Inhibition of Gsk3 beta in cartilage induces steoarthritic features through activation of the canonical Wnt signaling pathway

Objective: In the past years, the canonical Wnt/\u3b2-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3\u3b2 (GSK3\u3b2) down-regulates transduction of the canonical Wnt signal by promoting degradation of \u3b2-catenin. In this study we wanted to further investigate the role of Gsk3\u3b2 in cartilage maintenance. Design: Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3\u3b2 inhibitor. Results: In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3. days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7. days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of \u3b2-catenin in chondrocytes 72. h later. Three intra-articular GIN injections with a 2. days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6. weeks later. Conclusions: These results suggest that, by down-regulating \u3b2-catenin, Gsk3\u3b2 preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term \u3b2-catenin up-regulation in cartilage secondary to Gsk3\u3b2 inhibition may be sufficient to induce osteoarthritis-like features in vivo

Role of Glycosylation in Conformational Stability, Activity, Macromolecular Interaction and Immunogenicity of Recombinant Human Factor VIII

Factor VIII (FVIII) is a multi-domain glycoprotein that is an essential cofactor in the blood coagulation cascade. Its deficiency or dysfunction causes hemophilia A, a bleeding disorder. Replacement using exogenous recombinant human factor VIII (rFVIII) is the first line of therapy for hemophilia A. The role of glycosylation on the activity, stability, protein–lipid interaction, and immunogenicity of FVIII is not known. In order to investigate the role of glycosylation, a deglycosylated form of FVIII was generated by enzymatic cleavage of carbohydrate chains. The biochemical properties of fully glycosylated and completely deglycosylated forms of rFVIII (degly rFVIII) were compared using enzyme-linked immunosorbent assay, size exclusion chromatography, and clotting activity studies. The biological activity of degly FVIII decreased in comparison to the fully glycosylated protein. The ability of degly rFVIII to interact with phosphatidylserine containing membranes was partly impaired. Data suggested that glycosylation significantly influences the stability and the biologically relevant macromolecular interactions of FVIII. The effect of glycosylation on immunogenicity was investigated in a murine model of hemophilia A. Studies showed that deletion of glycosylation did not increase immunogenicity