Embry Flory Jennifer J, Fosang Amanda J, Knudson Warren
Rush Medical College, Rush University Medical Center, Chicago, Illinois 60612, USA.
Arthritis Rheum. 2006 Feb;54(2):443-54. doi: 10.1002/art.21623.
A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44.
ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface.
Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44.
One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.
软骨中聚集蛋白聚糖蛋白多糖的显著丢失与骨关节炎相关。聚集蛋白聚糖残余G1结构域的命运尚不清楚，但这些结构域的低效周转可能会阻碍新合成的聚集蛋白聚糖的后续修复和保留。因此，本研究的目的是确定原位产生的含ITEGE和DIPEN的G1结构域是否被关节软骨细胞内化，以及这些事件是否依赖于透明质酸（HA）及其受体CD44。
通过对用或不用白细胞介素-1α（IL-1α）处理的牛关节软骨细胞进行免疫荧光染色来检测ITEGE和DIPEN新表位。此外，将纯化的含ITEGE或DIPEN的G1结构域与HA聚集，然后添加到关节软骨细胞、用CD44δ67转染的关节软骨细胞或用全长CD44转染或未转染的COS-7细胞中。通过对细胞表面进行广泛胰蛋白酶消化后内化表位的抗性来区分内化表位。
ITEGE和DIPEN在软骨细胞的细胞外相关基质以及细胞内囊泡中均可见。胰蛋白酶消化后，两种表位的细胞内积累清晰可见。IL-1处理增加了细胞外以及细胞内ITEGE表位的积累。一旦内化，ITEGE新表位就定位于细胞核内，与HA、DIPEN或其他G1结构域表位几乎没有共定位。ITEGE和DIPEN G1结构域的内化均依赖于HA和CD44的存在。
聚集蛋白聚糖细胞外降解后消除残余G1结构域的一个重要机制是CD44介导的与HA共内化。
