Identification of Combinatorial Signaling Mechanisms that regulate CTGF Induction

描述（由申请人提供）： 全身或局部形式的骨丢失由多种疾病或病症引起，包括衰老，并且所产生的骨质减少导致许多临床表现，包括骨折发生率增加。新的骨合成代谢药物的鉴定，也许更重要的是，深入了解其作用机制，是临床上非常感兴趣的主题。CTGF最近已成为骨生成中的一个重要因素，我们最近证明了骨生长因子TGF-21是CTGF表达的有效诱导剂，并且CTGF是成骨细胞中TGF-21诱导的ECM产生的下游介质。控制TGF-21诱导CTGF的机制是细胞类型特异性的，并且对成骨细胞中的这种机制知之甚少。我们目前的初步数据表明，第一次的独特要求的两个启动子基序的转化生长因子-β反应元件（TRE）和Smad结合元件（SBE）的CTGF启动子的反式激活。这些结果导致我们的假设，在成骨细胞的TRE和SBE功能同源图案，这两个元素合作，以实现诱导CTGF表达的TGF-21。在成骨细胞中尚未研究与SBE、TRE或两者结合的核蛋白的身份，我们在目标1中建议研究这些蛋白，重点是需要这两种基序的蛋白，因为它们可能是成骨细胞所独有的。在我们的初步数据中，我们证明了成骨细胞中TGF-21上调CTGF需要Smad和Erk信号传导，并且我们还证明了转录因子Ets-1的表达与TGF-21协同诱导成骨细胞中CTGF启动子活化。MAPK如Erk可通过直接介导Smads的磷酸化（活化）或间接通过介导Smad DNA结合的所需核转录因子的活化/失活来调节Smad途径。我们的初步数据表明，Erk信号不是Smad激活所直接需要的，而是CTGF启动子上的SBE和TRE上的转录复合物形成所需要的，这表明Erk信号对SBE和TRE反式激活是重要的。已知Ets-1在某些细胞类型中作为Erk信号传导的下游介体起作用，并且最近Ets-1已显示以与Smad蛋白组合的方式起作用。在目的2中，我们提出研究Erk和Ets-1信号传导在成骨细胞中TGF-21诱导CTGF中的作用，特别是Erk是否易位到细胞核并且是SBE的Smad反式激活所需的，Ets-1在TGF-21诱导的CTGF中的作用，以及Ets-1是否是TGF-21诱导Erk介导的CTGF的下游效应物。鉴定和表征促进成骨细胞分化和功能的分子机制可能有助于开发新的治疗策略，以选择性地增强显著骨丢失患者的骨形成。 公共卫生相关性： 骨质疏松症是一个主要的卫生保健问题，因为大约有1000万50岁以上的人被诊断患有这种疾病，估计还有3360万人骨质疏松。低骨量伴随着骨折发生率的增加，据估计，与骨量减少有关的骨折的直接医疗保健费用（住院、急诊、医生就诊等）每年120亿至180亿美元。CTGF是一种新的骨生长因子，并且所提出的研究将产生关于成骨细胞中TGF-21对其调控的新信息。了解成骨细胞的生长控制机制，特别是TGF-21和CTGF调节成骨细胞分化和功能的机制的鉴定和表征，将有助于确定新的治疗靶点，以增强临床显著骨丢失患者的骨形成。

DESCRIPTION (provided by applicant): Systemic or localized forms of bone loss are caused by a variety of diseases or conditions, including aging, and the resulting osteopenia leads to numerous clinical manifestations including increased incidence of fracture. The identification of novel anabolic agents in bone, and perhaps even more importantly, gaining insights into their mechanisms of action, are subjects of intense clinical interest. CTGF has recently emerged as an important factor in osteogenesis and we recently demonstrated that the bone growth factor, TGF-21, is a potent inducer of CTGF expression and that CTGF is a downstream mediator of TGF-21-induced ECM production in osteoblasts. The mechanisms that govern CTGF induction by TGF-21 are cell type specific and little is known about this mechanism in osteoblasts. We present preliminary data that demonstrates for the first time the unique requirement of two promoter motifs the Transforming growth factor-beta response element (TRE) and the Smad binding element (SBE) for CTGF promoter trans-activation. These results led to our hypothesis that in osteoblasts the TRE and SBE function as cognate motifs and that both elements cooperate to achieve induction of CTGF expression by TGF-21. The identities of nuclear proteins that bind to the SBE, the TRE, or both have not been investigated in osteoblasts, and we propose in Aim 1 to investigate these proteins with a focus on proteins that require both motifs, since they may be unique to osteoblasts. In our preliminary data, we demonstrated the requirement of Smad and Erk signaling for TGF-21 up-regulation of CTGF in osteoblasts and we also demonstrate that expression of the transcription factor Ets-1 synergizes with TGF-21 to induce CTGF promoter activation in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation (activation) of Smads or indirectly through activation/ inactivation of required nuclear transcription factors that mediate Smad DNA binding. Our preliminary data demonstrates that Erk signaling is not required directly for Smad activation but is required for transcriptional complex formation on the SBE and TRE on the CTGF promoter, demonstrating that Erk signaling is important for SBE and TRE trans-activation. Ets-1 is known to function as a downstream mediator of Erk signaling in some cell types and recently Ets-1 has been shown to function in a combinatorial manner with Smad proteins. In Aim 2, we propose to investigate the role of Erk and Ets-1 signaling for CTGF induction by TGF-21 in osteoblasts, specifically if Erk translocates to the nucleus and is required for Smad trans-activation of the SBE, the role of Ets-1 in TGF-21 induced CTGF, and if Ets-1 is a downstream effector for Erk mediated CTGF induction by TGF-21. Identification and characterization of molecular mechanisms that work to promote osteoblast differentiation and function may be helpful in developing new therapeutic strategies to selectively enhance bone formation in patients with significant bone loss. PUBLIC HEALTH RELEVANCE: Osteoporosis is a major health care problem since approximately 10 million people over the age of 50 have been diagnosed with the disease and 33.6 million more are estimated to have low bone mass (osteopenia). Low bone mass is accompanied by an increased incidence of fracture, and it is estimated that the direct health care costs from fractures related to osteopenia (hospitalizations, ER visits, physician visits, etc.) ranges from $12-$18 billion annually. CTGF is a novel growth factor in bone and the proposed studies will generate new information regarding its regulation by TGF-21 in osteoblasts. Developing an understanding of the growth control mechanisms in osteoblasts, and particularly the identification and characterization of the mechanisms of TGF-21 and CTGF in regulating osteoblast differentiation and function, will help identify new therapeutic targets to enhance bone formation in patients with clinically significant bone loss.