Hunziker E B, Kapfinger E, Martin J, Buckwalter J, Morales T I
ITI Research Institute for Dental and Skeletal Biology, Bern, Switzerland.
Osteoarthritis Cartilage. 2008 Feb;16(2):185-94. doi: 10.1016/j.joca.2007.06.008. Epub 2007 Aug 13.
Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells.
Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included.
LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining.
IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.
胰岛素样生长因子-I（IGF-I）在软骨基质代谢的调控中起关键作用。众所周知，骨关节炎（OA）期间胰岛素样生长因子结合蛋白-3（IGFBP-3）水平升高，但其功能尚不清楚。在其他细胞中，IGFBP-3可在细胞外环境中调节IGF-I的作用，也可在细胞内独立发挥作用；这包括细胞核中的转录基因调控。本研究旨在确定IGFBP-3在人关节软骨中的定位，尤其是在细胞内的定位。
从因OA行关节置换术获取的人股骨头中分离软骨，并通过组织学评估OA分级。通过IGF配体印迹（LB）和酶联免疫吸附测定（ELISA）对组织切片进行进一步分析，以提取和检测IGFBP。对轻度、中度和重度OA供体的软骨进行IGF-I和IGFBP-3的免疫组织化学（IHC）检测。包括间接荧光和免疫金标记IHC研究。
软骨细胞裂解物的LB显示IGFBP-3有强烈信号。所有OA阶段的股骨软骨切片的IHC显示IGF-I和IGFBP-3在基质中染色，特别是在表层区域，且与大多数细胞紧密相关。可见明显的核周/核IGFBP-3信号。使用非免疫血清或抗原封闭抗体的对照显示染色阴性或显著减弱。在人踝关节软骨冰冻切片中，免疫荧光IGFBP-3染色与核4',6-二脒基-2-苯基吲哚（DAPI）染色在90%以上的细胞中共定位。薄切片的免疫金IHC和超薄切片的透射电子免疫金显微镜显示明显的核内染色。
人软骨中的IGFBP-3位于基质以及软骨细胞的细胞质和细胞核内。这一新发现表明，IGFBP-3在关节软骨中的作用范围可能包括不依赖IGF的作用，并为研究其核作用打开了大门，包括可能对激素受体或转录复合物的调控以控制基因作用。
