Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Melbourne, Victoria 3052, Australia; Central Science Laboratory, University of Tasmania, Hobart, Tasmania 7001, Australia.
Protein Discovery Center, Queensland Institute of Medical Research, Royal Brisbane Hospital, Herston, Queensland 4029, Australia.
Mol Cell Proteomics. 2012 Jan;11(1):M111.014159. doi: 10.1074/mcp.M111.014159. Epub 2011 Oct 11.
Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.
软骨内骨化的骨骼生长涉及到紧密协调的软骨细胞分化，在生长板中形成储备、增殖、预肥大和肥大软骨区。许多人类骨骼疾病是由于软骨细胞外基质 (ECM) 成分的突变引起的，这些突变会影响 ECM 结构和软骨细胞功能。因此，了解正常软骨的发育、组成和结构对于揭示这些疾病机制至关重要。为了通过蛋白质组学在体内研究这个复杂的过程，我们分析了在富含未成熟软骨细胞或成熟/肥大软骨细胞的发育阶段的小鼠股骨头软骨（分别为出生后第 3 天和第 21 天）。使用 LTQ-Orbitrap 串联质谱，我们鉴定了 703 种软骨蛋白。差异丰度蛋白（q < 0.01）包括早期和晚期软骨细胞分化的典型标志物（分别为软骨蛋白聚糖和胶原 X）以及新型 ECM 和细胞黏附蛋白，它们在软骨发育中没有先前描述的作用（腱糖蛋白 X、玻连蛋白、Urb、弹力蛋白 1 和富含 sushi 重复的蛋白 SRPX 和 SRPX2）。体内软骨发育的荟萃分析和体外软骨细胞培养模型（Wilson，R.，Diseberg，A. F.，Gordon，L.，Zivkovic，S.，Tatarczuch，L.，Mackie，E. J.，Gorman，J. J.和 Bateman，J. F.（2010）使用顺序提取和无标记定量蛋白质组学全面分析软骨细胞外基质形成和成熟。Mol. Cell. Proteomics 9, 1296-1313）确定了涉及两个系统的成分，如 Urb，以及在体内具有特定作用的成分，包括玻连蛋白和 CILP-2（软骨中间层蛋白-2）。Urb、玻连蛋白和 CILP-2 的免疫定位表明它们在不同的成熟阶段具有特定的作用。除了与 ECM 相关的变化外，我们还提供了第一个关于软骨发育过程中内质网功能变化的生化证据。尽管多功能伴侣蛋白 BiP 没有差异表达，但胶原蛋白生物合成所需的酶和伴侣蛋白，如脯氨酰 3-羟化酶 1、软骨相关蛋白和肽基脯氨酰顺反异构酶 B 复合物，在成熟过程中下调。相反，腔蛋白钙网蛋白、肌钙蛋白 1 和肌钙蛋白 2 的表达显著增加，表明向钙结合功能的转变。这是对体内软骨发育的第一次蛋白质组学分析，揭示了在小鼠股骨头中软骨细胞成熟和 ECM 重塑过程中广泛的蛋白质表达变化。
