Interleukin (IL)-17 is a recently described cytokine involved in the amplification of inflammatory responses and pathologies. A hallmark feature of IL-17 is its ability to induce expression of other cytokines and chemokines. In addition, IL-17 potently synergizes with tumor necrosis factor-alpha (TNFalpha) to up-regulate expression of many target genes, particularly IL-6. Despite the many observations of IL-17 signaling synergy observed to date, little is known about the molecular mechanisms that underlie this phenomenon. In the osteoblastic cell line MC-3T3, we have found that IL-17 and TNFalpha exhibit potent synergy in mediating IL-6 secretion. Here, we show that at least part of the functional cooperation between IL-17 and TNFalpha occurs at the level of IL-6 gene transcription. Both the NF-kappaB and CCAAT/enhancer-binding protein (C/EBP; NF-IL6) sites in the IL-6 promoter are important for cooperative gene expression, but NF-kappaB does not appear to be the direct target of the combined signal. Microarray analysis using the Affymetrix mouse MG-U74v2 chip identified C/EBPdelta as another gene target of combined IL-17- and TNFalpha-induced signaling. Because C/EBP family members are known to control IL-6, we examined whether enhanced C/EBPdelta expression is involved in the cooperative up-regulation of IL-6 by IL-17 and TNFalpha. Accordingly, we show that C/EBPdelta (or the related transcription factor C/EBPbeta) is essential for expression of IL-6. Moreover, overexpression of C/EBPdelta (and, to a lesser extent, C/EBPbeta) could substitute for the IL-17 signal at the level of IL-6 transcription. Thus, C/EBP family members, particularly C/EBPdelta, appear to be important for the functional cooperation between IL-17 and TNFalpha.

RUNX2, a key transcription factor for osteoblast differentiation, is regulated by ERK1/2 and p38 MAP kinase-mediated phosphorylation. However, the specific contribution of each kinase to RUNX2-dependent transcription is not known. Here we investigate ERK and p38 regulation of RUNX2 using a unique P-RUNX2-specific antibody. Both MAP kinases stimulated RUNX2 Ser319 phosphorylation and transcriptional activity. However, a clear preference for ERK1 versus p38α/β was found when the ability of these MAPKs to phosphorylate and activate RUNX2 was compared. Similarly, ERK1 preferentially bound to a consensus MAPK binding site on RUNX2 that was essential for the activity of either kinase. To assess the relative contribution of ERK1/2 and p38 to osteoblast gene expression, MC3T3-E1 preosteoblast cells were grown in control or ascorbic acid (AA)-containing medium ± BMP2/7. AA-induced gene expression, which requires collagen matrix synthesis, was associated with parallel increases in P-ERK and RUNX2-S319-P in the absence of any changes in P-p38. This response was blocked by ERK, but not p38, inhibition. Significantly, in the presence of AA, BMP2/7 synergistically stimulated RUNX2 S319 phosphorylation and transcriptional activity without affecting total RUNX2 and this response was totally dependent on ERK/MAPK activity. In contrast, although p38 inhibition partially blocked BMP-dependent transcription, it did not affect RUNX2 S319 phosphorylation, suggesting the involvement of other phosphorylation sites and/or transcription factors in this response. Based on this work, we conclude that extracellular matrix and BMP regulation of RUNX2 phosphorylation and transcriptional activity in osteoblasts is predominantly mediated by ERK rather than p38 MAPKs. © 2012 American Society for Bone and Mineral Research.