Cartilage Development and Disease

软骨含有广泛的细胞外基质，并提供机械强度，以帮助抵抗关节中的压缩。软骨也是大多数骨骼生长和发育的模板。ECM分子如串珠蛋白聚糖、连接蛋白、聚集蛋白聚糖和II型胶原蛋白在软骨细胞分化期间表达。这些基因和调节因子的突变导致软骨形成受损以及四肢、颅面骨和无骨骨骼的畸形。软骨形成是由间充质细胞凝聚形成原始软骨开始的，随后是软骨细胞分化，包括静息、增殖、肥大前和肥大软骨细胞。作为软骨内骨形成的最后一步，肥大的软骨被血管和成骨细胞侵入，钙化的软骨随后被骨取代。因此，软骨细胞分化的空间和时间调节在决定骨骼组成部分的长度和宽度方面是必不可少的。 我们以前报道，转录因子epiprofin（Epfn/Sp6）是必不可少的牙齿形态发生，毛囊，和正常的数字形成。与Maria Ros博士合作，我们使用小鼠模型研究了Epfn在手指形成中的作用。外胚层顶嵴（AER）是肢体发育的信号中心，其形成和维持对脊椎动物肢体的生长和形成模式至关重要。AER的建立是由FGF、WNT/β-连环蛋白和BMP信号通路之间的复杂相互作用指导的，这些信号通路在外胚层内以及在早期肢芽的外胚层和中胚层组分之间起作用。β 这个过程也与肢芽的形成和背腹（DV）图案的建立有关。Epfn在肢体发育过程中在肢体外胚层和AER中瞬时表达。Epfn缺乏导致前肢中轴并指畸形，后肢部分双背趾尖骨性结合（骨融合）。 我们发现Epfn突变小鼠在AER成熟中表现出缺陷，其表现为平坦和宽的双脊表型。使用遗传分析，我们表明，Epfn是一个目标的WNT/-连环蛋白信号在肢体外胚层，它的表达是独立的FGF信号。β 我们已经确定，泛连接蛋白3（Panx3）是高度表达在发育中的软骨。Panx3是泛连接蛋白间隙连接蛋白家族的新成员，最初是通过GenBank数据库搜索确定的。在脊椎动物中，间隙连接蛋白包括连接蛋白超家族的20多个成员和泛连接蛋白家族的3个成员。间隙连接调节细胞形态和生理学，并与细胞增殖和分化有关。间隙连接通过允许小分子如离子以及低分子量代谢物和其他信使分子在相邻细胞之间（经由间隙连接）和细胞与细胞外空间之间（经由半通道）交换来发挥其作用。许多参与软骨发育的生长因子和转录因子已被鉴定。然而，控制从增殖到分化的转换或维持分化状态的调节机制仍然不清楚。我们推测Panx3可能在软骨细胞分化过程中的这种转换中发挥作用。我们还证明了Panx3在生长板的prehypertrophic区强烈表达，软骨细胞停止增殖并分化为肥大软骨细胞。Panx3在软骨细胞系ATDC 5的分化过程中被诱导。Panx3的过表达促进ATDC 4细胞的分化，而内源性Panx3表达的shRNA抑制抑制分化。我们发现Panx3抑制甲状旁腺激素（PTH）介导的ATDC 5细胞增殖。此外，Panx3通过其半通道活性促进ATP从ATDC 5细胞释放到细胞外空间，并且该ATP释放被Panx3的细胞外结构域的抗体抑制。我们还发现Panx3表达降低了细胞内cAMP水平，以及CREB（PKA下游效应物）的激活，其激活增殖所需的基因。我们的研究结果表明，Panx3的功能切换软骨细胞的命运从增殖到分化，通过调节细胞内ATP/cAMP水平。这反过来又抵消了PTH/PTHrP信号通路。

Cartilage contains an extensive extracellular matrix and provides mechanical strength to help resist compression in joints. Cartilage also serves as the template for growth and development of most bones. ECM molecules such as perlecan, link protein, aggrecan, and type II collagen are expressed during chondrocyte differentiation. Mutations of these genes and regulatory factors result in impaired cartilage formation and malformation of the limbs, craniofacial bones, and appendicular skeleton. Cartilage formation is initiated by mesenchymal cell condensation to form primordial cartilage and is followed by chondrocyte differentiation, which includes resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As the final step in endochondral bone formation, hypertrophic cartilage is invaded by blood vessels and osteoblasts, and the calcified cartilage is subsequently replaced by bone. Thus, spatial and temporal regulation of chondrocyte differentiation is essential in determining the length and width of skeletal components. We previously reported that the transcription factor epiprofin (Epfn/Sp6) is essential for tooth morphogenesis, hair follicles, and normal digit formation. In collaboration with Dr. Maria Ros, we studied the role of Epfn in digit formation using mouse models. The apical ectodermal ridge (AER) functions as a signaling center for limb development, and the formation and maintenance of the AER is critical for the outgrowth and patterning of the vertebrate limb. The establishment of the AER is directed by complex interactions between the FGF, WNT/β-catenin and BMP signaling pathways that operate within the ectoderm, as well as between the ectoderm and mesoderm components of the early limb bud. This process is also linked to the initiation of the limb bud and the establishment of dorsoventral (DV) patterning. Epfn is transiently expressed in the limb ectoderm and in the AER during limb development. Epfn deficiency resulted in a defective autopod with mesoaxial syndactyly in the forelimb, and synostosis (bone fusion) in the hindlimb with partial bidorsal digital tips. We found that Epfn mutant mice display a defect in AER maturation, which appears flat and broad with a double-ridge phenotype. Using genetic analysis, we showed that Epfn is a target of WNT/β-catenin signaling in the limb ectoderm and that its expression is independent of FGF signaling. We have identified that pannexin 3 (Panx3) is highly expressed in developing cartilage. Panx3 is a new member of the pannexin gap junction family of proteins, and was originally identified through a GenBank database search. In vertebrates, gap junction proteins comprise more than 20 members of the connexin superfamily and 3 members of the pannexin family. Gap junctions regulate cell morphology and physiology and are implicated in cell proliferation and differentiation. Gap junctions exert their actions by allowing the exchange of small molecules such as ions, as well as low molecular weight metabolites and other messenger molecules, between adjacent cells (via gap junctions) and between cells and extracellular space (via hemichannels). Many of the growth factors and transcription factors involved in cartilage development have been identified. However, the regulatory mechanisms that control the switch from proliferation to differentiation, or that maintain the differentiated state, are still unclear. We hypothesized that Panx3 may play a role in this switch during chondrocyte differentiation. We also demonstrated that Panx3 is strongly expressed in the prehypertrophic zone of the growth plate, where chondrocytes stop proliferation and differentiate into hypertrophic chondrocytes. Panx3 was induced during differentiation of the chondrogenic cell line ATDC5. Overexpression of Panx3 promoted ATDC4 cell differentiation, while suppression of endogenous Panx3 expression by shRNA inhibited that differentiation. We found that Panx3 inhibited parathyroid hormone (PTH)-mediated ATDC5 cell proliferation. In addition, Panx3 promoted the release of ATP from ATDC5 cells to the extracellular space through its hemichannel activity, and this ATP release was inhibited by an antibody to the extracellular domain of Panx3. We also found that Panx3 expression reduced intracellular cAMP levels, as well as the activation of CREB, a PKA downstream effector, which activates the genes necessary for proliferation. Our results suggest that Panx3 functions to switch chondrocyte cell fate from proliferation to differentiation by regulating intracellular ATP/cAMP levels. This in turn counteracts the PTH/PTHrP signal pathway.