br Material and methods br Results br Discussion In

Material and methods
Results
Discussion In the present study, we attempted to unravel the mechanism underlying the statin-mediated inhibition of FGFR3 signaling in chondrocytes, reported earlier by Yamashita and colleagues. We evaluated the role of statins in four different experimental systems which were previously used to model the pathological FGFR signaling in a chondrocyte environment. Altogether, we did not observe any significant statin effect on any parameter of the FGFR signaling studied here. Specifically, statins did not produce any rescue effect on FGFR-mediated inhibition of chondrocyte proliferation and hypertrophic differentiation in cultured chondrocyte cell lines and embryonal tibias, or limb bud micromass cultures. We observed some variations in the level of endogenous FGFR3 in RCS KPT-185 [Fig. 4(B)] or human chondrocytes [Fig. 4(C)] after statin treatment but these changes were not consistent among individual statins and among used control and TD cell lines. Thus, we assessed the effect of statins on the level of easily detectable Flag-FGFR3 in F@F cells [Fig. 4(E)]. Since we observed no change in Flag-FGFR3 level, we concluded that abovementioned variations might be attributed to poor antibody quality and that statins do not affect the cellular amount of FGFR3. We also noted some changes in the intensity ratio of upper and lower band of transfected FGFR3 protein on the immunoblot (Supp. Fig. 3), representing the mature (glycosylated) and immature (non-glycosylated) FGFR3 variants, respectively32, 33, 34. This phenotype might be caused by statin effect on protein glycosylation35, 36. However, changes in the ratio of mature vs immature FGFR3 also occur in untreated control duplicates (Supp. Fig. 3) and are not obvious in endogenous Flag-FGFR3 in F@F cells [Fig. 4(E)], thus arguing against statin effect on FGFR3 glycosylation. In any case, in contrast to BGJ398 and AZD4547, statins had no obvious impact on activation of FGFR downstream signaling, or on phenotypes induced by FGFR activation (Fig. 1, Fig. 2, Fig. 3; Supp. Figs. 1,2). At the highest concentrations, statins lowered expression of FGF2-induced genes [Fig. 1(C)], however this effect may be attributed to cytotoxicity [Fig. 1(A)], rather than direct influence of statins on FGFR signaling. The most pronounced statin effect was on FGF2-mediated expression of caveolin-1 which, however, was described to be downregulated upon statin treatment37, 38, 39. Furthermore, the high statin concentrations used here outrange therapeutic statin concentrations in human serum by one order of magnitude, are cytotoxic [Fig. 1, Fig. 3; Supp. Fig. S1], and cause stunted growth and myopathy in developing rats40, 41. Statins did not rescue the collagen II downregulation mediated by FGF2 [Fig. 1(C)], although upregulated collagen II was detected in cells treated with atorvastatin, lovastatin and pravastatin without FGF2 [Fig. 1(C)]. This effect is in concordance with previous findings demonstrating statin-mediated upregulation of collagen II expression, and appears independent of FGFR signaling, since there is no endogenous FGFR signaling taking place in RCS cells. Statins also failed to rescue the FGF-mediated effects in cultured tibias and micromass cultures (Fig. 3; Supp. Fig. 1). These results are contradictory to the observations of positive statin effect on iPS-to-chondrocyte differentiation in vitro and improved growth and cartilage formation in statin-treated iPS cells derived from TD skin fibroblasts3, 44. In contrast to patient derived chondrocytes, the RCS chondrocyte induce their growth-arrest and ECM degradation by FGFR2 and FGFR3, thus leaving a theoretical possibility that while FGFR3 signaling was inhibited by statins, the activation of expressed FGFR2 could still mediate the effects on proliferation and ECM to the full extent. Similarly, the lack of statin activity towards other FGFRs could account for their failure to rescue the FGF-mediated inhibition of cartilaginous differentiation in micromass cultures, since this process is driven mostly by FGFR1 and FGFR2.