Role of Zfp521 in bone formation and anabolic responses

描述（由申请人提供）：维持骨量对于预防老年人骨质疏松症和骨折至关重要。除了抗吸收疗法，唯一批准的合成代谢疗法是间歇性PTH，其作用机制仍不完全清楚。我们已经鉴定了Zfp 521，一种含有30个锌指的蛋白质，作为有助于骨形成的转录过程中的重要参与者（2）。Zfp 521在培养的成骨细胞（OBs）中的表达随时间增加，并且受BMP-2和PTH/PTHrP调节。在体内，Zfp 521在转基因小鼠的成熟OB中的靶向过表达导致骨形成增加（2）。相反，成骨细胞靶向的Zfp 521-/-小鼠是骨质减少的，具有受损的OB成熟和减少的骨形成。此外，Zfp 521结合Runx 2和Ebf 1并抑制它们的转录活性以及Rankl启动子上CREB的转录活性，从而抑制OB诱导的骨吸收。因此，我们的初步数据显示，Zfp 521 1）与几种关键的OB转录因子相互作用，2）响应骨形成的几种关键调节因子，以及3）当在小鼠体内过表达或缺失时影响骨形成和骨量。这确立了Zfp 521作为调节骨形成和体内平衡的新的重要贡献者，值得进一步探索。因此，我们建议进一步探讨Zfp 521影响成骨细胞分化和骨稳态的机制，重点是它与Runx 2，Ebf 1和CREB的相互作用。由于我们已经生成了重要的初步数据，并且已经生成了本次调查所需的大部分遗传模型，（OG2-Zfp521; 2.3kb-Col1a1-Zfp521;全局Zfp 521-/-和浮动Zfp 521，以及OC-Cre-Zfp 521和Osx-Cre-Zfp 521线，Ebf 1-/-和浮动Ebf 1，2. 3 kb-Col 1a 1-Ebf 1），本研究计划的具体目的是：目的1：进一步分析Zfp 521的完全和OB靶向条件性、阶段特异性缺失对体内和体外OB分化和骨形成的影响;目的2：进一步表征Zfp 521对OB中的转录因子发挥作用的分子机制，从而影响骨形成，特别强调了它与Runx 2、Ebf 1和CREB在靶基因上的相互作用。目标3：通过全基因组筛选确定Zfp 521新的合作伙伴，结合位点和靶基因这一新发现的骨形成辅助调节因子的功能特性将促进我们对调节骨稳态的关键生理过程的理解，最终允许探索骨质疏松症和其他低骨量综合征药物发现的新途径。 公共卫生相关性：维持骨量对于预防老年人骨质疏松症和骨折至关重要，除了抗再吸收疗法外，唯一批准的合成代谢疗法是甲状旁腺激素（PTH），其作用机制仍不完全清楚。我们已经确定了Zfp 521，一种富含锌指的蛋白质，作为骨形成和骨稳态的重要参与者，并建议探索Zfp 521影响骨密度的机制，研究细胞和基因工程小鼠。这种新的骨形成调节剂的功能特性可能允许发现新的途径，用于骨质疏松症和其他低骨量综合征的合成代谢药物的发现。

DESCRIPTION (provided by applicant): The maintenance of bone mass is essential for the prevention of osteoporosis and fractures in the elderly. Besides anti-resorptive therapies, the only approved anabolic therapy is intermittent PTH, the mechanism of action of which is still incompletely understood. We have identified Zfp521, a 30 zinc-finger containing protein, as a prominent player in the transcriptional processes that contribute to bone formation (2). Zfp521 expression in cultured osteoblasts (OBs) increases with time, and is regulated by BMP-2 and PTH/PTHrP. In vivo, targeted over-expression of Zfp521 in mature OBs in transgenic mice resulted in an increase in bone formation (2). In contrast, osteoblast-targeted Zfp521-/- mice are osteopenic with impaired maturation of OBs, and decreased bone formation. Furthermore Zfp521 binds Runx2 and Ebf1 and repress their transcriptional activity as well as that of CREB on the Rankl promoter, repressing OB-induced bone resorption. Thus, our preliminary data shows that Zfp521 1) interacts with several key OB transcription factors, 2) responds to several key regulators of bone formation and 3) affects bone formation and bone mass when over-expressed or deleted in vivo in mice. This establishes Zfp521 as a novel and important contributor to the regulation of bone formation and homeostasis, and one that deserves further exploration. Accordingly, we propose to further explore the mechanism by which Zfp521 affects OB differentiation and bone homeostasis, focusing on its interplay with Runx2, Ebf1 and CREB. Since we have generated significant preliminary data and have already generated most of the genetic models required for this investigation (OG2-Zfp521; 2.3kb-Col1a1-Zfp521; both global Zfp521-/- and floxed Zfp521, as well as the OC- Cre-Zfp521 and Osx-Cre-Zfp521 lines, Ebf1 -/- and floxed Ebf1, 2.3kb-Col1a1-Ebf1), the specific aims of this research proposal are: Aim1: Further analyze the effects of full and OB-targeted conditional, stage-specific deletion of Zfp521 on OB differentiation and bone formation in vivo and in vitro; Aim2: Further characterize the molecular mechanisms by which Zfp521 exerts its effects on transcription factors in OBs, affecting bone formation, with particular emphasis on its interplay with Runx2 , Ebf1 and CREB on target genes. Aim3: Identify Zfp521 new partners, binding sites and target genes by genome-wide screening Functional characterization of this newly discovered co-regulator of bone formation will advance our understanding of key physiological processes that regulate bone homeostasis, ultimately allowing the exploration of novel pathways for drug discovery in osteoporosis and other low bone mass syndromes. PUBLIC HEALTH RELEVANCE: The maintenance of bone mass is essential for the prevention of osteoporosis and fractures in the elderly and aside from anti-resorptive therapies, the only approved anabolic therapy is Parathyroid Hormone (PTH), the mechanism of action of which is still incompletely understood. We have identified Zfp521, a protein rich in Zinc Fingers, as a prominent player in bone formation and bone homeostasis and propose to explore the mechanism by which Zfp521 affects bone density, studying cells and genetically engineered mice. Functional characterization of this new regulator of bone formation may allow the discovery of novel pathways for anabolic drug discovery in osteoporosis and other low bone mass syndromes.