A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor-β-driven SMAD3/4-specific gene expression

Abstract

We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-β (TGF-β)/SMAD signaling. Using jnkl/2-knockout (jnk-/-) and IκB kinase-γ/nemo-/- fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-κB/Rel pathways in this phenomenon. We demonstrate that, in a cellular context devoid of JNK activity (i.e. jnk-/- fibroblasts), interleukin-1 and tumor necrosis factor-α (TNF-α) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-α. On the other hand, lack of NF-κB activity in nemo-/- fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-β. In the latter cell type, overexpression of antisense c-jun mRNA or of a dominant-negative form of MKK4 blocked the inhibitory activity of TNF-α, similar to what was observed in normal human dermal fibroblasts. Among JNK substrates, c-Jun and JunB (but not activating transcription factor-2) antagonized TGF-β/SMAD signaling in a JNK-dependent manner. Overexpression of JNK1 in jnk-/- fibroblasts restored the ability of cytokines and Jun proteins to interfere with SMAD signaling. In junAA mouse embryo fibroblasts, in which c-Jun can no longer be phosphorylated by JNK, JunB substituted for c-Jun in mediating the cytokine effect against SMAD-driven transcription in a JNK-dependent manner. These results suggest a critical role for JNK-mediated c-Jun and JunB phosphorylation in transmitting the inhibitory effect of pro-inflammatory cytokines against TGF-β-induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-α against TGF-β-induced up-regulation of type I and III collagens in fibroblasts.