Transcriptional Regulation of Indian Hedgehog Expression in Chondrocytes

描述(申请人提供)：在软骨内骨形成过程中，成骨和软骨生成是紧密联系和高度协调的。然而，负责协调成骨和软骨形成的调控机制仍不清楚。小鼠遗传模型表明，Hedgehog(HH)途径控制着软骨形成中的靶基因，如PTHrP和BMP2，对于正常的软骨内骨形成是必不可少的。然而，尽管它的重要性显而易见，但人们对负责控制Hedgehog信号的调控机制知之甚少。这项建议旨在提高对Hedgehog途径，特别是IHH在软骨形成过程中的转录控制以及它可能如何与成骨联系的理解。由于软骨细胞分化也发生在软骨退化的早期阶段以及骨再生过程中，因此这项研究直接适用于骨关节炎和骨折等疾病。我们假设转录因子ATF4在软骨形成过程中是Hedgehog信号的主要调节因子，可能在成骨过程中也是如此。此外，我们假设ATF4是IHH转录的直接调节因子。我们将这一假设建立在以下理论基础之上。1.我们发现ATF4是成骨细胞分化和骨形成所必需的(Yang，et al.，2004)。2.ATF4基因缺陷(ATF4-/-)小鼠为侏儒，提示生长板软骨细胞存在缺陷。3.ATF4-/-生长板软骨细胞表现出几种异常，包括肥大矿化延迟，肥大带扩张，最显著的是IHH基因表达和增殖减少。这些缺陷使人联想到IHH-/-、PPR-/-(甲状旁腺激素和甲状旁腺激素相关蛋白受体，PTHrP)和PTHrP-/-肢体表型。4.ATF4通过直接与IHH启动子的顺式元件结合反式激活IHH。5.在器官培养系统中，激活IHH信号可恢复In ATF4-/-肢体的大小。为了验证我们的假设，即ATF4是Hedgehog信号在软骨形成中的调节因子，我们计划了以下具体目标。1.确定ATF4调控IHH转录的分子机制。2.探讨ATF4在体内对生长板软骨细胞增殖和分化的调节作用。3.证实IHH是ATF4在体内的直接转录靶点。与公共卫生相关。印度刺猬(India Hedgehog，IHH)是一种研究较多的形态原，它调节软骨形成和成骨之间的协调，这是骨骼发育和骨折修复过程中的两个过程。令人惊讶的是，对IHH本身的调控缺乏研究，特别是在转录水平上。在这项建议中，我们打算通过系统的分子和遗传学研究来研究这一重要的生物学问题。

Description (provided by applicant): During endochondral bone formation, osteogenesis and chondrogenesis are linked and highly coordinated. However, the regulatory mechanisms responsible for coordinating osteogenesis and chondrogenesis remain unclear. Genetic mouse models have shown that the Hedgehog (Hh) pathway, which controls target genes in chondrogenesis such as PTHrP and osteogenesis such as BMP2, is essential for normal endochondral bone formation. However, despite its obvious importance, little is known of the regulatory mechanisms that are responsible for controlling Hedgehog signaling. This proposal is directed at improving understanding of the transcriptional control of the Hedgehog pathway, and specifically of Ihh, during chondrogenesis and how it may be linked to osteogenesis. Since chondrocyte differentiation occurs also at early stages of cartilage degeneration as well as during bone regeneration, this study is directly applicable to diseases such as osteoarthritis and fractures. We hypothesize that the transcription factor ATF4 is a major regulator of Hedgehog signaling during chondrogenesis, and possibly during osteogenesis as well. Further, we hypothesize that ATF4 is a direct regulator of Ihh transcription. We base this hypothesis on the following rationale. 1. We have found that ATF4 is essential for osteoblast differentiation and bone formation (Yang, et al., 2004). 2. Atf4-deficient (Atf4-/-) mice are dwarfs, suggesting a defect in growth plate chondrocytes. 3. Atf4-/- growth plate chondrocytes show several abnormalities including a delay in hypertrophic mineralization, an expansion of the hypertrophic zone, and most notably a decrease in Ihh gene expression and proliferation. These defects are reminiscent of the Ihh-/-, PPR-/-(receptor for parathyroid hormone, PTH, and PTH related protein, PTHrP), and PTHrP-/- limb phenotypes. 4. ATF4 transactivates Ihh through directly binding to a cis element of the Ihh promoter. 5. Activation of Ihh signaling restores the size of in Atf4-/- limb in an organ culture system. To test our hypothesis that ATF4 is a regulatory factor for Hedgehog signaling in chondrogenesis, we plan the following specific aims. 1. To determine the molecular mechanisms whereby ATF4 regulates Ihh transcription. 2. To address the role of ATF4 in regulating growth plate chondrocytes proliferation and differentiation in vivo. 3. To demonstrate that Ihh is a direct transcriptional target of ATF4 in vivo. PUBLIC HEALTH RELEVANCE. Indian hedgehog (Ihh) is a well-studied morphogen that regulates the coordination between chondrogenesis and osteogenesis, two processes during skeletal development and bone fracture repair. Surprisingly, investigations on the regulation of Ihh itself are lacking, particularly at the transcriptional level. In this proposal, we intend to investigate this important biological question through systematic molecular and genetic studies.