Yuji Tani

Upregulation of CDKN2A and suppression of cyclin D1 gene expressions in ACTH-secreting pituitary adenomas

OBJECTIVE Cushings disease (CD) is usually caused by ACTH-secreting pituitary microadenomas, while silent corticotroph adenomas (SCA) are macroadenomas without Cushingoid features. However, the molecular mechanism(s) underlying their different tumor growth remains unknown. The aim of the current study was to evaluate and compare the gene expression profile of cell cycle regulators and cell growth-related transcription factors in CD, SCA, and non-functioning adenomas (NFA). DESIGN AND METHODS Tumor tissue specimens resected from 43 pituitary tumors were studied: CD (n=10), SCA (n=11), and NFA (n=22). The absolute transcript numbers of the following genes were quantified with real-time quantitative PCR assays: CDKN2A (or p16(INK4a)), cyclin family (A1, B1, D1, and E1), E2F1, RB1, BUB1, BUBR1, ETS1, and ETS2. Protein expressions of p16 and cyclin D1 were semi-quantitatively evaluated by immunohistochemical study. RESULTS AND CONCLUSION CDKN2A gene expression was about fourfold greater in CD than in SCA and NFA. The gene expressions of cyclins D1, E1, and B1, but not of A1, in CD were significantly suppressed than those in NFA. Cyclin D1 gene expression positively correlated with cyclins B1 and E1. The gene expressions of E2F1, RB1, BUB1, BUBR1, ETS1, and ETS2 did not differ between each group. Positive immunostaining for p16 and negative immunostaining for cyclin D1 were more frequent in CD than in NFA; there were positive correlations between mRNA and protein expressions of p16 and cyclin D1. Thus, it is suggested that upregulated CDKN2A with the concomitant downregulated cyclin gene family is partly involved in the small size of ACTH-secreting adenoma.

Prolonged effects of intracerebroventricular angiotensin II on drinking, eating and locomotor behavior in mice

The effects of centrally administered Angiotensin II (Ang II) on water and food intake in rodent models are well known. However, most studies have focused on the acute effects of intracranial Ang II. In the current study, we evaluated the effects of intracerebroventricular Ang II on food and water intake as well as locomotor activity over the entire dark phase of the murine diurnal cycle. Consistent with the previous reports, centrally administered Ang II rapidly stimulated water intake over the initial 1-hour period following treatment. However, this acute increase was immediately followed by a marked reduction in water intake resulting in decreased cumulative water intake approximately 7h after Ang II treatment. Pretreating animals with an Ang II type 1 receptor blocker, Losartan, completely antagonized the acute effect of Ang II and abolished initial water intake. In contrast, application of an Ang II type 2 receptor blocker, PD123319, abrogated the prolonged inhibitory effect of Ang II on drinking behavior and partially suppressed the initial increases in water intake. The suppressive effects of Ang II on cumulative food intake and spontaneous physical activity were also evident throughout the entire dark phase of diurnal cycle. These experiments are the first to suggest that the stimulatory effect of central Ang II treatment on water consumption is very temporary and that it causes a sustained suppressive effect on voluntary locomotion and food intake behavior in mice.

Regulation of growth hormone secretion by (pro)renin receptor

(Pro)renin receptor (PRR) has a single transmembrane domain that co-purifies with the vacuolar H+-ATPase (V-ATPase). In addition to its role in cellular acidification, V-ATPase has been implicated in membrane fusion and exocytosis via its Vo domain. Results from the present study show that PRR is expressed in pituitary adenoma cells and regulates growth hormone (GH) release via V-ATPase-induced cellular acidification. Positive PRR immunoreactivity was detected more often in surgically resected, growth hormone-producing adenomas (GHomas) than in nonfunctional pituitary adenomas. GHomas strongly expressing PRR showed excess GH secretion, as evidenced by distinctly high plasma GH and insulin-like growth factor-1 levels, as well as an elevated nadir GH in response to the oral glucose tolerance test. Suppression of PRR expression in rat GHoma-derived GH3 cells using PRR siRNA resulted in reduced GH secretion and significantly enhanced intracellular GH accumulation. GH3 treatment with bafilomycin A1, a V-ATPase inhibitor, also blocked GH release, indicating mediation via impaired cellular acidification of V-ATPase. PRR knockdown decreased Atp6l, a subunit of the Vo domain that destabilizes V-ATPase assembly, increased intracellular GH, and decreased GH release. To our knowledge, this is the first report demonstrating a pivotal role for PRR in a pituitary hormone release mechanism.

Processing of high-molecular-weight form adrenocorticotropin in human adrenocorticotropin-secreting tumor cell line (DMS-79) after transfection of prohormone convertase 1/3 gene

Ectopic ACTH-producing tumors preferentially secrete biologically inactive ACTH precursors and ACTH-related fragments. DMS-79 is known to secrete unprocessed high-molecular-weight (HMW) form ACTH. To determine whether prohormone convertase (PC) 1/3 is involved in the abnormal processing of proopiomelanocortin (POMC), we studied whether PC1/3 and 2 genes are expressed in DMS-79, and whether overexpression of PC1/3 gene affects POMC processing pattern. Steady-state mRNA levels of PC1/3 and 2 were determined by real-time RT-PCR. Molecular weights of ACTH-related peptides were determined by chromatographical analyses coupled with ACTH and β-endorphin (β-END) radioimmunoassays. PC1/3 gene was transfected into DMS-79 by retrovirus transduction using pMX-IP vector encoding PC1/3 cDNA. The steady-state mRNA levels of PC1/3 and 2 in DMS-79 were lower than those in ACTH-secreting and nonfunctioning pituitary tumors. DMS-79 predominantly secreted HMW form with both ACTH and β-END immunoreactivities by size-exclusion chromatography. After purification by immunoaffinity chromatography with anti-ACTH antibody, the apparent molecular weight of HMW form ACTH was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. After retroviral transfection of PC1/3 cDNA into DMS-79 and puromycin selection, PC1/3 stably-expressing cell line (DMS-79T) secreted two immunoreactive ACTH components, a major one coeluting with ACTH(1–39) and a minor one as a HMW form as well as two β-END immunoreactive components coeluting with β-lipotropic hormone and β-END, respectively. Thus, we have established PC1/3 stably-expressing cell line (DMS-79T) capable of proteolytically processing ACTH precursor molecule(s) into mature ACTH and β-END.

Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis

The transglutaminase (TG) family comprises eight isozymes that form the isopeptide bonds between glutamine and lysine residues and contribute to the fibrotic diseases via crosslinking-mediated stabilization of ECM and the activation of TGF-β in several tissues. However, despite a growing body of evidence implicating TG2 as a key enzyme in fibrosis, the causative role of TG2 and the involvement of the other isozymes have not yet been fully elucidated. Therefore, here we clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific possible substrates for both TG1 and TG2 using their substrate peptides in mouse fibrotic liver. We found that TG1 activity was markedly enhanced intracellularly over a widespread area, whereas TG2 activity increased in the extracellular space. In total, 43 and 42 possible substrates were identified for TG1 and TG2, respectively, as involved in chromatin organization and cellular component morphogenesis. These included keratin 18, a biomarker for hepatic injury, which was accumulated in the fibrotic liver and showed the partly similar distribution with TG1 activity. These findings suggest that TG1 activity may be involved in the functional modification of intracellular proteins, whereas TG2 activity contributes to the stabilization of extracellular proteins during liver fibrosis.

Salusin-β as a powerful endogenous antidipsogenic neuropeptide.

Salusin-β is an endogenous parasympathomimetic peptide, predominantly localized to the hypothalamus and posterior pituitary. Subcutaneously administered salusin-β (50 nmol/mouse) significantly increased water intake but did not affect locomotor activity or food intake. The salusin-β-induced increase in water intake was completely abrogated by pretreatment with muscarinic antagonist, atropine sulphate. In contrast, intracerebroventricular injection of salusin-β, at lower doses (10–100 fmol/mouse) caused a long-lasting decrease in water intake and locomotor activity throughout the entire dark phase of the diurnal cycle. Pre-injection of intracerebroventricular anti-salusin-β IgG completely abrogated the central salusin-β mediated suppression of water intake and locomotor activity. These results demonstrate contrasting actions of salusin-β in the control of water intake via the central and peripheral systems and highlight it as a potent endogenous antidipsogenic neuropeptide.