Sprifermin (rhfgf18) has an anabolic effect on human osteoarthritic chondrocytes involving fgfr3 and erk1/2 but not p38α and JNKS

Abstract

Purpose: Restoring the articular surface of degraded or largely injured cartilage necessitates cartilage re-cellularization and the stimulation of cartilage matrix molecule production. Sprifermin (currently in a phase II clinical trial for knee osteoarthritis) is a growth factor that is involved in chondrogenesis and exerts an anabolic effect on cartilage. Using a 3D cell culture system, we previously demonstrated the ability of Sprifermin to stimulate cartilage matrix molecule expression and to promote the chondrocyte phenotype in bovine and porcine articular chondrocytes. In the present work, we aimed to confirm this effect in osteoarthritic chondrocytes of human origin (hOA). In parallel, the pathways involved in Sprifermin signaling were investigated in porcine chondrocytes. Methods: Primary osteoarthritic chondrocytes were isolated from the cartilage of patients who underwent total knee replacement. Cells were cultivated for a few days in monolayer first and then for one week in scaffold-free 3D culture before starting the treatment. The latter consisted in the incubation with Sprifermin [100 ng/mL] permanently or one day/week for a total period of four weeks. Results were compared to a control culture without Sprifermin. Biochemical assays, quantitative PCR (qPCR) and histology were used to characterize the 3D constructs. FGFR expression by qPCR and activation of various kinases (with the human phospho-MAPK array kit from R&D Systems) upon Sprifermin stimulation were assessed on freshly isolated hOA chondrocytes. The effects of a panel of inhibitors (MAPK inhibitors and one pan-FGFR inhibitor) were tested in porcine cells. For this, freshly isolated chondrocytes from a pig hip were cultivated for 7 days in monolayer with 100 ng/mL of Sprifermin in presence of three concentrations of the inhibitors (0.1, 0.3 and 1 μM or 1, 3 and 10 μM depending on their potency). Sprifermin has been previously shown to strongly decrease the collagen I expression (monitored by qPCR) and to promote a round cell morphology (evaluated by actin staining) of porcine chondrocytes in monolayer. These parameters were used to evaluate the impact of the various inhibitors. Results: A 3D scaffold-free culture was used to test the effect of Sprifermin on hOA chondrocytes. In this setting Sprifermin [1day/week] has been found to have a positive effect on the cell content and to greatly increase the size and matrix content of the 3D constructs. In accordance with the literature, FGFR1 and 3 exhibited the highest expression among FGFRs in hOA chondrocytes. In addition, Sprifermin effects were suppressed by FGFR inhibition. In porcine chondrocytes cultivated in monolayer, Sprifermin strongly inhibited collagen I expression and promoted a round cell shape. Both effects were completely inhibited in presence of the pan-FGFR inhibitor PD173074 in a dose-dependent manner. Because FGF18 does not bind FGFR1, in chondrocytes Sprifermin signaling involves mainly FGFR3. Upon incubation with Sprifermin [100 ng/mL], ERK1/2 was activated as well as p38γ, but not p38α or the JNKs. The ERK inhibitor PD325901 completely inhibited the effect of Sprifermin on cell morphology at 100 nM. The JNK inhibitor (SR3306) and p38 inhibitors (BIRB-796 and SB203580) did not have any effect. The effect of these inhibitors on collagen I expression is still under investigation. Taken together these results indicate that ERK1/2 is involved in Sprifermin signaling whereas p38α and JNKs are not. Conclusions: As observed in previous studies with bovine and porcine chondrocytes, Sprifermin was found to exert anabolic effects in hOA chondrocytes. These effects seem to be mediated by FGFR3 and ERK1/2 activation. Others demonstrated that ERK1/2 is also involved in FGF2-FGFR1 signaling but in this case led to a catabolic response. This difference can be possibly explained by the fact that JNK and p38 are also activated by FGF2-FGFR1 signaling but not by FGF18-FGFR3.