Transcriptional Regulation of Indian Hedgehog Expression in Chondrocytes

描述（由申请方提供）：在软骨内骨形成过程中，骨生成和软骨生成是相互联系和高度协调的。然而，负责协调成骨和软骨形成的调节机制仍不清楚。遗传小鼠模型已经表明，Hedgehog（Hh）通路控制软骨形成（如PTHrP）和骨形成（如BMP 2）中的靶基因，对正常的软骨内骨形成至关重要。然而，尽管其明显的重要性，很少有人知道的监管机制，负责控制刺猬信号。该建议旨在提高对Hedgehog通路的转录控制的理解，特别是在软骨形成过程中的Ihh，以及它如何与骨形成相关。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期间，因此该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ATF 4是软骨形成过程中Hedgehog信号的主要调节因子，也可能是骨形成过程中的调节因子。此外，我们假设ATF 4是Ihh转录的直接调节因子。我们基于以下理由提出这一假设。1.我们已经发现，ATF 4对于成骨细胞分化和骨形成是必需的（Yang，et al.，2004年）。2. Atf 4-缺陷（Atf 4-/-）小鼠是侏儒，表明生长板软骨细胞的缺陷。3. Atf 4-/-生长板软骨细胞显示出几种异常，包括肥大矿化延迟，肥大区扩大，最明显的是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、PTH和PTH相关蛋白的受体，PTHrP）和PTHrP-/-肢体表型。4. ATF 4通过直接结合Ihh启动子的顺式元件反式激活Ihh。5. Ihh信号传导的激活恢复器官培养系统中Atf 4-/-肢的大小。为了验证我们的假设，即ATF 4是软骨形成中Hedgehog信号传导的调节因子，我们计划以下具体目标。1.确定ATF 4调控Ihh转录的分子机制。2.探讨ATF 4在体内对生长板软骨细胞增殖和分化的调控作用。3.证明Ihh是ATF 4在体内的直接转录靶点。公共卫生相关性。印度刺猬（Ihh）是一个研究充分的形态发生调节软骨和骨形成，骨骼发育和骨折修复过程中的两个过程之间的协调。令人惊讶的是，缺乏对Ihh本身调控的研究，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调查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.