Cartilage Development and Disease

软骨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。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，在软骨细胞分化过程中表达。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。软骨形成起始于间充质细胞凝聚形成原始软骨，随后是软骨细胞分化，包括静止、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。 转化生长因子-β（TGF-β）及其相关因子，包括骨形态发生蛋白（BMP）和激活素，调节胚胎发生期间的多种细胞过程，如增殖、分化、凋亡和细胞外基质形成。TGF-β信号传导由两种类型的跨膜丝氨酸/苏氨酸激酶受体介导，I型（ALK 5）和II型受体，其形成异聚复合物。在该信号传导复合物中，在TGF-β与II型受体结合后，II型受体磷酸化并激活ALK 5。活化的ALK 5通过Smad依赖性和Smad非依赖性途径诱导信号级联。在Smad依赖性途径中，TGF-β受体复合物激活Smad 2/3，而BMP-受体复合物激活Smad 1/5/8。TGF-β参与软骨细胞和成骨细胞的增殖和分化。然而，TGF-β在骨骼发育中的体内功能尚不清楚，主要是因为其多种活性和多种TGF-β蛋白（TGF-β 1，-β 2和-β 3）的冗余表达。TGF-β I型受体ALK 5是骨骼组织中TGF-β家族成员最主要的受体之一。ALK 5的缺乏应消除所有TGF-β亚型和其他潜在TGF-β超家族蛋白的Smad依赖性和Smad非依赖性信号传导。为了研究TGF-β信号传导在生长板发育中的作用，我们建立了条件性敲除小鼠，其中通过小鼠中的Dermo 1-Cre表达和体外他莫昔芬诱导的Cre表达使骨骼祖细胞中的ALK 5失活。条件性ALK 5敲除（ALK 5CKO）小鼠具有短而宽的长骨、减少的骨领和短的骨小梁。在ALK 5CKO生长板中，软骨细胞增殖分化并形成软骨，但异位软骨组织在Ranvier骨化沟处突出到软骨膜中。在对照生长板中，ALK 5蛋白在软骨周围的软骨膜祖细胞中强烈表达，其最终分化成成骨细胞。突变生长板有一个异常薄的软骨膜细胞层，以及减少增殖和分化的成骨细胞。这些软骨膜的缺陷可能导致生长板中的短骨和异位软骨突起。使用可诱导的ALK 5缺陷的原代颅骨细胞培养，我们发现TGF-β信号促进骨祖细胞增殖和早期分化。我们还发现它通过选择性MAPK和Smad 2/3途径调节成骨细胞谱系的定型。我们的研究结果揭示了TGF-β信号在软骨膜形成和分化中的关键作用，以及在骨骼发育过程中生长板的完整性。 虽然已知几种因子，如PTH/PTHrP，在软骨细胞增殖中起重要作用，但仍不清楚细胞增殖信号是如何关闭的，并致力于分化。在我们寻找一种调节骨骼祖细胞从增殖到分化的过渡阶段的因子时，我们发现最近鉴定的泛连接蛋白间隙连接家族的成员泛连接蛋白3（Panx 3）在软骨细胞分化中执行这样的功能。我们证明，Panx 3在生长板的前肥大区强烈表达，软骨细胞停止增殖并分化为肥大软骨细胞。Panx 3在软骨细胞系ATDC 5的分化过程中被诱导。Panx 3的过表达促进ATDC 4细胞的分化，而内源性Panx 3表达的shRNA抑制抑制分化。我们发现Panx 3抑制PTH介导的ATDC 5细胞增殖。另外，Panx 3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Panx 3的细胞外结构域的抗体抑制。我们还发现Panx 3表达降低了细胞内cAMP水平和CREB的激活，CREB是PKA下游效应物，其激活增殖所必需的基因。我们的研究结果表明，Panx 3的功能，通过调节细胞内ATP/cAMP水平，从增殖到分化的软骨细胞的命运。

Cartilage contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. Cartilage also serves as the template for the 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 followed by chondrocyte differentiation, which includes resting, proliferative, prehypertrophic, and hypertrophic chondrocytes. As a 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. Transforming growth factor-beta (TGF-beta) and its related factors, including bone morphogenetic proteins (BMPs) and activins, regulate diverse cellular processes such as proliferation, differentiation, apoptosis, and extracellular matrix formation during embryogenesis. TGF-beta signaling is mediated by two types of transmembrane serine/threonine kinase receptors, type I (ALK5) and type II receptors, which form a heteromeric complex. In this signaling complex, following TGF-beta binding to the type II receptor, the type II receptor phosphorylates and activates ALK5. Activated ALK5 induces signaling cascades through Smad-dependent and Smad-independent pathways. In the Smad-dependent pathway, the TGF-beta receptor complex activates Smad2/3, whereas the BMP-receptor complex activates Smad1/5/8. TGF-beta is implicated in proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-beta in skeletal development is not clear, primarily because of its diverse activities and redundant expression of multiple TGF-beta proteins (TGF-beta1, -beta2 and -beta3). The TGF-beta type I receptor ALK5 is one of the most prominent receptors for TGF-beta family members in skeletal tissues. Deficiency of ALK5 should eliminate Smad-dependent and Smad-independent signaling for all TGF-beta isoforms and other potential TGF-beta superfamily proteins. To investigate the role of TGF-beta signaling in growth plate development, we have created conditional knockout mice in which ALK5 was inactivated in skeletal progenitor cells by Dermo1-Cre expression in mice, and tamoxifen-inducible Cre expression in vitro. Conditional ALK5 knockout (ALK5CKO) mice had short and wide long bones, reduced bone collars, and short trabecular bones. In ALK5CKO growth plates, chondrocytes proliferated and differentiated and cartilage was formed, but ectopic cartilaginous tissues protruded into the perichondrium at the ossification groove of Ranvier. In control growth plates, ALK5 protein was strongly expressed in the perichondrial progenitor cells surrounding cartilage, which eventually differentiated into osteoblasts. Mutant growth plates had an abnormally thin perichondrial cell layer as well as reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions in the growth plate. Using inducible ALK5-deficient primary calvarial cell cultures, we found that TGF-beta signaling promoted osteoprogenitor proliferation and early differentiation. We also found that it regulated commitment to the osteoblastic lineage through selective MAPK and Smad2/3 pathways. Our results have uncovered critical roles of TGF-beta signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development. Although several factors, such as PTH/PTHrP, are also known to play an essential role in chondrocyte proliferation, it is still unclear how cell proliferation signals are turned off and a commitment to differentiation is made. In our search for a factor which regulates the transitional stage from proliferation to differentiation of skeletal progenitors, we found that pannexin3 (Panx3), a member of the recently identified pannexin gap junction family, performs such functions in chondrocyte differentiation. We demonstrated that Panx3 was strongly expressed in the prehypertrophic zone in 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 PTH-mediated ATDC5 cell proliferation. In addition, Panx3 promoted release of ATP from ATDC5 cells to the extracellular space by 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 and the activation of CREB, a PKA downstream effector, which activates the genes necessary for proliferation. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating intracellular ATP/cAMP levels.