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1．神経科学シリーズ cDNA full-length cloneの導入およびsiRNA法を用いた解析による培養下垂体腺腫細胞におけるシグナル伝達経路の研究（5）
吉田 大蔵1, 寺本 明2
1日本医科大学武蔵小杉病院脳神経外科
2日本医科大学大学院医学研究科神経病態解析学
1. Neuroscience Series Investigation of Signaling Pathways in Cultured Pituitary Adenoma Cell by Full-length Clone Transfection and Gene Silencing with the Small Interfering RNA Method (5)
Daizo Yoshida1 and Akira Teramoto2
1Department of Neurosurgery, Nippon Medical School Musashi Kosugi Hospital
2Department of Clinical Neuroscience, Graduate School of Medicine, Nippon Medical School
Recently, complementary (c) DNA full-length clone transfection and gene silencing with the small interfering (si) RNA method have emerged as new methods for elucidating intracellular signaling pathways in cultured cell lines. However, these methods have rarely been used in the field of neuroendocrinology. Recent reports have shown the presence of a novel matrix metalloproteinase (MMP)-inhibiting cell membrane-anchored glycoprotein designated "reversion-inducing cysteine-rich protein with Kazal motifs" (RECK). The purpose of this study was to elucidate the role of RECK in the cell invasion of pituitary adenomas and its contribution to signal transduction. The function of RECK in cell invasion was investigated by comparing data obtained from full-length RECK clone transfection and gene silencing with RECK messenger RNA-targeting siRNA. RECK expression was confirmed by means of real-time reverse transcription polymerase chain reaction and Western blotting. Levels of MMP-2 and MMP-9 and of tissue inhibitor of metalloproteinases-1 were measured with zymography and reverse zymography, respectively. Cell invasion was examined with a 3-dimensional invasion assay. The signal cascade was investigated with cDNA microarray analysis. As expected, expression of RECK was elevated upon cDNA transfection and decreased with siRNA. We observed elevations of MMP-2 and MMP-9 expression and consequent 3-dimensional cell invasion in cells under-expressing RECK. However, expression of tissue inhibitor of metalloproteinases was not affected by RECK. cDNA microarray analysis revealed that RECK additionally up-regulates growth hormone-releasing hormone receptor and latrophilin 2 at the transcriptional level. Our findings collectively suggest that RECK regulates the cell-signalling pathway and plays a critical neuroendocrinological role in the pituitary adenoma cell line.
日医大医会誌 2008; 4(4), 201-204
Key words
growth hormone releasing hormone receptor, matrix metalloproteinase, pituitary adenoma, reversion-inducing cysteine-rich protein with kazal motifs
Correspondence to
Akira Teramoto, MD, PhD, Department of Clinical Neuroscience, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan
E-mail：a-tera@nms.ac.jp
受付：2008年7月31日 受理：2008年8月19日 |
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