Ashton Charles, Rhie Suhn K, Carmichael John D, Zada Gabriel
Departments of1Neurosurgery.
2Biochemistry and Molecular Medicine, and.
J Neurosurg. 2020 Feb 28;134(3):787-793. doi: 10.3171/2019.12.JNS192435. Print 2021 Mar 1.
Prior profiling of the human pituitary adenoma (PA) DNA methylome showed the potassium channel subunit-encoding gene KCNAB2 to be highly differentially methylated between nonfunctional PAs (NFPAs) and growth hormone (GH)-secreting PAs, with greater KCNAB2 methylation detected in secretory PAs. KCNAB2 encodes an aldo-keto reductase that, among other things, negatively regulates members of the voltage-gated potassium channel (Kv) family. In this study, the authors aimed to determine whether modulation of Kcnab2 expression would alter GH secretion in the GH3 mammosomatotroph rat cell line. In addition, they examined whether dosing GH3 cells with the antiarrhythmic drug quinidine, a known inhibitor of Kv and voltage-gated sodium channels, would affect hormonal secretion.
Previously generated RNA-seq data were reanalyzed to compare KCNAB2 expression levels in human NFPAs and GH-secreting PAs. Kcnab2 was overexpressed in GH3 cells using plasmid transfection and knocked down using shRNA, with confirmation by quantitative polymerase chain reaction (qPCR). GH concentrations in cell culture supernatants collected 24 hours after cell seeding were measured using enzyme-linked immunosorbent assay (ELISA). Separately, quinidine was administered to GH3 cells at graduated doses. GH and prolactin concentrations in supernatants collected 48 hours after quinidine treatment were measured by fluorometric immunoassay.
Modulation of expression at the transcript level in GH3 cells resulted in proportionate changes in the expression of GH mRNA and secretion of GH peptide, as confirmed by qPCR and ELISA. Specifically, partial knockdown of Kcnab2 was associated with fewer GH RNA transcripts and less GH secretion compared with controls, while augmentation of Kcnab2 expression was associated with more GH transcripts and secretion than the controls. Administration of quinidine (≥ 50 µM) reduced both GH and prolactin secretion in a dose-dependent fashion (p ≤ 0.05).
GH secretion in a somatotroph cell line is partially dependent on KCNAB2 gene expression and may be mitigated in vitro by quinidine. These results collectively suggest a potential new target and pharmacological candidate to be considered in the development of clinical therapeutics for acromegaly.
先前对人垂体腺瘤（PA）DNA甲基化组的分析表明，钾通道亚基编码基因KCNAB2在无功能垂体腺瘤（NFPA）和生长激素（GH）分泌性垂体腺瘤之间存在高度差异甲基化，在分泌性垂体腺瘤中检测到更高的KCNAB2甲基化水平。KCNAB2编码一种醛糖酮还原酶，除其他功能外，它对电压门控钾通道（Kv）家族成员具有负调节作用。在本研究中，作者旨在确定Kcnab2表达的调节是否会改变GH3乳腺生长激素分泌大鼠细胞系中的GH分泌。此外，他们还研究了用抗心律失常药物奎尼丁（一种已知的Kv和电压门控钠通道抑制剂）处理GH3细胞是否会影响激素分泌。
重新分析先前生成的RNA测序数据，以比较人NFPA和GH分泌性垂体腺瘤中KCNAB2的表达水平。使用质粒转染在GH3细胞中过表达Kcnab2，并使用短发夹RNA（shRNA）敲低，通过定量聚合酶链反应（qPCR）进行验证。在细胞接种24小时后收集细胞培养上清液中的GH浓度，使用酶联免疫吸附测定（ELISA）进行测量。另外，以递增剂量向GH3细胞施用奎尼丁。在奎尼丁处理48小时后收集上清液中的GH和催乳素浓度，通过荧光免疫测定法进行测量。
如qPCR和ELISA所证实，GH3细胞中转录水平的表达调节导致GH mRNA表达和GH肽分泌成比例变化。具体而言，与对照相比，Kcnab2的部分敲低与更少的GH RNA转录本和更少的GH分泌相关，而Kcnab2表达的增加与比对照更多的GH转录本和分泌相关。施用奎尼丁（≥50μM）以剂量依赖性方式降低了GH和催乳素的分泌（p≤0.05）。
生长激素分泌细胞系中的GH分泌部分依赖于KCNAB2基因表达，并且在体外可能被奎尼丁减轻。这些结果共同表明，在肢端肥大症临床治疗药物开发中可能需要考虑一个潜在的新靶点和药理学候选物。
