The accumulation of intracellular ITEGE and DIPEN neoepitopes in bovine articular chondrocytes is mediated by CD44 internalization of hyaluronan.
OBJECTIVE
A dramatic loss of aggrecan proteoglycan from cartilage is associated with osteoarthritis. The fate of residual G1 domains of aggrecan is unknown, but inefficient turnover of these domains may impede subsequent repair and retention of newly synthesized aggrecan. Thus, the objective of this study was to determine whether ITEGE- and DIPEN-containing G1 domains, generated in situ, are internalized by articular chondrocytes, and whether these events are dependent on hyaluronan (HA) and its receptor, CD44.


METHODS
ITEGE and DIPEN neoepitopes were detected by immunofluorescence staining of bovine articular cartilage chondrocytes treated with or without interleukin-1alpha (IL-1alpha). Additionally, purified ITEGE- or DIPEN-containing G1 domains were aggregated with HA and then added to articular chondrocytes, articular chondrocytes transfected with CD44delta67, or COS-7 cells transfected with or without full-length CD44. Internalized epitopes were distinguished by their resistance to extensive trypsinization of the cell surface.


RESULTS
Both ITEGE and DIPEN were visualized within the extracellular cell-associated matrix of chondrocytes as well as within intracellular vesicles. Following trypsinization, the intracellular accumulation of both epitopes was clearly visible. IL-1 treatment increased extracellular as well as intracellular ITEGE epitope accumulation. Once internalized, the ITEGE neoepitope became localized within the nucleus and displayed little colocalization with HA, DIPEN, or other G1 domain epitopes. The internalization of both ITEGE and DIPEN G1 domains was dependent on the presence of HA and CD44.


CONCLUSIONS
One important mechanism for the elimination of residual G1 domains following extracellular degradation of aggrecan is CD44-mediated co-internalization with HA.
Embry Flory
Jennifer J
JJ
Rush Medical College, Rush University Medical Center, Chicago, Illinois 60612, USA.
Fosang
Amanda J
AJ
Knudson
Warren
W
D053674:Aggrecans; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D019556:COS Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D004705:Endocytosis; D000939:Epitopes; D016326:Extracellular Matrix Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology; D037181:Lectins, C-Type / Q000276:immunology / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments; D021381:Protein Transport; D014162:Transfection
D053674:Aggrecans; D019138:Antibodies, Blocking; D011508:Chondroitin Sulfate Proteoglycans; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D007375:Interleukin-1; D037181:Lectins, C-Type; D010446:Peptide Fragments; D006820:Hyaluronic Acid