Ras-association domain family 1 protein, RASSF1C, is an IGFBP-5 binding partner and a potential regulator of osteoblast cell proliferation
Ras-association domain family 1 protein, RASSF1C, is an IGFBP-5 binding partner and a potential regulator of osteoblast cell proliferation
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DOI：
10.1359/jbmr.050311
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发表时间：
2005-08-01
影响因子：
6.2
通讯作者：
Mohan, S
中科院分区：
文献类型：
--
作者：
Amaar, YG;Baylink, DJ;Mohan, S
简介：据预测，胰岛素样生长因子结合蛋白（IGFBP）-5的内在生长因子作用涉及IGFBP-5与推定受体的结合以诱导下游信号传导途径和/或IGFBP-5的细胞内易位以结合参与成骨细胞调节的潜在信号传导分子。本研究报告了亚型的表征。材料与方法：以IGFBP-5为诱饵，用酵母双杂交技术筛选人骨肉瘤cDNA文库，并与RASSF 1C基因进行同源性比较。RT-PCR和/或北方印迹法检测RASSF 1C的表达水平。通过免疫印迹分析评估IGFBP-5对ERK磷酸化的影响。结果：在高严谨度条件下，RASSF 1C siRNA与诱饵有较强的相互作用，其中一个克隆对应于RASSF 1C。RASSF 1C和IGFBP-5之间的相互作用通过体外免疫共沉淀研究证实。北方印迹和RT-PCR分析表明，RASSF 1C在多种产生IGFBP-5的成骨细胞类型中表达。添加合成的RASSF 1C特异性小干扰（si）RNA双链体或使用RASSF 1C特异性si-hairpin质粒导致细胞数量减少，并取消IGFBP-5诱导的细胞外信号调节激酶（ERK）-1/2磷酸化，但对IGFBP-5诱导的碱性磷酸酶（ALP）activity.Conclusions增加没有影响：我们已经显示了一种新的IGFBP-5和RASSF 1C之间的相互作用。我们的研究发现，RASSF 1C沉默导致成骨细胞增殖减少，IGFBP-5处理增加ERK-1/2磷酸化，这增加了RASSF 1C（Ras效应子）可能部分介导IGFBP-5对ERK磷酸化的影响，从而介导细胞增殖的可能性。
Introduction: It has been predicted that the intrinsic growth factor action of insulin-like growth factor binding protein (IGFBP)-5 involves either the binding of IGFBP-5 to a putative receptor to induce downstream signaling pathways and/or intracellular translocation of IGFBP-5 to bind to potential signaling molecules involved in osteoblast cell regulation. This study reports the characterization of isoform. C of the Ras association family 1 (RASSF1C) gene as an interacting partner of IGFBP-5.Materials and Methods: IGFBP-5 was used as bait in a yeast two-hybrid screen of a human osteosarcoma cDNA library. Expression levels of RASSF1C were measured by RT-PCR and/or Northern blot. IGFBP-5 effects on ERK phosphorylation were evaluated by immunoblot analysis. The effect of RASSF1C siRNA on cell proliferation was measured by the AlamarBlue assay.Results: One of the clones that interacted strongly with the bait under high stringency conditions corresponded to RASSF1C. The interaction between RASSF1C and IGFBP-5 was confirmed by in vitro coimmunoprecipitation studies. Northern blot and RT-PCR analysis showed that RASSF1C was expressed in a variety of osteoblast cell types that produce IGFBP-5. Addition of synthetic RASSF1C-specific small interfering (si) RNA duplex or use of a RASSF1C-specific si-hairpin plasmid caused a decrease in cell number and abolished IGFBP-5-induced extracellular signal-regulated kinase (ERK)-1/2 phosphorylation but had no effect on IGFBP-5-induced increases in alkaline phosphatase (ALP) activity.Conclusions: We have shown a novel interaction between IGFBP-5 and RASSF1C. Our findings that silencing of RASSF1C results in the reduction of osteoblast cell proliferation and that IGFBP-5 treatment increases phosphorylation of ERK-1/2 raise the possibility that RASSF1C, a Ras effector, could, in part, contribute to mediating the effects of IGFBP-5 on ERK phosphorylation and, consequently, cell proliferation.