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Kenji Fujieda and Tsutomu Hiroshige


The changes in the hypothalamic content of corticotrophin-releasing factor (CRF), the plasma concentrations of ACTH and corticosterone were studied in the intact male rat. CRF activity was estimated by bioassay and plasma ACTH levels by radioimmunoassay. The variation in CRF hypothalamic content reflected CRF secretion since plasma ACTH and corticosterone increased concomitantly. The activity of CRF separated into 2 peaks, one occurring immediately (2 min) and a second about 80 min later after the start of stress. The elevation of plasma ACTH and corticosterone followed the first CRF activity peak, and both showed a sustained time course. Cycloheximide pre-treatment (1 mg/100 g ip) was used to test whether protein synthesis was required for secretion of CRF. Treatment with cycloheximide reduced the incorporation of [14C]L-alanine into hypothalamic and pituitary proteins. The second CRF activity peak was completely abolished while the first activity peak was essentially intact. Plasma ACTH increased in response to the first CRF activity peak, but then declined. Plasma corticosterone remained low throughout, possibly as a consequence of inhibition of adrenal protein synthesis. When a second stress stimulus (fracturing of one leg without anaesthesia) was superimposed, CRF activity and plasma ACTH did not show any incremental response. These results show that the second CRF activity peak is sensitive to cycloheximide inhibition of protein synthesis, while the first activity peak appears to be cycloheximide resistant.

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Shigeru Suzuki, Kumihiro Matsuo, Yoshiya Ito, Atsushi Kobayashi, Takahide Kokumai, Akiko Furuya, Osamu Ueda, Tokuo Mukai, Koichi Yano, Kenji Fujieda, Akimasa Okuno, Yusuke Tanahashi, and Hiroshi Azuma

Background: POU1F1 encodes both PIT-1α, which plays pivotal roles in pituitary development and GH, PRL and TSHB expression, and the alternatively spliced isoform PIT-1β, which contains an insertion of 26-amino acids (β-domain) in the transactivation domain of PIT-1α due to the use of an alternative splice acceptor at the end of the first intron. PIT-1β is expressed at much lower levels than PIT-1α and represses endogenous PIT-1α transcriptional activity. Although POU1F1 mutations lead to combined pituitary hormone deficiency (CPHD), no patients with β-domain mutations have been reported.

Results: Here, we report that a three-generation family exhibited different degrees of CPHD, including growth hormone deficiency with intrafamilial variability of prolactin/TSH insufficiency and unexpected prolactinoma occurrence. The CPHD was due to a novel POU1F1 heterozygous variant (c.143-69T>G) in intron 1 of PIT-1α (RefSeq number NM_000306) or as c.152T>G (p.Ile51Ser) in exon 2 of PIT-1β (NM_001122757). Gene splicing experiments showed that this mutation yielded the PIT-1β transcript without other transcripts. Lymphocyte PIT-1β mRNA expression was significantly higher in the patients with the heterozygous mutation than a control. A luciferase reporter assay revealed that the PIT-1β-Ile51Ser mutant repressed PIT-1α and abolished transactivation capacity for the rat prolactin promoter in GH3 pituitary cells.

Conclusions: We describe, for the first time, that PIT-1β mutation can cause CPHD through a novel genetic mechanism, such as PIT-1β overexpression, and that POU1F1 mutation might be associated with a prolactinoma. Analysis of new patients and long-term follow-up are needed to clarify the characteristics of PIT-1β mutations.