G1 domain of aggrecan cointernalizes with hyaluronan via a CD44‐mediated mechanism in bovine articular chondrocytes

Abstract

Objective. To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization.Methods. Binding and internalization of a variety of fluorescein isothiocyanate (FITC)-or biotin-labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wildtype CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface.Results. Biotinylated aggrecan fragments bound to FITC-HA were cointernalized in bovine articular chondrocytes or COS-7 cells transfected with CD44. Intracellular vesicles containing FITC-HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC-HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized.Conclusion. Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44-mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA-bound fragments.One of the features of osteoarthritis is the loss of the proteoglycan aggrecan from the cartilage extracellular matrix (1). It has been suggested that this loss is due to the extracellular proteolytic cleavage of aggrecan by enzymes such as matrix metalloproteinases (2) and aggrecanases (3). Following proteolytic cleavage, the C-terminal, chondroitin sulfate-rich portion of degraded aggrecan is readily lost from the cartilage, and newly generated neoepitopes can be detected within synovial fluid (4). The fate of the N-terminal domain of the aggrecan degradation product, presumably still bound to HA and stabilized by link protein, is less clear. In previous studies using organ culture of normal bovine articular cartilage slices, the turnover kinetics of newly synthesized, radiolabeled aggrecan and HA were found to be nearly identical, with half-lives in the range of 17-25 days (5,6). However, whereas released aggrecan monomers could be quantitatively recovered in the culture medium, little intact HA and no small HA fragments were recovered in the medium or within guanidine HCl extracts of the tissue. These data suggested that HA was not being degraded extracellularly, but rather undergoing turnover by another distinct mechanism such as receptor-mediated endocytosis. Recently, Fosang and colleagues demonstrated by confocal micros...

The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan

“…Fosang et al (9) demonstrated, using confocal microscopy, that the G1 domain derived from aggrecanase-mediated cleavage of aggrecan (containing a new C-terminal neoepitope termed ITEGE) was localized intracellularly, inside chondrocytes present within sections of intact porcine articular cartilage. After their study, we demonstrated that a commercially purified and biotinylated aggrecan G1 domain-link protein complex (isolated following trypsin digest of aggrecan) could be co-internalized with HA via a CD44-mediated endocytosis event (18). Together, these results suggest that the final pathway for complete catabolism of aggrecan includes receptormediated co-internalization of HA, the G1 domain of aggrecan, and other HA-bound proteins such as link protein.…”

Hyaluronan Oligosaccharides Induce Matrix Metalloproteinase 13 via Transcriptional Activation of NFκB and p38 MAP Kinase in Articular Chondrocytes

“…These donor tissues were obtained, with appropriate institutional approval, less than 24 h from the time of death and were from donors with no history of arthritic disease. Full thickness slices of articular cartilages were dissected, and chondrocytes were isolated by use of a sequential Pronase/collagenase digestion as described previously (28). The primary chondrocytes were cultured as a high density monolayer (5 ϫ 106 cells/35-mm diameter dish) in DMEM supplemented with 50 units/ml penicillin and 50 g/ml streptomycin containing 10% FBS.…”

“…We have used a variety of methods to selectively interfere with the clustering potential of high molecular mass HA. These include the use of testicular or Streptomyces hyaluronidase (11, 26), pCD44⌬67 (a dominant negative recombinant CD44 that cannot bind HA) (18,27,28), and small HA oligosaccharides * This work was supported in part by National Institutes of Health Grants RO1-AR43384, T32-AR07590, and P50-AR39239. The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…We have used a variety of methods to selectively interfere with the clustering potential of high molecular mass HA. These include the use of testicular or Streptomyces hyaluronidase (11,26), pCD44⌬67 (a dominant negative recombinant CD44 that cannot bind HA) (18,27,28), and small HA oligosaccharides (HA oligo ) such as HA hexasaccharides (HA 6 ) (29 -31). Streptomyces hyaluronidase and pCD44⌬67 block the ability of CD44 to interact with Smad1 (18).…”

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

“…Finally, it has recently been reported that a proportion of aggrecanase-generated G1 domains can be cointernalized with HA by chondrocytes via a CD44-dependent mechanism (58,59), indicating that loss of G1-NITEGE 373 (as well as HA) from the cartilage matrix may occur through a variety of mechanisms. Nevertheless, levels of G1-NITEGE 373 in human synovial fluid have been shown to increase during aging, following acute joint injury, and with progression of OA (13), suggesting that aggrecanase-actuated loss of HA and hyaladherins may be part of a physiologic process that occurs in vivo.…”