Sarkar D, Imai T, Kambe F, Shibata A, Ohmori S, Siddiq A, Hayasaka S, Funahashi H, Seo H
Department of Endocrinology and Metabolism, and Genetics Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
J Clin Endocrinol Metab. 2001 Nov;86(11):5130-7. doi: 10.1210/jcem.86.11.8032.
To elucidate the molecular mechanism of the pathogenesis of benign functioning adrenocortical adenomas causing Cushing's syndrome, we employed suppression PCR-based cDNA subtractive hybridization to identify novel genes that are differentially expressed in the adenoma. In this report we describe the adenoma-specific overexpression of the human homolog of the Diminuto/Dwarf1 (hDiminuto) gene. Northern blot analysis revealed that hDiminuto mRNA was overexpressed in the adenoma tissue of 14 patients with Cushing's syndrome in comparison to the adjacent nontumorous adrenal gland. In situ hybridization using hDiminuto cRNA probe showed its abundant expression in the tumor cells, whereas the nontumorous cells showed a low level of expression. As the atrophic adjacent gland may not represent the normal architecture, we examined the expression pattern of hDiminuto mRNA in normal human adrenal cortex. In situ hybridization revealed that it was expressed in all layers of the normal adrenal cortex. In situ apoptosis detection by the TUNEL method revealed that a low level of hDiminuto expression in the atrophic, adjacent gland was associated with numerous TUNEL-positive cells in all layers of cortex. In contrast almost no apoptotic cell was detected in the tumor or in the normal adrenal cortex where hDiminuto expression was abundant. These results are compatible with a recent report that hDiminuto acts as an antiapoptotic factor in neurons. The expression of hDiminuto in the normal adrenal cortex was most abundant in the zona fasciculata, suggesting its possible regulation by ACTH/cAMP. Indeed, forskolin treatment of H295R human adrenocortical cells resulted in a significant induction of the mRNA in a time- and dose-dependent manner. To further demonstrate the physiological regulation, an in vivo experiment was carried out in dexamethasone-treated rats. ACTH administration to these rats increased the mRNA expression. These results led us to speculate that the overexpression of hDiminuto in the adenoma could be due to the abundant expression of ACTH receptor, as we previously described. Diminuto is involved in steroid synthesis and cell elongation in plants. We, therefore, hypothesize that hDiminuto might be involved in the molecular events of adrenocortical tumorigenesis by facilitating steroid synthesis and cell growth.
为阐明导致库欣综合征的良性功能性肾上腺皮质腺瘤发病机制的分子机制，我们采用基于抑制性PCR的cDNA消减杂交技术来鉴定腺瘤中差异表达的新基因。在本报告中，我们描述了Diminuto/Dwarf1（hDiminuto）基因人类同源物在腺瘤中的特异性过表达。Northern印迹分析显示，与相邻的非肿瘤性肾上腺相比，hDiminuto mRNA在14例库欣综合征患者的腺瘤组织中过表达。使用hDiminuto cRNA探针进行原位杂交显示其在肿瘤细胞中大量表达，而非肿瘤细胞表达水平较低。由于萎缩的相邻腺体可能不代表正常结构，我们检测了hDiminuto mRNA在正常人肾上腺皮质中的表达模式。原位杂交显示它在正常肾上腺皮质的所有层中均有表达。通过TUNEL法进行的原位凋亡检测显示，萎缩的相邻腺体中hDiminuto低水平表达与皮质各层中大量TUNEL阳性细胞相关。相反，在肿瘤或hDiminuto表达丰富的正常肾上腺皮质中几乎未检测到凋亡细胞。这些结果与最近一份关于hDiminuto在神经元中作为抗凋亡因子的报告一致。hDiminuto在正常肾上腺皮质中的表达在束状带最为丰富，提示其可能受促肾上腺皮质激素/环磷酸腺苷（ACTH/cAMP）调节。事实上，用福司可林处理H295R人肾上腺皮质细胞导致mRNA以时间和剂量依赖性方式显著诱导。为进一步证明其生理调节作用，在接受地塞米松治疗的大鼠中进行了体内实验。对这些大鼠给予ACTH可增加mRNA表达。这些结果使我们推测，如我们之前所描述，腺瘤中hDiminuto的过表达可能是由于ACTH受体的大量表达。Diminuto参与植物中的类固醇合成和细胞伸长。因此，我们假设hDiminuto可能通过促进类固醇合成和细胞生长参与肾上腺皮质肿瘤发生的分子事件。
