Jennifer J Embry1, Warren Knudson. 1. Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA.
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 wild-type 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.
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 bovinearticular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wild-type 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.
Authors: Ahmad Abu-Hakmeh; Amy Kung; Benjamin R Mintz; Sarah Kamal; James A Cooper; X Lucas Lu; Leo Q Wan Journal: Med Biol Eng Comput Date: 2016-04-07 Impact factor: 2.602