Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.
Development and optimization of a cell-based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein-2 activity.
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DOI：
10.1002/cbf.1615
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发表时间：
2009-12
影响因子：
3.6
通讯作者：
Boden, Scott D.
中科院分区：
文献类型：
--
作者：
Okada, Motohiro;Sangadala, Sreedhara;Liu, Yunshan;Yoshida, Munehito;Reddy, Boojala Vijay B.;Titus, Louisa;Boden, Scott D.
重组人骨形态发生蛋白-2(BMP-2)的大量需求为其临床应用带来了巨大的翻译障碍。这导致迫切需要开发替代方法来降低使用BMP-2的成本和/或提高其效率。在这项研究中，我们描述了一种基于细胞的分析的发展和优化，该方法灵敏、可重复性和可靠地鉴定能增强BMP-2在诱导C2C12成肌细胞向成骨表型转分化中的作用的试剂。该检测是基于BMP反应的Smad1驱动的荧光素酶报告基因。LIM矿化蛋白-1(LMP-1)是本课题组发现的一种新的细胞内LIM结构域蛋白，可增强细胞对BMP-2的反应性。我们以前的报道阐明，LMP-1与Smad泛素调节因子-1(Smad Ubiquitin Regulating Factor-1，SMurf1)中的WW2结构域的结合可以使成骨Smads免于降解。在这里，我们首先使用优化的基于细胞的分析方法，评估了重组制备的蛋白质LMP-1及其缺乏SMurf1-WW2结构域结合基序的突变体(LMP-1ΔS-1)的活性。野生型和突变型蛋白都被设计成含有11个氨基酸的HIV-Tat蛋白衍生的膜转导结构域，以帮助重组蛋白的细胞递送。基于细胞的报告分析证实，LMP-1增强了BMP诱导的C2C12细胞向成骨细胞表型的刺激。当已知与SMurf1相互作用的特定基序发生突变时，LMP-1的增强作用显著降低。我们还通过监测骨钙素和碱性磷酸酶(ALP)的mRNA表达来验证报告实验中的结果，碱性磷酸酶是广泛接受的成骨细胞分化标志基因。最后，我们通过测量碱性磷酸酶的活性来进一步证实我们的结果，以支持我们基于细胞的检测的准确性和可靠性。合成蛋白质的直接传递可能受到高成本、不稳定或翻译后修饰不充分的限制。因此，一种低成本、可穿透细胞的化合物将有明显的好处。我们成功地使用了我们的基于基因表达的分析，从一组经过计算筛选确定为最有可能模拟LMP-1功能的化合物中选择了一种活性化合物。其中，我们选择了SVAK-3，这是一种在诱导C2C12细胞成骨分化方面显示出剂量依赖的BMP-2活性增强的化合物。我们发现，无论是全长LMP-1蛋白还是其潜在的模拟化合物，即使在并行监测成骨细胞表型的多个标记物时，BMP-2活性的增强作用也是相似的。
The requirement of large amounts of the recombinant human bone morphogenetic protein-2 (BMP-2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP-2. In this study, we describe the development and optimization of a cell-based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP-2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP-responsive Smad1-driven luciferase reporter gene. LIM mineralization protein-1 (LMP-1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP-2. Our previous report elucidated that the binding of LMP-1 with the WW2 domain in Smad ubiquitin regulatory factor-1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell-based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP-1, and its mutant (LMP-1ΔSmurf1) that lacks the Smurf1-WW2 domain-binding motif. Both the wild type and the mutant proteins were engineered to contain an 11-amino acid HIV-TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell-based reporter assay confirmed that LMP-1 potentiates the BMP-induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP-1 was significantly reduced when a specific-motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell-based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post-translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression-based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP-1. Among them, we selected SVAK-3, a compound that showed a dose-dependent potentiation of BMP-2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP-1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP-2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored.
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DOI：
10.1080/07391102.2007.10507151
发表时间：
2007-08-01
影响因子：
4.4
作者：
Sangadala, Sreedhara;Metpally, Raghu Prasad Rao;Reddy, Boojala Vijay B.
通讯作者：
Reddy, Boojala Vijay B.
影响因子：
3
作者：
Boden, SD;Schimandle, JH;Hutton, WC
通讯作者：
Hutton, WC
影响因子：
4.8
作者：
Boden, SD;Liu, YS;Titus, L
通讯作者：
Titus, L
影响因子：
3
作者：
Boden, SD;Kang, J;Heller, JG
通讯作者：
Heller, JG
影响因子：
6.2
作者：
Liu, YS;Hair, GA;Titus, L
通讯作者：
Titus, L