title: CD44 knock-down in bovine and human chondrocytes results in release of bound HYAL2.
abstract: 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.
authors: 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.
authors: Daisuke
authors: D
authors: Hida
authors: Department of Anatomy and Cell Biology, East Carolina University, The Brody School of Medicine, Greenville, NC 27834, USA.
authors: Ben T
authors: BT
authors: Danielson
authors: Department of Anatomy and Cell Biology, East Carolina University, The Brody School of Medicine, Greenville, NC 27834, USA.
authors: Cheryl B
authors: CB
authors: Knudson
authors: Department of Anatomy and Cell Biology, East Carolina University, The Brody School of Medicine, Greenville, NC 27834, USA. Electronic address: knudsonw@ecu.edu.
authors: Warren
authors: W
authors: Knudson
mesh_terms: D000368:Aged; D000818:Animals; D019645:Arthroplasty, Replacement, Knee; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005260:Female; D058851:GPI-Linked Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism*; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D053583:Interleukin-1beta / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D061251:Primary Cell Culture; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology
chemical_list: C497483:CD44 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D018960:Hyaluronan Receptors; D053583:Interleukin-1beta; D034741:RNA, Small Interfering; D006820:Hyaluronic Acid; C473096:Hyal2 protein, human; D006821:Hyaluronoglucosaminidase; D014357:Trypsin
keywords: CD44; Chondrocyte; Hyaluronan; Hyaluronidase