Estrogen receptors and estrogen responsive genes in the pathogenesis of osteoporosis

骨质疏松症发病机制中的雌激素受体和雌激素反应基因

• 批准号: 12470219
• 负责人: INOUE Satoshi
• 金额: $ 8.58万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470219/
• 关键词: Osteoporosis; Estrogen; SNP; Osteoblast; Target gene; Receptor; エストロゲン受容体; トランスジェニックラット; DNAチップ; 性差; 応答遺伝子

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also ERbeta. We observed bone mineral density (BMD) of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E2) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E2, suggesting that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways. We then isolated primary osteoblasts derived from transgenic rats and from their wild-type littermates. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Besides, we identified estrogen responsive pathways including Efp and EBAG9 pathways. Especially, we have revealed the molecular mechanism of Efp action as ubiquitin ligase in the cell cycle control. Functional analyzes of these genes in osteoporosis are underway. We have also shown associations between BMD and each SNP in human genes involved in the bone metabolism such as VDP, IL-6, TNFalpa, Klotho, BNP, TNFR1, LRP5/6.

雌激素在维持骨密度和预防骨质疏松中起着重要作用，但雌激素通过骨中的雌激素受体（ER）发挥作用的潜在机制仍有待阐明。在本研究中，我们已经产生了大鼠窝藏一个显性负ER α，这不仅抑制ER α的行动，但也ER β。我们观察到，在卵巢切除后，即使给予17 β-雌二醇（E2），转基因雌性大鼠的骨密度（BMD）仍然下降，而相反，同窝仔BMD的下降被E2完全阻止，这表明雌激素预防卵巢切除引起的骨丢失是通过ER途径介导的。然后，我们从转基因大鼠及其野生型同窝仔中分离出原代成骨细胞。利用基因芯片分析发现，转基因大鼠成骨细胞中cyclin D2的mRNA水平较低。17 β-雌二醇处理的野生型成骨细胞中，D型细胞周期蛋白（包括细胞周期蛋白D2和细胞周期蛋白D3，但不包括细胞周期蛋白D1）的蛋白水平升高，导致细胞周期蛋白依赖性激酶4和6（Cdk 4/6）活性的激活和细胞生长的促进。此外，我们确定了雌激素反应途径，包括Efp和EBAG 9途径。特别是，我们揭示了Efp作为泛素连接酶在细胞周期调控中的分子机制。这些基因在骨质疏松症中的功能分析正在进行中。我们还显示了BMD与参与骨代谢的人类基因中的每个SNP之间的关联，例如VDP，IL-6，TNFalpa，Klotho，BNP，TNFR 1，LRP 5/6。

项目成果

Ogawa S, Emi M, Inoue S et al.: "Association of amino acid variation (Trp64Arg) in the beta3-adrenergic receptor gene with bone mineral density"Geriatric Gerontol Int. 2. 138-142 (2002)

Okawa S、Emi M、Inoue S 等：“β3-肾上腺素受体基因中的氨基酸变异 (Trp64Arg) 与骨矿物质密度的关联”Geriatric Gerontol Int.

Inoue S, Ogawa S, Horie K, Hoshino S, Goto W, Hosoi T, Tsutsumi O, Muramatsu M, Ouchi Y: "An estrogen receptor beta (ER beta) Isoform that lacks exon 5 has dominant negative activity on both ER alpha and ER beta"Biochem Biophys Res Commun. 279. 814-819 (2

Inoue S、Okawa S、Horie K、Hoshino S、Goto W、Hosoi T、Ttsutsumi O、Muramatsu M、Ouchi Y：“缺乏外显子 5 的雌激素受体 β (ER β) 异构体对 ER α 和 ER α 均具有显性负活性。

Ogawa S, Emi M, Shiraki M, Hosoi T, Orimo H, Ouchi Y, Inoue S: "Association of amino acid variation (Trp64Arg) in the beta3-adrenergic receptor gene with bone mineral density"Geriatric Gerontol Int. 2. 138-142 (2002)

Okawa S、Emi M、Shiraki M、Hosoi T、Orimo H、Ouchi Y、Inoue S：“β3-肾上腺素受体基因中的氨基酸变异 (Trp64Arg) 与骨矿物质密度的关联”Geriatric Gerontol Int。

Fujita M, Ogawa S, Fukuoka H, Tsukui T, Nemoto N, Tsutsumi O, Ouchi Y, Inoue S: "Differential expression of secreted frizzled related protein 4 (sFRP4) in decidual cells during pregnancy"J Mol Endocrinol. 28. 213-223 (2000)

Fujita M、Okawa S、Fukuoka H、Tsukui T、Nemoto N、Ttsutsumi O、Ouchi Y、Inoue S：“妊娠期间蜕膜细胞中分泌的卷曲相关蛋白 4 (sFRP4) 的差异表达”J Mol Endocrinol。

Fujita M, Urano T, Horie K, Ikeda K, Tsukui T, Fukuoka H, Tsutsui O, Ouchi Y, Inoue S: "Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts"Biochem Biophys Res Commun. 299. 222-228 (2002)

Fujita M、Urano T、Horie K、Ikeda K、Tsukui T、Fukuoka H、Ttsutsui O、Ouchi Y、Inoue S：“雌激素通过诱导大鼠原代成骨细胞中的细胞周期蛋白 D 激活细胞周期蛋白依赖性激酶 4 和 6”Biochem Biophys Res