Transforming growth factor-beta (TGF-beta) is a potent anabolic factor for fibroblasts, stimulating extracellular matrix component synthesis and inhibiting the activity of matrix metallo-proteinases. On the other hand, pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) have potent catabolic activities, inhibiting the expression of several extracellular matrix components and activating that of matrix metalloproteinases. The authors aimed to reveal the molecular mechanisms underlying the antagonistic activities of pro-inflammatory cytokines against TGF-beta/SMAD signaling. To further reveal the role of c-Jun, wild type mouse embryonic fibroblasts were nucleofected with a c-Jun expression vector or an empty vector. 24 hours post nucleofection cells were treated with TNF-alpha and cell lysates were analyzed by Western blotting. Significantly higher amounts of c-Jun protein were detected in extracts from fibroblasts transfected with the c-Jun compared to mock transfected cells. Together with other results, it was found that c-Jun over-expression antagonizes TGF-beta/SMAD signaling.
We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-beta (TGF-beta)/SMAD signaling. Using jnk1/2-knockout (jnk(-/-)) and I kappa B kinase-gamma/nemo(-/-) fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-kappa B/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-alpha (TNF-alpha) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-beta. On the other hand, lack of NF-kappa B activity in nemo(-/-) fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-beta. 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-alpha, 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-beta/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-beta-induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-alpha against TGF-beta-induced up-regulation of type I and III collagens in fibroblasts.