TGFbeta inducible early gene-1 directly binds to, and represses, the OPG promoter in osteoblasts.
TGFbeta inducible early gene-1 directly binds to, and represses, the OPG promoter in osteoblasts.
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DOI：
10.1016/j.bbrc.2009.12.171
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发表时间：
2010-01-29
影响因子：
3.1
通讯作者：
Spelsberg, T. C.
中科院分区：
文献类型：
--
作者：
Subramaniam, M.;Hawse, J. R.;Bruinsma, E. S.;Grygo, S. B.;Cicek, M.;Oursler, M. J.;Spelsberg, T. C.
关键词：
TGFβ诱导早期基因1（TIEG）是Krüppel样转录因子家族（KLF 10）的成员，在TGFβ介导的Smad信号转导中起重要作用。为了更好地理解TIEG在骨中的作用，我们产生了TIEG敲除（KO）小鼠。从这些小鼠中分离的颅骨成骨细胞（OB）在体外表现出支持破骨细胞生成的能力降低。基因表达研究显示，TIEG KO OB中NF-κ B配体受体激活因子（RANKL）表达降低，骨保护素（OPG）表达增加，表明TIEG在调节这些基因表达中的潜在作用。由于OPG和RANKL是破骨细胞（OC）分化的两个重要调节因子，我们试图确定TIEG是否直接调节其表达。将含有小鼠OPG启动子（−1486至+133 bp）或RANKL启动子（−2000至+1 bp）片段的荧光素酶构建体分别克隆到pGL 3基本报告载体中，并在有和没有TIEG表达载体的情况下瞬时转染到TIEG KO颅骨OB中。在存在TIEG的情况下，未检测到RANKL启动子活性的显著变化。然而，OPG启动子活性在TIEG蛋白的存在下被抑制，这表明TIEG直接抑制OPG在OB中的表达。为了确定TIEG通过其起作用的该启动子的区域，产生连续的5′-缺失构建体。这些构建体的瞬时转染揭示了TIEG调控元件位于OPG启动子的200 bp区域内。使用TIEG特异性抗体的瞬时ChIP分析显示，TIEG结合到OPG启动子的该区域。由于我们以前已经表明，TIEG通过Sp-1位点调节靶基因表达，我们检查了OPG启动子的这一区域的潜在TIEG结合元件，并确定了四个潜在的Sp-1结合位点。定点诱变用于确定TIEG是否利用这些Sp-1元件来调节OPG启动子的活性。数据表明，两个Sp-1位点可能参与TIEG对OPG启动子的抑制。总之，这些结果证实了TIEG直接结合并抑制OB中的OPG启动子活性，部分解释了TIEG KO OB不能完全支持破骨细胞分化。
TGFβ Inducible Early Gene-1 (TIEG) is a member of the Krüppel-like family of transcription factors (KLF10) that plays an important role in TGFβ mediated Smad signaling. In order to better understand the role of TIEG in bone, we generated TIEG knockout (KO) mice. Calvarial osteoblasts (OBs) isolated from these mice exhibit a reduced ability to support osteoclastogenesis in vitro. Gene expression studies revealed decreased receptor activator of NF-kB ligand (RANKL) and increased osteoprotegerin (OPG) expression in TIEG KO OBs, suggesting a potential role for TIEG in regulating the expression of these genes. Since OPG and RANKL are two important regulators of osteoclast (OC) differentiation, we sought to determine if TIEG directly regulates their expression. Luciferase constructs, containing fragments of either the mouse OPG promoter (−1486 to +133 bp) or the RANKL promoter (−2000 to +1 bp) were each cloned into the pGL3 basic reporter vector and transiently transfected into TIEG KO calvarial OBs with and without a TIEG expression vector. No significant changes in the activity of the RANKL promoter were detected in the presence of TIEG. However, OPG promoter activity was inhibited in the presence of TIEG protein suggesting that TIEG directly represses the expression of OPG in OBs. In order to determine the region of this promoter through which TIEG acts, sequential 5′-deletion constructs were generated. Transient transfection of these constructs revealed that the TIEG regulatory element(s) reside within a 200 bp region of the OPG promoter. Transient ChIP analyses, using a TIEG-specific antibody, revealed that TIEG binds to this region of the OPG promoter. Since we have previously shown that TIEG regulates target gene expression through Sp-1 sites, we examined this region of the OPG promoter for potential TIEG binding elements and identified four potential Sp-1 binding sites. Site directed mutagenesis was used to determine if TIEG utilizes these Sp-1 elements to regulate the activity of the OPG promoter. The data demonstrate that two Sp-1 sites are likely to be involved in TIEG’s repression of the OPG promoter. Taken together, these results confirm that TIEG directly binds to and inhibits OPG promoter activity in OBs, partially explaining the inability of TIEG KO OBs to fully support osteoclast differentiation.
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影响因子：
4.1
作者：
Bensamoun, Sabine F.;Hawse, John R.;Spelsberg, Thomas C.
通讯作者：
Spelsberg, Thomas C.
影响因子：
4.8
作者：
Alvarez-Rodiguez, Ruben;Barzi, Mercedes;Pons, Sebastian
通讯作者：
Pons, Sebastian
影响因子：
4
作者：
Mezquita-Raya, P;de la Higuera, M;Muñoz-Torres, M
通讯作者：
Muñoz-Torres, M
影响因子：
--
作者：
Hawse, John R.;Subramaniam, Malayannan;Spelsberg, Thomas C.
通讯作者：
Spelsberg, Thomas C.
影响因子：
4
作者：
Sundaram, Kumaran;Mani, Santhosh K.;Reddy, Salkamuri V.
通讯作者：
Reddy, Salkamuri V.