ABSTRACT
OBJECTIVE: Osteoarthritis (OA) is a progressive degenerative disease of the articular cartilage caused by an unbalanced activity of proteases, cytokines and other secreted proteins. Since heparan sulfate (HS) determines the activity of many extracellular factors, we investigated its role in OA progression. METHODS: To analyze the role of the HS level, OA was induced by anterior cruciate ligament transection (ACLT) in transgenic mice carrying a loss-of-function allele of Ext1 in clones of chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl). To study the impact of the HS sulfation pattern, OA was surgically induced in mice with a heterozygous (Ndst1+/-) or chondrocyte-specific (Col2-Cre;Ndst1fl/fl) loss-of-function allele of the sulfotransferase Ndst1. OA progression was evaluated using the OARSI scoring system. To investigate expression and activity of cartilage degrading proteases, femoral head explants of Ndst1+/- mutants were analyzed by qRT-PCR, Western Blot and gelatin zymography. RESULTS: All investigated mouse strains showed reduced OA scores (Col2-rtTA-Cre;Ext1e2fl/e2fl: 0.83; 95% HDI 0.72-0.96; Ndst1+/-: 0.83, 95% HDI 0.74-0.9; Col2-Cre;Ndst1fl/fl: 0.87, 95% HDI 0.76-1). Using cartilage explant cultures of Ndst1 animals, we detected higher amounts of aggrecan degradation products in wildtype samples (NITEGE 4.24-fold, 95% HDI 1.05-18.55; VDIPEN 1.54-fold, 95% HDI 1.54-2.34). Accordingly, gelatin zymography revealed lower Mmp2 activity in mutant samples upon RA-treatment (0.77-fold, 95% HDI: 0.60-0.96). As expression of major proteases and their inhibitors was not altered, HS seems to regulate cartilage degeneration by affecting protease activity. CONCLUSION: A decreased HS content or a reduced sulfation level protect against OA progression by regulating protease activity rather than expression.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Heparitin Sulfate/metabolism , Matrix Metalloproteinase 2/metabolism , Osteoarthritis/metabolism , Aggrecans/metabolism , Animals , Anterior Cruciate Ligament/surgery , Blotting, Western , Cartilage, Articular/pathology , Disease Models, Animal , Disease Progression , Loss of Function Mutation , Mice , Mice, Transgenic , N-Acetylglucosaminyltransferases/genetics , Osteoarthritis/genetics , Osteoarthritis/pathology , Real-Time Polymerase Chain Reaction , Sulfotransferases/geneticsABSTRACT
The majority of bones in the vertebrate skeleton develop by endochondral ossification, a process during which an intermediate cartilage template is successively replaced by bone. Many aspects of this process are relatively well understood; nevertheless, the origin of trabecular bone-forming osteoblasts and mesenchymal stem cells of the stroma has long remained under debate. Until recently, progenitors of these cell types were thought to enter the bone-forming structures from the periosteum together with the invading vasculature. Recent unexpected results revealed, however, that under physiological conditions differentiated hypertrophic chondrocytes give rise to both, osteoblasts and mesenchymal progenitor cells, thereby contributing to the formation of trabecular bone and bone marrow.