Cartilage Development and Disease

软骨发育和疾病

• 批准号: 7967043
• 负责人: Yoshihiko Yamada
• 金额: $ 74.4万
• 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/7967043
• 关键词: Activins; Apoptosis; Binding; Blood Vessels; Bone Morphogenetic Proteins; CREB1 gene; Calcified; Calvaria; Cartilage; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cell Proliferation; Cell physiology; Cells; Chondrocytes; Collagen Type II; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Defect; Development; Disease; Embryonic Development; Epiphysial cartilage; Extracellular Domain; Extracellular Matrix; Extracellular Space; Family; Family member; Gap Junctions; Gene Mutation; Gene Targeting; Genes; Goals; Growth and Development function; Hereditary Disease; Human Genetics; In Vitro; Invaded; Joints; Knock-out; Knockout Mice; Length; Limb structure; MAP Kinase Gene; Mechanics; Mediating; Mesenchymal; Modeling; Molecular; Molecular Biology; Mus; Osteoblasts; Osteogenesis; Pathway interactions; Physical condensation; Play; Proliferating; Protein Isoforms; Proteins; Receptor Serine/Threonine Kinase; Regulation; Regulator Genes; Rest; Role; Signal Transduction; Skeletal Development; Skeleton; Staging; Stem cells; TGF-beta type I receptor; Tamoxifen; Tissues; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Width; aggrecan; base; bone; bone morphogenetic protein receptors; cartilage development; clavicle; craniofacial; human TGFB1 protein; in vivo; inhibiting antibody; link protein; long bone; malformation; member; mutant; osteoblast differentiation; osteoprogenitor cell; overexpression; parathyroid hormone-related protein; perlecan; progenitor; receptor; short bone; skeletal; skeletal tissue; small hairpin RNA; substantia spongiosa

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.

软骨含有广泛的细胞外基质，提供抗关节压缩的机械强度。软骨也是大多数骨骼生长发育的模板。ECM分子，如perlecan、连接蛋白、聚集蛋白和II型胶原，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，四肢、颅面骨和尾骨畸形。软骨的形成始于间充质细胞凝聚形成原始软骨，然后是软骨细胞分化，包括静息软骨细胞、增殖软骨细胞、前增生性软骨细胞和增生性软骨细胞。作为软骨内成骨的最后一步，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，空间和时间调节的软骨细胞分化是至关重要的，以确定骨骼成分的长度和宽度。