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是由各种疾病或条件引起的，包括衰老，由此导致的骨量减少会导致许多临床表现，包括骨折发生率的增加。鉴定骨骼中的新型合成代谢制剂，也许更重要的是，深入了解它们的作用机制，是临床上非常感兴趣的课题。CTGF是新近出现的一种重要的成骨因子，我们最近证实，骨生长因子--转化生长因子-21是CTGF表达的有力诱导者，CTGF是转化生长因子-21诱导的成骨细胞产生细胞外基质的下游介质。转化生长因子-21诱导CTGF的机制是细胞类型特异性的，对成骨细胞的这一机制知之甚少。我们提供的初步数据首次证明了CTGF启动子反式激活需要两个启动子基序：转化生长因子-β反应元件(TrE)和Smad结合元件(SBE)。这些结果导致了我们的假设，即在成骨细胞中，TrE和SBE作为同源基序发挥作用，这两个元件协同作用，实现了转化生长因子-21诱导CTGF的表达。在成骨细胞中，结合SBE和/或TRE的核蛋白的特性还没有被研究，我们在目标1中建议研究这些蛋白质，重点是需要这两个基序的蛋白质，因为它们可能是成骨细胞所独有的。在我们的初步数据中，我们证实了Smad和Erk信号对成骨细胞中CTGF上调的需求，我们还证明了转录因子Ets-1的表达与转化生长因子21协同诱导成骨细胞中CTGF启动子的激活。ERK等MAPK可以通过直接介导Smads的磷酸化(激活)或间接通过激活/失活介导Smad DNA结合所需的核转录因子来调节Smad途径。我们的初步数据表明，ERK信号不是Smad激活直接所必需的，而是SBE和CTGF启动子上TrE转录复合体形成所必需的，表明Erk信号对SBE和TrE反式激活是重要的。Ets-1在某些细胞类型中作为ERK信号的下游介体发挥作用，最近研究表明Ets-1与Smad蛋白结合发挥作用。在目的2中，我们建议研究ERK和Ets-1信号在转化生长因子-21诱导的成骨细胞CTGF中的作用，特别是如果ERK转位到细胞核并且是Smad反式激活SBE所必需的，Ets-1在转化生长因子-21诱导的CTGF中的作用，以及Ets-1是否是ERK介导的转化生长因子的下游效应因子。识别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，选择性地促进明显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岁以上的人被诊断出患有这种疾病，估计还有3360万人患有低骨量(骨量减少)。低骨量伴随着骨折发生率的增加，据估计，与骨量减少相关的骨折的直接医疗费用(住院、急诊室就诊、医生就诊等)。每年从120亿至180亿美元不等。CTGF是一种新的骨生长因子，目前的研究将为其在成骨细胞中的调控提供新的信息。了解成骨细胞的生长控制机制，特别是对转化生长因子-21和结缔组织生长因子调控成骨细胞分化和功能的机制的识别和表征，将有助于确定新的治疗靶点，以促进临床上有意义的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.