Molecular Control of MSC differentiation and Bone Formation by KDM4B

描述(申请人提供)：本申请的长期目标是了解分子和表观遗传学机制如何控制间充质干细胞/基质细胞(MSCs)的成骨分化、成骨细胞功能和骨形成。MSCs是具有自我更新能力和多向分化潜能的多潜能前体细胞，包括成骨、软骨生成和脂肪生成。虽然在理解MSC分化的转录调控方面已经取得了重大进展，但关于骨形成是如何表观遗传调控的知之甚少。组蛋白甲基化是一个重要的过程，与基因表达的激活和抑制有关，因此它在细胞分化的表观遗传调控中起着关键作用。虽然越来越多的证据表明，组蛋白去甲基酶在表观遗传学上调节胚胎干细胞的性质和功能，但在很大程度上还不清楚去甲基酶对骨髓间充质干细胞分化和骨形成的影响。为了探讨去甲基酶在MSC分化中的作用，我们系统地研究了BMP刺激的骨髓间充质干细胞中组蛋白去甲基酶的表达，因为BMP是有效的成骨分化诱导剂。我们发现，BMPs能迅速诱导赖氨酸(K)特异性去甲基酶(KDM4B；也称为JMJD2B)的表达，该酶可使组蛋白H3在赖氨酸9(H3K9me3)处去甲基化。H3K9me3是参与生长发育的基因沉默的标志。通常，当干细胞分化程序被触发时，一组特定的基因被激活。我们的初步研究表明，KDM4B促进了MSCs的成骨分化，而抑制了成脂分化。此外，我们发现Kdm4b在老年小鼠分离的MSCs中的表达明显低于年轻小鼠。巧合的是，H3K9me3标记在衰老小鼠或去卵巢小鼠的成骨细胞中显著增加。基于这些新发现，在这一应用中，我们假设KDM4B清除H3K9me3标记在MSCs体外成骨分化和体内成骨过程中起着不可或缺的作用。 为了检验我们的假设，本文提出了三个具体目标。目的1是确定KDM4B是否通过诱导DLX5对MSC谱系承诺进行表观遗传调控。目的2探讨KDM4B清除H3K9me3对骨髓间充质干细胞成骨分化的协同调节作用。目的3是确定KDM4B是否是体内骨形成所必需的，以及KDM4B的失调是否会损害骨质疏松时的成骨细胞功能和骨形成。由于组蛋白去甲基酶在化学上是可修饰的，KDM4B可能成为再生医学中特异性控制MSCs分化的新的治疗靶点，也为骨质疏松症等代谢性骨病的新治疗提供线索。

DESCRIPTION (provided by applicant): The long-term objectives of this application are to understand how molecular and epigenetic mechanisms control osteogenic differentiation of mesenchymal stem/stromal cells (MSCs), osteoblast function, and bone formation. MSCs are multipotent progenitor cells with self-renewal capabilities and multilineage differentiation potentials including osteogenesis, chondrogenesis and adipogenesis. Although significant progress has been made in understanding transcriptional control of MSC differentiation, little is known about how bone formation is epigenetically regulated. Histone methylation is an important process linked to the activation and repression of gene expression, thus it plays a critical role in epigenetic regulation of cell differentiation. While growing evidence indicates tht histone demethylases epigenetically regulate embryonic stem cell properties and functions, it is largely unknown what affect demethylases have on MSC differentiation and bone formation. To explore the role of demethylases in MSC differentiation, we systemically profiled the expression of histone demethylases in BMP-stimulated MSCs from bone marrow, as BMPs are potent inducers of osteogenic differentiation. We found that BMPs rapidly induced the expression of the lysine (K)-specific demethylase (KDM4B; also known as JMJD2B) that demethylates trimethylated histone H3 at lysine 9 (H3K9me3). H3K9me3 is a hallmark for gene silencing involved in growth and development. In general, a group of specific genes are activated when the stem cell differentiation program is triggered. Our preliminary studies demonstrated that KDM4B promoted osteogenic differentiation of MSCs while inhibiting adipogenic differentiation. Moreover, we found that the expression of Kdm4b was significantly down regulated in MSCs isolated from aging mice compared to young mice. Co-incidentally, H3K9me3 marks were significantly increased in osteoblasts of aging mice or ovariectomized mice. Based on these novel discoveries, in this application, we hypothesize that erasing H3K9me3 marks by KDM4B plays integral roles in osteogenic differentiation of MSCs in vitro and bone formation and in vivo. Three specific aims are proposed to test our hypothesis. Aim 1 is to determine whether KDM4B epigenetically regulate MSC lineage commitment through induction of DLX5. Aim 2 is to explore how erasing H3K9me3 by KDM4B coordinately regulates osteogenic differentiation of MSCs. Aim 3 is to determine whether KDM4B is required for bone formation in vivo and whether dysregulation of KDM4B impairs osteoblast function and bone formation in osteoporosis. Since histone demethylases are chemically modifiable, KDM4B may present as a novel therapeutic target for specifically controlling the differentiation of MSCs in regenerative medicine, and also lead to clues for new treatment in metabolic bone diseases such as, osteoporosis.