Fukuda Takashi, Tanaka Tomoko, Hamaguchi Yuriko, Kawanami Takako, Nomiyama Takashi, Yanase Toshihiko
Department of Endocrinology and Diabetes Mellitus, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
Department of Bioregulatory Science of Life-related Diseases, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
PLoS One. 2016 Oct 5;11(10):e0164131. doi: 10.1371/journal.pone.0164131. eCollection 2016.
Aryl hydrocarbon receptor interacting protein (AIP) is thought to be a tumor suppressor gene, as indicated by a mutational analysis of pituitary somatotroph adenomas. However, the physiological significance of AIP inactivation in somatotroph cells remains unclear. Using CRISPR/Cas9, we identified a GH3 cell clone (termed GH3-FTY) in which Aip was genetically disrupted, and subsequently investigated its character with respect to growth hormone (Gh) synthesis and proliferation. Compared with GH3, GH3-FTY cells showed remarkably increased Gh production and a slight increase in cell proliferation. Gh-induced Stat3 phosphorylation is known to be a mechanism of Gh oversecretion in GH3. Interestingly, phosphorylated-Stat3 expression in GH3-FTY cells was increased more compared with GH3 cells, suggesting a stronger drive for this mechanism in GH3-FTY. The phenotypes of GH3-FTY concerning Gh overproduction, cell proliferation, and increased Stat3 phosphorylation were significantly reversed by the exogenous expression of Aip. GH3-FTY cells were less sensitive to somatostatin than GH3 cells in the suppression of cell proliferation, which might be associated with the reduced expression of somatostatin receptor type 2. GH3-FTY xenografts in BALB/c nude mice (GH3-FTY mice) formed more mitotic somatotroph tumors than GH3 xenografts (GH3 mice), as also evidenced by increased Ki67 scores. GH3-FTY mice were also much larger and had significantly higher plasma Gh levels than GH3 mice. Furthermore, GH3-FTY mice showed relative insulin resistance compared with GH3 mice. In conclusion, we established a somatotroph cell line, GH3-FTY, which possessed prominent Gh secretion and mitotic features associated with the disruption of Aip.
芳香烃受体相互作用蛋白（AIP）被认为是一种肿瘤抑制基因，垂体生长激素细胞腺瘤的突变分析表明了这一点。然而，生长激素细胞中AIP失活的生理意义仍不清楚。我们使用CRISPR/Cas9技术鉴定出一个Aip基因被破坏的GH3细胞克隆（称为GH3-FTY），随后研究了其在生长激素（Gh）合成和增殖方面的特性。与GH3细胞相比，GH3-FTY细胞的Gh分泌显著增加，细胞增殖略有增加。已知Gh诱导的Stat3磷酸化是GH3细胞中Gh分泌过多的一种机制。有趣的是，与GH3细胞相比，GH3-FTY细胞中磷酸化-Stat3的表达增加得更多，这表明在GH3-FTY细胞中这种机制的驱动更强。Aip的外源性表达显著逆转了GH3-FTY细胞在Gh过度产生、细胞增殖和Stat3磷酸化增加方面的表型。在抑制细胞增殖方面，GH3-FTY细胞对生长抑素的敏感性低于GH3细胞，这可能与生长抑素2型受体表达降低有关。BALB/c裸鼠中的GH3-FTY异种移植物（GH3-FTY小鼠）比GH3异种移植物（GH3小鼠）形成更多有丝分裂的生长激素细胞肿瘤，Ki67评分增加也证明了这一点。GH3-FTY小鼠也比GH3小鼠大得多，血浆Gh水平显著更高。此外，与GH3小鼠相比，GH3-FTY小鼠表现出相对胰岛素抵抗。总之，我们建立了一种生长激素细胞系GH3-FTY，其具有与Aip破坏相关的显著Gh分泌和有丝分裂特征。
