Cartilage and Bone Development and Disease

软骨和骨骼发育与疾病

• 批准号: 8929667
• 负责人: Yoshihiko Yamada
• 金额: $ 58.61万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8929667
• 关键词: ATP Receptors; Adherens Junction; Adhesions; Apical Ectodermal Ridge; Atherosclerosis; BMP2 gene; Bone Development; CREB1 gene; Calmodulin; Calvaria; Cartilage; Cell Culture Techniques; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cells; Chondrocytes; Collaborations; Complex; Connective Tissue; Connexins; Cues; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Development; Disease; Distal; Dose; Ectoderm; Endoplasmic Reticulum; Environment; Exhibits; Extracellular Matrix; Extracellular Space; Family; Gap Junctions; Gene Targeting; Genes; Genetic Transcription; Goals; Growth and Development function; Hereditary Disease; Homeostasis; Human Genetics; Hypertension; Joints; Knockout Mice; Limb Bud; Limb Development; Limb structure; Link; Malignant Neoplasms; Mechanics; Mediating; Mediator of activation protein; Mesenchyme; Modeling; Molecular; Molecular Biology; Morphology; Mus; Mutation; Organelles; Osteoblasts; Osteogenesis; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Proteins; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; Stem cells; Stimulus; Syndactyly; Tight Junctions; Tissues; Tooth structure; Work; Zinc Fingers; autocrine; base; beta catenin; bone; cartilage development; cell growth; extracellular; gap junction channel; human disease; intramembranous bone formation; member; mineralization; novel; osteoblast differentiation; osteogenic; osteoprogenitor cell; overexpression; soft tissue; transcription factor

Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. Our focus has been on protein factors that regulate differentiation of chondrocytes and osteoblasts to understand the molecular mechanisms of these factors in tissue formation and diseases. In tissue development and homeostasis, cells perceive micro-environmental cues from soluble mediators, the extracellular matrix (ECM), and neighboring cells. Adhesion complexes, such as adherens junctions, tight junctions, and gap junctions, mediate cell-cell and cell-ECM interactions and links between the cell and its environment, which integrate signaling cascades for cell proliferation and differentiation. Gap junction proteins consist of two families, connexins (Cxs) and pannexins (Panxs). The Cx family has more than 20 members. Mutations of Cxs cause human diseases, such as cancer, hypertension, atherosclerosis, and developmental abnormalities. The Panx family consists of three members, Panx1, 2, and 3. Although Panx3 is expressed in certain soft tissues, we found high levels of Panx3 expression in developing hard tissues, including cartilage, bone, and teeth. We previously showed that Panx3 functions to promote differentiation of chondrocytes and osteoblasts. Osteoblasts differentiate from mesenchyme stem cells and form bone through endochondral and intramembranous ossification. BMP2 induces the master osteogenic transcription factors Runx2 and osterix. This leads to the activation of osteogenic marker genes and subsequently to terminal differentiation of osteoblasts and mineralization. Ca2+ is a universal intracellular signaling molecule that regulates cell proliferation, differentiation, morphology, and function. Intracellular Ca2+ concentrations can rise more than five-fold via Ca2+ influx from the extracellular space and/or release from the endoplasmic reticulum (ER), an intracellular Ca2+ storage organelle. This occurs when cells are activated by extracellular stimuli such as ATP. We previously showed that Panx3 functions as a unique Ca2+ channel in the endoplasmic reticulum (ER), which was activated by ATP receptor/PI3K/Akt signaling for differentiation. Panx3 also forms hemichannels that allow the release of ATP into the extracellular space. ATP in the extracellular space activates ATP receptors via autocrine and non-autocrine mechanism following the activation of PI3K/Akt signaling. In addition, Panx3 forms gap junctions and propagates Ca2+ waves between adjacent cells. Blocking the Panx3 Ca2+ channel and gap junction activities inhibits osteoblast differentiation. These findings reveal that Panx3 promotes osteoblast differentiation by functioning as an ER Ca2+ channel, a hemichannel, and by forming gap junctions. The Panx3 hemichannel triggers these Panx3 signaling pathways. Because Panx3 is induced in the transitional stage from proliferation to differentiation during osteogenesis, we hypothesized that Panx3 may also play a role in the inhibition of osteoprogenitor cell proliferation. Canonical Wnt/beta-catenin signaling and BMP promote the proliferation and differentiation of osteoprogenitors, respectively. However, the regulatory mechanism involved in the transition from proliferation to differentiation is unclear. We show that Panx3 plays a key role in this transition by inhibiting proliferation and promoting cell cycle exit. Using cell cultures and calvaria explants, we found that Panx3 overexpression inhibited cell growth, whereas the inhibition of endogenous Panx3 expression increased it. We found that the Panx3 hemichannel inhibited cell growth by promoting beta-catenin degradation through GSK3beta, which was activated by reduced cAMP/PKA signaling. The Panx3 hemichannel also reduced CREB signaling, which inhibited cyclin D1 transcription and Rb phosphorylation. In addition, the Panx3 ER Ca2+ channel induced transcription and phosphorylation of p21 through the calmodulin/Smad pathway, resulting in cell cycle exit. Thus, Panx3 is a novel regulator that promotes the switch from proliferation to differentiation of osteoprogenitors. The apical ectodermal ridge (AER) at the distal edge of the developing limb bud is a major signaling center for limb development. Epiprofin (Epfn)/Sp6 and Buttonhead/Sp8 are two members of the Sp zinc-finger transcription family that are expressed in the limb bud ectoderm. Sp6 and Sp8 function downstream of Wnt/beta-catenin signaling for Fgf 8 induction. Sp6 knockout (KO) mice show a mild syndactyly phenotype, while Sp8 KO mice exhibit severe limb truncations. In collaboration with Dr. Marian Ros and her group, we studied the role of Sp6 and Sp8 in limb development by creating double KO mice for Sp6 and Sp8. We found that Sp6 and Sp8 worked together as indispensable mediators of Wnt/beta-catenin and Bmp signaling in the limb ectoderm. Our results suggest that Sp6 and Sp8 are required in a dose-dependent manner for Fgf8 and En1 induction and function as an important link between the induction of the AER and the establishment of dorso-ventral patterning during limb development.

软骨是一种高度特化的结缔组织，含有广泛的细胞外基质，并提供机械强度以抵抗关节中的压缩。在发育过程中，软骨作为大多数骨骼生长和发育的模板。我们的重点是调节软骨细胞和成骨细胞分化的蛋白质因子，以了解这些因子在组织形成和疾病中的分子机制。 在组织发育和稳态中，细胞从可溶性介质、细胞外基质（ECM）和邻近细胞感知微环境线索。粘附复合物，如粘附连接、紧密连接和间隙连接，介导细胞-细胞和细胞-ECM相互作用以及细胞与其环境之间的联系，其整合用于细胞增殖和分化的信号级联。 缝隙连接蛋白由两个家族组成，连接蛋白（Cxs）和泛连接蛋白（Panxs）。Cx家族有20多个成员。Cxs突变会导致癌症、高血压、动脉粥样硬化和发育异常等人类疾病。Panx家族由三个成员组成：Panx 1、2和3。虽然Panx 3在某些软组织中表达，但我们发现Panx 3在发育中的硬组织中表达水平很高，包括软骨，骨骼和牙齿。我们先前表明Panx 3的功能是促进软骨细胞和成骨细胞的分化。 成骨细胞由间充质干细胞分化而来，通过软骨内成骨和膜内成骨形成骨。BMP 2诱导主要成骨转录因子Runx 2和osterix。这导致成骨标志物基因的激活，随后导致成骨细胞的终末分化和矿化。Ca 2+是一种调节细胞增殖、分化、形态和功能的通用细胞内信号分子。细胞内Ca 2+浓度可以通过Ca 2+从细胞外空间流入和/或从内质网（ER）（细胞内Ca 2+储存细胞器）释放而升高超过5倍。这发生在细胞被细胞外刺激物如ATP激活时。我们先前表明，Panx 3作为内质网（ER）中独特的Ca 2+通道发挥作用，其通过ATP受体/PI 3 K/Akt信号转导激活以进行分化。Panx 3还形成允许ATP释放到细胞外空间的半通道。细胞外间隙中的ATP在PI 3 K/Akt信号转导激活后通过自分泌和非自分泌机制激活ATP受体。此外，Panx 3形成间隙连接并在相邻细胞之间传播Ca 2+波。阻断Panx 3 Ca 2+通道和间隙连接活性抑制成骨细胞分化。这些发现表明Panx 3通过作为ER Ca 2+通道、半通道和形成间隙连接来促进成骨细胞分化。Panx 3半通道触发这些Panx 3信号通路。 由于Panx 3在骨生成过程中从增殖到分化的过渡阶段被诱导，我们假设Panx 3也可能在抑制骨祖细胞增殖中发挥作用。典型的Wnt/β-catenin信号传导和BMP分别促进骨祖细胞的增殖和分化。然而，从增殖到分化的过渡所涉及的调控机制尚不清楚。我们发现Panx 3通过抑制增殖和促进细胞周期退出在这种转变中起着关键作用。使用细胞培养物和颅骨外植体，我们发现Panx 3过表达抑制细胞生长，而内源性Panx 3表达的抑制则增加了它。我们发现Panx 3半通道通过促进通过GSK 3 β的β-连环蛋白降解来抑制细胞生长，GSK 3 β通过减少cAMP/PKA信号传导而被激活。Panx 3半通道还减少CREB信号传导，其抑制细胞周期蛋白D1转录和Rb磷酸化。此外，Panx 3 ER Ca 2+通道通过钙调蛋白/Smad途径诱导p21的转录和磷酸化，导致细胞周期退出。因此，Panx 3是一种新的调节剂，促进骨祖细胞从增殖到分化的转变。 肢芽发育过程中的顶端外胚层嵴（AER）是肢发育的主要信号中心。Epiprofin（Epfn）/Sp 6和Buttonhead/Sp 8是Sp锌指转录家族的两个成员，在肢芽外胚层中表达。Sp 6和Sp 8在Wnt/β-连环蛋白信号传导的下游起Fgf 8诱导的作用。Sp 6基因敲除（KO）小鼠表现出轻度并指表型，而Sp 8基因敲除小鼠表现出严重的肢体截短。与Marian Ros博士及其团队合作，我们通过创建Sp 6和Sp 8的双KO小鼠来研究Sp 6和Sp 8在肢体发育中的作用。我们发现，Sp 6和Sp 8一起工作的Wnt/β-catenin和BMP信号在肢体外胚层的不可或缺的介质。我们的研究结果表明，Sp 6和Sp 8所需的FGF 8和En 1诱导剂量依赖性的方式和功能作为一个重要的联系之间的诱导AER和建立背腹图案在肢体发育。