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 (postnatal 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. Cartilage is a unique tissue characterized by an abundant extracellular matrix (ECM) 1 and a single cell type, the chondrocyte. However, the permanent hyaline cartilage, which provides the articulating surfaces of long bones and vertebrae, and the transient growth plate cartilage responsible for endochondral bone growth are uniform in neither cellular phenotype nor protein composition. In articular cartilage, the chondrocytes form morphologically distinct regions comprising a superficial region of flattened cells, a sparsely populated middle layer, and a deep zone of hypertrophic chondrocytes embedded in calcified cartilage at the chondro-osseous junc-

Background: Collagen IX is an integral cartilage extracellular matrix component important in skeletal development and joint function. Results: Proteomic analysis and validation studies revealed novel alterations in collagen IX null cartilage. Conclusion: Matrilin-4, collagen XII, thrombospondin-4, fibronectin, ␤ig-h3, and epiphycan are components of the in vivo collagen IX interactome. Significance: We applied a proteomics approach to advance our understanding of collagen IX ablation in cartilage.

Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate. ß

Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.Discrimination of viable from dying cells is a prerequisite for the efficient clearance of dying and dead cells and to suppress uncontrolled cell lysis and the release of potentially harmful cellular compounds to the local environment. During development and under normal physiological conditions, cells are removed through the process of apoptosis and undergo a strictly defined series of morphological and biochemical changes, before being engulfed by professional phagocytes such as macrophages or even by neighboring cells. A signature of specific "eat me" signals are exposed at the cell surface of apoptotic cells, which enable direct or indirect interactions with phagocytes and dying cells to promote the efficient clearance of apoptotic cells. This reduces the risk of accumulation of secondarily necrotic cells and the release of cellular content to the microenvironment. Exposure of apoptotic-cell associated molecular patterns together with recognition and interpretation of these by phagocytes are crucial steps in the appropriate response of phagocytes toward the engulfed cells (1).A hallmark of early apoptotic cells is the loss of membrane asymmetry and the exposure of anionic phosphatidylserine (PS) 3 on the outer lipid layer of the cells. In most cells PS predominantly resides in the inner leaflet of the plasma membrane, regulating membrane charge and protein localization (2). This membrane asymmetry is actively maintained by an inward transporting aminophospholipid translocase (3). Upon induction of apotosis, rising cytoplasmic Ca 2ϩ levels cause a loss of translocase activity and an activ...

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle-mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X-deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1 À/À and AnxA5 À/À AnxA6 À/À Col10a1 À/À newborns, whereas endochondral ossification and mineralization were not affected in 13-day-and 1-month-old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1-month-and 1-year-old mutants even though the diaphyseal circumference was reduced in Col10a1 À/À and AnxA5 cells and peripheral T-helper cellswere increased in Col10a1 À/À and AnxA5Col10a1 À/À mutants, and activated splenic T cells isolated from Col10a1 À/À mice secreted elevated levels of IL-4 and GM-CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process. ß