Abstract
In patients who were treated with exogenous BMP-2 to repair bone fractures or defects, the levels of the inflammatory cytokines such as TNF-α and IL-1β in sera are significantly elevated, which may affect the outcome of bone regeneration. Mitogen-activated protein kinase (MAPK) cascades such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase 1/2 (JNK1/2) have a crucial role in osteogenic differentiation and are activated by both BMP-2 and TNF-α/IL-1β. However, previous studies suggested that the effects of BMP-2 and TNF-α/IL-1β in osteoblastic differentiation are opposite. Here, we investigated the exact role of MAPKs in a BMP-2 and TNF-α/IL-1β co-existed condition. Treatment with TNF-α/IL-1β inhibited BMP-2-induced alkaline phosphatase activity, calcium deposition, osteogenic transcriptional factor Runx2, and the expression of osteogenic markers in C2C12 and MC3T3-E1 cells. This inhibitory effect was independent of the canonical BMP/Smad pathway, suggesting the presence of an alternate regulatory pathway for BMP-2-induced Runx2 activity and subsequent osteoblastic differentiation. We then confirmed that BMP-2, TNF-α, and IL-1β alone can activate p38, ERK1/2, and JNK1/2, respectively. However, only inhibition of p38 and ERK1/2 signaling were required to modulate BMP-2-induced Runx2 expression. Finally, we determined that TNF-α/IL-1β decreased BMP-2-induced Runx2 expression through the activation of p38 and ERK1/2 signaling. Furthermore, strong activation of p38 and ERK1/2 signaling by transfection with CA-MKK3 or CA-MEK1 inhibited BMP-2-induced Runx2 expression and osteoblastic differentiation in C2C12 and MC3T3-E1 cells. Based on these results, we conclude that TNF-α/IL-1β- and BMP-2-activated p38 and ERK1/2 signaling have opposing roles that converge on Runx2 to regulate osteoblastic differentiation. The elucidation of these mechanisms may hasten the development of new strategies and improve the osteoinductive efficacy of BMP-2 in the clinic to enhance osteoblastic differentiation and bone formation.
Highlights
Extended clinical use of Bone morphogenetic protein-2 (BMP-2)/absorbable collagen sponge (ACS) has revealed increasing concerns regarding inflammation-related adverse events such as soft-tissue swelling,[5,6] seroma,[7,8] bone resorption,[9] and the low osteoinductive efficacy of BMP-2.4,10–12 These complications have resulted in unsatisfactory long-term outcomes in clinical applications.[4,13,14]
We previously demonstrated that an inflammatory environment inhibits BMP-2-induced bone mass in vivo and osteoblastic differentiation of BMSCs in vitro through inflammatory cytokines, including TNF-a and IL-1b
We cultured the multipotent C2C12 cells or preosteoblastic MC3T3-E1 cells in BMP-2and/or TNF-a/IL-1b-supplemented medium for several days to study the effects of these inflammatory cytokines on BMP-2-induced osteoblastic differentiation
Summary
The presence of TNF-a or IL-1b significantly suppressed the transcriptional activity of Runx[2] but did not affect the phosphorylation of Smad1/5/8 (Figures 2c and d) These data indicate that TNF-a/IL-1b alone inhibits BMP-2-induced Runx[2] expression and activation but does not affect the BMP/ Smad signaling. Pretreatment with TNF-a/IL-1b did not affect the nuclear translocation of Smad[1] These data indicate that the inhibitory effect of TNF-a/IL-1b on BMP2-induced Runx[2] expression and activation is independent of the BMP/Smad signaling. Similar results were observed in C2C12 cells stimulated with BMP-2 and IL-1b (Figures 5c and d) These results indicated that the BMP-2- and TNF-a/IL-1b-activated p38 and ERK1/2 signaling pathways may have an opposing roles in the regulation of Runx[2] expression and activation.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
