheromone precursor in vertebrates.

Peptide pheromones in newts

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.