S Shortkroff1, K E Yates. 1. Department of Orthopedic Surgery, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
Abstract
OBJECTIVE: Although Wnt signaling is a key regulator of the chondrocyte life cycle during embryonic development, little is known about Wnt activity in articular cartilage. Recent studies have suggested an association between excess signaling through the canonical Wnt pathway and osteoarthritis (OA). Genetic and in vitro studies with Drosophila have shown that signaling by the orthologous protein, Wingless (Wg), is regulated by glycosaminoglycans (GAGs) found at the cell surface. The objective of this study was to determine whether alteration in GAG sulfation or matrix content, such as that occurs in OA cartilage, would affect articular chondrocytes' response to a canonical Wnt stimulus. METHODS: Cells were isolated from shoulder joints of young calves (bovine articular chondrocytes, bACs) and from human cartilage (human articular chondrocytes, hACs) discarded during total knee replacement for OA. Conditioned media from a cell line that is stably transfected with Wnt3a was used as a source of Wnt protein that activates the canonical signaling pathway. Conditioned media from the parental cell line was used as a control. beta-catenin levels were measured by immunoblot. In some experiments, chondrocyte cultures were treated with sodium chlorate (NaClO3) to inhibit GAG sulfation, or with chondroitinase ABC (ChABC) to digest chondroitin sulfate (CS) in the matrix. RESULTS: Cultured bACs showed low steady-state levels of beta-catenin that increased upon stimulation with Wnt3a. A decrease in either GAG sulfation or CS content diminished bACs' response to Wnt3a (approximately 40% and 37% of control, respectively). Similar effects on the response to Wnt3a via beta-catenin were observed for cultured hACs with undersulfation of GAGs (16% of control) and decreased CS content (20% of control). CONCLUSION: This study demonstrates that articular chondrocytes respond to canonical Wnt stimulation, and that reduced sulfation or CS content diminishes that response.
OBJECTIVE: Although Wnt signaling is a key regulator of the chondrocyte life cycle during embryonic development, little is known about Wnt activity in articular cartilage. Recent studies have suggested an association between excess signaling through the canonical Wnt pathway and osteoarthritis (OA). Genetic and in vitro studies with Drosophila have shown that signaling by the orthologous protein, Wingless (Wg), is regulated by glycosaminoglycans (GAGs) found at the cell surface. The objective of this study was to determine whether alteration in GAG sulfation or matrix content, such as that occurs in OA cartilage, would affect articular chondrocytes' response to a canonical Wnt stimulus. METHODS: Cells were isolated from shoulder joints of young calves (bovine articular chondrocytes, bACs) and from humancartilage (human articular chondrocytes, hACs) discarded during total knee replacement for OA. Conditioned media from a cell line that is stably transfected with Wnt3a was used as a source of Wnt protein that activates the canonical signaling pathway. Conditioned media from the parental cell line was used as a control. beta-catenin levels were measured by immunoblot. In some experiments, chondrocyte cultures were treated with sodium chlorate (NaClO3) to inhibit GAG sulfation, or with chondroitinase ABC (ChABC) to digest chondroitin sulfate (CS) in the matrix. RESULTS: Cultured bACs showed low steady-state levels of beta-catenin that increased upon stimulation with Wnt3a. A decrease in either GAG sulfation or CS content diminished bACs' response to Wnt3a (approximately 40% and 37% of control, respectively). Similar effects on the response to Wnt3a via beta-catenin were observed for cultured hACs with undersulfation of GAGs (16% of control) and decreased CS content (20% of control). CONCLUSION: This study demonstrates that articular chondrocytes respond to canonical Wnt stimulation, and that reduced sulfation or CS content diminishes that response.
Authors: Andre F Steinert; Steven C Ghivizzani; Axel Rethwilm; Rocky S Tuan; Christopher H Evans; Ulrich Nöth Journal: Arthritis Res Ther Date: 2007 Impact factor: 5.156