Background Interleukin-6 (IL-6), a potent inflammatory cytokine, plays a key role in the pathogenesis of rheumatoid arthritis (RA), including osteoporosis not only in inflamed joints but possibly also in whole body. Since increase of type 1 collagen N-propeptide, a bone formation marker, is recognized in RA patients treated with tocilizumab, a humanized anti- IL-6 receptor antibody, IL-6 could be thought to have negative effect on osteoblast differentiation. However, previous reports regarding the effects of IL-6 on osteoblast differentiation in vitro are not consistent.
Objectives The objectives of this study was to clarify the effect of IL-6 on osteoblast differentiation in vitro, with consideration of intracellular signaling pathways. Cross-talk between these pathways was also investigated.
Methods Osteoblast differentiation was induced in murine MC3T3-E1 osteoblastic cells with or without addition of IL-6 (soluble IL-6 receptor was used to enhance the effect of IL-6). Osteoblast differentiation was assessed by alkaline phosphatase (ALP) activity, expression of osteoblastic gene such as Runx2 and osteocalcin, and mineralization. ERK and STAT3 pathway phosphorylation was evaluated by Western blotting in the presence or absence of IL-6 as well as each specific inhibitor, U0126 and Stattic, respectively, and their effect on osteoblast differentiation were assessed.
Results IL-6 significantly reduced ALP activity, and expression of osteoblastic gene, Runx2 and osteocalcin, and also inhibited mineralization in a dose-dependent manner, which indicates a negative effect of IL-6 on osteoblast differentiation. Signal transduction analyses by Western blotting demonstrated that IL-6 significantly promoted phosphorylation of ERK and STAT3. The negative effect of IL-6 on ALP activity was restored by inhibition of ERK in a dose-dependent manner. On the other hand, the negative effect of IL-6 on ALP activity was enhanced by inhibition of STAT3. These results indicate that ERK has negative effect on osteoblast differentiation, whereas STAT3 has positive one. Moreover, ERK inhibitor enhanced IL-6-triggered STAT3 phosphorylation, and STAT3 inhibitor enhanced IL-6-triggered ERK phosphorylation, suggesting that ERK and STAT3 signaling pathway negatively regulates each other.
Conclusions IL-6 can be thought to negatively regulate osteoblast differentiation through ERK pathway. On the other hand, STAT3 pathway could have a positive regulatory role in osteoblast differentiation. Predominant activation of ERK signaling pathway compared to STAT3 signaling pathway may result in inhibiting osteoblastic differentiation. Inhibition of ERK might be of potential use in the treatment of osteoporosis in RA.
Garnero, P., et al., Arthritis Rheum, 2010. 62(1): p. 33-43.
Ernst, M. and B.J. Jenkins,Trends Genet, 2004. 20(1): p. 23-32.
Disclosure of Interest None Declared