Hida Daisuke, Danielson Ben T, Knudson Cheryl B, Knudson Warren
Department of Anatomy and Cell Biology, East Carolina University, The Brody School of Medicine, Greenville, NC 27834, USA; Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.
Department of Anatomy and Cell Biology, East Carolina University, The Brody School of Medicine, Greenville, NC 27834, USA.
Matrix Biol. 2015 Oct;48:42-54. doi: 10.1016/j.matbio.2015.04.002. Epub 2015 Apr 9.
CD44 shedding occurs in osteoarthritic chondrocytes. Previous work of others has suggested that the hyaluronidase isoform HYAL2 has the capacity to bind to CD44, a binding that may itself induce CD44 cleavage. Experiments were developed to elucidate whether chondrocyte HYAL2: (1) was exposed on the extracellular plasma membrane of chondrocytes, (2) bound to CD44, (3) underwent shedding together with CD44 and lastly, (4) exhibited hyaluronidase activity within a near-neutral pH range. Enhancing CD44 shedding by IL-1β resulted in a proportional increase in HYAL2 released from human and bovine chondrocytes into the medium. CD44 knockdown by siRNA also resulted in increased accumulation of HYAL2 in the media of chondrocytes. By hyaluronan zymography only activity at pH3.7 was observed and this activity was reduced by pre-treatment of chondrocytes with trypsin. CD44 and HYAL2 were found to co-immunoprecipitate, and to co-localize within intracellular vesicles and at the plasma membrane. Degradation of hyaluronan was visualized by agarose gel electrophoresis. With this approach, hyaluronidase activity could be observed at pH4.8 under assay conditions in which CD44 and HYAL2 binding remained intact; additionally, weak hyaluronidase activity could be observed at pH6.8 under these conditions. This study suggests that CD44 and HYAL2 are bound at the surface of chondrocytes. The release of HYAL2 when CD44 is shed could provide a mechanism for weak hyaluronidase activity to occur within the more distant extracellular matrix of cartilage.
CD44裂解发生在骨关节炎软骨细胞中。其他人之前的研究表明，透明质酸酶同工型HYAL2具有与CD44结合的能力，这种结合本身可能诱导CD44裂解。开展实验以阐明软骨细胞HYAL2是否：(1)暴露于软骨细胞的细胞外质膜上，(2)与CD44结合，(3)与CD44一起发生裂解，最后，(4)在接近中性的pH范围内表现出透明质酸酶活性。IL-1β增强CD44裂解导致从人和牛软骨细胞释放到培养基中的HYAL2成比例增加。通过siRNA敲低CD44也导致软骨细胞培养基中HYAL2的积累增加。通过透明质酸酶谱法仅观察到pH3.7时的活性，并且用胰蛋白酶预处理软骨细胞可降低该活性。发现CD44和HYAL2可共免疫沉淀，并在细胞内囊泡和质膜内共定位。通过琼脂糖凝胶电泳观察到透明质酸的降解。采用这种方法，在CD44和HYAL2结合保持完整的检测条件下，可在pH4.8观察到透明质酸酶活性；此外，在这些条件下，在pH6.8时可观察到微弱的透明质酸酶活性。本研究表明，CD44和HYAL2在软骨细胞表面结合。当CD44裂解时HYAL2的释放可能为在软骨更远端的细胞外基质中发生微弱的透明质酸酶活性提供一种机制。
