Vertel B M, Walters L M, Grier B, Maine N, Goetinck P F
Department of Cell Biology and Anatomy, University of Health Sciences, Chicago Medical School, IL 60064.
J Cell Sci. 1993 Mar;104 ( Pt 3):939-48. doi: 10.1242/jcs.104.3.939.
Cartilage extracellular matrix (ECM) is composed primarily of type II collagen and large, link stabilized aggregates of hyaluronic acid and chondroitin sulfate proteoglycan (aggrecan). Maturation and function of these complex macromolecules are dependent upon sequential processing events which occur during their movements through specific subcellular compartments in the constitutive secretory pathway. Failure to complete these events successfully results in assembly of a defective ECM and may produce skeletal abnormalities. Nanomelia is a lethal genetic mutation of chickens characterized by shortened and malformed limbs. Previous biochemical studies have shown that cultured nanomelic chondrocytes synthesize a truncated aggrecan core protein precursor that disappears with time; however, the protein does not appear to be processed by the Golgi or secreted. The present study investigates the intracellular trafficking of the defective aggrecan precursor using immunofluorescence, immunoelectron microscopy and several inhibitors. Results indicate that nanomelic chondrocytes assemble an ECM that contains type II collagen, but lacks aggrecan. Instead, aggrecan precursor was localized intracellularly, within small cytoplasmic structures corresponding to extensions of the endoplasmic reticulum (ER). At no time were precursor molecules observed in the Golgi. In contrast, normal and nanomelic chondrocytes exhibited no difference in the intracellular or extracellular distribution of type II procollagen. Therefore, retention of the aggrecan precursor appears to be selective. Incubation of chondrocytes at 15 degrees C resulted in the retention and accumulation of product in the ER. After a return to 37 degrees C, translocation of the product to the Golgi was observed for normal, but not for nanomelic, chondrocytes, although the precursors disappeared with time. Ammonium chloride, an inhibitor of lysosomal function, had no effect on protein loss, suggesting that the precursor was removed by a non-lysosomal mechanism, possibly by ER-associated degradation. Based on these studies, we suggest that nanomelic chondrocytes are a useful model for examining cellular trafficking and sorting events and the processes by which abnormal products are targeted for retention or degradation. Further investigations should provide insight into the mechanisms underlying chondrodystrophies and other related diseases.
软骨细胞外基质（ECM）主要由II型胶原蛋白以及由透明质酸和硫酸软骨素蛋白聚糖（聚集蛋白聚糖）构成的大型、连接稳定的聚集体组成。这些复杂大分子的成熟和功能取决于它们在组成型分泌途径中通过特定亚细胞区室移动时发生的一系列加工事件。未能成功完成这些事件会导致有缺陷的ECM组装，并可能产生骨骼异常。短肢畸形是鸡的一种致死性基因突变，其特征是肢体缩短和畸形。先前的生化研究表明，培养的短肢畸形软骨细胞合成一种截短的聚集蛋白聚糖核心蛋白前体，该前体随时间消失；然而，该蛋白似乎未被高尔基体加工或分泌。本研究使用免疫荧光、免疫电子显微镜和几种抑制剂来研究有缺陷的聚集蛋白聚糖前体的细胞内运输。结果表明，短肢畸形软骨细胞组装的ECM含有II型胶原蛋白，但缺乏聚集蛋白聚糖。相反，聚集蛋白聚糖前体定位于细胞内，位于对应于内质网（ER）延伸的小细胞质结构内。在高尔基体中从未观察到前体分子。相比之下，正常和短肢畸形软骨细胞在II型前胶原的细胞内或细胞外分布上没有差异。因此，聚集蛋白聚糖前体的保留似乎是选择性的。将软骨细胞在15摄氏度下孵育导致产物在内质网中保留和积累。恢复到37摄氏度后，观察到正常软骨细胞的产物转运到高尔基体，但短肢畸形软骨细胞没有，尽管前体随时间消失。氯化铵是一种溶酶体功能抑制剂，对蛋白质损失没有影响，这表明前体是通过非溶酶体机制去除的，可能是通过内质网相关降解。基于这些研究，我们认为短肢畸形软骨细胞是用于研究细胞运输和分选事件以及异常产物被靶向保留或降解的过程的有用模型。进一步的研究应该能够深入了解软骨发育不良和其他相关疾病的潜在机制。
