Cartilage and Bone Development and Disease

软骨是一种高度专业化的结缔组织，含有广泛的细胞外基质，并提供抵抗关节压缩的机械强度。在发育过程中，软骨是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。 在组织发育和动态平衡中，细胞从可溶性介质、细胞外基质(ECM)和邻近细胞感知微环境信号。黏附复合体，如黏附连接、紧密连接和缝隙连接，调节细胞与细胞和细胞外基质的相互作用以及细胞与环境之间的联系，整合了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)和连接蛋白(Panxs)。CX家族有20多名成员。CXS基因突变会导致人类疾病，如癌症、高血压、动脉粥样硬化和发育异常。Panx家族由Panx1、Panx2和Panx3三个成员组成。虽然Panx3在某些软组织中表达，但我们发现Panx3在发育中的硬组织中高水平表达，包括软骨、骨和牙齿。我们之前已经证明Panx3具有促进软骨细胞和成骨细胞分化的功能。 成骨细胞由间充质干细胞分化而来，通过软骨内和膜内成骨形成骨。BMP2诱导主要的成骨转录因子Runx2和Osterix。这导致成骨标记基因的激活，进而导致成骨细胞的终末分化和矿化。钙离子是一种普遍存在的细胞内信号分子，调节细胞的增殖、分化、形态和功能。细胞内钙离子浓度可通过细胞外空间的钙离子内流和/或细胞内钙离子储存细胞器内质网(ER)的释放而增加五倍以上。当细胞被细胞外刺激如三磷酸腺苷激活时，就会发生这种情况。我们先前已经证明Panx3在内质网(ER)中是一个独特的钙通道，该通道被ATP受体/PI3K/Akt信号激活以进行分化。Panx3还形成半通道，允许ATP释放到细胞外空间。PI3K/Akt信号通路激活后，细胞外间隙中的ATP通过自分泌和非自分泌机制激活ATP受体。此外，Panx3形成缝隙连接，并在相邻细胞之间传播钙波。阻断Panx3钙通道和缝隙连接活性可抑制成骨细胞分化。这些发现表明，Panx3通过作为内质网钙通道、半通道和形成缝隙连接来促进成骨细胞的分化。Panx3半通道触发了这些Panx3信号通路。 由于Panx3在成骨过程中处于从增殖向分化的过渡阶段，因此我们推测Panx3也可能在抑制骨祖细胞的增殖中发挥作用。规范的Wnt/β-catenin信号和BMP分别促进骨祖细胞的增殖和分化。然而，从增殖到分化的过程中涉及的调控机制尚不清楚。我们发现Panx3通过抑制细胞增殖和促进细胞周期退出在这一转变中发挥关键作用。利用细胞培养和颅骨外植体，我们发现Panx3的过度表达抑制了细胞的生长，而抑制内源Panx3的表达则增加了细胞的生长。我们发现，Panx3半通道通过减少cAMP/PKA信号激活GSK3β，促进β-连环蛋白的降解，从而抑制细胞生长。Panx3半通道也减少了CREB信号，从而抑制了细胞周期蛋白D1的转录和Rb的磷酸化。此外，Panx3 ER钙离子通道通过钙调素/Smad途径诱导p21转录和磷酸化，导致细胞周期退出。因此，Panx3是一种促进骨祖细胞从增殖到分化的新型调控因子。 位于发育中的肢芽远端边缘的顶端外胚层脊(AER)是肢体发育的主要信号中枢。Epiproin(Epfn)/Sp6和Buttonhead/Sp8是Sp锌指转录家族中的两个成员，在肢芽外胚层中表达。Sp6和Sp8位于Wnt/β-catenin信号的下游，参与成纤维细胞生长因子8的诱导。SP6基因敲除(KO)小鼠表现出轻微的并指表型，而SP8KO小鼠则表现出严重的肢体截断。在玛丽安·罗斯博士和她的团队的合作下，我们创造了SP6和SP8的双KO小鼠，研究了SP6和SP8在肢体发育中的作用。我们发现，在肢体外胚层中，Sp6和Sp8共同作用于Wnt/β-catenin和BMP信号通路。我们的结果表明，Sp6和Sp8在Fgf8和EN1的诱导中是剂量依赖的，并在肢体发育过程中作为AER的诱导和背腹模式建立之间的重要联系。

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.